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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR & BIOCHEMICAL DETECTION & INTERVENTION METHODS FOR BACTERIAL AND VIRAL PATHOGENS IN AQUACULTURE PRODUCTS Title: New targets for expedient detection of viruses within shellfish

Author
item Kingsley, David

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: December 17, 2010
Publication Date: February 20, 2011
Citation: Kingsley, D.H. 2011. New targets for expedient detection of viruses within shellfish [abstract]. 1st International Symposium on Fisheries, Agriculture and Food Hygiene. February 20-28, 2011, Lima,Peru. p.1.

Technical Abstract: Previously our laboratory developed an expedient method for extraction of viral RNA from food-borne virus contaminated bivalve shellfish, termed the GPTT protocol. This protocol utilizes either whole shellfish or dissected digestive diverticula. This four step protocol utilizes a high pH glycine or phosphate buffer to elute the viruses from shellfish tissues, followed by concentration of the intact viruses by polyethylene glycol precipitation (PEG). Subsequently the PEG pellet is resuspended in Tri-reagent, a mixture of phenol and guanidinium isothiocyanate, to liberate the RNA genome from the virus capsid followed by addition of chloroform and isopropanol precipitation. After resuspension of the precipitate, the poly A-containing viral RNA is annealed to poly dT magnetic beads. This method is easy to perform, sensitive, and uses commercially available reagents for detection of hepatitis A virus (HAV) and human norovirus. The GPTT procedure has been successfully used in a number of different laboratories worldwide for shellfish testing. However one drawback for this method it that is takes approximately 8 hr to process a handful of samples, effectively limiting application of this technique to outbreak tracking and research uses. Our recent research indicates that viable viruses sequesters themselves within the hemocytes, or oyster blood cells, of live shellfish. We have recently utilized this finding to demonstrate that purifying HAV or murine norovirus RNA directly from hemocytes is an efficient and rapid method for testing shellfish. Essentially this test simply involves removing or draining hemocytes from oyster tissues, centrifuging, and lysing the pellet and purifying virus RNA using commercial RNA purification kits. We anticipate that this newly developed method will lead to a practical method for routine testing of shellfish.

Last Modified: 10/22/2014
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