title: A quadruplex real-time PCR assay for the detection of Yersinia pestis and its plasmids.
abstract: 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.
authors.affiliation: 851 WIDB, Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA.
authors.forename: Alvin
authors.initials: A
authors.lastname: Stewart
authors.affiliation: 851 WIDB, Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Satterfield
authors.affiliation: 851 WIDB, Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA.
authors.forename: Marissa
authors.initials: M
authors.lastname: Cohen
authors.affiliation: 851 WIDB, Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA.
authors.forename: Kim
authors.initials: K
authors.lastname: O'Neill
authors.affiliation: 851 WIDB, Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Robison
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D015342:DNA Probes; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase; D014774:Virulence / Q000235:genetics; D015010:Yersinia pestis / Q000145:classification / Q000235:genetics / Q000472:pathogenicity
chemical_list: D001426:Bacterial Proteins; D015342:DNA Probes; D019914:Taq Polymerase