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

Siyi Chen,Beilei Ge

doi:10.1186/1471-2180-10-41

Abstract

BackgroundVibrio 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 108 CFU/ml to extinction. The assay was also tested in experimentally inoculated oyster samples.ResultsThe 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 × 105 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.ConclusionsThe 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.

Highlights

Vibrio parahaemolyticus is a leading cause of seafood-related bacterial gastroenteritis and outbreaks worldwide
Parahaemolyticus strains in the environment and seafood samples lack these two hemolysin genes [8,9,10], the number of total V. parahaemolyticus has been used as an indicator for preventing V. parahaemolyticus infections from seafood consumption [11,12]
Specificity of the loop-mediated isothermal amplification (LAMP) assay The V. parahaemolyticus toxR-based LAMP assay run on two platforms by using either a real-time PCR machine or a real-time turbidimeter successfully detected 36 V. parahaemolyticus strains while showing negative results for 39 non- V. parahaemolyticus strains (Table 1), indicating that the toxR-based LAMP assay was highly specific

Summary

Introduction

Vibrio parahaemolyticus is a leading cause of seafood-related bacterial gastroenteritis and outbreaks worldwide. Traditional culture-based methods for isolating and enumerating V. parahaemolyticus from seafood samples involve the most probable number (MPN) technique [13]. Widely used, such methods are laborintensive and time-consuming (4-7 days). Molecularbased methods such as DNA probe hybridization and PCR assays have been developed for V. parahaemolyticus and yielded rapid and specific results [14,15,16,17,18]. Several real-time PCR assays have been developed for the detection of V. parahaemolyticus with increased speed and sensitivity [12,19,20,21]. These assays require a dedicated real-time PCR machine, which is rather expensive and not yet widely available

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