Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains

For the detection of pathogenic Yersinia enterocolitica strains, a duplex P CR has been developed based on differences observed between the fingerprint profiles of pathogenic and non-pathogenic strains. The profiles were obtai ned by using a primer derived from the Enterobacterial Repetitive Intergeni c Consensus (ERIC) sequences. From the sequence of one patho.-en-specific a mplified fragment, a discriminative primer has been designed bridging the s equence of the highly conserved core region and 3' end of the ERIC element. In combination with three other primers, all located within the detected o pen reading frame that resembled the sequence of the bipA gene, this primer was applied in a duplex PCR assay to simultaneously detect Y. enterocoliti ca and to discriminate between pathogenic and non-pathogenic strains. The s ame primer combinations were used in an on line rapid cycling real-time PCR assay. The used SYBR Green I format allowed for the easy translation of th e PCR conditions and confirmation of the resulting amplicons. The time of a nalysis was reduced to approximately 60 min. (C) 2001 Elsevier Science B.V. All rights reserved.