Kit and method for HPV (human papillomavirus) parting detection

A technology of kits and molecular beacons, applied in biochemical equipment and methods, and microbial determination/inspection, etc., can solve the problems of low sensitivity, high cost, and poor specificity of HPV typing, and achieve high sensitivity, low requirements, and specificity strong effect

Pending Publication Date: 2019-09-27
HUBEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a kit and method for HPV typing detection, aiming to solve the current problems of HPV typing detection or low sensitivity or poor specificity or high cost

Method used

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  • Kit and method for HPV (human papillomavirus) parting detection
  • Kit and method for HPV (human papillomavirus) parting detection
  • Kit and method for HPV (human papillomavirus) parting detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 is used for the kit and method of HPV typing detection

[0039] 1. Quadruple PCR amplification to obtain PCR amplification products

[0040] 1. Design of PCR amplification primers

[0041] The upstream and downstream primer sets of the four HPVs are mainly designed for the E6 genes of HPV6 and HPV11 and the L1 genes of HPV16 and HPV18, so that the corresponding DNA fragments can be respectively amplified in the same PCR system.

[0042] The upstream and downstream primer sets of HPV6: the upstream primers of HPV6 are shown in SEQ ID NO.1, and the downstream primers of HPV6 are shown in SEQ ID NO.2;

[0043] The upstream and downstream primer sets of HPV11: the upstream primer of HPV11 is shown in SEQ ID NO.3, and the downstream primer of HPV11 is shown in SEQ ID NO.4;

[0044] The upstream and downstream primer sets of HPV16: the upstream primers of HPV16 are shown in SEQ ID NO.5, and the downstream primers of HPV16 are shown in SEQ ID NO.6;

[0045] The u...

Embodiment 2

[0101] The detection of embodiment 2 positive standard substance

[0102] 1. Acquisition of positive standard products

[0103] Take the positive sample determined to contain at least one of HPV6, HPV11, HPV16, and HPV18 as a positive standard, and use Anbiping’s human papillomavirus nucleic acid detection kit to detect the sample. Positive samples (including high-risk human papilloma virus samples and low-risk human papillomavirus samples) as the positive standard of the present invention.

[0104] 2, quadruple PCR amplification obtains PCR amplification product: adopt the method for embodiment 1 to carry out quadruple PCR amplification to obtain amplification product after above-mentioned positive standard substance; image 3 It is the result figure of the agarose gel electrophoresis experiment of the amplified product obtained after performing quadruple PCR amplification on the positive standard provided by the example of the present invention, wherein, 1 swimming lane is ...

Embodiment 3

[0108] Embodiment 3 sensitivity analysis and specificity analysis

[0109] 1. Using the positive standard as a template, take the detection sensitivity analysis of HPV6 upstream and downstream primers and molecular beacon probes as an example.

[0110] 1. Acquisition of different gradient positive standards

[0111] Take 20 μL of HPV6 positive standard and add 180 μL of DEPC-treated water, and perform serial dilutions to obtain positive standards with different gradients. 10 respectively 9 aM, 10 8 aM, 10 7 aM, 10 6 aM, 10 5 aM, 10 4 aM, 10 3 aM, 10 2 aM, 10 1 aM, 10 0 aM, 10 -1 aM, 10 -2 aM, 10 -3 aM, 10 -4 aM;

[0112] 2, adopt the method for embodiment 1 to carry out quadruple PCR amplification to the positive standard substance of above-mentioned different concentrations and obtain the amplified product;

[0113] 3. The product obtained by PCR amplification is mixed with the above-mentioned DNA guide and molecular beacon, and then added with Pfago protease ...

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Abstract

The invention provides a kit and a method for HPV (human papillomavirus) parting detection. The kit comprises four upstream and downstream primer groups for HPV; DNA guides, four molecular beacons marked by different fluorophores and DNA editase PfAgo. Highly specific primers, single-stranded DNA guides and the molecular beacons marked by different fluorophores are designed according to an E6 gene or L1 gene target sequence of the HPV, primary signal amplification can be performed by PCR, then, digestion of PfAgo is mediated by the single-stranded DNA guides after phosphorylation treatment, finally, secondary signal amplification is performed by the molecular beacons, and a fluorophotometer is used for measuring intensity of florescence signals to perform HPV parting. The method has the characteristics of being high in specificity, high in sensitivity, low in requirement for original materials, simple, rapid and accurate to operate and the like and can realize HPV parting detection of multiple subtypes simultaneously.

Description

technical field [0001] The invention relates to the technical field of virus detection, in particular to a kit and method for HPV typing detection. Background technique [0002] Human papillomavirus (HPV) belongs to the papillomavirus genus of the papillomaviridae family (papovaviridae), and is a virus that can cause benign and malignant tumors in human mucosal tissues. HPV is very easy to spread and spread in the crowd, and it can be cross-infected through direct or indirect contact. The main pathogenic factor of cervical cancer, 99.7% of cervical cancer patients have HPV infection. Screening is currently the main method for the prevention and early diagnosis of cervical cancer, while HPV detection is often used for early screening of cervical cancer, so as to concentrate high-risk groups, facilitate effective monitoring and early detection of cervical cancer. HPV detection is of great value in the diagnosis of condyloma acuminatum and female cervical cancer. It can not o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686
CPCC12Q1/708C12Q1/686C12Q2521/301C12Q2563/107
Inventor 马立新何如怡王珑瑜翟超王亚平
Owner HUBEI UNIV
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