Duplex RT-PCR for simultaneous detection of hepatitis A and hepatitis E virus isolated from drinking water samples.

Abstract

A duplex reverse transcription polymerase chain reaction (RT-PCR) protocol for simultaneous detection of hepatitis A virus (HAV) and hepatitis E virus (HEV) in water samples has been developed and demonstrated. Both HAV and HEV were concentrated from drinking water samples through a one-step concentration protocol. Different cDNA could be produced in the RT step carried out with a random primer in a single reaction tube. Two different sets of primers specific for HAV-cDNA and HEV-cDNA were used for PCR amplification. Amplified DNA products representing HAV and HEV were identified by gel electrophoresis at 247 and 327 bp (base pair) sequences, respectively. Specific sets of primers amplified a single type of virus and no cross-reactivity of the primers was noticed in duplex RT-PCR. The protocol was used for direct isolation and detection of HAV and HEV from 23 water samples in urban areas of Chennai city. Out of these, nine water samples were positive for HAV, and three for HEV. All three samples positive for HEV were also positive for HAV. The test provides a rapid and economical means of water quality surveillance to specifically detect HAV and HEV.