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Hepatitis C sequencing and typing kit and detection method thereof

A detection method and kit technology, which is applied in the field of hepatitis C sequencing typing kit and its detection, can solve the problems of poor stability and low accuracy, and achieve high accuracy, low cost, and high throughput

Active Publication Date: 2010-12-22
CHONGQING DIAN SRAB CENT FOR CLINICAL LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because sometimes only a few nucleotides vary between each type, there are stricter requirements on the conditions of hybridization, so this method is not accurate and stable

Method used

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  • Hepatitis C sequencing and typing kit and detection method thereof
  • Hepatitis C sequencing and typing kit and detection method thereof
  • Hepatitis C sequencing and typing kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Preparation of the kit.

[0062] (1) RNA extraction reagent:

[0063] Trizol: purchased from Invitrogen,

[0064] Chloroform: purchased from Hangzhou Gaojing Fine Chemical Co., Ltd.

[0065] Isopropanol: purchased from Hangzhou Shuanglin Chemical Reagent Factory,

[0066] Absolute ethanol: purchased from Hangzhou Changzheng Chemical Reagent Co., Ltd.

[0067] DEPC treated water: purchased from Fermentas Company.

[0068] (2) Reagents for reverse transcription:

[0069] 200U / μL M-MLV reverse transcriptase: purchased from Fermentas Company,

[0070] 20U / μL RNase inhibitor: purchased from Fermentas Company,

[0071] 5×RT-PCR Buffer: 250mM Tris-HCl (pH 8.3), 250mM KCl, 20mM MgCl 2 , 50mMDTT (purchased from fermentas company),

[0072] 10mM dNTP mix: purchased from Shanghai Dingguo Biotechnology Co., Ltd.,

[0073] 100 μM random hexamer primer: purchased from Invitrogen Company,

[0074] DEPC treated water: purchased from Fermentas Company.

[0075] (3)...

Embodiment 2

[0092] Embodiment 2: detection method.

[0093] (1) The extraction of hepatitis C HCV RNA template specifically includes the following steps:

[0094] (1a) Mix 200 μL of sample serum with 1 mL Trizol, add 200 μL of chloroform, mix for 15 seconds, room temperature for 5 minutes, and centrifuge at 12000 g for 15 minutes at 4°C (the centrifuge is Microfuge 22R Centrinofuge, BECKMAN Company, the same below);

[0095] (1b) Transfer the upper aqueous phase into another centrifuge tube, add isopropanol equal to the volume of the upper aqueous, mix for 15 seconds, room temperature for 10 minutes, and centrifuge at 12000g for 10 minutes at 4°C;

[0096] (1c) Discard the supernatant obtained in step (1b), add ice-precooled 75% (v / v) ethanol (mixed with DEPC water) 1mL, and centrifuge at 7500g for 5min at 4°C;

[0097] (1d) Discard the supernatant obtained in step (1c), air-dry for 5 min, add 20 uLDEPC-treated water, and obtain HCV RNA at -70°C for later use.

[0098] (2) the RNA templ...

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PUM

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Abstract

The invention discloses a hepatitis C sequencing and typing kit which comprises: (1) primers of specific amplified hepatitis C virus RNA reverse transcription product c DNA and (2) hepatitis C virus specific sequencing primers, wherein the nucleotide sequences of the primers of specific amplified hepatitis C virus RNA reverse transcription product c DNA are shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, the SEQ ID NO:1 and the SEQ ID NO:2 are a primer pair and the SEQ ID NO:3 and the SEQ ID NO:4 are another primer pair; and the nucleotide sequences of the hepatitis C virus specific sequencing primers are shown as SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; the SEQ ID NO:4 is marked with a biotin at a 5' end; and all the primers are all used in a detection. The invention also discloses a detection method of the hepatitis C sequencing and typing kit. The hepatitis C sequencing and typing kit can be used for sequencing and typing hepatitis C, thereby providing more comprehensive reference information for clinical diagnosis and treatment.

Description

technical field [0001] The invention relates to a hepatitis C sequencing typing kit and a detection method thereof, belonging to the technical field of detection of virus subtypes. Background technique [0002] There are regional differences in HCV genotypes, and different genotypes are related to disease progression, liver cancer and interferon efficacy, and are of great significance to the transmission route of sporadic hepatitis C and the development of vaccines. [0003] There were obvious regional differences in the distribution of HCV genotypes. Five common genotypes (1a, 1b, 2a, 2b, 3a) are widely distributed, among which, type 1a predominates in the United States, Brazil, and northwestern Europe, type 1b is more common in the Far East, east and west of Europe, and types 2a and 2b are more common Distributed in Asia and Europe, type 3a is more common in some western countries, Thailand, and Singapore, and becomes the only genotype of HCV infection in some populations...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 任绪义虞闰六吕江峰王栋梁
Owner CHONGQING DIAN SRAB CENT FOR CLINICAL LAB CO LTD
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