Development of a Real-time Quantitative PCR Assay to Enumerate Yersinia pestis in Fleas

A real-time quantitative polymerase chain reaction (qPCR) assay was developed for Yersina pestis . The qPCR assay was developed utilizing a conserved region of the Y. pestis ferric iron uptake regulator gene ( fur ) to design primers and a fluorescent (FAM-labeled) Taq Man probe. The assay was optimized using cultured Y. pestis (UG05-0454) and was confirmed to work with strains from 3 Y. pestis biovars. The optimized assay was capable of detecting a single organism of cultured Y. pestis and as little as 300 bacteria in infected flea triturates. This qPCR assay enables rapid enumeration of Y. pestis bacterium in laboratory-infected fleas when compared with conventional serial dilution plating.