Real-Time PCR Assays Targeting a Unique Chromosomal Sequence of Yersinia pestis

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Abstract
Background: Yersinia pestis, the causative agent of the zoonotic infection plague, is a major concern as a potential bioweapon. Current real-time PCR assays used for Y. pestis detection are based on plasmid targets, some of which may generate false-positive results.Methods: Using the yp48 gene of Y. pestis, we designed and tested 2 real-time TaqMan® minor groove binder (MGB) assays that allowed us to use chromosomal genes as both confirmatory and differential targets for Y. pestis. We also designed several additional assays using both Simple-Probe® and MGB Eclipse™ probe technologies for the selective differentiation of Yersinia pseudotuberculosis from Y. pestis. These assays were designed around a 25-bp insertion site recently identified within the yp48 gene of Y. pseudotuberculosis.Results: The Y. pestis-specific assay distinguished this bacterium from other Yersinia species but had unacceptable low-level detection of Y. pseudotuberculosis, a closely related species. Simple-Probe and MGB Eclipse probes specific for the 25-bp insertion detected only Y. pseudotuberculosis DNA. Probes that spanned the deletion site detected both Y. pestis and Y. pseudotuberculosis DNA, and the 2 species were clearly differentiated by a post-PCR melting temperature (Tm) analysis. The Simple-Probe assay produced an almost 7 °C Tm difference and the MGB Eclipse probe a slightly more than 4 °C difference.Conclusions: Our method clearly discriminates Y. pestis DNA from all other Yersinia species tested and from the closely related Y. pseudotuberculosis. These chromosomal assays are important both to verify the presence of Y. pestis based on a chromosomal target and to easily distinguish it from Y. pseudotuberculosis.