The Establishment Of Hcv Rna Detection Methods & The Analysis Of Variation Characteristic On Drug Target

Hepatitis C virus (HCV) is a global problem. It is estimated that 170 million persons (about 3% world's population) were infected with HCV. Once infected, the 55%-85% infected man might become chronic hepatitis, and most of them would be able to get liver necrosis, liver cirrhosis, and liver cancer.Being that lack of an efficient HCV cell culture replication system and no small-animal model, This hampers us to undersdand the pathogentic characters of virus and chronic disease mechanism.It is also an impediment in developing anti-virus drug and vaccine.The laboratory detection of HCV infection is a necessary means in clinical diagnosis,disease prediction and drug efficacy evaluation.In this study,an efficient and specific HCV qualitative detection method was set up based on conventional polymerase chain reaction technology, and which combined with nucleic acid hybridization technology. Another,an real time PCR assay for quantitation of HCV RNA was newly developed by Using the luminescence/quenching principle of taqman probe.The qualitative and the quantitative detections targeting the HCV RNA have a high general applicability, specificity, sensitivity and repeatabilityFirst of all, PCR primers and Digoxin-labeled probe were designed and synthesized,and the length of the amplified product was 678bp.After separated by agarose gel electrophoresis, the product was transferred to positively charged nylon membrane, and then was hybridized with the digoxin-probe. The image was obtained through the chemical staining. Then, both the PCR and the hybridization procedures were optimized. Repetition and sensitivity of this method was evaluated and it possesses an universal detection applicability in different HCV genotypes.Secondly, fluorescence real time PCR method, which based on TaqMan technique, was used to quantitatively detect HCV. In order to build the standard curve, PCR product of HCV 5 NCR-C gene was cloned into pMD-18T vector. Then, sequencing and followed analysis was conducted. The real-time PCR procedure was optimized according to produced fluorescence level. Repetition and sensitivity of this method was evaluated. As a result, the linear relationship between the values of cycle threshold (Ct) and the copy number was significant (R2=0.9985). Its repetition is qualified (CV<5%). The 102 copies of HCV genome in 25μl reaction system could be detected. Obviously, this method has high sensitivity.The dada of present study indicated that this method can be used to detect the different genotype of HCV(1,3,6).Current therapy using a combination of a-interferon and ribavirin is effective only in about 50% of cases and exhibits severe adverse side effects,Novel and more novel efficacious therapies for HCV infection are urgently needed.HCV NS3 serine protease, as an key protease in HCV life cycle, was consider as an potential HCV drug target.To analyze the genetic variation characteristic of the region,The sequences of different HCV genotype were downloaded from the HCV database and the genetic variation was researched in the amino acids level. Then the structure of HCV NS3 serine protease was modeled to analyze the mechanism the inhibitors interacting with the proteas. According to the result, fifty amino acids was selected from the 120aa to 170aa to survey the variation for further study. Statistical approach was used to illustrate relationship between the mutations of key amino acids and the drug resistance of inhibitors to the protease. At last, we find that the sequence of HCV NS3 serine protease is conservative between the genotypes in entire evolution and the mutation of key amino acids possesses a characteristic of type specificity.In summary, the PCR-southern blotting qualitative detection method and fluorescence quantitative PCR detection method with high reproducibility, sensitivity and specificity, were established to detection HCV at the RNA level. At the same time, the genetic variation of HCV NS3 serine protease was surveyed by statistical approach. The establishment of HCV detection method provides a technology platform for further research, and the genetic variation analysis of the target in NS3 serine protease provides a reference data for future drug development.