A kit for detecting hepatitis C virus nucleic acid using one-step real-time fluorescent quantitative RT-qPCR

Abstract

The invention discloses a kit for detecting hepatitis C virus nucleic acid by one-step real-time fluorescence quantitative RT-qPCR, which includes virus nucleic acid extraction reagents, probes for detecting target polynucleotides and internal controls and for amplifying target polynuclei RT‑qPCR reaction buffer for nucleotide primers with hot start DNA polymerase, Revert Aid TM M‑MLV Reverse Transcriptase and RiboLock TM RT-PCR enzyme mixture of ribonuclease inhibitors, quantitative standard reference substance containing pseudovirions; wherein the novel hepatitis C virus target sequence probe and primers disclosed by the present invention avoid the hepatitis C virus 5' innovatively The secondary structure and endonuclease site in the UTR region are easy to cause degradation, so that even when the viral nucleic acid is unstable, it can still reflect its original viral load to the maximum extent, and it is very important for sample collection, transportation, storage, Hepatitis C virus nucleic acid RNA in improperly processed samples can detect all six genotypes, which provides an accurate basis for the auxiliary diagnosis of hepatitis C virus infection and the monitoring of drug treatment of infected persons.

Description

technical field [0001] The invention relates to a kit for rapidly detecting hepatitis C virus nucleic acid by using one-step real-time fluorescence quantitative RT-qPCR technology, belonging to the field of biomedical detection. Background technique [0002] Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus discovered in the last ten years. In 1991, the International Committee on Taxonomy of Viruses classified HCV into the Flaviviridae family. [0003] The amount of hepatitis C virus in the serum and liver tissue of the infected person is small, and it is difficult to see it with an electron microscope. According to HCV-cDNA analysis, the full length of HCV genome is 9419bp. Recent nucleotide sequence analysis shows that there are at least 6 HCV genotypes and more than 90 subtypes. The HCV genotypes in my country are mainly type II and type III. [0004] Compared with hepatitis B virus (HBV), HCV has many differences in biological characteristics. RNA v...

AI Technical Summary

Problems solved by technology

At present, among the HCV nucleic acid quantitative detection kits on the domestic market, taking Guangzhou Daan Gene’s kit as an example, the sensitivity is the lowest at 250IU / mL, which is far behind the international standard of 50IU / mL proposed by the European Society for the Study of Liver Diseases. Provide an accurate basis for clinical prognosis; although the sensitivity of the kits of Shanghai Kehua and Hunan Shengxiang reaches or even lower than 50IU / mL, considering that the viral nucleic acid is easily degraded, these kits have extremely high requirements for virus samples, and there are strict samples Collection, Shipping, Storage and Processing Processes

However, under different regions, environments, and personnel factors, it is impossible for samples to meet such strict requirements, and nucleic acid degradation is extremely prone to occur

For such unstable samples, the detection efficiency of existing commercial kits is greatly reduced, and even false negative results may appear, which cannot accurately reflect the original virus load in the patient's blood, which greatly misleads clinical treatment.

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