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Application of integrated high-frequency gene loci of high and medium risk type HPV related to cervical cancer generation

A technology of integration sites and cervical cancer, applied in the field of molecular genetics, to achieve the effect of low false positive rate

Pending Publication Date: 2021-10-15
WUHAN KDWS BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can detect HPV integration sites, they all need to pre-design the fixed target gene loci or regions to be detected.

Method used

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  • Application of integrated high-frequency gene loci of high and medium risk type HPV related to cervical cancer generation
  • Application of integrated high-frequency gene loci of high and medium risk type HPV related to cervical cancer generation
  • Application of integrated high-frequency gene loci of high and medium risk type HPV related to cervical cancer generation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] The cervical exfoliated cells of 988 LSIL patients, 358 HSIL patients, and 126 cervical cancer patients were selected as samples.

[0082] LSIL in the present invention refers to low-grade squamous intraepithelial lesion, and HSIL refers to high-grade squamous intraepithelial lesion, which are different pathological stages of cervical intraepithelial neoplasia.

[0083] 1. Centrifuge the exfoliated cells of the cervix for 5 minutes to collect the cells, discard the supernatant; add 200 μl PBS and 20 μl Proteinase K in sequence, shake and mix well;

[0084] 2. Add 200 μl of Buffer BCL, oscillate and mix well, bathe in 56°C water for 10 minutes, mix up and down several times during this period, and pass through the column for purification; add 150 μl of absolute ethanol, oscillate to mix, and briefly centrifuge to collect the liquid on the inner wall of the tube cap;

[0085]3. Put the FastPure gDNA Mini Columns II adsorption column in 2ml Collection Tubes, transfer the a...

Embodiment 2

[0092] Detection of HPV integration sites in genomic DNA of cervical tissue by reversible end-termination sequencing.

[0093] 1. Virus integration detection of genomic DNA samples of cervical exfoliated cells: Design HPV full-length probes containing 18 subtypes (HPV16, HPV18, HPV26, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV53, HPV56 , HPV58, HPV59, HPV66, HPV68, HPV73, HPV82), for the detailed content of the probe sequence, please refer to the applicant's patent application CN112195287A on November 12, 2020 - a human papillomavirus HPV typing and integration detection Probe sets and kits thereof;

[0094] Construct a DNA sequencing library according to the requirements of Illumina, use enzyme digestion to digest the sample genomic DNA into small fragments, and then repair and add A to the ends of these small fragments; ensure that the total amount of the constructed DNA library should be ≥ 1500ng, and The main peak of the library fragment length should be about 3...

Embodiment 3

[0099] The HPV integration sites were verified by PCR amplification and Sanger sequencing.

[0100] 1. PCR amplification

[0101] Design the upstream and downstream primers that can amplify the HPV integration site, and the primers are as shown in Table 2, and are prepared according to the system shown in Table 4 below:

[0102] Table 4 Primer System

[0103]

[0104] Mix each tube separately, put it into a standard PCR instrument for amplification, and perform amplification according to the procedures shown in Table 5 below:

[0105] Table 5 PCR amplification program

[0106]

[0107] 2. Send the amplified PCR product to Wuhan Qingke Biotechnology Co., Ltd. for Sanger sequencing.

[0108] (1) The PCR product obtained after amplification (content greater than 200ng, volume greater than 20ul) was sent to Wuhan Qingke Biotechnology Co., Ltd. for Sanger sequencing;

[0109] (2) Purify the PCR sample and add it to an ABI3730XL sequencer to detect the sequence of the PCR ...

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Abstract

The invention discloses application of integrated high-frequency gene loci of high-and-medium-risk HPV related to cervical cancer occurrence, and belongs to the technical field of molecular genetics. Corresponding PCR amplification upstream and downstream primers are designed aiming at integration sites CCAT1, CSMD3, PABPC1P2, RAD51B, TENM2, CSMD1, GRIA3, DPP10, SLC25A51P1, CDH6 and CTNND2 of high and medium risk HPV, and are assembled to obtain the corresponding kit for detecting cervical cancer high risk groups, and the kit has the advantages of rapidness, convenience, accuracy, low false positive rate and the like, is good in innovation and is helpful for clinical diagnosis.

Description

technical field [0001] The invention belongs to the technical field of molecular genetics, and specifically relates to the application of an integration site of a high-risk type HPV on a human genome in a kit for detecting high-risk groups of cervical cancer. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) is a non-encapsulated circular double-stranded small molecule DNA virus with a genome length of about 8000 base pairs (base pair, bp). According to the research results of the WHO International Agency for Research on Cancer (IARC) and other international organizations, it is recommended that the 13 genotypes of HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 be listed as The five genotypes of 26, 53, 66, 73, and 82 are listed as medium risk types. Persistent high-risk HPV infection is the main cause of cervical intraepithelial neoplasia and cervical cancer. Research results worldwide have shown that high-risk HPV DNA was detected ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6869C12Q1/686C12N15/11
CPCC12Q1/708C12Q1/6869C12Q1/686G01N2333/025C12Q2535/101
Inventor 陈世民杨帆黄晓园
Owner WUHAN KDWS BIOLOGICAL TECH CO LTD
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