Quadruple fluorescence quantitive PCR typing detection method for common human papillomavirus infection

A human papillomavirus and fluorescence quantitative technology, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of simultaneous detection of multiple subtypes, expensive reagents, complex technology, etc. , to achieve the effects of monitoring clinical curative effect, improving detection sensitivity, and simple and fast operation

Active Publication Date: 2010-05-19
ZHEJIANG UNIV
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Problems solved by technology

However, the currently reported fluorescent quantitative PCR detection of HPV is either low-throughput, or the technology is complex, the reagents are expensive, or the simultaneous quantitative detection of four types of HPV in a single tube is not realized, which is difficult to meet the clinical needs of simultaneous detection of multiple subtypes

Method used

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  • Quadruple fluorescence quantitive PCR typing detection method for common human papillomavirus infection
  • Quadruple fluorescence quantitive PCR typing detection method for common human papillomavirus infection
  • Quadruple fluorescence quantitive PCR typing detection method for common human papillomavirus infection

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Embodiment

[0083] 1. Construction of type four HPV target gene plasmid cloning vector

[0084] Using the T-A vector cloning scheme, the PCR products of the four target genes were electrophoresed to confirm the molecular weight of the amplified fragments, and then the amplified fragments were cloned into the PMD-19T plasmid and ligated overnight at 16°C. The ligation product was transformed into competent Escherichia coli DH5α, positive colonies were screened on LB medium (containing Amp100g / ml), and the plasmid was recovered, extracted, purified, and recombined with the plasmid mini-extraction kit. Sequencing verification by the biological company, the target plasmid whose sequence is completely correct after Blast is quantified with a UV spectrophotometer, and diluted to 10 with 1 × TE (pH8.0) buffer 10 copies / μl as a storage solution and stored at -20°C for future use.

[0085]2. DNA template extraction: Dissolve the secretion swab specimen with physiological saline, put the tissue bl...

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Abstract

The invention discloses a quadruple fluorescence quantitive PCR typing detection method for common human papillomavirus infection, which comprises a fluorescence quantitive PCR technology-based quadruple PCR reaction system and contains forward and reverse primers of four kinds of human papillomavirus (HPV) genotypes and AllGlo fluorescence probes, wherein the capacity of carrying DNA at most comprising HPV-6,-11, -16 and -18 can be synchronously detected in a reaction tube under a proper PCR condition. The method comprises the following steps of: designing specific primers and the fluorescence probes; building a standard molecule; preparing a standard curve; and detecting a clinical sample. The method can conveniently, quickly and synchronously detect four types of clinically common HPV infection in the reaction tube, realizes typing detection of the four types of HPV by a single-tube PCR at the same time, can quantitatively detect, is simple and quick in operation, high in sensitivity, good in specificity and repeatability and accurate and reliable in the result, and has very high clinical value on early preventing and treating woman cervical carcinogenesis caused by pointed condyloma and the common HPV, blocking infectious source, decreasing HPV infection and monitoring clinical treatment effect.

Description

technical field [0001] The present invention relates to a kind of human papillomavirus common type quadruple fluorescent quantitative PCR typing detection method, specifically, the present invention relates to the application of quadruple real-time fluorescent quantitative PCR technology to quickly and parallelly detect four types in one PCR reaction tube Methods for Human Papillomaviruses (HPV-6, -11, -16, -18). Background technique [0002] Human papillomavirus (Humanpapillomavirus, HPV) is a double-stranded DNA virus that spreads through human contact. It is more common in various benign and malignant hyperplasia of skin and mucous membranes and genital infections. So far, more than 120 species have been found in the world. HPV genotypes, various types of HPV are highly correlated with their biological behavior, and different genotypes of HPV have different pathogenic risks, which are generally divided into high-risk HPV and low-risk HPV. [0003] High-risk HPV infection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 李兰娟余道军
Owner ZHEJIANG UNIV
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