CN118256653A - Composition and kit for human papilloma virus detection and typing - Google Patents

Composition and kit for human papilloma virus detection and typing Download PDF

Info

Publication number
CN118256653A
CN118256653A CN202211692589.7A CN202211692589A CN118256653A CN 118256653 A CN118256653 A CN 118256653A CN 202211692589 A CN202211692589 A CN 202211692589A CN 118256653 A CN118256653 A CN 118256653A
Authority
CN
China
Prior art keywords
seq
primer pair
type
specific probe
specific
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202211692589.7A
Other languages
Chinese (zh)
Inventor
彭凯秀
朱威
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Getein Biotech Inc
Original Assignee
Getein Biotech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Getein Biotech Inc filed Critical Getein Biotech Inc
Priority to CN202211692589.7A priority Critical patent/CN118256653A/en
Publication of CN118256653A publication Critical patent/CN118256653A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/708—Specific hybridization probes for papilloma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to a composition for human papillomavirus detection and typing, which is characterized by comprising a primer pair and probes corresponding to the primer pair, wherein the probes comprise a specific probe 1 aiming at HPV16 type, a specific probe 2 aiming at HPV18 type and a specific probe 3 aiming at other high-risk types of HPV, fluorescent marking groups are connected to the specific probe 1, the specific probe 2 and the specific probe 3, and fluorescent signals generated by the fluorescent marking groups on the specific probe 1, the specific probe 2 and the specific probe 3 in a PCR process are different. The invention also relates to a kit adopting the composition. The kit provided by the invention adopts a fluorescent PCR method to carry out HPV typing detection, can cover more than 98% of 14 types of HPV clinically infected at one time, can detect HPV16 type, HPV18 type and other 12 types of high-risk HPVs (namely 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) at one time, and has high sensitivity and high accuracy of detection results.

