title: Detection of hepatitis a virus from oyster by nested PCR using efficient extraction and concentration method.
abstract: The molecular methods using polymerase chain reaction have been proposed as useful tools for the identification of viral pathogens in food and water. However, the PCR-based methods are highly dependent on the methods of virus concentration and nucleic acid purification due to the low sensitivity of PCR in the presence of PCR inhibitors. We developed TPTT [tris elution buffer-PEG-TRIzol-poly(dT) magnetic bead] protocol in order to detect hepatitis A virus (HAV) inoculated in oyster digestive glands. The detection limit of HAV precipitated with zirconium hydroxide was 10(5) fold less sensitive in a nested PCR than that precipitated the HAV supernatant twice with PEG/NaCl (16% polyethylene glycol 6,000, 0.525 M NaCl) in a 1:2 (v/v) ratio, which provided an efficient detection of 0.0148 PFU/g from approximately 0.05 g of oyster homogenate. This method is efficient for potential use in the detection of HAV from shellfish and is more sensitive than most currently published tests.
authors.affiliation: Division of Food Science and Aqualife Medicine, Chonnam National University, Yeosu 550-749, Republic of Korea.
authors.forename: Duwoon
authors.initials: D
authors.lastname: Kim
authors.forename: Seok-Ryel
authors.initials: SR
authors.lastname: Kim
authors.forename: Ki-Sung
authors.initials: KS
authors.lastname: Kwon
authors.forename: Ji-Won
authors.initials: JW
authors.lastname: Lee
authors.forename: Myung-Joo
authors.initials: MJ
authors.lastname: Oh