Novel single-step multiplex real-time polymerase chain reaction assay for simultaneous quantification of hepatitis virus A, B, C, and E in serum.

BACKGROUND AND AIM: Viral hepatitis needs an earliest diagnosis for its proper and timely treatment. Although serodiagnosis of viral hepatitis is in regular practice, however, it has certain limitations and points to alternate procedures of diagnosis. Present study was designed to develop a single-step multiplex real-time polymerase chain reaction (PCR) assay for detection of hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV) and hepatitis E virus (HEV) related nucleic acids in sera from infected patients.
METHODS: The PCR was standardized to detect HAV, HBV, HCV and HEV in serum using variables including annealing temperature, extension temperature, MgCl2 , and primer concentrations. The conserved regions of all viral genomes were used as targets for amplification.
RESULTS: This novel assay was found to be a fast, sensitive, specific, and reproducible system for detection of HAV, HBV, HCV, and HEV in serum. The detection limit for different viral genomes at 100% level was found to be 280 copies/mL for HAV, 290 copies/mL for HBV, 30 copies/mL for HCV, and 300 copies/mL for HEV in a single-tube assay system.
CONCLUSION: Present multiplex real-time PCR is the first report on single-step nucleic acid detection of HAV, HBV, HCV, and HEV in sera samples. It is an alternate diagnostic assay for common use in laboratories analyzing viral hepatitis cases.