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

A high-throughput, labeling technology, applied in the field of human papillomavirus detection, can solve the problems of high cost, low throughput, time-consuming and labor-intensive, and achieve the effect of low-cost detection

Active Publication Date: 2012-11-14
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
Applying the above methods is time-consuming, labor-intensive, and costly when performing HPV detection on large-scale samples

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, DNA was extracted from 190 exfoliated cells with known HC-II results using a KingFisher automatic extractor (ThermoScientific Kingfisher Flex automatic magnetic bead extraction and purification system, USA). Nucleic acid extraction was performed using the program "Bioeasy-200 μl BloodDNA-KF.msz". After the program is finished, about 100 μl of the eluted product (extracted DNA) is obtained, which is used as a template in the next step of PCR amplification.

Embodiment 2

[0103] Embodiment 2: PCR amplification

[0104] The 190 copies of DNA obtained in Example 1 were numbered 1-190 in sequence, and were evenly divided into 2 groups (HPV-1 group: numbering 1-95; HPV-2 group: numbering 96-190). According to the sequence (Table 2, SEQ ID NO:96-107) of each primer of the primer set (including 6 forward primers and 5 reverse primers) used to amplify HPV DNA, design a set of tags, a total of 95 (Table 1, SEQ ID NO: 1-95). Add each designed tag to the 5' end of the sequence of each primer in the primer set to obtain 95 tag primer sets, where each tag primer set includes corresponding 6 forward tag primers and 5 reverse Index primers, and different index primer sets use different tags (ie, 95 index primer sets correspond to 95 tags one-to-one).

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

Embodiment 3

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

[0122] Mix the remaining PCR products of the HPV-1 group and the HPV-2 group in a 3ml EP tube (also marked as HPV-1 group and HPV-2 group), and shake to mix. 500 μl of DNA was taken from each of the 2 tubes of mixture, and purified by column using Qiagen DNA Purification kit according to the manufacturer's instructions to obtain 200 μl of DNA. Using Nanodrop 8000 (Thermo Fisher Scientific), the DNA concentrations of the purified mixtures were determined to be 98 ng / μl (HPV-1 group) and 102 ng / μl (HPV-2 group), respectively.

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PUM

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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 detection method for human papilloma virus (Human Papilloma Virus, HPV), in particular to a detection method for HPV based on the Solexa sequencing method. Background technique [0002] Cervical cancer is the second leading cause of cancer among women in the world, after breast cancer. There are about 500,000 new cases and 250,000 deaths in the world every year, of which two-thirds are in developing countries. 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, is an important carcinogen, and is also one of the necessary conditions for cervical cancer. More than 100 HPV species have been shown to infect the skin (cutaneous types) or mucous membranes of the respiratory and anogenital tracts (mucosal types), and more than 40 HPV species to infect the cervix. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/68
CPCC12Q1/6869C12Q1/708
Inventor 易鑫刘涛徐佳佳杨晓楠
Owner 上海华大基因科技有限公司
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