Method for amplifying and genotyping nucleic acid genes of human papilloma virus and assay kit for same

A viral nucleic acid and gene amplification technology, which is applied in the field of human papillomavirus nucleic acid gene amplification and typing, can solve the problems of high cost, cross-hybridization risk, and only typing, etc., and achieves a high degree of automation and eliminates Effects of Nucleic Acid Cross Contamination

Inactive Publication Date: 2011-11-23
JIANGYIN TAIKANG BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In short, the existing HPV detection methods, whether it is hybridization method, PCR method, gene chip, or the latest liquid chip, high-resolution mass spectrometry and other technologies, can only quantify (or semi-quantify) the HPV in the sample, or It can only be typed, and there are also disadvantages such as cross-hybridization risk, high cost, etc. that are not conducive to clinical application

Method used

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  • Method for amplifying and genotyping nucleic acid genes of human papilloma virus and assay kit for same
  • Method for amplifying and genotyping nucleic acid genes of human papilloma virus and assay kit for same
  • Method for amplifying and genotyping nucleic acid genes of human papilloma virus and assay kit for same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of primers and probes

[0041] Primer (primer sequence: SEQ.ID.NO: 1-26) and probe (quantitative probe sequence: SEQ.ID.NO: 27-39; typing probe sequence: SEQ.ID.NO: 40-41) All were synthesized by an automatic nucleic acid synthesizer, and were synthesized by Shanghai Sangon Biotechnology Co., Ltd. in this example.

[0042] Among them, the 5' end of the quantitative probe is labeled with a fluorescent luminescent group FAM (emission wavelength: 522nm), and the 3' end is modified with a BHQ-2 fluorescence quenching group.

[0043] The 5' end of the typing probe is labeled with fluorescent groups HEX (emission wavelength: 555nm) and ROX (emission wavelength: 608nm), and the 3' end is modified with BHQ-2 fluorescence quenching group.

[0044] The above primers and probes were all dissolved in double-distilled water and made into a 4 μM solution for later use.

Embodiment 2

[0045] Example 2 Sample Collection

[0046] Use a special cervical brush for sampling. The central bristle part of the cervical brush is gently inserted into the cervical canal, so that the shorter bristles can fully touch the cervix, gently press the cervical brush forward, and rotate in the same clock direction Cervical brush for 3-5 weeks, scrape cervical cells without leaving a dead angle, and stay for 10 seconds. The sampling range included the entire transformation zone (Transformation Zone, that is, the junction area between cervical squamous epithelium and cervical columnar epithelium).

[0047] Put the cervical brush that has collected the sample into the sample preservation tube (contains 1ml of sample preservation solution, which can make the DNA intact and inhibit the growth of bacteria), cover it tightly and make a label (indicate the name and date).

[0048] Sample preservation solution formula: phosphate buffer solution 10mM, NaCl 138mM, KCl 2.7mM, 0.05% NaN3, ...

Embodiment 3

[0049] Example 3 Extraction of viral DNA

[0050] The DNA of HPV virus was extracted by magnetic bead method automatic nucleic acid extractor (or silica gel column method). Its working principle and steps include cracking, adsorption, cleaning, elution and recovery.

[0051] The buffer formulations are as follows:

[0052] Lysis buffer formulation: 0.5mg / ml proteinase K, 0.01M Tris-HCl (trishydroxymethylaminomethane), 0.001M EDTA (ethylenediaminetetraacetic acid), pH=8.0.

[0053] Adsorption buffer formulation: 5.5M GuSCN (guanidine isothiocyanate), 20mM EDTA, 10mM Tris-HCl, 65mM dithiothreitol, 40g / L silica-based magnetic beads, pH=6.5.

[0054] Rinse solution I formula: 5.5M GuSCN (guanidine isothiocyanate), 20mM EDTA, 10mM Tris-HCl, 65mM dithiothreitol, pH=6.5.

[0055] Rinse solution II formula: 25% (v / v) isopropanol, 25% (v / v) ethanol (96%), 50% double distilled water, 0.1M NaCl.

[0056] Eluent formula: 10mM Tris-HCl, pH=8.0, or double distilled water. Finally, 30-5...

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Abstract

The invention belongs to the technical field of diagnostic reagents, and particularly provides a method for amplifying and genotyping nucleic acid genes of HPV (Human Papilloma Virus) and an assay kit for the same. The method comprises the following steps of: carrying out multiple real-time quantitative fluorescence gene amplifications (QPCR) by using a hybrid primer; quantitatively determining high-risk subtype HPVs by using a quantitative probe; and differentiating HPV 16 and 18 subtypes in types by using a typing probe. The hybrid primer is designed by conservative sequences at two sides of a special area of an E1 gene encoded by the HPV; and the genotyping probe is designed according to a type-specific sequence at the center of the area. Gradient dilutions of recombinant plasmids containing 16 and 18 subtype E1-area genes are taken as standard reference. The kit comprises a cervical brush, a sample storage tube/liquid, a primer, a probe, a Taq enzyme & reaction buffer solution, and a standard contrast reference. The system can be used for quantitatively detecting 13 high-risk subtype HPVs (HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59, HPV-68) and differentiating two subtypes HPV-16 and HPV-18 in types.

Description

technical field [0001] The invention relates to a method for amplifying and typing human papillomavirus nucleic acid gene and a detection kit for the method. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) can cause genital warts and cervical cancer, and is currently the most sexually transmitted disease that affects the physical and mental health of patients except AIDS. At present, more than 100 HPV genotypes have been discovered, which can be divided into cutaneous HPV and genital tract epithelial HPV. Genital tract epithelial HPV can be divided into high-risk HPV (such as HPV-16 subtype and HPV-18 subtype, etc.) and low-risk HPV (such as HPV-6 subtype and HPV-11 subtype, etc.) based on whether it is related to reproductive tract tumors. subtypes, etc.), high-risk HPV is associated with the occurrence of cervical cancer and cervical intraepithelial neoplasia (CIN2 / 3), and low-risk HPV often causes the same amount of lesions as genital warts...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 张宇杲
Owner JIANGYIN TAIKANG BIOLOGICAL TECH
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