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BMC Microbiol. 2010 Feb 10;10:41. doi: 10.1186/1471-2180-10-41.

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

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1
Department of Food Science, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803, USA.

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.

PMID:
20146814
PMCID:
PMC2838873
DOI:
10.1186/1471-2180-10-41
[Indexed for MEDLINE]
Free PMC Article
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