RNA extraction method for the PCR detection of hepatitis A virus in shellfish

Viruses are the leading cause of foodborne illness associated with the consumption of raw or slightly-cooked contaminated shellfish. This study evaluated the E.Z.N.A. Mollusc RNA extraction and purification kit for the detection of HAV in shellfish. The E.Z.N.A. method, based on the cationic detergent, cetyltrimethyl ammonium bromide, in conjunction with a selective RNA binding silica matrix, efficiently isolated viral RNA with a detection limit of 1 TCID50/ml by hemi-nested PCR. This method proved to be faster and less expensive than PEG precipitation-based procedures. It is also technically undemanding, requiring no extensive processing steps or excess manipulation, minimizing RNA degradation and ensuring the absence of PCR inhibitors. The EZ.N.A. method applied to HAV screening of shellfish samples from the Apulian region, revealed a high level of contamination. These results confirm that conventional faecal indicators are unreliable for demonstrating the presence or absence of viruses. (C) 2010 Elsevier B.V. All rights reserved.