Kit for real-time fluorescence quantification RT-qPCR detection of hepatis c viral nucleic acid by means of one-step method

Abstract

The invention discloses a kit for real-time fluorescence quantification RT-qPCR detection of hepatis c viral nucleic acid by means of a one-step method. The kit comprises a viral nucleic acid extraction reagent, a probe containing detection target polynucleotide and an internal control, RT-qPCR reaction buffer used for amplification of a target polynucleotide primer, an RT-PCR enzyme mixture containing warm start DNA polymerase, Revert AidTM M-MLV reverse transcriptase and RiboLockTM ribonuclease inhibitor, and a quantitative criterion reference containing virus-like particles. By the adoption of the novel hepatitis c virus target sequence probe and primer, a secondary structure and an incision enzyme site which cause degradation easily in a hepatis c virus 5'-UTR area are avoided innovatively, original viral load can still be reflected to the maximum degree even if viral nucleic acid is unstable, all six genotypes of hepatis c viral nucleic acid RNA in samples subjected to improper acquisition, transportation, storage and treatment can be tested, and accurate basis is provided for auxiliary diagnosis of hepatis c virus infection and monitoring of drug therapy of infectors.

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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