One-step semi-nested human papilloma virus screening method

A human papillomavirus, semi-nested technology, applied in the field of one-step semi-nested screening of human papillomavirus, can solve the problems of cumbersome operation, easy pollution, and difficulty in designing common primers, and achieve good specificity and high sensitivity Effect

Inactive Publication Date: 2012-08-15
SHANGHAI ADICON CLINICAL LAB LNC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In clinical laboratories, common methods for detecting HPV are common polymerase chain reaction (PCR) and multicolor fluorescent real-time PCR. Because there are many types of HPV and it is difficult to design common primers, two-step nested PCR is often used. method, that is, to do the outer PCR first, and then use the outer PCR product as a template to do the second PCR, which is cumbersome to operate and easy to contaminate
Therefore, it is not conducive to the detection of large quantities of samples in the laboratory.

Method used

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  • One-step semi-nested human papilloma virus screening method
  • One-step semi-nested human papilloma virus screening method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Take a portion of human endocervical cell lysate DNA, and use SYBR Green method for real-time fluorescent PCR for HPV virus gene detection.

[0021] The HPV gene was amplified using the following primers:

[0022] HPV-out1-Forward: TGT CCA / CAA / GA / G GGA A / TAC TGA GG;

[0023] HPV-out2-Reverse: AAGCA / C CAG GGA / T CAT AAC / T AAT GC;

[0024] HPV-in: AAGAA AAA TAA ACT GTA AAT CAT ATT.

[0025] The amplification conditions are:

[0026] Real-time fluorescent PCR reaction solution includes: 1.5mM MgCl 2 , 200mM dNTP mixture, 10pmol HPV-out1-Reverse and HPV-in primers, 2.5pmol HPV-ou1-Forward primers, 20ul PCR reaction system containing 1U polymerase and 1ul 10×SYBR Green.

[0027] The fluorescent PCR amplification conditions are as follows: 95°C for 3 minutes; 95°C for 30 seconds, 53°C for 30 seconds, 72°C for 30 seconds, a total of 10 cycles; 95°C for 30 seconds, 40°C for 30 seconds, 72°C for 30 seconds, a total of 30 cycles Cycle; final extension at 72°C for 10 minutes....

Embodiment 2

[0029] Embodiment 2: clinical specimen comparative experiment

[0030] 400 cases of clinical specimens were detected in the early stage, and HPV genes were detected by traditional double-tube nested PCR and single-tube semi-nested PCR of the present invention. The results are shown in the table below:

[0031] Number of specimens Double-tube nested PCR (traditional method) Single tube semi-nested PCR (method of the present invention) 142 positive positive 3 positive feminine 5 feminine positive 250 feminine feminine

[0032] The results showed that the coincidence rate was about 98%.

[0033] The results of electrophoresis running gel bands are as follows: figure 2 shown. The traditional double-tube nested PCR and the single-tube semi-nested PCR of the present invention are used to detect HPV genes, and the results are basically consistent.

[0034] The invention can detect the HPV gene by a one-step method, avoids the dis...

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Abstract

The invention discloses a one-step semi-nested human papilloma virus screening method, which uses SYBRGreen real-time fluorescence PCR (polymerase chain reaction) to detect HPV (human papilloma virus) genes. The one-step semi-nested human papilloma virus screening method is characterized in that the HPV genes are amplified by following degenerate primers including HPV-out1-Forward: TGTCCA / CAA / GA / GGGAA / TACTGAGG, HPV-out2-Reverse: AAGCA / CCAGGGA / TCATAAC / TAATGC and HPV-in: AAGAAAAATAAACTGTAAATCATATT. The method can detect the HPV genes by a one-step process, avoids shortcomings that a traditional two-step nested PCR method needs twice PCR and is troublesome in operation and easy to cause pollution, and is fine in specificity and high in sensitivity.

Description

technical field [0001] The invention belongs to the fields of life science and biotechnology, and relates to a one-step semi-nested method for screening human papillomaviruses, which can detect HPV virus genes, has good specificity and high sensitivity. Background technique [0002] Human papillomavirus (Human papillomavirlls, HPV) belongs to the Papillomavirus genus of Papovaviridae. HPV was discovered by Strauss under the electron microscope in 1949. It is a small circular double-stranded DNA virus with a genome length of about 8000bp. . At present, there are more than 100 HPV subtypes identified, which are divided into skin type HPV and genital tract epithelial HPV according to the epithelial site of infection. About 30 types can infect women's genital tract, and about 20 of them are related to tumors. . According to the different subtypes of HPV and the risk of tumor occurrence, they are divided into low-risk HPV and high-risk HPV. Because the pathogenic mechanism and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 王淑一方国伟薛群周晓犊孙翠莲
Owner SHANGHAI ADICON CLINICAL LAB LNC
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