Description

Composition and kit for human papilloma virus detection and typing
Technical Field
The invention relates to the field of molecular biology detection, in particular to a composition and a kit for human papilloma virus detection and typing.
Background
Human papillomaviruses (huamn papilloma virus, HPV) are a mucosal and cutaneous epithelial virus, a major factor leading to cervical epithelial hyperplasia and cervical cancer, and up to 100 HPV viruses have been identified to date, with more than 30 being able to infect the human genital mucosa. The occurrence of cervical cancer has a close relationship with human papillomavirus (hereinafter referred to as HPV) infection. At present, about 20 ten thousand women die from the disease every year, and the trend of younger age is more obvious, thereby attracting general attention of all communities. The World Health Organization (WHO) survey results show that 99.9% of cervical cancer patients can detect HPV infection; the international association of cancer research (IARC) announced in 1995: HPV infection is the main cause of cervical cancer. Among the 30 kinds of HPV which can infect the mucous membrane of human genital tract, some of them are very easy to cause cervical cancer, and are called high-risk HPV; the WHO international cancer research center classifies HPV types into three types according to the size of oncogenic potential, namely high-risk HPV (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, 82), assumed high-risk type (HPV 26, 53, 66) and low-risk type (HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81), of which HPV types that are most commonly oncogenic are HPV16, 18 types.
Currently, detection of HPV infection is largely divided into cytological detection and HPV nucleic acid detection. Compared with cytological examination, the nucleic acid detection has the characteristics of high sensitivity, low technical requirements on operators, specific typing on HPV infection types and the like. In China, 38 companies and 75 HPV detection products currently obtain CFDA registration certificates, wherein 46 products use a fluorescent PCR method, 11 products use a PCR-reverse dot hybridization method (including a gene chip method), 6 products use a hybridization capture method, and the rest products use the following methods: an enzyme digestion signal amplification method, a branched DNA signal amplification method, a constant temperature amplification fluorescent method, a flow hybridization method, a surface plasmon resonance method, a semiconductor sequencing method and the like. In the HPV nucleic acid detection kit, pretreatment is complex and detection time is long.
Disclosure of Invention
The invention overcomes the defects in the prior art and provides a composition and a kit for human papillomavirus detection typing, which can realize rapid and accurate detection.
The specific technical scheme of the invention is as follows:
The composition for human papillomavirus detection and typing comprises a primer pair and probes corresponding to the primer pair, wherein the probes comprise a specific probe 1 aiming at HPV16 type, a specific probe 2 aiming at HPV18 type and a specific probe 3 aiming at other high-risk types of HPV, fluorescent marker groups are connected to the specific probe 1, the specific probe 2 and the specific probe 3, and fluorescent signals generated by the fluorescent marker groups on the specific probe 1, the specific probe 2 and the specific probe 3 in a PCR process are different.
Preferably, the primer pair comprises a primer pair 1 for HPV16 type, a primer pair 2 for HPV18 type and a primer pair 3 for other high-risk types;
The nucleotide sequences of the primer pair 1 are shown as SEQ ID NO.01 and SEQ ID NO.02, and the nucleotide sequence of the specific probe 1 is shown as SEQ ID NO. 29; the nucleotide sequences of the primer pair 2 are shown in SEQ ID NO.03 and SEQ ID NO.04, and the nucleotide sequence of the specific probe 2 is shown in SEQ ID NO. 30;
SEQ ID NO.01：5′-CGCCATGAGACTGAAACACCA-3′；
SEQ ID NO.02：5′-TCATTATCATTTACTATA-3′；
SEQ ID NO.29：5′-TGGTTTTATGTAGAGGCTGTAG-3′；
SEQ ID NO.03：5′-TACAGTAGTGATACTG-3′；
SEQ ID NO.04：5′-ATTCCTCCGCTTCCT-3′；
SEQ ID NO.30：5′-TGTAATGTATACAACAATCAGGCAG-3′。
Preferably, the concentration of each primer in the primer pair 1 and the primer pair 2 is 0.05-0.08 mu M, and the concentration of the specific probe 1 and the specific probe 2 is 0.03-0.06 mu M.
Preferably, the primer pair 3 comprises a primer pair 4 for HPV31 type, a primer pair 5 for HPV33 type, a primer pair 6 for HPV35 type, a primer pair 7 for HPV39 type, a primer pair 8 for HPV45 type, a primer pair 9 for HPV51 type, a primer pair 10 for HPV52 type, a primer pair 11 for HPV56 type, a primer pair 12 for HPV58 type, a primer pair 13 for HPV59 type, a primer pair 14 for HPV66 type, a primer pair 15 for HPV68 type,
The specific probes 3 comprise specific probes 4 for HPV31 type, specific probes 5 for HPV33 type, specific probes 6 for HPV35 type, specific probes 7 for HPV39 type, specific probes 8 for HPV45 type, specific probes 9 for HPV51 type, specific probes 10 for HPV52 type, specific probes 11 for HPV56 type, specific probes 12 for HPV58 type, specific probes 13 for HPV59 type, specific probes 14 for HPV66 type and specific probes 15 for HPV68 type,
The nucleotide sequences of the primer pair 4 are shown as SEQ ID NO.05 and SEQ ID NO.06, and the nucleotide sequence of the specific probe 4 is shown as SEQ ID NO. 31; the nucleotide sequences of the primer pair 5 are shown as SEQ ID NO.07 and SEQ ID NO.08, and the nucleotide sequence of the specific probe 5 is shown as SEQ ID NO. 32; the nucleotide sequences of the primer pair 6 are shown as SEQ ID NO.09 and SEQ ID NO.10, and the nucleotide sequence of the specific probe 6 is shown as SEQ ID NO. 33; the nucleotide sequences of the primer pair 7 are shown as SEQ ID NO.11 and SEQ ID NO.12, and the nucleotide sequence of the specific probe 7 is shown as SEQ ID NO. 34; the nucleotide sequence of the primer pair 8 is shown as SEQ ID NO.13 and SEQ ID NO.14, and the nucleotide sequence of the specific probe 8 is shown as SEQ ID NO. 35; the nucleotide sequences of the primer pair 9 are shown as SEQ ID NO.15 and SEQ ID NO.16, and the nucleotide sequence of the specific probe 9 is shown as SEQ ID NO. 36; the nucleotide sequences of the primer pair 10 are shown as SEQ ID NO.17 and SEQ ID NO.18, and the nucleotide sequence of the specific probe 10 is shown as SEQ ID NO. 37; the nucleotide sequences of the primer pair 11 are shown as SEQ ID NO.19 and SEQ ID NO.20, and the nucleotide sequence of the specific probe 11 is shown as SEQ ID NO. 38; the nucleotide sequences of the primer pair 12 are shown as SEQ ID NO.21 and SEQ ID NO.22, and the nucleotide sequence of the specific probe 12 is shown as SEQ ID NO. 39; the nucleotide sequences of the primer pair 13 are shown as SEQ ID NO.23 and SEQ ID NO.24, and the nucleotide sequence of the specific probe 13 is shown as SEQ ID NO. 40; the nucleotide sequences of the primer pair 14 are shown as SEQ ID NO.25 and SEQ ID NO.26, and the nucleotide sequence of the specific probe 14 is shown as SEQ ID NO. 41; the nucleotide sequences of the primer pair 15 are shown as SEQ ID NO.27 and SEQ ID NO.28, and the nucleotide sequence of the specific probe 15 is shown as SEQ ID NO. 42;
SEQ ID NO.05：5′-ATGACACAACATTTGATTTGTCCC-3′；
SEQ ID NO.06：5′-GCATTACTATCACTGTCAGCT-3′；
SEQ ID NO.31：5′-AATGGTACAATGGGCATATGACAAT-3′；
SEQ ID NO.07：5′-CAGCCCGGGCATTGTTTAAT-3′；
