Kit for detecting genotyping and IL28-site polymorphism of hepatitis C virus (HCV)

A kit and genotype technology, applied in the field of kits for detecting hepatitis C virus genotype and IL28 site polymorphism, can solve the problems of missed detection of the population, regional and ethnic differences in virus distribution, etc.

Active Publication Date: 2011-07-20
DAAN GENE CO LTD
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Trugene HCV 5'NCGenotyping kit (Canada), this kit is completed by sequencing, but the cost of sequencing and the ability of sequencing to distinguish mixed infections can only reach about 20% and still cannot meet the clinical requirements; Inno LiPA HCV II ( The reverse hybridization method used by Innogenetics, but the virus has regional and ethnic differences in distribution, and there may be missed detection in the Chinese population

Method used

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  • Kit for detecting genotyping and IL28-site polymorphism of hepatitis C virus (HCV)
  • Kit for detecting genotyping and IL28-site polymorphism of hepatitis C virus (HCV)
  • Kit for detecting genotyping and IL28-site polymorphism of hepatitis C virus (HCV)

Examples

Experimental program
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Effect test

Embodiment 1

[0040] The preparation of embodiment 1 sample target nucleic acid

[0041] Three cases of EDTA anticoagulated whole blood samples confirmed by sequencing were selected as HCV type 1b simultaneous IL28B polymorphic site rs12979860C, HCV type 2a simultaneous IL28B polymorphic site rs12979860C, and 3b type simultaneous IL28B polymorphic site rs12979860T, according to Roche company's extraction reagent High Pure Viral Nucleic Acid Kit instructions require the extraction of nucleic acids.

Embodiment 2

[0042] Embodiment 2 one-step RT-PCR reaction

[0043] The volume of RT-PCR reaction reagent per person is 50 μl, prepared according to Qiagen’s Onestep RT-PCR Kit (operated according to the instructions), including 1 μl 10mM HCV-RT, 1 μl 0.5mM HCV-F, 1 μl 5mM HCV-R, 1 μl 0.5 mM IL28B-F, 1 μl of 5 mM IL28B-R. Add 5 μl of the nucleic acid extracted from the specimen of Example 1 into the RT-PCR reaction reagent respectively, and perform amplification under one-step reaction conditions. 72°C for 45 seconds, run 45 cycles, and finally 72°C for 7min.

Embodiment 3

[0044] Embodiment 3 Two-step reverse transcription reaction

[0045] The volume of reverse transcription reagent per person is 25 μl, according to Ambion's RETROscript TM The kit (operated according to the instructions) was prepared, including 0.5 μl of 10mM HCV-RT, and 5 μl of the nucleic acid extracted from the specimen of Example 1 was added to the reverse transcription reagent respectively, and the reverse transcription reaction conditions were used for amplification. The specific conditions were 44°C 60 minutes, 10 minutes at 92°C.

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Abstract

The invention relates to a kit for detecting the genotype and IL28-site polymorphism of a hepatitis C virus (HCV), in particular to a kit for detecting the genotype and IL28-site polymorphism of an HCV, which is prepared by using a nucleic acid reverse dot hybridization technology. The kit comprises an amplification reagent, a low-density chip using a nylon membrane as a carrier and a hybridization reagent, and can simultaneously detect the genotype of the HCV and the IL28B gene polymorphism closely related to the HCV treatment, thereby providing a more reliable basis for the individual hepatitis C treatment.

Description

technical field [0001] The invention relates to a kit for detecting hepatitis C virus genotype and IL28 site polymorphism, in particular to preparing a hepatitis C virus genotype and IL28 site polymorphism detection kit using nucleic acid reverse dot hybridization technology , the kit of the invention can quickly and accurately distinguish the genotype of hepatitis C virus and the polymorphism of IL28 in the sample. Background technique [0002] Hepatitis C virus (HCV) is one of the hepatitis viruses that seriously endanger human health and is an important pathogenic factor of liver diseases. At present, there are more than 170 million people infected with HCV in the world, and more than 100,000 cases of hepatitis C patients develop liver cancer every year, followed by gastrointestinal bleeding and ascites. According to the WHO 2002 Annual Report, in 2001, chronic liver diseases caused 14 million deaths, of which 796,000 were caused by liver cirrhosis and 616,000 were cause...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 程钢林炳生张帆陈华云
Owner DAAN GENE CO LTD
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