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Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus
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Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus
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Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus
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Journal Article
Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus
2010
Overview
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 × 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.
Publisher
BioMed Central,BioMed Central Ltd,BMC
Subject
/ Bacterial Proteins - genetics
/ Biomedical and Life Sciences
/ DNA-Binding Proteins - genetics
/ Humans
/ Identification and classification
/ Mycology
/ Nephelometry and Turbidimetry
/ Nucleic Acid Amplification Techniques - methods
/ Transcription Factors - genetics
/ Vibrio
/ Vibrio Infections - microbiology
/ Vibrio parahaemolyticus - genetics
/ Vibrio parahaemolyticus - isolation & purification
/ Virology
