Molecular Detection of Hepatitis C Virus (HCV) by Conventional One-step RT-PCR Coupled with Nested PCR

Abstract

Aims: HCV causes both acute and chronic infections and can be easily transmitted through contaminated blood or other body fluids. The present study deals with the molecular detection of HCV with help of one-step RT-PCR assay followed by nested PCR and agarose gel electrophoresis.  Study Design: RNA extracted from the confirmed positive samples of HCV was utilized for the standardization of the one-step RT-PCR assay and nested PCR assay for diagnosis of HCV. Place and Duration of Study: Centre for Biotechnology, Maharshi Dayanand University, Rohtak Haryana, India, during period of one year (January-December 2015). Methodology: HCV positive samples were obtained from Department of Medicine, Maulana Azad Medical College (MAMC), New Delhi, India. Published primers from most conserved regions of HCV were taken and these primers were able to amplify all the strains of HCV. One-step RT-PCR kits, primers, extracted RNA from these positive samples were used for standardization of molecular diagnostic assays. The results were checked by 2% agarose gel electrophoresis. Results: Positive samples of HCV were detected by nested PCR. Positive samples showed sharp band of 405bp while there was no amplification in the negative control. Conclusion: Rapid tests have low sensitivity and specificity while molecular assays are rapid, sensitive and specific. Conventional one-step RT-PCR assay followed by nested PCR is rapid, specific, sensitive and it is also less costly than real-time RT-PCR. Cost of an assay is an important factor in controlling a disease in resource limited settings of developing countries.