Multiplexed real-time PCR amplification of tlh, tdh and trh genes in Vibrio parahaemolyticus and its rapid detection in shellfish and Gulf of Mexico water.
Antonie Van Leeuwenhoek
; 98(3): 279-90, 2010 Oct.
Article
em En
| MEDLINE
| ID: mdl-20376562
In this study, we have developed a SYBR Green 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 C(t) 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 (approximately 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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ostreidae
/
Água do Mar
/
Frutos do Mar
/
Proteínas de Bactérias
/
Vibrio parahaemolyticus
/
Proteínas Hemolisinas
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
/
Humans
País/Região como assunto:
Mexico
Idioma:
En
Revista:
Antonie Van Leeuwenhoek
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Estados Unidos
País de publicação:
Holanda