A hepatitis C virus one-step-method nucleic acid quantification/genotype RT-PCR detection kit

Abstract

A hepatitis C virus one-step-method nucleic acid quantification / genotype RT-PCR detection kit is provided. The kit comprises an RT-PCR reaction liquid, an RT-PCR enzyme mixture, HCV quantification / genotype primer probes, an inner reference substance, a negative control, a critical positive control, a strong positive control, and number 1 to 5 calibrators. The kit can directly subject extracted HCV RNA to a one-step-method RT-PCR reaction. The kit subjects the HCV RNA in a sample to fluorescence quantitative detection, and subjects the HCV RNA to genotype detection at the same time. The kit adopts reference genes as an inner control, and prevents contamination by utilization of UNG enzyme. The kit is simple in one-step-method amplification, short and simple in processes, simple and convenient in operation and capable of preventing contamination. A detection result is high in specificity, high in sensitivity, clear and high in reliability. The kit can be used for nucleic acid quantitative detection of hepatitis C viruses in serum and can subject HCV-1 and HCV-2 to genotype detection at the same time.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a biological detection technology, in particular to a reagent kit for the fluorescence quantitative detection of hepatitis C virus nucleic acid and genotyping detection for samples. Background technique [0002] Viral hepatitis is caused by a variety of hepatitis viruses, a group of systemic infectious diseases with liver damage. Hepatitis C virus (HCV) is the main pathogenic virus of hepatitis C virus. HCV genome sequence is highly variable. According to the variation of gene sequence in different regions, HCV can be divided into 6 genotypes including 1, 2, 3, 4, 5 and 6 types and more than 80 genotypes. The genetic diversity of HCV has a strong impact on disease progression and drug treatment responsiveness. Studies in recent years have shown that the genotype of HCV is closely related to the treatment plan and treatment cycle of hepatitis C. [0003] At present, the combi...

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Problems solved by technology

[0009] At the same time, in order to avoid false positives in the reaction results, using the anti-pollution characteristics of UNG enzyme, dUTP is used instead of dTTP in the PCR amplification reaction, so that only dUTP is contained in the amplified fragment; Pre-DNA is easily degraded by UNG enzyme: this PCR product containing dUTP is incubated with UNG enzyme, because UDG enzyme can cleave the N-glycosyl bond between uracil base and sugar phosphate backbone, and can be obtained from single-stranded or double-stranded DNA Elimination of dUTP prevents the extension of Taq DNA polymerase and thus loses the ability to be reamplified

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