SEQ ID NO.08：5′-ATCTTCTGTCGCCTATACCGA-3′；
SEQ ID NO.32：5′-TGTGTGTGCACTAAAACGAAAGT-3′；
SEQ ID NO.09：5′-CAGAATGGATTCAAAGACAAACAG-3′；
SEQ ID NO.10：5′-TTCTAACACGTTGTTACAC-3′；
SEQ ID NO.33：5′-ACAATGGGCATATGACAATGAT-3′；
SEQ ID NO.11：5′-GCAACAGATACAGGTTCAG-3′；
SEQ ID NO.12：5′-ATGTCCGTCTCGCACTCTG-3′；
SEQ ID NO.34：5′-TACAGGCAGAGCGTGAGACA-3′；
SEQ ID NO.13：5′-ATGTAGATCCGCATTGCAGT-3′；
SEQ ID NO.14：5′-TGATTTCCTCGATAATGTTC-3′；
SEQ ID NO.35：5′-AGGCTGCAATGCTGGCAGTATT-3′；
SEQ ID NO.15：5′-TAAGTACATTAGTAAATAT-3′；
SEQ ID NO.16：5′-AATATCTTGTCCGTATAGTTC-3′；
SEQ ID NO.36：5′-TATAGAACCACCAAAATTACGTAGT-3′；
SEQ ID NO.17：5′-AGTAGATTGGTCGTGTTT-3′；
SEQ ID NO.18：5′-TCCTTCTCCTGGTTTTGTA-3′；
SEQ ID NO.37：5′-CTCAAGGACGTGGTGCAAATTAG-3′；
SEQ ID NO.19：5′-ATTGTGGAATAATGTGTAGA-3′；
SEQ ID NO.20：5′-TCTATAGTTCCCGAGCTA-3′；
SEQ ID NO.38：5′-TGAATATGTGCCAGTGGATAA-3′；
SEQ ID NO.21：5′-TACCTCAAATACAAATGCA-3′；
SEQ ID NO.22：5′-TCCTTCTCCTGTTCCTT-3′；
SEQ ID NO.39：5′-CTCAAGGACGTGGTGCAAATTAG-3′；
SEQ ID NO.23：5′-TGCAACAGATACAGGTTCAG-3′；
SEQ ID NO.24：5′-CGGGTTTCCCTACGTGC-3′；
SEQ ID NO.40：5′-TACAGGCAGAGCGCGAGACAGC-3′；
SEQ ID NO.25：5′-ATACAAAGACAGACACA-3′；
SEQ ID NO.26：5′-GTTGTGTCAAATGTTCTG-3′；
SEQ ID NO.41：5′-CAACACAGTTTACAAGACAATCAAT-3′；
SEQ ID NO.27：5′-ACGCAACAGATACAGGTTCAG-3′；
SEQ ID NO.28：5′-ACGTTCTCCGGGTTTC-3′；
SEQ ID NO.42：5′-TACAGGCAGAGCGTGAGACA-3′。
Preferably, the concentration of each primer in the primer pair 3 is 0.05-0.1 mu M, and the concentration of each probe in the specific probe 3 is 0.02-0.06 mu M.
Preferably, the specific probe 1 is connected with a CY5 fluorescent labeling group, the specific probe 2 is connected with a FAM fluorescent labeling group, and the specific probe 3 is connected with a JOE fluorescent labeling group.
A kit for human papillomavirus detection typing, the kit comprising: a PCR reaction system, taq DNA polymerase, a positive reference substance, a negative reference substance and preservation solution; the PCR reaction system comprises a buffer, a composition according to any one of claims 1 to 6.
Preferably, the buffer comprises 70-120 mM Tris-HCl, 30-60 mM KCl, 4-7 mM MgCl2 and 0.5-0.8 mM dNTPs.
Preferably, the preservation solution comprises 35-60 mM of isoguanidine salt, 0.1-0.5 w% of trehalose, 1-10 mM of Tris-HCl, 1-8 v% of DMSO and 2-8 mM of betaine.
The kit provided by the invention adopts a fluorescent PCR method to carry out HPV typing detection, can cover more than 98% of 14 types of HPV clinically infected at one time, can detect HPV16 type, 18 type and other 12 types of high-risk HPVs (namely 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) by one-time sampling, and can finish screening and shunting in one step. The sample extraction step can be omitted by innovatively adding the nucleic acid extraction reagent into the preservation solution of the sampling tube, the sample can be directly sampled and put on the machine after being sampled, the result can be obtained after the rapid amplification only needs 30 minutes, the sensitivity is high, and the detection result is highly accurate. The kit can meet the requirement that the detection limit is not higher than 1copies/reaction, and the kit prepared from the composition has stronger anti-interference capability.
Drawings
FIG. 1 is a graph showing the test results of the positive control of example 2 of the present invention;
FIG. 2 is a graph showing the test results of the negative control of example 2 of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art without the exercise of inventive faculty, are intended to be within the scope of the invention. The present invention is not mentioned in part in the prior art.
The concentration of each primer and the concentration of the probe in the primer pair refer to the concentration in a PCR reaction system.
The invention discloses a composition for human papillomavirus detection and typing, which comprises a primer pair and probes corresponding to the primer pair, wherein the probes comprise specific probes 1 and 2 for HPV16 type and other high-risk types of HPV, and fluorescent signals generated by the fluorescent marking groups on the specific probes 1, 2 and 3 in the PCR process are different.
Further, the primer pair comprises a primer pair 1 aiming at HPV16 type, a primer pair 2 aiming at HPV18 type and a primer pair 3 aiming at other high-risk types;
The nucleotide sequences of the primer pair 1 are shown as SEQ ID NO.01 and SEQ ID NO.02, and the nucleotide sequence of the specific probe 1 is shown as SEQ ID NO. 29; the nucleotide sequences of the primer pair 2 are shown in SEQ ID NO.03 and SEQ ID NO.04, and the nucleotide sequence of the specific probe 2 is shown in SEQ ID NO. 30;
SEQ ID NO.01：5′-CGCCATGAGACTGAAACACCA-3′；
SEQ ID NO.02：5′-TCATTATCATTTACTATA-3′；
SEQ ID NO.29：5′-TGGTTTTATGTAGAGGCTGTAG-3′；
SEQ ID NO.03：5′-TACAGTAGTGATACTG-3′；
SEQ ID NO.04：5′-ATTCCTCCGCTTCCT-3′；
SEQ ID NO.30：5′-TGTAATGTATACAACAATCAGGCAG-3′。
Further, the concentration of each primer in the primer pair 1 and the primer pair 2 is 0.05-0.08 mu M, and the concentration of the specific probe 1 and the specific probe 2 is 0.03-0.06 mu M.
Further, the primer pair 3 comprises a primer pair 4 for HPV31 type, a primer pair 5 for HPV33 type, a primer pair 6 for HPV35 type, a primer pair 7 for HPV39 type, a primer pair 8 for HPV45 type, a primer pair 9 for HPV51 type, a primer pair 10 for HPV52 type, a primer pair 11 for HPV56 type, a primer pair 12 for HPV58 type, a primer pair 13 for HPV59 type, a primer pair 14 for HPV66 type, a primer pair 15 for HPV68 type,
The specific probes 3 comprise specific probes 4 for HPV31 type, specific probes 5 for HPV33 type, specific probes 6 for HPV35 type, specific probes 7 for HPV39 type, specific probes 8 for HPV45 type, specific probes 9 for HPV51 type, specific probes 10 for HPV52 type, specific probes 11 for HPV56 type, specific probes 12 for HPV58 type, specific probes 13 for HPV59 type, specific probes 14 for HPV66 type and specific probes 15 for HPV68 type,
The nucleotide sequences of the primer pair 4 are shown as SEQ ID NO.05 and SEQ ID NO.06, and the nucleotide sequence of the specific probe 4 is shown as SEQ ID NO. 31; the nucleotide sequences of the primer pair 5 are shown as SEQ ID NO.07 and SEQ ID NO.08, and the nucleotide sequence of the specific probe 5 is shown as SEQ ID NO. 32; the nucleotide sequences of the primer pair 6 are shown as SEQ ID NO.09 and SEQ ID NO.10, and the nucleotide sequence of the specific probe 6 is shown as SEQ ID NO. 33; the nucleotide sequences of the primer pair 7 are shown as SEQ ID NO.11 and SEQ ID NO.12, and the nucleotide sequence of the specific probe 7 is shown as SEQ ID NO. 34; the nucleotide sequence of the primer pair 8 is shown as SEQ ID NO.13 and SEQ ID NO.14, and the nucleotide sequence of the specific probe 8 is shown as SEQ ID NO. 35; the nucleotide sequences of the primer pair 9 are shown as SEQ ID NO.15 and SEQ ID NO.16, and the nucleotide sequence of the specific probe 9 is shown as SEQ ID NO. 36; the nucleotide sequences of the primer pair 10 are shown as SEQ ID NO.17 and SEQ ID NO.18, and the nucleotide sequence of the specific probe 10 is shown as SEQ ID NO. 37; the nucleotide sequences of the primer pair 11 are shown as SEQ ID NO.19 and SEQ ID NO.20, and the nucleotide sequence of the specific probe 11 is shown as SEQ ID NO. 38; the nucleotide sequences of the primer pair 12 are shown as SEQ ID NO.21 and SEQ ID NO.22, and the nucleotide sequence of the specific probe 12 is shown as SEQ ID NO. 39; the nucleotide sequences of the primer pair 13 are shown as SEQ ID NO.23 and SEQ ID NO.24, and the nucleotide sequence of the specific probe 13 is shown as SEQ ID NO. 40; the nucleotide sequences of the primer pair 14 are shown as SEQ ID NO.25 and SEQ ID NO.26, and the nucleotide sequence of the specific probe 14 is shown as SEQ ID NO. 41; the nucleotide sequences of the primer pair 15 are shown as SEQ ID NO.27 and SEQ ID NO.28, and the nucleotide sequence of the specific probe 15 is shown as SEQ ID NO. 42;
