WO2023072076A1 - Reagent and method for diagnosis of nasopharyngeal carcinoma - Google Patents

Reagent and method for diagnosis of nasopharyngeal carcinoma Download PDF

Info

Publication number
WO2023072076A1
WO2023072076A1 PCT/CN2022/127393 CN2022127393W WO2023072076A1 WO 2023072076 A1 WO2023072076 A1 WO 2023072076A1 CN 2022127393 W CN2022127393 W CN 2022127393W WO 2023072076 A1 WO2023072076 A1 WO 2023072076A1
Authority
WO
WIPO (PCT)
Prior art keywords
content
solution
epstein
mirna
barr virus
Prior art date
Application number
PCT/CN2022/127393
Other languages
French (fr)
Chinese (zh)
Inventor
王纯婷
马璐
隋亚鑫
皮潇特
马昕怡
刘刚
贾卫华
郑小辉
李锡照
周婷
吕宁
陈一友
Original Assignee
杭州诺辉健康科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 杭州诺辉健康科技有限公司 filed Critical 杭州诺辉健康科技有限公司
Publication of WO2023072076A1 publication Critical patent/WO2023072076A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the present invention relates to the technical field of diagnosis of nasopharyngeal carcinoma, in particular, to reagents and methods for diagnosis of nasopharyngeal carcinoma, and related biomarkers.
  • Nasopharyngeal carcinoma refers to malignant tumors that occur in the posterior wall of the nasopharynx and pharyngeal recesses. It is one of the most frequently occurring malignant tumors in China, and its incidence rate is the first among malignant tumors of the ear, nose and throat.
  • the five-year survival rate of early nasopharyngeal carcinoma after treatment can reach more than 90%, while the five-year survival rate of middle and advanced nasopharyngeal carcinoma is less than 50%.
  • the disease has progressed to the middle and late stage when the first visit to the doctor, so promoting early screening and early diagnosis and treatment of nasopharyngeal carcinoma is the key to improving the prognosis of nasopharyngeal carcinoma.
  • Epstein-Barr virus Epstein-Barr virus
  • WHO type III undifferentiated nasopharyngeal carcinoma
  • WHO type II non-angular nasopharyngeal carcinoma
  • EBV infection of nasopharyngeal epithelial cells is an important feature of nasopharyngeal carcinoma.
  • nasopharyngeal carcinoma The gold standard for clinical diagnosis of nasopharyngeal carcinoma is tissue biopsy under nasopharyngoscope, followed by pathological diagnosis by pathologists. Due to the relatively traumatic nature of the specimens, the small size of the specimens, and the need to observe with the naked eye under a microscope, about 10% of the patients failed to be diagnosed when the specimens were first drawn. Delayed illness. In addition, nasopharyngeal biopsy strictly relies on the clinical experience of clinicians and medical devices such as nasopharyngeal electronic mirrors, making it difficult to carry out extensive screening of nasopharyngeal carcinoma in high-incidence sites and early diagnosis in primary medical units.
  • EBV markers based on blood samples also relies on clinical nurses to take blood samples, which limits its promotion and detection of the whole population in high-incidence sites of nasopharyngeal carcinoma, and the lack of accuracy also reduces the compliance of the public. Therefore, it is still of great significance to develop sampling techniques that are easy to promote and apply in high-occurrence sites and efficient diagnostic molecular markers.
  • nasopharyngeal carcinoma originates from the nasopharynx
  • the non-invasive and efficient sampling of nasopharynx and the detection of corresponding molecular markers can more directly reflect the condition of the lesion, and theoretically, early primary or recurrent nasopharynx can be detected in time cancer patients.
  • the pathological biopsy sampling of the nasopharynx is traumatic, but it is a feasible technical solution to take nasopharyngeal samples by nasopharyngeal brush or nasopharyngeal swab.
  • nasopharyngeal brush is a method that can directly obtain samples of nasopharyngeal lesions, it not only has the advantages of painlessness and non-invasiveness, but also the obtained exfoliated cells can directly reflect the characteristics of nasopharyngeal tumors.
  • nasopharyngeal brush examination has the advantages of non-invasive and repeatable sampling, but in order to improve the accuracy of sampling, previous studies still rely on clinicians and nasopharyngoscope guidance, which hinders This technical solution is widely used in the screening of nasopharyngeal carcinoma in the field. Therefore, nasopharyngeal brush or nasopharyngeal swab detection (referred to as nasopharyngeal blind inspection) that does not rely on clinicians and nasopharyngoscope guidance has natural advantages that are conducive to popularization.
  • the current disadvantage of nasopharyngeal blind inspection is the sampling accuracy. Therefore, it is of great significance to develop molecular markers and their detection systems suitable for nasopharyngeal blind brush sampling system.
  • kits for detecting Epstein-Barr virus characterized in that, the kit includes nucleic acid methylation detection reagents, miRNA detection reagents, miRNA extraction reagents, and/or sample preservation One or more reagents of the liquid.
  • kits for nasopharyngeal carcinoma detection characterized in that, the kit includes nucleic acid methylation detection reagents, miRNA detection reagents, miRNA extraction reagents, and/or samples One or more reagents of the preservation solution.
  • the kit includes a nucleic acid methylation detection reagent, which can perform methylation detection for methylation sites in the Epstein-Barr virus genome.
  • the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) (NCBI Reference Sequence: NC_007605.1; ACCESSION 10717bp, 11029bp, 14015bp, 14566bp, 45850bp , 57946bp, 662 27bp and 128103bp sites.
  • the methylation sites in the Epstein-Barr virus genome comprise sites corresponding to 10717bp, 11029bp, and 14015bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: 11029bp and 45850bp of the Epstein-Barr virus genome DNA sequence corresponding to GenBank LOCUS NC_007605 (SEQ ID NO: 94) , 57946bp, 66227bp and 128103bp sites.
  • the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: 11029 bp and 57946 bp of the Epstein-Barr virus genome DNA sequence corresponding to GenBank LOCUS NC_007605 (SEQ ID NO: 94) , and the 66227bp site.
  • the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 11029 bp of the Epstein-Barr virus genome DNA sequence in GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 10717 bp of the Epstein-Barr virus genome DNA sequence in GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 14015 bp of the Epstein-Barr virus genome DNA sequence in GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 14566 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 45850 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 57946 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 66227 bp of the Epstein-Barr virus genome DNA sequence in GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 128103 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • the kit includes the miRNA detection reagent, and the miRNA detection reagent can detect the content of one or more miRNAs from Epstein-Barr virus.
  • the miRNA comprises one or more of the following miRNAs: EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR-BART6-3p, EBV-miR-BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p. In some embodiments, all six of said miRNAs are included.
  • the miRNAs comprise EBV-miR-BART1-5p and EBV-miR-BART7-3p.
  • the miRNA comprises EBV-miR-BART1-5p. In some embodiments, the miRNA comprises EBV-miR-BART2-5p. In some embodiments, the miRNA comprises EBV-miR-BART6-3p. In some embodiments, the miRNA comprises EBV-miR-BART7-3p. In some embodiments, the miRNA comprises EBV-miR-BART13-3p. In some embodiments, the miRNA comprises EBV-miR-BART17-5p.
  • the kit includes the sample preservation solution, and the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and/or trisodium phosphate one or more components.
  • the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
  • One aspect of the present invention provides a kit for preserving biological samples containing nucleic acid, which is characterized in that it includes a sample preservation solution, and the sample preservation solution includes EDTA, NaCl, absolute ethanol, sodium citrate, NP40, gluten Glutathione, and Trisodium Phosphate.
  • the content range of the EDTA is 0.7%-1.5% (w/w); the content range of the NaCl is 0.01%-0.1% (w/w); The concentration range of the dehydrated alcohol is 15%-35% (w/w); the content range of the sodium citrate is 0.1%-1.0% (w/w); the concentration range of the NP40 is 0.01%-0.1 % (v/v); said glutathione content ranges from 0.07% to 0.7% (w/w); and/or said trisodium phosphate content ranges from 0.07% to 0.4% (w/w) .
  • the content of the EDTA is about 1.05% (w/w); the content of the NaCl is about 0.035% (w/w); the concentration of the absolute ethanol is about 27.6% (w/w); the content of the sodium citrate is about 0.21% (w/w); the concentration of the NP40 is about 0.035% (v/v); the content of the glutathione is about 0.21% (w/w); and/or said trisodium phosphate is present in an amount of about 0.14% (w/w).
  • the sample preservation solution comprises antibiotics.
  • the antibiotic comprises double penicillin-streptomycin.
  • the pH value of the sample preservation solution is 7.6-8.0.
  • the pH value of the sample preservation solution is 7.8.
  • the sample preservation solution has a better preservation effect on DNA, miRNA, and/or DNA methylation than the control sample preservation solution.
  • the DNA is derived from Epstein-Barr virus
  • the miRNA is derived from Epstein-Barr virus
  • the DNA methylation is methylation of Epstein-Barr virus DNA.
  • the kit comprises the miRNA extraction reagent, and the miRNA extraction reagent comprises a lysis solution, a binding solution, a washing solution 1, and/or a washing solution 2.
  • One aspect of the present invention provides a kit comprising miRNA extraction reagents, characterized in that the miRNA extraction reagents comprise a lysing solution, a binding solution, a washing solution 1, and/or a washing solution 2.
  • the miRNA extraction reagent comprises the lysate comprising one or more components selected from guanidine thiocyanate, citric acid, Triton-100, and/or EDTA . In some embodiments, the miRNA extraction reagent comprises the lysate comprising guanidine thiocyanate, citric acid, Triton-100, and EDTA.
  • the content range of the guanidine thiocyanate is 28%-40% (w/w); the content range of the citric acid is 1.8%-2.6% (w/w ); the content range of the Triton-100 is 0.8%-1.6% (w/w); the content range of the EDTA is 0.5%-0.9% (w/w); and/or the lysate
  • the pH range is 3.8-4.2.
  • the content of the guanidine thiocyanate is about 32% (w/w); the content of the citric acid is about 2.2% (w/w); The content of Tong-100 is about 1.1% (w/w); the content of the EDTA is about 0.7% (w/w); and/or the pH value of the lysate is about 4.0.
  • the miRNA extraction reagent comprises the binding solution, which comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, lauryl sarcosine Sodium, and/or one or more components of isopropanol.
  • the binding solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, sodium sarcosine, and isopropanol.
  • the content range of the magnesium chloride is 0.1%-0.4% (w/w); the content range of the disodium edetate is 0.5%-1.0% (w/w).
  • the content range of the sodium bicarbonate is 0.6%-1.0% (w/w); the content range of the guanidine thiocyanate is 13%-16% (w/w); the dodecane
  • the content range of sodium sarcosinate is 1.0%-1.4% (w/w); the content range of the isopropanol is 60%-80% (w/w); and/or the pH value of the combination solution The range is 4.8-5.2.
  • the magnesium chloride content is about 0.24% (w/w); the disodium edetate content is about 0.71% (w/w); the The content of sodium bicarbonate is about 0.83% (w/w); The content of said guanidine thiocyanate is about 14.9% (w/w); The content of said sodium lauryl sarcosinate is about 1.2% ( w/w); the content of the isopropanol is about 70% (w/w); and/or the pH value of the combination solution is about 5.0.
  • the miRNA extraction reagent comprises the washing solution 1, and the washing solution 1 comprises disodium edetate, sodium bicarbonate, guanidinium thiocyanate, and/or dodecyl One or more components of sodium sarcosinate.
  • the miRNA extraction reagent comprises the rinse solution 1 comprising disodium edetate, sodium bicarbonate, guanidinium thiocyanate, and sodium lauryl sarcosinate .
  • the content range of the disodium edetate is 0.4%-0.9% (w/w); the content range of the sodium bicarbonate is 0.5%-0.9% % (w/w); The content scope of described guanidinium thiocyanate is 50%-55% (w/w); The content scope of described sodium lauryl sarcosinate is 0.8%-1.2% (w/ w); and/or the pH range of the rinse solution 1 is 6.2-6.6.
  • the disodium edetate content is about 0.5% (w/w); the sodium bicarbonate content is about 0.6% (w/w) the content of the guanidine thiocyanate is about 53% (w/w); the content of the sodium lauryl sarcosinate is about 0.9% (w/w); and/or the rinse solution 1
  • the pH is about 6.4.
  • the miRNA extraction reagent comprises the rinsing solution 2, and the rinsing solution 2 comprises tris (Tris) or sodium chloride.
  • the miRNA extraction reagent comprises the rinse solution 2 comprising tris (Tris) and sodium chloride.
  • the content range of the tris (Tris) is 0.5%-0.7% (w/w); the content range of the sodium chloride is 0.6% -1.8% (w/w); and/or the pH value of the rinse solution 2 is in the range of 6.6-7.0.
  • the content of the tris (Tris) is about 0.6% (w/w); the content of the sodium chloride is about 1.2% (w/ w); and/or the pH value of the rinse solution 2 is about 6.8.
  • the miRNA extraction reagent comprises RNA-adsorbing magnetic beads.
  • the magnetic core of the magnetic beads is ferric hydroxide, coated with silicon dioxide, and embedded with hydroxyl groups on the surface.
  • the particle diameter of the magnetic beads is 50-100 nm.
  • the kit further comprises a nasopharyngeal brush or nasopharyngeal swab.
  • the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab that does not rely on the guidance of a clinician and a nasopharyngoscope.
  • the kit further includes Epstein-Barr virus load detection reagents.
  • the Epstein-Barr virus load detection reagent comprises a detection reagent for the BamHI-W fragment in the Epstein-Barr virus genome and/or the LMP-2 gene.
  • the EB virus load detection reagent comprises a detection reagent for LMP-2 gene.
  • the kit is a nasopharyngeal carcinoma screening kit. In some embodiments, the kit is a nasopharyngeal carcinoma diagnostic kit.
  • One aspect of the present invention provides a use of the kit described in the present invention in preparing products for screening nasopharyngeal carcinoma.
  • One aspect of the present invention provides a use of the kit described in the present invention in preparing products for nasopharyngeal carcinoma diagnosis.
  • One aspect of the present invention provides a method for detecting Epstein-Barr virus-related nucleic acids in a sample, characterized in that the method comprises the following steps:
  • step (b-2) the miRNA is firstly extracted from the sample preservation solution, and then the content of the miRNA is detected.
  • the method includes step (b-1), and the methylation site detected by the Epstein-Barr virus genome nucleic acid methylation comprises one or more of the following sites: equivalent to GenBank LOCUS The sites of 10717bp, 11029bp, 14015bp, 14566bp, 45850bp, 57946bp, 66227bp and 128103bp of the Epstein-Barr virus genome DNA sequence of NC_007605 (SEQ ID NO:94).
  • the methylation site comprises sites corresponding to 10717bp, 11029bp, and 14015bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • the methylation site comprises a site corresponding to 10717bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 11029 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 14015 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • the methylation site comprises a site corresponding to 14566 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 45850 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 57946 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • the methylation site comprises a site corresponding to 66227bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 128103bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • the method comprises step (b-2), and the miRNA comprises one or more of the following miRNAs: EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR- BART6-3p, EBV-miR-BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p. In some embodiments, all six of said miRNAs are included. In some embodiments, the miRNAs comprise EBV-miR-BART1-5p and EBV-miR-BART7-3p. In some embodiments, the miRNA comprises EBV-miR-BART1-5p.
  • the miRNA comprises EBV-miR-BART2-5p. In some embodiments, the miRNA comprises EBV-miR-BART6-3p. In some embodiments, the miRNA comprises EBV-miR-BART7-3p. In some embodiments, the miRNA comprises EBV-miR-BART13-3p. In some embodiments, the miRNA comprises EBV-miR-BART17-5p.
  • the step of extracting the Epstein-Barr virus-related miRNA includes: i) adding a lysate to the sample preservation solution; in some embodiments, proteinase K is also added; ii) adding a binding solution and a nucleic acid adsorption material ; iii) removing the liquid portion, adding a rinsing solution to the nucleic acid adsorption material for cleaning; and iv) removing the liquid portion, adding an eluent to the nucleic acid adsorption material, and adsorbing the nucleic acid and/or the miRNA from the nucleic acid material eluted.
  • One aspect of the present invention provides a method for extracting miRNA, comprising the following steps: i) adding a lysate to the sample preservation solution; in some embodiments, proteinase K is also added; ii) adding a binding solution and a nucleic acid adsorption material ; iii) removing the liquid portion, adding a rinsing solution to the nucleic acid adsorption material for cleaning; and iv) removing the liquid portion, adding an eluent to the nucleic acid adsorption material, and adsorbing the nucleic acid and/or the miRNA from the nucleic acid material eluted.
  • the lysate comprises one or more components selected from guanidine thiocyanate, citric acid, Triton-100, and/or EDTA. In some embodiments, the lysate comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA.
  • the content range of the guanidine thiocyanate is 28%-40% (w/w); the content range of the citric acid is 1.8%-2.6% (w/w ); the content range of the Triton-100 is 0.8%-1.6% (w/w); the content range of the EDTA is 0.5%-0.9% (w/w); and/or the lysate
  • the pH range is 3.8-4.2.
  • the content of the guanidine thiocyanate is about 32% (w/w); the content of the citric acid is about 2.2% (w/w); The content of Tong-100 is about 1.1% (w/w); the content of the EDTA is about 0.7% (w/w); and/or the pH value of the lysate is about 4.0.
  • the binding solution comprises one selected from magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, sodium lauryl sarcosine, and/or isopropanol or multiple components.
  • the binding solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, sodium sarcosine, and isopropanol.
  • the content range of the magnesium chloride is 0.1%-0.4% (w/w); the content range of the disodium edetate is 0.5%-1.0% (w/w).
  • the content range of the sodium bicarbonate is 0.6%-1.0% (w/w); the content range of the guanidine thiocyanate is 13%-16% (w/w); the dodecane
  • the content range of sodium sarcosinate is 1.0%-1.4% (w/w); the content range of the isopropanol is 60%-80% (w/w); and/or the pH value of the combination solution The range is 4.8-5.2.
  • the magnesium chloride content is about 0.24% (w/w); the disodium edetate content is about 0.71% (w/w); the The content of sodium bicarbonate is about 0.83% (w/w); The content of said guanidine thiocyanate is about 14.9% (w/w); The content of said sodium lauryl sarcosinate is about 1.2% ( w/w); the content of the isopropanol is about 70% (w/w); and/or the pH value of the combination solution is about 5.0.
  • step iii) of the method rinse solution 1 and rinse solution 2 are sequentially added to the nucleic acid adsorption material for washing, and the liquid part is removed after each wash and then the next wash is performed.
  • the nucleic acid adsorption material is washed at least twice with the rinsing solution 2 .
  • the rinse solution 1 comprises one or more components selected from disodium edetate, sodium bicarbonate, guanidine thiocyanate, and/or sodium lauryl sarcosine .
  • the rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidine thiocyanate, and sodium lauryl sarcosinate.
  • the content range of the disodium edetate is 0.4%-0.9% (w/w); the content range of the sodium bicarbonate is 0.5%-0.9% % (w/w); The content scope of described guanidinium thiocyanate is 50%-55% (w/w); The content scope of described sodium lauryl sarcosinate is 0.8%-1.2% (w/ w); and/or the pH range of the rinse solution 1 is 6.2-6.6.
  • the disodium edetate content is about 0.5% (w/w); the sodium bicarbonate content is about 0.6% (w/w) the content of the guanidine thiocyanate is about 53% (w/w); the content of the sodium lauryl sarcosinate is about 0.9% (w/w); and/or the rinse solution 1
  • the pH is about 6.4.
  • ethanol is added to the rinsing solution 1, and the volume ratio of the rinsing solution 1:ethanol is 1:1-1:2. In some embodiments, the volume ratio is about 2:3.
  • the rinse solution 2 includes tris (Tris) or sodium chloride. In some embodiments, the rinse solution 2 comprises tris (Tris) and sodium chloride. In some embodiments, in the rinse liquid 2: the content range of the tris (Tris) is 0.5%-0.7% (w/w); the content range of the sodium chloride is 0.6% -1.8% (w/w); and/or the pH value of the rinse solution 2 is in the range of 6.6-7.0. In some embodiments, in the rinse solution 2: the content of the tris (Tris) is about 0.6% (w/w); the content of the sodium chloride is about 1.2% (w/ w); and/or the pH value of the rinse solution 2 is about 6.8.
  • ethanol is added to the rinsing solution 2, and the volume ratio of the rinsing solution 2:ethanol is 1:1.6-1:3. In some embodiments, the volume ratio is about 1:2.3.
  • the eluent of the method is DEPC water.
  • the nucleic acid adsorption material is magnetic beads capable of adsorbing RNA.
  • the magnetic core of the magnetic beads is ferric hydroxide, coated with silicon dioxide, and embedded with hydroxyl groups on the surface.
  • the particle size of the magnetic beads is 50-100 nm.
  • the sample preservation solution used in the method comprises one or more groups selected from EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and/or trisodium phosphate point.
  • the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
  • One aspect of the present invention provides a method for preserving a nucleic acid-containing biological sample, characterized in that the sample is placed in a sample preservation solution, and the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
  • the content range of the EDTA is 0.7%-1.5% (w/w); the content range of the NaCl is 0.01%-0.1% (w/w ); the concentration range of the dehydrated alcohol is 15%-35% (w/w); the content range of the sodium citrate is 0.1%-1.0% (w/w); the concentration range of the NP40 is 0.01 %-0.1% (v/v); said glutathione content range is 0.07%-0.7% (w/w); and/or said trisodium phosphate content range is 0.07%-0.4% (w /w).
  • the content of the EDTA is about 1.05% (w/w); the content of the NaCl is about 0.035% (w/w); the concentration of the absolute ethanol is about 27.6% (w/w); the content of the sodium citrate is about 0.21% (w/w); the concentration of the NP40 is about 0.035% (v/v); the content of the glutathione is about 0.21% (w/w); and/or said trisodium phosphate is present in an amount of about 0.14% (w/w).
  • the sample preservation solution comprises antibiotics.
  • the antibiotic comprises double penicillin-streptomycin.
  • the pH value of the sample preservation solution is 7.6-8.0.
  • the pH value of the sample preservation solution is 7.8.
  • the sample preservation solution has a better preservation effect on DNA, miRNA, and/or DNA methylation than the control sample preservation solution.
  • the method includes step (b-3), and the detection of the Epstein-Barr virus load includes the detection of the BamHI-W fragment in the Epstein-Barr virus genome, and/or the LMP-2 gene.
  • the detection of the EB virus load comprises the detection of the LMP-2 gene.
  • the method is used for screening for nasopharyngeal carcinoma. In some embodiments, the method is used for the diagnosis of nasopharyngeal carcinoma. In some embodiments, the sample is a nasopharyngeal sample.
  • the method before step (a), further comprises using a nasopharyngeal brush and/or a nasopharyngeal swab to obtain the sample.
  • the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab that does not rely on the guidance of a clinician and a nasopharyngoscope.
  • One aspect of the present invention provides a method for treating nasopharyngeal carcinoma in an individual in need, comprising providing the individual with drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus therapy, wherein the individual's The sample was tested by the method of the present invention and judged to be positive for nasopharyngeal carcinoma.
  • One aspect of the present invention provides a method for treating nasopharyngeal carcinoma in an individual in need, comprising 1) performing detection according to the method of the present invention on a sample from the individual; 2) if the detection result shows that the nasal Pharyngeal cancer positive, provide nasopharyngeal cancer treatment.
  • the nasopharyngeal carcinoma is treated with drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus therapy.
  • the object of the present invention is to provide a nasopharyngeal carcinoma diagnostic marker in order to overcome the above-mentioned deficiencies of the prior art.
  • the first object of the present invention is to provide the use/application of a reagent for detecting Epstein-Barr virus methylation sites and/or Epstein-Barr virus-related miRNAs in the preparation of nasopharyngeal carcinoma screening and/or diagnostic kits.
  • the second object of the present invention is to provide a product for screening and/or diagnosing nasopharyngeal carcinoma.
  • the third object of the present invention is to provide a product and method for nucleic acid sample preservation.
  • the fourth object of the present invention is to provide a product and method for RNA extraction, especially miRNA extraction.
  • the diagnosis is an auxiliary diagnosis.
  • the screening is early screening, that is, early screening or pre-onset screening of nasopharyngeal carcinoma.
  • the nasopharyngeal sample used is a nasopharyngeal brush and/or nasopharyngeal swab sample.
  • the nasopharyngeal brush and/or nasopharyngeal swab sample is a nasopharyngeal brush and/or nasopharyngeal swab sample that does not rely on the guidance of a clinician and a nasopharyngoscope.
  • Epstein-Barr virus DNA methylation sites and/or Epstein-Barr virus-related miRNAs in nasopharyngeal samples can be used as molecular markers for the diagnosis of nasopharyngeal carcinoma, and the methylation difference and/or miRNA content can be used for nasopharyngeal cancer. Cancer diagnosis and prognosis.
  • the sensitivity and specificity of Epstein-Barr virus DNA methylation sites as molecular markers for the diagnosis of nasopharyngeal carcinoma are related to the Epstein-Barr virus DNA load Equivalent; diagnostic sensitivity of Epstein-Barr virus DNA load as a diagnostic molecular marker for nasopharyngeal carcinoma when the sample is a nasopharyngeal brush or nasopharyngeal swab (blind examination of the nasopharynx) guided by a clinician and nasopharyngoscope
  • the sensitivity and specificity of Epstein-Barr virus methylation site diagnosis were significantly better than Epstein-Barr virus DNA load.
  • Epstein-Barr virus methylation sites and/or Epstein-Barr virus-related miRNAs can be better Applied to the prediction of nasopharyngeal carcinoma.
  • This solution has the characteristics of non-invasive, non-destructive and repeatable sampling, and does not rely on clinicians and medical devices such as nasopharyngoscopes.
  • the accuracy of identifying nasopharyngeal carcinoma is above 90%, so the overall technical solution is very suitable for screening nasopharyngeal carcinoma among large-scale populations in high-incidence sites.
  • kits for nasopharyngeal carcinoma detection characterized in that, the kit includes nucleic acid methylation detection reagents, miRNA detection reagents, miRNA extraction reagents, and/or samples One or more reagents of the preservation solution.
  • the kit includes a nucleic acid methylation detection reagent, which can perform methylation detection for methylation sites in the Epstein-Barr virus genome.
  • the kit comprises miRNA detection reagents.
  • the miRNA detection reagent can detect the content of one or more miRNAs from Epstein-Barr virus.
  • the kit comprises a sample preservation solution.
  • the kit comprises miRNA extraction reagents.
  • the kit comprises Epstein-Barr viral load detection reagents.
  • One aspect of the present invention provides a method for detecting Epstein-Barr virus-related nucleic acids in a sample, characterized in that the method comprises the following steps:
  • (b-2) extract Epstein-Barr virus-related miRNA from described sample preservation solution and detect the content of described miRNA; And/or
  • the method includes the step (b-1) of detecting methylation of Epstein-Barr virus genome nucleic acid in the sample preservation solution containing the sample.
  • the method includes step (b-2) extracting Epstein-Barr virus-related miRNA from the sample preservation solution and detecting the content of the miRNA.
  • the method includes step (b-3) performing detection of Epstein-Barr virus nucleic acid load on the sample preservation solution containing the sample.
  • the method is for screening and/or diagnosis of nasopharyngeal carcinoma.
  • the sample is a nasopharyngeal sample.
  • the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab that does not rely on the guidance of a clinician and a nasopharyngoscope.
  • the process of obtaining a sample is independent of clinician and nasopharyngoscopy guidance.
  • the terms “about” and “approximately” are used as equivalents. Any numerical value with or without about/approximately used in this application is meant to cover any normal fluctuations understood by those of ordinary skill in the relevant art. In some embodiments, the term “about” or “approximately” refers to a range of 10% in either direction (greater than or less than) of the stated reference value, unless otherwise stated or otherwise evident from the context (except for such numerical will exceed 100% of the possible value).
  • the methylation sites in the EBV genome include sites located in the C promoter region of the EBV genome. In some embodiments, the methylation sites in the EBV genome include sites in the w promoter region on the EBV genome.
  • the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) (NCBI Reference Sequence: NC_007605.1; ACCESSION : NC_007605; VERSION: NC_007605.1; 10717bp, 11029bp, 14015bp, 14566bp, 45850bp, 57946bp, 66227 of the Epstein-Barr virus genomic DNA sequence obtained from: https://www.ncbi.nlm.nih.gov/nuccore/NC_007605) bp and 128103bp sites.
  • the methylation sites in the Epstein-Barr virus genome include 1, 2, 3, 4, 5, 6, 7, or 8 of these sites. In some embodiments, the methylation sites in the Epstein-Barr virus genome comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, and at least 7 of these sites , or at least 8 sites. Preferably, the methylation site comprises sites corresponding to 10717bp, 11029bp, and 14015bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 10717 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 11029 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 14015 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 14566 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 45850 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 57946 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 66227 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 128103 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: 10717bp, 11029bp of the Epstein-Barr virus genome DNA sequence equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) , 14015bp, 14566bp, 45850bp, 57946bp, 66227bp and 128103bp sites.
  • the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: 11029bp and 45850bp of the Epstein-Barr virus genome DNA sequence equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) , 57946bp, 66227bp and 128103bp sites. In some embodiments, the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: 11029bp and 57946bp of the Epstein-Barr virus genome DNA sequence equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) , and the 66227bp site.
  • the miRNA detected comprises one or more of the following miRNAs: EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR-BART6-3p, EBV-miR-BART7-3p , EBV-miR-BART13-3p, and EBV-miR-BART17-5p.
  • the miRNA comprises 1, 2, 3, 4, 5, or 6 selected from these miRNAs.
  • the miRNA comprises at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 miRNAs selected from these miRNAs.
  • the miRNAs comprise EBV-miR-BART1-5p and EBV-miR-BART7-3p. In some embodiments, the miRNAs comprise these six miRNAs.
  • the miRNA comprises EBV-miR-BART1-5p.
  • the sequence of the EBV-miR-BART1-5p is UCUUAGUGGAAGUGACGUGCUGUG (SEQ ID NO: 39).
  • the miRNA comprises EBV-miR-BART2-5p.
  • the sequence of the EBV-miR-BART2-5p is UAUUUUCUGCAUUCGCCCUUGC (SEQ ID NO: 40).
  • the miRNA comprises EBV-miR-BART6-3p.
  • the sequence of the EBV-miR-BART6-3p is CGGGGAUCGGACUAGCCUUAGA (SEQ ID NO: 41).
  • the miRNA comprises EBV-miR-BART7-3p.
  • the sequence of the EBV-miR-BART7-3p is CAUCAUAGUCCAGUGUCCAGGG (SEQ ID NO: 42).
  • the miRNA comprises EBV-miR-BART13-3p.
  • the sequence of the EBV-miR-BART13-3p is UGUAACUUGCCAGGGACGGCUGA (SEQ ID NO: 43).
  • the miRNA comprises EBV-miR-BART17-5p.
  • the sequence of the EBV-miR-BART17-5p is UAAGAGGACGCAGGCAUACAAG (SEQ ID NO: 44).
  • the detection reagents herein can detect the methylation of sites in the Epstein-Barr virus genome and perform methylation differential analysis.
  • the methylation difference analysis is to detect the Ct value after amplification of each site amplification primer and methylated probe and the Ct value after amplification of amplification primer and unmethylated probe Difference.
  • the formula for calculating the methylation difference (Methylation Difference, MD) value of each site is:
  • CT m is the Ct value after amplification with the amplification primers and methylated probes at this site
  • CT u is the Ct value after amplification with the amplification primers and unmethylated probes at this site.
  • diagnosis of nasopharyngeal carcinoma can be carried out by using the MD value of each site to construct a model.
  • the combination of detection primers and probes for the methylation site is selected from any combination of primers and probes shown in Table 2, Table 12, and Table 30.
  • the detection primer and probe combination of each methylation site is selected from any one of the following combinations (the site information of Epstein-Barr virus genomic DNA is relative to SEQ ID NO: 94):
  • Reverse primer TTAAACTCTCTTTATTAACTATAATC (SEQ ID NO: 8),
  • Methylation probe FAM-AGATTTAGGTTCGTGGGGTATTTAG-BHQ 1 (SEQ ID NO: 13),
  • Methylation probe FAM-AGGAAGATGTCGCGCGGGTTAG-BHQ1 (SEQ ID NO: 21),
  • Reverse primer CTCCAAATTCAAACTTCTTTAAC (SEQ ID NO: 24),
  • Methylation probe FAM-TGTTTTCGTCGCGTAGGTTAGTA-BHQ1 (SEQ ID NO: 25),
  • Unmethylated probe HEX-TGTTTTTGTTGTGTAGGTTAGTAATG-BHQ1 (SEQ ID NO: 26).
  • Reverse primer TAATTATTCGCAAAACCGCCCT (SEQ ID NO: 34),
  • Reverse primer AACAAAATAACGCCCATTCG (SEQ ID NO: 37),
  • Reverse primer TCTCTTATTAACTATAATCCGTCGC (SEQ ID NO: 80),
  • Reverse primer CTTACCTCTAACCCGATACCG (SEQ ID NO: 83),
  • Probe FAM-ACCTTCACTTCGATCTCCCCTA-MGB (SEQ ID NO: 84),
  • Reverse primer AACAAAACGTAATTAATCCCGC (SEQ ID NO: 77)
  • Probe FAM-TACTCCACCTCTAAAATCCCA-MGB (SEQ ID NO: 78)
  • the detection reagents/methods herein can detect and differentially analyze miRNA content.
  • the detection primer and probe combination of the miRNA is selected from any combination of the following:
  • miR-BART1-5p forward primer GCCGTCTTAGTGGAAGTGACG (SEQ ID NO: 46),
  • miR-BART1-5p reverse primer TGCAGGGTCCGAGGTATTC (SEQ ID NO: 47),
  • miR-BART1-5p probe FAM-ACCAGAGCCCACAGCA-MGB (SEQ ID NO: 48).
  • miR-BART2-5p forward primer GCCGCTATTTTCTGCATTCG (SEQ ID NO: 50),
  • miR-BART2-5p reverse primer TGCAGGGTCCGAGGTATTC (SEQ ID NO: 51),
  • miR-BART2-5p probe FAM-CCAGAGCCGCAAGG-MGB (SEQ ID NO: 52),
  • miR-BART6-3p forward primer GCCGGGGATCGGACTAG (SEQ ID NO: 54),
  • miR-BART6-3p reverse primer CAGTGCAGGGTCCGAGGTAT (SEQ ID NO: 55),
  • miR-BART6-3p probe VIC-ACTGGATACGACTCTAAGG-MGB (SEQ ID NO: 56),
  • miR-BART7-3p forward primer CGGCCATCATAGTCCAGTGT (SEQ ID NO: 58),
  • miR-BART7-3p reverse primer TGCAGGGTCCGAGGTATTC (SEQ ID NO: 59),
  • miR-BART7-3p probe FAM-ACCAGAGCCCCCTGGA-MGB (SEQ ID NO: 60),
  • miR-BART13-3p forward primer CGGTGTAACTTGCCAGGGA (SEQ ID NO: 62),
  • miR-BART13-3p reverse primer CAGTGCAGGGTCCGAGGTAT (SEQ ID NO: 63),
  • miR-BART13-3p probe VIC-TGGATACGACTCAGCCG-MGB (SEQ ID NO: 64),
  • miR-BART17-5p forward primer CGGTAAGAGGACGCAGGC (SEQ ID NO: 66),
  • miR-BART17-5p reverse primer CAGTGCAGGGTCCGAGGTAT (SEQ ID NO: 67),
  • miR-BART17-5p probe VIC-ACTGGATACGACCTTGTATG-MGB (SEQ ID NO: 68), primer/probe combination for miR-16-5p:
  • miR-16-5p forward primer CGGGCTAGCAGCACGTAAA (SEQ ID NO: 70),
  • miR-16-5p reverse primer TGCAGGGTCCGAGGTATTC (SEQ ID NO: 71),
  • miR-16-5p probe CY5-CAGAGCCCGCCAATA-MGB (SEQ ID NO: 72).
  • the detection reagent/method herein can detect Epstein-Barr virus load.
  • the EB virus load detection comprises a nucleotide sequence such as primers shown in SEQ ID NO: 1 to 2 and a nucleotide sequence such as a probe shown in SEQ ID NO: 3 for quantitative
  • the amplified target gene is the BamHI-w fragment of Epstein-Barr virus.
  • forward primer 5'-CCCAACACT CCACCACACC-3' (SEQ ID NO: 1)
  • reverse primer 5'-TCTTAGGAGCTGTCCGAGGG-3'
  • probe 5'-FAM -CACACACTACACACACCCACCCGTCTC-TAMRA-3' SEQ ID NO: 3
  • the primers and probes include forward primer TGCCAAAGAGCCAGATCTAAG (SEQ ID NO: 27), reverse primer AGTGTCAGATTTCGGGTCCAAA (SEQ ID NO: 28), probe FAM-CAGCCCCAAAGCGGGTGCA-BHQ 1 (SEQ ID NO: 29)
  • the detection of the EB virus load comprises primers and probes for detecting the housekeeping gene ⁇ -globin gene.
  • the primer nucleotide sequence of the housekeeping gene ⁇ -globin gene is shown in SEQ ID NO: 4 to 5
  • the probe nucleotide sequence is shown in SEQ ID NO: 6.
  • the two primer sequences are 5'-GTGCACCTGACTCCTGAGGAGA-3' (SEQ ID NO: 4) and 5'-CCTTGATACCAACCTGCCCAG-3' (SEQ ID NO: 5)
  • the probe sequence is 5'-FAM-AAGGTGAACGTGGATGAAGTTGGTGG- TAMRA-3' (SEQ ID NO: 6).
  • the primers and probes include forward primer GTGCATCTGACTCCTGAGGAGA (SEQ ID NO: 73), reverse primer CCTTGATACCAACCTGCCCAG (SEQ ID NO: 74), probe VIC-AAGGTGAACGTGGATGAAGTTGGTGG-BHQ1 (SEQ ID NO: 75 ).
  • the detection of the EB virus load comprises primers and probes for detecting LMP-2.
  • the primers and probes include forward primer AGCTGTAACTGTGGTTTCCATGAC (SEQ ID NO: 30), reverse primer GCCCCCTGGCGAAGAG (SEQ ID NO: 31), probe FAM-CTGCTGCTACTGGCTTTCGTCCTCTGG-BHQ 1 (SEQ ID NO: 32).
  • the detecting comprises primers and probes for detecting GAPDH3.
  • the primers and probes include forward primer GGTGGAGGAAGTTAGGGTTCGT (SEQ ID NO: 91), reverse primer CAACTCAACTCCAAACTAAACTCCACTA (SEQ ID NO: 92), probe CY5-TTGGGTTTTGACGTTGATT-MGB (SEQ ID NO: 93 )
  • the 5' of the probe described herein carries a fluorescent group, and the 3' carries a fluorescent quenching group.
  • the 5' of the probe described herein carries a FAM fluorophore, a HEX fluorophore, a VIC fluorophore, or a CY5 fluorophore.
  • the 3' of the probe described herein carries a TAMRA fluorescent quencher, a BHQ1 fluorescent quencher, or an MGB fluorescent quencher.
  • a sample preservation solution is provided.
  • One aspect of the present invention provides a kit for preserving a nucleic acid-containing biological sample comprising a sample preservation solution.
  • One aspect of the present invention provides a method for preserving a nucleic acid-containing biological sample using a sample preservation solution.
  • the sample preservation solution comprises one or more components selected from EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and/or trisodium phosphate. In some embodiments, the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
  • the content range of the EDTA is 0.7%-1.5% (w/w);
  • the content range of the NaCl is 0.01%-0.1% (w/w);
  • the concentration range of the absolute ethanol is 15%-35% (w/w);
  • the content range of the sodium citrate is 0.1%-1.0% (w/w);
  • the concentration range of the NP40 is 0.01%-0.1% (v/v);
  • said glutathione content ranges from 0.07% to 0.7% (w/w);
  • trisodium phosphate ranges from 0.07% to 0.4% (w/w).
  • said EDTA content is about 1.05% (w/w);
  • said sodium citrate content is about 0.21% (w/w);
  • NP40 concentration is about 0.035% (v/v);
  • said glutathione content is about 0.21% (w/w);
  • the content of said trisodium phosphate is about 0.14% (w/w).
  • the sample preservation solution includes antibiotics; preferably, the antibiotics include penicillin-streptomycin double antibody.
  • the pH value of the sample preservation solution is 7.6-8.0. In some embodiments, the pH value of the sample preservation solution is 7.7-7.9. In some embodiments, the pH value of the sample preservation solution is 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the pH value of the sample preservation solution is 7.8.
  • the sample preservation solution has a better preservation effect on DNA, miRNA, and/or DNA methylation than the control sample preservation solution.
  • the DNA is derived from Epstein-Barr virus
  • the miRNA is derived from Epstein-Barr virus
  • the DNA methylation is methylation of Epstein-Barr virus DNA.
  • One aspect of the present invention provides a miRNA extraction reagent.
  • One aspect of the present invention provides a kit comprising miRNA extraction reagents.
  • One aspect of the present invention provides a method for extracting miRNA from a sample using an miRNA extraction reagent.
  • the step of extracting miRNA comprises:
  • the miRNA is Epstein-Barr virus-associated miRNA.
  • the miRNA extraction reagents and methods include a lysis solution, a binding solution, a washing solution 1, and/or a washing solution 2. In some embodiments, the miRNA extraction reagents and methods include a lysis solution, a binding solution, a washing solution 1, and/or a washing solution 2.
  • rinse solution 1 and rinse solution 2 are sequentially added to the nucleic acid adsorption material for washing, and the liquid part is removed after each wash and then the next wash is performed; preferably, the nucleic acid adsorption material is The material was washed at least twice with the rinse solution 2.
  • the miRNA extraction reagents/methods comprise a lysate.
  • the lysate comprises one or more components selected from guanidine thiocyanate, citric acid, Triton-100, and/or EDTA.
  • the lysate comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA.
  • the content range of the guanidinium thiocyanate is 28%-40% (w/w);
  • Triton-100 the content range of the Triton-100 is 0.8%-1.6% (w/w);
  • said EDTA content ranges from 0.5% to 0.9% (w/w);
  • the pH range of the lysate is 3.8-4.2.
  • citric acid content is about 2.2% (w/w);
  • Triton-100 the content of said Triton-100 is about 1.1% (w/w);
  • said EDTA content is about 0.7% (w/w);
  • the pH of the lysate is about 4.0.
  • the miRNA extraction reagent/method comprises a binding solution.
  • the binding solution comprises one selected from magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, sodium lauryl sarcosine, and/or isopropanol or multiple components.
  • the binding solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, sodium sarcosine, and isopropanol.
  • the content range of the magnesium chloride is 0.1%-0.4% (w/w);
  • the content range of the sodium bicarbonate is 0.6%-1.0% (w/w);
  • the content range of the guanidinium thiocyanate is 13%-16% (w/w);
  • the content range of the sodium lauryl sarcosine is 1.0%-1.4% (w/w);
  • said isopropanol is present in an amount ranging from 60% to 80% (w/w);
  • the pH range of the binding solution is 4.8-5.2.
  • said magnesium chloride content is about 0.24% (w/w);
  • said sodium bicarbonate content is about 0.83% (w/w);
  • the pH value of the binding solution is about 5.0.
  • the miRNA extraction reagent/method comprises Wash 1.
  • the rinse solution 1 comprises one or more components selected from disodium edetate, sodium bicarbonate, guanidine thiocyanate, and/or sodium lauryl sarcosine .
  • the rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidine thiocyanate, and sodium lauryl sarcosinate.
  • the content range of the sodium bicarbonate is 0.5%-0.9% (w/w);
  • the content range of the guanidinium thiocyanate is 50%-55% (w/w);
  • the pH range of the rinse solution 1 is 6.2-6.6.
  • the pH value of the rinse solution 1 is about 6.4.
  • ethanol is added to the rinse solution 1 before contacting the nucleic acid adsorption material, and the volume ratio of the rinse solution 1:ethanol is 1:1-1:2; preferably, the volume The ratio is about 2:3.
  • the miRNA extraction reagent/method comprises the wash solution 2.
  • the rinse solution 2 includes tris (Tris) or sodium chloride.
  • the rinse solution 2 comprises tris (Tris) and sodium chloride.
  • Tris the content range of the tris
  • said sodium chloride content ranges from 0.6% to 1.8% (w/w);
  • the pH range of the rinse solution 2 is 6.6-7.0.
  • Tris tris
  • said sodium chloride content is about 1.2% (w/w);
  • the pH value of the rinse solution 2 is about 6.8.
  • ethanol is added to the rinse solution 2 before contacting the nucleic acid adsorption material, and the volume ratio of the rinse solution 2:ethanol is 1:1.6-1:3; preferably, the volume The ratio is about 1:2.3.
  • the eluent is DEPC water.
  • the miRNA extraction reagent/method comprises a nucleic acid adsorbent material.
  • the nucleic acid adsorption material is magnetic beads that adsorb RNA.
  • the magnetic core of the magnetic beads is ferric hydroxide.
  • the magnetic beads are coated with silica.
  • the surface of the magnetic beads is embedded with hydroxyl groups.
  • the particle size of the magnetic beads is 50-100 nm.
  • the detection of the Epstein-Barr virus load comprises the detection of the BamHI-W fragment in the Epstein-Barr virus genome, and/or the LMP-2 gene. In some embodiments, the detection of the Epstein-Barr virus load comprises the detection of the BamHI-W fragment in the Epstein-Barr virus genome. In some embodiments, the detection of the Epstein-Barr virus load comprises the detection of the LMP-2 gene in the Epstein-Barr virus genome.
  • the kit of the present invention further comprises a nasopharyngeal brush or a nasopharyngeal swab.
  • the uses and methods of the present invention further comprise obtaining a sample using a nasopharyngeal brush or a nasopharyngeal swab.
  • the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab that does not rely on the guidance of a clinician and a nasopharyngoscope.
  • the present invention provides a method for treating nasopharyngeal carcinoma in an individual in need thereof, comprising providing drug, radiotherapy, surgery, cell therapy and/or oncolytic virus therapy to the individual, wherein the Individual samples are tested by the screening and/or diagnosing method for nasopharyngeal carcinoma according to the present invention and judged to be positive for nasopharyngeal carcinoma.
  • the present invention provides a method for treating nasopharyngeal carcinoma in an individual in need thereof, comprising 1) performing the screening and/or diagnosing method for nasopharyngeal carcinoma according to the present invention on a sample from said individual 2) If the test result is positive for nasopharyngeal carcinoma, provide nasopharyngeal carcinoma treatment; preferably, the nasopharyngeal carcinoma treatment adopts drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus treatment.
  • the nasopharyngeal carcinoma screening and/or diagnosis method of the present invention comprises:
  • the individual is a human.
  • the method comprises providing a therapeutically effective amount of a drug, radiation therapy, surgery, cell therapy, and/or oncolytic virus therapy.
  • Relevant treatment methods include but are not limited to those mentioned in the following literature: Liu et al., Front Oncol. 2021; 11: 635737 (doi: 10.3389/fonc.2021.635737). This application incorporates these documents by reference in their entirety.
  • the method of treating nasopharyngeal carcinoma comprises radiation therapy.
  • the radiotherapy is intensity-modulated radiotherapy (IMRT).
  • the method of treating nasopharyngeal carcinoma comprises proton therapy.
  • the method of treating nasopharyngeal carcinoma comprises chemotherapy.
  • the chemotherapy uses a platinum-containing chemotherapy drug.
  • the method of treating nasopharyngeal carcinoma comprises targeted therapy.
  • the targeted therapy comprises an antibody drug or a small molecule drug.
  • the method of treating nasopharyngeal carcinoma comprises immunotherapy.
  • the immunotherapy comprises cell therapy or oncolytic virotherapy.
  • the cell therapy comprises chimeric antigen receptor modified T cell (CAR-T) therapy or chimeric antigen receptor modified NK cell (CAR-NK) therapy.
  • the present invention has the following beneficial effects:
  • Epstein-Barr virus methylation site/miRNA can be used as a diagnostic marker for nasopharyngeal carcinoma, which is independent of clinicians and nasopharyngoscope-guided nasopharyngeal brush or nasopharyngeal swab (nasopharyngeal blind examination) samples It has the advantages of high sensitivity and high specificity, and is conducive to the promotion of nasopharyngeal brushes or nasopharyngeal swabs (blind nasopharyngeal examination) that do not rely on clinicians and nasopharyngoscope guidance.
  • the present invention can solve the technical problems existing in the prior art through the following technical solutions:
  • Item 1 A kit for detecting Epstein-Barr virus, characterized in that, the kit includes one or more selected from nucleic acid methylation detection reagents, miRNA detection reagents, miRNA extraction reagents, and/or sample preservation solutions reagent.
  • Item 2 The kit as described in Item 1, characterized in that, the kit includes a nucleic acid methylation detection reagent, and the nucleic acid methylation detection reagent can carry out methylation for the methylation site in the Epstein-Barr virus genome chemical detection.
  • Item 3 The kit as described in item 2, wherein the methylation site in the Epstein-Barr virus genome comprises one or more of the following sites: equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 10717bp, 11029bp, 14015bp, 14566bp, 45850bp, 57946bp, 66227bp and 128103bp sites of the Epstein-Barr virus genome DNA sequence.
  • Item 4 The kit according to any one of items 2-3, wherein the methylation site in the Epstein-Barr virus genome comprises EB equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 10717bp, 11029bp, and 14015bp sites of the viral genome DNA sequence.
  • Item 5 The kit as described in any one of items 2-4, wherein the methylation site in the Epstein-Barr virus genome comprises one or more of the following sites: equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) the sites of 11029bp, 45850bp, 57946bp, 66227bp and 128103bp of the Epstein-Barr virus genome DNA sequence.
  • Item 6 The kit as described in any one of items 2-5, wherein the methylation site in the Epstein-Barr virus genome comprises one or more of the following sites: equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) the 11029bp, 57946bp and 66227bp sites of the Epstein-Barr virus genome DNA sequence.
  • Item 7 The kit as described in any one of items 2-6, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 11029bp site of the sequence.
  • Item 7.1 The kit as described in any one of items 2-7, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 10717bp site of the sequence.
  • Item 7.2 The kit as described in any one of item 2-7.1, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 14015bp site of the sequence.
  • Item 7.3 The kit as described in any one of item 2-7.2, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 14566bp site of the sequence.
  • Item 7.4 The kit as described in any one of item 2-7.3, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 45850bp site of the sequence.
  • Item 7.5 The kit as described in any one of item 2-7.4, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 57946bp site of the sequence.
  • Item 7.6 The kit as described in any one of item 2-7.5, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 66227bp site of the sequence.
  • Item 7.7 The kit as described in any one of item 2-7.6, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 128103bp site of the sequence.
  • Item 8 The kit according to any one of Items 1-7.7, characterized in that, the kit includes the miRNA detection reagent, and the miRNA detection reagent can be used for one or more miRNAs from Epstein-Barr virus. detection.
  • Item 9 The kit as described in Item 8, wherein the miRNA comprises one or more of the following miRNAs: EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR-BART6- 3p, EBV-miR-BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p; preferably comprising all six of said miRNAs.
  • miRNAs EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR-BART6- 3p, EBV-miR-BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p; preferably comprising all six of said miRNAs.
  • Item 10 The kit according to Item 9, wherein the miRNA comprises EBV-miR-BART1-5p and EBV-miR-BART7-3p.
  • Item 11 The kit according to any one of Items 9-10, wherein the miRNA comprises EBV-miR-BART 1-5p.
  • Item 12 The kit according to any one of Items 9-11, wherein the miRNA comprises EBV-miR-BART2-5p.
  • Item 13 The kit according to any one of Items 9-12, wherein the miRNA comprises EBV-miR-BART6-3p.
  • Item 14 The kit according to any one of Items 9-13, wherein the miRNA comprises EBV-miR-BART7-3p.
  • Item 15 The kit according to any one of Items 9-14, wherein the miRNA comprises EBV-miR-BART13-3p.
  • Item 16 The kit according to any one of Items 9-15, wherein the miRNA comprises EBV-miR-BART 17-5p.
  • Item 17 The kit as described in any one of Items 1-16, wherein the kit includes the sample preservation solution, and the sample preservation solution comprises EDTA, NaCl, absolute ethanol, citric acid One or more components of sodium, NP40, glutathione, and/or trisodium phosphate.
  • Item 18 The kit according to Item 17, wherein the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
  • a kit for preserving a nucleic acid-containing biological sample characterized in that it comprises a sample preservation solution comprising EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and Trisodium Phosphate.
  • Item 20 The kit according to any one of Items 17-19, wherein in the sample preservation solution:
  • the content range of the EDTA is 0.7%-1.5% (w/w);
  • the content range of the NaCl is 0.01%-0.1% (w/w);
  • the concentration range of the absolute ethanol is 15%-35% (w/w);
  • the content range of the sodium citrate is 0.1%-1.0% (w/w);
  • the concentration range of the NP40 is 0.01%-0.1% (v/v);
  • the glutathione content ranges from 0.07% to 0.7% (w/w); and/or
  • the content range of the trisodium phosphate is 0.07%-0.4% (w/w).
  • Item 21 The kit according to any one of Items 17-20, wherein in the sample preservation solution:
  • the content of said EDTA is about 1.05% (w/w);
  • the content of said NaCl is about 0.035% (w/w);
  • the concentration of absolute ethanol is about 27.6% (w/w);
  • the content of the sodium citrate is about 0.21% (w/w);
  • the concentration of said NP40 is about 0.035% (v/v);
  • the glutathione content is about 0.21% (w/w); and/or
  • the content of the trisodium phosphate is about 0.14% (w/w).
  • Item 22 The kit according to item 17-21, wherein the sample preservation solution includes antibiotics; preferably, the antibiotics include penicillin-streptomycin double antibody.
  • Item 23 The kit according to any one of Items 17-22, wherein the pH value of the sample preservation solution is 7.6-8.0; preferably, the pH value of the sample preservation solution is 7.8.
  • Item 24 The kit according to any one of Items 17-23, characterized in that, compared with the control sample preservation solution, the sample preservation solution has a better preservation effect on DNA, miRNA, and/or DNA methylation. good.
  • Item 25 The kit according to Item 24, wherein the DNA is derived from Epstein-Barr virus, the miRNA is derived from Epstein-Barr virus, and/or the DNA methylation is methylation of Epstein-Barr virus DNA.
  • Item 26 The kit according to any one of Items 1-25, wherein the kit includes the miRNA extraction reagent, and the miRNA extraction reagent includes a lysate, a binding solution, a rinse solution 1, and/or or rinse solution 2.
  • Item 27 A kit comprising miRNA extraction reagents, characterized in that the miRNA extraction reagents comprise lysis solution, binding solution, washing solution 1, and/or washing solution 2.
  • Item 28 The kit according to Item 26 or 27, wherein the miRNA extraction reagent comprises the lysate, and the lysate comprises guanidinium thiocyanate, citric acid, Triton-100, and/or one or more components of EDTA.
  • Item 29 The kit according to Item 26 or 27, wherein the miRNA extraction reagent comprises the lysate, which comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA .
  • the miRNA extraction reagent comprises the lysate, which comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA .
  • Item 30 The kit according to any one of Items 26-29, wherein in the lysate:
  • guanidinium thiocyanate The content scope of described guanidinium thiocyanate is 28%-40% (w/w);
  • the content range of the citric acid is 1.8%-2.6% (w/w);
  • the content range of the Triton-100 is 0.8%-1.6% (w/w);
  • the content range of the EDTA is 0.5%-0.9% (w/w); and/or
  • the pH range of the lysate is 3.8-4.2.
  • Item 31 The kit according to any one of Items 26-30, wherein in the lysate:
  • the content of the guanidinium thiocyanate is about 32% (w/w);
  • the content of said citric acid is about 2.2% (w/w);
  • the content of the Triton-100 is about 1.1% (w/w);
  • the content of said EDTA is about 0.7% (w/w);
  • the pH of the lysate is about 4.0.
  • Item 32 The kit according to any one of Items 26-31, wherein the miRNA extraction reagent comprises the binding liquid, and the binding liquid comprises magnesium chloride, disodium edetate, carbonic acid One or more components of sodium hydrogen, guanidinium thiocyanate, sodium lauryl sarcosinate, and/or isopropanol.
  • Item 33 The kit according to any one of Items 26-32, wherein the miRNA extraction reagent comprises the binding solution, and the binding solution comprises magnesium chloride, disodium edetate, and sodium bicarbonate , guanidinium thiocyanate, sodium lauryl sarcosinate, and isopropanol.
  • Item 34 The kit according to any one of Items 26-33, wherein in the binding solution:
  • the content range of the magnesium chloride is 0.1%-0.4% (w/w);
  • the content range of the disodium edetate is 0.5%-1.0% (w/w);
  • the content scope of described sodium bicarbonate is 0.6%-1.0% (w/w);
  • the content range of the guanidine thiocyanate is 13%-16% (w/w);
  • the content range of the sodium lauryl sarcosinate is 1.0%-1.4% (w/w);
  • the content range of the isopropanol is 60%-80% (w/w); and/or
  • the pH range of the binding solution is 4.8-5.2.
  • Item 35 The kit according to any one of Items 26-34, wherein in the binding solution:
  • the content of said magnesium chloride is about 0.24% (w/w);
  • the content of disodium edetate is about 0.71% (w/w);
  • the content of the sodium bicarbonate is about 0.83% (w/w);
  • the content of guanidine thiocyanate is about 14.9% (w/w);
  • the content of sodium sarcosyl is about 1.2% (w/w);
  • the content of said isopropanol is about 70% (w/w); and/or
  • the pH of the binding solution is about 5.0.
  • Item 36 The kit according to any one of Items 26-35, wherein the miRNA extraction reagent comprises the rinse solution 1, and the rinse solution 1 comprises disodium edetate, carbonic acid One or more components of sodium hydrogen, guanidinium thiocyanate, and/or sodium lauryl sarcosinate.
  • Item 37 The kit according to any one of items 26-36, wherein the miRNA extraction reagent comprises the rinse solution 1, and the rinse solution 1 comprises disodium edetate, sodium bicarbonate , guanidinium thiocyanate, and sodium lauryl sarcosinate.
  • Item 38 The kit according to any one of Items 26-37, wherein in the rinse solution 1:
  • the content range of the disodium edetate is 0.4%-0.9% (w/w);
  • the content scope of described sodium bicarbonate is 0.5%-0.9% (w/w);
  • the content range of the guanidinium thiocyanate is 50%-55% (w/w);
  • the content range of the sodium lauryl sarcosyl is 0.8%-1.2% (w/w); and/or
  • the pH range of the rinse solution 1 is 6.2-6.6.
  • Item 39 The kit according to any one of Items 26-38, wherein in the rinse solution 1:
  • the content of disodium edetate is about 0.5% (w/w);
  • the content of the sodium bicarbonate is about 0.6% (w/w);
  • the content of the guanidinium thiocyanate is about 53% (w/w);
  • the content of sodium sarcosyl is about 0.9% (w/w);
  • the pH of the Rinse 1 was about 6.4.
  • Item 40 The kit according to any one of Items 26-39, wherein the miRNA extraction reagent comprises the rinse solution 2, and the rinse solution 2 comprises tris (Tris) or chlorine sodium chloride.
  • the miRNA extraction reagent comprises the rinse solution 2
  • the rinse solution 2 comprises tris (Tris) or chlorine sodium chloride.
  • Item 41 The kit according to any one of Items 26-40, wherein the miRNA extraction reagent comprises the rinse solution 2, and the rinse solution 2 comprises tris (Tris) and chlorine sodium chloride.
  • the miRNA extraction reagent comprises the rinse solution 2
  • the rinse solution 2 comprises tris (Tris) and chlorine sodium chloride.
  • Item 42 The kit according to any one of Items 26-41, wherein in the rinse solution 2:
  • Tris The content range of the tris (Tris) is 0.5%-0.7% (w/w);
  • the content range of the sodium chloride is 0.6%-1.8% (w/w); and/or
  • the pH range of the rinse solution 2 is 6.6-7.0.
  • Item 43 The kit according to any one of Items 26-42, wherein in the rinse solution 2:
  • Tris tris
  • the sodium chloride content is about 1.2% (w/w); and/or
  • the pH value of the rinse solution 2 is about 6.8.
  • Item 44 The kit according to any one of Items 26-43, wherein the miRNA extraction reagent comprises magnetic beads that adsorb RNA.
  • Item 45 The kit according to Item 44, wherein the magnetic core of the magnetic beads is ferric hydroxide, coated with silicon dioxide, and hydroxy groups are embedded on the surface.
  • Item 46 The kit according to Item 44 or 45, wherein the magnetic beads have a particle diameter of 50-100 nm.
  • Item 47 The kit according to any one of Items 26-46, further comprising a nasopharyngeal brush or a nasopharyngeal swab.
  • Item 48 The kit according to Item 47, wherein the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab independent of the guidance of a clinician and a nasopharyngoscope.
  • kits according to any one of Items 1-48, characterized in that, the kit also includes EB virus load detection reagents; preferably, the EB virus load detection reagents contain In the BamHI-W fragment, and/or the detection reagent of LMP-2 gene.
  • Item 50 The kit according to Item 49, wherein the EB virus load detection reagent includes a detection reagent for LMP-2 gene.
  • Item 51 The kit according to any one of Items 1-50, wherein the kit is a nasopharyngeal carcinoma screening kit.
  • Item 51.1 The kit according to any one of Items 1-51, wherein the kit is a diagnostic kit for nasopharyngeal carcinoma.
  • Item 52 A method for detecting Epstein-Barr virus-related nucleic acids in a sample, characterized in that the method comprises the following steps:
  • it also includes extracting the miRNA from the sample preservation solution; and/or
  • step (b-1) is included, and the methylation site detected for methylation of the Epstein-Barr virus genome nucleic acid includes one or more of the following sites : the sites equivalent to 10717bp, 11029bp, 14015bp, 14566bp, 45850bp, 57946bp, 66227bp and 128103bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  • Item 54 The method as described in Item 53, wherein the methylation site comprises positions equivalent to 10717bp, 11029bp, and 14015bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
  • Item 54.1 The method according to any one of Item 53-54, wherein the methylation site comprises a position equivalent to 10717bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
  • Item 54.2 The method according to any one of Item 53-54.1, wherein the methylation site comprises a position equivalent to 11029bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
  • Item 54.3 The method according to any one of Item 53-54.2, wherein the methylation site comprises a position equivalent to 14015bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
  • Item 54.4 The method according to any one of Item 53-54.3, wherein the methylation site comprises a position corresponding to 14566 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
  • Item 54.5 The method according to any one of Item 53-54.4, wherein the methylation site comprises a position equivalent to 45850bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
  • Item 54.6 The method according to any one of Item 53-54.5, wherein the methylation site comprises a position corresponding to 57946 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
  • Item 54.7 The method according to any one of Item 53-54.6, wherein the methylation site comprises a position corresponding to 66227bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
  • Item 54.8 The method according to any one of Item 53-54.7, wherein the methylation site comprises a position corresponding to 128103 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
  • Item 55 The method according to any one of Items 52-54.8, characterized in that step (b-2) is included, and the miRNA includes one or more of the following miRNAs: EBV-miR-BART1-5p, EBV- miR-BART2-5p, EBV-miR-BART6-3p, EBV-miR-BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p; preferably comprising all six of said miRNAs.
  • Item 56 The method according to Item 55, wherein the miRNA comprises EBV-miR-BART1-5p and EBV-miR-BART7-3p.
  • Item 56.1 The method according to any one of Items 55-56, wherein the miRNA comprises EBV-miR-BART1-5p.
  • Item 56.2 The method according to any one of Items 55-56.1, wherein the miRNA comprises EBV-miR-BART2-5p.
  • Item 56.3 The method according to any one of Items 55-56.2, wherein the miRNA comprises EBV-miR-BART6-3p.
  • Item 56.4 The method according to any one of Items 55-56.3, wherein the miRNA comprises EBV-miR-BART7-3p.
  • Item 56.5 The method according to any one of Items 55-56.4, wherein the miRNA comprises EBV-miR-BART13-3p.
  • Item 56.6 The method according to any one of Items 55-56.5, wherein the miRNA comprises EBV-miR-BART17-5p.
  • Item 58 A method for extracting miRNA, comprising the steps of:
  • Item 59 The method according to any one of Items 57-58, wherein the lysate comprises one or more compounds selected from guanidine thiocyanate, citric acid, Triton-100, and/or EDTA A component; Preferably, the lysate comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA.
  • Item 60 The method according to Item 59, wherein in the lysate:
  • guanidinium thiocyanate The content scope of described guanidinium thiocyanate is 28%-40% (w/w);
  • the content range of the citric acid is 1.8%-2.6% (w/w);
  • the content range of the Triton-100 is 0.8%-1.6% (w/w);
  • the content range of the EDTA is 0.5%-0.9% (w/w); and/or
  • the pH range of the lysate is 3.8-4.2.
  • Item 61 The method according to any one of Items 59-60, wherein in the lysate:
  • the content of the guanidinium thiocyanate is about 32% (w/w);
  • the content of said citric acid is about 2.2% (w/w);
  • the content of the Triton-100 is about 1.1% (w/w);
  • the content of said EDTA is about 0.7% (w/w);
  • the pH of the lysate is about 4.0.
  • Item 62 The method according to any one of Items 57-61, wherein the binding solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, dodecyl Sodium sarcosinate, and/or one or more components of isopropanol; preferably, the combined solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, dodecane Sodium sarcosinate, and isopropanol.
  • the binding solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, dodecyl Sodium sarcosinate, and/or one or more components of isopropanol; preferably, the combined solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, dodecane Sodium sarcosinate, and isopropanol
  • Item 63 The method according to Item 62, wherein in the binding solution:
  • the content range of the magnesium chloride is 0.1%-0.4% (w/w);
  • the content range of the disodium edetate is 0.5%-1.0% (w/w);
  • the content scope of described sodium bicarbonate is 0.6%-1.0% (w/w);
  • the content range of the guanidine thiocyanate is 13%-16% (w/w);
  • the content range of the sodium lauryl sarcosinate is 1.0%-1.4% (w/w);
  • the content range of the isopropanol is 60%-80% (w/w); and/or
  • the pH range of the binding solution is 4.8-5.2.
  • Item 64 The method according to any one of Items 62-63, wherein in the binding solution:
  • the content of said magnesium chloride is about 0.24% (w/w);
  • the content of disodium edetate is about 0.71% (w/w);
  • the content of the sodium bicarbonate is about 0.83% (w/w);
  • the content of the guanidinium thiocyanate is about 14.9% (w/w);
  • the content of sodium sarcosyl is about 1.2% (w/w);
  • the content of said isopropanol is about 70% (w/w); and/or
  • the pH of the binding solution is about 5.0.
  • Item 65 The method according to any one of Items 57-64, characterized in that in step iii), rinse solution 1 and rinse solution 2 are sequentially added to the nucleic acid adsorption material for washing, and the liquid part is removed after each washing Then perform the next wash; preferably, wash the nucleic acid adsorption material with the rinse solution 2 at least twice.
  • Item 66 The method according to Item 65, wherein the rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidinium thiocyanate, and/or lauryl sarcosine One or more components of sodium; preferably, the rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidinium thiocyanate, and sodium lauryl sarcosinate.
  • Item 67 The method according to Item 66, wherein in the rinse solution 1:
  • the content range of the disodium edetate is 0.4%-0.9% (w/w);
  • the content scope of described sodium bicarbonate is 0.5%-0.9% (w/w);
  • the content range of the guanidinium thiocyanate is 50%-55% (w/w);
  • the content range of the sodium lauryl sarcosyl is 0.8%-1.2% (w/w); and/or
  • the pH range of the rinse solution 1 is 6.2-6.6.
  • Item 68 The method according to any one of Items 66-67, wherein in the rinse solution 1:
  • the content of disodium edetate is about 0.5% (w/w);
  • the content of the sodium bicarbonate is about 0.6% (w/w);
  • the content of the guanidinium thiocyanate is about 53% (w/w);
  • the content of sodium sarcosyl is about 0.9% (w/w);
  • the pH of the Rinse 1 was about 6.4.
  • Item 69 The method according to any one of Items 66-68, wherein ethanol is added to the rinse solution 1 before contacting the nucleic acid adsorption material, and the volume ratio of the rinse solution 1:ethanol is 1:1-1:2; preferably, the volume ratio is about 2:3.
  • Item 70 The method according to any one of Items 65-69, wherein the rinse solution 2 comprises Tris (Tris) or sodium chloride; preferably, the rinse solution 2 comprises Tris Hydroxymethylaminomethane (Tris) and Sodium Chloride.
  • the rinse solution 2 comprises Tris (Tris) or sodium chloride; preferably, the rinse solution 2 comprises Tris Hydroxymethylaminomethane (Tris) and Sodium Chloride.
  • Item 71 The method according to Item 70, wherein in the rinse solution 2:
  • Tris The content range of the tris (Tris) is 0.5%-0.7% (w/w);
  • the content range of the sodium chloride is 0.6%-1.8% (w/w); and/or
  • the pH range of the rinse solution 2 is 6.6-7.0.
  • Item 72 The method according to any one of Items 70-71, wherein in the rinse solution 2:
  • Tris tris
  • the sodium chloride content is about 1.2% (w/w); and/or
  • the pH value of the rinse solution 2 is about 6.8.
  • Item 73 The method according to any one of Items 70-72, wherein ethanol is added to the rinse solution 2 before contacting the nucleic acid adsorption material, and the volume ratio of the rinse solution 2:ethanol is 1:1.6-1:3; preferably, the volume ratio is about 1:2.3.
  • Item 74 The method according to any one of Item 57-73, wherein the eluent is DEPC water.
  • Item 75 The method according to any one of Items 57-74, wherein the nucleic acid adsorption material is a magnetic bead capable of adsorbing RNA.
  • Item 76 The method as described in Item 75, wherein the magnetic core of the magnetic bead is ferric hydroxide, coated with silicon dioxide, and the surface is embedded with hydroxyl groups; preferably, the magnetic bead The diameter is 50-100nm.
  • Item 77 The method according to any one of Items 52-76, wherein the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and/or One or more components of trisodium phosphate; preferably, the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
  • Item 78 A method for preserving a nucleic acid-containing biological sample, characterized in that the sample is placed in a sample preservation solution, and the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, and glutathione peptides, and trisodium phosphate.
  • Item 79 The method according to any one of Items 77-78, wherein in the sample preservation solution:
  • the content range of the EDTA is 0.7%-1.5% (w/w);
  • the content range of the NaCl is 0.01%-0.1% (w/w);
  • the concentration range of the absolute ethanol is 15%-35% (w/w);
  • the content range of the sodium citrate is 0.1%-1.0% (w/w);
  • the concentration range of the NP40 is 0.01%-0.1% (v/v);
  • the glutathione content ranges from 0.07% to 0.7% (w/w); and/or
  • the content range of the trisodium phosphate is 0.07%-0.4% (w/w).
  • Item 80 The method according to any one of Items 77-79, wherein in the sample preservation solution:
  • the content of said EDTA is about 1.05% (w/w);
  • the content of said NaCl is about 0.035% (w/w);
  • the concentration of absolute ethanol is about 27.6% (w/w);
  • the content of the sodium citrate is about 0.21% (w/w);
  • the concentration of said NP40 is about 0.035% (v/v);
  • the glutathione content is about 0.21% (w/w); and/or
  • the content of the trisodium phosphate is about 0.14% (w/w).
  • Item 81 The method according to Item 77-80, wherein the sample preservation solution contains antibiotics; preferably, the antibiotics contain penicillin-streptomycin double antibody.
  • Item 82 The method according to any one of Items 77-81, wherein the pH value of the sample preservation solution is 7.6-8.0; preferably, the pH value of the sample preservation solution is 7.8.
  • Item 83 The method according to any one of Items 77-82, characterized in that, compared with the control sample preservation solution, the sample preservation solution has a better preservation effect on DNA, miRNA, and/or DNA methylation .
  • Item 84 The method according to any one of Items 52-83, characterized in that step (b-3) is included, and the detection of the EB virus load comprises targeting the BamHI-W fragment in the Epstein-Barr virus genome, and/or LMP -2 gene detection.
  • Item 85 The method according to Item 84, wherein the detection of the EB virus load comprises detection of the LMP-2 gene.
  • Item 86 The method according to any one of Items 52-85, wherein the method is used for screening nasopharyngeal carcinoma.
  • Item 86.1 The method according to any one of Items 52-86, wherein said method is used for the diagnosis of nasopharyngeal carcinoma.
  • Item 87 The method according to any one of Items 52-86.1, wherein the sample is a nasopharyngeal sample.
  • Item 88 The method according to any one of Item 52-87, characterized in that, before step (a), it also includes using a nasopharyngeal brush and/or a nasopharyngeal swab to obtain the sample; preferably, the Nasopharyngeal brushes and/or nasopharyngeal swabs are nasopharyngeal brushes and/or nasopharyngeal swabs that do not rely on the guidance of clinicians and nasopharyngoscopes.
  • Item 89 A method for treating nasopharyngeal carcinoma in an individual in need thereof, comprising providing the individual with drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus therapy, wherein a sample of the individual has received the The method described in any one of 52-88 was detected and judged to be positive for nasopharyngeal carcinoma.
  • Item 90 A method for treating nasopharyngeal carcinoma in an individual in need thereof, comprising 1) performing the detection according to the method according to any one of Items 52-88 on a sample from the individual; 2) if the detection result shows that the nasal If the pharyngeal cancer is positive, provide nasopharyngeal cancer treatment; preferably, the nasopharyngeal cancer treatment uses drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus treatment.
  • Item 91 Use of the kit according to any one of claims 1-51.1 in the preparation of products for nasopharyngeal cancer screening.
  • Item 92 Use of the kit according to any one of claims 1-51.1 in the preparation of products for the diagnosis of nasopharyngeal carcinoma.
  • test kit for nasopharyngeal carcinoma detection characterized in that the test kit comprises:
  • nucleic acid methylation detection reagent can carry out methylation detection at methylation site in Epstein-Barr virus genome, and described methylation site comprises the equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) the 10717bp, 11029bp and 14015bp sites of the Epstein-Barr virus genome DNA sequence;
  • miRNA detection reagents can detect the content of multiple miRNAs from Epstein-Barr virus, and the miRNAs include EBV-miR-BART1-5p and EBV-miR-BART7-3p; and
  • said EB virus load detection reagent comprises a detection reagent for LMP-2 gene.
  • Claim 2 The kit as claimed in claim 1, wherein the miRNA comprises EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR-BART6-3p, EBV-miR- BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p.
  • Claim item 3 The test kit according to any one of claim items 1-2, characterized in that, the test kit comprises the sample preservation solution, and the sample preservation solution comprises EDTA, NaCl, dehydrated alcohol, citric acid Sodium, NP40, Glutathione, and Trisodium Phosphate, of which,
  • the content range of the EDTA is 0.7%-1.5% (w/w);
  • the content range of the NaCl is 0.01%-0.1% (w/w);
  • the concentration range of the absolute ethanol is 15%-35% (w/w);
  • the content range of the sodium citrate is 0.1%-1.0% (w/w);
  • the concentration range of the NP40 is 0.01%-0.1% (v/v);
  • the glutathione content ranges from 0.07% to 0.7% (w/w);
  • the content range of the trisodium phosphate is 0.07%-0.4% (w/w),
  • the pH value of the sample preservation solution is 7.6-8.0.
  • Claim item 4 The kit as described in any one of claim items 1-3, characterized in that, the kit includes miRNA extraction reagents, and the miRNA extraction reagents include lysate, binding solution, rinse solution 1, and rinse Liquid 2.
  • Claim item 5 The test kit as claimed in claim item 4, wherein the lysate comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA, wherein:
  • guanidinium thiocyanate The content scope of described guanidinium thiocyanate is 28%-40% (w/w);
  • the content range of the citric acid is 1.8%-2.6% (w/w);
  • the content range of the Triton-100 is 0.8%-1.6% (w/w);
  • the content range of said EDTA is 0.5%-0.9% (w/w);
  • the pH range of the lysate is 3.8-4.2
  • the combination solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidine thiocyanate, sodium lauryl sarcosine, and isopropanol, wherein:
  • the content range of the magnesium chloride is 0.1%-0.4% (w/w);
  • the content range of the disodium edetate is 0.5%-1.0% (w/w);
  • the content scope of described sodium bicarbonate is 0.6%-1.0% (w/w);
  • the content range of the guanidine thiocyanate is 13%-16% (w/w);
  • the content range of the sodium lauryl sarcosinate is 1.0%-1.4% (w/w);
  • the content range of the isopropanol is 60%-80% (w/w);
  • the pH range of the binding solution is 4.8-5.2
  • the rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidine thiocyanate, and sodium lauryl sarcosine, wherein,
  • the content range of the disodium edetate is 0.4%-0.9% (w/w);
  • the content scope of described sodium bicarbonate is 0.5%-0.9% (w/w);
  • the content range of the guanidinium thiocyanate is 50%-55% (w/w);
  • the content range of the sodium sarcosyl is 0.8%-1.2% (w/w);
  • the pH range of the rinse solution 1 is 6.2-6.6
  • the rinse solution 2 comprises tris (Tris) and sodium chloride, wherein:
  • Tris The content range of the tris (Tris) is 0.5%-0.7% (w/w);
  • the sodium chloride content ranges from 0.6% to 1.8% (w/w);
  • the pH range of the rinse solution 2 is 6.6-7.0.
  • kit according to any one of claims 1-5, wherein the kit also comprises a nasopharyngeal brush or a nasopharyngeal swab, and the nasopharyngeal brush and/or nasopharyngeal swab Nasopharyngeal brushes and/or nasopharyngeal swabs are clinician-independent and nasopharyngoscope-guided.
  • Claim 7 Use of the kit as described in any one of claims 1-6 in the preparation of products for nasopharyngeal carcinoma detection.
  • a method for detecting Epstein-Barr virus-related nucleic acids in a sample characterized in that the method comprises the following steps:
  • the methylation sites detected by the methylation include sites equivalent to 10717bp, 11029bp, and 14015bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94), and the miRNA includes EBV- miR-BART1-5p and EBV-miR-BART7-3p, the EB virus load detection reagent includes the detection of LMP-2 gene.
  • Claim 9 The method as claimed in claim 8, wherein the miRNA comprises EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR-BART6-3p, EBV-miR-BART7 -3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p.
  • Claim item 10 The method as described in any one of claim items 8-9, wherein the step of extracting the Epstein-Barr virus-related miRNA comprises:
  • Claim 11 The method of claim 10, wherein:
  • the lysate comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA, wherein:
  • guanidinium thiocyanate The content scope of described guanidinium thiocyanate is 28%-40% (w/w);
  • the content range of the citric acid is 1.8%-2.6% (w/w);
  • the content range of the Triton-100 is 0.8%-1.6% (w/w);
  • the content range of said EDTA is 0.5%-0.9% (w/w);
  • the pH range of the lysate is 3.8-4.2
  • the combination solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidine thiocyanate, sodium lauryl sarcosine, and isopropanol, wherein:
  • the content range of the magnesium chloride is 0.1%-0.4% (w/w);
  • the content range of the disodium edetate is 0.5%-1.0% (w/w);
  • the content scope of described sodium bicarbonate is 0.6%-1.0% (w/w);
  • the content range of the guanidine thiocyanate is 13%-16% (w/w);
  • the content range of the sodium lauryl sarcosinate is 1.0%-1.4% (w/w);
  • the content range of the isopropanol is 60%-80% (w/w);
  • the pH range of the binding solution is 4.8-5.2
  • the rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidine thiocyanate, and sodium lauryl sarcosine, wherein,
  • the content range of the disodium edetate is 0.4%-0.9% (w/w);
  • the content scope of described sodium bicarbonate is 0.5%-0.9% (w/w);
  • the content range of the guanidinium thiocyanate is 50%-55% (w/w);
  • the content range of the sodium sarcosyl is 0.8%-1.2% (w/w);
  • the pH range of the rinse solution 1 is 6.2-6.6
  • the rinse solution 2 comprises tris (Tris) and sodium chloride, wherein:
  • Tris The content range of the tris (Tris) is 0.5%-0.7% (w/w);
  • the sodium chloride content ranges from 0.6% to 1.8% (w/w);
  • the pH range of the rinse solution 2 is 6.6-7.0.
  • Claim 12 The method according to claim 11, wherein ethanol is added to the rinse solution 1 before contacting the nucleic acid adsorption material, and the volume ratio of the rinse solution 1:ethanol is 1:1 - 1:2; and, before contacting the nucleic acid adsorption material, adding ethanol to the rinse liquid 2, the volume ratio of the rinse liquid 2:ethanol is 1:1.6-1:3.
  • Claim item 13 The method according to any one of claim items 8-12, wherein the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and triphosphate Sodium, of which:
  • the content range of the EDTA is 0.7%-1.5% (w/w);
  • the content range of the NaCl is 0.01%-0.1% (w/w);
  • the concentration range of the absolute ethanol is 15%-35% (w/w);
  • the content range of the sodium citrate is 0.1%-1.0% (w/w);
  • the concentration range of the NP40 is 0.01%-0.1% (v/v);
  • the glutathione content ranges from 0.07% to 0.7% (w/w);
  • the content range of the trisodium phosphate is 0.07%-0.4% (w/w),
  • the pH value of the sample preservation solution is 7.6-8.0.
  • Claim 14 The method according to any one of claims 8-13, characterized in that the method is used for the detection of nasopharyngeal carcinoma.
  • Claim item 15 The method according to any one of claim items 8-14, wherein the sample is a nasopharyngeal sample, and before step (a), it also includes using a nasopharyngeal brush and/or a nasopharyngeal swab
  • the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab independent of the guidance of a clinician and a nasopharyngoscope.
  • Claim 16 A method for treating nasopharyngeal carcinoma in an individual in need thereof, comprising providing the individual with drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus therapy, wherein the sample of the individual has received as The detection by the method described in any one of Claims 8-15 is judged to be positive for nasopharyngeal carcinoma.
  • Claim 17 A method for treating nasopharyngeal carcinoma in an individual in need, comprising 1) performing detection according to the method described in any one of Claims 8-15 on a sample from the individual; 2) the detection result If it is positive for nasopharyngeal carcinoma, treatment for nasopharyngeal carcinoma is provided; preferably, the treatment for nasopharyngeal carcinoma adopts drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus treatment.
  • Figure 1 shows the detection of Epstein-Barr virus DNA load and Epstein-Barr virus DNA methylation in nasopharyngeal swab samples independent of clinicians and guided by nasopharyngoscope, where NPC is nasopharyngeal carcinoma (NPC) and MD is A Methylation difference (methylation difference, MD), EBV DNA load is EBV DNA load.
  • NPC nasopharyngeal carcinoma
  • MD A Methylation difference (methylation difference, MD)
  • EBV DNA load is EBV DNA load.
  • Figure 2 is the detection of Epstein-Barr virus DNA load and Epstein-Barr virus DNA methylation in nasopharyngeal swab samples guided by clinicians and nasopharyngoscopes.
  • Figure 3 is a side-by-side comparison of EBV DNA load and EBV DNA methylation detection in the same patient in dependent and independent clinician-guided and nasopharyngoscope-guided nasopharyngeal brush samples.
  • brush refers to nasopharyngeal brushing samples that rely on clinicians and nasopharyngoscope guidance
  • blind brush refers to nasopharyngeal brushing samples that do not rely on clinicians and nasopharyngoscope guidance.
  • Figure 4 is a parallel comparison of the DNA preservation effect of the self-prepared sample preservation solution, the commercial DNA preservation solution and the commercial RNA preservation solution.
  • Figure 5 is a parallel comparison of the DNA methylation preservation effects of the self-prepared sample preservation solution, the commercial DNA preservation solution and the commercial RNA preservation solution.
  • Figure 6 is a parallel comparison of self-made sample preservation solution, commercially available DNA preservation solution and commercial RNA preservation solution for the miRNA preservation effects.
  • Figure 7 is a parallel comparison of the extraction effects of self-prepared RNA extraction reagents and commercially available RNA extraction reagents on the miRNAs shown.
  • Figure 8 shows the detection results related to Epstein-Barr virus DNA.
  • Figure 9 is the detection structure related to DNA methylation of Epstein-Barr virus.
  • Figure 10 shows the detection results related to miRNA.
  • test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.
  • Example 1 A sampling method for nasopharyngeal swabbing that does not rely on clinicians and nasopharyngoscope guidance
  • nasopharyngeal brush or nasopharyngeal swab which is directly operated by a clinical nurse.
  • the nasopharyngeal brush or nasopharyngeal swab directly enters the nasopharynx through the nasal cavity and rotates, brushes the sample and takes it back.
  • Example 2 A sampling method that relies on clinicians and nasopharyngoscope-guided nasopharyngeal brushing samples
  • the nasopharyngeal brush Under the guidance of the electronic nasopharyngoscope, operated by a clinician or resident doctor, the nasopharyngeal brush enters the nasopharynx through the middle meatus with the nasopharyngoscope, rolls samples at suspicious parts of the nasopharynx, and then quickly retrieve.
  • nasopharyngeal brush sample is first clamped with tweezers after autoclaving and rinsed back and forth in PBS solution for five times, and then scraped the brush with tweezers to scrape off the exfoliated cells that may remain on the brush .
  • the concentration of DNA was quantified using the Nanodrop 1000 absorbance method (NanoDrop Technologies, Waltham, MA, USA).
  • the DNA solution was stored at -20°C, and the RNA solution was stored at -80°C for subsequent use.
  • the method of fluorescent quantitative PCR was used to quantitatively detect the Epstein-Barr virus DNA load in nasopharyngeal brush samples, and the detection primers and probes are shown in Table 1.
  • the target gene amplified for the quantification of EBV DNA is the BamHI-W fragment of EB virus, which consists of 76 nucleotides.
  • the forward and reverse primer sequences are: 5'-CCCAACACTCCACCACACC-3 ' (SEQ ID NO: 1), and 5'-TCTTAGGAGCTGTCCGAGGG-3' (SEQ ID NO: 2).
  • the fluorescent quantitative PCR system also includes primers and probes for the housekeeping gene ⁇ -globin gene.
  • the sequences of the two primers are 5'-GTGCACCTGACTCCTGAGGAGA-3' (SEQ ID NO: 4); 5'-CCTTGATACCAACCTGCCCAG-3' (SEQ ID NO: 5), the amplified fragment is 102bp.
  • the sequence of the probe for this gene is 5'-FAM-AAGGTGAACGTGGATGAAGTTGGTGG-TAMRA-3' (SEQ ID NO: 6).
  • the two ends of the same probe have fluorescent groups and quenching groups respectively.
  • the sequences of primers and probes are provided by life technology company synthesis.
  • the volume of each gene Q-PCR reaction system is 8 ⁇ L, including 4 ⁇ L of 2 ⁇ mix solution, 1 ⁇ L of primers (including forward and reverse primers), 0.2 ⁇ L of probe, 0.8 ⁇ L of water and 2 ⁇ L of DNA template.
  • the Q-PCR reaction conditions are denaturation at 95°C for 5 minutes; 45 reaction cycles, each cycle condition is 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 15 seconds.
  • the genomic DNA of 500ng samples was first treated with bisulfite and purified, and the modified DNA was converted as a template for q-PCR detection.
  • EBV DNA methylation detection aiming at five methylation sites of 11029bp, 45850bp, 57946bp, 66227bp and 128103bp on the EBV genomic DNA (the EBV genome is GenBank LOCUS is NC_007605), these sites are located in EBV C promoter region (11029bp), latent infection phase gene (57946bp), early lytic infection phase gene (45850bp), late lytic infection phase gene (66227bp and 128103bp) on the genome. For each site, design a pair of primers and a pair of fluorescent probes.
  • probes 1 One of the fluorescent probes is methylated for this site, and the 5' and 3' ends are labeled with FAM and BHQ1 respectively, named as probes 1 (Probe1), a fluorescent probe is not methylated at this site, and the 5' and 3' ends are labeled with HEX and BHQ1 respectively, named as probe 2 (Probe2).
  • probes 1 a fluorescent probe is not methylated at this site, and the 5' and 3' ends are labeled with HEX and BHQ1 respectively, named as probe 2 (Probe2).
  • the sequences of primers and probes are shown in Table 2 .
  • the primer and probe sequences for the five methylation sites are listed in Table 2.
  • reaction system including 10 ⁇ L mix, 1.5 ⁇ L primer, 0.8 ⁇ L each of probe 1 and probe 2, 4.9 ⁇ L water, 2 ⁇ L DNA.
  • the reaction conditions were 95 °C for 5 min, followed by 45 cycles with each cycle set to 95 °C for 15 s, 58 °C for 60 s, and 40 °C for 30 s.
  • MD Method for calculating the MD value
  • CT m is the Ct value after amplification with the amplification primers and methylated probes at this site
  • CT u is the Ct value after amplification with the amplification primers and unmethylated probes at this site.
  • COV cut off value
  • Youden index also known as the correct index, is a method to evaluate the authenticity of screening tests, assuming that its false negative (missed diagnosis rate) and false positive (misdiagnosed rate) are equally harmful, it can be applied Youden index. Indicates the total ability of the screening method to detect true patients and non-patients. The larger the index, the better the effect of the screening experiment and the greater the accuracy.
  • the MD value of the methylation site located at 57946bp of Epstein-Barr virus genomic DNA has a sensitivity of 92.30%, a specificity of 78.60%, and a Youden index of 70.90% in the diagnosis of nasopharyngeal carcinoma; it is located at 128103bp of Epstein-Barr virus genomic DNA
  • the sensitivity of the MD value of the methylation site in the diagnosis of nasopharyngeal carcinoma was 81.60%, the specificity was 79.80%, and the Youden index was 61.40%.
  • the accuracy of the diagnosis of nasopharyngeal carcinoma is significantly better than that of Epstein-Barr virus DNA load (the sensitivity is 75.60%, the specificity is 85.70%, and the Youden index is 61.30%).
  • the decrease in sampling precision resulted in a decrease in the Epstein-Barr virus load, resulting in low sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma.
  • NPC nasopharyngeal carcinoma
  • Control control
  • EB virus DNA load greater than the Cut off value is judged as nasopharyngeal carcinoma
  • EB virus DNA methylation site MD value less than the Cut off value is judged as nasopharyngeal carcinoma
  • AUC(Area Under Curve) The area under the ROC curve and the coordinate axis.
  • each methylation site was used to construct a nasopharyngeal carcinoma diagnostic model, and the sensitivity and specificity of each model in the diagnosis of nasopharyngeal carcinoma were analyzed. The results are shown in Table 4.
  • bamHI is the Epstein-Barr virus DNA load, and its unit is copy/ng DNA; in each model, the judgment standard is that the model Pd score is greater than the cut off value, which is judged as nasopharyngeal carcinoma; AUC (Area Under Curve): the area under the ROC curve and the coordinate axis circumference into the area.
  • models 1 to 6 are nasopharyngeal carcinoma diagnostic models constructed only using the MD values calculated by Epstein-Barr virus DNA methylation. Sex is the best.
  • models 7 to 13 were constructed by combining the EBV DNA load and the MD value of the EBV DNA methylation site. Except for model 12, the diagnostic accuracy is also better than using the EBV DNA load alone as a nasopharyngeal carcinoma. diagnostic markers. Among them, model 9 has the best accuracy.
  • At least one of the five sites had an MD value exceeding the detection line and was judged to be nasopharyngeal carcinoma.
  • the data suggested that the detection of Epstein-Barr virus DNA methylation had a positive effect on the occurrence of nasopharyngeal carcinoma. Good predictive power.
  • Epstein-Barr virus DNA methylation MD, 11029, 45850, 57946, 66227 and 128103 cut off values were -0.39, -0.04, -1.07, - 2.91 and -2.19, less than the Cut off value is judged as nasopharyngeal carcinoma;
  • Example 5 Relying on the detection of methylation of Epstein-Barr virus DNA and the load of Epstein-Barr virus DNA in nasopharyngeal swab samples guided by clinicians and nasopharyngoscope
  • the median value of the Epstein-Barr virus DNA 11029 locus MD value was -2.79 in the nasopharyngeal brush sampling samples guided by clinicians and nasopharyngoscopes, ranging from -4.53 to -1.13, In contrast, the median value was -2.77 in the clinician-guided and nasopharyngoscope-guided nasopharyngeal brush sampling samples, and the range was -4.77 to 0, with no significant change between the two groups.
  • Example 6 Nasopharyngeal sample preservation solution capable of simultaneously preserving DNA and RNA
  • a comparison of two groups of preservation solutions was set up, namely self-preparation and a commercially available DNA preservation solution, and self-preparation and a commercially available RNA preservation solution.
  • Each group selected 6 patients with nasopharyngeal carcinoma, with one swab on the left and right. Place them in self-prepared and commercially available preservation solutions, and then use Tiangen DNA extraction reagent to extract DNA, Qiagen RNA extraction reagent to extract RNA, DNA/RNA is used for the detection of downstream targets, the targets include BamHI-W, LMP -2, C4, W2, miR-BART1-5p, miR-BART7-3p.
  • Reagent component name Formula content Deionized water 100mL EDTA-2Na 1.5g NaCl 0.05g
  • the DNA was extracted using the Magnetic Beads Method Universal Genomic DNA Extraction Kit, manufactured by Tiangen Biochemical Technology (Beijing) Co., Ltd., article number: DP705, and extracted using Tianlong NP968 Automatic Nucleic Acid Extractor (Xi’an Tianlong Technology Co., Ltd.). The process is as follows:
  • the magnetic beads were eluted with 900 ⁇ L buffer GDZ, and then eluted once with 500 ⁇ L buffer GDZ.
  • washing solution PWD Use 900 ⁇ L of washing solution PWD to elute the magnetic beads, and then use 300 ⁇ L of washing solution PWD to elute once.
  • the method of fluorescent quantitative PCR was used to qualitatively detect the Epstein-Barr virus DNA in the nasopharyngeal brush sample, and the detection primers and probes are shown in Table 8.
  • LMP-2 primers are from Lao, T.D., Nguyen, T.A.H., Ngo, K.D., et al. Molecular Screening of Nasopharyngeal Carcinoma: Detection of LMP-1, LMP-2 Gene Expression in Vietnamese Nasopharyngeal Swab Samples.2019, Asian Pac J Cancer Prev, 20(9): 2757-2761.
  • the genomic DNA of the 5 ⁇ L sample was first treated and purified with bisulfite, and 22 ⁇ L was eluted to obtain the converted modified DNA as a template for q-PCR detection.
  • EBV-miR-BART1-5p UCUUAGUGGAAGUGACGUGCUGUG (SEQ ID NO: 39)
  • EBV-miR-BART7-3p CAUCAUAGUCCAGUGUCCAGGG (SEQ ID NO: 42)
  • the primers and probes used are listed in Table 16.
  • the reverse transcription kit uses TaqMan TM MicroRNA Reverse Transcription Kit (Applied Biosystems), catalog number: 4366596.
  • RNA 5 ⁇ L
  • reverse transcription master mix 7 ⁇ L
  • reverse transcription primer 3 ⁇ L
  • the reaction system is shown in Table 17.
  • qPCR was performed using TaqMan TM Fast Advanced Master Mix (Applied Biosystems), catalog number: 4444557.
  • TaqMan TM Fast Advanced Master Mix 10.00 ⁇ L cDNA 1.00 ⁇ L Primer F (final concentration 300nM) 0.60 ⁇ L Primer R (final concentration 300nM) 0.60 ⁇ L Probe (final concentration 200nM) 0.40 ⁇ L RNase-free water x ⁇ L Total volume 20.00 ⁇ L
  • EBV-miR-BART1-5p UCUUAGUGGAAGUGACGUGCUGUG (SEQ ID NO: 39)
  • EBV-miR-BART7-3p CAUCAUAGUCCAGUGUCCAGGG (SEQ ID NO: 42)
  • the primers and probes involved are shown in Table 25.
  • RNA extraction reagent ng/ ⁇ L
  • Self-prepared RNA extraction reagent 1 4.1 28.6 2 1.06 2.37 3 0.649 2.03 4 0.518 1.98 5 6.24 17.9 6 0.841 3.04
  • RNA extraction reagent ng/ ⁇ L
  • Self-prepared RNA extraction reagent ng/ ⁇ L 1 18 32.8 2 6.1 11.5 3 3.1 11.2 4 2.9 12.4 5 19.2 23.9 6 4.6 11.6
  • Example 8 Detection of nasopharyngeal carcinoma based on miRNA as a supplementary biomarker
  • EBV-miR-BART1-5p UCUUAGUGGAAGUGACGUGCUGUG (SEQ ID NO: 39)
  • EBV-miR-BART2-5p UAUUUUCUGCAUUCGCCCUUGC (SEQ ID NO: 40)
  • EBV-miR-BART6-3p CGGGGAUCGGACUAGCCUUAGA (SEQ ID NO: 41)
  • EBV-miR-BART7-3p CAUCAUAGUCCAGUGUCCAGGG (SEQ ID NO: 42)
  • EBV-miR-BART13-3p UGUAACUUGCCAGGGACGGCUGA (SEQ ID NO: 43)
  • EBV-miR-BART17-5p UAAGAGGACGCAGGCAUACAAG (SEQ ID NO: 44)
  • the miRNA primer probe sequences are shown in Table 28.
  • DNA load primer probes are shown in Table 29.
  • LMP-2 primers Lao, T.D., Nguyen, T.A.H., Ngo, K.D., et al. Molecular Screening of Nasopharyngeal Carcinoma: Detection of LMP-1, LMP-2 Gene Expression in Vietnamese Nasopharyngeal Swab Samples.2019, Asian Pac J Cancer Prev, 20(9): 2757-2761.
  • the primer probes for the methylation detection of Epstein-Barr virus DNA are shown in Table 30.
  • the methylation sites were located in the C promoter region and the W promoter region of the EBV genome, respectively.
  • C1 upstream and downstream primer sequences are from Zheng Xiaohui's doctoral thesis: Zheng Xiaohui. 2020. Xinjiang Medical University.
  • the C10 probe is derived from the probe of this patent 11029.
  • the nasopharyngeal carcinoma diagnosis model 1 was constructed by combining the Epstein-Barr virus DNA load and each methylation site, and the sensitivity and specificity of the model for nasopharyngeal carcinoma diagnosis were analyzed, in which A1 is BamHI-W, A2 is LMP-2, B1 is methylated C1, B2 is methylated C4, B3 is methylated W1, B4 is methylated C10, and B5 is methylated W2.
  • the results are shown in Table 31.
  • the model includes sites A2, B2, B3 and B4, with a sensitivity of 92.6%, a specificity of 93.1%, and a Youden index of 0.857.
  • E1 is miR-BART1-5p
  • E2 is miR-BART17-5p
  • E3 is miR-BART6-3p
  • E4 is miR-BART7-3p
  • E5 is miR-BART2-5p
  • E6 is miR-BART13-3p.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pathology (AREA)
  • Hospice & Palliative Care (AREA)
  • Neurosurgery (AREA)
  • Virology (AREA)
  • Oncology (AREA)
  • Plant Pathology (AREA)
  • Neurology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Psychiatry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

