A kit for genotyping hepatitis C virus

A hepatitis C virus and genotyping technology, applied in the field of medical devices, can solve the problems of inability to meet clinical needs, low sensitivity, low specificity, insufficient subtyping ability, etc., achieving a high degree of automation, accurate results, Actionable fast effects

Active Publication Date: 2011-12-21
HUAXIN SCI & TECH PANYU CITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have low sensitivity and specificity, can only distinguish several main g

Method used

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  • A kit for genotyping hepatitis C virus
  • A kit for genotyping hepatitis C virus
  • A kit for genotyping hepatitis C virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of the kit of the invention

[0041] The composition of the kit of the present invention is as follows:

[0042] (1) Hepatitis C virus RNA extraction reagent: TRIzol lysate, chloroform, isopropanol and absolute ethanol

[0043] (2) Primer:

[0044] The sequence of the upstream primer of HCV NS5B gene is: 5’ -TTAACCACATCMRCTCCGTGTG-3’

[0045] The downstream primer sequence of HCV NS5B gene is: 5’-GTACCTGGTCATAGCYTCCGTRAA-3’

[0046] The sequence of the upstream primer of HCV NCR conserved region gene is: 5’-GCGGAACCGGTGAGTACA-3’

[0047] The downstream primer sequence of HCV NCR conserved region gene is: 5’-CCTATCAGGCAGTACCACAAGG-3’

[0048] The above primers were synthesized by Shanghai Life Technology Company.

[0049] (3) Negative control and positive control: deionized water is used as a negative control, and a sample containing HCV RNA is used as a positive control.

[0050] (4) Reverse transcription PCR reagents: 200 U / μL M-MLV, 40 U / μL RNase, 50 μM Oligo(...

Embodiment 2

[0057] Example 2 Using the kit prepared in Example 1 to detect the genotype of HCV

[0058] Take the detection of HCV genotype in peripheral blood samples of 20 hepatitis C patients as an example.

[0059] Detection process: First, design specific primers based on the HCV nucleic acid sequence provided by the HCV nucleic acid database. Obtain peripheral blood samples of clinical hepatitis C patients, quickly extract HCV RNA, use the extracted RNA as a template, and perform reverse transcription reaction with synthetic oligonucleotide reverse transcription primers to obtain cDNA, and then use this cDNA as a template and synthesis PCR primers are used for PCR amplification. The amplified product is directly recovered by the gel recovery kit for rapid gel recovery, the recovered DNA is used for the sequencing reaction, the sequencing reaction product is purified and then sequenced, and finally the nucleic acid sequence is compared in the NCBI nucleic acid database to determine the H...

Embodiment 3

[0070] Example 3 Evaluation of the detection capability of the kit of the present invention

[0071] According to Example 1, 45 specimens of HCV patients who were genotyped by restriction fragment length polymorphism method were tested using the kit of the present invention. The detection capabilities of the two were compared. The sensitivity and specificity of the kit were compared. Compared with the restriction fragment length polymorphism method for typing and sensitivity, this kit is more accurate and fully meets the current practical requirements of clinical diagnosis and treatment:

[0072]

[0073] among them:

[0074] ① Specificity: 100%;

[0075] ② Sensitivity: 97%;

[0076] ③ Positive predictive value: the positive predictive value reaches 100%;

[0077] ④ Negative predictive value: Negative predictive value reaches 94%;

[0078] ⑤ Repeatability: the results of repeated experiments are consistent;

[0079] ⑥ Time-consuming: The testing time for a clinical specimen is about 8-1...

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Abstract

The invention relates to a kit for genotyping hepatitis C virus (HCV), which comprises HCVNS5B gene amplification primers, HCVNCR conserved region gene amplification primers, an HCVRNA extraction reagent, a negative control, a positive control, a cDNA (complementary deoxyribonucleic acid) synthetic reagent, a PCR (polymerase chain reaction) solution and a PCR sequencing reagent, wherein the sequence of the HCVNS5B gene forward primer is disclosed as SEQ ID NO.1, the sequence of the HCVNS5B gene reverse primer is disclosed as SEQ ID NO.2, the sequence of the HCVNCR conserved region gene forward primer is disclosed as SEQ ID NO.3, and the sequence of the HCVNCR conserved region gene forward primer is disclosed as SEQ ID NO.4. The kit provided by the invention has the advantages of high detection sensitivity and good specificity, and can be used for detecting all the reported HCV genotypes (subtypes) at high speed (within 12-14 hours).

Description

technical field [0001] The invention relates to a kit for genotyping hepatitis C virus, which is used for rapidly detecting the type and subtype of hepatitis C virus in blood samples of patients, and belongs to the field of medical equipment. [0002] Background technique [0003] Hepatitis C virus (hepatitis C virμs, HCV) is a single-stranded linear positive-sense RNA virus, which is one of the important pathogenic factors of liver disease and seriously threatens human health. At present, there are more than 170 million people infected with HCV in the world, and more than 100,000 HCV-infected patients develop liver cancer every year, which in turn causes gastrointestinal bleeding and ascites. Deaths from HCV infection in patients with liver disease are steadily increasing. [0004] The hepatitis C virus consists of about 9400 amino acids. The HCV genome has a single open reading frame, which encodes a polyprotein body with 3010 amino acids. After translation, it is divide...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 李艳童永清顾剑郑红云
Owner HUAXIN SCI & TECH PANYU CITY
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