Development and Application of an In-house Line Probe Assay for Hepatitis C Virus Genotyping

摘要

Hepatitis C virus (HCV) is the major cause of chronic liver disease. HCV is a single stranded positive sense RNA of approximately 9.6 Kb. Because of high conservativeness of 5?untranslated region of HCV genome, it is widely used for virus genotyping. Different methods are used for the virus genotyping, but all involve some difficulties. The aim of the present study was to develop an in-house reverse hybridization method as a line probe assay, for HCV genotyping. Sixty serum samples were collected with newly diagnosis of HCV infection. Genotyping process had already been performed for the samples using RT-PCR RFLP method. After total RNA extraction from the samples and cDNA synthesis, nested PCR method was applied for amplification of the target sequence on the 5?UTR. In the nested PCR, biotinylated oligonucleotides were used as inner primers. Optimized concentrations of the biotinylated inner primers (as positive control), two universal and seven specific probes were spotted onto nylon membrane stripes in a defined pattern. Hybridization process was conducted between the probes and the denaturized biotin labeled PCR products. Finally, the stripes were developed by using streptavidin conjugated alkaline phosphate as a signal generating agent. To determine the diagnostic sensitivity and specificity of the home made LiPA, a panel containing 60 confirmed sera with positive results for HCV (and PCR-RFLP genotyped) was subjected to evaluate. Agarose gel electrophoresis of the nested PCR products using the outer and inner primers showed 305 and 234 bp fragments respectively. After performing hybridization and detection processes on the prepared strips, the colored bands were formed for the positive control, universal probes and the corresponding genotypes. HCV genotype results were found to be in 100% concordance through studying 60 sera that were successfully typed by the two methods. P-value of 0.045 conveys that the two methods were the same and had no significant difference. The most common genotyping method in Iran is RT-PCR RFLP. Given the results and advantages of this homemade technique, such as high specificity and sensitivity, ability for detection of most genotypes, it provides possibility of evaluating much of the isolates without needing electrophoresis stage.