An application of a reagent for EB virus methylation site typing and EB virus-related miRNA detection in the preparation of a nasopharyngeal carcinoma diagnostic kit. An EB virus methylation site and/or an EB virus-related miRNA can serve as a nasopharyngeal carcinoma diagnostic marker, which has the advantages of high sensitivity and high specificity in a nasopharyngeal brush or nasopharyngeal swab (nasopharynx blind detection) sample that does not rely on a clinician and a nasopharyngoscope.

Description

用于鼻咽癌诊断的试剂和方法Reagents and methods for diagnosis of nasopharyngeal carcinoma 技术领域technical field
本发明涉及鼻咽癌的诊断技术领域，具体地，涉及用于鼻咽癌诊断的试剂和方法，以及相关的生物标志物。The present invention relates to the technical field of diagnosis of nasopharyngeal carcinoma, in particular, to reagents and methods for diagnosis of nasopharyngeal carcinoma, and related biomarkers.
背景技术Background technique
鼻咽癌是指发生于鼻咽顶后壁和咽隐窝的恶性肿瘤，是在中国高发恶性肿瘤之一，发病率为耳鼻咽喉恶性肿瘤之首。早期鼻咽癌治疗后的五年生存率能达到90％以上，而中晚期鼻咽癌的五年生存率只有不到50％，由于早期疾病基本没有任何症状，70％以上的鼻咽癌病人在首次就诊时疾病已经进展到中晚期，因此推动鼻咽癌的早筛以及早诊早治是提高鼻咽癌预后的关键。Nasopharyngeal carcinoma refers to malignant tumors that occur in the posterior wall of the nasopharynx and pharyngeal recesses. It is one of the most frequently occurring malignant tumors in China, and its incidence rate is the first among malignant tumors of the ear, nose and throat. The five-year survival rate of early nasopharyngeal carcinoma after treatment can reach more than 90%, while the five-year survival rate of middle and advanced nasopharyngeal carcinoma is less than 50%. The disease has progressed to the middle and late stage when the first visit to the doctor, so promoting early screening and early diagnosis and treatment of nasopharyngeal carcinoma is the key to improving the prognosis of nasopharyngeal carcinoma.
已有充分的研究表明，EB病毒(Epstein-Barr virus)感染在鼻咽癌的发病中扮演着重要的角色。在鼻咽癌流行地区，几乎100％的未分化型鼻咽癌(WHO III型)的病变部位都可以检测到EBV基因组的存在；此外，鼻咽鳞状细胞癌(WHO I型)和非角化型鼻咽癌(WHO II型)的发生也与EBV感染密切相关，可以说鼻咽上皮细胞的EBV感染是鼻咽癌的一个重要特征。Sufficient studies have shown that Epstein-Barr virus (Epstein-Barr virus) infection plays an important role in the pathogenesis of nasopharyngeal carcinoma. In nasopharyngeal carcinoma endemic areas, almost 100% of undifferentiated nasopharyngeal carcinoma (WHO type III) lesions can detect the presence of EBV genome; in addition, nasopharyngeal squamous cell carcinoma (WHO type I) and non-angular The occurrence of nasopharyngeal carcinoma (WHO type II) is also closely related to EBV infection. It can be said that EBV infection of nasopharyngeal epithelial cells is an important feature of nasopharyngeal carcinoma.
鼻咽癌临床诊断的金标准是鼻咽镜下进行组织活检取材，之后由病理医生进行病理诊断。由于取材创伤性较大、取材组织小、且依赖镜下肉眼观察，因此约有10％的患者在首次取材时诊断失败，医生和患者需面临再次或多次取材、或随访观察的抉择，易延误病情。此外，鼻咽活检严格依赖临床医生的临床经验，以及鼻咽电子镜等医疗器械，难以在高发现场开展鼻咽癌广泛筛查和在基层医疗单位开展早期诊断。基于血液样本的EBV标志物的检测还依赖临床护士进行采血取样，限制了其在鼻咽癌高发现场进行全人群的推广检测，因准确性不足也降低了民众的依从性。所以开发易于在高发现场推广应用的取样技术以及高效的诊断分子标志物仍然意义重大。The gold standard for clinical diagnosis of nasopharyngeal carcinoma is tissue biopsy under nasopharyngoscope, followed by pathological diagnosis by pathologists. Due to the relatively traumatic nature of the specimens, the small size of the specimens, and the need to observe with the naked eye under a microscope, about 10% of the patients failed to be diagnosed when the specimens were first drawn. Delayed illness. In addition, nasopharyngeal biopsy strictly relies on the clinical experience of clinicians and medical devices such as nasopharyngeal electronic mirrors, making it difficult to carry out extensive screening of nasopharyngeal carcinoma in high-incidence sites and early diagnosis in primary medical units. The detection of EBV markers based on blood samples also relies on clinical nurses to take blood samples, which limits its promotion and detection of the whole population in high-incidence sites of nasopharyngeal carcinoma, and the lack of accuracy also reduces the compliance of the public. Therefore, it is still of great significance to develop sampling techniques that are easy to promote and apply in high-occurrence sites and efficient diagnostic molecular markers.
由于鼻咽癌起源于鼻咽部，因此对于鼻咽部的无创，高效取材及相应分子标志物的检测能更为直接反应病变部位的状况，理论上能及时发现早期原发或复发的鼻咽癌患者。对鼻咽部的病理活检取样创伤大，但通过鼻咽刷或鼻咽拭子刷取鼻咽部样本是可行的技术方案，既往已经在中国香港地区，中国台湾地区，印度尼西亚，加拿大等国家或地区开展了相应研究，即在电子鼻咽镜的引导下，利用鼻咽刷或鼻咽拭子对鼻咽部可疑部位刷取取样， 随后通过对EB病毒DNA载量进行q-PCR的检测，来判断是否患有鼻咽癌。由于鼻咽刷是可以直接获取鼻咽部病变部位的样本而采用的方法，不仅具有无痛、无侵袭性的优点，而且获取的脱落细胞可以直接反映鼻咽部肿瘤的特性。Since nasopharyngeal carcinoma originates from the nasopharynx, the non-invasive and efficient sampling of nasopharynx and the detection of corresponding molecular markers can more directly reflect the condition of the lesion, and theoretically, early primary or recurrent nasopharynx can be detected in time cancer patients. The pathological biopsy sampling of the nasopharynx is traumatic, but it is a feasible technical solution to take nasopharyngeal samples by nasopharyngeal brush or nasopharyngeal swab. It has been used in Hong Kong, Taiwan, Indonesia, Canada and other countries or Corresponding research has been carried out in the region, that is, under the guidance of an electronic nasopharyngoscope, use a nasopharyngeal brush or a nasopharyngeal swab to brush and sample suspicious parts of the nasopharynx, and then perform q-PCR detection of the EB virus DNA load. To determine whether there is nasopharyngeal carcinoma. Because the nasopharyngeal brush is a method that can directly obtain samples of nasopharyngeal lesions, it not only has the advantages of painlessness and non-invasiveness, but also the obtained exfoliated cells can directly reflect the characteristics of nasopharyngeal tumors.
但是，对鼻咽部的鼻咽刷检相对鼻咽病理活检，虽然具有无创，可重复取样等优势，但为了提高取样的准确度，过往研究中仍然依赖临床医生和鼻咽镜引导开展，阻碍了这一技术方案在现场广泛应用于鼻咽癌的筛查。因此，不依赖临床医生和鼻咽镜引导的鼻咽刷或鼻咽拭子检测(简称为鼻咽部盲检)具有利于推广的天然优势，但是，目前鼻咽部盲检的劣势是取样精度的降低，所以开发适用于鼻咽盲刷取样体系下的分子标志物及其检测体系意义重大。However, compared with nasopharyngeal biopsy, nasopharyngeal brush examination has the advantages of non-invasive and repeatable sampling, but in order to improve the accuracy of sampling, previous studies still rely on clinicians and nasopharyngoscope guidance, which hinders This technical solution is widely used in the screening of nasopharyngeal carcinoma in the field. Therefore, nasopharyngeal brush or nasopharyngeal swab detection (referred to as nasopharyngeal blind inspection) that does not rely on clinicians and nasopharyngoscope guidance has natural advantages that are conducive to popularization. However, the current disadvantage of nasopharyngeal blind inspection is the sampling accuracy. Therefore, it is of great significance to develop molecular markers and their detection systems suitable for nasopharyngeal blind brush sampling system.
概述overview
本发明的一方面，提供了一种用于检测EB病毒的试剂盒，其特征在于，所述试剂盒包含选自核酸甲基化检测试剂、miRNA检测试剂、miRNA提取试剂，和/或样本保存液的一种或多种试剂。One aspect of the present invention provides a kit for detecting Epstein-Barr virus, characterized in that, the kit includes nucleic acid methylation detection reagents, miRNA detection reagents, miRNA extraction reagents, and/or sample preservation One or more reagents of the liquid.
本发明的一方面，提供了一种用于鼻咽癌检测的试剂盒，其特征在于，所述试剂盒包含选自核酸甲基化检测试剂、miRNA检测试剂、miRNA提取试剂，和/或样本保存液的一种或多种试剂。One aspect of the present invention provides a kit for nasopharyngeal carcinoma detection, characterized in that, the kit includes nucleic acid methylation detection reagents, miRNA detection reagents, miRNA extraction reagents, and/or samples One or more reagents of the preservation solution.
在一些实施例中，所述试剂盒包含核酸甲基化检测试剂，所述核酸甲基化检测试剂可针对EB病毒基因组中甲基化位点进行甲基化检测。在一些实施例中，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)(NCBI Reference Sequence：NC_007605.1；ACCESSION：NC_007605；VERSION：NC_007605.1；获得自： https：//www.ncbi.nlm.nih.gov/nuccore/NC_007605)的EB病毒基因组DNA序列的10717bp、11029bp、14015bp、14566bp、45850bp、57946bp、66227bp和128103bp的位点。在一些实施例中，所述EB病毒基因组中的所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、和14015bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp、45850bp、57946bp、66227bp和128103bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的 11029bp、57946bp、和66227bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的14015bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的14566bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的45850bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的57946bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的66227bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的128103bp的位点。 In some embodiments, the kit includes a nucleic acid methylation detection reagent, which can perform methylation detection for methylation sites in the Epstein-Barr virus genome. In some embodiments, the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) (NCBI Reference Sequence: NC_007605.1; ACCESSION 10717bp, 11029bp, 14015bp, 14566bp, 45850bp , 57946bp, 662 27bp and 128103bp sites. In some embodiments, the methylation sites in the Epstein-Barr virus genome comprise sites corresponding to 10717bp, 11029bp, and 14015bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: 11029bp and 45850bp of the Epstein-Barr virus genome DNA sequence corresponding to GenBank LOCUS NC_007605 (SEQ ID NO: 94) , 57946bp, 66227bp and 128103bp sites. In some embodiments, the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: 11029 bp and 57946 bp of the Epstein-Barr virus genome DNA sequence corresponding to GenBank LOCUS NC_007605 (SEQ ID NO: 94) , and the 66227bp site. In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 11029 bp of the Epstein-Barr virus genome DNA sequence in GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 10717 bp of the Epstein-Barr virus genome DNA sequence in GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 14015 bp of the Epstein-Barr virus genome DNA sequence in GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 14566 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 45850 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 57946 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 66227 bp of the Epstein-Barr virus genome DNA sequence in GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 128103 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
在一些实施例中，所述试剂盒包含所述miRNA检测试剂，所述miRNA检测试剂可针对一个或多个来自EB病毒的miRNA进行含量检测。在一些实施例中，所述miRNA包含以下miRNA的一个或几个：EBV-miR-BART1-5p、EBV-miR-BART2-5p、EBV-miR-BART6-3p、EBV-miR-BART7-3p、EBV-miR-BART13-3p、和EBV-miR-BART17-5p。在一些实施例中，包含全部六个所述miRNA。在一些实施例中，所述miRNA包含EBV-miR-BART1-5p和EBV-miR-BART7-3p。在一些实施例中，所述miRNA包含EBV-miR-BART1-5p。在一些实施例中，所述miRNA包含EBV-miR-BART2-5p。在一些实施例中，所述miRNA包含EBV-miR-BART6-3p。在一些实施例中，所述miRNA包含EBV-miR-BART7-3p。在一些实施例中，所述miRNA包含EBV-miR-BART13-3p。在一些实施例中，所述miRNA包含EBV-miR-BART17-5p。In some embodiments, the kit includes the miRNA detection reagent, and the miRNA detection reagent can detect the content of one or more miRNAs from Epstein-Barr virus. In some embodiments, the miRNA comprises one or more of the following miRNAs: EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR-BART6-3p, EBV-miR-BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p. In some embodiments, all six of said miRNAs are included. In some embodiments, the miRNAs comprise EBV-miR-BART1-5p and EBV-miR-BART7-3p. In some embodiments, the miRNA comprises EBV-miR-BART1-5p. In some embodiments, the miRNA comprises EBV-miR-BART2-5p. In some embodiments, the miRNA comprises EBV-miR-BART6-3p. In some embodiments, the miRNA comprises EBV-miR-BART7-3p. In some embodiments, the miRNA comprises EBV-miR-BART13-3p. In some embodiments, the miRNA comprises EBV-miR-BART17-5p.
在一些实施例中，所述试剂盒包含所述样本保存液，所述样本保存液包含选自EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和/或磷酸三钠的一种或多种组分。在一些实施例中，，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。In some embodiments, the kit includes the sample preservation solution, and the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and/or trisodium phosphate one or more components. In some embodiments, the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
本发明的一方面，提供了一种保存含核酸的生物样本的试剂盒，其特征在于，包含样 本保存液，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。One aspect of the present invention provides a kit for preserving biological samples containing nucleic acid, which is characterized in that it includes a sample preservation solution, and the sample preservation solution includes EDTA, NaCl, absolute ethanol, sodium citrate, NP40, gluten Glutathione, and Trisodium Phosphate.
在一些实施例中，所述样本保存液中：所述EDTA的含量范围是0.7％-1.5％(w/w)；所述NaCl的含量范围是0.01％-0.1％(w/w)；所述无水乙醇的浓度范围是15％-35％(w/w)；所述柠檬酸钠的含量范围是0.1％-1.0％(w/w)；所述NP40的浓度范围是0.01％-0.1％(v/v)；所述谷胱甘肽的含量范围是0.07％-0.7％(w/w)；和/或所述磷酸三钠的含量范围是0.07％-0.4％(w/w)。在一些实施例中，所述样本保存液中：所述EDTA的含量是约1.05％(w/w)；所述NaCl的含量是约0.035％(w/w)；所述无水乙醇的浓度是约27.6％(w/w)；所述柠檬酸钠的含量是约0.21％(w/w)；所述NP40的浓度是约0.035％(v/v)；所述谷胱甘肽的含量是约0.21％(w/w)；和/或所述磷酸三钠的含量是约0.14％(w/w)。在一些实施例中，所述样本保存液包含抗生素。在一些实施例中，所述抗生素包含青霉素-链霉素双抗。在一些实施例中，所述样本保存液的pH值为7.6-8.0。在一些实施例中，所述样本保存液的pH值为7.8。在一些实施例中，与对照样本保存液相比，所述样本保存液对DNA，miRNA，和/或DNA甲基化的保存效果更好。In some embodiments, in the sample preservation solution: the content range of the EDTA is 0.7%-1.5% (w/w); the content range of the NaCl is 0.01%-0.1% (w/w); The concentration range of the dehydrated alcohol is 15%-35% (w/w); the content range of the sodium citrate is 0.1%-1.0% (w/w); the concentration range of the NP40 is 0.01%-0.1 % (v/v); said glutathione content ranges from 0.07% to 0.7% (w/w); and/or said trisodium phosphate content ranges from 0.07% to 0.4% (w/w) . In some embodiments, in the sample preservation solution: the content of the EDTA is about 1.05% (w/w); the content of the NaCl is about 0.035% (w/w); the concentration of the absolute ethanol is about 27.6% (w/w); the content of the sodium citrate is about 0.21% (w/w); the concentration of the NP40 is about 0.035% (v/v); the content of the glutathione is about 0.21% (w/w); and/or said trisodium phosphate is present in an amount of about 0.14% (w/w). In some embodiments, the sample preservation solution comprises antibiotics. In some embodiments, the antibiotic comprises double penicillin-streptomycin. In some embodiments, the pH value of the sample preservation solution is 7.6-8.0. In some embodiments, the pH value of the sample preservation solution is 7.8. In some embodiments, the sample preservation solution has a better preservation effect on DNA, miRNA, and/or DNA methylation than the control sample preservation solution.
在一些实施例中，所述DNA来源于EB病毒，所述miRNA来源于EB病毒，和/或所述DNA甲基化是EB病毒DNA的甲基化。In some embodiments, the DNA is derived from Epstein-Barr virus, the miRNA is derived from Epstein-Barr virus, and/or the DNA methylation is methylation of Epstein-Barr virus DNA.
在一些实施例中，所述试剂盒包含所述miRNA提取试剂，所述miRNA提取试剂包含裂解液，结合液，漂洗液1，和/或漂洗液2。In some embodiments, the kit comprises the miRNA extraction reagent, and the miRNA extraction reagent comprises a lysis solution, a binding solution, a washing solution 1, and/or a washing solution 2.
本发明的一方面，提供了一种包含miRNA提取试剂的试剂盒，其特征在于，所述miRNA提取试剂包含裂解液，结合液，漂洗液1，和/或漂洗液2。One aspect of the present invention provides a kit comprising miRNA extraction reagents, characterized in that the miRNA extraction reagents comprise a lysing solution, a binding solution, a washing solution 1, and/or a washing solution 2.
在一些实施例中，所述miRNA提取试剂包含所述裂解液，所述裂解液包含选自硫氰酸胍，柠檬酸，曲拉通-100，和/或EDTA的一种或多种组分。在一些实施例中，所述miRNA提取试剂包含所述裂解液，所述裂解液包含硫氰酸胍，柠檬酸，曲拉通-100，和EDTA。在一些实施例中，所述裂解液中：所述硫氰酸胍的含量范围是28％-40％(w/w)；所述柠檬酸的含量范围是1.8％-2.6％(w/w)；所述曲拉通-100的含量范围是0.8％-1.6％(w/w)；所述EDTA的含量范围是0.5％-0.9％(w/w)；和/或所述裂解液的pH值范围为3.8-4.2。在一些实施例中，所述裂解液中：所述硫氰酸胍的含量是约32％(w/w)；所述柠檬酸的含量是约2.2％(w/w)；所述曲拉通-100的含量是约1.1％(w/w)；所述EDTA的含量是约0.7％(w/w)；和/或所述裂解液的pH值为约4.0。In some embodiments, the miRNA extraction reagent comprises the lysate comprising one or more components selected from guanidine thiocyanate, citric acid, Triton-100, and/or EDTA . In some embodiments, the miRNA extraction reagent comprises the lysate comprising guanidine thiocyanate, citric acid, Triton-100, and EDTA. In some embodiments, in the lysate: the content range of the guanidine thiocyanate is 28%-40% (w/w); the content range of the citric acid is 1.8%-2.6% (w/w ); the content range of the Triton-100 is 0.8%-1.6% (w/w); the content range of the EDTA is 0.5%-0.9% (w/w); and/or the lysate The pH range is 3.8-4.2. In some embodiments, in the lysate: the content of the guanidine thiocyanate is about 32% (w/w); the content of the citric acid is about 2.2% (w/w); The content of Tong-100 is about 1.1% (w/w); the content of the EDTA is about 0.7% (w/w); and/or the pH value of the lysate is about 4.0.
在一些实施例中，所述miRNA提取试剂包含所述结合液，所述结合液包含选自氯化 镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和/或异丙醇的一种或多种组分。在一些实施例中，所述结合液包含氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和异丙醇。在一些实施例中，所述结合液中：所述氯化镁的含量范围是0.1％-0.4％(w/w)；所述乙二胺四乙酸二钠的含量范围是0.5％-1.0％(w/w)；所述碳酸氢钠的含量范围是0.6％-1.0％(w/w)；所述硫氰酸胍的含量范围是13％-16％(w/w)；所述十二烷基肌氨酸钠的含量范围是1.0％-1.4％(w/w)；所述异丙醇的含量范围是60％-80％(w/w)；和/或所述结合液的pH值范围是4.8-5.2。在一些实施例中，所述结合液中：所述氯化镁的含量是约0.24％(w/w)；所述乙二胺四乙酸二钠的含量是约0.71％(w/w)；所述碳酸氢钠的含量是约0.83％(w/w)；所述硫氰酸胍的含量是约14.9％(w/w)；所述十二烷基肌氨酸钠的含量是约1.2％(w/w)；所述异丙醇的含量是约70％(w/w)；和/或所述结合液的pH值是约5.0。In some embodiments, the miRNA extraction reagent comprises the binding solution, which comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, lauryl sarcosine Sodium, and/or one or more components of isopropanol. In some embodiments, the binding solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, sodium sarcosine, and isopropanol. In some embodiments, in the combined solution: the content range of the magnesium chloride is 0.1%-0.4% (w/w); the content range of the disodium edetate is 0.5%-1.0% (w/w). /w); the content range of the sodium bicarbonate is 0.6%-1.0% (w/w); the content range of the guanidine thiocyanate is 13%-16% (w/w); the dodecane The content range of sodium sarcosinate is 1.0%-1.4% (w/w); the content range of the isopropanol is 60%-80% (w/w); and/or the pH value of the combination solution The range is 4.8-5.2. In some embodiments, in the combined solution: the magnesium chloride content is about 0.24% (w/w); the disodium edetate content is about 0.71% (w/w); the The content of sodium bicarbonate is about 0.83% (w/w); The content of said guanidine thiocyanate is about 14.9% (w/w); The content of said sodium lauryl sarcosinate is about 1.2% ( w/w); the content of the isopropanol is about 70% (w/w); and/or the pH value of the combination solution is about 5.0.
在一些实施例中，所述miRNA提取试剂包含所述漂洗液1，所述漂洗液1包含选自乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和/或十二烷基肌氨酸钠的一种或多种组分。在一些实施例中，所述miRNA提取试剂包含所述漂洗液1，所述漂洗液1包含乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和十二烷基肌氨酸钠。在一些实施例中，所述漂洗液1中：所述乙二胺四乙酸二钠的含量范围是0.4％-0.9％(w/w)；所述碳酸氢钠的含量范围是0.5％-0.9％(w/w)；所述硫氰酸胍的含量范围是50％-55％(w/w)；所述十二烷基肌氨酸钠的含量范围是0.8％-1.2％(w/w)；和/或所述漂洗液1的pH值范围是6.2-6.6。在一些实施例中，所述漂洗液1中：所述乙二胺四乙酸二钠的含量是约0.5％(w/w)；所述碳酸氢钠的含量是约0.6％(w/w)；所述硫氰酸胍的含量是约53％(w/w)；所述十二烷基肌氨酸钠的含量是约0.9％(w/w)；和/或所述漂洗液1的pH值是约6.4。In some embodiments, the miRNA extraction reagent comprises the washing solution 1, and the washing solution 1 comprises disodium edetate, sodium bicarbonate, guanidinium thiocyanate, and/or dodecyl One or more components of sodium sarcosinate. In some embodiments, the miRNA extraction reagent comprises the rinse solution 1 comprising disodium edetate, sodium bicarbonate, guanidinium thiocyanate, and sodium lauryl sarcosinate . In some embodiments, in the rinse liquid 1: the content range of the disodium edetate is 0.4%-0.9% (w/w); the content range of the sodium bicarbonate is 0.5%-0.9% % (w/w); The content scope of described guanidinium thiocyanate is 50%-55% (w/w); The content scope of described sodium lauryl sarcosinate is 0.8%-1.2% (w/ w); and/or the pH range of the rinse solution 1 is 6.2-6.6. In some embodiments, in the rinse solution 1: the disodium edetate content is about 0.5% (w/w); the sodium bicarbonate content is about 0.6% (w/w) the content of the guanidine thiocyanate is about 53% (w/w); the content of the sodium lauryl sarcosinate is about 0.9% (w/w); and/or the rinse solution 1 The pH is about 6.4.
在一些实施例中，所述miRNA提取试剂包含所述漂洗液2，所述漂洗液2包含三羟甲基氨基甲烷(Tris)或氯化钠。在一些实施例中，所述miRNA提取试剂包含所述漂洗液2，所述漂洗液2包含三羟甲基氨基甲烷(Tris)和氯化钠。在一些实施例中，所述漂洗液2中：所述三羟甲基氨基甲烷(Tris)的含量范围是0.5％-0.7％(w/w)；所述氯化钠的含量范围是0.6％-1.8％(w/w)；和/或所述漂洗液2的pH值范围是6.6-7.0。在一些实施例中，所述漂洗液2中：所述三羟甲基氨基甲烷(Tris)的含量是约0.6％(w/w)；所述氯化钠的含量是约1.2％(w/w)；和/或所述漂洗液2的pH值是约6.8。In some embodiments, the miRNA extraction reagent comprises the rinsing solution 2, and the rinsing solution 2 comprises tris (Tris) or sodium chloride. In some embodiments, the miRNA extraction reagent comprises the rinse solution 2 comprising tris (Tris) and sodium chloride. In some embodiments, in the rinse liquid 2: the content range of the tris (Tris) is 0.5%-0.7% (w/w); the content range of the sodium chloride is 0.6% -1.8% (w/w); and/or the pH value of the rinse solution 2 is in the range of 6.6-7.0. In some embodiments, in the rinse solution 2: the content of the tris (Tris) is about 0.6% (w/w); the content of the sodium chloride is about 1.2% (w/ w); and/or the pH value of the rinse solution 2 is about 6.8.
在一些实施例中，所述miRNA提取试剂包含吸附RNA的磁珠。在一些实施例中，所述磁珠的磁核是氢氧化三铁，包被二氧化硅，且表面镶嵌羟基基团。在一些实施例中， 所述磁珠的粒径为50-100nm。In some embodiments, the miRNA extraction reagent comprises RNA-adsorbing magnetic beads. In some embodiments, the magnetic core of the magnetic beads is ferric hydroxide, coated with silicon dioxide, and embedded with hydroxyl groups on the surface. In some embodiments, the particle diameter of the magnetic beads is 50-100 nm.
在一些实施例中，所述试剂盒还包含鼻咽刷或鼻咽拭子。在一些实施例中，所述鼻咽刷和/或鼻咽拭子为不依赖临床医生和鼻咽镜引导的鼻咽刷和/或鼻咽拭子。In some embodiments, the kit further comprises a nasopharyngeal brush or nasopharyngeal swab. In some embodiments, the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab that does not rely on the guidance of a clinician and a nasopharyngoscope.
在一些实施例中，所述试剂盒还包含EB病毒载量检测试剂。在一些实施例中，所述EB病毒载量检测试剂包含针对EB病毒基因组中BamHI-W片段，和/或LMP-2基因的检测试剂。在一些实施例中，所述EB病毒载量检测试剂包含针对LMP-2基因的检测试剂。In some embodiments, the kit further includes Epstein-Barr virus load detection reagents. In some embodiments, the Epstein-Barr virus load detection reagent comprises a detection reagent for the BamHI-W fragment in the Epstein-Barr virus genome and/or the LMP-2 gene. In some embodiments, the EB virus load detection reagent comprises a detection reagent for LMP-2 gene.
在一些实施例中，所述试剂盒为鼻咽癌筛查试剂盒。在一些实施例中，所述试剂盒为鼻咽癌诊断试剂盒。In some embodiments, the kit is a nasopharyngeal carcinoma screening kit. In some embodiments, the kit is a nasopharyngeal carcinoma diagnostic kit.
本发明的一方面，提供了一种本发明所述的试剂盒在制备鼻咽癌筛查的产品的用途。One aspect of the present invention provides a use of the kit described in the present invention in preparing products for screening nasopharyngeal carcinoma.
本发明的一方面，提供了一种本发明所述的试剂盒在制备鼻咽癌诊断的产品的用途。One aspect of the present invention provides a use of the kit described in the present invention in preparing products for nasopharyngeal carcinoma diagnosis.
本发明的一方面，提供了一种检测样本中EB病毒相关核酸的方法，其特征在于，所述方法包含以下步骤：One aspect of the present invention provides a method for detecting Epstein-Barr virus-related nucleic acids in a sample, characterized in that the method comprises the following steps:
(a)将所述样本置于样本保存液中；(a) placing the sample in a sample preservation solution;
(b)进行选自以下的一项或多项检测：(b) Conduct one or more tests selected from the following:
(b-1)对含有所述样本的所述样本保存液进行EB病毒基因组核酸甲基化检测；(b-1) performing Epstein-Barr virus genomic nucleic acid methylation detection on the sample preservation solution containing the sample;
(b-2)对含有所述样本的所述样本保存液的EB病毒相关miRNA的含量进行检测；和/或(b-2) detecting the content of Epstein-Barr virus-related miRNA in the sample preservation solution containing the sample; and/or
(b-3)对含有所述样本的所述样本保存液进行EB病毒核酸载量检测。(b-3) Detection of Epstein-Barr virus nucleic acid load on the sample preservation solution containing the sample.
在一些实施例中，步骤(b-2)中先从所述样本保存液中提取所述miRNA，然后对所述miRNA的含量进行检测。In some embodiments, in step (b-2), the miRNA is firstly extracted from the sample preservation solution, and then the content of the miRNA is detected.
在一些实施例中，所述的方法包含步骤(b-1)，且所述EB病毒基因组核酸甲基化检测的甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、14015bp、14566bp、45850bp、57946bp、66227bp和128103bp的位点。在一些实施例中，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、和14015bp的位点。在一些实施例中，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp的位点。在一些实施例中，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp的位点。在一些实施例中，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的14015 bp的位点。在一些实施例中，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的14566bp的位点。在一些实施例中，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的45850bp的位点。在一些实施例中，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的57946bp的位点。在一些实施例中，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的66227bp的位点。在一些实施例中，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的128103bp的位点。In some embodiments, the method includes step (b-1), and the methylation site detected by the Epstein-Barr virus genome nucleic acid methylation comprises one or more of the following sites: equivalent to GenBank LOCUS The sites of 10717bp, 11029bp, 14015bp, 14566bp, 45850bp, 57946bp, 66227bp and 128103bp of the Epstein-Barr virus genome DNA sequence of NC_007605 (SEQ ID NO:94). In some embodiments, the methylation site comprises sites corresponding to 10717bp, 11029bp, and 14015bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 10717bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 11029 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 14015 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 14566 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 45850 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 57946 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 66227bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site comprises a site corresponding to 128103bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
在一些实施例中，所述的方法包含步骤(b-2)，所述miRNA包含以下miRNA的一个或几个：EBV-miR-BART1-5p、EBV-miR-BART2-5p、EBV-miR-BART6-3p、EBV-miR-BART7-3p、EBV-miR-BART13-3p、和EBV-miR-BART17-5p。在一些实施例中，包含全部六个所述miRNA。在一些实施例中，所述miRNA包含EBV-miR-BART1-5p和EBV-miR-BART7-3p。在一些实施例中，所述miRNA包含EBV-miR-BART1-5p。在一些实施例中，所述miRNA包含EBV-miR-BART2-5p。在一些实施例中，所述miRNA包含EBV-miR-BART6-3p。在一些实施例中，所述miRNA包含EBV-miR-BART7-3p。在一些实施例中，所述miRNA包含EBV-miR-BART13-3p。在一些实施例中，所述miRNA包含EBV-miR-BART17-5p。In some embodiments, the method comprises step (b-2), and the miRNA comprises one or more of the following miRNAs: EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR- BART6-3p, EBV-miR-BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p. In some embodiments, all six of said miRNAs are included. In some embodiments, the miRNAs comprise EBV-miR-BART1-5p and EBV-miR-BART7-3p. In some embodiments, the miRNA comprises EBV-miR-BART1-5p. In some embodiments, the miRNA comprises EBV-miR-BART2-5p. In some embodiments, the miRNA comprises EBV-miR-BART6-3p. In some embodiments, the miRNA comprises EBV-miR-BART7-3p. In some embodiments, the miRNA comprises EBV-miR-BART13-3p. In some embodiments, the miRNA comprises EBV-miR-BART17-5p.
在一些实施例中，提取所述EB病毒相关miRNA的步骤包含：i)向所述样本保存液中加入裂解液；在一些实施例中，还加入蛋白酶K；ii)加入结合液与核酸吸附材料；iii)去除液体部分，向核酸吸附材料加入漂洗液清洗；和iv)去除液体部分，向所述核酸吸附材料中加入洗脱液，将所述核酸和/或所述miRNA从所述核酸吸附材料中洗脱。In some embodiments, the step of extracting the Epstein-Barr virus-related miRNA includes: i) adding a lysate to the sample preservation solution; in some embodiments, proteinase K is also added; ii) adding a binding solution and a nucleic acid adsorption material ; iii) removing the liquid portion, adding a rinsing solution to the nucleic acid adsorption material for cleaning; and iv) removing the liquid portion, adding an eluent to the nucleic acid adsorption material, and adsorbing the nucleic acid and/or the miRNA from the nucleic acid material eluted.
本发明的一方面，提供了一种提取miRNA的方法，包含以下步骤：i)向样本保存液中加入裂解液；在一些实施例中，还加入蛋白酶K；ii)加入结合液与核酸吸附材料；iii)去除液体部分，向核酸吸附材料加入漂洗液清洗；和iv)去除液体部分，向所述核酸吸附材料中加入洗脱液，将所述核酸和/或所述miRNA从所述核酸吸附材料中洗脱。One aspect of the present invention provides a method for extracting miRNA, comprising the following steps: i) adding a lysate to the sample preservation solution; in some embodiments, proteinase K is also added; ii) adding a binding solution and a nucleic acid adsorption material ; iii) removing the liquid portion, adding a rinsing solution to the nucleic acid adsorption material for cleaning; and iv) removing the liquid portion, adding an eluent to the nucleic acid adsorption material, and adsorbing the nucleic acid and/or the miRNA from the nucleic acid material eluted.
在一些实施例中，所述裂解液包含选自硫氰酸胍，柠檬酸，曲拉通-100，和/或EDTA的一种或多种组分。在一些实施例中，所述裂解液包含硫氰酸胍，柠檬酸，曲拉通-100，和EDTA。在一些实施例中，所述裂解液中：所述硫氰酸胍的含量范围是28％-40％(w/w)；所述柠檬酸的含量范围是1.8％-2.6％(w/w)；所述曲拉通-100的含量范围是0.8％-1.6％ (w/w)；所述EDTA的含量范围是0.5％-0.9％(w/w)；和/或所述裂解液的pH值范围为3.8-4.2。在一些实施例中，所述裂解液中：所述硫氰酸胍的含量是约32％(w/w)；所述柠檬酸的含量是约2.2％(w/w)；所述曲拉通-100的含量是约1.1％(w/w)；所述EDTA的含量是约0.7％(w/w)；和/或所述裂解液的pH值为约4.0。In some embodiments, the lysate comprises one or more components selected from guanidine thiocyanate, citric acid, Triton-100, and/or EDTA. In some embodiments, the lysate comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA. In some embodiments, in the lysate: the content range of the guanidine thiocyanate is 28%-40% (w/w); the content range of the citric acid is 1.8%-2.6% (w/w ); the content range of the Triton-100 is 0.8%-1.6% (w/w); the content range of the EDTA is 0.5%-0.9% (w/w); and/or the lysate The pH range is 3.8-4.2. In some embodiments, in the lysate: the content of the guanidine thiocyanate is about 32% (w/w); the content of the citric acid is about 2.2% (w/w); The content of Tong-100 is about 1.1% (w/w); the content of the EDTA is about 0.7% (w/w); and/or the pH value of the lysate is about 4.0.
在一些实施例中，所述结合液包含选自氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和/或异丙醇的一种或多种组分。在一些实施例中，所述结合液包含氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和异丙醇。在一些实施例中，所述结合液中：所述氯化镁的含量范围是0.1％-0.4％(w/w)；所述乙二胺四乙酸二钠的含量范围是0.5％-1.0％(w/w)；所述碳酸氢钠的含量范围是0.6％-1.0％(w/w)；所述硫氰酸胍的含量范围是13％-16％(w/w)；所述十二烷基肌氨酸钠的含量范围是1.0％-1.4％(w/w)；所述异丙醇的含量范围是60％-80％(w/w)；和/或所述结合液的pH值范围是4.8-5.2。在一些实施例中，所述结合液中：所述氯化镁的含量是约0.24％(w/w)；所述乙二胺四乙酸二钠的含量是约0.71％(w/w)；所述碳酸氢钠的含量是约0.83％(w/w)；所述硫氰酸胍的含量是约14.9％(w/w)；所述十二烷基肌氨酸钠的含量是约1.2％(w/w)；所述异丙醇的含量是约70％(w/w)；和/或所述结合液的pH值是约5.0。In some embodiments, the binding solution comprises one selected from magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, sodium lauryl sarcosine, and/or isopropanol or multiple components. In some embodiments, the binding solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, sodium sarcosine, and isopropanol. In some embodiments, in the combined solution: the content range of the magnesium chloride is 0.1%-0.4% (w/w); the content range of the disodium edetate is 0.5%-1.0% (w/w). /w); the content range of the sodium bicarbonate is 0.6%-1.0% (w/w); the content range of the guanidine thiocyanate is 13%-16% (w/w); the dodecane The content range of sodium sarcosinate is 1.0%-1.4% (w/w); the content range of the isopropanol is 60%-80% (w/w); and/or the pH value of the combination solution The range is 4.8-5.2. In some embodiments, in the combined solution: the magnesium chloride content is about 0.24% (w/w); the disodium edetate content is about 0.71% (w/w); the The content of sodium bicarbonate is about 0.83% (w/w); The content of said guanidine thiocyanate is about 14.9% (w/w); The content of said sodium lauryl sarcosinate is about 1.2% ( w/w); the content of the isopropanol is about 70% (w/w); and/or the pH value of the combination solution is about 5.0.
在一些实施例中，所述的方法在步骤iii)中，向所述核酸吸附材料依次加入漂洗液1和漂洗液2清洗，每次清洗后去除液体部分然后进行下一个清洗。在一些实施例中，向所述核酸吸附材料用所述漂洗液2清洗至少两次。In some embodiments, in step iii) of the method, rinse solution 1 and rinse solution 2 are sequentially added to the nucleic acid adsorption material for washing, and the liquid part is removed after each wash and then the next wash is performed. In some embodiments, the nucleic acid adsorption material is washed at least twice with the rinsing solution 2 .
在一些实施例中，所述漂洗液1包含选自乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和/或十二烷基肌氨酸钠的一种或多种组分。在一些实施例中，所述漂洗液1包含乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和十二烷基肌氨酸钠。在一些实施例中，所述漂洗液1中：所述乙二胺四乙酸二钠的含量范围是0.4％-0.9％(w/w)；所述碳酸氢钠的含量范围是0.5％-0.9％(w/w)；所述硫氰酸胍的含量范围是50％-55％(w/w)；所述十二烷基肌氨酸钠的含量范围是0.8％-1.2％(w/w)；和/或所述漂洗液1的pH值范围是6.2-6.6。在一些实施例中，所述漂洗液1中：所述乙二胺四乙酸二钠的含量是约0.5％(w/w)；所述碳酸氢钠的含量是约0.6％(w/w)；所述硫氰酸胍的含量是约53％(w/w)；所述十二烷基肌氨酸钠的含量是约0.9％(w/w)；和/或所述漂洗液1的pH值是约6.4。In some embodiments, the rinse solution 1 comprises one or more components selected from disodium edetate, sodium bicarbonate, guanidine thiocyanate, and/or sodium lauryl sarcosine . In some embodiments, the rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidine thiocyanate, and sodium lauryl sarcosinate. In some embodiments, in the rinse liquid 1: the content range of the disodium edetate is 0.4%-0.9% (w/w); the content range of the sodium bicarbonate is 0.5%-0.9% % (w/w); The content scope of described guanidinium thiocyanate is 50%-55% (w/w); The content scope of described sodium lauryl sarcosinate is 0.8%-1.2% (w/ w); and/or the pH range of the rinse solution 1 is 6.2-6.6. In some embodiments, in the rinse solution 1: the disodium edetate content is about 0.5% (w/w); the sodium bicarbonate content is about 0.6% (w/w) the content of the guanidine thiocyanate is about 53% (w/w); the content of the sodium lauryl sarcosinate is about 0.9% (w/w); and/or the rinse solution 1 The pH is about 6.4.
在一些实施例中，所述的方法在接触所述核酸吸附材料前，向所述漂洗液1中加入乙醇，所述漂洗液1∶乙醇的体积比为1∶1-1∶2。在一些实施例中，所述体积比为约2∶3。In some embodiments, in the method, before contacting the nucleic acid adsorption material, ethanol is added to the rinsing solution 1, and the volume ratio of the rinsing solution 1:ethanol is 1:1-1:2. In some embodiments, the volume ratio is about 2:3.
在一些实施例中，所述漂洗液2包含三羟甲基氨基甲烷(Tris)或氯化钠。在一些实施例中，所述漂洗液2包含三羟甲基氨基甲烷(Tris)和氯化钠。在一些实施例中，所述漂洗液2中：所述三羟甲基氨基甲烷(Tris)的含量范围是0.5％-0.7％(w/w)；所述氯化钠的含量范围是0.6％-1.8％(w/w)；和/或所述漂洗液2的pH值范围是6.6-7.0。在一些实施例中，所述漂洗液2中：所述三羟甲基氨基甲烷(Tris)的含量是约0.6％(w/w)；所述氯化钠的含量是约1.2％(w/w)；和/或所述漂洗液2的pH值是约6.8。In some embodiments, the rinse solution 2 includes tris (Tris) or sodium chloride. In some embodiments, the rinse solution 2 comprises tris (Tris) and sodium chloride. In some embodiments, in the rinse liquid 2: the content range of the tris (Tris) is 0.5%-0.7% (w/w); the content range of the sodium chloride is 0.6% -1.8% (w/w); and/or the pH value of the rinse solution 2 is in the range of 6.6-7.0. In some embodiments, in the rinse solution 2: the content of the tris (Tris) is about 0.6% (w/w); the content of the sodium chloride is about 1.2% (w/ w); and/or the pH value of the rinse solution 2 is about 6.8.
在一些实施例中，所述的方法在接触所述核酸吸附材料前，向所述漂洗液2中加入乙醇，所述漂洗液2∶乙醇的体积比为1∶1.6-1∶3。在一些实施例中，所述体积比为约1∶2.3。In some embodiments, before the method contacts the nucleic acid adsorption material, ethanol is added to the rinsing solution 2, and the volume ratio of the rinsing solution 2:ethanol is 1:1.6-1:3. In some embodiments, the volume ratio is about 1:2.3.
在一些实施例中，所述的方法的所述洗脱液为DEPC水.In some embodiments, the eluent of the method is DEPC water.
在一些实施例中，所述核酸吸附材料为能够吸附RNA的磁珠。在一些实施例中，所述磁珠的磁核是氢氧化三铁，包被二氧化硅，且表面镶嵌羟基基团。在一些实施例中，所述磁珠的粒径为50-100nm。In some embodiments, the nucleic acid adsorption material is magnetic beads capable of adsorbing RNA. In some embodiments, the magnetic core of the magnetic beads is ferric hydroxide, coated with silicon dioxide, and embedded with hydroxyl groups on the surface. In some embodiments, the particle size of the magnetic beads is 50-100 nm.
在一些实施例中，所述的方法使用的样本保存液包含选自EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和/或磷酸三钠的一种或多种组分。在一些实施例中，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。In some embodiments, the sample preservation solution used in the method comprises one or more groups selected from EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and/or trisodium phosphate point. In some embodiments, the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
本发明的一方面，提供了一种保存含核酸的生物样本的方法，其特征在于，将样本置于样本保存液中，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。One aspect of the present invention provides a method for preserving a nucleic acid-containing biological sample, characterized in that the sample is placed in a sample preservation solution, and the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
在一些实施例中，所述的方法的样本保存液中：所述EDTA的含量范围是0.7％-1.5％(w/w)；所述NaCl的含量范围是0.01％-0.1％(w/w)；所述无水乙醇的浓度范围是15％-35％(w/w)；所述柠檬酸钠的含量范围是0.1％-1.0％(w/w)；所述NP40的浓度范围是0.01％-0.1％(v/v)；所述谷胱甘肽的含量范围是0.07％-0.7％(w/w)；和/或所述磷酸三钠的含量范围是0.07％-0.4％(w/w)。在一些实施例中，所述样本保存液中：所述EDTA的含量是约1.05％(w/w)；所述NaCl的含量是约0.035％(w/w)；所述无水乙醇的浓度是约27.6％(w/w)；所述柠檬酸钠的含量是约0.21％(w/w)；所述NP40的浓度是约0.035％(v/v)；所述谷胱甘肽的含量是约0.21％(w/w)；和/或所述磷酸三钠的含量是约0.14％(w/w)。在一些实施例中，所述样本保存液包含抗生素。在一些实施例中，所述抗生素包含青霉素-链霉素双抗。在一些实施例中，所述样本保存液的pH值为7.6-8.0。在一些实施例中，所述样本保存液的pH值为7.8。在一些实施例中，与对照样本保存液相比，所述样本保存液对DNA，miRNA，和/或DNA甲基化的保存效 果更好。In some embodiments, in the sample preservation solution of the method: the content range of the EDTA is 0.7%-1.5% (w/w); the content range of the NaCl is 0.01%-0.1% (w/w ); the concentration range of the dehydrated alcohol is 15%-35% (w/w); the content range of the sodium citrate is 0.1%-1.0% (w/w); the concentration range of the NP40 is 0.01 %-0.1% (v/v); said glutathione content range is 0.07%-0.7% (w/w); and/or said trisodium phosphate content range is 0.07%-0.4% (w /w). In some embodiments, in the sample preservation solution: the content of the EDTA is about 1.05% (w/w); the content of the NaCl is about 0.035% (w/w); the concentration of the absolute ethanol is about 27.6% (w/w); the content of the sodium citrate is about 0.21% (w/w); the concentration of the NP40 is about 0.035% (v/v); the content of the glutathione is about 0.21% (w/w); and/or said trisodium phosphate is present in an amount of about 0.14% (w/w). In some embodiments, the sample preservation solution comprises antibiotics. In some embodiments, the antibiotic comprises double penicillin-streptomycin. In some embodiments, the pH value of the sample preservation solution is 7.6-8.0. In some embodiments, the pH value of the sample preservation solution is 7.8. In some embodiments, the sample preservation solution has a better preservation effect on DNA, miRNA, and/or DNA methylation than the control sample preservation solution.
在一些实施例中，所述的方法包含步骤(b-3)，所述EB病毒载量检测包含针对EB病毒基因组中BamHI-W片段，和/或LMP-2基因的检测。在一些实施例中，所述EB病毒载量检测包含针对LMP-2基因的检测。In some embodiments, the method includes step (b-3), and the detection of the Epstein-Barr virus load includes the detection of the BamHI-W fragment in the Epstein-Barr virus genome, and/or the LMP-2 gene. In some embodiments, the detection of the EB virus load comprises the detection of the LMP-2 gene.
在一些实施例中，所述方法用于鼻咽癌的筛查。在一些实施例中，所述方法用于鼻咽癌的诊断。在一些实施例中，所述样本是鼻咽部样本。In some embodiments, the method is used for screening for nasopharyngeal carcinoma. In some embodiments, the method is used for the diagnosis of nasopharyngeal carcinoma. In some embodiments, the sample is a nasopharyngeal sample.
在一些实施例中，所述的方法在步骤(a)之前，还包括使用鼻咽刷和/或鼻咽拭子获取所述样本。在一些实施例中，所述鼻咽刷和/或鼻咽拭子为不依赖临床医生和鼻咽镜引导的鼻咽刷和/或鼻咽拭子。In some embodiments, before step (a), the method further comprises using a nasopharyngeal brush and/or a nasopharyngeal swab to obtain the sample. In some embodiments, the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab that does not rely on the guidance of a clinician and a nasopharyngoscope.
本发明的一方面，提供了一种对有需要的个体治疗鼻咽癌的方法，包含对所述个体提供药物、放疗、手术、细胞疗法和/或溶瘤病毒治疗，其中，所述个体的样本接受了如本发明所述的方法的检测并被判断为鼻咽癌阳性。One aspect of the present invention provides a method for treating nasopharyngeal carcinoma in an individual in need, comprising providing the individual with drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus therapy, wherein the individual's The sample was tested by the method of the present invention and judged to be positive for nasopharyngeal carcinoma.
