DNA molecular label for high-throughput detection of human papilloma virus

A high-throughput, labeling technology, applied in the direction of recombinant DNA technology, DNA/RNA fragments, microbial determination/inspection, etc., can solve the problems of high cost, time-consuming and labor-consuming, low throughput, etc., and achieve low-cost detection Effect

Active Publication Date: 2010-12-22
上海华大基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] On the other hand, currently known HPV detection methods, such as those described above, are low throughput
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Method used

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  • DNA molecular label for high-throughput detection of human papilloma virus
  • DNA molecular label for high-throughput detection of human papilloma virus
  • DNA molecular label for high-throughput detection of human papilloma virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1: Sample extraction

[0102] According to the manufacturer's instructions, a KingFisher automatic extractor (American ThermoScientific Kingfisher Flex automatic magnetic bead extraction and purification system) was used to extract DNA from 190 exfoliated cells with known HC-II results. Use the program "Bioeasy-200μl BloodDNA-KF.msz" for nucleic acid extraction. After the program is over, about 100 μl of eluted product (extracted DNA) is obtained, which is used as a template in the next PCR amplification.

Embodiment 2

[0103] Example 2: PCR amplification

[0104] The 190 DNAs obtained in Example 1 were sequentially numbered 1-190, and were equally divided into 2 groups (HPV-1 group: No. 1-95; HPV-2 group: No. 96-190). According to the sequence of each primer (Table 2, SEQ ID NO: 96-107) of the primer set (including 6 forward primers and 5 reverse primers) used to amplify HPV DNA, a set of tags was designed, a total of 95 (Table 1, SEQ ID NO: 1-95). Each designed tag is added to the 5'end of the sequence of each primer of the primer set, so as to obtain 95 tag primer sets, wherein each tag primer set includes corresponding 6 forward tag primers and 5 reverse primers Tag primers, and different tag primer sets use different tags (ie, 95 tag primer sets correspond to 95 tags one to one).

[0105] PCR reactions were performed on all samples in 96-well plates, using 2 plates (1 plate each for HPV-1 group and HPV-2 group). The DNA obtained in Example 1 was used as a template, and in the HPV-1 group ...

Embodiment 3

[0121] Example 3: Mixing and purification of PCR products

[0122] Mix the remaining PCR products in the HPV-1 group and the HPV-2 group in a 3ml EP tube (also labeled as the HPV-1 group and the HPV-2 group), and shake and mix. 500μl DNA was taken out from each of the two tubes of the mixture, and column purification was performed using Qiagen DNA Purification kit according to the manufacturer's instructions to obtain 200μl DNA. Using Nanodrop8000 (Thermo Fisher Scientific), the DNA concentration of the purified mixture was determined to be 98 ng / μl (HPV-1 group) and 102 ng / μl (HPV-2 group), respectively.

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Abstract

The invention relates to a detection method of human papilloma virus (HPV), in particular to the HPV detection method based on the Solexa sequencing method.

Description

Technical field [0001] The invention relates to a method for detecting human papilloma virus (Human Papilloma Virus, HPV), in particular to a method for detecting HPV based on the Solexa sequencing method. Background technique [0002] Cervical cancer is the second killer of female cancer in the world, second only to breast cancer. There are about 500,000 new cases and 250,000 deaths worldwide each year, of which developing countries account for 2 / 3. my country is a high incidence area of ​​cervical cancer, accounting for 10% of the total number of cervical cancers in the world. Studies have shown that human papillomavirus (HPV) is closely related to cervical cancer. It is an important carcinogen and one of the necessary conditions for cervical cancer. It has been shown that more than 100 types of HPV can infect the skin (skin type) or mucous membranes of the respiratory and anogenital tracts (mucosa type), and more than 40 types of HPV can infect the cervix. Based on induced ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
CPCC12Q1/6869C12Q1/708
Inventor 易鑫刘涛徐佳佳
Owner 上海华大基因科技有限公司
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