SEQ ID NO.05：5′-ATGACACAACATTTGATTTGTCCC-3′；
SEQ ID NO.06：5′-GCATTACTATCACTGTCAGCT-3′；
SEQ ID NO.31：5′-AATGGTACAATGGGCATATGACAAT-3′；
SEQ ID NO.07：5′-CAGCCCGGGCATTGTTTAAT-3′；
SEQ ID NO.08：5′-ATCTTCTGTCGCCTATACCGA-3′；
SEQ ID NO.32：5′-TGTGTGTGCACTAAAACGAAAGT-3′；
SEQ ID NO.09：5′-CAGAATGGATTCAAAGACAAACAG-3′；
SEQ ID NO.10：5′-TTCTAACACGTTGTTACAC-3′；
SEQ ID NO.33：5′-ACAATGGGCATATGACAATGAT-3′；
SEQ ID NO.11：5′-GCAACAGATACAGGTTCAG-3′；
SEQ ID NO.12：5′-ATGTCCGTCTCGCACTCTG-3′；
SEQ ID NO.34：5′-TACAGGCAGAGCGTGAGACA-3′；
SEQ ID NO.13：5′-ATGTAGATCCGCATTGCAGT-3′；
SEQ ID NO.14：5′-TGATTTCCTCGATAATGTTC-3′；
SEQ ID NO.35：5′-AGGCTGCAATGCTGGCAGTATT-3′；
SEQ ID NO.15：5′-TAAGTACATTAGTAAATAT-3′；
SEQ ID NO.16：5′-AATATCTTGTCCGTATAGTTC-3′；
SEQ ID NO.36：5′-TATAGAACCACCAAAATTACGTAGT-3′；
SEQ ID NO.17：5′-AGTAGATTGGTCGTGTTT-3′；
SEQ ID NO.18：5′-TCCTTCTCCTGGTTTTGTA-3′；
SEQ ID NO.37：5′-CTCAAGGACGTGGTGCAAATTAG-3′；
SEQ ID NO.19：5′-ATTGTGGAATAATGTGTAGA-3′；
SEQ ID NO.20：5′-TCTATAGTTCCCGAGCTA-3′；
SEQ ID NO.38：5′-TGAATATGTGCCAGTGGATAA-3′；
SEQ ID NO.21：5′-TACCTCAAATACAAATGCA-3′；
SEQ ID NO.22：5′-TCCTTCTCCTGTTCCTT-3′；
SEQ ID NO.39：5′-CTCAAGGACGTGGTGCAAATTAG-3′；
SEQ ID NO.23：5′-TGCAACAGATACAGGTTCAG-3′；
SEQ ID NO.24：5′-CGGGTTTCCCTACGTGC-3′；
SEQ ID NO.40：5′-TACAGGCAGAGCGCGAGACAGC-3′；
SEQ ID NO.25：5′-ATACAAAGACAGACACA-3′；
SEQ ID NO.26：5′-GTTGTGTCAAATGTTCTG-3′；
SEQ ID NO.41：5′-CAACACAGTTTACAAGACAATCAAT-3′；
SEQ ID NO.27：5′-ACGCAACAGATACAGGTTCAG-3′；
SEQ ID NO.28：5′-ACGTTCTCCGGGTTTC-3′；
SEQ ID NO.42：5′-TACAGGCAGAGCGTGAGACA-3′。
Further, the concentration of each primer in the primer pair 3 is 0.05-0.1 mu M, and the concentration of each probe in the specific probe 3 is 0.02-0.06 mu M.
Furthermore, the specific probe 1 is connected with a CY5 fluorescent marking group, the specific probe 2 is connected with a FAM fluorescent marking group, and the specific probe 3 is connected with a JOE fluorescent marking group.
A kit for human papillomavirus detection typing, the kit comprising: a PCR reaction system, taq DNA polymerase, a positive reference substance, a negative reference substance and preservation solution; the PCR reaction system comprises a buffer solution and the composition.
Further, the buffer solution comprises 70-120 mM Tris-HCl, 30-60 mM KCl, 4-7 mM MgCl2 and 0.5-0.8 mM dNTPs.
Further, the preservation solution comprises 35-60 mM of isoguanidine salt, 0.1-0.5 w% of trehalose, 1-10 mM of Tris-HCl, 1-8 v% of DMSO and 2-8 mM of betaine.
The invention provides a kit for 14 kinds of high-risk human papilloma virus typing detection by adopting a fluorescence PCR method, which comprises the following components: design and synthesis of primer pairs and probes corresponding to the primer pairs: the E1 region of human papillomavirus gene is used as a target region, 14 subtype specific primer pairs and probes corresponding to the primer pairs are designed for amplifying corresponding nucleic acid fragments, and FAM, JOY, CY fluorescent markers are respectively used for corresponding subtypes. The probe adopts a high-specificity TaqMan probe, can be combined with a corresponding nucleic acid fragment, is hydrolyzed under the action of the Taq enzyme exonuclease activity, generates a fluorescent signal, and can obtain a real-time amplification curve according to the relationship between the fluorescent signal and the amplification cycle number.
The amplification primer pair comprises primer pair sequences corresponding to HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 subtypes, and the nucleotide sequences of the primer pair comprise SEQ ID NO.01-SEQ ID NO.28.
The amplification probes corresponding to the primer pairs comprise probe sequences corresponding to subtypes HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68, and the probe sequences comprise SEQ ID NO.29-SEQ ID NO.42.
The primer pairs and the probes corresponding to the primer pairs are designed by adopting Ologo7.0 software, the Tm value of each primer is close to 60 ℃, the length range of an amplified product is 100-150bp, the Tm value of each probe is close to 68 ℃, and each pair of primers and probes are subjected to amplification test and optimized until an amplification curve with smooth curve and higher signal value is obtained. And through a multiplex amplification test, non-specific amplification and primer dimer are not generated, and other interactions or cross reactions are not generated.
The invention also discloses a using method of the detection parting kit, which comprises the following steps: filling the collected sample into a preservation solution of a kit, sucking the preservation solution filled with the sample, filling the sample into a PCR amplification tube, and then placing the sample on a fluorescent PCR instrument for PCR amplification, wherein the amplification procedure of the PCR amplification is that the pre-denaturation is carried out at 95 ℃ for 30 seconds; 95℃for 10 seconds and 60℃for 18 seconds, 42 cycles; finally, the temperature is kept at 4-16 ℃.
The amplification adopts polymerase chain reaction, the reaction can be carried out in a PCR reaction system, the DNA polymerase required by the reaction is a hot start DNA polymerase, the antibody is subjected to blocking modification or chemical modification, and each amplification system (25 mu L) needs 2.5U to 5U of DNA polymerase. The amplification system of the invention comprises a positive control and a negative control besides a primer pair, a probe mixture corresponding to the primer pair, a reaction buffer solution and a hot start DNA polymerase.
The kit uses a specific primer pair and a probe designed by a human beta-globin gene as an internal standard (IC), and monitors the amplification detection process of a sample by detecting the endogenous beta-globin gene, so that false negative results are avoided.
The invention provides a composition for detecting and typing human papilloma virus, which comprises a primer pair for detecting human genome beta-globin and a probe besides a primer pair for detecting HPV 14 types and a novel fluorescent marked probe, wherein the nucleotide sequence (5 '-3') of the primer pair is shown as SEQ ID NO. 43: GCTTACATTTGCTTCTGACA, SEQ ID NO. 44: AGTAACGGCAGACTTCTCC; the nucleotide sequence (5 '-3') of the probe is shown as SEQ ID NO. 45: : ROX-ACTAGCAACCTCAAACAGACACC-BHQ).
The kit provided by the invention has a larger annealing temperature range, and an amplification system can be used on various general fluorescent PCR instruments (such as ABI7500, SLAN, rocgene and the like) and rapid amplification fluorescent PCR instruments (GN 7120 rapid nucleic acid analyzer), and the optimal amplification program is pre-denaturation at 95 ℃ for 30 seconds; 95℃for 10 seconds and 60℃for 18 seconds, 42 cycles; finally, the temperature is kept at 4-16 ℃.
