Comparison of multiplex PCR, PCR-ELISA and fluorogenic 5' nuclease PCR assays for detection of plasmid-bearing virulent Yersinia enterocolitica in swine feces.
Swine are implicated as the principal animal reservoir for plasmid-bearing Yersinia enterocolitica (YEP(+)) strains that are pathogenic to humans. To evaluate the utility of the PCR for detection of YEP(+) strains in naturally-contaminated pig feces, samples were first enriched in Irgasan ticarcillin potassium chlorate broth for 48 h at 25 degrees C and then tested by multiplex PCR, PCR-ELISA, and fluorogenic 5' nuclease PCR assays. Three different primer sets for amplification of the ail gene sequences were used in these three assays. Three out of 50 (6%) samples were positive for YEP(+) strains using the multiplex PCR targeting the chromosomal ail (170 bp) and plasmid virF (591 bp) genes. Two of the 3 samples positive by the multiplex PCR were also positive by the PCR-ELISA method using primers targeting the ail gene (425 bp). In contrast, the fluorogenic 5' nuclease PCR assay failed to detect an ail gene sequence (118 bp) in any of the 50 samples. These results indicate that the multiplex PCR was the most reliable and sensitive assay for detecting YEP(+) strains in feces among the three assays evaluated.
Bhaduri
S
S
Microbial Food Safety Research Unit, Eastern Regional Research Center, USDA, Agricultural Research Service, Wyndmoor, PA 19038, USA. sbhaduri@arserrc.gov
D000818:Animals; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D005243:Feces / Q000382:microbiology; D005456:Fluorescent Dyes / Q000592:standards; D006801:Humans; D010957:Plasmids / Q000032:analysis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012680:Sensitivity and Specificity; D013552:Swine; D014774:Virulence; D015009:Yersinia Infections / Q000175:diagnosis*; D015008:Yersinia enterocolitica / Q000235:genetics* / Q000472:pathogenicity
D017931:DNA Primers; D004269:DNA, Bacterial; D005456:Fluorescent Dyes