DETECTION OF PATHOGENIC YERSINIA-ENTEROCOLITICA USING THE MULTIPLEX POLYMERASE CHAIN-REACTION

A multiplex polymerase chain reaction (PCR) was developed to detect th e presence of the ail, yst, and virF genes of Yersinia enterocolitica simultaneously, quickly and accurately. The amplified fragment sizes w ere 356 base-pairs (bp) for the ail gene, 134 bp for the yst gene, and 231 bp for the virF gene. The specificity of the amplified products w as confirmed by hybridization with digoxigenin-labelled oligonucleotid e probes. Amplification was successful whether the template was derive d from a single colony of bacteria, aliquots of boiled bacterial suspe nsions, from DNA extracted from pure or mixed cultures or from stool s pecimens. Amplification of the virF gene was also achieved from strain s of Y. pseudotuberculosis carrying the 70 kb plasmid but not with pre parations from other related Yersinia species or from other members of the family Enterobacteriaceae. The detection limit we established was 5-10 colony forming units per millilitre (cfu/ml) and 1.0 pg of DNA.