Detection of hepatitis A virus in Mercenaria mercenaria by coupled reverse transcription and polymerase chain reaction

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Abstract
Hepatitis A virus (HAV) is a major cause of infectious hepatitis in humans. In this respect, bivalve mollusks pose a major health concern because they are filter feeders and can concentrate the virus up to 900-fold from contaminated water. Detection of HAV has been hampered because wild-type HAV grows poorly if at all in cell culture. Here we describe a technique for the detection of HAV in shellfish based on reverse transcription coupled with the polymerase chain reaction. RNA is isolated from hard-shell clam tissue and reverse transcribed with avian myeloblastosis virus reverse transcriptase. A portion of the cDNA pool is then amplified with primers specific for HAV. In experiments with an in vitro-synthesized HAV transcript, we were able to detect HAV sequence in the presence of a 200-million-fold excess of shellfish RNA. When intact virus was added to shellfish tissue before the isolation of RNA, the method was capable of detecting 10 viral RNA molecules in a reaction mixture.