Serum Hcv Rna Detect And Microarray Genotyping Of Study

ObjectiveThe common method adopted in clinical detection for HCV infection is the serology detection method. Though the sensitivity and speciality of enzyme immune assay method can reach to 99% now, the results may show difference in different examine time due to window-period of anti-body and other factors in immune response. Furthermore, the anti-virus drug can eliminate virus and forbid virus replicating inside body, which results in unsteady and non-detection of anti-body. It also has certain effect on the amount of antibody. HCV-RNA detection is an ideal substitute way in this point. It can detect virus RNA either they are replicating or not, free of the body immune condition. This research designs an experiment to detect virus RNA in peripheral serum, utilize reverse translation- nest polymerase chain reaction (RT-nPCR) to examine the existence of hepatitis C virus.HCV has a great variation in its genome, which can be divided into different genotypes and sub-genotypes. The different genotypes have a certain relation with the severity of disease, sensitivity to interferon, and prognosis. Genotype differentiation has some important significance in epidemiology and anti-virus therapy. There are several methods in genotyping. This research designs a method which combines the nucleic acid molecular hybridization and the microarray gene chip. The aim is to find out a rapid, convenient and cheap method, which can be used widely for large number samples.MethodStudy the diversity of the HCV genome sequence and compare nucleotide homologous to analysis the different genotypes according to research papers. Consult in gene bank at NCBI to compare the homologous sequences, and then design common PCR primers in HCV 5-non-coding region with digoxin labels. Design different genotype probes in this region, fix them on modified glass to form gene array. Extract HCV-RNA in peripheral serum, PCR amplification, and hybrid PCR products with probes on the chip. Use NBT/BCIP to print the blue precipitate in nylon membrane.ResultsAll 60 cases in HCV infection group have clear strips in electrophoresis, which means the HCV-RNA is positive. Obvious blue precipitates can be seen in gene chip hybridization. The blue precipitates in membrane are clear even to naked eyes. The results can be judged and saved expediently. 56 cases of them show consistent results in standard nucleotide sequencing. x~2 test (P>0.05) shows no obvious difference in statistics. All 20 cases in non HCV infection group have no strips in electrophoresis, which means the HCV-RNA is negative. Hybridization in gene chip shows negative results.ConclusionsGene chip can detect HCV-RNA in peripheral serum exactly. In clinical application, the result is free of immune response condition and virus variation. Gene chip can differentiate the different genotypes of HCV at the same time. In this research, four genotypes and five sub-genotypes are detected. The results in gene chip consist with those in standard sequencing at 93. 3%.Clinically, the operation is convenient, rapid, accurate and no florescence label and single detection are needed. It can be used for large number samples.