Simultaneous detection of both hepatitis B virus and hepatitis C virus using multiplex polymerase chain reaction technique

Abstract

The hepatitis B virus (HBV) DNA polymerase chain reaction (PCR) and hepatitis c virus (HCV) RNA PCR is widely used for diagnosis of infection and evaluation of therapy. Application of both PCR techniques is limited by labor-intensity, potential for contamination, and substantial time required for analysis. In this study, a multiplex PCR method was developed to detect HBV DNA and HCV RNA in pooled specimen using primers from the HBV precore and core gene and the HCV 5; noncoding region. The method was performed for both RT-PCR and PCR in single tube. HCV RNA required prior reverse transcription into cDNA and then amplified with both HBV and HCV primers. From the various known standard dilution analysis of 10 replications of each showed that sensitivity of the multiplex HBV DNA/HCV RNA PCR for detecting HBV DNA was approximately 19,629 copies/ml (392 copies/assay), while HCV RNA was approximately 4,506 copies/ml (90 copies/assay). In this study, internal control template for nested primer PCR of HBV DNA was constructed and coamplified with clinical specimen for excluding of false negative result. The internal control construct shares the primer recognition sequences with the target template and yields a 200-bp in length of the fragmented amplified product whereas the product of the target template yields 266-bp. The optimal copy numbers of internal control in PCR run were 25 copies. The potential clinical and epidemological applications of this candidate multiplex HBV DNA/ HCV RNA PCR assay are warranted for further evaluation.