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Title: Improvement of the quantitation method for the tdh (+) Vibrio parahaemolyticus in molluscan shellfish based on most-probable- number, immunomagnetic separation, and loop-mediated isothermal amplification.
Authors: Escalante-Maldonado, Oscar
Kayali, Ahmad Y
Yamazaki, Wataru  kyouindb  KAKEN_id  orcid (unconfirmed)
Vuddhakul, Varaporn
Nakaguchi, Yoshitsugu
Nishibuchi, Mitsuaki  kyouindb  KAKEN_id
Author's alias: 西渕, 光昭
Keywords: Vibrioparahaemolyticus
loop-mediated isothermalamplification
Issue Date: 9-Apr-2015
Publisher: Frontiers Research Foundation
Journal title: Frontiers in microbiology
Volume: 6
Thesis number: 270
Abstract: Vibrio parahaemolyticus is a marine microorganism that can cause seafood-borne gastroenteritis in humans. The infection can be spread and has become a pandemic through the international trade of contaminated seafood. Strains carrying the tdh gene encoding the thermostable direct hemolysin (TDH) and/or the trh gene encoding the TDH-related hemolysin (TRH) are considered to be pathogenic with the former gene being the most frequently found in clinical strains. However, their distribution frequency in environmental isolates is below 1%. Thus, very sensitive methods are required for detection and quantitation of tdh (+) strains in seafood. We previously reported a method to detect and quantify tdh (+) V. parahaemolyticus in seafood. This method consists of three components: the most-probable-number (MPN), the immunomagnetic separation (IMS) targeting all established K antigens, and the loop-mediated isothermal amplification (LAMP) targeting the tdh gene. However, this method faces regional issues in tropical zones of the world. Technicians have difficulties in securing dependable reagents in high-temperature climates where we found MPN underestimation in samples having tdh (+) strains as well as other microorganisms present at high concentrations. In the present study, we solved the underestimation problem associated with the salt polymyxin broth enrichment for the MPN component and with the immunomagnetic bead-target association for the IMS component. We also improved the supply and maintenance of the dependable reagents by introducing a dried reagent system to the LAMP component. The modified method is specific, sensitive, quick and easy and applicable regardless of the concentrations of tdh (+) V. parahaemolyticus. Therefore, we conclude this modified method is useful in world tropical, sub-tropical, and temperate zones.
Rights: © 2015 Escalante-Maldonado, Kayali, Yamazaki, Vuddhakul, Nakaguchi and Nishibuchi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
DOI(Published Version): 10.3389/fmicb.2015.00270
PubMed ID: 25914681
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