ArticleApplied and environmental microbiology2007
Development of a multiplex real-time PCR assay with an internal amplification control for the detection of total and pathogenic Vibrio parahaemolyticus bacteria in oysters.
Article in Applied and environmental microbiology, 2007. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 104 papers.
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104 citing papers in PubMed.
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- Innovative Multiplex PCR Assay for Detection ofPathogens (Basel, Switzerland) · 2024Article
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- Detection of non-pathogenic and pathogenic populations of Vibrio parahaemolyticus in various samples by the conventional, quantitative and droplet digital PCRs.Scientific reports · 2024Article
- Development of Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay to Detect Species-SpecificPathogens (Basel, Switzerland) · 2024Article
- Antimicrobial resistance, virulence factors and phylogenetic profiles ofFrontiers in microbiology · 2024Article
- Evaluation of the relationships between physico-chemical parameters and the abundance ofFrontiers in microbiology · 2024Article
- Tools to Enumerate and Predict Distribution Patterns of EnvironmentalMicroorganisms · 2023Review
44 more citing papers are in PubMed but not listed here.
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5 authors.
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Abstract
Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >10(4) CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh(+) and trh(+) strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.
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