Development of a method for detection of enteroviruses in shellfish by PCR with poliovirus as a model.
The application of the PCR to complex samples is hindered by amplification inhibitors. We describe a reverse transcription-PCR-based method capable of inhibitor removal for the detection of enteroviruses in shellfish. Initial virus extraction stages based on a modified polyethylene glycol precipitation technique (G.D. Lewis and T.G. Metcalf, Appl. Environ. Microbiol. 54:1983-1988, 1988) were followed by virus purification with 1,1,2-trichloro,2,2,1-trifluoroethane and concentration by ultrafiltration. A guanidine isothiocyanate-glass powder extraction system was utilized for sample lysis, RNase protection, and nucleic acid purification. Removal of PCR inhibitors and method sensitivity were quantified in shellfish (oysters and mussels) seeded with poliovirus. PCR sample tolerance exceeded 4 g for depurated shellfish; however, polluted field samples were more inhibitory. Virus recoveries of 31% for oyster extracts and 17% for mussel extracts and nucleic acid extraction reverse transcription-PCR detection limits down to 1 PFU yielded an overall sensitivity limit of < 10 PFU of poliovirus in up to 5 g of shellfish. PCR-positive results were obtained from a variety of polluted field samples naturally contaminated with human enteroviruses. The methods developed for virus recovery and PCR inhibitor removal should be equally applicable to detection of other RNA viruses such as hepatitis A virus, Norwalk virus, and other small round-structured viruses in shellfish.
Lees
D N
DN
Fish Diseases Laboratory, Ministry of Agriculture, Fisheries and Food, Weymouth, Dorset, England.
Henshilwood
K
K
Doré
W J
WJ
D001483:Base Sequence; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D005516:Food Microbiology; D006801:Humans; D008969:Molecular Sequence Data; D017955:Poliovirus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D012722:Sewage; D012758:Shellfish / Q000382:microbiology*
D012367:RNA, Viral; D012722:Sewage; D012194:RNA-Directed DNA Polymerase