本发明的一方面，提供了一种对有需要的个体治疗鼻咽癌的方法，包含1)对来自所述个体的样本进行如本发明所述的方法的检测；2)如检测结果显示鼻咽癌阳性，提供鼻咽癌治疗。在一些实施例中，所述鼻咽癌治疗采用药物、放疗、手术、细胞疗法和/或溶瘤病毒治疗。One aspect of the present invention provides a method for treating nasopharyngeal carcinoma in an individual in need, comprising 1) performing detection according to the method of the present invention on a sample from the individual; 2) if the detection result shows that the nasal Pharyngeal cancer positive, provide nasopharyngeal cancer treatment. In some embodiments, the nasopharyngeal carcinoma is treated with drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus therapy.
发明详述Detailed description of the invention
本发明的目的是为了克服现有技术的上述不足，提供一种鼻咽癌诊断标志物。The object of the present invention is to provide a nasopharyngeal carcinoma diagnostic marker in order to overcome the above-mentioned deficiencies of the prior art.
本发明的第一个目的是提供检测EB病毒甲基化位点和/或EB病毒相关miRNA的试剂在制备鼻咽癌筛查和/或诊断试剂盒中的用途/应用。The first object of the present invention is to provide the use/application of a reagent for detecting Epstein-Barr virus methylation sites and/or Epstein-Barr virus-related miRNAs in the preparation of nasopharyngeal carcinoma screening and/or diagnostic kits.
本发明的第二个目的是提供一种鼻咽癌筛查和/或诊断产品。The second object of the present invention is to provide a product for screening and/or diagnosing nasopharyngeal carcinoma.
本发明的第三个目的是提供一种用于核酸样本保存的产品和方法。The third object of the present invention is to provide a product and method for nucleic acid sample preservation.
本发明的第四个目的是提供一种用于RNA提取，尤其是miRNA提取的产品和方法。The fourth object of the present invention is to provide a product and method for RNA extraction, especially miRNA extraction.
在一些实施例中，所述诊断为辅助诊断。在一些实施例中，所述筛查为早期筛查，即为鼻咽癌早期或者发病前的筛查。在一些实施例中，所用的鼻咽部样本为鼻咽刷和/或鼻咽拭子样本。在一些实施例中，所述鼻咽刷和/或鼻咽拭子样本为不依赖临床医生和鼻咽镜引导的鼻咽刷和/或鼻咽拭子样本。In some embodiments, the diagnosis is an auxiliary diagnosis. In some embodiments, the screening is early screening, that is, early screening or pre-onset screening of nasopharyngeal carcinoma. In some embodiments, the nasopharyngeal sample used is a nasopharyngeal brush and/or nasopharyngeal swab sample. In some embodiments, the nasopharyngeal brush and/or nasopharyngeal swab sample is a nasopharyngeal brush and/or nasopharyngeal swab sample that does not rely on the guidance of a clinician and a nasopharyngoscope.
为了实现上述目的，本发明是通过以下方案予以实现的：In order to achieve the above object, the present invention is achieved through the following schemes:
发明人公开了鼻咽部样本的EB病毒DNA甲基化位点和/或EB病毒相关miRNA可以作为鼻咽癌诊断的分子标记物，甲基化差异值和/或miRNA含量可以用于鼻咽癌的诊断和预测。当样本为活检样本，或者依赖临床医生和鼻咽镜引导的鼻咽刷样本时，EB病毒DNA甲基化位点作为鼻咽癌诊断分子标记物的灵敏度和特异性均与EB病毒DNA载量相当；当样本为不依赖临床医生和鼻咽镜引导的鼻咽刷或鼻咽拭子(鼻咽部盲检)样本时，作为鼻咽癌诊断分子标记物，EB病毒DNA载量的诊断灵敏度和特异性显著下降，EB病毒甲基化位点诊断的灵敏度和特异性均显著优于EB病毒DNA载量。进一步当样本为不依赖临床医生和鼻咽镜引导的鼻咽刷或鼻咽拭子(鼻咽部盲检)样本时，EB病毒甲基化位点和/或EB病毒相关miRNA可以更好的应用于鼻咽癌的预测。The inventors disclosed that the Epstein-Barr virus DNA methylation sites and/or Epstein-Barr virus-related miRNAs in nasopharyngeal samples can be used as molecular markers for the diagnosis of nasopharyngeal carcinoma, and the methylation difference and/or miRNA content can be used for nasopharyngeal cancer. Cancer diagnosis and prognosis. When the samples are biopsy samples, or rely on clinicians and nasopharyngoscope-guided nasopharyngeal brush samples, the sensitivity and specificity of Epstein-Barr virus DNA methylation sites as molecular markers for the diagnosis of nasopharyngeal carcinoma are related to the Epstein-Barr virus DNA load Equivalent; diagnostic sensitivity of Epstein-Barr virus DNA load as a diagnostic molecular marker for nasopharyngeal carcinoma when the sample is a nasopharyngeal brush or nasopharyngeal swab (blind examination of the nasopharynx) guided by a clinician and nasopharyngoscope The sensitivity and specificity of Epstein-Barr virus methylation site diagnosis were significantly better than Epstein-Barr virus DNA load. Further, when the sample is a nasopharyngeal brush or nasopharyngeal swab (nasopharyngeal blind test) sample that does not rely on clinicians and nasopharyngoscope guidance, Epstein-Barr virus methylation sites and/or Epstein-Barr virus-related miRNAs can be better Applied to the prediction of nasopharyngeal carcinoma.
这一方案既有无创，无损可反复取样的特点，又不依赖临床医生和鼻咽镜等医疗器械，整体技术方案简单，便捷，同时EB病毒DNA甲基化和/或EB病毒相关miRNA的检测对于鼻咽癌识别的准确度在90％以上，所以整体技术方案非常适合在高发现场大规模人群中开展鼻咽癌的筛检。This solution has the characteristics of non-invasive, non-destructive and repeatable sampling, and does not rely on clinicians and medical devices such as nasopharyngoscopes. The accuracy of identifying nasopharyngeal carcinoma is above 90%, so the overall technical solution is very suitable for screening nasopharyngeal carcinoma among large-scale populations in high-incidence sites.
本发明的一方面，提供了一种用于鼻咽癌检测的试剂盒，其特征在于，所述试剂盒包含选自核酸甲基化检测试剂、miRNA检测试剂、miRNA提取试剂，和/或样本保存液的一种或多种试剂。One aspect of the present invention provides a kit for nasopharyngeal carcinoma detection, characterized in that, the kit includes nucleic acid methylation detection reagents, miRNA detection reagents, miRNA extraction reagents, and/or samples One or more reagents of the preservation solution.
在一些实施例中，所述试剂盒包含核酸甲基化检测试剂，所述核酸甲基化检测试剂可针对EB病毒基因组中甲基化位点进行甲基化检测。In some embodiments, the kit includes a nucleic acid methylation detection reagent, which can perform methylation detection for methylation sites in the Epstein-Barr virus genome.
在一些实施例中，所述试剂盒包含miRNA检测试剂。在一些实施例中，所述miRNA检测试剂可针对一个或多个来自EB病毒的miRNA进行含量检测。In some embodiments, the kit comprises miRNA detection reagents. In some embodiments, the miRNA detection reagent can detect the content of one or more miRNAs from Epstein-Barr virus.
在一些实施例中，所述试剂盒包含样本保存液。In some embodiments, the kit comprises a sample preservation solution.
在一些实施例中，所述试剂盒包含miRNA提取试剂。In some embodiments, the kit comprises miRNA extraction reagents.
在一些实施例中，所述试剂盒包含EB病毒载量检测试剂。In some embodiments, the kit comprises Epstein-Barr viral load detection reagents.
本发明的一方面，提供了一种检测样本中EB病毒相关核酸的方法，其特征在于，所述方法包含以下步骤：One aspect of the present invention provides a method for detecting Epstein-Barr virus-related nucleic acids in a sample, characterized in that the method comprises the following steps:
(a)将所述样本置于样本保存液中；(a) placing the sample in a sample preservation solution;
(b)进行选自以下的一项或多项检测：(b) Conduct one or more tests selected from the following:
(b-1)对含有所述样本的所述样本保存液进行EB病毒基因组核酸甲基化检测；(b-1) performing Epstein-Barr virus genomic nucleic acid methylation detection on the sample preservation solution containing the sample;
(b-2)从所述样本保存液中提取EB病毒相关miRNA并对所述miRNA的含量进 行检测；和/或(b-2) extract Epstein-Barr virus-related miRNA from described sample preservation solution and detect the content of described miRNA; And/or
(b-3)对含有所述样本的所述样本保存液进行EB病毒核酸载量检测。(b-3) Detection of Epstein-Barr virus nucleic acid load on the sample preservation solution containing the sample.
在一些实施例中，所述的方法包含步骤(b-1)对含有所述样本的所述样本保存液进行EB病毒基因组核酸甲基化检测。In some embodiments, the method includes the step (b-1) of detecting methylation of Epstein-Barr virus genome nucleic acid in the sample preservation solution containing the sample.
在一些实施例中，所述的方法包含步骤(b-2)从所述样本保存液中提取EB病毒相关miRNA并对所述miRNA的含量进行检测。In some embodiments, the method includes step (b-2) extracting Epstein-Barr virus-related miRNA from the sample preservation solution and detecting the content of the miRNA.
在一些实施例中，所述的方法包含步骤(b-3)对含有所述样本的所述样本保存液进行EB病毒核酸载量检测。In some embodiments, the method includes step (b-3) performing detection of Epstein-Barr virus nucleic acid load on the sample preservation solution containing the sample.
在一些实施例中，所述方法是用于鼻咽癌的筛查和/或诊断。在一些实施例中，所述样本是鼻咽部样本。在一些实施例中，在步骤(a)之前，还包括使用鼻咽刷和/或鼻咽拭子获取所述样本。在一些实施例中，所述鼻咽刷和/或鼻咽拭子为不依赖临床医生和鼻咽镜引导的鼻咽刷和/或鼻咽拭子。在一些实施例中，获取样本的过程不依赖临床医生和鼻咽镜引导。In some embodiments, the method is for screening and/or diagnosis of nasopharyngeal carcinoma. In some embodiments, the sample is a nasopharyngeal sample. In some embodiments, before step (a), further comprising using a nasopharyngeal brush and/or a nasopharyngeal swab to obtain the sample. In some embodiments, the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab that does not rely on the guidance of a clinician and a nasopharyngoscope. In some embodiments, the process of obtaining a sample is independent of clinician and nasopharyngoscopy guidance.
如本申请中使用的，术语“约”和“大约”用作等同物。本申请中使用的有或无约/大约的任何数值意指涵盖相关领域普通技术人员所了解的任何正常波动。在一些实施例中，术语“大约”或“约”是指在陈述的参考值的任一方向(大于或小于)的10％的范围，除非另外陈述或另外从上下文明显可见(除了这种数字将会超过可能值的100％的情况)。As used in this application, the terms "about" and "approximately" are used as equivalents. Any numerical value with or without about/approximately used in this application is meant to cover any normal fluctuations understood by those of ordinary skill in the relevant art. In some embodiments, the term "about" or "approximately" refers to a range of 10% in either direction (greater than or less than) of the stated reference value, unless otherwise stated or otherwise evident from the context (except for such numerical will exceed 100% of the possible value).
EB病毒基因组甲基化Epstein-Barr virus genome methylation
在一些实施例中，所述EB病毒基因组中甲基化位点包含位于EBV基因组上的C启动子区的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含位于EBV基因组上的w启动子区的位点。In some embodiments, the methylation sites in the EBV genome include sites located in the C promoter region of the EBV genome. In some embodiments, the methylation sites in the EBV genome include sites in the w promoter region on the EBV genome.
在一些实施例中，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)(NCBI Reference Sequence：NC_007605.1；ACCESSION：NC_007605；VERSION：NC_007605.1；获得自：https：//www.ncbi.nlm.nih.gov/nuccore/NC_007605)的EB病毒基因组DNA序列的10717bp、11029bp、14015bp、14566bp、45850bp、57946bp、66227bp和128103bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含这些位点中的1，2，3，4，5，6，7，或8个位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含这些位点中的至少1个，至少2个，至少3个，至少4个，至少5个，至少6个，至少7个，或至少8 个位点。优选地，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、和14015bp的位点。In some embodiments, the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) (NCBI Reference Sequence: NC_007605.1; ACCESSION : NC_007605; VERSION: NC_007605.1; 10717bp, 11029bp, 14015bp, 14566bp, 45850bp, 57946bp, 66227 of the Epstein-Barr virus genomic DNA sequence obtained from: https://www.ncbi.nlm.nih.gov/nuccore/NC_007605) bp and 128103bp sites. In some embodiments, the methylation sites in the Epstein-Barr virus genome include 1, 2, 3, 4, 5, 6, 7, or 8 of these sites. In some embodiments, the methylation sites in the Epstein-Barr virus genome comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, and at least 7 of these sites , or at least 8 sites. Preferably, the methylation site comprises sites corresponding to 10717bp, 11029bp, and 14015bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp的位点。In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 10717 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的14015bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的14566bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的45850bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的57946bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的66227bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的128103bp的位点。In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 11029 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 14015 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 14566 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 45850 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 57946 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 66227 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94). In some embodiments, the methylation site in the Epstein-Barr virus genome comprises a site corresponding to 128103 bp of the Epstein-Barr virus genome DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
在一些实施例中，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、14015bp、14566bp、45850bp、57946bp、66227bp和128103bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp、45850bp、57946bp、66227bp和128103bp的位点。在一些实施例中，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp、57946bp、和66227bp的位点。In some embodiments, the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: 10717bp, 11029bp of the Epstein-Barr virus genome DNA sequence equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) , 14015bp, 14566bp, 45850bp, 57946bp, 66227bp and 128103bp sites. In some embodiments, the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: 11029bp and 45850bp of the Epstein-Barr virus genome DNA sequence equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) , 57946bp, 66227bp and 128103bp sites. In some embodiments, the methylation sites in the Epstein-Barr virus genome include one or more of the following sites: 11029bp and 57946bp of the Epstein-Barr virus genome DNA sequence equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) , and the 66227bp site.
miRNAmiRNA
在一些实施例中，检测所述miRNA包含以下miRNA的一个或几个：EBV-miR-BART1-5p、EBV-miR-BART2-5p、EBV-miR-BART6-3p、EBV-miR-BART7-3p、EBV-miR-BART13-3p、和EBV-miR-BART17-5p。在一些实施例中，所述miRNA包含选自这些miRNA的1，2，3，4，5，或6个。在一些实施例中，所述miRNA包含选自这些 miRNA的至少1个，至少2个，至少3个，至少4个，至少5个，或至少6个miRNA。在一些实施例中，所述miRNA包含EBV-miR-BART1-5p和EBV-miR-BART7-3p。在一些实施例中，所述miRNA包含这六个miRNA。In some embodiments, the miRNA detected comprises one or more of the following miRNAs: EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR-BART6-3p, EBV-miR-BART7-3p , EBV-miR-BART13-3p, and EBV-miR-BART17-5p. In some embodiments, the miRNA comprises 1, 2, 3, 4, 5, or 6 selected from these miRNAs. In some embodiments, the miRNA comprises at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 miRNAs selected from these miRNAs. In some embodiments, the miRNAs comprise EBV-miR-BART1-5p and EBV-miR-BART7-3p. In some embodiments, the miRNAs comprise these six miRNAs.
在一些实施例中，所述miRNA包含EBV-miR-BART1-5p。在一些实施例中，所述EBV-miR-BART1-5p的序列为UCUUAGUGGAAGUGACGUGCUGUG(SEQ ID NO：39)。In some embodiments, the miRNA comprises EBV-miR-BART1-5p. In some embodiments, the sequence of the EBV-miR-BART1-5p is UCUUAGUGGAAGUGACGUGCUGUG (SEQ ID NO: 39).
在一些实施例中，所述miRNA包含EBV-miR-BART2-5p。在一些实施例中，所述EBV-miR-BART2-5p的序列为UAUUUUCUGCAUUCGCCCUUGC(SEQ ID NO：40)。In some embodiments, the miRNA comprises EBV-miR-BART2-5p. In some embodiments, the sequence of the EBV-miR-BART2-5p is UAUUUUCUGCAUUCGCCCUUGC (SEQ ID NO: 40).
在一些实施例中，所述miRNA包含EBV-miR-BART6-3p。在一些实施例中，所述EBV-miR-BART6-3p：的序列为CGGGGAUCGGACUAGCCUUAGA(SEQ ID NO：41)。In some embodiments, the miRNA comprises EBV-miR-BART6-3p. In some embodiments, the sequence of the EBV-miR-BART6-3p: is CGGGGAUCGGACUAGCCUUAGA (SEQ ID NO: 41).
在一些实施例中，所述miRNA包含EBV-miR-BART7-3p。在一些实施例中，所述EBV-miR-BART7-3p的序列为CAUCAUAGUCCAGUGUCCAGGG(SEQ ID NO：42)。In some embodiments, the miRNA comprises EBV-miR-BART7-3p. In some embodiments, the sequence of the EBV-miR-BART7-3p is CAUCAUAGUCCAGUGUCCAGGG (SEQ ID NO: 42).
在一些实施例中，所述miRNA包含EBV-miR-BART13-3p。在一些实施例中，所述EBV-miR-BART13-3p的序列为UGUAACUUGCCAGGGACGGCUGA(SEQ ID NO：43)。In some embodiments, the miRNA comprises EBV-miR-BART13-3p. In some embodiments, the sequence of the EBV-miR-BART13-3p is UGUAACUUGCCAGGGACGGCUGA (SEQ ID NO: 43).
在一些实施例中，所述miRNA包含EBV-miR-BART17-5p。在一些实施例中，所述EBV-miR-BART17-5p的序列为UAAGAGGACGCAGGCAUACAAG(SEQ ID NO：44)。In some embodiments, the miRNA comprises EBV-miR-BART17-5p. In some embodiments, the sequence of the EBV-miR-BART17-5p is UAAGAGGACGCAGGCAUACAAG (SEQ ID NO: 44).
核酸甲基化，miRNA，和病毒载量的检测；引物和探针组合Detection of nucleic acid methylation, miRNA, and viral load; primer and probe sets
在一些实施例中，本文的检测试剂可以对EB病毒基因组中位点进行甲基化检测，并进行甲基化差异分析。在一些实施例中，所述甲基化差异分析为检测各个位点扩增引物和甲基化探针扩增之后的Ct值与扩增引物和非甲基化探针扩增之后的Ct值之差。In some embodiments, the detection reagents herein can detect the methylation of sites in the Epstein-Barr virus genome and perform methylation differential analysis. In some embodiments, the methylation difference analysis is to detect the Ct value after amplification of each site amplification primer and methylated probe and the Ct value after amplification of amplification primer and unmethylated probe Difference.
具体地，各个位点的甲基化差异(Methylation Difference，MD)值的计算公式为：Specifically, the formula for calculating the methylation difference (Methylation Difference, MD) value of each site is:
MD＝CT m-CT u， MD= CTm - CTu ,
CT m为该位点的扩增引物和甲基化探针扩增之后的Ct值，CT u为该位点的扩增引物和非甲基化探针扩增之后的Ct值。进一步，可通过利用各位点的MD值构建模型进行鼻咽癌的诊断。 CT m is the Ct value after amplification with the amplification primers and methylated probes at this site, and CT u is the Ct value after amplification with the amplification primers and unmethylated probes at this site. Furthermore, the diagnosis of nasopharyngeal carcinoma can be carried out by using the MD value of each site to construct a model.
在一些实施例中，所述甲基化位点的检测引物和探针的组合选自表2，表12，表30中所示的任意一个引物和探针组合。In some embodiments, the combination of detection primers and probes for the methylation site is selected from any combination of primers and probes shown in Table 2, Table 12, and Table 30.
在一些实施例中，所述各个甲基化位点的检测引物和探针组合选自如下的任意一个组合(EB病毒基因组DNA的位点信息相对于SEQ ID NO：94)：In some embodiments, the detection primer and probe combination of each methylation site is selected from any one of the following combinations (the site information of Epstein-Barr virus genomic DNA is relative to SEQ ID NO: 94):
EB病毒基因组DNA的11029bp：11029bp of Epstein-Barr virus genomic DNA:
正向引物：GAGTGTTATTTTTGGAATAGTAG(SEQ ID NO：7)，Forward primer: GAGTGTTATTTTTGGAATAGTAG (SEQ ID NO: 7),
反向引物：TTAAACTCTCTTATTAACTATAATC(SEQ ID NO：8)，Reverse primer: TTAAACTCTCTTTATTAACTATAATC (SEQ ID NO: 8),
甲基化探针FAM-TGAATTTTGTTGGCGGGAGAAGGA-BHQ 1(SEQ ID NO：9)，Methylation probe FAM-TGAATTTTGTTGGCGGGAGAAGGA-BHQ 1 (SEQ ID NO: 9),
非甲基化探针HEX-TGAATTTTGTTGGTGGGAGAAGGA-BHQ 1(SEQ ID NO：10)；Unmethylated probe HEX-TGAATTTTGTTGGTGGGAGAAGGA-BHQ 1 (SEQ ID NO: 10);
EB病毒基因组DNA的45850bp：45850bp of Epstein-Barr virus genomic DNA:
正向引物：AGTTTATGTTTGTGATGAGGTT(SEQ ID NO：11)，Forward primer: AGTTTATGTTTGTGATGAGGTT (SEQ ID NO: 11),
反向引物：CCTAATCAATCACTCAAATACC(SEQ ID NO：12)，Reverse primer: CCTAATCAATCACTCAAATACC (SEQ ID NO: 12),
甲基化探针：FAM-AGATTTAGGTTCGTGGGGTATTTAG-BHQ 1(SEQ ID NO：13)，Methylation probe: FAM-AGATTTAGGTTCGTGGGGTATTTAG-BHQ 1 (SEQ ID NO: 13),
非甲基化探针：HEX-AGATTTAGGTTTGTGGGGTATTTAG-BHQ1(SEQ ID NO：14)；Unmethylated probe: HEX-AGATTTAGGTTTGTGGGGTATTTAG-BHQ1 (SEQ ID NO: 14);
EB病毒基因组DNA的57946bp：57946bp of Epstein-Barr virus genomic DNA:
正向引物TGATTATTTTTTGTGTTAGTGGT(SEQ ID NO：15)，Forward primer TGATTATTTTTTGTGTTAGTGGT (SEQ ID NO: 15),
反向引物AAATAACCCCCACAAAAAACC(SEQ ID NO：16)，Reverse primer AAATAACCCCCACAAAAAACC (SEQ ID NO: 16),
甲基化探针FAM-AGGATAGAGGGAGGCGGCGGT-BHQ1(SEQ ID NO：17)，Methylation probe FAM-AGGATAGAGGGAGGCGGCGGT-BHQ1 (SEQ ID NO: 17),
非甲基化探针HEX-AGGATAGAGGGAGGTGGTGGTT-BHQ1(SEQ ID NO：18)；Unmethylated probe HEX-AGGATAGAGGGAGGTGGTGGTT-BHQ1 (SEQ ID NO: 18);
EB病毒基因组DNA的66227bp：66227bp of Epstein-Barr virus genomic DNA:
正向引物：GGATAGGGTTTTTTTTGTGGAT(SEQ ID NO：19)，Forward primer: GGATAGGGTTTTTTTTGTGGAT (SEQ ID NO: 19),
反向引物：CATAATAATCTTCAAACCCAAC(SEQ ID NO：20)，Reverse primer: CATAATAATCTTTCAAACCCAAC (SEQ ID NO: 20),
甲基化探针：FAM-AGGAAGATGTCGCGCGGGTTAG-BHQ1(SEQ ID NO：21)，Methylation probe: FAM-AGGAAGATGTCGCGCGGGTTAG-BHQ1 (SEQ ID NO: 21),
非甲基化探针：HEX-AGGAAGATGTTGTGTGGGTTAGT-BHQ1(SEQ ID NO：22)；Unmethylated probe: HEX-AGGAAGATGTTGTGTGGGTTAGT-BHQ1 (SEQ ID NO: 22);
EB病毒基因组DNA的128103bp：128103bp of Epstein-Barr virus genomic DNA:
正向引物：GATAGGTGTTTAAGTTGGGTA(SEQ ID NO：23)，Forward primer: GATAGGTGTTTAAGTTGGGTA (SEQ ID NO: 23),
反向引物：CTCCAAATTCAAACTCTTTAAC(SEQ ID NO：24)，Reverse primer: CTCCAAATTCAAACTTCTTTAAC (SEQ ID NO: 24),
甲基化探针：FAM-TGTTTTCGTCGCGTAGGTTAGTA-BHQ1(SEQ ID NO：25)，Methylation probe: FAM-TGTTTTCGTCGCGTAGGTTAGTA-BHQ1 (SEQ ID NO: 25),
非甲基化探针：HEX-TGTTTTTGTTGTGTAGGTTAGTAATG-BHQ1(SEQ ID NO：26)。Unmethylated probe: HEX-TGTTTTTGTTGTGTAGGTTAGTAATG-BHQ1 (SEQ ID NO: 26).
EB病毒基因组DNA的10717bp：10717bp of Epstein-Barr virus genomic DNA:
正向引物：AAGGGGTATAAGAATGTCGGC(SEQ ID NO：33)，Forward primer: AAGGGGTATAAGAATGTCGGC (SEQ ID NO: 33),
反向引物：TAATTATTCGCAAAACCGCCCT(SEQ ID NO：34)，Reverse primer: TAATTATTCGCAAAACCGCCCT (SEQ ID NO: 34),
探针：VIC-TTAACCGAACCGCCCGACT-MGB(SEQ ID NO：35)，Probe: VIC-TTAACCGAACCGCCCGACT-MGB (SEQ ID NO: 35),
EB病毒基因组DNA的14015bp：14015bp of Epstein-Barr virus genomic DNA:
正向引物：TCGGGAGAGGTAGTTTTAAAGC(SEQ ID NO：36)，Forward primer: TCGGGAGAGGTAGTTTTTAAAGC (SEQ ID NO: 36),
反向引物：AACAAAATAACGCCCATTCG(SEQ ID NO：37)，Reverse primer: AACAAAATAACGCCCATTCG (SEQ ID NO: 37),
探针：FAM-TTGGTAGTGATTTGGATTCGAA-MGB(SEQ ID NO：38)，Probe: FAM-TTGGTAGTGATTTGGATTCGAA-MGB (SEQ ID NO: 38),
EB病毒基因组DNA的11029bp：11029bp of Epstein-Barr virus genomic DNA:
正向引物：AATTTATGGTTTAGTGCGTCG(SEQ ID NO：79)，Forward primer: AATTTATGGTTTAGTGCGTCG (SEQ ID NO: 79),
反向引物：TCTCTTATTAACTATAATCCGTCGC(SEQ ID NO：80)，Reverse primer: TCTCTTATTAACTATAATCCGTCGC (SEQ ID NO: 80),
探针：FAM-TGAATTTTGTTGGCGGGAGAAGGA-BHQ1(SEQ ID NO：81)，Probe: FAM-TGAATTTTGTTGGCGGGAGAAGGA-BHQ1 (SEQ ID NO: 81),
EB病毒基因组DNA的14566bp：14566bp of Epstein-Barr virus genomic DNA:
正向引物：TTTCGTTTTGTTTTGCGTTCGG(SEQ ID NO：82)，Forward primer: TTTCGTTTTGTTTTGCGTTCGG (SEQ ID NO: 82),
反向引物：CTTACCTCTAACCCGATACCG(SEQ ID NO：83)，Reverse primer: CTTACCTCTAACCCGATACCG (SEQ ID NO: 83),
探针：FAM-ACCTTCACTTCGATCTCCCCTA-MGB(SEQ ID NO：84)，Probe: FAM-ACCTTCACTTCGATCTCCCCTA-MGB (SEQ ID NO: 84),
EB病毒基因组DNA的11062位点：Site 11062 of Epstein-Barr virus genomic DNA:
正向引物：TAACGTTTTATTTGGGAGGAGC(SEQ ID NO：76)Forward primer: TAACGTTTTATTTGGGAGGAGC (SEQ ID NO: 76)
反向引物：AACAAAACGTAATTAATCCCGC(SEQ ID NO：77)Reverse primer: AACAAAACGTAATTAATCCCGC (SEQ ID NO: 77)
探针：FAM-TACTCCACCTCTAAAATCCCA-MGB(SEQ ID NO：78)Probe: FAM-TACTCCACCTCTAAAATCCCA-MGB (SEQ ID NO: 78)
在一些实施例中，本文的检测试剂/方法可以对miRNA含量进行检测和差异分析。在一些实施例中，所述miRNA的检测引物和探针组合选自如下的任意一个组合：In some embodiments, the detection reagents/methods herein can detect and differentially analyze miRNA content. In some embodiments, the detection primer and probe combination of the miRNA is selected from any combination of the following:
针对miR-BART1-5p的引物/探针组合：Primer/probe combinations targeting miR-BART1-5p:
miR-BART1-5p逆转录颈环引物：miR-BART1-5p reverse transcription neck loop primer:
GGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCCACAGC(SEQ ID NO：45)，GGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCCACAGC (SEQ ID NO: 45),
miR-BART1-5p正向引物：GCCGTCTTAGTGGAAGTGACG(SEQ ID NO：46)，miR-BART1-5p forward primer: GCCGTCTTAGTGGAAGTGACG (SEQ ID NO: 46),
miR-BART1-5p反向引物：TGCAGGGTCCGAGGTATTC(SEQ ID NO：47)，miR-BART1-5p reverse primer: TGCAGGGTCCGAGGTATTC (SEQ ID NO: 47),
miR-BART1-5p探针：FAM-ACCAGAGCCCACAGCA-MGB(SEQ ID NO：48)。miR-BART1-5p probe: FAM-ACCAGAGCCCACAGCA-MGB (SEQ ID NO: 48).
针对miR-BART2-5p的引物/探针组合：Primer/probe combinations targeting miR-BART2-5p:
miR-BART2-5p逆转录颈环引物：miR-BART2-5p reverse transcription neck loop primer:
GGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCGCAAGGG(SEQ ID NO：49)，GGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCGCAAGGG (SEQ ID NO: 49),
miR-BART2-5p正向引物：GCCGCTATTTTCTGCATTCG(SEQ ID NO：50)，miR-BART2-5p forward primer: GCCGCTATTTTCTGCATTCG (SEQ ID NO: 50),
miR-BART2-5p反向引物：TGCAGGGTCCGAGGTATTC(SEQ ID NO：51)，miR-BART2-5p reverse primer: TGCAGGGTCCGAGGTATTC (SEQ ID NO: 51),
miR-BART2-5p探针：FAM-CCAGAGCCGCAAGG-MGB(SEQ ID NO：52)，miR-BART2-5p probe: FAM-CCAGAGCCGCAAGG-MGB (SEQ ID NO: 52),
针对BART6-3p的引物/探针组合：Primer/probe combinations for BART6-3p:
miR-BART6-3p逆转录颈环引物：miR-BART6-3p reverse transcription neck loop primer:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCTAAGG(SEQ ID NO：53)，GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCTAAGG (SEQ ID NO: 53),
miR-BART6-3p正向引物：GCCGGGGATCGGACTAG(SEQ ID NO：54)，miR-BART6-3p forward primer: GCCGGGGATCGGACTAG (SEQ ID NO: 54),
miR-BART6-3p反向引物：CAGTGCAGGGTCCGAGGTAT(SEQ ID NO：55)，miR-BART6-3p reverse primer: CAGTGCAGGGTCCGAGGTAT (SEQ ID NO: 55),
miR-BART6-3p探针：VIC-ACTGGATACGACTCTAAGG-MGB(SEQ ID NO：56)，miR-BART6-3p probe: VIC-ACTGGATACGACTCTAAGG-MGB (SEQ ID NO: 56),
针对miR-BART7-3p的引物/探针组合：Primer/probe combinations targeting miR-BART7-3p:
miR-BART7-3p逆转录颈环引物：miR-BART7-3p reverse transcription neck loop primer:
GGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCCCCTGGA(SEQ ID NO：57)，GGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCCCCTGGA (SEQ ID NO: 57),
miR-BART7-3p正向引物：CGGCCATCATAGTCCAGTGT(SEQ ID NO：58)，miR-BART7-3p forward primer: CGGCCATCATAGTCCAGTGT (SEQ ID NO: 58),
miR-BART7-3p反向引物：TGCAGGGTCCGAGGTATTC(SEQ ID NO：59)，miR-BART7-3p reverse primer: TGCAGGGTCCGAGGTATTC (SEQ ID NO: 59),
miR-BART7-3p探针：FAM-ACCAGAGCCCCCTGGA-MGB(SEQ ID NO：60)，miR-BART7-3p probe: FAM-ACCAGAGCCCCCTGGA-MGB (SEQ ID NO: 60),
针对miR-BART13-3p的引物/探针组合：Primer/probe combinations targeting miR-BART13-3p:
miR-BART13-3p逆转录颈环引物：miR-BART13-3p reverse transcription neck loop primer:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGCC(SEQ ID NO：61)，GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAGCC (SEQ ID NO: 61),
miR-BART13-3p正向引物：CGGTGTAACTTGCCAGGGA(SEQ ID NO：62)，miR-BART13-3p forward primer: CGGTGTAACTTGCCAGGGA (SEQ ID NO: 62),
miR-BART13-3p反向引物：CAGTGCAGGGTCCGAGGTAT(SEQ ID NO：63)，miR-BART13-3p reverse primer: CAGTGCAGGGTCCGAGGTAT (SEQ ID NO: 63),
miR-BART13-3p探针：VIC-TGGATACGACTCAGCCG-MGB(SEQ ID NO：64)，miR-BART13-3p probe: VIC-TGGATACGACTCAGCCG-MGB (SEQ ID NO: 64),
针对BART17-5p的引物/探针组合：Primer/probe combinations for BART17-5p:
miR-BART17-5p逆转录颈环引物：miR-BART17-5p reverse transcription neck loop primer:
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTTGTAT(SEQ ID NO：65)，GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTTGTAT (SEQ ID NO: 65),
miR-BART17-5p正向引物：CGGTAAGAGGACGCAGGC(SEQ ID NO：66)，miR-BART17-5p forward primer: CGGTAAGAGGACGCAGGC (SEQ ID NO: 66),
miR-BART17-5p反向引物：CAGTGCAGGGTCCGAGGTAT(SEQ ID NO：67)，miR-BART17-5p reverse primer: CAGTGCAGGGTCCGAGGTAT (SEQ ID NO: 67),
miR-BART17-5p探针：VIC-ACTGGATACGACCTTGTATG-MGB(SEQ ID NO：68)，针对miR-16-5p的引物/探针组合：miR-BART17-5p probe: VIC-ACTGGATACGACCTTGTATG-MGB (SEQ ID NO: 68), primer/probe combination for miR-16-5p:
miR-16-5p逆转录颈环引物：miR-16-5p reverse transcription neck loop primer:
GGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCCGCCAAT(SEQ ID NO：69)，GGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCCGCCAAT (SEQ ID NO: 69),
miR-16-5p正向引物：CGGGCTAGCAGCACGTAAA(SEQ ID NO：70)，miR-16-5p forward primer: CGGGCTAGCAGCACGTAAA (SEQ ID NO: 70),
miR-16-5p反向引物：TGCAGGGTCCGAGGTATTC(SEQ ID NO：71)，miR-16-5p reverse primer: TGCAGGGTCCGAGGTATTC (SEQ ID NO: 71),
miR-16-5p探针：CY5-CAGAGCCCGCCAATA-MGB(SEQ ID NO：72)。miR-16-5p probe: CY5-CAGAGCCCGCCAATA-MGB (SEQ ID NO: 72).
在一些实施例中，本文的检测试剂/方法可以对EB病毒载量进行检测。In some embodiments, the detection reagent/method herein can detect Epstein-Barr virus load.
在一些实施例中，所述EB病毒载量检测包含核苷酸序列如SEQ ID NO：1到2所示的引物和核苷酸序列如SEQ ID NO：3所示的探针，用于定量所扩增的目的基因为EB病毒的BamHI-w片段。在一些实施例中，正向引物5’-CCCAACACT CCACCACACC-3’(SEQ ID NO：1)，反向引物：5’-TCTTAGGAGCTGTCCGAGGG-3’(SEQ ID NO：2)，探针5’-FAM-CACACACTACACACACCCACCCGTCTC-TAMRA-3’(SEQ ID NO：3)。在一些实施例中，所述引物和探针包含正向引物TGCCAAAGAGCCAGATCTAAG(SEQ ID NO：27)，反向引物AGTGTCAGATTTCGGGTCCAAA(SEQ ID NO：28)，探针FAM-CAGCCCCAAAGCGGGTGCA-BHQ 1(SEQ ID NO：29)In some embodiments, the EB virus load detection comprises a nucleotide sequence such as primers shown in SEQ ID NO: 1 to 2 and a nucleotide sequence such as a probe shown in SEQ ID NO: 3 for quantitative The amplified target gene is the BamHI-w fragment of Epstein-Barr virus. In some embodiments, forward primer 5'-CCCAACACT CCACCACACC-3' (SEQ ID NO: 1), reverse primer: 5'-TCTTAGGAGCTGTCCGAGGG-3' (SEQ ID NO: 2), probe 5'-FAM -CACACACTACACACACCCACCCGTCTC-TAMRA-3' (SEQ ID NO: 3). In some embodiments, the primers and probes include forward primer TGCCAAAGAGCCAGATCTAAG (SEQ ID NO: 27), reverse primer AGTGTCAGATTTCGGGTCCAAA (SEQ ID NO: 28), probe FAM-CAGCCCCAAAGCGGGTGCA-BHQ 1 (SEQ ID NO: 29)
在一些实施例中，所述EB病毒载量检测包含检测看家基因β-globin基因的引物和探针。在一些实施例中，所述看家基因β-globin基因的引物核苷酸序列如SEQ ID NO：4到5所示，探针核苷酸序列如SEQ ID NO：6所示。具体地，两条引物序列分别是5’-GTGCACCTGACTCCTGAGGAGA-3’(SEQ ID NO：4)和5’-CCTTGATACCAACCTGCCCAG-3’(SEQ ID NO：5)，探针序列为5’-FAM-AAGGTGAACGTGGATGAAGTTGGTGG-TAMRA-3’(SEQ ID NO：6)。在一些实施例中，所述引物和探针包含正向引物GTGCATCTGACTCCTGAGGAGA(SEQ ID NO：73)，反向引物CCTTGATACCAACCTGCCCAG(SEQ ID NO：74)，探针VIC-AAGGTGAACGTGGATGAAGTTGGTGG-BHQ1(SEQ ID NO：75)。In some embodiments, the detection of the EB virus load comprises primers and probes for detecting the housekeeping gene β-globin gene. In some embodiments, the primer nucleotide sequence of the housekeeping gene β-globin gene is shown in SEQ ID NO: 4 to 5, and the probe nucleotide sequence is shown in SEQ ID NO: 6. Specifically, the two primer sequences are 5'-GTGCACCTGACTCCTGAGGAGA-3' (SEQ ID NO: 4) and 5'-CCTTGATACCAACCTGCCCAG-3' (SEQ ID NO: 5), and the probe sequence is 5'-FAM-AAGGTGAACGTGGATGAAGTTGGTGG- TAMRA-3' (SEQ ID NO: 6). In some embodiments, the primers and probes include forward primer GTGCATCTGACTCCTGAGGAGA (SEQ ID NO: 73), reverse primer CCTTGATACCAACCTGCCCAG (SEQ ID NO: 74), probe VIC-AAGGTGAACGTGGATGAAGTTGGTGG-BHQ1 (SEQ ID NO: 75 ).
在一些实施例中，所述EB病毒载量检测包含检测LMP-2的引物和探针。在一些实施例中，所述引物和探针包含正向引物AGCTGTAACTGTGGTTTCCATGAC(SEQ ID NO：30)，反向引物GCCCCCTGGCGAAGAG(SEQ ID NO：31)，探针FAM-CTGCTGCTACTGGCTTTCGTCCTCTGG-BHQ 1(SEQ ID NO：32)。In some embodiments, the detection of the EB virus load comprises primers and probes for detecting LMP-2. In some embodiments, the primers and probes include forward primer AGCTGTAACTGTGGTTTCCATGAC (SEQ ID NO: 30), reverse primer GCCCCCTGGCGAAGAG (SEQ ID NO: 31), probe FAM-CTGCTGCTACTGGCTTTCGTCCTCTGG-BHQ 1 (SEQ ID NO: 32).
在一些实施例中，所述检测包含检测GAPDH3的引物和探针。在一些实施例中，所述引物和探针包含正向引物GGTGGAGGAAGTTAGGGTTCGT(SEQ ID NO：91)，反向引物CAACTCAACTCCAAACTAAACTCCACTA(SEQ ID NO：92)，探针 CY5-TTGGGTTTTGACGTTGATT-MGB(SEQ ID NO：93)In some embodiments, the detecting comprises primers and probes for detecting GAPDH3. In some embodiments, the primers and probes include forward primer GGTGGAGGAAGTTAGGGTTCGT (SEQ ID NO: 91), reverse primer CAACTCAACTCCAAACTAAACTCCACTA (SEQ ID NO: 92), probe CY5-TTGGGTTTTGACGTTGATT-MGB (SEQ ID NO: 93 )
在一些实施例中，本文所述探针的5’携带荧光基团，3’携带荧光淬基团。在一些实施例中，本文所述探针的5’携带FAM荧光基团，HEX荧光基团，VIC荧光基团，或CY5荧光基团。在一些实施例中，本文所述探针的3’携带TAMRA荧光淬基团，BHQ1荧光淬基团，或MGB荧光淬基团。In some embodiments, the 5' of the probe described herein carries a fluorescent group, and the 3' carries a fluorescent quenching group. In some embodiments, the 5' of the probe described herein carries a FAM fluorophore, a HEX fluorophore, a VIC fluorophore, or a CY5 fluorophore. In some embodiments, the 3' of the probe described herein carries a TAMRA fluorescent quencher, a BHQ1 fluorescent quencher, or an MGB fluorescent quencher.
核酸样本保存液和保存方法Nucleic acid sample preservation solution and preservation method
本发明的一方面，提供了一种样本保存液。本发明的一方面，提供了一种包含样本保存液的保存含核酸的生物样本的试剂盒。本发明的一方面，提供了一种使用样本保存液保存含核酸的生物样本的方法。In one aspect of the present invention, a sample preservation solution is provided. One aspect of the present invention provides a kit for preserving a nucleic acid-containing biological sample comprising a sample preservation solution. One aspect of the present invention provides a method for preserving a nucleic acid-containing biological sample using a sample preservation solution.
在一些实施例中，所述样本保存液包含选自EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和/或磷酸三钠的一种或多种组分。在一些实施例中，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。In some embodiments, the sample preservation solution comprises one or more components selected from EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and/or trisodium phosphate. In some embodiments, the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
在一些实施例中，所述样本保存液中：In some embodiments, in the sample preservation solution:
-所述EDTA的含量范围是0.7％-1.5％(w/w)；- the content range of the EDTA is 0.7%-1.5% (w/w);
-所述NaCl的含量范围是0.01％-0.1％(w/w)；- the content range of the NaCl is 0.01%-0.1% (w/w);
-所述无水乙醇的浓度范围是15％-35％(w/w)；- the concentration range of the absolute ethanol is 15%-35% (w/w);
-所述柠檬酸钠的含量范围是0.1％-1.0％(w/w)；- the content range of the sodium citrate is 0.1%-1.0% (w/w);
-所述NP40的浓度范围是0.01％-0.1％(v/v)；- the concentration range of the NP40 is 0.01%-0.1% (v/v);
-所述谷胱甘肽的含量范围是0.07％-0.7％(w/w)；和/或- said glutathione content ranges from 0.07% to 0.7% (w/w); and/or
-所述磷酸三钠的含量范围是0.07％-0.4％(w/w)。- The content of trisodium phosphate ranges from 0.07% to 0.4% (w/w).
在一些实施例中，所述样本保存液中：In some embodiments, in the sample preservation solution:
-所述EDTA的含量是约1.05％(w/w)；- said EDTA content is about 1.05% (w/w);
-所述NaCl的含量是约0.035％(w/w)；- said NaCl content is about 0.035% (w/w);
-所述无水乙醇的浓度是约27.6％(w/w)；- said concentration of absolute ethanol is about 27.6% (w/w);
-所述柠檬酸钠的含量是约0.21％(w/w)；- said sodium citrate content is about 0.21% (w/w);
-所述NP40的浓度是约0.035％(v/v)；- said NP40 concentration is about 0.035% (v/v);
-所述谷胱甘肽的含量是约0.21％(w/w)；和/或- said glutathione content is about 0.21% (w/w); and/or
-所述磷酸三钠的含量是约0.14％(w/w)。- The content of said trisodium phosphate is about 0.14% (w/w).
在一些实施例中，所述样本保存液包含抗生素；优选地，所述抗生素包含青霉素-链霉 素双抗。In some embodiments, the sample preservation solution includes antibiotics; preferably, the antibiotics include penicillin-streptomycin double antibody.
在一些实施例中，所述样本保存液的pH值为7.6-8.0。在一些实施例中，所述样本保存液的pH值为7.7-7.9。在一些实施例中，所述样本保存液的pH值为7.6，7.7，7.8，7.9，或8.0。在一些实施例中，述样本保存液的pH值为7.8。In some embodiments, the pH value of the sample preservation solution is 7.6-8.0. In some embodiments, the pH value of the sample preservation solution is 7.7-7.9. In some embodiments, the pH value of the sample preservation solution is 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the pH value of the sample preservation solution is 7.8.
在一些实施例中，与对照样本保存液相比，所述样本保存液对DNA，miRNA，和/或DNA甲基化的保存效果更好。在一些实施例中，所述DNA来源于EB病毒，所述miRNA来源于EB病毒，和/或所述DNA甲基化是EB病毒DNA的甲基化。In some embodiments, the sample preservation solution has a better preservation effect on DNA, miRNA, and/or DNA methylation than the control sample preservation solution. In some embodiments, the DNA is derived from Epstein-Barr virus, the miRNA is derived from Epstein-Barr virus, and/or the DNA methylation is methylation of Epstein-Barr virus DNA.
miRNA提取试剂和方法miRNA extraction reagents and methods
本发明的一方面，提供了一种miRNA提取试剂。本发明的一方面，提供了一种包含miRNA提取试剂的试剂盒。本发明的一方面，提供了一种使用miRNA提取试剂从样本中提取miRNA的方法。One aspect of the present invention provides a miRNA extraction reagent. One aspect of the present invention provides a kit comprising miRNA extraction reagents. One aspect of the present invention provides a method for extracting miRNA from a sample using an miRNA extraction reagent.
在一些实施例中，提取miRNA的步骤包含：In some embodiments, the step of extracting miRNA comprises:
i)向所述样本保存液中加入裂解液；优选地，还加入蛋白酶K；i) adding a lysate to the sample preservation solution; preferably, proteinase K is also added;
ii)加入结合液与核酸吸附材料；ii) adding binding solution and nucleic acid adsorption material;
iii)去除液体部分，向核酸吸附材料加入漂洗液清洗；和iii) removing the liquid portion, adding a rinse solution to the nucleic acid adsorbing material for washing; and
iv)去除液体部分，向所述核酸吸附材料中加入洗脱液，将所述核酸和/或所述miRNA从所述核酸吸附材料中洗脱。iv) removing the liquid part, adding an eluent to the nucleic acid adsorption material, and eluting the nucleic acid and/or the miRNA from the nucleic acid adsorption material.
在一些实施例中，所述miRNA为EB病毒相关miRNA。In some embodiments, the miRNA is Epstein-Barr virus-associated miRNA.
在一些实施例中，所述miRNA提取试剂、方法包含裂解液，结合液，漂洗液1，和/或漂洗液2。在一些实施例中，所述miRNA提取试剂、方法包含裂解液，结合液，漂洗液1，和/或漂洗液2。In some embodiments, the miRNA extraction reagents and methods include a lysis solution, a binding solution, a washing solution 1, and/or a washing solution 2. In some embodiments, the miRNA extraction reagents and methods include a lysis solution, a binding solution, a washing solution 1, and/or a washing solution 2.
在一些实施例中，在步骤iii)中，向所述核酸吸附材料依次加入漂洗液1和漂洗液2清洗，每次清洗后去除液体部分然后进行下一个清洗；优选地，向所述核酸吸附材料用所述漂洗液2清洗至少两次。In some embodiments, in step iii), rinse solution 1 and rinse solution 2 are sequentially added to the nucleic acid adsorption material for washing, and the liquid part is removed after each wash and then the next wash is performed; preferably, the nucleic acid adsorption material is The material was washed at least twice with the rinse solution 2.
在一些实施例中，所述miRNA提取试剂/方法包含裂解液。在一些实施例中，所述裂解液包含选自硫氰酸胍，柠檬酸，曲拉通-100，和/或EDTA的一种或多种组分。在一些实施例中，所述裂解液包含硫氰酸胍，柠檬酸，曲拉通-100，和EDTA。In some embodiments, the miRNA extraction reagents/methods comprise a lysate. In some embodiments, the lysate comprises one or more components selected from guanidine thiocyanate, citric acid, Triton-100, and/or EDTA. In some embodiments, the lysate comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA.
在一些实施例中，所述裂解液中：In some embodiments, in the lysate:
-所述硫氰酸胍的含量范围是28％-40％(w/w)；- the content range of the guanidinium thiocyanate is 28%-40% (w/w);
-所述柠檬酸的含量范围是1.8％-2.6％(w/w)；- the content range of the citric acid is 1.8%-2.6% (w/w);
-所述曲拉通-100的含量范围是0.8％-1.6％(w/w)；- the content range of the Triton-100 is 0.8%-1.6% (w/w);
-所述EDTA的含量范围是0.5％-0.9％(w/w)；和/或- said EDTA content ranges from 0.5% to 0.9% (w/w); and/or
-所述裂解液的pH值范围为3.8-4.2。- The pH range of the lysate is 3.8-4.2.
在一些实施例中，所述裂解液中：In some embodiments, in the lysate:
-所述硫氰酸胍的含量是约32％(w/w)；- said content of guanidinium thiocyanate is about 32% (w/w);
-所述柠檬酸的含量是约2.2％(w/w)；- said citric acid content is about 2.2% (w/w);
-所述曲拉通-100的含量是约1.1％(w/w)；- the content of said Triton-100 is about 1.1% (w/w);
-所述EDTA的含量是约0.7％(w/w)；和/或- said EDTA content is about 0.7% (w/w); and/or
-所述裂解液的pH值为约4.0。- The pH of the lysate is about 4.0.
在一些实施例中，所述miRNA提取试剂/方法包含结合液。在一些实施例中，所述结合液包含选自氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和/或异丙醇的一种或多种组分。在一些实施例中，所述结合液包含氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和异丙醇。In some embodiments, the miRNA extraction reagent/method comprises a binding solution. In some embodiments, the binding solution comprises one selected from magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, sodium lauryl sarcosine, and/or isopropanol or multiple components. In some embodiments, the binding solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, sodium sarcosine, and isopropanol.
在一些实施例中，所述结合液中：In some embodiments, in the binding solution:
-所述氯化镁的含量范围是0.1％-0.4％(w/w)；- the content range of the magnesium chloride is 0.1%-0.4% (w/w);
-所述乙二胺四乙酸二钠的含量范围是0.5％-1.0％(w/w)；- the content range of disodium edetate is 0.5%-1.0% (w/w);
-所述碳酸氢钠的含量范围是0.6％-1.0％(w/w)；- the content range of the sodium bicarbonate is 0.6%-1.0% (w/w);
-所述硫氰酸胍的含量范围是13％-16％(w/w)；- the content range of the guanidinium thiocyanate is 13%-16% (w/w);
-所述十二烷基肌氨酸钠的含量范围是1.0％-1.4％(w/w)；- the content range of the sodium lauryl sarcosine is 1.0%-1.4% (w/w);
-所述异丙醇的含量范围是60％-80％(w/w)；和/或- said isopropanol is present in an amount ranging from 60% to 80% (w/w); and/or
-所述结合液的pH值范围是4.8-5.2。- The pH range of the binding solution is 4.8-5.2.
在一些实施例中，所述结合液中：In some embodiments, in the binding solution:
-所述氯化镁的含量是约0.24％(w/w)；- said magnesium chloride content is about 0.24% (w/w);
-所述乙二胺四乙酸二钠的含量是约0.71％(w/w)；- the content of disodium edetate is about 0.71% (w/w);
-所述碳酸氢钠的含量是约0.83％(w/w)；- said sodium bicarbonate content is about 0.83% (w/w);
-所述硫氰酸胍的含量是约14.9％(w/w)；- said content of guanidinium thiocyanate is about 14.9% (w/w);
-所述十二烷基肌氨酸钠的含量是约1.2％(w/w)；- the content of sodium sarcosyl is about 1.2% (w/w);
-所述异丙醇的含量是约70％(w/w)；和/或- said isopropanol is present in an amount of about 70% (w/w); and/or
-所述结合液的pH值是约5.0。- The pH value of the binding solution is about 5.0.
在一些实施例中，所述miRNA提取试剂/方法包含漂洗液1。在一些实施例中，所述漂洗液1包含选自乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和/或十二烷基肌氨酸钠的一种或多种组分。在一些实施例中，所述漂洗液1包含乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和十二烷基肌氨酸钠。In some embodiments, the miRNA extraction reagent/method comprises Wash 1. In some embodiments, the rinse solution 1 comprises one or more components selected from disodium edetate, sodium bicarbonate, guanidine thiocyanate, and/or sodium lauryl sarcosine . In some embodiments, the rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidine thiocyanate, and sodium lauryl sarcosinate.
