Detection of Yersinia pestis by pesticin fluorogenic probe-coupled PCR

The 'TaqMan' assay (PE Applied Biosystems) combines PCR with concomitant re lease of fluorogenic nucleotides for immediate product detection by fluorom etry. Yersinia pestis, the etiological agent of bubonic plague, expresses s pecies-specific genes known to be located on two unique plasmids (9.6-kb pP CP and 100.9-kb pMT). Pesticin (pst) is a unique gene located on pPCP which encodes for a bacteriocin. Using fluorogenic probe coupled PCR as few as t hree copies of pst targets were detected from total Y. pestis genomic DNA. The pst probe used in this report was positive only for pesticinogenic isol ates and did not show complementarity with Yersiniae nor with other bacteri a targeted in this study suggesting, that the pst probe is very specific fo r Y. pestis. Under optimal conditions of Mg2+ concentration and thermal cyc le number, addition of extraneous DNA to respective assay mixtures had no e ffect on detection. (C) 2000 Academic Press.