Improved method for the recovery of hepatitis A virus from oysters

Abstract

Hepatitis A is one of the major infectious diseases epidemiologically associated with worldwide shellfish consumption. Molecular detection using polymerase chain reaction (PCR) to detect hepatitis A virus (HAV) in contaminated shellfish can be hindered by low virus recoveries during the concentration process and by natural PCR inhibitors in shellfish. This study evaluated and modified two major steps of a processing procedure for virus concentration from oysters: acid adsorption–elution and solvent extraction. With the addition of second and third elutions, the acid adsorption–elution step doubled the recovery to 46% of HAV seeded initially. Extraction with chloroform or chloroform–butanol resulted in lower HAV detection limits by reverse transcription–PCR (RT-PCR)-oligoprobing than extraction with the fluorocarbon, Freon. These results led to the following modified procedure: HAV was acid 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-extracted, and RT–PCR (single round) amplified. Using the modified procedure, HAV was detected by RT–PCR in all trials with a seeding density of ≥1 plaque forming unit (PFU)/g of oyster, and in which the equivalent detection limit was 0.33 PFU of HAV seeded per RT–PCR reaction (corresponding to 111 PCR units). The method developed is capable of detecting low levels of HAV in oysters environmentally contaminated.