在一些实施例中，所述漂洗液1中：In some embodiments, in the rinse solution 1:
-所述乙二胺四乙酸二钠的含量范围是0.4％-0.9％(w/w)；- the content range of disodium edetate is 0.4%-0.9% (w/w);
-所述碳酸氢钠的含量范围是0.5％-0.9％(w/w)；- the content range of the sodium bicarbonate is 0.5%-0.9% (w/w);
-所述硫氰酸胍的含量范围是50％-55％(w/w)；- the content range of the guanidinium thiocyanate is 50%-55% (w/w);
-所述十二烷基肌氨酸钠的含量范围是0.8％-1.2％(w/w)；和/或- the content of sodium sarcosyl is in the range of 0.8%-1.2% (w/w); and/or
-所述漂洗液1的pH值范围是6.2-6.6。- The pH range of the rinse solution 1 is 6.2-6.6.
在一些实施例中，所述漂洗液1中：In some embodiments, in the rinse solution 1:
-所述乙二胺四乙酸二钠的含量是约0.5％(w/w)；- the content of disodium edetate is about 0.5% (w/w);
-所述碳酸氢钠的含量是约0.6％(w/w)；- the content of said sodium bicarbonate is about 0.6% (w/w);
-所述硫氰酸胍的含量是约53％(w/w)；- said content of guanidinium thiocyanate is about 53% (w/w);
-所述十二烷基肌氨酸钠的含量是约0.9％(w/w)；和/或- the content of sodium sarcosyl is about 0.9% (w/w); and/or
-所述漂洗液1的pH值是约6.4。- The pH value of the rinse solution 1 is about 6.4.
在一些实施例中，在接触所述核酸吸附材料前，向所述漂洗液1中加入乙醇，所述漂洗液1∶乙醇的体积比为1∶1-1∶2；优选地，所述体积比为约2∶3。In some embodiments, ethanol is added to the rinse solution 1 before contacting the nucleic acid adsorption material, and the volume ratio of the rinse solution 1:ethanol is 1:1-1:2; preferably, the volume The ratio is about 2:3.
在一些实施例中，所述miRNA提取试剂/方法包含所述漂洗液2。在一些实施例中，所述漂洗液2包含三羟甲基氨基甲烷(Tris)或氯化钠。在一些实施例中，所述漂洗液2包含三羟甲基氨基甲烷(Tris)和氯化钠。In some embodiments, the miRNA extraction reagent/method comprises the wash solution 2. In some embodiments, the rinse solution 2 includes tris (Tris) or sodium chloride. In some embodiments, the rinse solution 2 comprises tris (Tris) and sodium chloride.
在一些实施例中，所述漂洗液2中：In some embodiments, in the rinse solution 2:
-所述三羟甲基氨基甲烷(Tris)的含量范围是0.5％-0.7％(w/w)；- the content range of the tris (Tris) is 0.5%-0.7% (w/w);
-所述氯化钠的含量范围是0.6％-1.8％(w/w)；和/或- said sodium chloride content ranges from 0.6% to 1.8% (w/w); and/or
-所述漂洗液2的pH值范围是6.6-7.0。- The pH range of the rinse solution 2 is 6.6-7.0.
在一些实施例中，所述漂洗液2中：In some embodiments, in the rinse solution 2:
-所述三羟甲基氨基甲烷(Tris)的含量是约0.6％(w/w)；- the content of tris (Tris) is about 0.6% (w/w);
-所述氯化钠的含量是约1.2％(w/w)；和/或- said sodium chloride content is about 1.2% (w/w); and/or
-所述漂洗液2的pH值是约6.8。- The pH value of the rinse solution 2 is about 6.8.
在一些实施例中，在接触所述核酸吸附材料前，向所述漂洗液2中加入乙醇，所述漂 洗液2∶乙醇的体积比为1∶1.6-1∶3；优选地，所述体积比为约1∶2.3。In some embodiments, ethanol is added to the rinse solution 2 before contacting the nucleic acid adsorption material, and the volume ratio of the rinse solution 2:ethanol is 1:1.6-1:3; preferably, the volume The ratio is about 1:2.3.
在一些实施例中，所述洗脱液为DEPC水.In some embodiments, the eluent is DEPC water.
在一些实施例中，所述miRNA提取试剂/方法包含核酸吸附材料。在一些实施例中，所述核酸吸附材料是吸附RNA的磁珠。在一些实施例中，在一些实施例中，所述磁珠的磁核是氢氧化三铁。在一些实施例中，所述磁珠包被二氧化硅。在一些实施例中，所述磁珠表面镶嵌羟基基团。在一些实施例中，所述磁珠的粒径为50-100nm。In some embodiments, the miRNA extraction reagent/method comprises a nucleic acid adsorbent material. In some embodiments, the nucleic acid adsorption material is magnetic beads that adsorb RNA. In some embodiments, in some embodiments, the magnetic core of the magnetic beads is ferric hydroxide. In some embodiments, the magnetic beads are coated with silica. In some embodiments, the surface of the magnetic beads is embedded with hydroxyl groups. In some embodiments, the particle size of the magnetic beads is 50-100 nm.
EB病毒载量检测试剂Epstein-Barr Viral Load Detection Reagent
在一些实施例中，所述EB病毒载量检测包含针对EB病毒基因组中BamHI-W片段，和/或LMP-2基因的检测。在一些实施例中，所述EB病毒载量检测包含针对EB病毒基因组中BamHI-W片段的检测。在一些实施例中，所述EB病毒载量检测包含针对EB病毒基因组中LMP-2基因的检测。In some embodiments, the detection of the Epstein-Barr virus load comprises the detection of the BamHI-W fragment in the Epstein-Barr virus genome, and/or the LMP-2 gene. In some embodiments, the detection of the Epstein-Barr virus load comprises the detection of the BamHI-W fragment in the Epstein-Barr virus genome. In some embodiments, the detection of the Epstein-Barr virus load comprises the detection of the LMP-2 gene in the Epstein-Barr virus genome.
鼻咽刷或鼻咽拭子Nasopharyngeal brush or nasopharyngeal swab
在一些实施例中，本发明涉及的试剂盒还包含鼻咽刷或鼻咽拭子。在一些实施例中，本发明涉及的用途和方法还包含使用鼻咽刷或鼻咽拭子获得样本。在一些实施例中，所述鼻咽刷和/或鼻咽拭子为不依赖临床医生和鼻咽镜引导的鼻咽刷和/或鼻咽拭子。In some embodiments, the kit of the present invention further comprises a nasopharyngeal brush or a nasopharyngeal swab. In some embodiments, the uses and methods of the present invention further comprise obtaining a sample using a nasopharyngeal brush or a nasopharyngeal swab. In some embodiments, the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab that does not rely on the guidance of a clinician and a nasopharyngoscope.
鼻咽癌治疗Nasopharyngeal Cancer Treatment
在一些实施例中，本发明提供了一种对有需要的个体治疗鼻咽癌的方法，包含对所述个体提供药物、放疗、手术、细胞疗法和/或溶瘤病毒治疗，其中，所述个体的样本接受了本发明所述的鼻咽癌筛查和/或诊断方法的检测并被判断为鼻咽癌阳性。In some embodiments, the present invention provides a method for treating nasopharyngeal carcinoma in an individual in need thereof, comprising providing drug, radiotherapy, surgery, cell therapy and/or oncolytic virus therapy to the individual, wherein the Individual samples are tested by the screening and/or diagnosing method for nasopharyngeal carcinoma according to the present invention and judged to be positive for nasopharyngeal carcinoma.
在一些实施例中，本发明提供了一种对有需要的个体治疗鼻咽癌的方法，包含1)对来自所述个体的样本进行本发明所述的鼻咽癌筛查和/或诊断方法的检测；2)如检测结果显示鼻咽癌阳性，提供鼻咽癌治疗；优选地，所述鼻咽癌治疗采用药物、放疗、手术、细胞疗法和/或溶瘤病毒治疗。In some embodiments, the present invention provides a method for treating nasopharyngeal carcinoma in an individual in need thereof, comprising 1) performing the screening and/or diagnosing method for nasopharyngeal carcinoma according to the present invention on a sample from said individual 2) If the test result is positive for nasopharyngeal carcinoma, provide nasopharyngeal carcinoma treatment; preferably, the nasopharyngeal carcinoma treatment adopts drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus treatment.
在一些实施例中，本发明所述的鼻咽癌筛查和/或诊断方法包含：In some embodiments, the nasopharyngeal carcinoma screening and/or diagnosis method of the present invention comprises:
(1)EB病毒基因组核酸甲基化检测；(1) Epstein-Barr virus genome nucleic acid methylation detection;
(2)EB病毒相关miRNA的含量检测；和/或(2) Detection of Epstein-Barr virus-related miRNA content; and/or
(3)EB病毒核酸载量检测。(3) Detection of EB virus nucleic acid load.
在一些实施例中，所述个体为人。在一些实施例中，所述方法包含提供治疗有效量的 药物、放疗、手术、细胞疗法和/或溶瘤病毒治疗。相关的治疗方法包括并不限于以下文献中提到的方法：Liu et al.，Front Oncol.2021；11：635737(doi：10.3389/fonc.2021.635737)。本申请通过引用并入这些文献的全部内容。In some embodiments, the individual is a human. In some embodiments, the method comprises providing a therapeutically effective amount of a drug, radiation therapy, surgery, cell therapy, and/or oncolytic virus therapy. Relevant treatment methods include but are not limited to those mentioned in the following literature: Liu et al., Front Oncol. 2021; 11: 635737 (doi: 10.3389/fonc.2021.635737). This application incorporates these documents by reference in their entirety.
在一些实施例中，治疗鼻咽癌的方法包含放疗。在某些实施例中，所述放疗为调强放射疗法(Intensity-modulated radiotherapy；IMRT)。In some embodiments, the method of treating nasopharyngeal carcinoma comprises radiation therapy. In certain embodiments, the radiotherapy is intensity-modulated radiotherapy (IMRT).
在一些实施例中，治疗鼻咽癌的方法包含质子疗法。In some embodiments, the method of treating nasopharyngeal carcinoma comprises proton therapy.
在一些实施例中，治疗鼻咽癌的方法包含化疗。在一些实施例中，所述化疗使用含铂化疗药物。In some embodiments, the method of treating nasopharyngeal carcinoma comprises chemotherapy. In some embodiments, the chemotherapy uses a platinum-containing chemotherapy drug.
在一些实施例中，治疗鼻咽癌的方法包含靶向性疗法。在一些实施例中，所述靶向性疗法包含抗体药物或小分子药物。In some embodiments, the method of treating nasopharyngeal carcinoma comprises targeted therapy. In some embodiments, the targeted therapy comprises an antibody drug or a small molecule drug.
在一些实施例中，治疗鼻咽癌的方法包含免疫疗法。在一些实施例中，所述免疫疗法包含细胞疗法或溶瘤病毒疗法。在一些实施例中，所述细胞疗法包含嵌合抗原受体修饰T细胞(CAR-T)疗法或嵌合抗原受体修饰NK细胞(CAR-NK)疗法。In some embodiments, the method of treating nasopharyngeal carcinoma comprises immunotherapy. In some embodiments, the immunotherapy comprises cell therapy or oncolytic virotherapy. In some embodiments, the cell therapy comprises chimeric antigen receptor modified T cell (CAR-T) therapy or chimeric antigen receptor modified NK cell (CAR-NK) therapy.
有益效果Beneficial effect
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明提出EB病毒甲基化位点/miRNA能够作为鼻咽癌诊断标志物，其在不依赖临床医生和鼻咽镜引导的鼻咽刷或鼻咽拭子(鼻咽部盲检)样本中具有高灵敏度和高特异性的优点，有利于不依赖临床医生和鼻咽镜引导的鼻咽刷或鼻咽拭子(鼻咽部盲检)的推广。The present invention proposes that Epstein-Barr virus methylation site/miRNA can be used as a diagnostic marker for nasopharyngeal carcinoma, which is independent of clinicians and nasopharyngoscope-guided nasopharyngeal brush or nasopharyngeal swab (nasopharyngeal blind examination) samples It has the advantages of high sensitivity and high specificity, and is conducive to the promotion of nasopharyngeal brushes or nasopharyngeal swabs (blind nasopharyngeal examination) that do not rely on clinicians and nasopharyngoscope guidance.
实施项/技术方案Implementation items/technical solutions
具体地，本发明可通过如下各项技术方案解决现有技术中存在的技术问题：Specifically, the present invention can solve the technical problems existing in the prior art through the following technical solutions:
项1.一种检测EB病毒的试剂盒，其特征在于，所述试剂盒包含选自核酸甲基化检测试剂、miRNA检测试剂、miRNA提取试剂，和/或样本保存液的一种或多种试剂。 Item 1. A kit for detecting Epstein-Barr virus, characterized in that, the kit includes one or more selected from nucleic acid methylation detection reagents, miRNA detection reagents, miRNA extraction reagents, and/or sample preservation solutions reagent.
项2.如项1所述的试剂盒，其特征在于，所述试剂盒包含核酸甲基化检测试剂，所述核酸甲基化检测试剂可针对EB病毒基因组中甲基化位点进行甲基化检测。 Item 2. The kit as described in Item 1, characterized in that, the kit includes a nucleic acid methylation detection reagent, and the nucleic acid methylation detection reagent can carry out methylation for the methylation site in the Epstein-Barr virus genome chemical detection.
项3.如项2所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、14015bp、14566bp、45850bp、57946bp、66227bp和128103bp的位点。Item 3. The kit as described in item 2, wherein the methylation site in the Epstein-Barr virus genome comprises one or more of the following sites: equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 10717bp, 11029bp, 14015bp, 14566bp, 45850bp, 57946bp, 66227bp and 128103bp sites of the Epstein-Barr virus genome DNA sequence.
项4.如项2-3任一项所述的试剂盒，其特征在于，所述EB病毒基因组中的所述甲基 化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、和14015bp的位点。 Item 4. The kit according to any one of items 2-3, wherein the methylation site in the Epstein-Barr virus genome comprises EB equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 10717bp, 11029bp, and 14015bp sites of the viral genome DNA sequence.
项5.如项2-4任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp、45850bp、57946bp、66227bp和128103bp的位点。Item 5. The kit as described in any one of items 2-4, wherein the methylation site in the Epstein-Barr virus genome comprises one or more of the following sites: equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) the sites of 11029bp, 45850bp, 57946bp, 66227bp and 128103bp of the Epstein-Barr virus genome DNA sequence.
项6.如项2-5任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp、57946bp、和66227bp的位点。Item 6. The kit as described in any one of items 2-5, wherein the methylation site in the Epstein-Barr virus genome comprises one or more of the following sites: equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) the 11029bp, 57946bp and 66227bp sites of the Epstein-Barr virus genome DNA sequence.
项7.如项2-6任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp的位点。 Item 7. The kit as described in any one of items 2-6, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 11029bp site of the sequence.
项7.1.如项2-7任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp的位点。Item 7.1. The kit as described in any one of items 2-7, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 10717bp site of the sequence.
项7.2.如项2-7.1任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的14015bp的位点。Item 7.2. The kit as described in any one of item 2-7.1, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 14015bp site of the sequence.
项7.3.如项2-7.2任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的14566bp的位点。Item 7.3. The kit as described in any one of item 2-7.2, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 14566bp site of the sequence.
项7.4.如项2-7.3任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的45850bp的位点。Item 7.4. The kit as described in any one of item 2-7.3, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 45850bp site of the sequence.
项7.5.如项2-7.4任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的57946bp的位点。Item 7.5. The kit as described in any one of item 2-7.4, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 57946bp site of the sequence.
项7.6.如项2-7.5任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的66227bp的位点。Item 7.6. The kit as described in any one of item 2-7.5, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 66227bp site of the sequence.
项7.7.如项2-7.6任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的128103bp的位点。Item 7.7. The kit as described in any one of item 2-7.6, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genomic DNA equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 128103bp site of the sequence.
项8.如项1-7.7任一项所述的试剂盒，其特征在于，所述试剂盒包含所述miRNA检测试剂，所述miRNA检测试剂可针对一个或多个来自EB病毒的miRNA进行含量检测。Item 8. The kit according to any one of Items 1-7.7, characterized in that, the kit includes the miRNA detection reagent, and the miRNA detection reagent can be used for one or more miRNAs from Epstein-Barr virus. detection.
项9.如项8所述的试剂盒，其特征在于，所述miRNA包含以下miRNA的一个或几个：EBV-miR-BART1-5p、EBV-miR-BART2-5p、EBV-miR-BART6-3p、EBV-miR-BART7-3p、EBV-miR-BART13-3p、和EBV-miR-BART17-5p；优选地包含全部六个所述miRNA。Item 9. The kit as described in Item 8, wherein the miRNA comprises one or more of the following miRNAs: EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR-BART6- 3p, EBV-miR-BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p; preferably comprising all six of said miRNAs.
项10.如项9所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART1-5p和EBV-miR-BART7-3p。 Item 10. The kit according to Item 9, wherein the miRNA comprises EBV-miR-BART1-5p and EBV-miR-BART7-3p.
项11.如项9-10任一项所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART 1-5p。Item 11. The kit according to any one of Items 9-10, wherein the miRNA comprises EBV-miR-BART 1-5p.
项12.如项9-11任一项所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART2-5p。Item 12. The kit according to any one of Items 9-11, wherein the miRNA comprises EBV-miR-BART2-5p.
项13.如项9-12任一项所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART6-3p。Item 13. The kit according to any one of Items 9-12, wherein the miRNA comprises EBV-miR-BART6-3p.
项14.如项9-13任一项所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART7-3p。Item 14. The kit according to any one of Items 9-13, wherein the miRNA comprises EBV-miR-BART7-3p.
项15.如项9-14任一项所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART13-3p。 Item 15. The kit according to any one of Items 9-14, wherein the miRNA comprises EBV-miR-BART13-3p.
项16.如项9-15任一项所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART 17-5p。Item 16. The kit according to any one of Items 9-15, wherein the miRNA comprises EBV-miR-BART 17-5p.
项17.如项1-16任一项所述的试剂盒，其特征在于，所述试剂盒包含所述样本保存液，所述样本保存液包含选自EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和/或磷酸三钠的一种或多种组分。Item 17. The kit as described in any one of Items 1-16, wherein the kit includes the sample preservation solution, and the sample preservation solution comprises EDTA, NaCl, absolute ethanol, citric acid One or more components of sodium, NP40, glutathione, and/or trisodium phosphate.
项18.如项17所述的试剂盒，其特征在于，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。Item 18. The kit according to Item 17, wherein the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
项19.一种保存含核酸的生物样本的试剂盒，其特征在于，包含样本保存液，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。Item 19. A kit for preserving a nucleic acid-containing biological sample, characterized in that it comprises a sample preservation solution comprising EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and Trisodium Phosphate.
项20.如项17-19任一项所述的试剂盒，其特征在于，所述样本保存液中： Item 20. The kit according to any one of Items 17-19, wherein in the sample preservation solution:
所述EDTA的含量范围是0.7％-1.5％(w/w)；The content range of the EDTA is 0.7%-1.5% (w/w);
所述NaCl的含量范围是0.01％-0.1％(w/w)；The content range of the NaCl is 0.01%-0.1% (w/w);
所述无水乙醇的浓度范围是15％-35％(w/w)；The concentration range of the absolute ethanol is 15%-35% (w/w);
所述柠檬酸钠的含量范围是0.1％-1.0％(w/w)；The content range of the sodium citrate is 0.1%-1.0% (w/w);
所述NP40的浓度范围是0.01％-0.1％(v/v)；The concentration range of the NP40 is 0.01%-0.1% (v/v);
所述谷胱甘肽的含量范围是0.07％-0.7％(w/w)；和/或The glutathione content ranges from 0.07% to 0.7% (w/w); and/or
所述磷酸三钠的含量范围是0.07％-0.4％(w/w)。The content range of the trisodium phosphate is 0.07%-0.4% (w/w).
项21.如项17-20任一项所述的试剂盒，其特征在于，所述样本保存液中：Item 21. The kit according to any one of Items 17-20, wherein in the sample preservation solution:
所述EDTA的含量是约1.05％(w/w)；The content of said EDTA is about 1.05% (w/w);
所述NaCl的含量是约0.035％(w/w)；The content of said NaCl is about 0.035% (w/w);
所述无水乙醇的浓度是约27.6％(w/w)；The concentration of absolute ethanol is about 27.6% (w/w);
所述柠檬酸钠的含量是约0.21％(w/w)；The content of the sodium citrate is about 0.21% (w/w);
所述NP40的浓度是约0.035％(v/v)；The concentration of said NP40 is about 0.035% (v/v);
所述谷胱甘肽的含量是约0.21％(w/w)；和/或The glutathione content is about 0.21% (w/w); and/or
所述磷酸三钠的含量是约0.14％(w/w)。The content of the trisodium phosphate is about 0.14% (w/w).
项22.如项17-21一项所述的试剂盒，其特征在于，所述样本保存液包含抗生素；优选地，所述抗生素包含青霉素-链霉素双抗。 Item 22. The kit according to item 17-21, wherein the sample preservation solution includes antibiotics; preferably, the antibiotics include penicillin-streptomycin double antibody.
项23.如项17-22任一项所述的试剂盒，其特征在于，所述样本保存液的pH值为7.6-8.0；优选地，所述样本保存液的pH值为7.8。Item 23. The kit according to any one of Items 17-22, wherein the pH value of the sample preservation solution is 7.6-8.0; preferably, the pH value of the sample preservation solution is 7.8.
项24.如项17-23任一项所述的试剂盒，其特征在于，与对照样本保存液相比，所述样本保存液对DNA，miRNA，和/或DNA甲基化的保存效果更好。 Item 24. The kit according to any one of Items 17-23, characterized in that, compared with the control sample preservation solution, the sample preservation solution has a better preservation effect on DNA, miRNA, and/or DNA methylation. good.
项25.如项24所述的试剂盒，其特征在于，所述DNA来源于EB病毒，所述miRNA来源于EB病毒，和/或所述DNA甲基化是EB病毒DNA的甲基化。 Item 25. The kit according to Item 24, wherein the DNA is derived from Epstein-Barr virus, the miRNA is derived from Epstein-Barr virus, and/or the DNA methylation is methylation of Epstein-Barr virus DNA.
项26.如项1-25任一项所述的试剂盒，其特征在于，所述试剂盒包含所述miRNA提取试剂，所述miRNA提取试剂包含裂解液，结合液，漂洗液1，和/或漂洗液2。 Item 26. The kit according to any one of Items 1-25, wherein the kit includes the miRNA extraction reagent, and the miRNA extraction reagent includes a lysate, a binding solution, a rinse solution 1, and/or or rinse solution 2.
项27.一种包含miRNA提取试剂的试剂盒，其特征在于，所述miRNA提取试剂包含裂解液，结合液，漂洗液1，和/或漂洗液2。Item 27. A kit comprising miRNA extraction reagents, characterized in that the miRNA extraction reagents comprise lysis solution, binding solution, washing solution 1, and/or washing solution 2.
项28.如项26或27所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述裂解液，所述裂解液包含选自硫氰酸胍，柠檬酸，曲拉通-100，和/或EDTA的一种或多种组分。 Item 28. The kit according to Item 26 or 27, wherein the miRNA extraction reagent comprises the lysate, and the lysate comprises guanidinium thiocyanate, citric acid, Triton-100, and/or one or more components of EDTA.
项29.如项26或27所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述裂解液，所述裂解液包含硫氰酸胍，柠檬酸，曲拉通-100，和EDTA。Item 29. The kit according to Item 26 or 27, wherein the miRNA extraction reagent comprises the lysate, which comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA .
项30.如项26-29任一项所述的试剂盒，其特征在于，所述裂解液中： Item 30. The kit according to any one of Items 26-29, wherein in the lysate:
所述硫氰酸胍的含量范围是28％-40％(w/w)；The content scope of described guanidinium thiocyanate is 28%-40% (w/w);
所述柠檬酸的含量范围是1.8％-2.6％(w/w)；The content range of the citric acid is 1.8%-2.6% (w/w);
所述曲拉通-100的含量范围是0.8％-1.6％(w/w)；The content range of the Triton-100 is 0.8%-1.6% (w/w);
所述EDTA的含量范围是0.5％-0.9％(w/w)；和/或The content range of the EDTA is 0.5%-0.9% (w/w); and/or
所述裂解液的pH值范围为3.8-4.2。The pH range of the lysate is 3.8-4.2.
项31.如项26-30任一项所述的试剂盒，其特征在于，所述裂解液中：Item 31. The kit according to any one of Items 26-30, wherein in the lysate:
所述硫氰酸胍的含量是约32％(w/w)；The content of the guanidinium thiocyanate is about 32% (w/w);
所述柠檬酸的含量是约2.2％(w/w)；The content of said citric acid is about 2.2% (w/w);
所述曲拉通-100的含量是约1.1％(w/w)；The content of the Triton-100 is about 1.1% (w/w);
所述EDTA的含量是约0.7％(w/w)；和/或The content of said EDTA is about 0.7% (w/w); and/or
所述裂解液的pH值为约4.0。The pH of the lysate is about 4.0.
项32.如项26-31任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述结合液，所述结合液包含选自氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和/或异丙醇的一种或多种组分。 Item 32. The kit according to any one of Items 26-31, wherein the miRNA extraction reagent comprises the binding liquid, and the binding liquid comprises magnesium chloride, disodium edetate, carbonic acid One or more components of sodium hydrogen, guanidinium thiocyanate, sodium lauryl sarcosinate, and/or isopropanol.
项33.如项26-32任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述结合液，所述结合液包含氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和异丙醇。Item 33. The kit according to any one of Items 26-32, wherein the miRNA extraction reagent comprises the binding solution, and the binding solution comprises magnesium chloride, disodium edetate, and sodium bicarbonate , guanidinium thiocyanate, sodium lauryl sarcosinate, and isopropanol.
项34.如项26-33任一项所述的试剂盒，其特征在于，所述结合液中： Item 34. The kit according to any one of Items 26-33, wherein in the binding solution:
所述氯化镁的含量范围是0.1％-0.4％(w/w)；The content range of the magnesium chloride is 0.1%-0.4% (w/w);
所述乙二胺四乙酸二钠的含量范围是0.5％-1.0％(w/w)；The content range of the disodium edetate is 0.5%-1.0% (w/w);
所述碳酸氢钠的含量范围是0.6％-1.0％(w/w)；The content scope of described sodium bicarbonate is 0.6%-1.0% (w/w);
所述硫氰酸胍的含量范围是13％-16％(w/w)；The content range of the guanidine thiocyanate is 13%-16% (w/w);
所述十二烷基肌氨酸钠的含量范围是1.0％-1.4％(w/w)；The content range of the sodium lauryl sarcosinate is 1.0%-1.4% (w/w);
所述异丙醇的含量范围是60％-80％(w/w)；和/或The content range of the isopropanol is 60%-80% (w/w); and/or
所述结合液的pH值范围是4.8-5.2。The pH range of the binding solution is 4.8-5.2.
项35.如项26-34任一项所述的试剂盒，其特征在于，所述结合液中： Item 35. The kit according to any one of Items 26-34, wherein in the binding solution:
所述氯化镁的含量是约0.24％(w/w)；The content of said magnesium chloride is about 0.24% (w/w);
所述乙二胺四乙酸二钠的含量是约0.71％(w/w)；The content of disodium edetate is about 0.71% (w/w);
所述碳酸氢钠的含量是约0.83％(w/w)；The content of the sodium bicarbonate is about 0.83% (w/w);
所述硫氰酸胍的含量是约14.9％(w/w)；The content of guanidine thiocyanate is about 14.9% (w/w);
所述十二烷基肌氨酸钠的含量是约1.2％(w/w)；The content of sodium sarcosyl is about 1.2% (w/w);
所述异丙醇的含量是约70％(w/w)；和/或The content of said isopropanol is about 70% (w/w); and/or
所述结合液的pH值是约5.0。The pH of the binding solution is about 5.0.
项36.如项26-35任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述漂洗液1，所述漂洗液1包含选自乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和/或十二烷基肌氨酸钠的一种或多种组分。 Item 36. The kit according to any one of Items 26-35, wherein the miRNA extraction reagent comprises the rinse solution 1, and the rinse solution 1 comprises disodium edetate, carbonic acid One or more components of sodium hydrogen, guanidinium thiocyanate, and/or sodium lauryl sarcosinate.
项37.如项26-36任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述漂洗液1，所述漂洗液1包含乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和十二烷基肌氨酸钠。Item 37. The kit according to any one of items 26-36, wherein the miRNA extraction reagent comprises the rinse solution 1, and the rinse solution 1 comprises disodium edetate, sodium bicarbonate , guanidinium thiocyanate, and sodium lauryl sarcosinate.
项38.如项26-37任一项所述的试剂盒，其特征在于，所述漂洗液1中： Item 38. The kit according to any one of Items 26-37, wherein in the rinse solution 1:
所述乙二胺四乙酸二钠的含量范围是0.4％-0.9％(w/w)；The content range of the disodium edetate is 0.4%-0.9% (w/w);
所述碳酸氢钠的含量范围是0.5％-0.9％(w/w)；The content scope of described sodium bicarbonate is 0.5%-0.9% (w/w);
所述硫氰酸胍的含量范围是50％-55％(w/w)；The content range of the guanidinium thiocyanate is 50%-55% (w/w);
所述十二烷基肌氨酸钠的含量范围是0.8％-1.2％(w/w)；和/或The content range of the sodium lauryl sarcosyl is 0.8%-1.2% (w/w); and/or
所述漂洗液1的pH值范围是6.2-6.6。The pH range of the rinse solution 1 is 6.2-6.6.
项39.如项26-38任一项所述的试剂盒，其特征在于，所述漂洗液1中：Item 39. The kit according to any one of Items 26-38, wherein in the rinse solution 1:
所述乙二胺四乙酸二钠的含量是约0.5％(w/w)；The content of disodium edetate is about 0.5% (w/w);
所述碳酸氢钠的含量是约0.6％(w/w)；The content of the sodium bicarbonate is about 0.6% (w/w);
所述硫氰酸胍的含量是约53％(w/w)；The content of the guanidinium thiocyanate is about 53% (w/w);
所述十二烷基肌氨酸钠的含量是约0.9％(w/w)；和/或The content of sodium sarcosyl is about 0.9% (w/w); and/or
所述漂洗液1的pH值是约6.4。The pH of the Rinse 1 was about 6.4.
项40.如项26-39任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述漂洗液2，所述漂洗液2包含三羟甲基氨基甲烷(Tris)或氯化钠。 Item 40. The kit according to any one of Items 26-39, wherein the miRNA extraction reagent comprises the rinse solution 2, and the rinse solution 2 comprises tris (Tris) or chlorine sodium chloride.
项41.如项26-40任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述漂洗液2，所述漂洗液2包含三羟甲基氨基甲烷(Tris)和氯化钠。Item 41. The kit according to any one of Items 26-40, wherein the miRNA extraction reagent comprises the rinse solution 2, and the rinse solution 2 comprises tris (Tris) and chlorine sodium chloride.
项42.如项26-41任一项所述的试剂盒，其特征在于，所述漂洗液2中：Item 42. The kit according to any one of Items 26-41, wherein in the rinse solution 2:
所述三羟甲基氨基甲烷(Tris)的含量范围是0.5％-0.7％(w/w)；The content range of the tris (Tris) is 0.5%-0.7% (w/w);
所述氯化钠的含量范围是0.6％-1.8％(w/w)；和/或The content range of the sodium chloride is 0.6%-1.8% (w/w); and/or
所述漂洗液2的pH值范围是6.6-7.0。The pH range of the rinse solution 2 is 6.6-7.0.
项43.如项26-42任一项所述的试剂盒，其特征在于，所述漂洗液2中：Item 43. The kit according to any one of Items 26-42, wherein in the rinse solution 2:
所述三羟甲基氨基甲烷(Tris)的含量是约0.6％(w/w)；The content of tris (Tris) is about 0.6% (w/w);
所述氯化钠的含量是约1.2％(w/w)；和/或The sodium chloride content is about 1.2% (w/w); and/or
所述漂洗液2的pH值是约6.8。The pH value of the rinse solution 2 is about 6.8.
项44.如项26-43任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含吸附RNA的磁珠。Item 44. The kit according to any one of Items 26-43, wherein the miRNA extraction reagent comprises magnetic beads that adsorb RNA.
项45.如项44所述的试剂盒，其特征在于，所述磁珠的磁核是氢氧化三铁，包被二氧化硅，且表面镶嵌羟基基团。Item 45. The kit according to Item 44, wherein the magnetic core of the magnetic beads is ferric hydroxide, coated with silicon dioxide, and hydroxy groups are embedded on the surface.
项46.如项44或45所述的试剂盒，其特征在于，所述磁珠的粒径为50-100nm。Item 46. The kit according to Item 44 or 45, wherein the magnetic beads have a particle diameter of 50-100 nm.
项47.如项26-46任一项所述的试剂盒，其特征在于，所述试剂盒还包含鼻咽刷或鼻咽拭子。Item 47. The kit according to any one of Items 26-46, further comprising a nasopharyngeal brush or a nasopharyngeal swab.
项48.如项47所述的试剂盒，其特征在于，所述鼻咽刷和/或鼻咽拭子为不依赖临床医生和鼻咽镜引导的鼻咽刷和/或鼻咽拭子。Item 48. The kit according to Item 47, wherein the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab independent of the guidance of a clinician and a nasopharyngoscope.
项49.如项1-48任一项所述的试剂盒，其特征在于，所述试剂盒还包含EB病毒载量检测试剂；优选地，所述EB病毒载量检测试剂包含针对EB病毒基因组中BamHI-W片段，和/或LMP-2基因的检测试剂。Item 49. The kit according to any one of Items 1-48, characterized in that, the kit also includes EB virus load detection reagents; preferably, the EB virus load detection reagents contain In the BamHI-W fragment, and/or the detection reagent of LMP-2 gene.
项50.如项49所述的试剂盒，其特征在于，所述EB病毒载量检测试剂包含针对LMP-2基因的检测试剂。Item 50. The kit according to Item 49, wherein the EB virus load detection reagent includes a detection reagent for LMP-2 gene.
项51.如项1-50任一项所述的试剂盒，其特征在于，所述试剂盒为鼻咽癌筛查试剂盒。Item 51. The kit according to any one of Items 1-50, wherein the kit is a nasopharyngeal carcinoma screening kit.
项51.1.如项1-51任一项所述的试剂盒，其特征在于，所述试剂盒为鼻咽癌诊断试剂盒。Item 51.1. The kit according to any one of Items 1-51, wherein the kit is a diagnostic kit for nasopharyngeal carcinoma.
项52.一种检测样本中EB病毒相关核酸的方法，其特征在于，所述方法包含以下步骤：Item 52. A method for detecting Epstein-Barr virus-related nucleic acids in a sample, characterized in that the method comprises the following steps:
(a)将所述样本置于样本保存液中；(a) placing the sample in a sample preservation solution;
(b)进行选自以下的一项或多项检测：(b) Conduct one or more tests selected from the following:
(b-1)对含有所述样本的所述样本保存液进行EB病毒基因组核酸甲基化检测；(b-1) performing Epstein-Barr virus genomic nucleic acid methylation detection on the sample preservation solution containing the sample;
(b-2)对含有所述样本的所述样本保存液的EB病毒相关miRNA的含量进行检测；(b-2) detecting the content of Epstein-Barr virus-related miRNA in the sample preservation solution containing the sample;
优选地，还包括从所述样本保存液中提取所述miRNA；和/或Preferably, it also includes extracting the miRNA from the sample preservation solution; and/or
(b-3)对含有所述样本的所述样本保存液进行EB病毒核酸载量检测。(b-3) Detection of Epstein-Barr virus nucleic acid load on the sample preservation solution containing the sample.
项53.如项52所述的方法，其特征在于，包含步骤(b-1)，且所述EB病毒基因组核酸甲基化检测的甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、14015bp、14566bp、45850bp、57946bp、66227bp和128103bp的位点。Item 53. The method as described in Item 52, characterized in that, step (b-1) is included, and the methylation site detected for methylation of the Epstein-Barr virus genome nucleic acid includes one or more of the following sites : the sites equivalent to 10717bp, 11029bp, 14015bp, 14566bp, 45850bp, 57946bp, 66227bp and 128103bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
项54.如项53所述的方法，其特征在于，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、和14015bp的位点。Item 54. The method as described in Item 53, wherein the methylation site comprises positions equivalent to 10717bp, 11029bp, and 14015bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
项54.1.如项53-54任一项所述的方法，其特征在于，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp的位点。Item 54.1. The method according to any one of Item 53-54, wherein the methylation site comprises a position equivalent to 10717bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
项54.2.如项53-54.1任一项所述的方法，其特征在于，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp的位点。Item 54.2. The method according to any one of Item 53-54.1, wherein the methylation site comprises a position equivalent to 11029bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
项54.3.如项53-54.2任一项所述的方法，其特征在于，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的14015bp的位点。Item 54.3. The method according to any one of Item 53-54.2, wherein the methylation site comprises a position equivalent to 14015bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
项54.4.如项53-54.3任一项所述的方法，其特征在于，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的14566bp的位点。Item 54.4. The method according to any one of Item 53-54.3, wherein the methylation site comprises a position corresponding to 14566 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
项54.5.如项53-54.4任一项所述的方法，其特征在于，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的45850bp的位点。Item 54.5. The method according to any one of Item 53-54.4, wherein the methylation site comprises a position equivalent to 45850bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
项54.6.如项53-54.5任一项所述的方法，其特征在于，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的57946bp的位点。Item 54.6. The method according to any one of Item 53-54.5, wherein the methylation site comprises a position corresponding to 57946 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
项54.7.如项53-54.6任一项所述的方法，其特征在于，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的66227bp的位点。Item 54.7. The method according to any one of Item 53-54.6, wherein the methylation site comprises a position corresponding to 66227bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
项54.8.如项53-54.7任一项所述的方法，其特征在于，所述甲基化位点包含相当于 GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的128103bp的位点。Item 54.8. The method according to any one of Item 53-54.7, wherein the methylation site comprises a position corresponding to 128103 bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94) point.
项55.如项52-54.8任一项所述的方法，其特征在于，包含步骤(b-2)，所述miRNA包含以下miRNA的一个或几个：EBV-miR-BART1-5p、EBV-miR-BART2-5p、EBV-miR-BART6-3p、EBV-miR-BART7-3p、EBV-miR-BART13-3p、和EBV-miR-BART17-5p；优选地包含全部六个所述miRNA。Item 55. The method according to any one of Items 52-54.8, characterized in that step (b-2) is included, and the miRNA includes one or more of the following miRNAs: EBV-miR-BART1-5p, EBV- miR-BART2-5p, EBV-miR-BART6-3p, EBV-miR-BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p; preferably comprising all six of said miRNAs.
项56.如项55所述的方法，其特征在于，所述miRNA包含EBV-miR-BART1-5p和EBV-miR-BART7-3p。Item 56. The method according to Item 55, wherein the miRNA comprises EBV-miR-BART1-5p and EBV-miR-BART7-3p.
项56.1.如项55-56任一项所述的方法，其特征在于，所述miRNA包含EBV-miR-BART1-5p。Item 56.1. The method according to any one of Items 55-56, wherein the miRNA comprises EBV-miR-BART1-5p.
项56.2.如项55-56.1任一项所述的方法，其特征在于，所述miRNA包含EBV-miR-BART2-5p。Item 56.2. The method according to any one of Items 55-56.1, wherein the miRNA comprises EBV-miR-BART2-5p.
项56.3.如项55-56.2任一项所述的方法，其特征在于，所述miRNA包含EBV-miR-BART6-3p。Item 56.3. The method according to any one of Items 55-56.2, wherein the miRNA comprises EBV-miR-BART6-3p.
项56.4.如项55-56.3任一项所述的方法，其特征在于，所述miRNA包含EBV-miR-BART7-3p。Item 56.4. The method according to any one of Items 55-56.3, wherein the miRNA comprises EBV-miR-BART7-3p.
项56.5.如项55-56.4任一项所述的方法，其特征在于，所述miRNA包含EBV-miR-BART13-3p。Item 56.5. The method according to any one of Items 55-56.4, wherein the miRNA comprises EBV-miR-BART13-3p.
项56.6.如项55-56.5任一项所述的方法，其特征在于，所述miRNA包含EBV-miR-BART17-5p。Item 56.6. The method according to any one of Items 55-56.5, wherein the miRNA comprises EBV-miR-BART17-5p.
项57.如项52-56.6任一项所述的方法，其特征在于，提取所述EB病毒相关miRNA的步骤包含：Item 57. The method according to any one of Items 52-56.6, wherein the step of extracting the Epstein-Barr virus-related miRNA comprises:
i)向所述样本保存液中加入裂解液；优选地，还加入蛋白酶K；i) adding a lysate to the sample preservation solution; preferably, proteinase K is also added;
ii)加入结合液与核酸吸附材料；ii) adding binding solution and nucleic acid adsorption material;
iii)去除液体部分，向核酸吸附材料加入漂洗液清洗；和iii) removing the liquid portion, adding a rinse solution to the nucleic acid adsorbing material for washing; and
iv)去除液体部分，向所述核酸吸附材料中加入洗脱液，将所述核酸和/或所述miRNA从所述核酸吸附材料中洗脱。iv) removing the liquid part, adding an eluent to the nucleic acid adsorption material, and eluting the nucleic acid and/or the miRNA from the nucleic acid adsorption material.
项58.一种提取miRNA的方法，包含以下步骤：Item 58. A method for extracting miRNA, comprising the steps of:
i)向样本保存液中加入裂解液；优选地，还加入蛋白酶K；i) adding a lysate to the sample preservation solution; preferably, proteinase K is also added;
ii)加入结合液与核酸吸附材料；ii) adding binding solution and nucleic acid adsorption material;
iii)去除液体部分，向核酸吸附材料加入漂洗液清洗；和iii) removing the liquid portion, adding a rinse solution to the nucleic acid adsorbing material for washing; and
iv)去除液体部分，向所述核酸吸附材料中加入洗脱液，将所述核酸和/或所述miRNA从所述核酸吸附材料中洗脱。iv) removing the liquid part, adding an eluent to the nucleic acid adsorption material, and eluting the nucleic acid and/or the miRNA from the nucleic acid adsorption material.
项59.如项57-58任一项所述的方法，其特征在于，所述裂解液包含选自硫氰酸胍，柠檬酸，曲拉通-100，和/或EDTA的一种或多种组分；优选地，所述裂解液包含硫氰酸胍，柠檬酸，曲拉通-100，和EDTA。Item 59. The method according to any one of Items 57-58, wherein the lysate comprises one or more compounds selected from guanidine thiocyanate, citric acid, Triton-100, and/or EDTA A component; Preferably, the lysate comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA.
项60.如项59所述的方法，其特征在于，所述裂解液中：Item 60. The method according to Item 59, wherein in the lysate:
所述硫氰酸胍的含量范围是28％-40％(w/w)；The content scope of described guanidinium thiocyanate is 28%-40% (w/w);
所述柠檬酸的含量范围是1.8％-2.6％(w/w)；The content range of the citric acid is 1.8%-2.6% (w/w);
所述曲拉通-100的含量范围是0.8％-1.6％(w/w)；The content range of the Triton-100 is 0.8%-1.6% (w/w);
所述EDTA的含量范围是0.5％-0.9％(w/w)；和/或The content range of the EDTA is 0.5%-0.9% (w/w); and/or
所述裂解液的pH值范围为3.8-4.2。The pH range of the lysate is 3.8-4.2.
项61.如项59-60任一项所述的方法，其特征在于，所述裂解液中：Item 61. The method according to any one of Items 59-60, wherein in the lysate:
所述硫氰酸胍的含量是约32％(w/w)；The content of the guanidinium thiocyanate is about 32% (w/w);
所述柠檬酸的含量是约2.2％(w/w)；The content of said citric acid is about 2.2% (w/w);
所述曲拉通-100的含量是约1.1％(w/w)；The content of the Triton-100 is about 1.1% (w/w);
所述EDTA的含量是约0.7％(w/w)；和/或The content of said EDTA is about 0.7% (w/w); and/or
所述裂解液的pH值为约4.0。The pH of the lysate is about 4.0.
项62.如项57-61任一项所述的方法，其特征在于，所述结合液包含选自氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和/或异丙醇的一种或多种组分；优选地，所述结合液包含氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和异丙醇。Item 62. The method according to any one of Items 57-61, wherein the binding solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, dodecyl Sodium sarcosinate, and/or one or more components of isopropanol; preferably, the combined solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, dodecane Sodium sarcosinate, and isopropanol.
项63.如项62所述的方法，其特征在于，所述结合液中：Item 63. The method according to Item 62, wherein in the binding solution:
所述氯化镁的含量范围是0.1％-0.4％(w/w)；The content range of the magnesium chloride is 0.1%-0.4% (w/w);
所述乙二胺四乙酸二钠的含量范围是0.5％-1.0％(w/w)；The content range of the disodium edetate is 0.5%-1.0% (w/w);
所述碳酸氢钠的含量范围是0.6％-1.0％(w/w)；The content scope of described sodium bicarbonate is 0.6%-1.0% (w/w);
所述硫氰酸胍的含量范围是13％-16％(w/w)；The content range of the guanidine thiocyanate is 13%-16% (w/w);
所述十二烷基肌氨酸钠的含量范围是1.0％-1.4％(w/w)；The content range of the sodium lauryl sarcosinate is 1.0%-1.4% (w/w);
所述异丙醇的含量范围是60％-80％(w/w)；和/或The content range of the isopropanol is 60%-80% (w/w); and/or
所述结合液的pH值范围是4.8-5.2。The pH range of the binding solution is 4.8-5.2.
项64.如项62-63任一项所述的方法，其特征在于，所述结合液中：Item 64. The method according to any one of Items 62-63, wherein in the binding solution:
所述氯化镁的含量是约0.24％(w/w)；The content of said magnesium chloride is about 0.24% (w/w);
所述乙二胺四乙酸二钠的含量是约0.71％(w/w)；The content of disodium edetate is about 0.71% (w/w);
所述碳酸氢钠的含量是约0.83％(w/w)；The content of the sodium bicarbonate is about 0.83% (w/w);
所述硫氰酸胍的含量是约14.9％(w/w)；The content of the guanidinium thiocyanate is about 14.9% (w/w);
所述十二烷基肌氨酸钠的含量是约1.2％(w/w)；The content of sodium sarcosyl is about 1.2% (w/w);
所述异丙醇的含量是约70％(w/w)；和/或The content of said isopropanol is about 70% (w/w); and/or
所述结合液的pH值是约5.0。The pH of the binding solution is about 5.0.
项65.如项57-64任一项所述的方法，其特征在于，在步骤iii)中，向所述核酸吸附材料依次加入漂洗液1和漂洗液2清洗，每次清洗后去除液体部分然后进行下一个清洗；优选地，向所述核酸吸附材料用所述漂洗液2清洗至少两次。Item 65. The method according to any one of Items 57-64, characterized in that in step iii), rinse solution 1 and rinse solution 2 are sequentially added to the nucleic acid adsorption material for washing, and the liquid part is removed after each washing Then perform the next wash; preferably, wash the nucleic acid adsorption material with the rinse solution 2 at least twice.
项66.如项65所述的方法，其特征在于，所述漂洗液1包含选自乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和/或十二烷基肌氨酸钠的一种或多种组分；优选地，所述漂洗液1包含乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和十二烷基肌氨酸钠。Item 66. The method according to Item 65, wherein the rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidinium thiocyanate, and/or lauryl sarcosine One or more components of sodium; preferably, the rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidinium thiocyanate, and sodium lauryl sarcosinate.
