Detection kit, detection system and detection method for 19 high-risk human papilloma viruses (HPVs)

A technology of human papillomavirus and kit, applied in the biological field, can solve the problems of high false negative rate and false positive rate, poor specificity, and low sensitivity

Active Publication Date: 2015-08-05
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional cytology detection methods can only detect changes in cytology, which has low sensitivity, poor specificity, high false negative rate and false positive rate, and cannot type HPV

Method used

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  • Detection kit, detection system and detection method for 19 high-risk human papilloma viruses (HPVs)
  • Detection kit, detection system and detection method for 19 high-risk human papilloma viruses (HPVs)
  • Detection kit, detection system and detection method for 19 high-risk human papilloma viruses (HPVs)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] In this embodiment, 19 high-risk human papillomavirus DNA plasmids in the whole genome type reference product are used as templates, and the kit of the present invention is used for real-time fluorescent PCR detection:

[0049] (1) Establish PCR reaction system

[0050] Using the probes and specific primers designed above, take 5 μL of reference DNA as a reaction template, and use the following PCR reaction system for amplification. The PCR amplification reaction system is:

[0051]

[0052] Put the template-added PCR reaction tube into the real-time fluorescent PCR detector, and proceed according to the following amplification procedures: first stage: 50°C for 2 minutes, 95°C for 5 minutes, 1 cycle; second stage: 95°C for 5s, 40°C 30s, 72°C for 30s, 10 cycles; third stage: 95°C for 5s, 60°C for 35s, 72°C for 30s, 35 cycles; fluorescence collection: collect FAM, HEX (or VIC) and CY5 signals at 60°C in the third stage .

[0053] (2) Judge the detection result accordin...

Embodiment 2

[0061] The reaction system is as in Example 1, using the kit of the present invention to detect each component in the national reference product for the full genome type of human papillomavirus, and investigating the effect of the final determined reaction system on the national reference product for the full genome type of human papillomavirus The detection situation, the test results are as follows Figure 3-7 .

[0062] The test results show that this kit is negative for the negative reference products N1-N5 in the national reference products of the full genome type of human papillomavirus, and it is negative for the 7 low-risk samples in the national reference products of the full genome type of human papillomavirus. Types (HPV6, 11, 61, 67, 69, 71, 81) positive reference products were all tested negative, and the 13 high-risk types (HPV16, 18, 26, 31 . 100%.

Embodiment 3

[0064] Using the present invention to detect clinical samples, a total of 80 clinical samples were detected, and the human papillomavirus nucleic acid detection kit (PCR-fluorescent probe method) (type 17) of Yaneng Biotechnology (Shenzhen) Co., Ltd. was used as a contrast . The steps of using the real-time fluorescent PCR system of the present invention to detect 80 cases of clinical samples are as follows.

[0065] (1) Detection sample DNA extraction

[0066] Test samples include cotton swabs of urogenital secretions, genital scrapings, tissue biopsy specimens, etc.

[0067] Transfer 1 mL of the collected sample to a clean centrifuge tube, centrifuge at 12,000 rpm for 1 minute; carefully suck away 700 μL of supernatant, and add 350 μL of Buffer VDL12 (with absolute ethanol added) to the remaining precipitate; shake and mix for 10 seconds , centrifuge in a palm centrifuge for 5-10s, place in an incubator for digestion at 80°C for 3min; shake and mix for 10s, centrifuge in a...

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Abstract

The invention discloses a detection kit, detection system and detection method for 19 high-risk HPVs. The 19 high-risk HPVs comprise HPV16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82. According to the invention, a plurality of general primers are employed for amplification of target fragments, and specific fluorescence probes are used for real-time detection of the amplified fragments; since the general primers are employed, the primer amount of the system and complexity of the system are reduced, cost is saved, and all the high risk types are detected through combined action of 10 primers; and 20 specific fluorescence probes are used to distinguish different types. With the detection kit, detection system and detection method provided by the invention, the 19 high-risk HPVs can be detected in a reaction tube; high sensitivity and good specificity are obtained; operation is simple and fast; and for patients, screening cost is saved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a primer, a probe, a kit and a method for detecting 19 high-risk human papillomaviruses. Background technique [0002] Human papillomavirus (HPV) is a kind of papillomavacuolating virus A belonging to Papovaviridae. It is a spherical DNA virus that can cause squamous epithelial proliferation of human skin and mucous membranes. Humans are its only host. At present, more than 190 types of HPV have been identified, and about 50 types are directed to infect the stratified squamous epithelium of the human genital tract skin and mucous membrane. Clinically, HPV can be divided into high-risk and low-risk types according to the pathogenicity of different subtypes of HPV. In addition to causing external genital warts, the high-risk type is more important to cause external genital cancer, cervical cancer and high-grade cervical intraepithelial neoplasia. The high-risk type is the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/68C12Q1/708C12Q2600/16
Inventor 江春琴林清华施伟杰韩元龙郑立谋
Owner AMOY DIAGNOSTICS CO LTD
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