The template DNA in the invention is female cervical exfoliated cell DNA. The kit has stronger anti-interference capability, and the sample contains interfering substances in the following concentration range without affecting normal amplification. Hemoglobin (less than or equal to 200 g/L), white blood cells (less than or equal to 50/HPF), mucin (less than or equal to 0.9 mg/mL), econazole (less than or equal to 100 mg/mL), nonoxynol suppository (less than or equal to 20 mg/mL), baofukang suppository (less than or equal to 1000 mg/mL), nystatin vaginal effervescent tablet (less than or equal to 1 ten thousand IU/mL), and human lubricant (less than or equal to 0.5 g/mL).
Example 1 preparation of kit for human papillomavirus detection typing
1. Primer pair and probe special for high-risk type HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 typing detection system
The amplification primer pairs and probes corresponding thereto were designed for typing HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 respectively, and the primer pair probe sequences, primer pair probe concentrations and fluorescent labeling patterns are shown in Table 1. In addition, the kit selects human genome beta-globin as an internal standard, and the primer probe information is as follows (primer pair sequence (5 '-3'): GCTTACATTTGCTTCTGACA, AGTAACGGCAGACTTCTCC; probe sequence (5 '-3'): CY 5-ACTAGCAACCTCAAACAGACACC-BHQ).
TABLE 1 primer pair probe information
2. The preparation kit of the high-risk human papillomavirus detection typing detection system comprises the following components:
the PCR amplification system per 25. Mu.L was as follows:
Component (A) Addition (mu L)
PCR reaction system 19
Taq DNA polymerase 1
Preservation solution containing sample template 5
The following table shows components of the PCR reaction system
The following table shows the components of the preservation solution
Preservation solution component Concentration of
Isoguanidine salt 50mM
Trehalose 0.25w％
Tris-HCl 2mM
DMSO 2v％
Betaine (betaine) 5mM
The plasmid contained in the negative and positive reference substance is a 800bp fragment intercepted by the corresponding detection target area, the concentration is 20000copies/mL, and the plasmid is purchased to the biological engineering (Shanghai) Co. Hot start DNA polymerase was purchased from Nanjinouzan Biotechnology Inc. Example 2 application of kit for human papillomavirus detection typing
1. Amplification of positive control
20 Mu L of the PCR reaction system prepared in example 1 and 5 mu L of the positive control are prepared into a mixed solution, the mixed solution is centrifuged briefly, and the PCR reaction solution is placed in a fluorescent PCR instrument (model GN7120 rapid nucleic acid analyzer is used in this example and is taken to Nanjing's Basil Biotechnology Co., ltd.) for PCR amplification.
PCR amplification procedure: pre-denaturation at 95 ℃ for 30 seconds; 95℃for 10 seconds and 60℃for 18 seconds, 42 cycles; finally, the temperature is kept at 4-16 ℃.
After amplification, the data analysis is carried out by using GN7120, the detection results are shown in figure 1, HPV16, HPV18, HPV12+ and IC amplification curves are clear, standard S-shaped curves are formed, and the fluorescence background has no obvious fluctuation.
2. Amplification of negative control
20. Mu.L of the PCR reaction system prepared in example 1 and 5. Mu.L of a negative control were prepared into a mixed solution, and after mixing, the mixed solution was centrifuged briefly, and the PCR reaction solution was placed in a fluorescent PCR apparatus (model GN7120 Rapid nucleic acid Analyzer, nanjing, basil Biotech Co., ltd.) for PCR amplification.
PCR amplification procedure: pre-denaturation at 95 ℃ for 30 seconds; 95℃for 10 seconds and 60℃for 18 seconds, 42 cycles; finally, the temperature is kept at 4-16 ℃.
After amplification, the data analysis is carried out by using GN7120, the detection result is shown in figure 2, the IC amplification curve is clear and is a standard S-shaped curve, the fluorescence background has no obvious fluctuation, and no non-specific amplification occurs.
Example 3A kit for genotyping human papillomavirus detection compared to commercially available kits
1. Sample and commercial kit information
150 HPV clinical samples were provided by the women and child care facility in Hunan province and the second women and child care facility in Jinan city. Two samples were collected, one placed in the preservation solution of the kit of the invention and the other placed in the preservation solution of the comparison kit.
The comparison kit is a high-risk human papilloma virus nucleic acid detection kit (fluorescence PCR method), national mechanical standard 20163401763, purchased from Kappy biochemical Co., ltd.
2. Sample pretreatment
The cervical exfoliated cells are taken by a cervical brush and then placed in the preservation solution of each kit. The kit is directly sampled and put on the machine after being uniformly mixed, the comparative kit is operated according to the recommended method of the specification, and the specific operation steps and time are shown in the following table 2.
Table 2 comparison with pretreatment procedure of comparative kit
3. Amplification procedure
The kit adopts a rapid amplification procedure, and the comparison kit carries out amplification according to the recommended procedure of the specification, and the specific amplification procedure and time are shown in Table 3.
Table 3 shows comparison of amplification procedure with commercially available kit
4. Sample detection
Preparing a PCR reaction solution according to the sample addition of example 2, and performing rapid amplification on GN7120, wherein the GN7120 rapid nucleic acid analyzer is taken to Nanjing's Basil egg Biotech Co., ltd; the comparative kit was operated according to its instructions using the model ABI7500. The detection results are shown in table 4, and through data statistics, compared with a comparison kit, the positive coincidence rate of the kit provided by the invention is 100%, the negative coincidence rate is 100%, and the total coincidence rate is 100%.
Table 4 comparison of the results of the yin-yang detection with the comparative kit
Example 4 Performance verification of kit for human papillomaVirus detection typing
1. Precision verification
HPV national reference is selected for precision verification, and the reference batch number is 370060-201901 and purchased to China food and drug inspection institute. The following three concentrations (3 copies/reaction, 10opies/reaction, 200 copies/reaction) were obtained by gradient dilution of human papillomavirus type 16, type 18, and type 33 national references. The measurement was repeated 20 times to obtain the coefficient of variation CV. The model used in this precision verification was GN7120 rapid nucleic acid analyzer, which was obtained from Nanjing, basil Biotechnology Co., ltd.
The detection results are shown in Table 5, and the CV of the high value, the median value and the low value samples of the kit is less than 5% through data statistics.
TABLE 5 kit precision verification for human papillomavirus detection typing
2. Detection limit verification
HPV national reference is selected for detection limit verification, and the reference batch number is 370060-201901 and purchased to China food and drug inspection institute. Human papillomavirus type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 national references were subjected to gradient dilution to obtain 1copies/reaction, and the test was repeated 20 times. The model used for the detection limit verification is GN7120 rapid nucleic acid analyzer, which is obtained from Nanjing, kiku-ken Biotechnology Co., ltd.
