Detection of hepatitis A virus in Mercenaria mercenaria by coupled reverse transcription and polymerase chain reaction.
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.
Goswami
B B
BB
Division of Molecular Biological Research and Evaluation, Food and Drug Administration, Washington, D.C. 20204.
Koch
W H
WH
Cebula
T A
TA
D000818:Animals; D001483:Base Sequence; D049872:Bivalvia / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005516:Food Microbiology; D006506:Hepatitis A / Q000635:transmission; D006507:Hepatovirus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012367:RNA, Viral / Q000235:genetics; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral