High-risk human papilloma virus typing detection method and kit
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A technology of human papillomavirus and detection method, which is applied in the field of high-risk human papillomavirus typing detection methods and kits, and can solve the problems of restriction of types of gene sequence detection and the like
Inactive Publication Date: 2021-02-23
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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Single-probe real-time quantitative PCR, because each probe is designed for a specific target site, with high specificity, each probe can only detect one target sequence, if multiple target sequences need to be detected, Multiple probes need to be added. Due to the limitation of the absorption wavelength of the fluorescent label of the probe, the types of gene sequence detection are restricted.
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Embodiment 1
[0129] Embodiment 1 Human papillomavirus 33, 35, 45, 51, 56, 66 type nucleic acid typing detection kit (fluorescent PCR method)
[0130] 1. Primer Probe Synthesis
[0131] Entrust Yingwei Jieji (Shanghai) Trading Co., Ltd. to synthesize the primer probe. The sequence of the synthesized primer probe is as follows:
[0132] The upstream primers are:
[0133] 5'-CAACTATTTGTTACTGTTGTTGATACTAC-3' as shown in SEQ ID No.1;
[0134] 5'- CAATTATTTGTTACTGTGGTAGATACCAC -3' as shown in SEQ ID No.2;
[0135] 5'-CAGTTGTTTGTCACAGTTGTGGATACCAC-3' as shown in SEQ ID No.3;
[0136] 5'-CAATTGTTTTTAACAGTTGTAGATCCTAC-3', as shown in SEQ ID No.4;
[0137] Downstream primers are:
[0138] 5'-GAAATATAAACTGCAAATCAAATTCCTC-3', as shown in SEQ ID No.5;
[0139] 5'- GAAAAATAAATTGTAAATCAAATTCCTC-3', as shown in SEQ ID No.6;
[0140] 5'-GAAATATAAATTGTAAATCAAATTCCTC-3', as shown in SEQ ID No.7;
[0141] 5'- GAAAAATAAACTGCAAATCATATTCCTC-3', as shown in SEQ ID No.8;
[0142] 5'-GAAAAATAAACTGTAAATCATA...
Embodiment 2
[0174] Application of embodiment 2 human papillomavirus 33, 35, 45, 51, 56, 66 type nucleic acid typing detection kit (fluorescent PCR method)
[0175] Using the kit obtained in the present invention to test 20 cases of clinical samples, and at the same time use the human papillomavirus genotyping (type 25) detection kit (PCR-reverse dot hybridization method) produced by Aikang Biotechnology Co., Ltd. for parallel comparison Test and evaluate the effect of the kit.
[0176] 1. Human papillomavirus 33, 35, 45, 51, 56, 66 nucleic acid typing detection kit (fluorescent PCR method) for detection of clinical samples
[0177] (1) Reagent preparation
[0178] Prepare the reaction solution according to the number of samples n (number of samples = number of samples to be tested + 3 reference substances + 1):
[0179] Take PCR buffer n×9 μL, enzyme mixture n×3 μL, primer probe n×8 μL in a centrifuge tube and mix evenly; centrifuge at low speed for a few seconds, and dispense 20 μL / tub...
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Abstract
The invention provides a high-risk human papilloma virus (HPV) typing detection method and a kit. The method adopts a fluorescent nucleic acid amplification method of multiple pairs of primers and multiple probes, combines melting curve analysis, and can realize detection of multiple target nucleic acid sequences in a single-wavelength fluorescence channel. According to the method, an HPV specificprimer and a TaqMan probe are designed by taking an L1 segment sequence of an HPV genome as a target sequence, and a PCO hybridization probe which is incompletely complementary with the TaqMan probeis designed; and fluorescent quenching group labeling or non-fluorescent quenching group labeling can be performed on the 3'end of the hybridization probe according to the purpose, and the 3 'end of the hybridization probe cannot extend again after labeling, so that the HPV genotyping kit containing the primer probe and other fluorescent PCR components is developed, and the HPV33, 35, 45, 51, 56 and 66 types can be identified by combining fluorescent PCR amplification with single-wavelength melting curve analysis.
Description
technical field [0001] The invention relates to a high-risk human papilloma virus typing detection method and kit, which belong to the field of biotechnology. Background technique [0002] Human papillomavirus (Human Papillomavirus, HPV) belongs to the papillomaviridae family, is a small molecule, non-encapsulated, circular double-stranded DNA virus, the genome length is about 8000 base pairs (bp), divided into 3 There are 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. It can only infect human skin and mucosal epithelial cells, and can cause squamous epithelial proliferation of human skin and mucous membranes. More than 130 kinds of papillae that cause human skin have been isolated so far. ...
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