项67.如项66所述的方法，其特征在于，所述漂洗液1中：Item 67. The method according to Item 66, wherein in the rinse solution 1:
所述乙二胺四乙酸二钠的含量范围是0.4％-0.9％(w/w)；The content range of the disodium edetate is 0.4%-0.9% (w/w);
所述碳酸氢钠的含量范围是0.5％-0.9％(w/w)；The content scope of described sodium bicarbonate is 0.5%-0.9% (w/w);
所述硫氰酸胍的含量范围是50％-55％(w/w)；The content range of the guanidinium thiocyanate is 50%-55% (w/w);
所述十二烷基肌氨酸钠的含量范围是0.8％-1.2％(w/w)；和/或The content range of the sodium lauryl sarcosyl is 0.8%-1.2% (w/w); and/or
所述漂洗液1的pH值范围是6.2-6.6。The pH range of the rinse solution 1 is 6.2-6.6.
项68.如项66-67任一项所述的方法，其特征在于，所述漂洗液1中：Item 68. The method according to any one of Items 66-67, wherein in the rinse solution 1:
所述乙二胺四乙酸二钠的含量是约0.5％(w/w)；The content of disodium edetate is about 0.5% (w/w);
所述碳酸氢钠的含量是约0.6％(w/w)；The content of the sodium bicarbonate is about 0.6% (w/w);
所述硫氰酸胍的含量是约53％(w/w)；The content of the guanidinium thiocyanate is about 53% (w/w);
所述十二烷基肌氨酸钠的含量是约0.9％(w/w)；和/或The content of sodium sarcosyl is about 0.9% (w/w); and/or
所述漂洗液1的pH值是约6.4。The pH of the Rinse 1 was about 6.4.
项69.如项66-68任一项所述的方法，其特征在于，在接触所述核酸吸附材料前，向所述漂洗液1中加入乙醇，所述漂洗液1∶乙醇的体积比为1∶1-1∶2；优选地，所述体积比为约2∶3。Item 69. The method according to any one of Items 66-68, wherein ethanol is added to the rinse solution 1 before contacting the nucleic acid adsorption material, and the volume ratio of the rinse solution 1:ethanol is 1:1-1:2; preferably, the volume ratio is about 2:3.
项70.如项65-69任一项所述的方法，其特征在于，所述漂洗液2包含三羟甲基氨基 甲烷(Tris)或氯化钠；优选地，所述漂洗液2包含三羟甲基氨基甲烷(Tris)和氯化钠。Item 70. The method according to any one of Items 65-69, wherein the rinse solution 2 comprises Tris (Tris) or sodium chloride; preferably, the rinse solution 2 comprises Tris Hydroxymethylaminomethane (Tris) and Sodium Chloride.
项71.如项70所述的方法，其特征在于，所述漂洗液2中：Item 71. The method according to Item 70, wherein in the rinse solution 2:
所述三羟甲基氨基甲烷(Tris)的含量范围是0.5％-0.7％(w/w)；The content range of the tris (Tris) is 0.5%-0.7% (w/w);
所述氯化钠的含量范围是0.6％-1.8％(w/w)；和/或The content range of the sodium chloride is 0.6%-1.8% (w/w); and/or
所述漂洗液2的pH值范围是6.6-7.0。The pH range of the rinse solution 2 is 6.6-7.0.
项72.如项70-71任一项所述的方法，其特征在于，所述漂洗液2中：Item 72. The method according to any one of Items 70-71, wherein in the rinse solution 2:
所述三羟甲基氨基甲烷(Tris)的含量是约0.6％(w/w)；The content of tris (Tris) is about 0.6% (w/w);
所述氯化钠的含量是约1.2％(w/w)；和/或The sodium chloride content is about 1.2% (w/w); and/or
所述漂洗液2的pH值是约6.8。The pH value of the rinse solution 2 is about 6.8.
项73.如项70-72任一项所述的方法，其特征在于，在接触所述核酸吸附材料前，向所述漂洗液2中加入乙醇，所述漂洗液2∶乙醇的体积比为1∶1.6-1∶3；优选地，所述体积比为约1∶2.3。Item 73. The method according to any one of Items 70-72, wherein ethanol is added to the rinse solution 2 before contacting the nucleic acid adsorption material, and the volume ratio of the rinse solution 2:ethanol is 1:1.6-1:3; preferably, the volume ratio is about 1:2.3.
项74.如项57-73任一项所述的方法，其特征在于，所述洗脱液为DEPC水.Item 74. The method according to any one of Item 57-73, wherein the eluent is DEPC water.
项75.如项57-74任一项所述的方法，其特征在于，所述核酸吸附材料为能够吸附RNA的磁珠。Item 75. The method according to any one of Items 57-74, wherein the nucleic acid adsorption material is a magnetic bead capable of adsorbing RNA.
项76.如项75所述的方法，其特征在于，所述磁珠的磁核是氢氧化三铁，包被二氧化硅，且表面镶嵌羟基基团；优选地，所述磁珠的粒径为50-100nm。Item 76. The method as described in Item 75, wherein the magnetic core of the magnetic bead is ferric hydroxide, coated with silicon dioxide, and the surface is embedded with hydroxyl groups; preferably, the magnetic bead The diameter is 50-100nm.
项77.如项52-76任一项所述的方法，其特征在于，所述样本保存液包含选自EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和/或磷酸三钠的一种或多种组分；优选地，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。Item 77. The method according to any one of Items 52-76, wherein the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and/or One or more components of trisodium phosphate; preferably, the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
项78.一种保存含核酸的生物样本的方法，其特征在于，将样本置于样本保存液中，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。Item 78. A method for preserving a nucleic acid-containing biological sample, characterized in that the sample is placed in a sample preservation solution, and the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, and glutathione peptides, and trisodium phosphate.
项79.如项77-78任一项所述的方法，其特征在于，所述样本保存液中：Item 79. The method according to any one of Items 77-78, wherein in the sample preservation solution:
所述EDTA的含量范围是0.7％-1.5％(w/w)；The content range of the EDTA is 0.7%-1.5% (w/w);
所述NaCl的含量范围是0.01％-0.1％(w/w)；The content range of the NaCl is 0.01%-0.1% (w/w);
所述无水乙醇的浓度范围是15％-35％(w/w)；The concentration range of the absolute ethanol is 15%-35% (w/w);
所述柠檬酸钠的含量范围是0.1％-1.0％(w/w)；The content range of the sodium citrate is 0.1%-1.0% (w/w);
所述NP40的浓度范围是0.01％-0.1％(v/v)；The concentration range of the NP40 is 0.01%-0.1% (v/v);
所述谷胱甘肽的含量范围是0.07％-0.7％(w/w)；和/或The glutathione content ranges from 0.07% to 0.7% (w/w); and/or
所述磷酸三钠的含量范围是0.07％-0.4％(w/w)。The content range of the trisodium phosphate is 0.07%-0.4% (w/w).
项80.如项77-79任一项所述的方法，其特征在于，所述样本保存液中：Item 80. The method according to any one of Items 77-79, wherein in the sample preservation solution:
所述EDTA的含量是约1.05％(w/w)；The content of said EDTA is about 1.05% (w/w);
所述NaCl的含量是约0.035％(w/w)；The content of said NaCl is about 0.035% (w/w);
所述无水乙醇的浓度是约27.6％(w/w)；The concentration of absolute ethanol is about 27.6% (w/w);
所述柠檬酸钠的含量是约0.21％(w/w)；The content of the sodium citrate is about 0.21% (w/w);
所述NP40的浓度是约0.035％(v/v)；The concentration of said NP40 is about 0.035% (v/v);
所述谷胱甘肽的含量是约0.21％(w/w)；和/或The glutathione content is about 0.21% (w/w); and/or
所述磷酸三钠的含量是约0.14％(w/w)。The content of the trisodium phosphate is about 0.14% (w/w).
项81.如项77-80一项所述的方法，其特征在于，所述样本保存液包含抗生素；优选地，所述抗生素包含青霉素-链霉素双抗。Item 81. The method according to Item 77-80, wherein the sample preservation solution contains antibiotics; preferably, the antibiotics contain penicillin-streptomycin double antibody.
项82.如项77-81任一项所述的方法，其特征在于，所述样本保存液的pH值为7.6-8.0；优选地，所述样本保存液的pH值为7.8。Item 82. The method according to any one of Items 77-81, wherein the pH value of the sample preservation solution is 7.6-8.0; preferably, the pH value of the sample preservation solution is 7.8.
项83.如项77-82任一项所述的方法，其特征在于，与对照样本保存液相比，所述样本保存液对DNA，miRNA，和/或DNA甲基化的保存效果更好。Item 83. The method according to any one of Items 77-82, characterized in that, compared with the control sample preservation solution, the sample preservation solution has a better preservation effect on DNA, miRNA, and/or DNA methylation .
项84.如项52-83任一项所述的方法，其特征在于，包含步骤(b-3)，所述EB病毒载量检测包含针对EB病毒基因组中BamHI-W片段，和/或LMP-2基因的检测。Item 84. The method according to any one of Items 52-83, characterized in that step (b-3) is included, and the detection of the EB virus load comprises targeting the BamHI-W fragment in the Epstein-Barr virus genome, and/or LMP -2 gene detection.
项85.如项84所述的方法，其特征在于，所述EB病毒载量检测包含针对LMP-2基因的检测。Item 85. The method according to Item 84, wherein the detection of the EB virus load comprises detection of the LMP-2 gene.
项86.如项52-85任一项所述的方法，其特征在于，所述方法用于鼻咽癌的筛查。Item 86. The method according to any one of Items 52-85, wherein the method is used for screening nasopharyngeal carcinoma.
项86.1如项52-86任一项所述的方法，其特征在于，所述方法用于鼻咽癌的诊断。Item 86.1 The method according to any one of Items 52-86, wherein said method is used for the diagnosis of nasopharyngeal carcinoma.
项87.如项52-86.1任一项所述的方法，其特征在于，所述样本是鼻咽部样本。Item 87. The method according to any one of Items 52-86.1, wherein the sample is a nasopharyngeal sample.
项88.如项52-87任一项所述的方法，其特征在于，在步骤(a)之前，还包括使用鼻咽刷和/或鼻咽拭子获取所述样本；优选地，所述鼻咽刷和/或鼻咽拭子为不依赖临床医生和鼻咽镜引导的鼻咽刷和/或鼻咽拭子。Item 88. The method according to any one of Item 52-87, characterized in that, before step (a), it also includes using a nasopharyngeal brush and/or a nasopharyngeal swab to obtain the sample; preferably, the Nasopharyngeal brushes and/or nasopharyngeal swabs are nasopharyngeal brushes and/or nasopharyngeal swabs that do not rely on the guidance of clinicians and nasopharyngoscopes.
项89.一种对有需要的个体治疗鼻咽癌的方法，包含对所述个体提供药物、放疗、手术、细胞疗法和/或溶瘤病毒治疗，其中，所述个体的样本接受了如项52-88任一项所述的方法的检测并被判断为鼻咽癌阳性。Item 89. A method for treating nasopharyngeal carcinoma in an individual in need thereof, comprising providing the individual with drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus therapy, wherein a sample of the individual has received the The method described in any one of 52-88 was detected and judged to be positive for nasopharyngeal carcinoma.
项90.一种对有需要的个体治疗鼻咽癌的方法，包含1)对来自所述个体的样本进行如项52-88任一项所述的方法的检测；2)如检测结果显示鼻咽癌阳性，提供鼻咽癌治疗； 优选地，所述鼻咽癌治疗采用药物、放疗、手术、细胞疗法和/或溶瘤病毒治疗。Item 90. A method for treating nasopharyngeal carcinoma in an individual in need thereof, comprising 1) performing the detection according to the method according to any one of Items 52-88 on a sample from the individual; 2) if the detection result shows that the nasal If the pharyngeal cancer is positive, provide nasopharyngeal cancer treatment; preferably, the nasopharyngeal cancer treatment uses drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus treatment.
项91.如权利要求1-51.1任一项所述的试剂盒在制备鼻咽癌筛查的产品的用途。Item 91. Use of the kit according to any one of claims 1-51.1 in the preparation of products for nasopharyngeal cancer screening.
项92.如权利要求1-51.1任一项所述的试剂盒在制备鼻咽癌诊断的产品的用途。Item 92. Use of the kit according to any one of claims 1-51.1 in the preparation of products for the diagnosis of nasopharyngeal carcinoma.
本发明的一方面，可通过如下各项技术要求项解决现有技术中存在的技术问题：In one aspect of the present invention, the technical problems existing in the prior art can be solved by the following technical requirements:
要求项1.一种用于鼻咽癌检测的试剂盒，其特征在于，所述试剂盒包含：Claim item 1. A test kit for nasopharyngeal carcinoma detection, characterized in that the test kit comprises:
(i)核酸甲基化检测试剂，所述核酸甲基化检测试剂可针对EB病毒基因组中甲基化位点进行甲基化检测，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、和14015bp的位点；(i) nucleic acid methylation detection reagent, described nucleic acid methylation detection reagent can carry out methylation detection at methylation site in Epstein-Barr virus genome, and described methylation site comprises the equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) the 10717bp, 11029bp and 14015bp sites of the Epstein-Barr virus genome DNA sequence;
(ii)miRNA检测试剂，所述miRNA检测试剂可针对多个来自EB病毒的miRNA进行含量检测，所述miRNA包含EBV-miR-BART1-5p和EBV-miR-BART7-3p；和(ii) miRNA detection reagents, the miRNA detection reagents can detect the content of multiple miRNAs from Epstein-Barr virus, and the miRNAs include EBV-miR-BART1-5p and EBV-miR-BART7-3p; and
(iii)EB病毒载量检测试剂，所述EB病毒载量检测试剂包含针对LMP-2基因的检测试剂。(iii) EB virus load detection reagent, said EB virus load detection reagent comprises a detection reagent for LMP-2 gene.
要求项2.如要求项1所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART1-5p、EBV-miR-BART2-5p、EBV-miR-BART6-3p、EBV-miR-BART7-3p、EBV-miR-BART13-3p、和EBV-miR-BART17-5p。 Claim 2. The kit as claimed in claim 1, wherein the miRNA comprises EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR-BART6-3p, EBV-miR- BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p.
要求项3.如要求项1-2任一项所述的试剂盒，其特征在于，所述试剂盒包含所述样本保存液，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠，其中，Claim item 3. The test kit according to any one of claim items 1-2, characterized in that, the test kit comprises the sample preservation solution, and the sample preservation solution comprises EDTA, NaCl, dehydrated alcohol, citric acid Sodium, NP40, Glutathione, and Trisodium Phosphate, of which,
所述EDTA的含量范围是0.7％-1.5％(w/w)；The content range of the EDTA is 0.7%-1.5% (w/w);
所述NaCl的含量范围是0.01％-0.1％(w/w)；The content range of the NaCl is 0.01%-0.1% (w/w);
所述无水乙醇的浓度范围是15％-35％(w/w)；The concentration range of the absolute ethanol is 15%-35% (w/w);
所述柠檬酸钠的含量范围是0.1％-1.0％(w/w)；The content range of the sodium citrate is 0.1%-1.0% (w/w);
所述NP40的浓度范围是0.01％-0.1％(v/v)；The concentration range of the NP40 is 0.01%-0.1% (v/v);
所述谷胱甘肽的含量范围是0.07％-0.7％(w/w)；和The glutathione content ranges from 0.07% to 0.7% (w/w); and
所述磷酸三钠的含量范围是0.07％-0.4％(w/w)，The content range of the trisodium phosphate is 0.07%-0.4% (w/w),
所述样本保存液的pH值为7.6-8.0。The pH value of the sample preservation solution is 7.6-8.0.
要求项4.如要求项1-3任一项所述的试剂盒，其特征在于，所述试剂盒包含miRNA 提取试剂，所述miRNA提取试剂包含裂解液，结合液，漂洗液1，和漂洗液2。Claim item 4. The kit as described in any one of claim items 1-3, characterized in that, the kit includes miRNA extraction reagents, and the miRNA extraction reagents include lysate, binding solution, rinse solution 1, and rinse Liquid 2.
要求项5.如要求项4所述的试剂盒，其特征在于，所述裂解液包含硫氰酸胍，柠檬酸，曲拉通-100，和EDTA，其中：Claim item 5. The test kit as claimed in claim item 4, wherein the lysate comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA, wherein:
所述硫氰酸胍的含量范围是28％-40％(w/w)；The content scope of described guanidinium thiocyanate is 28%-40% (w/w);
所述柠檬酸的含量范围是1.8％-2.6％(w/w)；The content range of the citric acid is 1.8%-2.6% (w/w);
所述曲拉通-100的含量范围是0.8％-1.6％(w/w)；The content range of the Triton-100 is 0.8%-1.6% (w/w);
所述EDTA的含量范围是0.5％-0.9％(w/w)；和The content range of said EDTA is 0.5%-0.9% (w/w); and
所述裂解液的pH值范围为3.8-4.2，The pH range of the lysate is 3.8-4.2,
所述结合液包含氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和异丙醇，其中：The combination solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidine thiocyanate, sodium lauryl sarcosine, and isopropanol, wherein:
所述氯化镁的含量范围是0.1％-0.4％(w/w)；The content range of the magnesium chloride is 0.1%-0.4% (w/w);
所述乙二胺四乙酸二钠的含量范围是0.5％-1.0％(w/w)；The content range of the disodium edetate is 0.5%-1.0% (w/w);
所述碳酸氢钠的含量范围是0.6％-1.0％(w/w)；The content scope of described sodium bicarbonate is 0.6%-1.0% (w/w);
所述硫氰酸胍的含量范围是13％-16％(w/w)；The content range of the guanidine thiocyanate is 13%-16% (w/w);
所述十二烷基肌氨酸钠的含量范围是1.0％-1.4％(w/w)；The content range of the sodium lauryl sarcosinate is 1.0%-1.4% (w/w);
所述异丙醇的含量范围是60％-80％(w/w)；和The content range of the isopropanol is 60%-80% (w/w); and
所述结合液的pH值范围是4.8-5.2，The pH range of the binding solution is 4.8-5.2,
所述漂洗液1包含乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和十二烷基肌氨酸钠，其中，The rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidine thiocyanate, and sodium lauryl sarcosine, wherein,
所述乙二胺四乙酸二钠的含量范围是0.4％-0.9％(w/w)；The content range of the disodium edetate is 0.4%-0.9% (w/w);
所述碳酸氢钠的含量范围是0.5％-0.9％(w/w)；The content scope of described sodium bicarbonate is 0.5%-0.9% (w/w);
所述硫氰酸胍的含量范围是50％-55％(w/w)；The content range of the guanidinium thiocyanate is 50%-55% (w/w);
所述十二烷基肌氨酸钠的含量范围是0.8％-1.2％(w/w)；和The content range of the sodium sarcosyl is 0.8%-1.2% (w/w); and
所述漂洗液1的pH值范围是6.2-6.6，The pH range of the rinse solution 1 is 6.2-6.6,
所述漂洗液2包含三羟甲基氨基甲烷(Tris)和氯化钠，其中：The rinse solution 2 comprises tris (Tris) and sodium chloride, wherein:
所述三羟甲基氨基甲烷(Tris)的含量范围是0.5％-0.7％(w/w)；The content range of the tris (Tris) is 0.5%-0.7% (w/w);
所述氯化钠的含量范围是0.6％-1.8％(w/w)；和The sodium chloride content ranges from 0.6% to 1.8% (w/w); and
所述漂洗液2的pH值范围是6.6-7.0。The pH range of the rinse solution 2 is 6.6-7.0.
要求项6.如要求项1-5任一项所述的试剂盒，其特征在于，所述试剂盒还包含鼻咽刷 或鼻咽拭子，所述鼻咽刷和/或鼻咽拭子为不依赖临床医生和鼻咽镜引导的鼻咽刷和/或鼻咽拭子。Claim 6. The kit according to any one of claims 1-5, wherein the kit also comprises a nasopharyngeal brush or a nasopharyngeal swab, and the nasopharyngeal brush and/or nasopharyngeal swab Nasopharyngeal brushes and/or nasopharyngeal swabs are clinician-independent and nasopharyngoscope-guided.
要求项7.如要求项1-6任一项所述的试剂盒在制备鼻咽癌检测的产品的用途。 Claim 7. Use of the kit as described in any one of claims 1-6 in the preparation of products for nasopharyngeal carcinoma detection.
要求项8.一种检测样本中EB病毒相关核酸的方法，其特征在于，所述方法包含以下步骤：Requirement 8. A method for detecting Epstein-Barr virus-related nucleic acids in a sample, characterized in that the method comprises the following steps:
(a)将所述样本置于样本保存液中；(a) placing the sample in a sample preservation solution;
(b)进行以下的检测：(b) conduct the following tests:
(b-1)对含有所述样本的所述样本保存液进行EB病毒基因组核酸甲基化检测；(b-1) performing Epstein-Barr virus genomic nucleic acid methylation detection on the sample preservation solution containing the sample;
(b-2)从所述样本保存液中提取EB病毒相关miRNA并对所述miRNA的含量进行检测；和(b-2) extracting Epstein-Barr virus-related miRNA from the sample preservation solution and detecting the content of the miRNA; and
(b-3)对含有所述样本的所述样本保存液进行EB病毒核酸载量检测，(b-3) detecting the Epstein-Barr virus nucleic acid load on the sample preservation solution containing the sample,
其中，所述甲基化检测的甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、和14015bp的位点，所述miRNA包含EBV-miR-BART1-5p和EBV-miR-BART7-3p，所述EB病毒载量检测试剂包含针对LMP-2基因的检测。Wherein, the methylation sites detected by the methylation include sites equivalent to 10717bp, 11029bp, and 14015bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94), and the miRNA includes EBV- miR-BART1-5p and EBV-miR-BART7-3p, the EB virus load detection reagent includes the detection of LMP-2 gene.
要求项9.如要求项8所述的方法，其特征在于，所述miRNA包含EBV-miR-BART1-5p、EBV-miR-BART2-5p、EBV-miR-BART6-3p、EBV-miR-BART7-3p、EBV-miR-BART13-3p、和EBV-miR-BART17-5p。Claim 9. The method as claimed in claim 8, wherein the miRNA comprises EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR-BART6-3p, EBV-miR-BART7 -3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p.
要求项10.如要求项8-9任一项所述的方法，其特征在于，提取所述EB病毒相关miRNA的步骤包含：Claim item 10. The method as described in any one of claim items 8-9, wherein the step of extracting the Epstein-Barr virus-related miRNA comprises:
i)向所述样本保存液中加入裂解液；优选地，还加入蛋白酶K；i) adding a lysate to the sample preservation solution; preferably, proteinase K is also added;
ii)加入结合液与核酸吸附材料；ii) adding binding solution and nucleic acid adsorption material;
iii)去除液体部分，向核酸吸附材料加入漂洗液清洗；和iii) removing the liquid portion, adding a rinse solution to the nucleic acid adsorbing material for washing; and
iv)去除液体部分，向所述核酸吸附材料中加入洗脱液，将所述miRNA从所述核酸吸附材料中洗脱。iv) removing the liquid portion, adding an eluent to the nucleic acid adsorption material, and eluting the miRNA from the nucleic acid adsorption material.
要求项11.如要求项10所述的方法，其中：Claim 11. The method of claim 10, wherein:
所述裂解液包含硫氰酸胍，柠檬酸，曲拉通-100，和EDTA，其中：The lysate comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA, wherein:
所述硫氰酸胍的含量范围是28％-40％(w/w)；The content scope of described guanidinium thiocyanate is 28%-40% (w/w);
所述柠檬酸的含量范围是1.8％-2.6％(w/w)；The content range of the citric acid is 1.8%-2.6% (w/w);
所述曲拉通-100的含量范围是0.8％-1.6％(w/w)；The content range of the Triton-100 is 0.8%-1.6% (w/w);
所述EDTA的含量范围是0.5％-0.9％(w/w)；和The content range of said EDTA is 0.5%-0.9% (w/w); and
所述裂解液的pH值范围为3.8-4.2，The pH range of the lysate is 3.8-4.2,
所述结合液包含氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和异丙醇，其中：The combination solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidine thiocyanate, sodium lauryl sarcosine, and isopropanol, wherein:
所述氯化镁的含量范围是0.1％-0.4％(w/w)；The content range of the magnesium chloride is 0.1%-0.4% (w/w);
所述乙二胺四乙酸二钠的含量范围是0.5％-1.0％(w/w)；The content range of the disodium edetate is 0.5%-1.0% (w/w);
所述碳酸氢钠的含量范围是0.6％-1.0％(w/w)；The content scope of described sodium bicarbonate is 0.6%-1.0% (w/w);
所述硫氰酸胍的含量范围是13％-16％(w/w)；The content range of the guanidine thiocyanate is 13%-16% (w/w);
所述十二烷基肌氨酸钠的含量范围是1.0％-1.4％(w/w)；The content range of the sodium lauryl sarcosinate is 1.0%-1.4% (w/w);
所述异丙醇的含量范围是60％-80％(w/w)；和The content range of the isopropanol is 60%-80% (w/w); and
所述结合液的pH值范围是4.8-5.2，The pH range of the binding solution is 4.8-5.2,
所述漂洗液1包含乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和十二烷基肌氨酸钠，其中，The rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidine thiocyanate, and sodium lauryl sarcosine, wherein,
所述乙二胺四乙酸二钠的含量范围是0.4％-0.9％(w/w)；The content range of the disodium edetate is 0.4%-0.9% (w/w);
所述碳酸氢钠的含量范围是0.5％-0.9％(w/w)；The content scope of described sodium bicarbonate is 0.5%-0.9% (w/w);
所述硫氰酸胍的含量范围是50％-55％(w/w)；The content range of the guanidinium thiocyanate is 50%-55% (w/w);
所述十二烷基肌氨酸钠的含量范围是0.8％-1.2％(w/w)；和The content range of the sodium sarcosyl is 0.8%-1.2% (w/w); and
所述漂洗液1的pH值范围是6.2-6.6，The pH range of the rinse solution 1 is 6.2-6.6,
所述漂洗液2包含三羟甲基氨基甲烷(Tris)和氯化钠，其中：The rinse solution 2 comprises tris (Tris) and sodium chloride, wherein:
所述三羟甲基氨基甲烷(Tris)的含量范围是0.5％-0.7％(w/w)；The content range of the tris (Tris) is 0.5%-0.7% (w/w);
所述氯化钠的含量范围是0.6％-1.8％(w/w)；和The sodium chloride content ranges from 0.6% to 1.8% (w/w); and
所述漂洗液2的pH值范围是6.6-7.0。The pH range of the rinse solution 2 is 6.6-7.0.
要求项12.如要求项11所述的方法，其特征在于，在接触所述核酸吸附材料前，向所述漂洗液1中加入乙醇，所述漂洗液1∶乙醇的体积比为1∶1-1∶2；且，在接触所述核酸吸附材料前，向所述漂洗液2中加入乙醇，所述漂洗液2∶乙醇的体积比为1∶1.6-1∶3。Claim 12. The method according to claim 11, wherein ethanol is added to the rinse solution 1 before contacting the nucleic acid adsorption material, and the volume ratio of the rinse solution 1:ethanol is 1:1 - 1:2; and, before contacting the nucleic acid adsorption material, adding ethanol to the rinse liquid 2, the volume ratio of the rinse liquid 2:ethanol is 1:1.6-1:3.
要求项13.如要求项8-12任一项所述的方法，其特征在于，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠，其中：Claim item 13. The method according to any one of claim items 8-12, wherein the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and triphosphate Sodium, of which:
所述EDTA的含量范围是0.7％-1.5％(w/w)；The content range of the EDTA is 0.7%-1.5% (w/w);
所述NaCl的含量范围是0.01％-0.1％(w/w)；The content range of the NaCl is 0.01%-0.1% (w/w);
所述无水乙醇的浓度范围是15％-35％(w/w)；The concentration range of the absolute ethanol is 15%-35% (w/w);
所述柠檬酸钠的含量范围是0.1％-1.0％(w/w)；The content range of the sodium citrate is 0.1%-1.0% (w/w);
所述NP40的浓度范围是0.01％-0.1％(v/v)；The concentration range of the NP40 is 0.01%-0.1% (v/v);
所述谷胱甘肽的含量范围是0.07％-0.7％(w/w)；和The glutathione content ranges from 0.07% to 0.7% (w/w); and
所述磷酸三钠的含量范围是0.07％-0.4％(w/w)，The content range of the trisodium phosphate is 0.07%-0.4% (w/w),
所述样本保存液的pH值为7.6-8.0。The pH value of the sample preservation solution is 7.6-8.0.
要求项14.如要求项8-13任一项所述的方法，其特征在于，所述方法用于鼻咽癌的检测。Claim 14. The method according to any one of claims 8-13, characterized in that the method is used for the detection of nasopharyngeal carcinoma.
要求项15.如要求项8-14任一项所述的方法，其特征在于，所述样本是鼻咽部样本，在步骤(a)之前，还包括使用鼻咽刷和/或鼻咽拭子获取所述样本；优选地，所述鼻咽刷和/或鼻咽拭子为不依赖临床医生和鼻咽镜引导的鼻咽刷和/或鼻咽拭子。Claim item 15. The method according to any one of claim items 8-14, wherein the sample is a nasopharyngeal sample, and before step (a), it also includes using a nasopharyngeal brush and/or a nasopharyngeal swab Preferably, the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab independent of the guidance of a clinician and a nasopharyngoscope.
要求项16.一种对有需要的个体治疗鼻咽癌的方法，包含对所述个体提供药物、放疗、手术、细胞疗法和/或溶瘤病毒治疗，其中，所述个体的样本接受了如要求项8-15任一项所述的方法的检测并被判断为鼻咽癌阳性。Claim 16. A method for treating nasopharyngeal carcinoma in an individual in need thereof, comprising providing the individual with drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus therapy, wherein the sample of the individual has received as The detection by the method described in any one of Claims 8-15 is judged to be positive for nasopharyngeal carcinoma.
要求项17.一种对有需要的个体治疗鼻咽癌的方法，包含1)对来自所述个体的样本进行如要求项8-15任一项所述的方法的检测；2)如检测结果显示鼻咽癌阳性，提供鼻咽癌治疗；优选地，所述鼻咽癌治疗采用药物、放疗、手术、细胞疗法和/或溶瘤病毒治疗。Claim 17. A method for treating nasopharyngeal carcinoma in an individual in need, comprising 1) performing detection according to the method described in any one of Claims 8-15 on a sample from the individual; 2) the detection result If it is positive for nasopharyngeal carcinoma, treatment for nasopharyngeal carcinoma is provided; preferably, the treatment for nasopharyngeal carcinoma adopts drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus treatment.
附图说明Description of drawings
图1为不依赖于临床医生和鼻咽镜引导的鼻咽刷取样本中EB病毒DNA载量和EB病毒DNA甲基化检测，其中NPC为鼻咽癌(nasopharyngeal carcinoma，NPC)，MD为甲基化差异(methylation difference，MD)，EBV DNA载量为EBV DNA load。Figure 1 shows the detection of Epstein-Barr virus DNA load and Epstein-Barr virus DNA methylation in nasopharyngeal swab samples independent of clinicians and guided by nasopharyngoscope, where NPC is nasopharyngeal carcinoma (NPC) and MD is A Methylation difference (methylation difference, MD), EBV DNA load is EBV DNA load.
图2为依赖于临床医生和鼻咽镜引导的鼻咽刷取样本中EB病毒DNA载量和EB病毒DNA甲基化检测。Figure 2 is the detection of Epstein-Barr virus DNA load and Epstein-Barr virus DNA methylation in nasopharyngeal swab samples guided by clinicians and nasopharyngoscopes.
图3为同一例病人在依赖和不依赖临床医生和鼻咽镜引导的鼻咽刷取样本中EB病毒 DNA载量和EB病毒DNA甲基化检测的平行比较。其中，brush为依赖于临床医生和鼻咽镜引导的鼻咽刷取样本，blind brush为不依赖于临床医生和鼻咽镜引导的鼻咽刷取样本。Figure 3 is a side-by-side comparison of EBV DNA load and EBV DNA methylation detection in the same patient in dependent and independent clinician-guided and nasopharyngoscope-guided nasopharyngeal brush samples. Among them, brush refers to nasopharyngeal brushing samples that rely on clinicians and nasopharyngoscope guidance, and blind brush refers to nasopharyngeal brushing samples that do not rely on clinicians and nasopharyngoscope guidance.
图4为自配样本保存液与市售DNA保存液和市售RNA保存液对于DNA保存效果的平行比较。Figure 4 is a parallel comparison of the DNA preservation effect of the self-prepared sample preservation solution, the commercial DNA preservation solution and the commercial RNA preservation solution.
图5为自配样本保存液与市售DNA保存液和市售RNA保存液对于所示DNA甲基化保存效果的平行比较。Figure 5 is a parallel comparison of the DNA methylation preservation effects of the self-prepared sample preservation solution, the commercial DNA preservation solution and the commercial RNA preservation solution.
图6为自配样本保存液与市售DNA保存液和市售RNA保存液对于所示miRNA保存效果的平行比较。Figure 6 is a parallel comparison of self-made sample preservation solution, commercially available DNA preservation solution and commercial RNA preservation solution for the miRNA preservation effects.
图7为自配RNA提取试剂和市售RNA提取试剂对于所示miRNA的提取效果的平行比较。Figure 7 is a parallel comparison of the extraction effects of self-prepared RNA extraction reagents and commercially available RNA extraction reagents on the miRNAs shown.
图8为EB病毒DNA相关的检测结果。Figure 8 shows the detection results related to Epstein-Barr virus DNA.
图9为EB病毒DNA甲基化相关的检测结构。Figure 9 is the detection structure related to DNA methylation of Epstein-Barr virus.
图10为miRNA相关的检测结果。Figure 10 shows the detection results related to miRNA.
具体实施方式Detailed ways
下面结合说明书附图及具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are only used to explain the present invention, and are not intended to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.
实施例1 一种不依赖于临床医生和鼻咽镜引导的鼻咽刷取样本的采样方法Example 1 A sampling method for nasopharyngeal swabbing that does not rely on clinicians and nasopharyngoscope guidance
使用鼻咽刷或鼻咽拭子，直接由临床护士操作，鼻咽刷或鼻咽拭子直接通过鼻腔进入到鼻咽部转动，刷取样样本后取回。Use a nasopharyngeal brush or nasopharyngeal swab, which is directly operated by a clinical nurse. The nasopharyngeal brush or nasopharyngeal swab directly enters the nasopharynx through the nasal cavity and rotates, brushes the sample and takes it back.
取样完成后，用高压灭菌过的剪刀剪断鼻咽刷或鼻咽拭子的头部，放入装有500μL无菌PBS缓冲液的1.5mL离心管中，盖紧盖子并上下颠倒混匀，存入负八十度超低温冰箱保存，用于后续实验。After sampling, use high-pressure sterilized scissors to cut off the head of the nasopharyngeal brush or nasopharyngeal swab, put it into a 1.5mL centrifuge tube filled with 500μL sterile PBS buffer, close the lid tightly and mix it upside down. Store in a negative 80-degree ultra-low temperature refrigerator for subsequent experiments.
实施例2 一种依赖于临床医生和鼻咽镜引导的鼻咽刷取样本的采样方法Example 2 A sampling method that relies on clinicians and nasopharyngoscope-guided nasopharyngeal brushing samples
(1)使用1％的地卡因和0.5％的盐酸麻黄素浸湿无菌棉签，左右两侧各一只插入受检者的鼻腔，持续约3三分钟，已达到对受检者鼻咽粘膜进行表面麻醉及收缩鼻甲的作用。(1) Use 1% tetracaine and 0.5% ephedrine hydrochloride to soak sterile cotton swabs, insert one on each side into the nasal cavity of the subject, and last for about 3 to 3 minutes, until the nasopharynx of the subject is reached. The mucous membrane is topically anesthetized and the turbinates are contracted.
(2)在电子鼻咽镜的引导下，由临床医师或者住院医师操作，鼻咽刷随着鼻咽镜通过中鼻道进入到鼻咽部，在鼻咽部的可疑部位滚动取样，然后快速取回。(2) Under the guidance of the electronic nasopharyngoscope, operated by a clinician or resident doctor, the nasopharyngeal brush enters the nasopharynx through the middle meatus with the nasopharyngoscope, rolls samples at suspicious parts of the nasopharynx, and then quickly retrieve.
(3)取样完成后，用高压灭菌过的剪刀剪断鼻咽刷的刷子头，放入装有500μL无菌PBS缓冲液的1.5mL离心管中，盖紧盖子并上下颠倒混匀，存入负八十度超低温冰箱保存，用于后续实验。(3) After sampling, use high-pressure sterilized scissors to cut off the brush head of the nasopharyngeal brush, put it into a 1.5mL centrifuge tube containing 500 μL of sterile PBS buffer, close the lid tightly and mix it upside down, and store in Store in a negative 80-degree ultra-low temperature refrigerator for subsequent experiments.
实施例3 不依赖于临床医生和鼻咽镜引导的鼻咽刷取样本中EB病毒DNA的甲基化与EB病毒DNA载量的检测Example 3 Detection of Methylation of Epstein-Barr Virus DNA and Epstein-Barr Virus DNA Load in Nasopharyngeal Brushing Samples Not Dependent on Clinicians and Nasopharyngoscope Guidance
一、实验方法1. Experimental method
1、样本来源1. Sample source
共招募162例受试者，这其中包括78例的鼻咽癌患者和84例的未患有鼻咽癌的相对健康人员：78例鼻咽癌患者皆为在中山大学肿瘤防治中心鼻咽门诊就诊，经过病理活检确诊为鼻咽癌的病人；84例未患有鼻咽癌的相对健康人员中，其中25例人员是来中山大学肿瘤防治中心鼻咽门诊就诊，经过病理活检确诊为鼻咽慢性炎症的患者，另外59例是来自广东省四会市社区体检人员。A total of 162 subjects were recruited, including 78 patients with nasopharyngeal carcinoma and 84 relatively healthy people without nasopharyngeal carcinoma: all 78 patients with nasopharyngeal carcinoma were enrolled in the nasopharyngeal clinic of Sun Yat-sen University Cancer Center. Patients who were diagnosed with nasopharyngeal carcinoma by pathological biopsy; among the 84 relatively healthy people who did not suffer from nasopharyngeal carcinoma, 25 of them came to the nasopharyngeal clinic of Sun Yat-sen University Cancer Prevention and Control Center, and were diagnosed with nasopharyngeal carcinoma by pathological biopsy Patients with chronic inflammation, another 59 cases were from community health examiners in Sihui City, Guangdong Province.
2、鼻咽刷取样本的采集2. Collection of nasopharyngeal swab samples
同实施例1。With embodiment 1.
3、EB病毒DNA载量检测3. Epstein-Barr virus DNA load detection
(1)样本DNA的提取(1) Extraction of sample DNA
DNA的提取经过前处理后，使用自动化的核酸提取仪提取，过程如下：After the DNA extraction is pre-treated, it is extracted using an automated nucleic acid extractor. The process is as follows:
(a)鼻咽刷样本首先用高压灭菌后的镊子夹住刷子头在PBS溶液中来回涮洗五次，随后再用镊子对刷子进行刮取操作，把刷子上可能残留的脱落细胞刮下来。(a) The nasopharyngeal brush sample is first clamped with tweezers after autoclaving and rinsed back and forth in PBS solution for five times, and then scraped the brush with tweezers to scrape off the exfoliated cells that may remain on the brush .
(b)前处理之后的样本溶液，载入自动化提取工作站上机抽提总DNA，使用Hamilton自动化工作站(Chemagic Star，Hamilton Robotic)，按照标准的磁珠法抽提流程进行核酸提取。(b) The sample solution after pretreatment was loaded into the automated extraction workstation to extract the total DNA, and the nucleic acid was extracted according to the standard magnetic bead extraction process using the Hamilton automated workstation (Chemagic Star, Hamilton Robotic).
①、将前处理之后的样本溶液转移到试剂盒配套的96孔深孔板1中，加入350μl裂解缓冲液1在常温下裂解样本20分钟。①. Transfer the sample solution after pretreatment to the 96-well deep-well plate 1 provided with the kit, add 350 μl lysis buffer 1 to lyse the sample at room temperature for 20 minutes.
②、裂解之后每孔样本中加入50μl磁珠和950μl的结合缓冲液2，使裂解释放之后的核酸充分结合到磁珠上。②. After lysis, add 50 μl of magnetic beads and 950 μl of binding buffer 2 to each sample well to fully bind the released nucleic acid to the magnetic beads.
③、将样本混合液放在设备专用的磁力架上，磁棒从溶液上方采用顶部法使磁珠吸附到磁棒套上，先后用各800μl的洗脱缓冲液3、4、5对磁珠进行洗脱，随后用1.6ml的洗脱缓冲液6进行最后洗脱。③. Put the sample mixture on the special magnetic stand for the equipment, and use the top method from above the solution to make the magnetic beads adsorb to the magnetic rod cover. Use 3, 4, and 5 pairs of magnetic beads with 800 μl of elution buffer successively. Elution was performed followed by a final elution with 1.6 ml of elution buffer 6.
④、加入150μl溶解缓冲液7溶解磁珠上吸附的DNA，然后转移到500μL的eppendorf 管中保存。④. Add 150 μl of lysis buffer 7 to dissolve the DNA adsorbed on the magnetic beads, and then transfer to a 500 μL eppendorf tube for storage.
DNA的浓度均使用Nanodrop 1000吸光法进行定量(NanoDrop Technologies，Waltham，MA，USA)。DNA溶液于-20℃保存，RNA溶液于-80度保存备后续使用。The concentration of DNA was quantified using the Nanodrop 1000 absorbance method (NanoDrop Technologies, Waltham, MA, USA). The DNA solution was stored at -20°C, and the RNA solution was stored at -80°C for subsequent use.
(2)、EB病毒DNA载量的检测(2), detection of Epstein-Barr virus DNA load
采用荧光定量PCR的方法，对鼻咽刷样本中的EB病毒DNA载量进行定量检测，检测引物和探针如表1所示。The method of fluorescent quantitative PCR was used to quantitatively detect the Epstein-Barr virus DNA load in nasopharyngeal brush samples, and the detection primers and probes are shown in Table 1.
表1：Table 1:
Figure PCTCN2022127393-appb-000001
Figure PCTCN2022127393-appb-000001
在这个反应体系中，对EBV DNA的定量所扩增的目的基因为EB病毒的BamHI-W片段，由76个核苷酸组成，正向和反向引物序列分别是：5’-CCCAACACTCCACCACACC-3’(SEQ ID NO：1)，和5’-TCTTAGGAGCTGTCCGAGGG-3’(SEQ ID NO：2)。In this reaction system, the target gene amplified for the quantification of EBV DNA is the BamHI-W fragment of EB virus, which consists of 76 nucleotides. The forward and reverse primer sequences are: 5'-CCCAACACTCCACCACACC-3 ' (SEQ ID NO: 1), and 5'-TCTTAGGAGCTGTCCGAGGG-3' (SEQ ID NO: 2).
此外，体系中还有双标的杂交探针，探针两端分别携带FAM荧光集团和TAMRA荧光淬灭集团，探针序列是：5’-FAM-CACACACTACACACACCCACCCGTCTC-TAMRA-3’(SEQ ID NO：3)。为了加强取样过程的质量控制，荧光定量PCR体系中还包括针对看家基因β-globin基因的引物和探针，两条引物序列分别是5’-GTGCACCTGACTCCTGAGGAGA-3’(SEQ ID NO：4)；5’-CCTTGATACCAACCTGCCCAG-3’(SEQ ID NO：5)，扩增片段为102bp。针对该基因的探针序列 为5’-FAM-AAGGTGAACGTGGATGAAGTTGGTGG-TAMRA-3’(SEQ ID NO：6)，同样探针两端分别带有荧光集团和淬灭集团，引物和探针的序列由life technology公司合成。In addition, there are double-labeled hybridization probes in the system. The two ends of the probe carry FAM fluorescent group and TAMRA fluorescent quenching group respectively. The probe sequence is: 5'-FAM-CACACACTACACACACCCACCCGTCTC-TAMRA-3' (SEQ ID NO: 3 ). In order to strengthen the quality control of the sampling process, the fluorescent quantitative PCR system also includes primers and probes for the housekeeping gene β-globin gene. The sequences of the two primers are 5'-GTGCACCTGACTCCTGAGGAGA-3' (SEQ ID NO: 4); 5'-CCTTGATACCAACCTGCCCAG-3' (SEQ ID NO: 5), the amplified fragment is 102bp. The sequence of the probe for this gene is 5'-FAM-AAGGTGAACGTGGATGAAGTTGGTGG-TAMRA-3' (SEQ ID NO: 6). The two ends of the same probe have fluorescent groups and quenching groups respectively. The sequences of primers and probes are provided by life technology company synthesis.
每一个基因Q-PCR反应体系体积为8μL，包括4μL的2×mix溶液，1μL的引物(包括正向和反向引物)，0.2μL的探针，0.8μL的水以及2μL的DNA模板。The volume of each gene Q-PCR reaction system is 8 μL, including 4 μL of 2×mix solution, 1 μL of primers (including forward and reverse primers), 0.2 μL of probe, 0.8 μL of water and 2 μL of DNA template.
Q-PCR反应条件为变性95度，5分钟；45个反应循环，每一个循环条件为95度、15秒，60度、30秒，72度、15秒。The Q-PCR reaction conditions are denaturation at 95°C for 5 minutes; 45 reaction cycles, each cycle condition is 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 15 seconds.
通过质粒构建获得这两个基因的标准品，应用10 3，10 4，10 5，10 6and 10 7copies/μL来获得每个基因的标准曲线。待测样本PCR反应完成后，根据标准曲线，计算出EBVDNA和β-globin两个基因的表达水平，其单位为copy/ng DNA。 Standards for these two genes were obtained by plasmid construction, and 10 3 , 10 4 , 10 5 , 10 6 and 10 7 copies/μL were used to obtain a standard curve for each gene. After the PCR reaction of the sample to be tested is completed, the expression levels of the EBVDNA and β-globin genes are calculated according to the standard curve, and the unit is copy/ng DNA.
4、EB病毒DNA的甲基化检测4. Methylation detection of Epstein-Barr virus DNA
(1)DNA的重亚硫酸检测(1) DNA bisulfite detection
根据试剂盒说明书(ZYMO RESEARCH，CA，USA)，500ng样本的基因组DNA首先经过重亚硫酸盐处理和纯化，转化修饰的DNA作为q-PCR检测的模板。According to the kit instructions (ZYMO RESEARCH, CA, USA), the genomic DNA of 500ng samples was first treated with bisulfite and purified, and the modified DNA was converted as a template for q-PCR detection.
(2)EBV DNA甲基化检测，针对EBV基因组DNA上11029bp、45850bp、57946bp、66227bp和128103bp五个甲基化位点(EB病毒基因组为GenBank中的LOCUS为NC_007605)，这些位点分别位于EBV基因组上的C启动子区(11029bp)，潜伏感染期基因(57946bp)，早期裂解感染期基因(45850bp)，晚期裂解感染期基因(66227bp和128103bp)。针对每个位点，各设计对1对引物和1对荧光探针，其中一条荧光探针是针对该位点发生甲基化，5’和3’端分别标记FAM和BHQ1，命名为探针1(Probe1)，一条荧光探针是针对该位点未发生甲基化，5’和3’端分别标记HEX和BHQ1，命名为探针2(Probe2)，引物和探针的序列见表2。针对五个甲基化位点的引物和探针序列见表2。(2) EBV DNA methylation detection, aiming at five methylation sites of 11029bp, 45850bp, 57946bp, 66227bp and 128103bp on the EBV genomic DNA (the EBV genome is GenBank LOCUS is NC_007605), these sites are located in EBV C promoter region (11029bp), latent infection phase gene (57946bp), early lytic infection phase gene (45850bp), late lytic infection phase gene (66227bp and 128103bp) on the genome. For each site, design a pair of primers and a pair of fluorescent probes. One of the fluorescent probes is methylated for this site, and the 5' and 3' ends are labeled with FAM and BHQ1 respectively, named as probes 1 (Probe1), a fluorescent probe is not methylated at this site, and the 5' and 3' ends are labeled with HEX and BHQ1 respectively, named as probe 2 (Probe2). The sequences of primers and probes are shown in Table 2 . The primer and probe sequences for the five methylation sites are listed in Table 2.
表2：Table 2:
Figure PCTCN2022127393-appb-000002
Figure PCTCN2022127393-appb-000002
Figure PCTCN2022127393-appb-000003
Figure PCTCN2022127393-appb-000003
(3)Q-PCR的反应体系(3) Q-PCR reaction system
20μl反应体系，其中包含10μL mix，引物1.5μL，探针1和探针2各0.8μL，水4.9μL，DNA 2μL。反应条件为95℃、5min，随后是45个循环，每个循环设置为95℃、15s，58℃、60s，40℃、30s。20 μl reaction system, including 10 μL mix, 1.5 μL primer, 0.8 μL each of probe 1 and probe 2, 4.9 μL water, 2 μL DNA. The reaction conditions were 95 °C for 5 min, followed by 45 cycles with each cycle set to 95 °C for 15 s, 58 °C for 60 s, and 40 °C for 30 s.
(4)计算甲基化差值(4) Calculate methylation difference
q-PCR完成后，计算各个位点的甲基化差值(Methylation Difference，MD)，MD值的计算公式为：After q-PCR is completed, calculate the methylation difference (Methylation Difference, MD) of each site, and the formula for calculating the MD value is:
MD＝CT m-CT u， MD= CTm - CTu ,
CT m为该位点的扩增引物和甲基化探针扩增之后的Ct值，CT u为该位点的扩增引物和非甲基化探针扩增之后的Ct值。 CT m is the Ct value after amplification with the amplification primers and methylated probes at this site, and CT u is the Ct value after amplification with the amplification primers and unmethylated probes at this site.
并对各位点MD值和EB病毒DNA载量诊断鼻咽癌的cut off value(COV)、AUC、灵敏度、特异性、约登指数进行分析。The cut off value (COV), AUC, sensitivity, specificity, and Youden index of each site MD value and Epstein-Barr virus DNA load in the diagnosis of nasopharyngeal carcinoma were analyzed.
其中，约登指数(Youden index)：也称正确指数，是评价筛查试验真实性的方法，假设其假阴性(漏诊率)和假阳性(误诊率)的危害性同等意义时，即可应用约登指数。表示筛检方法发现真正的患者与非患者的总能力。指数越大说明筛查实验的效果越好，准确性越大。Among them, Youden index: also known as the correct index, is a method to evaluate the authenticity of screening tests, assuming that its false negative (missed diagnosis rate) and false positive (misdiagnosed rate) are equally harmful, it can be applied Youden index. Indicates the total ability of the screening method to detect true patients and non-patients. The larger the index, the better the effect of the screening experiment and the greater the accuracy.
约登指数＝灵敏度+特异度-1。Youden Index = Sensitivity + Specificity - 1.
二、实验结果2. Experimental results
结果如图1所示，表明无论是EB病毒DNA载量，还是各甲基化位点MD值在鼻咽刷检中均有显著查异。EB病毒DNA载量在鼻咽癌组显著高于非鼻咽癌的对照组；各甲基化位点MD值在鼻咽癌组均显著低于非鼻咽癌的对照组(鼻咽癌中位点发生显著的甲基化，CTm小于CTu)。The results are shown in Figure 1, indicating that both the Epstein-Barr virus DNA load and the MD value of each methylation site were significantly different in nasopharyngeal swabbing. The EB virus DNA load in the nasopharyngeal carcinoma group was significantly higher than that in the non-nasopharyngeal carcinoma control group; the MD value of each methylation site in the nasopharyngeal carcinoma group was significantly lower than that in the non-nasopharyngeal carcinoma control group (in nasopharyngeal carcinoma Significant methylation occurs at the site, CTm is smaller than CTu).
