Improved method for the recovery of hepatitis A virus from oysters

Hepatitis A is one of the major infectious diseases epidemiologically assoc iated with worldwide shellfish consumption. Molecular detection using polym erase chain reaction (PCR) to detect hepatitis A virus (HAV) in contaminate d shellfish can be hindered by low virus recoveries during the concentratio n process and by natural PCR inhibitors in shellfish. This study evaluated and modified two major steps of a processing procedure for virus concentrat ion from oysters: acid adsorption-elution and solvent extraction. With the addition of second and third elutions, the acid adsorption-elution step dou bled the recovery to 46% of HAV seeded initially. Extraction with chlorofor m or chloroform-butanol resulted in lower HAV detection limits by reverse t ranscription-PCR (RT-PCR)-oligoprobing than extraction with the fluorocarbo n, Freon. These results led to the following modified procedure: HAV was ac id adsorbed at pH 4.8, eluted first with 0.05 M glycine, second with 0.5 M threonine, PEG-precipitated twice, chloroform-extracted twice. RNA-extracte d. and RT-PCR(single round) amplified. Using the modified procedure, HAV wa s detected by RT-PCR in all trials with a seeding density of greater than o r equal to 1 plaque forming unit (PFU)/g of oyster, and in which the equiva lent detection limit was 0.33 PFU of HAV seeded per RT- PCR reaction (corre sponding to 111 PCR units). The method developed is capable of detecting lo w levels of HAV in oysters environmentally contaminated. (C) 2001 Elsevier Science B.V. All rights reserved.