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HPV-integrated gene sites related to occurrence of cervical carcinoma and application thereof

A gene locus, cervical cancer technology, applied in the fields of gynecological oncology and molecular genetics, can solve problems such as poor stability, low sensitivity, and cumbersome technical operations

Active Publication Date: 2015-08-12
武汉凯德维斯医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the detection methods and products for high-risk groups of cervical cancer are mainly for detection of HPV itself, that is, to detect the presence of HPV in samples, but in fact not all high-risk HPV-infected people develop cervical cancer. The detection methods and products for high-risk groups of cervical cancer have defects such as inaccurate detection results and low sensitivity
However, there is no method and product for detecting the high-risk population of cervical cancer against the integration site of HPV. This is mainly because the detection of HPV integration needs to detect the sequence of HPV and the sequence of the host human genome at the same time, and these two sequences form fusion sequence
Existing techniques for detecting HPV integration such as DIPS-PCR (Luft F et al., Int J Cancer, 2001, 92(1): 9-17) and APOT (Klaes R et al., Cancer Res, 1999, 59(24): 6132- 6136) The technical operation is cumbersome, the stability is poor, and the test results are inaccurate and incomplete. The detection technology only stays at the level of laboratory research and exploration, and cannot be applied to clinical testing.

Method used

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  • HPV-integrated gene sites related to occurrence of cervical carcinoma and application thereof
  • HPV-integrated gene sites related to occurrence of cervical carcinoma and application thereof
  • HPV-integrated gene sites related to occurrence of cervical carcinoma and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] The cervical tissues of 30 normal people (that is, human cervical tissues not infected by HPV virus), 10 cases of CINI patients, 9 cases of CINII patients, 7 cases of CINIII patients, and 52 cases of cervical cancer were respectively selected. Cervical tissue from stage I patients and 104 cervical cancer patients were used as samples.

[0104] CIN in the present invention refers to cervical intraepithelial neoplasia. CINI, CINII and CINIII refer to different pathological stages of cervical intraepithelial neoplasia.

[0105] 1) Mince 25 mg of cervical tissue, put it in a 1.5 ml tube, add 180 microliters of solution Buffer ATL, shake and mix;

[0106] 2) Add 20 microliters of proteinase K (Proteinase K), shake, and bathe in a 56°C water bath until the cervical tissue is completely dissolved;

[0107] 3) Centrifuge at low speed to shake off the liquid at the top of the tube, add 5 microliters of 100 mg / ml RNAase A, and shake for 15 seconds;

[0108] 4) Add 200 microlit...

Embodiment 2

[0115] Detection of HPV integration sites in genomic DNA of cervical tissue using a high-throughput viral integration assay.

[0116] 1) Take the genomic DNA sample of cervical tissue extracted in Example 1 from the -80°C refrigerator, put it in an insulated box with dry ice, and send it to Beijing Maijinuo Technology Gene Technology Co., Ltd. for high-throughput virus integration detection;

[0117] 2) Test the integrity of the sample: prepare an agarose gel with a mass fraction of 1%, take an appropriate amount (about 2 microliters) of the sample and mix it with Syber Green, add it to the agarose gel, and electrophoresis at 150V for 40 minutes in the electrophoresis tank . Exposure in the gel imager shows bands, and a single band without entrainment appears, indicating that the integrity of the sample is relatively good;

[0118] 3) Detection of sample concentration and content: DNA eluent was used as a blank control, and an appropriate amount of sample was taken to detect...

Embodiment 3

[0124] The HPV integration sites were verified by PCR amplification and Sanger sequencing.

[0125] 1. PCR amplification

[0126] Design the upstream and downstream primers that can amplify the HPV integration site, and the primers are as shown in Table 3, prepared according to the following system:

[0127]

[0128] Mix each tube separately, put it in a standard PCR machine and use the following procedure:

[0129]

[0130] 2. Send the amplified PCR product to Wuhan Tian-Huiyuan Biotechnology Co., Ltd. for Sanger sequencing.

[0131] 1) The PCR product obtained after amplification (content greater than 200ng, volume greater than 20ul) was sent to Wuhan Tian-Huiyuan Biotechnology Co., Ltd. for Sanger sequencing;

[0132] 2) The PCR sample is purified, and added to an ABI3730XL sequencer to detect the sequence of the PCR fragment.

[0133] 3. Analyze and detect the integration sites of HPV according to the sequencing results, and detect 50 integration sites.

[0134] ...

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Abstract

The invention relates to HPV-integrated gene sites related to occurrence of cervical carcinoma and a kit prepared based on the HPV-integrated gene sites related to occurrence of cervical carcinoma. Detection of high-risk populations of cervical carcinoma can be realized by extracting the DNA of cervical tissue of a subject, carrying out capturing and sequencing with a high flux virus, carrying out sequencing with PCR Sanger, detecting one or more arbitrary sites in the HPV-integrated gene sites in the DNA of the cervical tissue of the subject and calculating integration frequency. The kit prepared based on the HPV-integrated gene sites and used for detecting the high-risk populations of cervical carcinoma has the characteristics of accurate detection results, high sensitivity, etc.

Description

technical field [0001] The present invention relates to the fields of gynecological oncology and molecular genetics, in particular, the present invention relates to HPV integrated gene loci related to cervical cancer, and these HPV integrated gene loci in detection kits for cervical cancer high-risk groups application. Background technique [0002] Cervical cancer is one of the main malignant tumors that endanger women's health, and its mortality rate accounts for the third place among female malignant tumors worldwide. There are about 500,000 new cervical cancer patients every year in the world, and 268,000 people die from cervical cancer every year. There is a strong correlation between the incidence of cervical cancer and the degree of regional development. Nearly 86% of cervical cancer cases and 88% of deaths occur in underdeveloped regions (Arbyn M et al., Ann Oncol, 2011; 22: 2675-2683) . Cervical cancer has caused approximately 7.8 million years of life lost worldw...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12Q1/68
Inventor 马丁汪辉胡争祝达丁文成
Owner 武汉凯德维斯医学检验实验室有限公司
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