Title: AN RNA EXTRACTION PROTOCOL FOR SHELLFISH-BORNE VIRUSES Author
Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 23, 2006
Publication Date: January 30, 2007
Citation: Kingsley, D.H. 2007. An rna extraction protocol for shellfish-borne viruses. Journal of Virological Methods. Vol.141:58-62. Interpretive Summary: The GPPT method is a rapid viral RNA extraction procedure originally designed to facilitate the detection of hepatitis A virus and human norovirus within shellfish. In this publication, we demonstrate that the GPPT method can be used to extract a variety of enteric viruses which could potentially contaminate shellfish and subsequently cause human illness if the shellfish are not properly cooked. Because this method uses commercially available reagents and purifies viral RNA based on its chemical properties, this method is broadly applicable to almost any picornavirus, calicivirus or other related virus which could pose a threat to the health of shellfish consumers.
Technical Abstract: 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-PCR50 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-PCR50 for CAV9, CBV5, AiV, FCV, and MNV-1, respectively. These results indicate that the GPTT method can be used as a general viral RNA extraction method for multiple picornavirus and calicivirus that could potentially contaminate shellfish.