Rapid detection of the Vibrio cholerae ctx gene in food enrichments using real-time polymerase chain reaction.
A real-time polymerase chain reaction (qPCR) assay for the detection of the ctxA gene of toxigenic Vibrio cholerae (Vc) was validated against standard culture techniques. The first experimental phase determined optimal enrichment conditions for detection by culture and qPCR of Vc in shrimp, bottled water, milk, and potato salad. The conditions tested included temperature (35 and 42 degrees C), time (6 and 18 h), and effect of shaking (0 and 100 rpm). No definitive trends were found with enrichment temperature or shaking on Vc isolation frequency or detection by qPCR. Generally, Vc was detected by qPCR more frequently than Vc was isolated, but this difference was significant only in the 35 degrees C 6 h enrichment without shaking. In the second phase of experiments, shrimp, bottled water, milk, potato salad, and oysters were inoculated with each of 3 toxigenic Vc strains (Latin American O1 strain, an O139 strain, and an O1 strain from the U.S. Gulf Coast) and enriched under static conditions at 42OC for 6 and 18 h. Overall, detection frequency of ctx by qPCR was 98% (88/90) and 100% (90/90) after 6 and 18 h enrichments, respectively, while Vc isolation frequency was 87% (78/90) and 83% (75/90) after 6 and 18 h, respectively. Toxigenic Vc can be detected by qPCR within an 8 h work day using the 6 h enrichment procedure, assuming an initial level of at least 1-2 colony-forming units/g; however, overnight enrichment may be necessary to detect lower levels. These data indicate that the qPCR assay for ctx is a more reliable, sensitive, and rapid alternative to standard Vc culture methods and is applicable to diverse food products.
Fedio
Willis
W
New Mexico State University, Physical Science Laboratory, Las Cruces, NM 88003-8002, USA.
Blackstone
George M
GM
Kikuta-Oshima
Lynne
L
Wendakoon
Chitra
C
McGrath
Timothy H
TH
DePaola
Angelo
A
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D002623:Chemistry Techniques, Analytical / Q000379:methods; D002772:Cholera Toxin / Q000032:analysis* / Q000737:chemistry; D005504:Food Analysis / Q000379:methods*; D005506:Food Contamination; D008892:Milk; D033561:Penaeidae; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum; D013696:Temperature; D013997:Time Factors; D014734:Vibrio cholerae / Q000378:metabolism*; D014867:Water / Q000737:chemistry
D001426:Bacterial Proteins; D014867:Water; D002772:Cholera Toxin