Journal
ANTONIE VAN LEEUWENHOEK INTERNATIONAL JOURNAL OF GENERAL AND MOLECULAR MICROBIOLOGY
Volume 98, Issue 3, Pages 279-290Publisher
SPRINGER
DOI: 10.1007/s10482-010-9436-2
Keywords
Vibrio; Real-time PCR; SYBR Green I; Shellfish; Gulf of Mexico
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Funding
- Mississippi Alabama Sea Grant Consortium, National Oceanic and Atmospheric Administration, Department of Commerce
- University of Alabama at Birmingham [NA86RG0039-4, R/SP-8]
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In this study, we have developed a SYBR Green (TM) I-based real-time multiplexed PCR assay for the detection of Vibrio parahaemolyticus in Gulf of Mexico water (gulf water), artificially seeded and natural oysters targeting three hemolysin genes, tlh, tdh and trh in a single reaction. Post-amplification melt-temperature analysis confirmed the amplification of all three targeted genes with high specificity. The detection sensitivity was 10 cfu (initial inoculum) in 1 ml of gulf water or oyster tissue homogenate, following 5 h enrichment. The results showed 58% of the oysters to be positive for tlh, indicating the presence of V. parahaemolyticus; of which 21% were positive for tdh; and 0.7% for trh, signifying the presence of pathogenic strains. The Ct values showed that oyster tissue matrix had some level of inhibition, whereas the gulf water had negligible effect on PCR amplification. The assay was rapid (similar to 8 h), specific and sensitive, meeting the ISSC guidelines. Rapid detection using real-time multiplexed PCR will help reduce V. parahaemolyticus-related disease outbreaks, thereby increasing consumer confidence and economic success of the seafood industry.
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