Rapid and Specific Detection of the Thermostable Direct Hemolysin Gene in Vibrio parahaemolyticus by Loop-Mediated Isothermal Amplification

Authors: Nemoto, Jiro¹; Sugawara, Chiyo¹; Akahane, Kenji¹; Hashimoto, Keiji¹; Kojima, Tadashi¹; Ikedo, Masanari¹; Konuma, Hirotaka²; Hara-Kudo, Yukiko³

Source: Journal of Food Protection®, Volume 72, Number 4, April 2009 , pp. 748-754(7)

Publisher: International Association for Food Protection

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Abstract:

Several investigators have reported that thermostable direct hemolysin (TDH) and TDH-related hemolysin are important virulence factors of Vibrio parahaemolyticus, but it has been difficult to detect these factors rapidly in seafood and other environmental samples. A novel nucleic acid amplification method, termed the loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity and rapidity under isothermal conditions, was applied. In this study, we designed tdh gene-specific LAMP primers for detection of TDH-producing V. parahaemolyticus. The specificity of this assay was evaluated with 32 strains of TDH-producing V. parahaemolyticus, one strain of TDH-producing Grimontia hollisae, 10 strains of TDH-nonproducing V. parahaemolyticus, and 94 strains of TDH-nonproducing bacteria, and the sensitivity was high enough to detect one cell per test. Moreover, to investigate the detection of TDH-producing V. parahaemolyticus in oysters, the LAMP assay was performed with enrichment culture in alkaline peptone water of oyster samples inoculated with TDH-producing V. parahaemolyticus and TDH-nonproducing V. parahaemolyticus and V. alginolyticus after enrichment in alkaline peptone water. These results suggest that the LAMP assay targeting tdh gene has high sensitivity and specificity and is useful to detect TDH-producing V. parahaemolyticus in oyster after enrichment.

Document Type: Research article

Affiliations: 1: Eiken Chemical Company Ltd., 143, Nogi, Nogi-machi, shimotsuga-gun, Tochigi 329-0114, Japan 2: Department of Fisheries, School of Marine Science and Technology, Tokai University, 3-20-1 Shimizuorido, Shizuoka 424-8610, Japan 3: Division of Microbiology, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan

Publication date: 2009-04-01

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First published in 1937, the Journal of Food Protection^®, is a refereed monthly publication. Each issue contains scientific research and authoritative review articles reporting on a variety of topics in food science pertaining to food safety and quality. The Journal is internationally recognized as the leading publication in the field of food microbiology with a readership exceeding 11,000 scientists from 70 countries. The Journal of Food Protection^® is indexed in Index Medicus, Current Contents, BIOSIS, PubMed, Medline, and many others.

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Rapid and Specific Detection of the Thermostable Direct Hemolysin Gene in Vibrio parahaemolyticus by Loop-Mediated Isothermal Amplification

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