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A common type of human papillomavirus quadruple fluorescent quantitative PCR typing detection method

A human papillomavirus, fluorescence quantitative technology, applied in fluorescence/phosphorescence, microbial determination/inspection, biochemical equipment and methods, etc. , to achieve the effect of monitoring clinical efficacy, improving detection sensitivity, and simple and fast operation.

Active Publication Date: 2011-12-21
ZHEJIANG UNIV
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  • Claims
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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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  • A common type of human papillomavirus quadruple fluorescent quantitative PCR typing detection method
  • A common type of human papillomavirus quadruple fluorescent quantitative PCR typing detection method
  • A common type of human papillomavirus quadruple fluorescent quantitative PCR typing detection method

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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 fluorescent quantitative PCR typing detection method for common types of human papillomavirus. It includes a four-fold PCR reaction system based on fluorescent quantitative PCR technology, including forward and reverse primers and AllGlo fluorescent probes for four human papillomavirus (HPV) genotypes, which can be used under suitable PCR conditions. Simultaneously detect the DNA load of HPV-6, -11, -16, and -18 in one reaction tube at most, including the design of specific primers and fluorescent probes, the construction of standard molecules, the preparation of standard curves, and the detection of clinical specimens. The present invention can easily and quickly detect clinically common four-type HPV infection simultaneously and quickly in one reaction tube, realize simultaneous typing and detection of four-type HPV by single-tube PCR, and can quantitatively detect, simple and fast operation, high sensitivity and specificity , good repeatability, accurate and reliable results, and has great clinical value for early prevention and treatment of genital warts and cervical cancer in women caused by common types of HPV, blocking the source of infection, reducing HPV infection, and monitoring clinical efficacy.

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 Patents(China)
IPC IPC(8): C12Q1/68C12Q1/70G01N21/64
Inventor 李兰娟余道军
Owner ZHEJIANG UNIV
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