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Reagent kit for detecting and typing high-risk type human papilloma viruses and application of reagent kit

A technology of human papillomavirus and kit, applied in the field of virus diagnosis, can solve the problems of few types, inconvenient HPV typing, affecting the specificity and sensitivity of detection kits, etc.

Active Publication Date: 2016-07-13
SHANGHAI TELLGEN LIFE SCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has good sensitivity and specificity, but it has disadvantages such as cross-contamination, high cost, and inconvenient HPV typing.
[0009] PCR detection methods can not only confirm the diagnosis of HPV positive infection, but also perform HPV typing. The disadvantage is that there are relatively few types of one-time reaction detection, and it is still difficult to achieve high-throughput typing detection.
However, the reagents of these kits are only designed based on the sequence of the L1 region, which is consistent with people's understanding of HPV, because the L1 region is highly specific
However, it is precisely because of the high specificity of the L1 region that these kits have deficiencies. If you want to detect multiple genotypes at the same time with one PCR reaction, you need many pairs of primers and multiple probes. However, if the primers and Too many probes will affect the efficiency of the PCR reaction and seriously affect the specificity and sensitivity of the detection kit
Therefore, Sun Yat-sen University Daan Gene Co., Ltd. can only detect 9 kinds of HPV viruses

Method used

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  • Reagent kit for detecting and typing high-risk type human papilloma viruses and application of reagent kit
  • Reagent kit for detecting and typing high-risk type human papilloma viruses and application of reagent kit
  • Reagent kit for detecting and typing high-risk type human papilloma viruses and application of reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1, Screening of Specific Target Fragment, Primer and Probe Design

[0068] In order to develop a detection reagent suitable for simultaneously detecting 14 papillomavirus genotypes (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68), the inventors aimed at The gene sequences of each virus were studied in depth, and the segments suitable for the design of specific primers and probes were screened. After repeated research and comparison, the gene sequence of the overlapping part of the L1 region and the L2 region of HPV was selected as the main region of the design, and further, the specific target fragment (i.e. PCR amplification) for each HPV genotype was determined. Fragments), as shown in Table 1.

[0069] Table 1

[0070]

[0071]

[0072]

[0073] According to the regions determined in Table 1, the inventors further designed specific primers and probes, taking into account that the primers and probes at both ends have degeneracy, so as to ...

Embodiment 2

[0077] The optimization of Taq enzyme concentration in embodiment 2, PCR reaction system

[0078] Objective: To optimize the addition amount of PCR master mix, optimize the concentration of Taq enzyme in the reaction system, and then optimize the PCR reaction system.

[0079]Through the screening of existing relatively mature fluorescent quantitative PCR premixes on the market, the most suitable kit for the present invention was obtained from Kangwei Century Biotechnology Co., Ltd. 2×GoldStarTaqManMixture (Cat: CW0932). Therefore, the PCR master mix used is 2×GoldStarTaqManMixture from Kangwei Century Biotechnology Co., Ltd., which contains hot-start Taq enzyme.

[0080] Template: Use TE containing 1ng / μL human genome (extracted from normal human blood) to separate 14 HPV positive reference products (including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 ,59,66,68, the sequences of L1 and L2 regions of each virus were selected, and the plasmids synthesized by Shanghai Hanyu ...

Embodiment 3

[0092] Embodiment 3, the optimization of PCR amplification method

[0093] 1. Optimized from the annealing temperature

[0094] Template: In addition to the 14 positive references described in Example 2, add 2 negative references (HPV6 and HPV11, select the sequences of the L1 and L2 regions of each virus, and synthesize plasmids from Shanghai Hanyu Biotechnology Co., Ltd. ), using 1ng / μL human genome TE to dilute the above reference substance to a concentration of 1000 copies / μL.

[0095] Primer-probe mixture formula: Dilute the primers to 100 μM, and the final concentration of the primer reaction is about 0.08 μM. When preparing the mixture, a PCR reaction needs to add about 0.02 μL of 100 μM primer stock solution. Dilute the probe to 100 μM, and the final concentration of the probe reaction is 0.027 μM. When preparing the mixed solution, a PCR reaction needs to add about 0.007 uL of 100 μM probe storage solution. The volume of the primer-probe mixture added to a PCR react...

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Abstract

The invention relates to a reagent kit for detecting and typing high-risk type human papilloma viruses and application of the reagent kit, and discloses a detection reagent particularly applicable to diagnosing the high-risk type human papilloma viruses (PHV) and typing the human papilloma viruses HPV16 and PHV18.The reagent kit and the method have the advantages that the detection reagent is obtained by means of reasonable design and optimization, is excellent in specificity and high in amplification efficiency and sensitivity when used for PCR (polymerase chain reaction) amplification, diversified high-risk type viruses can be simultaneously detected by the detection reagent, and detection programs are simple and are easy to implement; PCR amplification reaction programs are further optimized by an inventor, and accordingly the amplification efficiency further can be improved.

Description

technical field [0001] The invention relates to the field of virus diagnosis, more specifically, the invention relates to a high-risk human papillomavirus detection and typing kit and application thereof. Background technique [0002] Cervical cancer is the most common malignant tumor in women worldwide after breast cancer, and there are about 500,000 new cases of cervical cancer every year worldwide. Cervical screening can greatly reduce the incidence of cervical cancer. [0003] A large number of epidemiological and molecular biology studies have proved that human papillomavirus (HumanPapillomaVirus, HPV) infection is the main factor in the occurrence of cervical cancer and precancerous lesions: the World Health Organization once conducted a survey in 22 countries, showing that 99.9% HPV infection can be detected in cervical cancer patients; the International Association for Cancer Research (IARC) announced in 1995 that HPV infection is the main cause of cervical cancer. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 白艳军姚见儿胡可
Owner SHANGHAI TELLGEN LIFE SCI CO LTD
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