The detection results are shown in Table 6, and through data statistics, the kit provided by the invention can meet the detection limit of not higher than 1copies/reaction.
TABLE 6 kit detection limit verification for human papillomavirus detection typing
3. Analytical specificity verification
The test sample is a parasitic microorganism or sexually transmitted pathogen of the urinary tract and the genital tract of a human. The bacteria are classified into 21 bacteria including lactobacillus acidophilus, staphylococcus epidermidis, staphylococcus aureus, streptococcus faecalis, streptococcus pyogenes, streptococcus agalactiae, candida, chlamydia trachomatis, neisseria gonorrhoeae, escherichia coli, enterococcus, clostridium, streptococcus, klebsiella, escherichia coli, bacillus proteus, pseudomonas, bacteroides, bifidobacterium, fusarium and pallidum; 17 pathogens, including 12 non-target HPV genotypes. HPV6, 11, 26, 30, 34, 53, 67, 69, 70, 73, 82, 85, and other 5 viruses: adenovirus, cytomegalovirus, epstein barr virus, herpes simplex virus 1, herpes simplex virus 2; candida albicans and trichomonas vaginalis.
Diluting 12 medium-low-risk human papillomavirus national references (HPV 6, 11, 26, 30, 34, 53, 67, 69, 70, 73, 82, 85) with a cervical sample of a healthy person to 106copies/reaction; cross-reactive pseudoviruses and bacteria were diluted to 105PFU/ml and 106CFU/ml, respectively, using a healthy human cervical sample.
HPV national reference lot number 370060-201901, purchased to China food and drug inspection institute. Pseudoviruses and bacteria used for cross-reactions were purchased from the biological sciences limited of nanking nuozan.
The model used for the analysis specificity verification is GN7120 rapid nucleic acid analyzer, which is obtained from Nanjing's Basil egg Biotechnology Co.
Cross-reactivity assessment is divided into two aspects, including gene sequence alignment and cross-reaction validation.
A) Alignment of Gene sequences
Full genomic sequences of cross-reactive samples were obtained from NCBI and compared in clusters with 14 detection target sequences. The detection results are shown in Table 7, and through data statistics, the target sequences of 14 HPV types in the kit have no homology with the parasitic microorganisms or sexually transmitted pathogen gene sequences of the human urinary tract and the genital tract.
TABLE 7 results of gene sequence alignment
B) Verification of Cross-reactivity
The detection result is shown in table 8 by using the kit of the invention, and through data statistics, parasitic microorganisms or sexually transmitted pathogens of common human urinary tract and genital tract have no cross interference with the kit of the invention.
TABLE 8 Cross-reaction validation results
4. Interference research
Endogenous interfering substances, including hemoglobin, leukocytes, and mucins; four concentration levels are set for each interfering substance, including 160g/L, 180g/L, 200g/L, and 220g/L of hemoglobin; white blood cells 40/HPF, 45/HPF, 50/HPF, 55/HPF; mucin 0.7mg/ml, 0.8mg/ml, 0.9mg/ml, 1mg/ml. The assay was repeated 20 times with samples of cervical swabs of healthy humans containing different endogenous interfering substances, with HPV16 and HPV18 plasmids diluted to a detection limit concentration (1 copies/reaction).
Exogenous interfering substances including nonanol ether suppository, mifepristone tablet, peony alkali vaginal effervescent tablet, compound clotrimazole cream, econazole, baofukang suppository and human body lubricant; four concentration levels were set for each interfering substance, including 6mg/mL, 8mg/mL, 10mg/mL, and 12mg/mL of nonanol-ether plug; 2.0mg/L, 2.2mg/L, 2.4mg/L and 2.6mg/L mifepristone tablets; 60000IU/mL, 80000IU/mL, 100000IU/mL, 120000IU/mL of nisetum Ding Yindao effervescent tablet; 0.06g/mL, 0.08g/mL, 0.1g/mL and 0.12g/mL of compound clotrimazole emulsifiable paste; 0.06g/mL, 0.08g/mL, 0.1g/mL, 0.12g/mL of econazole; 0.06g/mL, 0.08g/mL, 0.1g/mL, 0.12g/mL of Baofukang suppository; human body lubricant of 0.3g/mL, 0.4g/mL, 0.5g/mL, 0.6 g/mL. The assay was repeated 20 times with samples of cervical swabs of healthy humans containing different exogenous interfering substances, with HPV16 and HPV18 plasmids diluted to a detection limit concentration (1 copies/reaction).
HPV national reference lot number 370060-201901, purchased to China food and drug inspection institute.
The model used in the interference study was GN7120 rapid nucleic acid analyzer, which was obtained from Nanjing, basil Biotechnology Co.
A) Endogenous interfering substances
The detection results of the interfering substance detection by using the kit are shown in Table 9, and the detection results of the kit are not affected by the sample containing 200g/L hemoglobin, 50/HPF leucocytes and 0.9mg/mL mucin.
TABLE 9 endogenous interfering substance test results
B) Exogenous interfering substance
The detection results of the detection of the interfering substances by using the kit are shown in Table 10, and the detection results of the detection results are shown in Table 10, wherein the samples contain 10mg/mL of nonoxynol suppository, 2.4mg/L of mifepristone tablets, 100000IU/mL of nisin Ding Yindao effervescent tablets, 0.1g/mL of compound clotrimazole emulsifiable paste, 0.1g/mL of econazole, 0.1g/mL of Baofukang suppository and 0.5g/mL of human lubricant, and the detection results of the kit are not influenced.
TABLE 10 exogenous interfering substance test results
The invention uses a fluorescence PCR method to carry out HPV typing detection, can cover more than 98 percent of 14 HPV types clinically infected at one time, can detect HPV16 type, 18 type and other 12 high-risk HPVs (namely 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) by one-time sampling, and can finish screening and shunting in one step; the sample extraction step can be omitted by innovatively adding the nucleic acid extraction reagent into the sampling tube, the sample can be directly sampled and put on the machine after sampling, the result can be obtained after the rapid amplification only by 30 minutes, compared with other kits on the market, the time is greatly shortened, specific data are shown in Table 11, the sensitivity is high, and the detection result is highly accurate.
TABLE 11 comparison of detection time with commercial high-risk HPV nucleic acid detection kits currently available in the market
Manufacturer' s Principle of the technology Whether or not to need pretreatment Detection time
The invention is that Fluorescent PCR Whether or not 30 Minutes
Qiagen HR-HPV Hybrid capture Is that 2-3 Hours
River creature Fluorescent PCR Is that 2-3 Hours
Kaipu organism Fluorescent PCR Is that 2-3 Hours
Sub-energy organisms Reverse dot hybridization Is that 3-4 Hours
Port dragon Reverse dot hybridization Is that 3-4 Hours
Perspective view Fluorescent PCR Is that 2-3 Hours
Shengxiang tea Fluorescent PCR Is that 2-3 Hours
Roche 4800HPV test Fluorescent PCR Is that 2-3 Hours
Hologic Cervista HPV test Invader chemistry Is that 3-4 Hours
The invention provides a DNA detection method for qualitatively detecting human papilloma virus type 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68 in human cervical exfoliated cells in clinic, and the method is used for detecting type 16, 18 and other 12 high-risk HPV types in the human cervical exfoliated cells in vitro, and has the characteristics of simple operation, rapid detection, strong fluorescent signal and high sensitivity.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (9)

