Whole set reagent and method for detecting human papilloma virus

A technique for papilloma and human detection, applied in the fields of medical genetic engineering and molecular genetics, can solve problems such as low sensitivity, large number of samples, and inability to detect HPV integration status

Active Publication Date: 2018-10-19
重庆迈基诺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

[0009] However, these HPV nucleic acid detection methods either only detect HPV nucleic acid information, cannot detect HPV integration status, or have disadvantages such as low sensitivity, cumbersome operation, time-consuming, a large number of samples, few HPV genotypes, and uncertain judgment results. Detection methods such as hybridization, real-time quantitative PCR, gene chips, and amplification of HPV integrated oncogene transcripts

Method used

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  • Whole set reagent and method for detecting human papilloma virus
  • Whole set reagent and method for detecting human papilloma virus
  • Whole set reagent and method for detecting human papilloma virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1, the preparation of complete set of reagents and kit for detecting human papillomavirus

[0073] 1. A complete set of reagents for detecting human papillomavirus

[0074] The complete set of reagents for detecting human papillomavirus provided in this example is designed according to 27 HPV genotypes and used to identify these 27 HPV genotypes, these 27 HPV genotypes are HPV 6, 11, 16, 18 respectively , 31, 33, 35, 39, 45, 52, 56, 58, 59, 66, 68, 69, 82, 51, 26, 30, 34, 53, 67, 70, 73, 85, and 97. The complete set of reagents for detecting human papillomavirus is composed of 621 probes, the sequences of these 621 probes are sequences 1-621 in the sequence table, and the 16th-93rd positions of each sequence are used to identify human papillomavirus, each The 1-15th and 94th-108th positions of the sequence are identical and can be used to amplify probes, and the 5' end of each probe is labeled with biotin.

[0075] In this kit, all probes are packaged toget...

Embodiment 2

[0101] Embodiment 2, the method for detecting human papillomavirus

[0102] The method that utilizes the complete set of reagents of embodiment 1 and kit to detect human papillomavirus of the present invention is as follows:

[0103] 1. Preparation of whole genome library

[0104] With the informed consent of the patient, the genomic DNA of the peripheral blood of the HPV-infected individual was extracted, the concentration of the obtained genomic DNA was adjusted to 75ng / μl, and the total amount of 3-5μg DNA was randomly interrupted. ) to construct a genome-wide library. The sequences of the two single-stranded DNAs of the adapters used are as follows:

[0105] ssDNA1: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT

[0106] Single-stranded DNA2: AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNATCTCGTATGCCGTCTTCTGCTTG, wherein N is A, T, C or G.

[0107] 2. Specific capture and sequencing of HPV gene fragments

[0108] 1) Take 1 μg of the genome-wide library in step ...

Embodiment 3

[0134] Embodiment 3, utilize the kit of embodiment 1 to detect HPV typing result

[0135] The kit in Example 1 was used to detect the HPV genotypes of 12 patients with cervical cancer (all with the informed consent of the patients), and the method in Example 2 was followed.

[0136] The results confirm that the complete set of reagents in Example 1 has a high capture rate for HPV genes, the average effective sequencing READ amount of the target region (HPV gene) reaches 20Mb, and the average sequencing depth of the target region (HPV gene) is more than 1000X (see Table 1) The HPV genotypes detected in different patients are shown in Table 1. The HPV genotypes detected in the patients are all common HPV genotypes in mainland China.

[0137] Table 1, utilize kit of the present invention to the HPV typing result that 12 routine cervical cancer patients detect

[0138]

[0139] Detect the above samples with Kaipu 23 kinds of HPV fluorescent typing detection kits (fluorescent P...

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Abstract

The invention discloses a whole set reagent and a method for detecting a human papilloma virus. The whole set reagent for detecting the human papilloma virus disclosed by the invention consists of 621probes. The sequences of the 621 probes are sequences 1 to 621 in the sequence listing, and 1 to 15 positions and 94 to 108 positions of all sequences 1 to 621 in the sequence listing are the same and can be used for amplifying the 621 probes; the 5' end of each probe can be labeled with biotin. The whole set reagent provided by the invention can distinguish different types during capture of human papilloma virus genomes and sequencing, can also determine whether the DNA is integrated into human genomes or not, can also determine the copy number of the human papilloma virus, and have the characteristics of accurate typing, capability of obtaining integrated information and copy number, high sensitivity, and the like, thus meeting the development trend of precision medicine, and having great significance.

Description

technical field [0001] The invention relates to a complete set of reagents and a method for detecting human papillomavirus in the fields of medical genetic engineering and molecular genetics. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) belongs to the Papillomaviridae, is a small molecule, non-encapsulated circular double-stranded DNA virus, the genome is about 8000 base pairs (bp), divided into three Functional regions, namely early transcribed region (E region), late transcribed region (L region) and non-transcribed region (long control region, LCR). HPV infects humans through direct or indirect contact with contaminated objects or through sexual transmission. The virus is not only host-specific but also tissue-specific, and can only infect human skin and mucosal epithelial cells, causing various papillomas or warts on human skin and hyperplastic lesions of the genital tract epithelium. [0003] For HPV that infects the reproductive trac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/70C12Q1/6886C12Q1/6869
CPCC12Q1/6869C12Q1/6886C12Q1/708C12Q2535/122C12Q2537/165C12Q2537/16
Inventor 伍建姬晓雯刘娜王晓斌郑生伟
Owner 重庆迈基诺医学检验实验室有限公司
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