Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus

Abstract

Background: Vibrio parahaemolyticus is a leading cause of seafood-related bacterial gastroenteritis and outbreaks worldwide. Sensitive and specific detection methods are needed to better control V. parahaemolyticus infections. This study aimed at developing a highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting V. parahaemolyticus in oysters. A set of five LAMP primers, two outer, two inner, and one loop were designed based on the published V. parahaemolyticus toxR sequence. Specificity of the assay was evaluated using a panel of 36 V. parahaemolyticus and 39 other strains. The assay sensitivity was determined using serial dilutions of V. parahaemolyticus ATCC 27969 culture ranging from 10(8) CFU/ml to extinction. The assay was also tested in experimentally inoculated oyster samples.

Results: The toxR-based LAMP assay was able to specifically detect all of the 36 V. parahaemolyticus strains without amplification from 39 other strains. The detection limit was 47-470 cells per reaction in pure culture, up to 100-fold more sensitive than that of toxR-PCR. When applied in spiked oysters, the assay was able to detect 1.1 x 10(5) V. parahaemolyticus cells per gram of oyster without enrichment, up to 100-fold more sensitive than that of toxR-PCR. Standard curves generated for detecting V. parahaemolyticus in both pure culture and spiked oyster samples showed good linear relationship between cell numbers and the fluorescence or turbidity signals.

Conclusions: The toxR-based LAMP assay developed in this study was sensitive, specific, and quantitative, holding great potential for future field detection of V. parahaemolyticus in raw oysters.

Figure 1

Sensitivity of the LAMP assay when detecting Vibrio parahaemolyticus ATCC 27969 in pure culture. (A) A representative optic graph generated using the real-time PCR machine; (B) Corresponding melting curve analysis for samples in (A); (C) A representative turbidity graph generated using the real-time turbidimeter. Samples 1-7 corerspond to serial 10-fold dilutions of V. parahaemolyticus ATCC 27969 cells ranging from 4.7 × 10⁵ to 4.7 × 10^-1 CFU/reaction; sample 8 is water.

Figure 2

Standard curves generated when detecting Vibrio parahaemolyticus ATCC 27969 in pure culture. (A) Based on six independent repeats in a real-time PCR machine; (B) Based on six independent repeats in a real-time turbidimeter.

Figure 3

Standard curves generated when testing Vibrio parahaemolyticus ATCC 27969 in spiked oysters. Three sets of independent spiking experimetns were performed, and the LAMP reactions were repeated two times for each inoculation set. The data shown are for the inoculation set 3 ranging from 1.1 × 10⁵ to 1.1 × 10⁹ CFU/g. (A) The assay was run in a real-time PCR machine; (B) The assay was run in a real-time turbidimeter.