Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains.
For the detection of pathogenic Yersinia enterocolitica strains, a duplex PCR has been developed based on differences observed between the fingerprint profiles of pathogenic and non-pathogenic strains. The profiles were obtained by using a primer derived from the Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences. From the sequence of one pathogen-specific amplified fragment, a discriminative primer has been designed bridging the sequence of the highly conserved core region and 3' end of the ERIC element. In combination with three other primers, all located within the detected open reading frame that resembled the sequence of the bipA gene, this primer was applied in a duplex PCR assay to simultaneously detect Y. enterocolitica and to discriminate between pathogenic and non-pathogenic strains. The same 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 the PCR conditions and confirmation of the resulting amplicons. The time of analysis was reduced to approximately 60 min.
Aarts
H J
HJ
RIKILT Wageningen-UR, Bornsesteeg 45, 6708 PD Wageningen, The Netherlands. h.j.m.aarts@rikilt.wag-ur.nl
Joosten
R G
RG
Henkens
M H
MH
Stegeman
H
H
van Hoek
A H
AH
D000595:Amino Acid Sequence; D001483:Base Sequence; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism; D015009:Yersinia Infections / Q000382:microbiology; D015008:Yersinia enterocolitica / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity
D004269:DNA, Bacterial; D019914:Taq Polymerase