Development of novel triplex single-step real-time PCR assay for detection of Hepatitis Virus B and C simultaneously

Abstract

Multiplex RT-PCR assays are widely used tools for detection of hepatitis viruses, but none of them provide quality check of sample. In the present study we developed a single-step triplex real-time polymerase chain reaction (PCR) assay for detection of Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) with sample quality check, by using β-actin as housekeeping gene. The primers and probes were self-designed and assay was standardized. Assay was also destined to quantitate copy numbers of HBV and HCV. This novel assay was sensitive, specific, and reproducible for detection of HBV and HCV in serum/plasma. The assay also detected all genotypes of HBV and HCV. The detection limit was 60 IU/mL for HBV and 20 IU/mL for HCV. This assay is the first assay developed on single-step platform for nucleic acid detection of HBV and HCV with an extra edge over all other assays by providing inbuilt check for quality of sample.

Keywords: Hepatitis B Virus; Hepatitis C Virus; Triplex real time PCR assay; Validation; Viral load; β-Actin.