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. 2010 Feb;76(3):820-8.
doi: 10.1128/AEM.02284-09. Epub 2009 Dec 4.

Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of the tdh and trh genes of Vibrio parahaemolyticus and related Vibrio species

Wataru Yamazaki  1 , Yuko Kumeda, Naoaki Misawa, Yoshitsugu Nakaguchi, Mitsuaki Nishibuchi
Affiliations

Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of the tdh and trh genes of Vibrio parahaemolyticus and related Vibrio species

Wataru Yamazaki et al. Appl Environ Microbiol. 2010 Feb.

Abstract

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.

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Figures

FIG. 1.
FIG. 1.
Real-time monitoring of LAMP reactions with V. parahaemolyticus control strains for multiplex and individual detection of the tdh, trh1, and trh2 genes. Profiles for individual detection of tdh (a), trh1 (b), and trh2 (c) and for multiplex detection of tdh, trh1, and trh2 (d) and of trh1 and trh2 (e) are shown. Total DNAs extracted from AQ3815 (tdh1+, tdh2+, trh1 negative, trh2 negative) (□), AQ3776 (tdh3+, tdh4+, trh1+, trh2 negative) (▪), AQ3860 (tdh5+, trh1+, trh2 negative) (•), AQ4037 (tdh negative, trh1+, trh2 negative) (▴), AT4 (tdh negative, trh1 negative, trh2+) (○), and Vp02 (tdh negative, trh1 negative, trh2 negative [negative control]) (×) were examined.
FIG. 1.
FIG. 1.
Real-time monitoring of LAMP reactions with V. parahaemolyticus control strains for multiplex and individual detection of the tdh, trh1, and trh2 genes. Profiles for individual detection of tdh (a), trh1 (b), and trh2 (c) and for multiplex detection of tdh, trh1, and trh2 (d) and of trh1 and trh2 (e) are shown. Total DNAs extracted from AQ3815 (tdh1+, tdh2+, trh1 negative, trh2 negative) (□), AQ3776 (tdh3+, tdh4+, trh1+, trh2 negative) (▪), AQ3860 (tdh5+, trh1+, trh2 negative) (•), AQ4037 (tdh negative, trh1+, trh2 negative) (▴), AT4 (tdh negative, trh1 negative, trh2+) (○), and Vp02 (tdh negative, trh1 negative, trh2 negative [negative control]) (×) were examined.
FIG. 2.
FIG. 2.
Sensitivity test for detection of the tdh, trh1, trh2 genes in V. parahaemolyticus strains in spiked shrimp samples by real-time turbidimetry. The curves from left to right indicate decreasing concentrations of CFU from bacterial colonies. (a) Detection of the tdh gene of V. parahaemolyticus AQ3815. (b) Detection of the trh1 gene of V. parahaemolyticus AQ4037. (c) Detection of the trh2 gene of V. parahaemolyticus AT4. (d) Visual detection of the tdh gene by observation of precipitation. Tube 1, 84.4 CFU of AQ3815; tube 2, 0.1 CFU of AQ3815.
FIG. 2.
FIG. 2.
Sensitivity test for detection of the tdh, trh1, trh2 genes in V. parahaemolyticus strains in spiked shrimp samples by real-time turbidimetry. The curves from left to right indicate decreasing concentrations of CFU from bacterial colonies. (a) Detection of the tdh gene of V. parahaemolyticus AQ3815. (b) Detection of the trh1 gene of V. parahaemolyticus AQ4037. (c) Detection of the trh2 gene of V. parahaemolyticus AT4. (d) Visual detection of the tdh gene by observation of precipitation. Tube 1, 84.4 CFU of AQ3815; tube 2, 0.1 CFU of AQ3815.
FIG. 3.
FIG. 3.
Schematic representation of detection and differentiation of the tdh, trh1, and trh2 genes from a single V. parahaemolyticus colony on TCBS agar.
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