进一步对各个甲基化位点MD值和EB病毒DNA载量作为鼻咽癌诊断标志物的灵敏度和特异性进行分析。结果如表3所示，其中，位于EB病毒基因组DNA的11029bp的甲基化位点的MD值应用于鼻咽癌诊断的敏感度为85.90％，特异度为97.60％，约登指数为83.50％；位于EB病毒基因组DNA的57946bp的甲基化位点的MD值应用于鼻咽癌诊断的敏感度为92.30％，特异度为78.60％，约登指数为70.90％；位于EB病毒基因组DNA的128103bp的甲基化位点的MD值应用于鼻咽癌诊断的敏感度为81.60％，特异度为79.80％，约登指数为61.40％。对于鼻咽癌诊断的准确性均明显优于EB病毒DNA载量(其敏感度为75.60％，特异度为85.70％，约登指数为61.30％)，解决了不依赖于临床医生和鼻咽镜引导的鼻咽部刷取样本中，取样精度降低造成的EB病毒载量减少，导致对于鼻咽癌诊断的敏感度和特异度不高的问题。The sensitivity and specificity of each methylation site MD value and Epstein-Barr virus DNA load as diagnostic markers for nasopharyngeal carcinoma were further analyzed. The results are shown in Table 3, wherein the sensitivity of the MD value of the methylation site located at 11029bp of the Epstein-Barr virus genomic DNA in the diagnosis of nasopharyngeal carcinoma is 85.90%, the specificity is 97.60%, and the Youden index is 83.50%. The MD value of the methylation site located at 57946bp of Epstein-Barr virus genomic DNA has a sensitivity of 92.30%, a specificity of 78.60%, and a Youden index of 70.90% in the diagnosis of nasopharyngeal carcinoma; it is located at 128103bp of Epstein-Barr virus genomic DNA The sensitivity of the MD value of the methylation site in the diagnosis of nasopharyngeal carcinoma was 81.60%, the specificity was 79.80%, and the Youden index was 61.40%. The accuracy of the diagnosis of nasopharyngeal carcinoma is significantly better than that of Epstein-Barr virus DNA load (the sensitivity is 75.60%, the specificity is 85.70%, and the Youden index is 61.30%). In guided nasopharyngeal swabbing samples, the decrease in sampling precision resulted in a decrease in the Epstein-Barr virus load, resulting in low sensitivity and specificity for the diagnosis of nasopharyngeal carcinoma.
表3：table 3:
Figure PCTCN2022127393-appb-000004
Figure PCTCN2022127393-appb-000004
Figure PCTCN2022127393-appb-000005
Figure PCTCN2022127393-appb-000005
NPC：鼻咽癌(nasopharyngeal carcinoma，NPC)；Control：对照；EB病毒DNA载量大于Cut off value判定为鼻咽癌，EB病毒DNA甲基化位点MD值小于Cut off value判定为鼻咽癌；AUC(Area Under Curve)：ROC曲线下与坐标轴围成的面积。NPC: nasopharyngeal carcinoma (NPC); Control: control; EB virus DNA load greater than the Cut off value is judged as nasopharyngeal carcinoma, EB virus DNA methylation site MD value less than the Cut off value is judged as nasopharyngeal carcinoma ;AUC(Area Under Curve): The area under the ROC curve and the coordinate axis.
进一步，利用各个甲基化位点构建了鼻咽癌诊断模型，并对各模型诊断鼻咽癌的灵敏度和特异性进行分析，结果如表4所示。Furthermore, each methylation site was used to construct a nasopharyngeal carcinoma diagnostic model, and the sensitivity and specificity of each model in the diagnosis of nasopharyngeal carcinoma were analyzed. The results are shown in Table 4.
表4：Table 4:
Figure PCTCN2022127393-appb-000006
Figure PCTCN2022127393-appb-000006
bamHI为EB病毒DNA载量，其单位为copy/ng DNA；在各模型中判定标准为模型Pd score大于cut off value判定为鼻咽癌；AUC(Area Under Curve)：ROC曲线下与坐标轴围成的面积。bamHI is the Epstein-Barr virus DNA load, and its unit is copy/ng DNA; in each model, the judgment standard is that the model Pd score is greater than the cut off value, which is judged as nasopharyngeal carcinoma; AUC (Area Under Curve): the area under the ROC curve and the coordinate axis circumference into the area.
其中，模型1到6是只利用EB病毒DNA甲基化计算的MD值构建的鼻咽癌诊断模型，均优于单独使用EB病毒DNA载量作为鼻咽癌诊断标志物，其中模型2的准确性最好。Among them, models 1 to 6 are nasopharyngeal carcinoma diagnostic models constructed only using the MD values calculated by Epstein-Barr virus DNA methylation. Sex is the best.
同时，联合EB病毒DNA载量和EB病毒DNA甲基化位点的MD值构建了模型7到13，除模型12外诊断的准确性也都优于单独使用EB病毒DNA载量作为鼻咽癌诊断标志物。其中模型9的准确性最优。At the same time, models 7 to 13 were constructed by combining the EBV DNA load and the MD value of the EBV DNA methylation site. Except for model 12, the diagnostic accuracy is also better than using the EBV DNA load alone as a nasopharyngeal carcinoma. diagnostic markers. Among them, model 9 has the best accuracy.
实施例4 不依赖于临床医生和鼻咽镜引导的鼻咽刷取样本中EB病毒DNA的甲基化与EB病毒DNA载量检测对于鼻咽癌的预测Example 4 Prediction of Epstein-Barr Virus DNA Methylation and Epstein-Barr Virus DNA Load Detection in Nasopharyngeal Swipe Samples Independent of Clinicians and Nasopharyngoscope Guidance for Nasopharyngeal Carcinoma
一、实验方法1. Experimental method
1、样本来源1. Sample source
在广东省四会市招募社区体检人员中，有8例招募对象不同于其它84例未患有鼻咽癌的相对健康人员，这8个人员在后续随访过程中，于不同时间先后确诊为鼻咽癌。Among the recruited community physical examination personnel in Sihui City, Guangdong Province, 8 recruited subjects were different from the other 84 relatively healthy persons without nasopharyngeal carcinoma. These 8 persons were diagnosed with nasopharyngeal carcinoma at different times during follow-up. Pharyngeal cancer.
2、鼻咽刷样本的采集2. Collection of nasopharyngeal brush samples
所有受试者都按照实施例1的方式采集鼻咽部样本。All subjects collected nasopharyngeal samples according to the method of Example 1.
3、EB病毒DNA载量检测3. Epstein-Barr virus DNA load detection
同实施例3。With embodiment 3.
4、EB病毒DNA甲基化检测4. Epstein-Barr virus DNA methylation detection
同实施例3。With embodiment 3.
二、实验结果2. Experimental results
结果如表5所示，根据实施例3中构建的模型2和模型9的公式，计算这8例样本在模型2和模型9中的Pd score。虽然这8例人员在采样时并没有发现患有鼻咽癌，但在随后的数个月的随访中，这些人员相继确诊鼻咽癌。根据模型2的Pd score评分，2到5号共4人被判定为鼻咽癌；根据模型9的Pd score评分，1到5号共5人被判定为鼻咽癌。同时发现1到6号这6人中，五个位点中至少有一个位点MD值超过检测线，被判定为鼻咽癌，数据提示检测EB病毒DNA甲基化对于鼻咽癌的发生具有良好的预测能力。The results are shown in Table 5. According to the formulas of Model 2 and Model 9 constructed in Example 3, the Pd score of these 8 samples in Model 2 and Model 9 were calculated. Although these 8 patients were not found to have nasopharyngeal carcinoma at the time of sampling, they were successively diagnosed with nasopharyngeal carcinoma during follow-up visits in the following months. According to the Pd score of model 2, a total of 4 people from numbers 2 to 5 were judged as nasopharyngeal carcinoma; according to the Pd score of model 9, a total of 5 people from numbers 1 to 5 were judged to be nasopharyngeal carcinoma. At the same time, it was found that among the six people from No. 1 to No. 6, at least one of the five sites had an MD value exceeding the detection line and was judged to be nasopharyngeal carcinoma. The data suggested that the detection of Epstein-Barr virus DNA methylation had a positive effect on the occurrence of nasopharyngeal carcinoma. Good predictive power.
表5：table 5:
Figure PCTCN2022127393-appb-000007
Figure PCTCN2022127393-appb-000007
Figure PCTCN2022127393-appb-000008
Figure PCTCN2022127393-appb-000008
EB病毒DNA载量，大于Cut off value＝60.52判定为鼻咽癌；EB病毒DNA甲基化MD，11029，45850，57946 66227和128103的cut off value分别为-0.39，-0.04，-1.07，-2.91和-2.19，小于Cut off value判定为鼻咽癌；Epstein-Barr virus DNA load, greater than Cut off value = 60.52 is judged as nasopharyngeal carcinoma; Epstein-Barr virus DNA methylation MD, 11029, 45850, 57946, 66227 and 128103 cut off values were -0.39, -0.04, -1.07, - 2.91 and -2.19, less than the Cut off value is judged as nasopharyngeal carcinoma;
模型2 logit(Pd score)＝＝-1.94-1.04×11029MD-0.20×57946MD-0.23×66227MD，cut offvalue＝0.46，大于它判定为鼻咽癌； Model 2 logit(Pd score)＝＝-1.94-1.04×11029MD-0.20×57946MD-0.23×66227MD, cut offvalue＝0.46, greater than it is judged as nasopharyngeal carcinoma;
模型9 logit(Pd Score)＝-1.47+0.0018×bamHI-1.18×11029MD，cut off value＝0.22，大于它判定为鼻咽癌；Model 9 logit(Pd Score)＝-1.47+0.0018×bamHI-1.18×11029MD, cut off value＝0.22, greater than it is judged as nasopharyngeal carcinoma;
加粗为判读为鼻咽癌阳性。In bold, it was interpreted as positive for nasopharyngeal carcinoma.
实施例5 依赖于临床医生和鼻咽镜引导的鼻咽刷取样本中EB病毒DNA的甲基化与EB病毒DNA载量的检测Example 5 Relying on the detection of methylation of Epstein-Barr virus DNA and the load of Epstein-Barr virus DNA in nasopharyngeal swab samples guided by clinicians and nasopharyngoscope
一、实验方法1. Experimental method
1、样本来源1. Sample source
共招募170名受试者，其中98例经过病理活检确诊为鼻咽癌的患者，72例为未患有鼻咽癌的相对健康人(其中，24例经过病理活检确诊为鼻咽黏膜炎，48例在广东省四会市社区体检招募)A total of 170 subjects were recruited, including 98 patients with nasopharyngeal carcinoma diagnosed by pathological biopsy, and 72 relatively healthy people without nasopharyngeal carcinoma (among them, 24 cases were diagnosed with nasopharyngeal mucositis by pathological biopsy, 48 cases were recruited for physical examination in the community of Sihui City, Guangdong Province)
2、鼻咽刷样本的采集2. Collection of nasopharyngeal brush samples
所有受试者都按照实施例2的方式采集鼻咽部样本，其中的38例鼻咽癌患者还按照实施例1的方式采集了鼻咽部样本。All subjects collected nasopharyngeal samples according to the method of Example 2, and 38 nasopharyngeal cancer patients also collected nasopharyngeal samples according to the method of Example 1.
5、EB病毒DNA载量检测5. Epstein-Barr virus DNA load detection
同实施例3。With embodiment 3.
6、EB病毒DNA甲基化检测6. Epstein-Barr virus DNA methylation detection
同实施例3。With embodiment 3.
二、实验结果2. Experimental results
结果如图2和表6所示，对于依赖于临床医生和鼻咽镜引导的鼻咽刷取样本，EB病毒DNA载量和EB病毒DNA 11029bp处的MD值应用于鼻咽癌诊断的灵敏度和特异度相当，其余甲基化位点的MD值(即45850bp、57946bp、66227bp、和128103bp)诊断准 确度与之相似。The results are shown in Figure 2 and Table 6. For nasopharyngeal swab samples that rely on clinicians and nasopharyngoscope guidance, the Epstein-Barr virus DNA load and the MD value at 11029bp of Epstein-Barr virus DNA were applied to the sensitivity and sensitivity of nasopharyngeal carcinoma diagnosis. The specificity was comparable, and the MD values of the remaining methylated sites (ie, 45850bp, 57946bp, 66227bp, and 128103bp) had similar diagnostic accuracy.
表6：Table 6:
Figure PCTCN2022127393-appb-000009
Figure PCTCN2022127393-appb-000009
比较同一例病人依赖和不依赖临床医生和鼻咽镜引导的鼻咽刷采样样本，结果如图3所示，由于缺少内镜的引导，取样精度的降低，不依赖临床医生和鼻咽镜引导的鼻咽部样本EB病毒DNA载量显著下降，即中位值275，范围1到8744，而依赖临床医生和鼻咽镜引导的鼻咽刷样本中EB病毒DNA载量中位值为5144，范围94到40095。与EB病毒DNA载量不同的是，EB病毒DNA 11029位点MD值在不依赖临床医生和鼻咽镜引导的鼻咽刷采样样本中的中位值为-2.79，范围-4.53到-1.13，而在依赖临床医生和鼻咽镜引导的鼻咽刷采样样本中的中位值为-2.77，范围为-4.77到0，两组间没有显著变化。Comparing the nasopharyngeal brush sampling samples of the same patient relying on and not relying on the guidance of clinicians and nasopharyngoscopes, the results are shown in Figure 3. Due to the lack of guidance of endoscopes, the sampling accuracy is reduced, and the results are not dependent on clinicians and nasopharyngoscopes. The Epstein-Barr virus DNA load in nasopharyngeal samples decreased significantly, with a median value of 275, ranging from 1 to 8744, while the median EBV DNA load in nasopharyngeal brush samples guided by clinicians and nasopharyngoscopes was 5144, Range 94 to 40095. Different from the Epstein-Barr virus DNA load, the median value of the Epstein-Barr virus DNA 11029 locus MD value was -2.79 in the nasopharyngeal brush sampling samples guided by clinicians and nasopharyngoscopes, ranging from -4.53 to -1.13, In contrast, the median value was -2.77 in the clinician-guided and nasopharyngoscope-guided nasopharyngeal brush sampling samples, and the range was -4.77 to 0, with no significant change between the two groups.
实施例6 能够同时保存DNA和RNA的鼻咽样本保存液Example 6 Nasopharyngeal sample preservation solution capable of simultaneously preserving DNA and RNA
6.1实验设计：6.1 Experimental design:
设置了2组保存液的对比，分别是自配与一款市售DNA保存液，自配与一款市售RNA保存液，每组选取6位鼻咽癌患者，左右各一根拭子，分别放置于自配与市售的保存液中，之后分别采用天根DNA提取试剂提取DNA，Qiagen RNA提取试剂提取RNA，DNA/RNA用于下游靶点的检测，靶点包含BamHI-W，LMP-2，C4，W2，miR-BART1-5p，miR-BART7-3p。A comparison of two groups of preservation solutions was set up, namely self-preparation and a commercially available DNA preservation solution, and self-preparation and a commercially available RNA preservation solution. Each group selected 6 patients with nasopharyngeal carcinoma, with one swab on the left and right. Place them in self-prepared and commercially available preservation solutions, and then use Tiangen DNA extraction reagent to extract DNA, Qiagen RNA extraction reagent to extract RNA, DNA/RNA is used for the detection of downstream targets, the targets include BamHI-W, LMP -2, C4, W2, miR-BART1-5p, miR-BART7-3p.
6.2实验材料及方法：6.2 Experimental materials and methods:
6.2.1自配保存液配方如表7所示。6.2.1 The formulation of the self-made preservation solution is shown in Table 7.
表7：Table 7:
试剂组分名称Reagent component name 配方含量Formula content
去离子水Deionized water 100mL100mL
EDTA-2NaEDTA-2Na 1.5g1.5g
NaClNaCl 0.05g0.05g
无水乙醇Absolute ethanol 50mL50mL
柠檬酸钠Sodium citrate 0.3g0.3g
NP40NP40 50μL50μL
青霉素-链霉素双抗溶液Penicillin-Streptomycin Double Antibody Solution 100x，加1mL100x, add 1mL
谷胱甘肽Glutathione 0.3g谷胱甘肽0.3g glutathione
磷酸三钠trisodium phosphate 0.2g磷酸三钠0.2g trisodium phosphate
pHpH 7.87.8
6.2.2市售保存液6.2.2 Commercially available preservation solution
选取了两款市售保存液，其中一款为拭子DNA常温保存液，生产商为生工，货号：B641913，另一款为RNA保存液RNAstore，生产商为康为世纪，货号：CW0592。Two commercially available preservation solutions were selected, one of which was swab DNA storage solution at room temperature, the manufacturer was Sangon, product number: B641913, and the other was RNA storage solution RNAstore, the manufacturer was Kangwei Century, product number: CW0592.
6.2.3样本DNA的提取6.2.3 Extraction of sample DNA
DNA采用磁珠法通用型基因组DNA提取试剂盒提取，生产商为天根生化科技(北京)有限公司，货号：DP705，利用天隆NP968自动核酸提取仪(西安天隆科技有限公司)进行提取，过程如下：The DNA was extracted using the Magnetic Beads Method Universal Genomic DNA Extraction Kit, manufactured by Tiangen Biochemical Technology (Beijing) Co., Ltd., article number: DP705, and extracted using Tianlong NP968 Automatic Nucleic Acid Extractor (Xi’an Tianlong Technology Co., Ltd.). The process is as follows:
1.取出200μL样本溶液至1.5ml离心管中进行实验，加入100μL消化液GHA和20μL Proteinase K，涡旋10sec混匀。1. Take out 200 μL of sample solution to a 1.5ml centrifuge tube for experimentation, add 100 μL of digestion solution GHA and 20 μL of Proteinase K, and vortex for 10 sec to mix.
2.放入金属浴中，75℃放置15min，期间颠倒混匀3回，每回3-5次。2. Put it in a metal bath, place it at 75°C for 15 minutes, and mix it upside down 3 times during the period, 3-5 times each time.
3.将上述溶液转移加到配套的96孔深孔板1列中，并加入300μL裂解液GHL和300μL异丙醇，和15μL磁珠悬浮液GH，放在设备专用的磁力架上，磁棒从溶液上方使磁珠吸附到磁棒套上。3. Transfer the above solution to column 1 of the matching 96-well deep-well plate, add 300 μL of lysate GHL, 300 μL of isopropanol, and 15 μL of magnetic bead suspension GH, and place it on the special magnetic stand for the equipment. Attach the magnetic beads to the magnetic rod cover from above the solution.
4.用900μL缓冲液GDZ对磁珠进行洗脱，随后用500μL缓冲液GDZ再洗脱1次。4. The magnetic beads were eluted with 900 μL buffer GDZ, and then eluted once with 500 μL buffer GDZ.
5.用900μL漂洗液PWD对磁珠进行洗脱，随后用300μL漂洗液PWD在洗脱1次。5. Use 900 μL of washing solution PWD to elute the magnetic beads, and then use 300 μL of washing solution PWD to elute once.
6.加入80μL DEPC水溶解磁珠上吸附的DNA，然后转移到EP管中于-20℃保存。6. Add 80 μL DEPC water to dissolve the DNA adsorbed on the magnetic beads, then transfer to EP tubes and store at -20°C.
6.2.4样本RNA的提取6.2.4 Extraction of sample RNA
采用商品化的提取试剂miRNeasy Tissue/Cells Advanced Kits提取RNA，生产商为Qiagen，货号：217684，提取步骤如下：RNA was extracted using commercially available extraction reagent miRNeasy Tissue/Cells Advanced Kits, the manufacturer is Qiagen, product number: 217684, and the extraction steps are as follows:
1.200μL样本溶液中添加Buffer RLT 260μL，涡旋一分钟。1. Add 260 μL of Buffer RLT to the 200 μL sample solution, and vortex for one minute.
2.加入Buffer AL 80μL，搅拌均匀，室温孵育3分钟。2. Add 80 μL of Buffer AL, stir well, and incubate at room temperature for 3 minutes.
3.将裂解液转移到gDNA消除离心柱中，置于收集管内。≥8000x g(≥10,000rpm)离心30秒。丢弃的离心柱，留下离心下来的液体。3. Transfer the lysate to a gDNA depletion spin column and place it in a collection tube. Centrifuge at ≥8000x g (≥10,000rpm) for 30 seconds. Discard the spin column, leaving the centrifuged liquid.
4.加入1体积异丙醇，移液枪吹打混匀。4. Add 1 volume of isopropanol and mix by pipetting.
5.将700μL的样品转移到RNeasy Mini离心柱中，置于2mL收集管。关闭盖子并在≥8000x g的条件下离心15秒。5. Transfer 700μL of the sample to the RNeasy Mini spin column and place in a 2mL collection tube. Close the lid and centrifuge at ≥8000x g for 15 seconds.
6.加入700μL Buffer RWT至RNeasy Mini spin柱。盖上盖子，≥8000x g离心15秒，丢弃废液。6. Add 700μL Buffer RWT to the RNeasy Mini spin column. Close the lid, centrifuge at ≥8000x g for 15 seconds, and discard the waste.
7.添加500μL Buffer RPE到RNeasy Mini离心柱。盖上盖子，≥8000x g离心15秒，丢弃废液。7. Add 500μL Buffer RPE to the RNeasy Mini spin column. Close the lid, centrifuge at ≥8000x g for 15 seconds, and discard the waste.
8.向RNeasy Mini离心柱中加入500μL 80％乙醇。盖上盖子，≥8000x g离心15秒，弃掉废液和收集管。8. Add 500 μL of 80% ethanol to the RNeasy Mini spin column. Close the lid, centrifuge at ≥8000xg for 15 seconds, discard the waste liquid and collection tube.
9.将RNeasy Mini旋转柱置于一个新的2mL收集管中。关闭盖子全速离心1分钟，使膜干燥，丢弃废液和收集管。9. Place the RNeasy Mini spin column into a new 2mL collection tube. Centrifuge at full speed for 1 min with the lid closed to dry the membrane and discard the waste and collection tube.
10.将RNeasy Mini旋转柱置于一个新的1.5mL收集管中。添加50μL RNase-free水，孵育1分钟。盖上盖子，全速离心1分钟，以洗脱RNA。10. Place the RNeasy Mini spin column into a new 1.5mL collection tube. Add 50 μL of RNase-free water and incubate for 1 min. Cover and centrifuge at full speed for 1 min to elute RNA.
6.3靶点相关信息6.3 Target related information
6.3.1 EB病毒DNA的定性检测6.3.1 Qualitative detection of Epstein-Barr virus DNA
采用荧光定量PCR的方法，对鼻咽刷样本中的EB病毒DNA进行定性检测，检测引物和探针如表8所示。The method of fluorescent quantitative PCR was used to qualitatively detect the Epstein-Barr virus DNA in the nasopharyngeal brush sample, and the detection primers and probes are shown in Table 8.
表8：Table 8:
Figure PCTCN2022127393-appb-000010
Figure PCTCN2022127393-appb-000010
Figure PCTCN2022127393-appb-000011
Figure PCTCN2022127393-appb-000011
注：LMP-2引物出处Lao，T.D.，Nguyen，T.A.H.，Ngo，K.D.，et al.Molecular Screening of Nasopharyngeal Carcinoma：Detection of LMP-1，LMP-2 Gene Expression in Vietnamese Nasopharyngeal Swab Samples.2019，Asian Pac J Cancer Prev，20(9)：2757-2761.Note: LMP-2 primers are from Lao, T.D., Nguyen, T.A.H., Ngo, K.D., et al. Molecular Screening of Nasopharyngeal Carcinoma: Detection of LMP-1, LMP-2 Gene Expression in Vietnamese Nasopharyngeal Swab Samples.2019, Asian Pac J Cancer Prev, 20(9): 2757-2761.
qPCR反应体系如表10和表11：The qPCR reaction system is shown in Table 10 and Table 11:
Vazyme(诺唯赞Taq Pro HS Universal U+Probe Master Mix，货号QN114)Vazyme (Novazyme Taq Pro HS Universal U+Probe Master Mix, Item No. QN114)
表10：Table 10:
Figure PCTCN2022127393-appb-000012
Figure PCTCN2022127393-appb-000012
表11：Table 11:
Figure PCTCN2022127393-appb-000013
Figure PCTCN2022127393-appb-000013
6.3.2 EB病毒DNA的甲基化检测6.3.2 Methylation detection of Epstein-Barr virus DNA
(1)DNA的重亚硫酸检测(1) DNA bisulfite detection
根据试剂盒说明书(ZYMO RESEARCH，CA，USA)，5μL样本的基因组DNA首先经过重亚硫酸盐处理和纯化，22μL洗脱得到转化修饰的DNA作为q-PCR检测的模板。According to the kit instructions (ZYMO RESEARCH, CA, USA), the genomic DNA of the 5 μL sample was first treated and purified with bisulfite, and 22 μL was eluted to obtain the converted modified DNA as a template for q-PCR detection.
(2)采用荧光定量PCR的方法，对EBV DNA甲基化状态进行定性检测，检测引物和探针如表12所示。甲基化位点分别位于EBV基因组上的C启动子区和w启动子区。(2) The method of fluorescent quantitative PCR was used to qualitatively detect the methylation status of EBV DNA, and the detection primers and probes were shown in Table 12. The methylation sites were respectively located in the C promoter region and the w promoter region of the EBV genome.
表12：Table 12:
Figure PCTCN2022127393-appb-000014
Figure PCTCN2022127393-appb-000014
qPCR的反应体系如表13和表14所示：The reaction system of qPCR is shown in Table 13 and Table 14:
表13：Table 13:
Figure PCTCN2022127393-appb-000015
Figure PCTCN2022127393-appb-000015
表14：Table 14:
名称name 终浓度Final concentration 体积(μL)Volume (μL)
Platinum TM Taq DNA Polymerase Platinum TM Taq DNA Polymerase 2.5U/rxn2.5U/rxn 0.100.10
10X PCR Buffer 10X PCR Buffer 1×1× 2.002.00
50mM MgCl250mM MgCl2 1.5mM1.5mM 0.600.60
10mM dNTP10mM dNTPs 0.2mM each0.2mM each 0.400.40
ROXROX 50×50× 0.400.40
Forward primerForward primer 300nM300nM 0.600.60
Reverse primerReverse primer 300nM300nM 0.600.60
ProbeProbe 150nM150nM 0.300.30
细胞系DNAcell line DNA  the 2.002.00
WaterWater  the xx
TotalTotal // 2020
qPCR反应条件如表15所示：The qPCR reaction conditions are shown in Table 15:
表15：Table 15:
Figure PCTCN2022127393-appb-000016
Figure PCTCN2022127393-appb-000016
6.3.3 EB病毒miRNA的检测：6.3.3 Detection of EB virus miRNA:
本研究涉及的EBV编码microRNA的核苷酸序列：The nucleotide sequence of the EBV-encoded microRNA involved in this study:
EBV-miR-BART1-5p：UCUUAGUGGAAGUGACGUGCUGUG(SEQ ID NO：39)EBV-miR-BART1-5p: UCUUAGUGGAAGUGACGUGCUGUG (SEQ ID NO: 39)
EBV-miR-BART7-3p：CAUCAUAGUCCAGUGUCCAGGG(SEQ ID NO：42)EBV-miR-BART7-3p: CAUCAUAGUCCAGUGUCCAGGG (SEQ ID NO: 42)
所用引物和探针如表16所示。The primers and probes used are listed in Table 16.
表16：Table 16:
Figure PCTCN2022127393-appb-000017
Figure PCTCN2022127393-appb-000017
Figure PCTCN2022127393-appb-000018
Figure PCTCN2022127393-appb-000018
逆转录反应体系：Reverse transcription reaction system:
逆转录试剂盒采用TaqMan TM MicroRNA逆转录试剂盒(Applied Biosystems)，货号：4366596。 The reverse transcription kit uses TaqMan ™ MicroRNA Reverse Transcription Kit (Applied Biosystems), catalog number: 4366596.
(1)将Taqman MicroRNA逆转录试剂盒中的组分放在冰上融化后涡旋混匀。(1) Thaw the components in the Taqman MicroRNA Reverse Transcription Kit on ice and vortex to mix.
(2)准备一支无RNA酶的离心管，参照下表，在冰上配制反转录预混液。(2) Prepare an RNase-free centrifuge tube, refer to the table below, and prepare the reverse transcription master mix on ice.
(3)在冰上将逆转录引物和RNA模板融化，涡旋混匀。(3) Melt the reverse transcription primer and RNA template on ice, and vortex to mix.
(4)15μl的反转录体系中，加入RNA(5μL)，7μL反转录预混液，3μL反转录引物，涡旋混匀，离心，反应体系见表17。(4) Add RNA (5 μL), 7 μL reverse transcription master mix, and 3 μL reverse transcription primer to 15 μl reverse transcription system, vortex to mix, and centrifuge. The reaction system is shown in Table 17.
(5)将反应管置于冰上5min，之后进行逆转录实验；反应条件见表18。(5) Place the reaction tube on ice for 5 minutes, and then perform the reverse transcription experiment; see Table 18 for the reaction conditions.
表17：Table 17:
ComponentComponent Volume(μL)Volume(μL)
RNase-free waterRNase-free water 4.164.16
100mM dNTPs(with dTTP)100mM dNTPs(with dTTP) 0.150.15
10×RT Buffer10×RT Buffer 1.501.50
RNase Inhibitor 20U/μLRNase Inhibitor 20U/μL 0.190.19
Multiscribe TM Reverse Transcriptase 50U/μL Multiscribe ™ Reverse Transcriptase 50U/μL 1.001.00
RT primer(终浓度25nM)RT primer (final concentration 25nM) 3.003.00
RNARNA 5.005.00
Total volumeTotal volume 15.0015.00
表18：Table 18:
逆转录反应条件：Reverse transcription reaction conditions:
温度temperature 时间time 循环数number of cycles
16℃16°C 30min30min 11
42℃42°C 30min30min 11
85℃85°C 5min5min 11
qPCR反应体系：qPCR reaction system:
采用TaqMan TM Fast Advanced预混液(Applied Biosystems)进行qPCR，货号：4444557。 qPCR was performed using TaqMan ™ Fast Advanced Master Mix (Applied Biosystems), catalog number: 4444557.
(1)将反应用到的组分放在冰上融化，混匀后放置冰上备用。(1) Thaw the components used in the reaction on ice, mix well and place on ice for later use.
(2)按照20μL反应体系计算所需要的试剂量(表19)。将各组分加入离心管中，涡旋混匀，离心。(2) Calculate the required reagent volume according to the 20 μL reaction system (Table 19). Add each component to a centrifuge tube, vortex to mix, and centrifuge.
表19：Table 19:
TaqMan TM Fast Advanced Master Mix TaqMan ™ Fast Advanced Master Mix 10.00μL10.00 μL
cDNAcDNA 1.00μL1.00 μL
Primer F(终浓度300nM)Primer F (final concentration 300nM) 0.60μL0.60 μL
Primer R(终浓度300nM)Primer R (final concentration 300nM) 0.60μL0.60μL
探针(终浓度200nM)Probe (final concentration 200nM) 0.40μL0.40 μL
RNase-free waterRNase-free water xμLxμL
Total volumeTotal volume 20.00μL20.00μL
(3)在每个反应孔中加入20μl的反应液。(3) Add 20 μl of reaction solution to each reaction well.
(4)将反应板的膜封好或将八联管的管盖盖紧，离心，避免产生气泡。(4) Seal the film of the reaction plate or cover the cap of the eight-tube tube tightly, and centrifuge to avoid the generation of air bubbles.
(5)运行定量PCR反应程序，设置反应程序如表20：(5) Run the quantitative PCR reaction program, and set the reaction program as shown in Table 20:
表20：Table 20:
Figure PCTCN2022127393-appb-000019
Figure PCTCN2022127393-appb-000019
6.4 qPCR检测结果：6.4 qPCR test results:
6.4.1 DNAload检测结果如图4所示。6.4.1 The results of DNAload detection are shown in Figure 4.
6.4.2甲基化检测结果如图5所示。6.4.2 The results of methylation detection are shown in Figure 5.
6.4.3 miRNA检测结果如图6所示。6.4.3 miRNA detection results are shown in Figure 6.
这些结果显示，自配保存液对于DNA，DNA甲基化，以及miRNA的综合保存性能优于市售DNA保存液以及市售RNA保存液。These results show that the comprehensive preservation performance of the self-made preservation solution for DNA, DNA methylation, and miRNA is better than that of the commercial DNA preservation solution and the commercial RNA preservation solution.
实施例7 用于miRNA提取的试剂及方法Example 7 Reagents and methods for miRNA extraction
7.1实验设计：7.1 Experimental design:
选取6位鼻咽癌患者，采集拭子放入保存液中，采用自配RNA提取试剂提取保存液中的RNA，同时购买市售的一款RNA提取试剂作对比，提取完成后采用Nanodrop以及Qubit对total RNA以及miRNA分别进行浓度测定，结合下游miRNA靶点检测对miRNA的提取效果进行测评。Select 6 patients with nasopharyngeal carcinoma, collect swabs and put them in the preservation solution, use the self-prepared RNA extraction reagent to extract the RNA in the preservation solution, and purchase a commercially available RNA extraction reagent for comparison, use Nanodrop and Qubit after the extraction is completed The concentrations of total RNA and miRNA were measured separately, and the extraction effect of miRNA was evaluated in combination with downstream miRNA target detection.
7.2实验材料及方法：7.2 Experimental materials and methods:
7.2.1自配提取试剂配方如表21至表24所示：7.2.1 The formulas of self-prepared extraction reagents are shown in Table 21 to Table 24:
表21：Table 21:
Figure PCTCN2022127393-appb-000020
Figure PCTCN2022127393-appb-000020
表22：Table 22:
Figure PCTCN2022127393-appb-000021
Figure PCTCN2022127393-appb-000021
Figure PCTCN2022127393-appb-000022
Figure PCTCN2022127393-appb-000022
表23：Table 23:
Figure PCTCN2022127393-appb-000023
Figure PCTCN2022127393-appb-000023
表24：Table 24:
Figure PCTCN2022127393-appb-000024
Figure PCTCN2022127393-appb-000024
磁珠：南京瑞贝西生物科技有限公司，货号：NB0207。Magnetic beads: Nanjing Ruibeixi Biotechnology Co., Ltd., product number: NB0207.
7.2.2 RNA提取7.2.2 RNA extraction
采用商品化的提取试剂miRNeasy Tissue/Cells Advanced Kits提取RNA，生产商为Qiagen，货号：217684，提取步骤如下：RNA was extracted using commercially available extraction reagent miRNeasy Tissue/Cells Advanced Kits, the manufacturer is Qiagen, product number: 217684, and the extraction steps are as follows:
1.200μL样本溶液中添加Buffer RLT 260μL，涡旋一分钟。1. Add 260 μL of Buffer RLT to the 200 μL sample solution, and vortex for one minute.
2.加入Buffer AL 80μL，搅拌均匀，室温孵育3分钟。2. Add 80 μL of Buffer AL, stir well, and incubate at room temperature for 3 minutes.
3.将裂解液转移到gDNA消除离心柱中，置于收集管内。≥8000x g(≥10,000rpm)离心30秒。丢弃的离心柱，留下离心下来的液体。3. Transfer the lysate to a gDNA depletion spin column and place it in a collection tube. Centrifuge at ≥8000x g (≥10,000rpm) for 30 seconds. Discard the spin column, leaving the centrifuged liquid.
4.加入1体积异丙醇，移液枪吹打混匀。4. Add 1 volume of isopropanol and mix by pipetting.
5.将700μL的样品转移到RNeasy Mini离心柱中，置于2mL收集管。关闭盖子并在≥8000x g的条件下离心15秒。5. Transfer 700μL of the sample to the RNeasy Mini spin column and place in a 2mL collection tube. Close the lid and centrifuge at ≥8000x g for 15 seconds.
6.加入700μL Buffer RWT至RNeasy Mini spin柱。盖上盖子，≥8000x g离心15秒，丢弃废液。6. Add 700μL Buffer RWT to the RNeasy Mini spin column. Close the lid, centrifuge at ≥8000x g for 15 seconds, and discard the waste.
7.添加500μL Buffer RPE到RNeasy Mini离心柱。盖上盖子，≥8000x g离心15秒，丢弃废液。7. Add 500μL Buffer RPE to the RNeasy Mini spin column. Close the lid, centrifuge at ≥8000x g for 15 seconds, and discard the waste.
8.向RNeasy Mini离心柱中加入500μL 80％乙醇。盖上盖子，≥8000x g离心15秒，弃掉废液和收集管。8. Add 500 μL of 80% ethanol to the RNeasy Mini spin column. Close the lid, centrifuge at ≥8000xg for 15 seconds, discard the waste liquid and collection tube.
9.将RNeasy Mini旋转柱置于一个新的2mL收集管中。关闭盖子全速离心1分钟，使膜干燥，丢弃废液和收集管。9. Place the RNeasy Mini spin column into a new 2mL collection tube. Centrifuge at full speed for 1 min with the lid closed to dry the membrane and discard the waste and collection tube.
10.将RNeasy Mini旋转柱置于一个新的1.5mL收集管中。添加50μL RNase-free水，孵育1分钟。盖上盖子，全速离心1分钟，以洗脱RNA。10. Place the RNeasy Mini spin column into a new 1.5mL collection tube. Add 50 μL of RNase-free water and incubate for 1 min. Cover and centrifuge at full speed for 1 min to elute RNA.
采用自配提取试剂提取样本RNA，载入自动化核酸提取仪天隆NP968(西安天隆科技有限公司)进行提取，操作步骤如下：Use the self-prepared extraction reagent to extract sample RNA, load it into the automatic nucleic acid extraction instrument Tianlong NP968 (Xi'an Tianlong Technology Co., Ltd.) for extraction, the operation steps are as follows:
1吸取200uL拭子样本溶液，至于1.5mL离心管中。1 Pipette 200uL swab sample solution into a 1.5mL centrifuge tube.
2加入等量的裂解液(即200uL)，震荡混合25-30s，加入20μL蛋白酶K，室温静置10min。加入600uL的结合缓冲液与20uL的磁珠，涡旋混匀，使裂解释放之后的核酸充分结合到磁珠上。2 Add an equal amount of lysate (ie 200 uL), shake and mix for 25-30 s, add 20 μL proteinase K, and let stand at room temperature for 10 min. Add 600uL of binding buffer and 20uL of magnetic beads, vortex and mix well, so that the nucleic acid released after cleavage is fully bound to the magnetic beads.
3将混液加入96孔深孔板的第一列中，放在设备专用的磁力架上，磁棒从溶液上方使磁珠吸附到磁棒套上。3. Add the mixed solution into the first column of the 96-well deep-well plate, and place it on the special magnetic frame for the equipment. The magnetic rod is from above the solution to make the magnetic beads adsorb to the magnetic rod cover.
4用700uL漂洗液1/乙醇混合溶液(漂洗液1/无水乙醇体积比1∶1.5)对磁珠进行洗脱，随后用700uL漂洗液2/乙醇混合溶液(漂洗液2/无水乙醇体积比2∶2.3)洗脱两次。4. Use 700uL rinse liquid 1/ethanol mixed solution (wash liquid 1/absolute ethanol volume ratio 1:1.5) to elute the magnetic beads, and then use 700uL rinse liquid 2/ethanol mixed solution (rinse liquid 2/absolute ethanol volume ratio ratio 2:2.3) eluted twice.
5加入50μL DEPC水溶解磁珠上吸附的RNA，然后转移到EP管中于-80℃保存。5 Add 50 μL DEPC water to dissolve the RNA adsorbed on the magnetic beads, then transfer to EP tubes and store at -80°C.
7.2.3 Qubit检测7.2.3 Qubit detection
1.配置Qubit工作液，将Qubit microRNA reagent按照1∶200的比例稀释于Qubit  microRNA buffer中。1. Configure Qubit working solution, dilute Qubit microRNA reagent in Qubit microRNA buffer at a ratio of 1:200.
2.配置Qubit标准品反应液，将190μL的Qubit工作液装入薄壁透光的0.5mL PCR管中。2. Prepare the Qubit standard reaction solution, and put 190 μL of the Qubit working solution into a thin-walled light-transmitting 0.5mL PCR tube.
3.取Qubit microRNA标准品1以及2各10μL加入上一步配置的反应液中，涡旋2-3秒，使终体积为200μL，注意不要产生气泡。3. Add 10 μL each of Qubit microRNA standard 1 and 2 to the reaction solution prepared in the previous step, and vortex for 2-3 seconds to make the final volume 200 μL. Be careful not to generate air bubbles.
4.配置Qubit样本反应液，将198μL的Qubit工作液装入薄壁透光的0.5mL PCR管中。4. Prepare the Qubit sample reaction solution, and put 198 μL of the Qubit working solution into a thin-walled light-transmitting 0.5mL PCR tube.
5.将2μL的样本RNA加入上一步配置的反应液中，涡旋2-3秒，使终体积为200μL。5. Add 2 μL of sample RNA to the reaction solution prepared in the previous step, and vortex for 2-3 seconds to make the final volume 200 μL.
6.以上所有的样本于室温中放置2min。6. All the above samples were placed at room temperature for 2 minutes.
7.在Qubit 3.0荧光计主界面，将装有1号标准品的EP管插入样品室，盖上盖子。当读取完成，移除标准1。7. On the main interface of the Qubit 3.0 Fluorometer, insert the EP tube containing the No. 1 standard into the sample chamber and close the lid. When the read is complete, remove criterion 1.
8.将装有2号标准品的试管插入样品腔，当读取完成后，移除标准2。8. Insert the test tube containing standard 2 into the sample chamber, when the reading is complete, remove standard 2.
9.在检测屏幕上，选择样品体积和单位即2μL。将EP管插入样品腔，盖上盖子，读取完成，取出样本管。仪器将结果显示在化验屏幕上。9. On the Assay screen, select the sample volume and units, ie 2 μL. Insert the EP tube into the sample chamber, close the lid, and take out the sample tube after the reading is completed. The instrument displays the results on the assay screen.
7.3靶标信息及检测结果7.3 Target information and detection results
7.3.1本研究涉及的EBV编码microRNA的核苷酸序列：7.3.1 The nucleotide sequence of EBV-encoded microRNA involved in this study:
EBV-miR-BART1-5p：UCUUAGUGGAAGUGACGUGCUGUG(SEQ ID NO：39)EBV-miR-BART1-5p: UCUUAGUGGAAGUGACGUGCUGUG (SEQ ID NO: 39)
EBV-miR-BART7-3p：CAUCAUAGUCCAGUGUCCAGGG(SEQ ID NO：42)EBV-miR-BART7-3p: CAUCAUAGUCCAGUGUCCAGGG (SEQ ID NO: 42)
涉及的引物和探针如表25所示。The primers and probes involved are shown in Table 25.
表25：Table 25:
Figure PCTCN2022127393-appb-000025
Figure PCTCN2022127393-appb-000025
Figure PCTCN2022127393-appb-000026
Figure PCTCN2022127393-appb-000026
7.3.2 Qubit检测结果(检测miRNA含量)如表26所示。7.3.2 Qubit detection results (detection of miRNA content) are shown in Table 26.
表26：Table 26:
样本编号sample number 市售RNA提取试剂(ng/μL)Commercially available RNA extraction reagent (ng/μL) 自配RNA提取试剂(ng/μL)Self-prepared RNA extraction reagent (ng/μL)
11 4.14.1 28.628.6
22 1.061.06 2.372.37
33 0.6490.649 2.032.03
44 0.5180.518 1.981.98
55 6.246.24 17.917.9
66 0.8410.841 3.043.04
7.3.3 Nanodrop检测结果(检测Total RNA含量)如表27所示。7.3.3 Nanodrop detection results (detection of Total RNA content) are shown in Table 27.
表27：Table 27:
样本编号sample number 市售RNA提取试剂(ng/μL)Commercially available RNA extraction reagent (ng/μL) 自配RNA提取试剂(ng/μL)Self-prepared RNA extraction reagent (ng/μL)
11 1818 32.832.8
22 6.16.1 11.511.5
33 3.13.1 11.211.2
44 2.92.9 12.412.4
55 19.219.2 23.923.9
66 4.64.6 11.611.6
7.3.4 qPCR检测结果如图7所示。7.3.4 qPCR test results are shown in Figure 7.
这些实验结果显示，本实施例使用的自配提取试剂配方对RNA，包括miRNA的提取效果要显著好于市售试剂的提取效果。These experimental results show that the extraction effect of the self-prepared extraction reagent formula used in this example on RNA, including miRNA, is significantly better than that of commercially available reagents.
实施例8 基于miRNA作为补充生物标志物的鼻咽癌检测Example 8 Detection of nasopharyngeal carcinoma based on miRNA as a supplementary biomarker
8.1样本来源：8.1 Sample source:
共收集184例样本，其中包括88例鼻咽癌患者以及96例未患有鼻咽癌的人员。88例鼻咽癌患者皆经过病理活检确诊为鼻咽癌的病人；96例未患有鼻咽癌的人员中，有25例人员为鼻咽部其他疾病的患者，剩下的71例为健康人。A total of 184 samples were collected, including 88 patients with nasopharyngeal carcinoma and 96 persons without nasopharyngeal carcinoma. All 88 patients with nasopharyngeal carcinoma were diagnosed with nasopharyngeal carcinoma by pathological biopsy; among the 96 patients without nasopharyngeal carcinoma, 25 patients were patients with other nasopharyngeal diseases, and the remaining 71 patients were healthy people.
8.2靶点信息：8.2 Target information:
EBV miRNA的检测Detection of EBV miRNA
本研究涉及的EBV编码microRNA的核苷酸序列：The nucleotide sequence of the EBV-encoded microRNA involved in this study:
EBV-miR-BART1-5p：UCUUAGUGGAAGUGACGUGCUGUG(SEQ ID NO：39)EBV-miR-BART1-5p: UCUUAGUGGAAGUGACGUGCUGUG (SEQ ID NO: 39)
EBV-miR-BART2-5p：UAUUUUCUGCAUUCGCCCUUGC(SEQ ID NO：40)EBV-miR-BART2-5p: UAUUUUCUGCAUUCGCCCUUGC (SEQ ID NO: 40)
EBV-miR-BART6-3p：CGGGGAUCGGACUAGCCUUAGA(SEQ ID NO：41)EBV-miR-BART6-3p: CGGGGAUCGGACUAGCCUUAGA (SEQ ID NO: 41)
EBV-miR-BART7-3p：CAUCAUAGUCCAGUGUCCAGGG(SEQ ID NO：42)EBV-miR-BART7-3p: CAUCAUAGUCCAGUGUCCAGGG (SEQ ID NO: 42)
EBV-miR-BART13-3p：UGUAACUUGCCAGGGACGGCUGA(SEQ ID NO：43)EBV-miR-BART13-3p: UGUAACUUGCCAGGGACGGCUGA (SEQ ID NO: 43)
EBV-miR-BART17-5p：UAAGAGGACGCAGGCAUACAAG(SEQ ID NO：44)EBV-miR-BART17-5p: UAAGAGGACGCAGGCAUACAAG (SEQ ID NO: 44)
miRNA引物探针序列如表28所示。The miRNA primer probe sequences are shown in Table 28.
表28：Table 28:
Figure PCTCN2022127393-appb-000027
Figure PCTCN2022127393-appb-000027
Figure PCTCN2022127393-appb-000028
Figure PCTCN2022127393-appb-000028
DNA load引物探针如表29所示。DNA load primer probes are shown in Table 29.
表29：Table 29:
Figure PCTCN2022127393-appb-000029
Figure PCTCN2022127393-appb-000029
Figure PCTCN2022127393-appb-000030
Figure PCTCN2022127393-appb-000030
LMP-2引物出处：Lao，T.D.，Nguyen，T.A.H.，Ngo，K.D.，et al.Molecular Screening of Nasopharyngeal Carcinoma：Detection of LMP-1，LMP-2 Gene Expression in Vietnamese Nasopharyngeal Swab Samples.2019，Asian Pac J Cancer Prev，20(9)：2757-2761.Source of LMP-2 primers: Lao, T.D., Nguyen, T.A.H., Ngo, K.D., et al. Molecular Screening of Nasopharyngeal Carcinoma: Detection of LMP-1, LMP-2 Gene Expression in Vietnamese Nasopharyngeal Swab Samples.2019, Asian Pac J Cancer Prev, 20(9): 2757-2761.
EB病毒DNA的甲基化检测的引物探针如表30所示。甲基化位点分别位于EBV基因组上的C启动子区和W启动子区。The primer probes for the methylation detection of Epstein-Barr virus DNA are shown in Table 30. The methylation sites were located in the C promoter region and the W promoter region of the EBV genome, respectively.
表30：Table 30:
Figure PCTCN2022127393-appb-000031
Figure PCTCN2022127393-appb-000031
Figure PCTCN2022127393-appb-000032
Figure PCTCN2022127393-appb-000032
C1上下游引物序列来自于郑小辉的博士毕业论文：郑小辉.2020.新疆医科大学.鼻咽部EB病毒核酸标志物检测在鼻咽癌分子诊断中的应用研究.C1 upstream and downstream primer sequences are from Zheng Xiaohui's doctoral thesis: Zheng Xiaohui. 2020. Xinjiang Medical University. The application of detection of nucleic acid markers of Epstein-Barr virus in nasopharynx in the molecular diagnosis of nasopharyngeal carcinoma.
C10探针来自于本专利11029的探针。The C10 probe is derived from the probe of this patent 11029.
8.3实验结果8.3 Experimental results
结果如图8-图10所示，无论是EB病毒DNA load，还是各甲基化位点以及miRNA在鼻咽刷检中均有显著差异。各位点在鼻咽癌组的表达量显著高于非鼻咽癌的对照组。The results are shown in Figure 8-Figure 10, whether it is the Epstein-Barr virus DNA load, or each methylation site and miRNA in nasopharyngeal brushing examination, there are significant differences. The expression levels of each site in the nasopharyngeal carcinoma group were significantly higher than those in the non-nasopharyngeal carcinoma control group.
联合EB病毒DNA load以及各个甲基化位点构建了鼻咽癌诊断模型1，并对模型诊断鼻咽癌的灵敏度和特异性进行分析，其中A1为BamHI-W，A2为LMP-2，B1为甲基化C1，B2为甲基化C4，B3为甲基化W1，B4为甲基化C10，B5为甲基化W2。结果如表31所示，该模型包含位点A2，B2，B3以及B4，其敏感度为92.6％，特异度为93.1％，约登指数为0.857。The nasopharyngeal carcinoma diagnosis model 1 was constructed by combining the Epstein-Barr virus DNA load and each methylation site, and the sensitivity and specificity of the model for nasopharyngeal carcinoma diagnosis were analyzed, in which A1 is BamHI-W, A2 is LMP-2, B1 is methylated C1, B2 is methylated C4, B3 is methylated W1, B4 is methylated C10, and B5 is methylated W2. The results are shown in Table 31. The model includes sites A2, B2, B3 and B4, with a sensitivity of 92.6%, a specificity of 93.1%, and a Youden index of 0.857.
联合EB病毒DNA load，甲基化位点以及miRNA构建了鼻咽癌诊断模型2-9，其中E1为miR-BART1-5p，E2为miR-BART17-5p，E3为miR-BART6-3p，E4为miR-BART7-3p，E5为miR-BART2-5p，E6为miR-BART13-3p。在模型1的基础上单独添加E1，E4-6之后，模型性能得到了不同程度的提升。同时添加E1及E4，敏感度为96.3％，特异度为100.0％，约登指数为0.963，将6个miRNA全部加上之后，敏感度为100.0％，特异度为96.6％，约登指数为0.966。Combined with Epstein-Barr virus DNA load, methylation sites and miRNA to construct a nasopharyngeal carcinoma diagnostic model 2-9, in which E1 is miR-BART1-5p, E2 is miR-BART17-5p, E3 is miR-BART6-3p, E4 is miR-BART7-3p, E5 is miR-BART2-5p, and E6 is miR-BART13-3p. After adding E1 and E4-6 separately on the basis of model 1, the performance of the model has been improved to varying degrees. Add E1 and E4 at the same time, the sensitivity is 96.3%, the specificity is 100.0%, and the Youden index is 0.963. After adding all 6 miRNAs, the sensitivity is 100.0%, the specificity is 96.6%, and the Youden index is 0.966 .
表31：Table 31:
Figure PCTCN2022127393-appb-000033
Figure PCTCN2022127393-appb-000033
Figure PCTCN2022127393-appb-000034
Figure PCTCN2022127393-appb-000034
Figure PCTCN2022127393-appb-000035
Figure PCTCN2022127393-appb-000035
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它 不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the scope of the present invention. For those of ordinary skill in the art, on the basis of the above descriptions and ideas, they can also make There is no need to and cannot exhaustively list all the implementation manners for other changes or changes in different forms. All modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the claims of the present invention.
Figure PCTCN2022127393-appb-000036
Figure PCTCN2022127393-appb-000036
Figure PCTCN2022127393-appb-000037
Figure PCTCN2022127393-appb-000037
Figure PCTCN2022127393-appb-000038
Figure PCTCN2022127393-appb-000038