1. The composition for human papillomavirus detection and typing is characterized by comprising a primer pair and probes corresponding to the primer pair, wherein the probes comprise a specific probe 1 aiming at HPV16 type, a specific probe 2 aiming at HPV18 type and a specific probe 3 aiming at other high-risk types of HPV, fluorescent marker groups are connected to the specific probe 1, the specific probe 2 and the specific probe 3, and fluorescent signals generated by the fluorescent marker groups on the specific probe 1, the specific probe 2 and the specific probe 3 in a PCR process are different.
2. The composition of claim 1, wherein the primer pair comprises primer pair 1 for HPV type 16, primer pair 2 for HPV type 18, primer pair 3 for other high risk types;
The nucleotide sequences of the primer pair 1 are shown as SEQ ID NO.01 and SEQ ID NO.02, and the nucleotide sequence of the specific probe 1 is shown as SEQ ID NO. 29; the nucleotide sequences of the primer pair 2 are shown in SEQ ID NO.03 and SEQ ID NO.04, and the nucleotide sequence of the specific probe 2 is shown in SEQ ID NO. 30;
SEQ ID NO.01：5′-CGCCATGAGACTGAAACACCA-3′；
SEQ ID NO.02：5′-TCATTATCATTTACTATA-3′；
SEQ ID NO.29：5′-TGGTTTTATGTAGAGGCTGTAG-3′；
SEQ ID NO.03：5′-TACAGTAGTGATACTG-3′；
SEQ ID NO.04：5′-ATTCCTCCGCTTCCT-3′；
SEQ ID NO.30：5′-TGTAATGTATACAACAATCAGGCAG-3′。
3. The composition according to claim 2, wherein the concentration of each of the primer set 1 and the primer set 2 is 0.05 to 0.08. Mu.M, and the concentration of the specific probe 1 and the specific probe 2 is 0.03 to 0.06. Mu.M.
4. The composition of claim 1, wherein the primer pair 3 comprises primer pair 4 for HPV31 type, primer pair 5 for HPV33 type, primer pair 6 for HPV35 type, primer pair 7 for HPV39 type, primer pair 8 for HPV45 type, primer pair 9 for HPV51 type, primer pair 10 for HPV52 type, primer pair 11 for HPV56 type, primer pair 12 for HPV58 type, primer pair 13 for HPV59 type, primer pair 14 for HPV66 type, primer pair 15 for HPV68 type,
The specific probes 3 comprise specific probes 4 for HPV31 type, specific probes 5 for HPV33 type, specific probes 6 for HPV35 type, specific probes 7 for HPV39 type, specific probes 8 for HPV45 type, specific probes 9 for HPV51 type, specific probes 10 for HPV52 type, specific probes 11 for HPV56 type, specific probes 12 for HPV58 type, specific probes 13 for HPV59 type, specific probes 14 for HPV66 type and specific probes 15 for HPV68 type,
The nucleotide sequences of the primer pair 4 are shown as SEQ ID NO.05 and SEQ ID NO.06, and the nucleotide sequence of the specific probe 4 is shown as SEQ ID NO. 31; the nucleotide sequences of the primer pair 5 are shown as SEQ ID NO.07 and SEQ ID NO.08, and the nucleotide sequence of the specific probe 5 is shown as SEQ ID NO. 32; the nucleotide sequences of the primer pair 6 are shown as SEQ ID NO.09 and SEQ ID NO.10, and the nucleotide sequence of the specific probe 6 is shown as SEQ ID NO. 33; the nucleotide sequences of the primer pair 7 are shown as SEQ ID NO.11 and SEQ ID NO.12, and the nucleotide sequence of the specific probe 7 is shown as SEQ ID NO. 34; the nucleotide sequence of the primer pair 8 is shown as SEQ ID NO.13 and SEQ ID NO.14, and the nucleotide sequence of the specific probe 8 is shown as SEQ ID NO. 35; the nucleotide sequences of the primer pair 9 are shown as SEQ ID NO.15 and SEQ ID NO.16, and the nucleotide sequence of the specific probe 9 is shown as SEQ ID NO. 36; the nucleotide sequences of the primer pair 10 are shown as SEQ ID NO.17 and SEQ ID NO.18, and the nucleotide sequence of the specific probe 10 is shown as SEQ ID NO. 37; the nucleotide sequences of the primer pair 11 are shown as SEQ ID NO.19 and SEQ ID NO.20, and the nucleotide sequence of the specific probe 11 is shown as SEQ ID NO. 38; the nucleotide sequences of the primer pair 12 are shown as SEQ ID NO.21 and SEQ ID NO.22, and the nucleotide sequence of the specific probe 12 is shown as SEQ ID NO. 39; the nucleotide sequences of the primer pair 13 are shown as SEQ ID NO.23 and SEQ ID NO.24, and the nucleotide sequence of the specific probe 13 is shown as SEQ ID NO. 40; the nucleotide sequences of the primer pair 14 are shown as SEQ ID NO.25 and SEQ ID NO.26, and the nucleotide sequence of the specific probe 14 is shown as SEQ ID NO. 41; the nucleotide sequences of the primer pair 15 are shown as SEQ ID NO.27 and SEQ ID NO.28, and the nucleotide sequence of the specific probe 15 is shown as SEQ ID NO. 42;
SEQ ID NO.05：5′-ATGACACAACATTTGATTTGTCCC-3′；
SEQ ID NO.06：5′-GCATTACTATCACTGTCAGCT-3′；
SEQ ID NO.31：5′-AATGGTACAATGGGCATATGACAAT-3′；
SEQ ID NO.07：5′-CAGCCCGGGCATTGTTTAAT-3′；
SEQ ID NO.08：5′-ATCTTCTGTCGCCTATACCGA-3′；
SEQ ID NO.32：5′-TGTGTGTGCACTAAAACGAAAGT-3′；
SEQ ID NO.09：5′-CAGAATGGATTCAAAGACAAACAG-3′；
SEQ ID NO.10：5′-TTCTAACACGTTGTTACAC-3′；
SEQ ID NO.33：5′-ACAATGGGCATATGACAATGAT-3′；
SEQ ID NO.11：5′-GCAACAGATACAGGTTCAG-3′；
SEQ ID NO.12：5′-ATGTCCGTCTCGCACTCTG-3′；
SEQ ID NO.34：5′-TACAGGCAGAGCGTGAGACA-3′；
SEQ ID NO.13：5′-ATGTAGATCCGCATTGCAGT-3′；
SEQ ID NO.14：5′-TGATTTCCTCGATAATGTTC-3′；
SEQ ID NO.35：5′-AGGCTGCAATGCTGGCAGTATT-3′；
SEQ ID NO.15：5′-TAAGTACATTAGTAAATAT-3′；
SEQ ID NO.16：5′-AATATCTTGTCCGTATAGTTC-3′；
SEQ ID NO.36：5′-TATAGAACCACCAAAATTACGTAGT-3′；
SEQ ID NO.17：5′-AGTAGATTGGTCGTGTTT-3′；
SEQ ID NO.18：5′-TCCTTCTCCTGGTTTTGTA-3′；
SEQ ID NO.37：5′-CTCAAGGACGTGGTGCAAATTAG-3′；
SEQ ID NO.19：5′-ATTGTGGAATAATGTGTAGA-3′；
SEQ ID NO.20：5′-TCTATAGTTCCCGAGCTA-3′；
SEQ ID NO.38：5′-TGAATATGTGCCAGTGGATAA-3′；
SEQ ID NO.21：5′-TACCTCAAATACAAATGCA-3′；
SEQ ID NO.22：5′-TCCTTCTCCTGTTCCTT-3′；
SEQ ID NO.39：5′-CTCAAGGACGTGGTGCAAATTAG-3′；
SEQ ID NO.23：5′-TGCAACAGATACAGGTTCAG-3′；
SEQ ID NO.24：5′-CGGGTTTCCCTACGTGC-3′；
SEQ ID NO.40：5′-TACAGGCAGAGCGCGAGACAGC-3′；
SEQ ID NO.25：5′-ATACAAAGACAGACACA-3′；
SEQ ID NO.26：5′-GTTGTGTCAAATGTTCTG-3′；
SEQ ID NO.41：5′-CAACACAGTTTACAAGACAATCAAT-3′；
SEQ ID NO.27：5′-ACGCAACAGATACAGGTTCAG-3′；
SEQ ID NO.28：5′-ACGTTCTCCGGGTTTC-3′；
SEQ ID NO.42：5′-TACAGGCAGAGCGTGAGACA-3′。
5. The composition according to claim 4, wherein the concentration of each primer in the primer set 3 is 0.05 to 0.1. Mu.M, and the concentration of each probe in the specific probe 3 is 0.02 to 0.06. Mu.M.
6. The composition of claim 1 wherein specific probe 1 is attached to a CY5 fluorescent label group, specific probe 2 is attached to a FAM fluorescent label group, and specific probe 3 is attached to a JOE fluorescent label group.
7. A kit for human papillomavirus detection typing, said kit comprising: a PCR reaction system, taq DNA polymerase, a positive reference substance, a negative reference substance and preservation solution; the PCR reaction system comprises a buffer, a composition according to any one of claims 1 to 6.
8. The kit according to claim 7, wherein the buffer comprises 70-120 mM Tris-HCl, 30-60 mM KCl, 4-7 mM MgCl2 and 0.5-0.8 mM dNTPs.
9. The kit of claim 7, wherein the preservation solution comprises 35-60 mM of isoguanidine salt, 0.1-0.5 w% trehalose, 1-10 mM Tris-HCl, 1-8 v% DMSO, and 2-8 mM betaine.
CN202211692589.7A 2022-12-28 2022-12-28 Composition and kit for human papilloma virus detection and typing Pending CN118256653A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202211692589.7A CN118256653A (en) 2022-12-28 2022-12-28 Composition and kit for human papilloma virus detection and typing

