title: Development and evaluation of a 4-target multiplex real-time polymerase chain reaction assay for the detection and characterization of Yersinia pestis.
abstract: A multiplexed, 4-target real-time polymerase chain reaction (PCR) assay for the detection and characterization of Yersinia pestis was designed and optimized for respiratory and environmental samples. The target sequences include the entF3 gene of the chromosome, pla (plasminogen activator) on the pPCP1 virulence plasmid, caf1 (F1 capsule antigen) on the pMT1 virulence plasmid, and a region located on the pCD1 plasmid. The sensitivity of this assay was determined to be less than 85 CFU per reaction for each specimen type analyzed. This assay was also determined to be 100% specific with strains of Y. pestis, 9 additional Yersinia species, and related enteric and respiratory organisms. The results show that this multiplex real-time PCR assay using TaqMan(R) (Roche Molecular Systems, Inc., Alameda, CA) chemistry is sensitive and specific, requires minimal sample input, and can yield results in approximately 4 h. This assay is the first 4-target multiplex real-time PCR assay for Y. pestis in which detection and virulence assessment of Y. pestis can occur in one reaction, from clinical and environmental samples.
authors.affiliation: New York State Department of Health, Wadworth Center, Albany, NY 12208, USA.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Woron
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Nazarian
authors.forename: Christina
authors.initials: C
authors.lastname: Egan
authors.forename: Kathleen A
authors.initials: KA
authors.lastname: McDonough
authors.forename: Nick M
authors.initials: NM
authors.lastname: Cirino
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Limberger
authors.forename: Kimberlee A
authors.initials: KA
authors.lastname: Musser
mesh_terms: D000942:Antigens, Bacterial / Q000032:analysis* / Q000235:genetics; D001426:Bacterial Proteins; D005069:Evaluation Studies as Topic; D010957:Plasmids / Q000235:genetics; D010960:Plasminogen Activators / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014774:Virulence / Q000235:genetics; D015010:Yersinia pestis / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C078140:Pla protease, Yersinia pestis; D010960:Plasminogen Activators