An RNA extraction protocol for shellfish-borne viruses.
The GPTT virus RNA extraction method, originally developed for extraction of human norovirus and hepatitis A virus RNAs from contaminated shellfish, was evaluated for extraction of RNA from Aichi virus strain A846/88 (AiV), coxsackievirus strains A9 (CAV9) and B5 (CBV5), murine norovirus (strain MNV-1), and the norovirus surrogate, feline calicivirus (FCV) strain KCD, for the purpose of RT-PCR detection within seeded oyster (Crassostrea virginica) extracts. The RT-PCR equivalent sensitivities observed within seeded oysters as compared to virus stocks were 0.68, 6.8, 26, 5.6, and 14.5 RT-PCR(50) units when assaying 10% of total RNA extracted from seeded oyster extracts for CAV9, CBV5, AiV, FCV, and MNV-1, respectively. For oysters exposed to virus-contaminated seawater, the detection equivalent sensitivities observed were 680, 68, 2600, 560, and 14.5 RT-PCR(50) for CAV9, CBV5, AiV and FCV, and MNV-1, respectively. These results indicate that the GPTT method can be used as a general viral RNA extraction method for multiple picornaviruses and caliciviruses that could potentially contaminate shellfish.
Kingsley
David H
DH
U.S. Department of Agriculture, Agricultural Research Service, Microbial Food Safety Research Unit, James W.W. Baker Center, Delaware State University, Dover, DE 19901, USA. david.kingsley@ARS.USDA.GOV
D000818:Animals; D002139:Caliciviridae / Q000235:genetics / Q000302:isolation & purification*; D002415:Cats / Q000821:virology; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D052699:Kobuvirus / Q000235:genetics / Q000302:isolation & purification*; D029322:Norovirus / Q000235:genetics / Q000302:isolation & purification; D010125:Ostreidae / Q000821:virology*; D012367:RNA, Viral / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000821:virology*
D012367:RNA, Viral