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211692589.7A CN118256653A (en) 2022-12-28 2022-12-28 Composition and kit for human papilloma virus detection and typing

Publications (1)

Publication Number Publication Date
CN118256653A true CN118256653A (en) 2024-06-28

Family

ID=91603027

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202211692589.7A Pending CN118256653A (en) 2022-12-28 2022-12-28 Composition and kit for human papilloma virus detection and typing

Country Status (1)

Country Link
CN (1) CN118256653A (en)

Similar Documents

Publication Publication Date Title
CN109487013B (en) Herpes simplex virus type I and type II detection marker, primer probe pair, kit and detection method
Yang et al. Quantification of human papillomavirus DNA in the plasma of patients with cervical cancer
CN105087827B (en) Detection primer, probe and the kit of 16 kinds of type HPV viruses
EP3187596B1 (en) Method for detecting and typing high-risk human papillomaviruses
CN105755169B (en) Detection and typing kit for high-risk human papilloma virus and application thereof
CN110628953B (en) Kit for human papilloma virus typing detection
CN104818342B (en) Detection kit, detection architecture and method for 19 kinds of high-risk human mammilla papillomavirus (HPV)
CN112575123B (en) Primer combination, probe combination and human papillomavirus nucleic acid detection kit
CN108330215A (en) A kind of primer and probe compositions detecting high-risk 16 types of HPV using RPA technologies
CN105803110B (en) Kit for simultaneously typing and detecting multiple human papilloma viruses and application thereof
CN108179226B (en) Nucleic acid composition for detecting human papilloma virus, application thereof and kit
CN108546785B (en) Method for detecting DNA of pathogenic microorganisms in genital tract
CN106048081A (en) HPV (human papilloma virus) typing detection primers as well as detection method and application thereof
CN117625844A (en) Multiplex Q-PCR detection kit for HPV virus and preparation thereof
US8399652B2 (en) Primers and probes for detecting genital HPV genotypes
Mudhigeti et al. Evaluation of loop-mediated isothermal amplification assay for detection and typing of human papilloma virus 16 and 18 from endocervical samples
CN108085419B (en) probe and primer composition
CN118256653A (en) Composition and kit for human papilloma virus detection and typing
CN111172328A (en) Complete set of primers, complete set of probes, kit and method for HPV nucleic acid typing detection
CN110724761A (en) Reagent, method and application for detecting human papilloma virus
JP5898831B2 (en) Detection of human papillomavirus
MAGAJI et al. An overview of the general characteristics, pathogenicity, transmission, and diagnosis of human papilloma virus (HPV)
Abdulhamit et al. FDA-Approved Molecular Tests Used to Define Human Papillomavirus (HPV) Infections which Cause Cervix Cancer
Wang et al. Analytical performance evaluation of the HPV OncoCheck assay for detection of high-risk HPV infection in liquid-based cervical samples
CN113249508B (en) Specific primer and probe for detecting group B streptococcus and application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination