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Broad-spectrum, efficient and economical PCR (Polymerase Chain Reaction) detection method for high-risk human papilloma virus

A technology of papilloma virus and detection method, which is applied to the determination/testing of microorganisms, biochemical equipment and methods, etc., and can solve the problems of expensive real-time fluorescent PCR machines and not easy to popularize

Inactive Publication Date: 2013-11-27
潘晓静
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, real-time fluorescent PCR machines are very expensive and not easy to be popularized

Method used

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  • Broad-spectrum, efficient and economical PCR (Polymerase Chain Reaction) detection method for high-risk human papilloma virus
  • Broad-spectrum, efficient and economical PCR (Polymerase Chain Reaction) detection method for high-risk human papilloma virus
  • Broad-spectrum, efficient and economical PCR (Polymerase Chain Reaction) detection method for high-risk human papilloma virus

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Embodiment

[0034] The invention provides a method for detecting high-risk HPV. The method mainly includes the following steps: (1) extracting DNA from a sample; (2) performing PCR amplification on the extracted DNA using primers of SEQ ID NO: 1-26 (3) run 4% agarose gel identification test result.

[0035] (1) Sample collection and storage

[0036] The cervical exfoliated cells of patients were collected with sterile cotton test paper or cervical brush, placed in normal saline, and stored in a -20°C refrigerator.

[0037] (2) DNA extraction

[0038] Centrifuge the centrifuge tube containing the exfoliated cells of the cervix at 12000 RPM. After 5 minutes, remove the supernatant and precipitate DNA extraction using the sample DNA extraction kit from QIAGEN.

[0039] (3) PCR reaction

[0040] The total volume of each PCR reaction system is 50 μl, and the reaction system is shown in Table 1 below.

[0041] Table 1. PCR reaction system

[0042]

[0043] Thaw the components of t...

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Abstract

The invention discloses a broad-spectrum, efficient and economical PCR (Polymerase Chain Reaction) detection method for high-risk human papilloma virus. The detection method comprises the following steps of (1) designing primers aiming at 12 high-risk HPV (human papilloma virus) subtype L1 genes, wherein the melt points of the all genes are 60 DEG C, and the gas chromatography (GC) percents are 50%, so that the primers can amplify virus gene sequences in the same PCR tube under the same temperature, so as to save time and money; (2) taking glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a positive contrast, so as to avoid a false negative result; (3) avoiding a false positive result by utilizing an anti-pollution warm-start PCR system containing deoxyuridine triphosphate (dUTP) and uracil-N-glycosylase (UNG). According to the detection method, a PCR system is optimized based on the embodiment, and the specificity and the accuracy of a result are guaranteed, and according to the detection method, time saving and economy are realized, efficient and low-cost screening work is carried out on HPV viruses easily by a base hygiene department, and a purpose of preventing the cervical cancer is achieved.

Description

technical field [0001] The invention relates to a disease pathogen gene detection technology, in particular to a PCR detection technology for 12 high-risk human papillomaviruses. Background technique [0002] Human papillomavirus (human papillomavirus, HPV) is a kind of papillomavirus that can infect human beings. Its existence is closely related to the occurrence of various benign and malignant tumors of skin and mucous membranes, especially cervical cancer. There are more than 120 known types of HPV, and different subtypes of HPV have different pathogenic risks. Detection of HPV genes and analysis of subtypes are of great value in understanding related diseases, judging prognosis and guiding treatment. The International Association for Research on Cancer classifies 12 subtypes of HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81 as low-risk types, mainly causing genital warts and cervical intraepithelial neoplasia ( CIN I), mostly transient, can be cured naturally; HPV16,...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 潘晓静
Owner 潘晓静
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