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) TaqMan 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.
Gabitzsch
Elizabeth S
ES
Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, Fort Collins, Colorado 80521, USA.
Vera-Tudela
Rommelle
R
Eisen
Rebecca J
RJ
Bearden
Scott W
SW
Gage
Kenneth L
KL
Zeidner
Nordin S
NS
D000818:Animals; D017931:DNA Primers; D004269:DNA, Bacterial; D016133:Polymerase Chain Reaction / Q000379:methods*; D005423:Siphonaptera / Q000382:microbiology* / Q000502:physiology; D019914:Taq Polymerase; D015010:Yersinia pestis / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D004269:DNA, Bacterial; D019914:Taq Polymerase