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J Med Microbiol 57 (2008), 324-331; DOI: 10.1099/jmm.0.47485-0
© 2008 Society for General Microbiology
ISSN 1473-5644

A quadruplex real-time PCR assay for the detection of Yersinia pestis and its plasmids

Alvin Stewart, Benjamin Satterfield, Marissa Cohen, Kim O'Neill and Richard Robison

851 WIDB, Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA

Correspondence
Richard Robison
richard_robison{at}byu.edu

Received 29 June 2007
Accepted 3 December 2007

Yersinia pestis, the aetiological agent of the plague, causes sporadic disease in endemic areas of the world and is classified as a National Institute of Allergy and Infectious Diseases Category A Priority Pathogen because of its potential to be used as a bioweapon. Health departments, hospitals and government agencies need the ability to rapidly identify and characterize cultured isolates of this bacterium. Assays have been developed to perform this function; however, they are limited in their ability to distinguish Y. pestis from Yersinia pseudotuberculosis. This report describes the creation of a real-time PCR assay using Taqman probes that exclusively identifies Y. pestis using a unique target sequence of the yihN gene on the chromosome. As with other Y. pestis PCR assays, three major genes located on each of the three virulence plasmids were included: lcrV on pCD1, caf1 on pMT1 and pla on pPCP1. The quadruplex assay was validated on a collection of 192 Y. pestis isolates and 52 near-neighbour isolates. It was discovered that only 72 % of natural plague isolates from the states of New Mexico and Utah harboured all three virulence plasmids. This quadruplex assay proved to be 100 % successful in differentiating Y. pestis from all near neighbours tested and was able to reveal which of the three virulence plasmids a particular isolate possessed.

Abbreviations: NCBI, National Center for Biotechnology Information.






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