HCV gene typing detecting reagent kit

A hepatitis C virus and kit technology, applied in the direction of biological testing, material inspection products, microbial measurement/inspection, etc., can solve the problems of long detection time, cumbersome operation, low sensitivity, etc.

Inactive Publication Date: 2009-03-04
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently, HCV genotyping methods mainly include: restriction length polymorphism (RFLP), type-specific primer PCR, type-specific probe nucleic acid hybridization analysis, direct sequencing, etc. Long, expensive, low sensitivity, low specificity and other disadvantages, it is not suitable for the majority of primary hospitals

Method used

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  • HCV gene typing detecting reagent kit
  • HCV gene typing detecting reagent kit
  • HCV gene typing detecting reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1: Preparation of sample target nucleic acid

[0071] Use a disposable sterile syringe to extract 2 ml of venous blood from the subject, inject it into a sterile dry glass tube, and place it at room temperature (22-25°C) for 30-60 minutes or centrifuge to separate out the serum. Aspirate 100 μl of the upper serum, transfer it to a 1.5ml sterilized centrifuge tube, add 20 μl of well-mixed RNA extraction solution A, then add 400 μl of RNA extraction solution B and mix well. Place at room temperature for 5 minutes, centrifuge at 6000 rpm for 1 minute, carefully suck off the supernatant, and wash the precipitate twice with 75% pre-cooled ethanol (prepared with DEPC water). Dry the pellet at 65°C for 10 minutes. Add 18 μl of Hepatitis C Reverse Transcription System to the precipitation tube, and the next step of reverse transcription reaction can be carried out.

Embodiment 2

[0072] Example 2: Reverse transcription of RNA into cDNA

[0073] Take a 1.5mL centrifuge tube without RNase, take 19μL×(N+1) reverse transcription reaction solution, add 1μL×(N+1) reverse transcriptase, mix well to prepare a reverse transcription system, and dispense to N( N=Number of samples to be detected+2) In 0.2mL centrifuge tubes without RNase-free blanks, add 5 μL of the prepared RNA template to each centrifuge tube packed with the reverse transcription system, and centrifuge briefly (3 seconds), Put each reaction tube into the PCR instrument. The reverse transcription (RT) reaction was performed under the following conditions: constant temperature at 40°C for 60 minutes, then inactivation at 95°C for 3 minutes, and then constant temperature at 4°C.

Embodiment 3

[0074] Embodiment 3: the PCR amplification of target nucleic acid

[0075] Take several tubes of single-tube PCR reaction solution for one person, directly add 3 μl of the reverse-transcribed cNDA template, mix well, and centrifuge briefly (3 seconds), then put each reaction tube into the PCR machine. Amplify according to the following conditions: pre-denaturation at 93°C for 6 minutes, followed by 10 cycles of 93°C for 45 seconds, 57°C for 45 seconds, 72°C for 45 seconds, 93°C for 30 seconds, 55°C for 40 seconds, and 72°C for 45 seconds. Add 30 cycles, a total of 40 cycles; finally, keep warm at 72°C for 7 minutes. The amplified products were detected by 2% agarose gel electrophoresis (see attached figure 2 ).

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Abstract

The invention relates to a kit for detecting a hepatitis c virus genotype, particularly relates to that the nucleic acid reverse dot blot hybridization technology is used to prepare a kit for hepatitis c virus genotype detection. The invention is used for rapidly and accurately distinguishing the hepatitis c virus genotype in a clinical blood sample.

Description

technical field [0001] The invention relates to a kit for detecting the genotype of hepatitis C virus, in particular to using nucleic acid reverse dot hybridization technology to prepare a kit for detecting the genotype of hepatitis C virus, which is used for quickly and accurately distinguishing the genotype of hepatitis C virus in clinical blood samples. Hepatitis virus genotype. 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 HCV patients develop liver cancer every year, followed by gastrointestinal bleeding and ascites. According to the 2002 annual report of WHO, in 2001, chronic liver disease caused 14 million deaths, including 796,000 cases caused by liver cirrhosis and 616,000 cases caused by primary liver cancer. In 2001, 20% (more ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50C12Q1/68
Inventor 王敏王会龙马丽李明程钢何蕴韶
Owner DAAN GENE CO LTD
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