WO2023072076A1 - Réactif et procédé de diagnostic du carcinome nasopharyngien - Google Patents

Réactif et procédé de diagnostic du carcinome nasopharyngien Download PDF

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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
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content
solution
epstein
mirna
barr virus
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Chinese (zh)
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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
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杭州诺辉健康科技有限公司
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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.

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Abstract

Application d'un réactif pour le typage des sites de méthylation du virus d'Epstein-Barr (EB) et la détection des miARN liés au virus EB dans la préparation d'un kit de diagnostic du carcinome nasopharyngé. Un site de méthylation du virus EB et/ou un miARN lié au virus EB peut servir de marqueur de diagnostic du carcinome nasopharyngé, ce qui comporte les avantages d'une sensibilité et d'une spécificité élevées dans un échantillon de brosse nasopharyngée ou d'écouvillon nasopharyngé (détection aveugle du nasopharynx) ne nécessitant pas l'intervention d'un clinicien et d'un nasopharyngoscope.
PCT/CN2022/127393 2021-10-25 2022-10-25 Réactif et procédé de diagnostic du carcinome nasopharyngien WO2023072076A1 (fr)

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CN111902545A (zh) * 2019-01-03 2020-11-06 杭州诺辉健康科技有限公司 尿液样本保存及dna提取的组合物及方法
CN114457192A (zh) * 2021-07-07 2022-05-10 中山大学 对唾液中EB病毒CpG位点进行甲基化分型的试剂在制备鼻咽癌诊断试剂盒中的应用

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US20110151430A1 (en) * 2007-09-06 2011-06-23 University Of Massachusetts VIRUS-SPECIFIC miRNA SIGNATURES FOR DIAGNOSIS AND THERAPEUTIC TREATMENT OF VIRAL INFECTION
CN102268477A (zh) * 2011-06-15 2011-12-07 中山大学肿瘤防治中心 一种与鼻咽癌相关的EB病毒miRNA的应用
US20140235484A1 (en) * 2013-02-19 2014-08-21 The Johns Hopkins University Methods for detection and differentiation of origin of viral dna
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