Recovery and detection of enterovirus, hepatitis A virus and Norwalk virus in hardshell clams (Mercenaria mercenaria) by RT-PCR methods.
A method for recovery of enteric viruses from hardshell clams (Mercenaria mercenaria) has been developed and evaluated. Seeded 50-g samples of clam tissue homogenates were processed by adsorption elution precipitation, two fluorocarbon extractions and PEG precipitation. Clam concentrates were assayed by infectivity and by RT-PCR after guanidinium isothiocyanate (GIT) extraction and/or an indirect immunomagnetic capture (IC) of the virus using paramagnetic beads. GIT extraction removed PCR inhibitors and allowed a reliable RT-PCR detection of viral RNA. The detection sensitivity of GIT extraction-RT-PCR was < 1 PFU of poliovirus 1, < 10 PFU of HAV and 1-11 PCRU of Norwalk virus. IC was very effective for additional concentration and purification of enteric viruses from clam concentrates removing most RT-PCR inhibitors. The sensitivity of this method was comparable to the GIT extraction and the sample volume tolerance for PCR was increased about 10-fold. Both methods gave similar efficiency for virus detection in samples seeded with low virus levels. The procedure developed in this study is effective for enteric viruses detection in hardshell clams by RT-PCR.
Suñén
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Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill 27599-7400, USA.
Sobsey
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MD
D000818:Animals; D049872:Bivalvia / Q000821:virology*; D002460:Cell Line; D004770:Enterovirus / Q000302:isolation & purification* / Q000472:pathogenicity; D005069:Evaluation Studies as Topic; D006146:Guanidines; D006507:Hepatovirus / Q000302:isolation & purification* / Q000472:pathogenicity; D018189:Immunomagnetic Separation; D017879:Isothiocyanates; D009663:Norwalk virus / Q000302:isolation & purification* / Q000472:pathogenicity; D012367:RNA, Viral / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D006146:Guanidines; D017879:Isothiocyanates; D012367:RNA, Viral; C054435:guanidine isothiocyanate