Development A Real-time RT-PCR Assay With Partially Double-stranded Linear DNA Probe For The Detection Of Hepatitis C Viral RNA

Background Hepatitis C virus(HCV) is one of the major pathogens that causes chronic viral hepatitis worldwide. According to statistics, there are about 170 million people with chronic HCV infection worldwidely and more than 10 million in China. The symptoms after infection of HCV are not typical. It is easy to miss the best time for treatment if the infection is not early diagnosed. Chronic HCV infection can be accounted for the development of liver fibrosis, cirrhosis and liver cancer which could lead a serious threat for people’s health. HCV RNA is a reliable indicator for early diagnosis of HCV infection. It can be detected within 1-2 weeks after infection earlier than anti-HCV which can be detected about a month. At the same time, HCV RNA can also be used as a efficacy chronic HCV infection antiviral therapy evaluation index. Currently, there are a variety of domestic and foreign HCV RNA detection reagents but the detection performance varies differently. Due to the limitation of economic conditon, our clinical laboratories are dominated by domestic HCV RNA reagents, which is significantly different with imported reagent in the detection performance and can not meet the needs of clinical diagnosis and treatment. Therefore, development of a high sensitivity, good stability, reasonable cost of HCV RNA detection reagent is one of the main directions of the current study of HCV in China.Aims To develop a novel real-time RT-PCR assay for the detection of hepatitis C viral RNA which has high sensitivity, stability and specificity for the detection of various HCV genotypes and subtypes.Methods HCV RNA standards were prepared by invitro transcription technology in this study. We designed primers and probes in the highly conserved region. Meanwhile, we designed and optimized partially double-stranded linear DNA probe and compared with ordinary single probe. Concentration of components for HCV RNA detection system and the reaction conditions were optimized. This study set up and optimized an internal control system. To verify the performance of the new real-time RT-PCR method, a methodology detection for this method were applied, including sensitivity, linearity, specificity, intra-assay and inter-assay reproducibility, and the correlation and consistency of the detection results of 106 serum samples with the commercialization kit at the same time.Results This study successfully prepared the HCV RNA standards. Concentration of components for HCV RNA detection system and the reaction conditions were optimized. Partially double-stranded linear DNA probe was superior to the ordinary single probe. By optimizing internal control system, 100 copies internal control RNA were added to each HCV RNA detection system. The novel real-time RT-PCR assay has a wide linear dynamic range of HCV RNA quantification(100-1×107 IU/ml) and a limit of detection of 78 IU/ml. All kinds of common HCV genotypes and subtypes can be detected. When the HCV negative samples were detected, no positive amplification was observed. The assay exhibits an excellent reproducibility with 2.52% and 1.33% coefficients of variations for inter- and intra-assays, respectively. Compared with a commercial kit for HCV RNA detection using 106 anti-HCV antibody positive serum samples, results of two methods have great correlation(R2 = 0.940) and consistency with deviation of-0.05log(IU/ml).Conclusions This study developed a novel real-time RT-PCR assay for the detection of hepatitis C viral RNA with partially double-stranded linear DNA probe. It has high sensitivity, specificity, stability and can detect all kinds of common HCV genotypes and subtypes achieving precise quantitative detection of HCV RNA in serum samples. It is an appropriate way for HCV infection screening, diagnosis and chronic HCV infection monitoring during the antiviral therapy.