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Appl Environ Microbiol. 1993 September; 59(9): 2765-2770

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

Division of Molecular Biological Research and Evaluation, Food and Drug Administration, Washington, D.C. 20204.

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.

This article has been cited by other articles:

• Kingsley, D. H., Richards, G. P. (2001). Rapid and Efficient Extraction Method for Reverse Transcription-PCR Detection of Hepatitis A and Norwalk-Like Viruses in Shellfish. Appl. Environ. Microbiol. 67: 4152-4157 [Abstract] [Full Text]  
• Arnal, C., Ferre-Aubineau, V., Mignotte, B., Imbert-Marcille, B. M., Billaudel, S. (1999). Quantification of Hepatitis A Virus in Shellfish by Competitive Reverse Transcription-PCR with Coextraction of Standard RNA. Appl. Environ. Microbiol. 65: 322-326 [Abstract] [Full Text]