Detection of Hepatitis A Virus from Oyster by Nested PCR Using Efficient Extraction and Concentration Method

Duwoon Kim; Seok-Ryel Kim; Ki-Sung Kwon; Ji-Won Lee; Myung-Joo Oh

doi:10.1007/s12275-008-0131-1

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Research Support, Non-U.S. Gov't
Detection of Hepatitis A Virus from Oyster by Nested PCR Using Efficient Extraction and Concentration Method: Duwoon Kim ¹, Seok-Ryel Kim ¹, Ki-Sung Kwon ², Ji-Won Lee ², Myung-Joo Oh ¹; Journal of Microbiology 2008;46(4):436-440.
DOI: https://doi.org/10.1007/s12275-008-0131-1
Published online: August 31, 2008

¹Division of Food Science and Aqualife Medicine, Chonnam National University, Yeosu 550-749, Republic of Korea, ²Korea Food and Drug Administration, Seoul 122-704, Republic of Korea

Corresponding author: Myung-Joo Oh , Tel: 82-61-659-3173,

Received: 27 May 2008 • Accepted: 16 June 2008

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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 105 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.

Keywords: Hepatitis A virus;RT-PCR;oyster;nested PCR

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Detection of Hepatitis A Virus from Oyster by Nested PCR Using Efficient Extraction and Concentration Method

J. Microbiol. 2008;46(4):436-440. Published online August 31, 2008

DOI: https://doi.org/10.1007/s12275-008-0131-1

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