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HPV (human papillomavirus) DNA (deoxyribonucleic acid) genetic typing method and kit thereof

A technology of genotyping and treatment methods, applied in the field of HPV DNA genotyping, which can solve the problems of long detection time and the stability of results affected by the environment

Inactive Publication Date: 2013-09-25
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional capillary electrophoresis instrument is relatively large, and the detection takes a long time, and the stability of the result is also greatly affected by the environment.

Method used

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  • HPV (human papillomavirus) DNA (deoxyribonucleic acid) genetic typing method and kit thereof
  • HPV (human papillomavirus) DNA (deoxyribonucleic acid) genetic typing method and kit thereof
  • HPV (human papillomavirus) DNA (deoxyribonucleic acid) genetic typing method and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1. Establishment of PCR-RFLP method for HPV genotyping of the present invention

[0108] 1. Selection of PCR primers

[0109] So far, more than 120 types of HPV DNA have been found. For example, for different types of HPV-specific sequences, synthesizing specific primers for PCR reactions will require a lot of work and consumption. In order to avoid this disadvantage, the present invention uses PGMY09 / 11 universal primers to perform hot-start PCR on the sample genomic DNA based on the highly conserved capsid protein L1 gene of each type of HPV, and obtains a fragment with a length of 449bp-458bp .

[0110] The PGMY09 / 11 universal primer consists of an upstream primer and a downstream primer;

[0111] The upstream primer consists of primers PGMY11-A, PGMY11-B, PGMY11-C, PGMY11-D and PGMY11-E;

[0112] The downstream primer consists of primers PGMY09-F, PGMY09-G, PGMY09-H, PGMY09-I, PGMY09-J, PGMY09-K, PGMY09-L, PGMY09-M, PGMY09-N, PGMY09-P, PGMY09-Q, PGMY09-R and HMB01 ...

Embodiment 2

[0123] Example 2. Application of HPV genotyping detection method of the present invention

[0124] 1. Detection of samples with known HPV genotyping

[0125] 1. Cell culture

[0126] Culture Caski cells (containing type 16 HPV genomic DNA) (sample 1) and Hela cells (containing type 18 HPV genomic DNA) (sample 2) (purchased from the Tumor Cell Bank of the Chinese Academy of Medical Sciences). The former is DMEM medium with 10% fetal bovine serum, and the latter uses RPMI-1640 medium with 10% fetal bovine serum. Cultivation gas phase environment: air, 95%; carbon dioxide, 5%. Incubate at 37°C to the logarithmic growth phase.

[0127] 2. Preparation of DNA

[0128] Use conventional DNA extraction methods or DNA extraction kits to extract DNA from the cells in step 1, and store the product at 4°C for a short time and -20°C for a long time.

[0129] 3. PCR amplification

[0130] Take the DNA extracted in step 2, and perform PCR amplification according to the following reaction system and re...

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Abstract

The invention discloses an HPV(human papillomavirus) DNA (deoxyribonucleic acid) genetic typing method and a kit thereof. The method disclosed by the invention can be used for typing DNA of 46 types of HPVs, is simple to operate, is economical and practical and fast and efficient, and is combined with chip capillary electrophoresis testing to analyze the HPV DNA type with high throughput. When applied in the medical domain, the HPV / DNA genetic typing method is combined with the existing lamina ThinPrep cytology morphology diagnosis and is benefit to the early-stage prevention, prognosis and research of target gene vaccines for cervical cancer; and the method can be used for detecting multiple types and finding unknown types, thereby being especially suitable for research of pathogenesis and epidemiology of HPV-related diseases, such as hypopharyngeal carcinoma and head and neck cancer.

Description

Technical field [0001] The invention relates to a method for HPV DNA genotyping and its kit. Background technique [0002] Cervical cancer has become one of the main problems threatening women's health worldwide. According to statistics, approximately 231,000 women die from cervical cancer each year, and more than 80% of new cases occur in developing countries. The main cause of cervical cancer is infection with human papilloma virus (HPV for short). Approximately 99.7% of cervical cancer cases have been detected to carry HPV DNA, of which 16, 18, 31, and 45 are the most common. HPV can be divided into four categories in terms of its pathogenicity, namely high-risk types (16,18,26,31,33,35,39,45,51,52,56,58, and 59) and possibly high-risk types ( 26,53,66,68,73 and 82 types), low-risk types (6,11,13,40,42,43,44,54,61,70,72,81 and 89 types), and unspecified types (Type 30, 32, 34, 62, 67, 69, 71, 74, 83, 84, 85, 86, 87, 90, 91, etc.). Among them, high-risk types have attracte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 林金明易伶潞张炜飞刘建和
Owner TSINGHUA UNIV
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