Claims (91)

  1. 一种检测EB病毒的试剂盒，其特征在于，所述试剂盒包含选自核酸甲基化检测试剂、miRNA检测试剂、miRNA提取试剂，和/或样本保存液的一种或多种试剂。A kit for detecting Epstein-Barr virus, characterized in that the kit includes one or more reagents selected from nucleic acid methylation detection reagents, miRNA detection reagents, miRNA extraction reagents, and/or sample preservation solutions.
  2. 如权利要求1所述的试剂盒，其特征在于，所述试剂盒包含核酸甲基化检测试剂，所述核酸甲基化检测试剂可针对EB病毒基因组中甲基化位点进行甲基化检测。The kit according to claim 1, wherein the kit comprises a nucleic acid methylation detection reagent, and the nucleic acid methylation detection reagent can detect methylation at a methylation site in the Epstein-Barr virus genome .
  3. 如权利要求2所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、14015bp、14566bp、45850bp、57946bp、66227bp和128103bp的位点。The kit according to claim 2, wherein the methylation site in the Epstein-Barr virus genome comprises one or more of the following sites: EB equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) Sites of 10717bp, 11029bp, 14015bp, 14566bp, 45850bp, 57946bp, 66227bp and 128103bp of the viral genome DNA sequence.
  4. 如权利要求2-3任一项所述的试剂盒，其特征在于，所述EB病毒基因组中的所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、和14015bp的位点。The kit according to any one of claims 2-3, wherein the methylation site in the Epstein-Barr virus genome comprises the Epstein-Barr virus genome equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) The 10717bp, 11029bp, and 14015bp positions of the DNA sequence.
  5. 如权利要求2-4任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp、45850bp、57946bp、66227bp和128103bp的位点。The kit according to any one of claims 2-4, wherein the methylation site in the Epstein-Barr virus genome comprises one or more of the following sites: equivalent to GenBank LOCUS NC_007605 (SEQ ID NO : 94) the sites of 11029bp, 45850bp, 57946bp, 66227bp and 128103bp of the Epstein-Barr virus genome DNA sequence.
  6. 如权利要求2-5任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp、57946bp、和66227bp的位点。The kit according to any one of claims 2-5, wherein the methylation site in the Epstein-Barr virus genome comprises one or more of the following sites: equivalent to GenBank LOCUS NC_007605 (SEQ ID NO : 11029bp, 57946bp, and 66227bp of the Epstein-Barr virus genome DNA sequence of 94).
  7. 如权利要求2-6任一项所述的试剂盒，其特征在于，所述EB病毒基因组中甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的11029bp的位点。The kit according to any one of claims 2-6, wherein the methylation site in the Epstein-Barr virus genome comprises a part of the Epstein-Barr virus genome DNA sequence equivalent to GenBank LOCUS NC_007605 (SEQ ID NO: 94) 11029bp site.
  8. 如权利要求1-7任一项所述的试剂盒，其特征在于，所述试剂盒包含所述miRNA检测试剂，所述miRNA检测试剂可针对一个或多个来自EB病毒的miRNA进行含量检测。The kit according to any one of claims 1-7, characterized in that the kit contains the miRNA detection reagent, and the miRNA detection reagent can detect the content of one or more miRNAs from Epstein-Barr virus.
  9. 如权利要求8所述的试剂盒，其特征在于，所述miRNA包含以下miRNA的一个或几个：EBV-miR-BART1-5p、EBV-miR-BART2-5p、EBV-miR-BART6-3p、EBV-miR-BART7-3p、EBV-miR-BART13-3p、和EBV-miR-BART17-5p；优选地包含全部六个所述miRNA。The kit according to claim 8, wherein the miRNA comprises one or more of the following miRNAs: EBV-miR-BART1-5p, EBV-miR-BART2-5p, EBV-miR-BART6-3p, EBV-miR-BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p; preferably comprising all six of said miRNAs.
  10. 如权利要求9所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART1-5p 和EBV-miR-BART7-3p。The kit according to claim 9, wherein the miRNA comprises EBV-miR-BART1-5p and EBV-miR-BART7-3p.
  11. 如权利要求9-10任一项所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART1-5p。The kit according to any one of claims 9-10, wherein the miRNA comprises EBV-miR-BART1-5p.
  12. 如权利要求9-11任一项所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART2-5p。The kit according to any one of claims 9-11, wherein the miRNA comprises EBV-miR-BART2-5p.
  13. 如权利要求9-12任一项所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART6-3p。The kit according to any one of claims 9-12, wherein the miRNA comprises EBV-miR-BART6-3p.
  14. 如权利要求9-13任一项所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART7-3p。The kit according to any one of claims 9-13, wherein the miRNA comprises EBV-miR-BART7-3p.
  15. 如权利要求9-14任一项所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART13-3p。The kit according to any one of claims 9-14, wherein the miRNA comprises EBV-miR-BART13-3p.
  16. 如权利要求9-15任一项所述的试剂盒，其特征在于，所述miRNA包含EBV-miR-BART17-5p。The kit according to any one of claims 9-15, wherein the miRNA comprises EBV-miR-BART17-5p.
  17. 如权利要求1-16任一项所述的试剂盒，其特征在于，所述试剂盒包含所述样本保存液，所述样本保存液包含选自EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和/或磷酸三钠的一种或多种组分。The kit according to any one of claims 1-16, wherein the kit includes the sample preservation solution, and the sample preservation solution comprises selected from EDTA, NaCl, absolute ethanol, sodium citrate, One or more components of NP40, glutathione, and/or trisodium phosphate.
  18. 如权利要求17所述的试剂盒，其特征在于，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。The kit according to claim 17, wherein the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
  19. 一种保存含核酸的生物样本的试剂盒，其特征在于，包含样本保存液，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。A kit for preserving a nucleic acid-containing biological sample, characterized in that it comprises a sample preservation solution comprising EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate .
  20. 如权利要求17-19任一项所述的试剂盒，其特征在于，所述样本保存液中：The kit according to any one of claims 17-19, wherein in the sample preservation solution:
    所述EDTA的含量范围是0.7％-1.5％(w/w)；The content range of the EDTA is 0.7%-1.5% (w/w);
    所述NaCl的含量范围是0.01％-0.1％(w/w)；The content range of the NaCl is 0.01%-0.1% (w/w);
    所述无水乙醇的浓度范围是15％-35％(w/w)；The concentration range of the absolute ethanol is 15%-35% (w/w);
    所述柠檬酸钠的含量范围是0.1％-1.0％(w/w)；The content range of the sodium citrate is 0.1%-1.0% (w/w);
    所述NP40的浓度范围是0.01％-0.1％(v/v)；The concentration range of the NP40 is 0.01%-0.1% (v/v);
    所述谷胱甘肽的含量范围是0.07％-0.7％(w/w)；和/或The glutathione content ranges from 0.07% to 0.7% (w/w); and/or
    所述磷酸三钠的含量范围是0.07％-0.4％(w/w)。The content range of the trisodium phosphate is 0.07%-0.4% (w/w).
  21. 如权利要求17-20任一项所述的试剂盒，其特征在于，所述样本保存液中：The kit according to any one of claims 17-20, wherein in the sample preservation solution:
    所述EDTA的含量是约1.05％(w/w)；The content of said EDTA is about 1.05% (w/w);
    所述NaCl的含量是约0.035％(w/w)；The content of said NaCl is about 0.035% (w/w);
    所述无水乙醇的浓度是约27.6％(w/w)；The concentration of absolute ethanol is about 27.6% (w/w);
    所述柠檬酸钠的含量是约0.21％(w/w)；The content of the sodium citrate is about 0.21% (w/w);
    所述NP40的浓度是约0.035％(v/v)；The concentration of said NP40 is about 0.035% (v/v);
    所述谷胱甘肽的含量是约0.21％(w/w)；和/或The glutathione content is about 0.21% (w/w); and/or
    所述磷酸三钠的含量是约0.14％(w/w)。The content of the trisodium phosphate is about 0.14% (w/w).
  22. 如权利要求17-21一项所述的试剂盒，其特征在于，所述样本保存液包含抗生素；优选地，所述抗生素包含青霉素-链霉素双抗。The kit according to one of claims 17-21, wherein the sample preservation solution contains antibiotics; preferably, the antibiotics contain penicillin-streptomycin double antibody.
  23. 如权利要求17-22任一项所述的试剂盒，其特征在于，所述样本保存液的pH值为7.6-8.0；优选地，所述样本保存液的pH值为7.8。The kit according to any one of claims 17-22, wherein the pH value of the sample preservation solution is 7.6-8.0; preferably, the pH value of the sample preservation solution is 7.8.
  24. 如权利要求17-23任一项所述的试剂盒，其特征在于，与对照样本保存液相比，所述样本保存液对DNA，miRNA，和/或DNA甲基化的保存效果更好。The kit according to any one of claims 17-23, characterized in that, compared with the control sample preservation solution, the sample preservation solution has a better preservation effect on DNA, miRNA, and/or DNA methylation.
  25. 如权利要求24所述的试剂盒，其特征在于，所述DNA来源于EB病毒，所述miRNA来源于EB病毒，和/或所述DNA甲基化是EB病毒DNA的甲基化。The kit according to claim 24, wherein the DNA is derived from Epstein-Barr virus, the miRNA is derived from Epstein-Barr virus, and/or the DNA methylation is methylation of Epstein-Barr virus DNA.
  26. 如权利要求1-25任一项所述的试剂盒，其特征在于，所述试剂盒包含所述miRNA提取试剂，所述miRNA提取试剂包含裂解液，结合液，漂洗液1，和/或漂洗液2。The kit according to any one of claims 1-25, wherein the kit comprises the miRNA extraction reagent, and the miRNA extraction reagent comprises a lysate, a binding solution, a rinse solution 1, and/or a rinse Liquid 2.
  27. 一种包含miRNA提取试剂的试剂盒，其特征在于，所述miRNA提取试剂包含裂解液，结合液，漂洗液1，和/或漂洗液2。A kit comprising a miRNA extraction reagent, characterized in that the miRNA extraction reagent comprises a lysate, a binding solution, a washing solution 1, and/or a washing solution 2.
  28. 如权利要求26或27所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述裂解液，所述裂解液包含选自硫氰酸胍，柠檬酸，曲拉通-100，和/或EDTA的一种或多种组分。The kit according to claim 26 or 27, wherein the miRNA extraction reagent comprises the lysate, and the lysate comprises guanidinium thiocyanate, citric acid, Triton-100, and/or or one or more components of EDTA.
  29. 如权利要求26或27所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述裂解液，所述裂解液包含硫氰酸胍，柠檬酸，曲拉通-100，和EDTA。The kit according to claim 26 or 27, wherein the miRNA extraction reagent comprises the lysate, and the lysate comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA.
  30. 如权利要求26-29任一项所述的试剂盒，其特征在于，所述裂解液中：The kit according to any one of claims 26-29, wherein in the lysate:
    所述硫氰酸胍的含量范围是28％-40％(w/w)；The content scope of described guanidinium thiocyanate is 28%-40% (w/w);
    所述柠檬酸的含量范围是1.8％-2.6％(w/w)；The content range of the citric acid is 1.8%-2.6% (w/w);
    所述曲拉通-100的含量范围是0.8％-1.6％(w/w)；The content range of the Triton-100 is 0.8%-1.6% (w/w);
    所述EDTA的含量范围是0.5％-0.9％(w/w)；和/或The content range of the EDTA is 0.5%-0.9% (w/w); and/or
    所述裂解液的pH值范围为3.8-4.2。The pH range of the lysate is 3.8-4.2.
  31. 如权利要求26-30任一项所述的试剂盒，其特征在于，所述裂解液中：The kit according to any one of claims 26-30, wherein in the lysate:
    所述硫氰酸胍的含量是约32％(w/w)；The content of the guanidinium thiocyanate is about 32% (w/w);
    所述柠檬酸的含量是约2.2％(w/w)；The content of said citric acid is about 2.2% (w/w);
    所述曲拉通-100的含量是约1.1％(w/w)；The content of the Triton-100 is about 1.1% (w/w);
    所述EDTA的含量是约0.7％(w/w)；和/或The content of said EDTA is about 0.7% (w/w); and/or
    所述裂解液的pH值为约4.0。The pH of the lysate is about 4.0.
  32. 如权利要求26-31任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述结合液，所述结合液包含选自氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和/或异丙醇的一种或多种组分。The kit according to any one of claims 26-31, wherein the miRNA extraction reagent comprises the binding solution, and the binding solution comprises magnesium chloride, disodium edetate, sodium bicarbonate , guanidinium thiocyanate, sodium lauryl sarcosinate, and/or one or more components of isopropanol.
  33. 如权利要求26-32任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述结合液，所述结合液包含氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和异丙醇。The kit according to any one of claims 26-32, wherein the miRNA extraction reagent comprises the binding solution, and the binding solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, sulfur Guanidine Cyanate, Sodium Lauryl Sarcosinate, and Isopropanol.
  34. 如权利要求26-33任一项所述的试剂盒，其特征在于，所述结合液中：The kit according to any one of claims 26-33, wherein in the binding solution:
    所述氯化镁的含量范围是0.1％-0.4％(w/w)；The content range of the magnesium chloride is 0.1%-0.4% (w/w);
    所述乙二胺四乙酸二钠的含量范围是0.5％-1.0％(w/w)；The content range of the disodium edetate is 0.5%-1.0% (w/w);
    所述碳酸氢钠的含量范围是0.6％-1.0％(w/w)；The content scope of described sodium bicarbonate is 0.6%-1.0% (w/w);
    所述硫氰酸胍的含量范围是13％-16％(w/w)；The content range of the guanidine thiocyanate is 13%-16% (w/w);
    所述十二烷基肌氨酸钠的含量范围是1.0％-1.4％(w/w)；The content range of the sodium lauryl sarcosinate is 1.0%-1.4% (w/w);
    所述异丙醇的含量范围是60％-80％(w/w)；和/或The content range of the isopropanol is 60%-80% (w/w); and/or
    所述结合液的pH值范围是4.8-5.2。The pH range of the binding solution is 4.8-5.2.
  35. 如权利要求26-34任一项所述的试剂盒，其特征在于，所述结合液中：The kit according to any one of claims 26-34, wherein in the binding solution:
    所述氯化镁的含量是约0.24％(w/w)；The content of said magnesium chloride is about 0.24% (w/w);
    所述乙二胺四乙酸二钠的含量是约0.71％(w/w)；The content of disodium edetate is about 0.71% (w/w);
    所述碳酸氢钠的含量是约0.83％(w/w)；The content of the sodium bicarbonate is about 0.83% (w/w);
    所述硫氰酸胍的含量是约14.9％(w/w)；The content of guanidine thiocyanate is about 14.9% (w/w);
    所述十二烷基肌氨酸钠的含量是约1.2％(w/w)；The content of sodium sarcosyl is about 1.2% (w/w);
    所述异丙醇的含量是约70％(w/w)；和/或The content of said isopropanol is about 70% (w/w); and/or
    所述结合液的pH值是约5.0。The pH of the binding solution is about 5.0.
  36. 如权利要求26-35任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述漂洗液1，所述漂洗液1包含选自乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和/或十二烷基肌氨酸钠的一种或多种组分。The kit according to any one of claims 26-35, wherein the miRNA extraction reagent comprises the rinse solution 1, and the rinse solution 1 comprises disodium edetate, sodium bicarbonate , guanidinium thiocyanate, and/or one or more components of sodium lauryl sarcosinate.
  37. 如权利要求26-36任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述漂洗液1，所述漂洗液1包含乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和十二烷基肌氨酸钠。The kit according to any one of claims 26-36, wherein the miRNA extraction reagent comprises the rinse solution 1, and the rinse solution 1 comprises disodium edetate, sodium bicarbonate, sulfur Guanidine Cyanate, and Sodium Lauryl Sarcosinate.
  38. 如权利要求26-37任一项所述的试剂盒，其特征在于，所述漂洗液1中：The kit according to any one of claims 26-37, wherein in the rinse solution 1:
    所述乙二胺四乙酸二钠的含量范围是0.4％-0.9％(w/w)；The content range of the disodium edetate is 0.4%-0.9% (w/w);
    所述碳酸氢钠的含量范围是0.5％-0.9％(w/w)；The content scope of described sodium bicarbonate is 0.5%-0.9% (w/w);
    所述硫氰酸胍的含量范围是50％-55％(w/w)；The content range of the guanidinium thiocyanate is 50%-55% (w/w);
    所述十二烷基肌氨酸钠的含量范围是0.8％-1.2％(w/w)；和/或The content range of the sodium lauryl sarcosyl is 0.8%-1.2% (w/w); and/or
    所述漂洗液1的pH值范围是6.2-6.6。The pH range of the rinse solution 1 is 6.2-6.6.
  39. 如权利要求26-38任一项所述的试剂盒，其特征在于，所述漂洗液1中：The kit according to any one of claims 26-38, wherein in the rinse solution 1:
    所述乙二胺四乙酸二钠的含量是约0.5％(w/w)；The content of disodium edetate is about 0.5% (w/w);
    所述碳酸氢钠的含量是约0.6％(w/w)；The content of the sodium bicarbonate is about 0.6% (w/w);
    所述硫氰酸胍的含量是约53％(w/w)；The content of the guanidinium thiocyanate is about 53% (w/w);
    所述十二烷基肌氨酸钠的含量是约0.9％(w/w)；和/或The content of sodium sarcosyl is about 0.9% (w/w); and/or
    所述漂洗液1的pH值是约6.4。The pH of the Rinse 1 was about 6.4.
  40. 如权利要求26-39任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述漂洗液2，所述漂洗液2包含三羟甲基氨基甲烷(Tris)或氯化钠。The kit according to any one of claims 26-39, wherein the miRNA extraction reagent comprises the rinsing solution 2, and the rinsing solution 2 comprises Tris (Tris) or sodium chloride .
  41. 如权利要求26-40任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含所述漂洗液2，所述漂洗液2包含三羟甲基氨基甲烷(Tris)和氯化钠。The kit according to any one of claims 26-40, wherein the miRNA extraction reagent comprises the rinsing solution 2, and the rinsing solution 2 comprises tris (Tris) and sodium chloride .
  42. 如权利要求26-41任一项所述的试剂盒，其特征在于，所述漂洗液2中：The kit according to any one of claims 26-41, wherein in the rinse solution 2:
    所述三羟甲基氨基甲烷(Tris)的含量范围是0.5％-0.7％(w/w)；The content range of the tris (Tris) is 0.5%-0.7% (w/w);
    所述氯化钠的含量范围是0.6％-1.8％(w/w)；和/或The content range of the sodium chloride is 0.6%-1.8% (w/w); and/or
    所述漂洗液2的pH值范围是6.6-7.0。The pH range of the rinse solution 2 is 6.6-7.0.
  43. 如权利要求26-42任一项所述的试剂盒，其特征在于，所述漂洗液2中：The kit according to any one of claims 26-42, wherein in the rinse solution 2:
    所述三羟甲基氨基甲烷(Tris)的含量是约0.6％(w/w)；The content of tris (Tris) is about 0.6% (w/w);
    所述氯化钠的含量是约1.2％(w/w)；和/或The sodium chloride content is about 1.2% (w/w); and/or
    所述漂洗液2的pH值是约6.8。The pH value of the rinse solution 2 is about 6.8.
  44. 如权利要求26-43任一项所述的试剂盒，其特征在于，所述miRNA提取试剂包含吸附RNA的磁珠。The kit according to any one of claims 26-43, wherein the miRNA extraction reagent comprises magnetic beads that adsorb RNA.
  45. 如权利要求44所述的试剂盒，其特征在于，所述磁珠的磁核是氢氧化三铁，包被 二氧化硅，且表面镶嵌羟基基团。The test kit according to claim 44, wherein the magnetic core of the magnetic beads is ferric hydroxide, coated with silicon dioxide, and the surface is embedded with hydroxyl groups.
  46. 如权利要求44或45所述的试剂盒，其特征在于，所述磁珠的粒径为50-100nm。The kit according to claim 44 or 45, wherein the magnetic beads have a particle diameter of 50-100 nm.
  47. 如权利要求26-46任一项所述的试剂盒，其特征在于，所述试剂盒还包含鼻咽刷或鼻咽拭子。The kit according to any one of claims 26-46, wherein the kit further comprises a nasopharyngeal brush or a nasopharyngeal swab.
  48. 如权利要求47所述的试剂盒，其特征在于，所述鼻咽刷和/或鼻咽拭子为不依赖临床医生和鼻咽镜引导的鼻咽刷和/或鼻咽拭子。The kit according to claim 47, wherein the nasopharyngeal brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab independent of the guidance of a clinician and a nasopharyngoscope.
  49. 如权利要求1-48任一项所述的试剂盒，其特征在于，所述试剂盒还包含EB病毒载量检测试剂；优选地，所述EB病毒载量检测试剂包含针对EB病毒基因组中BamHI-W片段，和/或LMP-2基因的检测试剂。The kit according to any one of claims 1-48, wherein the kit also comprises an EB virus load detection reagent; preferably, the EB virus load detection reagent contains -W fragment, and/or detection reagent of LMP-2 gene.
  50. 如权利要求49所述的试剂盒，其特征在于，所述EB病毒载量检测试剂包含针对LMP-2基因的检测试剂。The kit according to claim 49, wherein the EB virus load detection reagent comprises a detection reagent for the LMP-2 gene.
  51. 如权利要求1-50任一项所述的试剂盒，其特征在于，所述试剂盒为鼻咽癌筛查和/或诊断试剂盒。The kit according to any one of claims 1-50, wherein the kit is a screening and/or diagnostic kit for nasopharyngeal carcinoma.
  52. 一种检测样本中EB病毒相关核酸的方法，其特征在于，所述方法包含以下步骤：A method for detecting Epstein-Barr virus-related nucleic acids in a sample, characterized in that the method comprises the following steps:
    (a)将所述样本置于样本保存液中；(a) placing the sample in a sample preservation solution;
    (b)进行选自以下的一项或多项检测：(b) Conduct one or more tests selected from the following:
    (b-1)对含有所述样本的所述样本保存液进行EB病毒基因组核酸甲基化检测；(b-1) performing Epstein-Barr virus genomic nucleic acid methylation detection on the sample preservation solution containing the sample;
    (b-2)从所述样本保存液中提取EB病毒相关miRNA并对所述miRNA的含量进行检测；和/或(b-2) extract Epstein-Barr virus-related miRNA from the sample preservation solution and detect the content of the miRNA; and/or
    (b-3)对含有所述样本的所述样本保存液进行EB病毒核酸载量检测。(b-3) Detection of Epstein-Barr virus nucleic acid load on the sample preservation solution containing the sample.
  53. 如权利要求52所述的方法，其特征在于，包含步骤(b-1)，且所述EB病毒基因组核酸甲基化检测的甲基化位点包含以下位点中的一个或几个：相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、14015bp、14566bp、45850bp、57946bp、66227bp和128103bp的位点。The method according to claim 52, characterized in that, step (b-1) is included, and the methylation site detected by the Epstein-Barr virus genome nucleic acid methylation comprises one or more of the following sites: equivalent Sites at 10717bp, 11029bp, 14015bp, 14566bp, 45850bp, 57946bp, 66227bp and 128103bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  54. 如权利要求53所述的方法，其特征在于，所述甲基化位点包含相当于GenBank LOCUS NC_007605(SEQ ID NO：94)的EB病毒基因组DNA序列的10717bp、11029bp、和14015bp的位点。The method according to claim 53, wherein the methylation site comprises sites corresponding to 10717bp, 11029bp, and 14015bp of the Epstein-Barr virus genomic DNA sequence of GenBank LOCUS NC_007605 (SEQ ID NO: 94).
  55. 如权利要求52-54任一项所述的方法，其特征在于，包含步骤(b-2)，所述miRNA包含以下miRNA的一个或几个：EBV-miR-BART1-5p、EBV-miR-BART2-5p、EBV-miR-BART6-3p、EBV-miR-BART7-3p、EBV-miR-BART13-3p、和 EBV-miR-BART17-5p；优选地包含全部六个所述miRNA。The method according to any one of claims 52-54, characterized in that, comprising step (b-2), the miRNA comprises one or more of the following miRNAs: EBV-miR-BART1-5p, EBV-miR- BART2-5p, EBV-miR-BART6-3p, EBV-miR-BART7-3p, EBV-miR-BART13-3p, and EBV-miR-BART17-5p; preferably comprise all six of said miRNAs.
  56. 如权利要求55所述的方法，其特征在于，所述miRNA包含EBV-miR-BART1-5p和EBV-miR-BART7-3p。The method of claim 55, wherein the miRNA comprises EBV-miR-BART1-5p and EBV-miR-BART7-3p.
  57. 如权利要求52-56任一项所述的方法，其特征在于，提取所述EB病毒相关miRNA的步骤包含：The method according to any one of claims 52-56, wherein the step of extracting the Epstein-Barr virus-related miRNA comprises:
    i)向所述样本保存液中加入裂解液；优选地，还加入蛋白酶K；i) adding a lysate to the sample preservation solution; preferably, proteinase K is also added;
    ii)加入结合液与核酸吸附材料；ii) adding binding solution and nucleic acid adsorption material;
    iii)去除液体部分，向核酸吸附材料加入漂洗液清洗；和iii) removing the liquid portion, adding a rinse solution to the nucleic acid adsorbing material for washing; and
    iv)去除液体部分，向所述核酸吸附材料中加入洗脱液，将所述核酸和/或所述miRNA从所述核酸吸附材料中洗脱。iv) removing the liquid part, adding an eluent to the nucleic acid adsorption material, and eluting the nucleic acid and/or the miRNA from the nucleic acid adsorption material.
  58. 一种提取miRNA的方法，包含以下步骤：A method for extracting miRNA, comprising the following steps:
    i)向样本保存液中加入裂解液；优选地，还加入蛋白酶K；i) adding a lysate to the sample preservation solution; preferably, proteinase K is also added;
    ii)加入结合液与核酸吸附材料；ii) adding binding solution and nucleic acid adsorption material;
    iii)去除液体部分，向核酸吸附材料加入漂洗液清洗；和iii) removing the liquid portion, adding a rinse solution to the nucleic acid adsorbing material for washing; and
    iv)去除液体部分，向所述核酸吸附材料中加入洗脱液，将所述核酸和/或所述miRNA从所述核酸吸附材料中洗脱。iv) removing the liquid part, adding an eluent to the nucleic acid adsorption material, and eluting the nucleic acid and/or the miRNA from the nucleic acid adsorption material.
  59. 如权利要求57-58任一项所述的方法，其特征在于，所述裂解液包含选自硫氰酸胍，柠檬酸，曲拉通-100，和/或EDTA的一种或多种组分；优选地，所述裂解液包含硫氰酸胍，柠檬酸，曲拉通-100，和EDTA。The method according to any one of claims 57-58, wherein the lysate comprises one or more groups selected from guanidine thiocyanate, citric acid, Triton-100, and/or EDTA points; preferably, the lysate comprises guanidine thiocyanate, citric acid, Triton-100, and EDTA.
  60. 如权利要求59所述的方法，其特征在于，所述裂解液中：The method according to claim 59, wherein in the lysate:
    所述硫氰酸胍的含量范围是28％-40％(w/w)；The content scope of described guanidinium thiocyanate is 28%-40% (w/w);
    所述柠檬酸的含量范围是1.8％-2.6％(w/w)；The content range of the citric acid is 1.8%-2.6% (w/w);
    所述曲拉通-100的含量范围是0.8％-1.6％(w/w)；The content range of the Triton-100 is 0.8%-1.6% (w/w);
    所述EDTA的含量范围是0.5％-0.9％( x/w)；和/或 The content range of the EDTA is 0.5%-0.9% ( x /w); and/or
    所述裂解液的pH值范围为3.8-4.2。The pH range of the lysate is 3.8-4.2.
  61. 如权利要求59-60任一项所述的方法，其特征在于，所述裂解液中：The method according to any one of claims 59-60, wherein in the lysate:
    所述硫氰酸胍的含量是约32％(w/w)；The content of the guanidinium thiocyanate is about 32% (w/w);
    所述柠檬酸的含量是约2.2％(w/w)；The content of said citric acid is about 2.2% (w/w);
    所述曲拉通-100的含量是约1.1％(w/w)；The content of the Triton-100 is about 1.1% (w/w);
    所述EDTA的含量是约0.7％(w/w)；和/或The content of said EDTA is about 0.7% (w/w); and/or
    所述裂解液的pH值为约4.0。The pH of the lysate is about 4.0.
  62. 如权利要求57-61任一项所述的方法，其特征在于，所述结合液包含选自氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和/或异丙醇的一种或多种组分；优选地，所述结合液包含氯化镁，乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，十二烷基肌氨酸钠，和异丙醇。The method according to any one of claims 57-61, wherein the binding solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, lauryl sarcosine sodium bicarbonate, and/or one or more components of isopropanol; preferably, the combined solution comprises magnesium chloride, disodium edetate, sodium bicarbonate, guanidinium thiocyanate, lauryl inosine Sodium Hydroxide, and Isopropanol.
  63. 如权利要求62所述的方法，其特征在于，所述结合液中：The method according to claim 62, characterized in that, in the binding solution:
    所述氯化镁的含量范围是0.1％-0.4％(w/w)；The content range of the magnesium chloride is 0.1%-0.4% (w/w);
    所述乙二胺四乙酸二钠的含量范围是0.5％-1.0％(w/w)；The content range of the disodium edetate is 0.5%-1.0% (w/w);
    所述碳酸氢钠的含量范围是0.6％-1.0％(w/w)；The content scope of described sodium bicarbonate is 0.6%-1.0% (w/w);
    所述硫氰酸胍的含量范围是13％-16％(w/w)；The content range of the guanidine thiocyanate is 13%-16% (w/w);
    所述十二烷基肌氨酸钠的含量范围是1.0％-1.4％(w/w)；The content range of the sodium lauryl sarcosinate is 1.0%-1.4% (w/w);
    所述异丙醇的含量范围是60％-80％(w/w)；和/或The content range of the isopropanol is 60%-80% (w/w); and/or
    所述结合液的pH值范围是4.8-5.2。The pH range of the binding solution is 4.8-5.2.
  64. 如权利要求62-63任一项所述的方法，其特征在于，所述结合液中：The method according to any one of claims 62-63, wherein in the binding solution:
    所述氯化镁的含量是约0.24％(w/w)；The content of said magnesium chloride is about 0.24% (w/w);
    所述乙二胺四乙酸二钠的含量是约0.71％(w/w)；The content of disodium edetate is about 0.71% (w/w);
    所述碳酸氢钠的含量是约0.83％(w/w)；The content of the sodium bicarbonate is about 0.83% (w/w);
    所述硫氰酸胍的含量是约14.9％(w/w)；The content of guanidine thiocyanate is about 14.9% (w/w);
    所述十二烷基肌氨酸钠的含量是约1.2％(w/w)；The content of sodium sarcosyl is about 1.2% (w/w);
    所述异丙醇的含量是约70％(w/w)；和/或The content of said isopropanol is about 70% (w/w); and/or
    所述结合液的pH值是约5.0。The pH of the binding solution is about 5.0.
  65. 如权利要求57-64任一项所述的方法，其特征在于，在步骤iii)中，向所述核酸吸附材料依次加入漂洗液1和漂洗液2清洗，每次清洗后去除液体部分然后进行下一个清洗；优选地，向所述核酸吸附材料用所述漂洗液2清洗至少两次。The method according to any one of claims 57-64, characterized in that, in step iii), rinse solution 1 and rinse solution 2 are sequentially added to the nucleic acid adsorption material for cleaning, and the liquid part is removed after each cleaning and then carried out Next washing; preferably, washing the nucleic acid adsorption material with the rinse solution 2 at least twice.
  66. 如权利要求65所述的方法，其特征在于，所述漂洗液1包含选自乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和/或十二烷基肌氨酸钠的一种或多种组分；优选地，所述漂洗液1包含乙二胺四乙酸二钠，碳酸氢钠，硫氰酸胍，和十二烷基肌氨酸钠。The method according to claim 65, wherein the rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidinium thiocyanate, and/or sodium lauryl sarcosinate One or more components; preferably, the rinse solution 1 comprises disodium edetate, sodium bicarbonate, guanidinium thiocyanate, and sodium lauryl sarcosinate.
  67. 如权利要求66所述的方法，其特征在于，所述漂洗液1中：The method according to claim 66, wherein in the rinse solution 1:
    所述乙二胺四乙酸二钠的含量范围是0.4％-0.9％(w/w)；The content range of the disodium edetate is 0.4%-0.9% (w/w);
    所述碳酸氢钠的含量范围是0.5％-0.9％(w/w)；The content scope of described sodium bicarbonate is 0.5%-0.9% (w/w);
    所述硫氰酸胍的含量范围是50％-55％(w/w)；The content range of the guanidinium thiocyanate is 50%-55% (w/w);
    所述十二烷基肌氨酸钠的含量范围是0.8％-1.2％(w/w)；和/或The content range of the sodium lauryl sarcosyl is 0.8%-1.2% (w/w); and/or
    所述漂洗液1的pH值范围是6.2-6.6。The pH range of the rinse solution 1 is 6.2-6.6.
  68. 如权利要求66-67任一项所述的方法，其特征在于，所述漂洗液1中：The method according to any one of claims 66-67, wherein in the rinse solution 1:
    所述乙二胺四乙酸二钠的含量是约0.5％(w/w)；The content of disodium edetate is about 0.5% (w/w);
    所述碳酸氢钠的含量是约0.6％(w/w)；The content of the sodium bicarbonate is about 0.6% (w/w);
    所述硫氰酸胍的含量是约53％(w/w)；The content of the guanidinium thiocyanate is about 53% (w/w);
    所述十二烷基肌氨酸钠的含量是约0.9％(w/w)；和/或The content of sodium sarcosyl is about 0.9% (w/w); and/or
    所述漂洗液1的pH值是约6.4。The pH of the Rinse 1 was about 6.4.
  69. 如权利要求66-68任一项所述的方法，其特征在于，在接触所述核酸吸附材料前，向所述漂洗液1中加入乙醇，所述漂洗液1：乙醇的体积比为1∶1-1∶2；优选地，所述体积比为约2∶3。The method according to any one of claims 66-68, wherein ethanol is added to the rinse liquid 1 before contacting the nucleic acid adsorption material, and the volume ratio of the rinse liquid 1:ethanol is 1: 1-1:2; preferably, said volume ratio is about 2:3.
  70. 如权利要求65-69任一项所述的方法，其特征在于，所述漂洗液2包含三羟甲基氨基甲烷(Tris)或氯化钠；优选地，所述漂洗液2包含三羟甲基氨基甲烷(Tris)和氯化钠。The method according to any one of claims 65-69, wherein the rinse solution 2 comprises tris (Tris) or sodium chloride; preferably, the rinse solution 2 comprises tris Trisaminomethane (Tris) and Sodium Chloride.
  71. 如权利要求70所述的方法，其特征在于，所述漂洗液2中：The method according to claim 70, wherein in the rinse solution 2:
    所述三羟甲基氨基甲烷(Tris)的含量范围是0.5％-0.7％(w/w)；The content range of the tris (Tris) is 0.5%-0.7% (w/w);
    所述氯化钠的含量范围是0.6％-1.8％(w/w)；和/或The content range of the sodium chloride is 0.6%-1.8% (w/w); and/or
    所述漂洗液2的pH值范围是6.6-7.0。The pH range of the rinse solution 2 is 6.6-7.0.
  72. 如权利要求70-71任一项所述的方法，其特征在于，所述漂洗液2中：The method according to any one of claims 70-71, wherein in the rinsing solution 2:
    所述三羟甲基氨基甲烷(Tris)的含量是约0.6％(w/w)；The content of tris (Tris) is about 0.6% (w/w);
    所述氯化钠的含量是约1.2％(w/w)；和/或The sodium chloride content is about 1.2% (w/w); and/or
    所述漂洗液2的pH值是约6.8。The pH value of the rinse solution 2 is about 6.8.
  73. 如权利要求70-72任一项所述的方法，其特征在于，在接触所述核酸吸附材料前，向所述漂洗液2中加入乙醇，所述漂洗液2：乙醇的体积比为1∶1.6-1∶3；优选地，所述体积比为约1∶2.3。The method according to any one of claims 70-72, wherein ethanol is added to the rinse liquid 2 before contacting the nucleic acid adsorption material, and the volume ratio of the rinse liquid 2:ethanol is 1: 1.6-1:3; preferably, said volume ratio is about 1:2.3.
  74. 如权利要求57-73任一项所述的方法，其特征在于，所述洗脱液为DEPC水.The method according to any one of claims 57-73, wherein the eluent is DEPC water.
  75. 如权利要求57-74任一项所述的方法，其特征在于，所述核酸吸附材料为能够吸附RNA的磁珠。The method according to any one of claims 57-74, wherein the nucleic acid adsorption material is a magnetic bead capable of adsorbing RNA.
  76. 如权利要求75所述的方法，其特征在于，所述磁珠的磁核是氢氧化三铁，包被二 氧化硅，且表面镶嵌羟基基团；优选地，所述磁珠的粒径为50-100nm。The method according to claim 75, wherein the magnetic core of the magnetic beads is ferric hydroxide, coated with silicon dioxide, and the surface is embedded with hydroxyl groups; preferably, the particle diameter of the magnetic beads is 50-100nm.
  77. 如权利要求52-76任一项所述的方法，其特征在于，所述样本保存液包含选自EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和/或磷酸三钠的一种或多种组分；优选地，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。The method according to any one of claims 52-76, wherein the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and/or triphosphate One or more components of sodium; preferably, the sample preservation solution comprises EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and trisodium phosphate.
  78. 一种保存含核酸的生物样本的方法，其特征在于，将样本置于样本保存液中，所述样本保存液包含EDTA，NaCl，无水乙醇，柠檬酸钠，NP40，谷胱甘肽，和磷酸三钠。A method for preserving a nucleic acid-containing biological sample, characterized in that the sample is placed in a sample preservation solution, the sample preservation solution comprising EDTA, NaCl, absolute ethanol, sodium citrate, NP40, glutathione, and Trisodium Phosphate.
  79. 如权利要求77-78任一项所述的方法，其特征在于，所述样本保存液中：The method according to any one of claims 77-78, wherein in the sample preservation solution:
    所述EDTA的含量范围是0.7％-1.5％(w/w)；The content range of the EDTA is 0.7%-1.5% (w/w);
    所述NaCl的含量范围是0.01％-0.1％(w/w)；The content range of the NaCl is 0.01%-0.1% (w/w);
    所述无水乙醇的浓度范围是15％-35％(w/w)；The concentration range of the absolute ethanol is 15%-35% (w/w);
    所述柠檬酸钠的含量范围是0.1％-1.0％(w/w)；The content range of the sodium citrate is 0.1%-1.0% (w/w);
    所述NP40的浓度范围是0.01％-0.1％(v/v)；The concentration range of the NP40 is 0.01%-0.1% (v/v);
    所述谷胱甘肽的含量范围是0.07％-0.7％(w/w)；和/或The glutathione content ranges from 0.07% to 0.7% (w/w); and/or
    所述磷酸三钠的含量范围是0.07％-0.4％(w/w)。The content range of the trisodium phosphate is 0.07%-0.4% (w/w).
  80. 如权利要求77-79任一项所述的方法，其特征在于，所述样本保存液中：The method according to any one of claims 77-79, wherein in the sample preservation solution:
    所述EDTA的含量是约1.05％(w/w)；The content of said EDTA is about 1.05% (w/w);
    所述NaCl的含量是约0.035％(w/w)；The content of said NaCl is about 0.035% (w/w);
    所述无水乙醇的浓度是约27.6％(w/w)；The concentration of absolute ethanol is about 27.6% (w/w);
    所述柠檬酸钠的含量是约0.21％(w/w)；The content of the sodium citrate is about 0.21% (w/w);
    所述NP40的浓度是约0.035％(v/v)；The concentration of said NP40 is about 0.035% (v/v);
    所述谷胱甘肽的含量是约0.21％(w/w)；和/或The glutathione content is about 0.21% (w/w); and/or
    所述磷酸三钠的含量是约0.14％(w/w)。The content of the trisodium phosphate is about 0.14% (w/w).
  81. 如权利要求77-80一项所述的方法，其特征在于，所述样本保存液包含抗生素；优选地，所述抗生素包含青霉素-链霉素双抗。The method according to one of claims 77-80, wherein the sample preservation solution contains antibiotics; preferably, the antibiotics contain penicillin-streptomycin double antibody.
  82. 如权利要求77-81任一项所述的方法，其特征在于，所述样本保存液的pH值为7.6-8.0；优选地，所述样本保存液的pH值为7.8。The method according to any one of claims 77-81, wherein the pH value of the sample preservation solution is 7.6-8.0; preferably, the pH value of the sample preservation solution is 7.8.
  83. 如权利要求77-82任一项所述的方法，其特征在于，与对照样本保存液相比，所述样本保存液对DNA，miRNA，和/或DNA甲基化的保存效果更好。The method according to any one of claims 77-82, characterized in that, compared with the control sample preservation solution, the preservation effect of the sample preservation solution on DNA, miRNA, and/or DNA methylation is better.
  84. 如权利要求52-83任一项所述的方法，其特征在于，包含步骤(b-3)，所述EB病 毒载量检测包含针对EB病毒基因组中BamHI-W片段，和/或LMP-2基因的检测。The method according to any one of claims 52-83, characterized in that, comprising step (b-3), the detection of the EB virus load comprises targeting the BamHI-W fragment in the Epstein-Barr virus genome, and/or LMP-2 genetic testing.
  85. 如权利要求84所述的方法，其特征在于，所述EB病毒载量检测包含针对LMP-2基因的检测。The method according to claim 84, wherein the detection of the EB virus load comprises the detection of the LMP-2 gene.
  86. 如权利要求52-85任一项所述的方法，其特征在于，所述方法用于鼻咽癌的筛查和/或诊断。The method according to any one of claims 52-85, wherein the method is used for screening and/or diagnosing nasopharyngeal carcinoma.
  87. 如权利要求52-86任一项所述的方法，其特征在于，所述样本是鼻咽部样本。The method of any one of claims 52-86, wherein the sample is a nasopharyngeal sample.
  88. 如权利要求52-87任一项所述的方法，其特征在于，在步骤(a)之前，还包括使用鼻咽刷和/或鼻咽拭子获取所述样本；优选地，所述鼻咽刷和/或鼻咽拭子为不依赖临床医生和鼻咽镜引导的鼻咽刷和/或鼻咽拭子。The method according to any one of claims 52-87, characterized in that, before step (a), further comprising using a nasopharyngeal brush and/or a nasopharyngeal swab to obtain the sample; preferably, the nasopharyngeal The brush and/or nasopharyngeal swab is a nasopharyngeal brush and/or nasopharyngeal swab that is independent of the clinician and nasopharyngoscope guidance.
  89. 一种对有需要的个体治疗鼻咽癌的方法，包含对所述个体提供药物、放疗、手术、细胞疗法和/或溶瘤病毒治疗，其中，所述个体的样本接受了如权利要求52-88任一项所述的方法的检测并被判断为鼻咽癌阳性。A method of treating nasopharyngeal carcinoma for an individual in need thereof, comprising providing the individual with drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus therapy, wherein the sample of the individual has received the method of claim 52- 88. The method described in any one of 88 is detected and judged to be positive for nasopharyngeal carcinoma.
  90. 一种对有需要的个体治疗鼻咽癌的方法，包含1)对来自所述个体的样本进行如权利要求52-88任一项所述的方法的检测；2)如检测结果显示鼻咽癌阳性，提供鼻咽癌治疗；优选地，所述鼻咽癌治疗采用药物、放疗、手术、细胞疗法和/或溶瘤病毒治疗。A method for treating nasopharyngeal carcinoma in an individual in need, comprising 1) performing detection according to the method according to any one of claims 52-88 on a sample from the individual; 2) if the detection result shows nasopharyngeal carcinoma If it is positive, provide nasopharyngeal carcinoma treatment; preferably, the nasopharyngeal carcinoma treatment adopts drugs, radiotherapy, surgery, cell therapy and/or oncolytic virus treatment.
  91. 如权利要求1-51任一项所述的试剂盒在制备鼻咽癌筛查和/或诊断的产品的用途。Use of the kit according to any one of claims 1-51 in preparing products for screening and/or diagnosing nasopharyngeal carcinoma.
PCT/CN2022/127393 2021-10-25 2022-10-25 Reagent and method for diagnosis of nasopharyngeal carcinoma WO2023072076A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202111251797 2021-10-25
CN202111251797.9 2021-10-25
CN202211272683.7A CN115521996A (en) 2021-10-25 2022-10-18 Reagent and method for diagnosing nasopharyngeal carcinoma
CN202211272683.7 2022-10-18

