DETECTION OF PATHOGENIC YERSINIA-ENTEROCOLITICA IN FOODS AND WATER BYIMMUNOMAGNETIC SEPARATION, NESTED POLYMERASE CHAIN-REACTIONS, AND COLORIMETRIC DETECTION OF AMPLIFIED DNA

A two-step polymerase chain reaction (PCR) procedure with two nested p airs of primers specific for the yadA gene of Yersinia enterocolitica was developed. The PCR assay identified all common pathogenic serogrou ps (O:3, O:5,27, O:8, O:9, O:13, and O:21) from three continents and d ifferentiated pathogenic Y. enterocolitica from Y. pseudotuberculosis and from a variety of nonpathogenic yersiniae representing 25 serogrou ps and four species. The performance of the method was evaluated with seeded food and water samples. We compared two procedures for sample p reparation prior to PCR: one was based on immunomagnetic separation of the target bacteria from the sample, using magnetic particles coated with immunoglobulin antibodies to Y. enterocolitica serogroup O:3, and the other method consisted of a series of centrifugation steps combin ed with proteinase treatment. Regardless of the method used, the PCR a ssay was capable of detecting 10 to 30 CFU/g of meat in 10(6)-fold exc ess of indigenous bacteria. When the samples were enriched overnight i n a nonselective medium, the sensitivity was increased to approximatel y 2 CFU/g, except for samples with an extremely high background flora (> 10(7) CFU/g). We compared gel electrophoretic detection of PCR prod ucts with a colorimetric detection method designated DIANA (detection of immobilized amplified nucleic acids), which enabled easy visualizat ion of amplified fragments in a microtiter plate format with an optica l density reader. DIANA and gel electrophoresis showed complete concor dance in their discrimination between positive and negative samples. T he combination of immunomagnetic separation, nested PCR, and DIANA mak es possible the development of a fully automated analytic process whic h requires a minimum of laboratory manipulations.