Publications (1)

Publication Number Publication Date
WO2023072076A1 true WO2023072076A1 (en) 2023-05-04

Family

ID=84703373

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/127393 WO2023072076A1 (en) 2021-10-25 2022-10-25 Reagent and method for diagnosis of nasopharyngeal carcinoma

Country Status (3)

Country Link
CN (1) CN115521996A (en)
TW (1) TW202340478A (en)
WO (1) WO2023072076A1 (en)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110151430A1 (en) * 2007-09-06 2011-06-23 University Of Massachusetts VIRUS-SPECIFIC miRNA SIGNATURES FOR DIAGNOSIS AND THERAPEUTIC TREATMENT OF VIRAL INFECTION
CN102268477A (en) * 2011-06-15 2011-12-07 中山大学肿瘤防治中心 Application of nasopharyngeal carcinoma related EB (Epstein-Barr) virus miRNAs (microribonucleic acids)
US20140235484A1 (en) * 2013-02-19 2014-08-21 The Johns Hopkins University Methods for detection and differentiation of origin of viral dna
US20170051359A1 (en) * 2014-05-01 2017-02-23 Stichting Vumc SMALL ncRNAS AS BIOMARKERS
CN106755590A (en) * 2017-01-22 2017-05-31 中南大学湘雅医院 One kind is for detecting leaflet patient's Epstein-Barr virus FISH detection probes
US20190085417A1 (en) * 2015-12-18 2019-03-21 Lucence Diagnostics Pte Ltd Detection and Quantification of Target Nucleic Acid Sequence of a Microorganism
CN110079603A (en) * 2019-06-14 2019-08-02 福建省肿瘤医院（福建省肿瘤研究所、福建省癌症防治中心） A kind of detection primer, probe and its application of nasopharynx carcinoma marker
CN111902545A (en) * 2019-01-03 2020-11-06 杭州诺辉健康科技有限公司 Composition and method for urine sample preservation and DNA extraction
CN114457192A (en) * 2021-07-07 2022-05-10 中山大学 Application of reagent for methylation typing of EB virus CpG sites in saliva in preparation of nasopharyngeal carcinoma diagnostic kit

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110151430A1 (en) * 2007-09-06 2011-06-23 University Of Massachusetts VIRUS-SPECIFIC miRNA SIGNATURES FOR DIAGNOSIS AND THERAPEUTIC TREATMENT OF VIRAL INFECTION
CN102268477A (en) * 2011-06-15 2011-12-07 中山大学肿瘤防治中心 Application of nasopharyngeal carcinoma related EB (Epstein-Barr) virus miRNAs (microribonucleic acids)
US20140235484A1 (en) * 2013-02-19 2014-08-21 The Johns Hopkins University Methods for detection and differentiation of origin of viral dna
US20170051359A1 (en) * 2014-05-01 2017-02-23 Stichting Vumc SMALL ncRNAS AS BIOMARKERS
US20190085417A1 (en) * 2015-12-18 2019-03-21 Lucence Diagnostics Pte Ltd Detection and Quantification of Target Nucleic Acid Sequence of a Microorganism
CN106755590A (en) * 2017-01-22 2017-05-31 中南大学湘雅医院 One kind is for detecting leaflet patient's Epstein-Barr virus FISH detection probes
CN111902545A (en) * 2019-01-03 2020-11-06 杭州诺辉健康科技有限公司 Composition and method for urine sample preservation and DNA extraction
CN110079603A (en) * 2019-06-14 2019-08-02 福建省肿瘤医院（福建省肿瘤研究所、福建省癌症防治中心） A kind of detection primer, probe and its application of nasopharynx carcinoma marker
CN114457192A (en) * 2021-07-07 2022-05-10 中山大学 Application of reagent for methylation typing of EB virus CpG sites in saliva in preparation of nasopharyngeal carcinoma diagnostic kit

Also Published As

Publication number Publication date
CN115521996A (en) 2022-12-27
TW202340478A (en) 2023-10-16

Similar Documents

Publication Publication Date Title
CN110904225B (en) Combined marker for liver cancer detection and application thereof
WO2021223349A1 (en) Protection sequence, primer, probe, composition, and kit for steady-state rapid detection of novel coronavirus, and use thereof and method therefor
WO2017124854A1 (en) Primers, probe and kit for detecting ndrg4 gene methylation for early diagnosis of intestinal cancer
US9732388B2 (en) Nucleic acid markers for rapid diagnosis of kawasaki disease and kit for detection of the nucleic acid markers
CN110387421A (en) DNA methylation qPCR kit and application method for lung cancer detection
WO2023280050A1 (en) Application of reagent for methylation typing of cpg site of eb virus in saliva in preparation of nasopharyngeal carcinoma diagnostic kit
WO2017054325A1 (en) Breast cancer combined diagnosis markers and detection kit
US20220259672A1 (en) Gene marker combination and use thereof
WO2020034583A1 (en) Set of genes for bladder cancer detection and application thereof
CN114875141A (en) Application of exosome miR-106b-3p, miR-450b-5p and the like in lung cancer diagnosis
CN108950003B (en) miRNA marker for diagnosing breast cancer and application of miRNA thereof
CN108866187B (en) Long-chain non-coding RNA marker related to lung cancer auxiliary diagnosis and application thereof
CN113549694A (en) Novel cervical cancer methylation gene detection method
CN111455053B (en) Exosome RNA molecular marker combination for colorectal adenoma diagnosis and application thereof
CN109402262A (en) The PCR detection kit of auxiliary diagnosis neuroblastoma and the method for detecting miR-199a-3p expression
CN109735612B (en) Biomolecule marker of Kawasaki disease coronary aneurysm complication and kit thereof
WO2023072076A1 (en) Reagent and method for diagnosis of nasopharyngeal carcinoma
TWI385252B (en) Cancer screening method
CN112779329A (en) Molecular marker for auxiliary diagnosis of viral meningitis and application and kit thereof
CN112522391B (en) Application of hsa_circ_0008961 as gout diagnosis marker
CN108913762A (en) The application of kit and CST1 gene as biomarker for detecting chronic nasosinusitis with nasal polyp hypotype
CN111733226B (en) Circular RNA marker for detecting Crohn's disease and application
WO2017167034A1 (en) Bladder cancer detection method and kit
WO2023273257A1 (en) Diagnostic or auxiliary diagnostic reagent for colorectal cancer or precancerous lesions and use thereof, nucleic acid combination, and kit
CN114410795A (en) Liver cancer early detection based on miRNA (micro ribonucleic acid) feature marker

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22885953

Country of ref document: EP

Kind code of ref document: A1