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Background  Although assays for detecting  Yersinia pestis  using TaqMan probe-based real-time PCR have been developed for years, little is reported on room-temperature-stable PCR reagents, which will be invaluable for field epidemic surveillance, immediate response to public health emergencies, counter-bioterrorism investigation, etc. In this work, a set of real-time PCR reagents for rapid detection of  Y. pestis  was developed with extraordinary stability at 37°C.    Methods/Principal Findings  TaqMan-based real-time PCR assays were developed using the primers and probes targeting the 3a sequence in the chromosome and the F1 antigen gene  caf1  in the plasmid pMT1of  Y. pestis , respectively. Then, carbohydrate mixtures were added to the PCR reagents, which were later vacuum-dried for stability evaluation. The vacuum-dried reagents were stable at 37°C for at least 49 days for a lower concentration of template DNA (10 copies/µl), and up to 79 days for higher concentrations (≥10 2  copies/µl). The reagents were used subsequently to detect soil samples spiked with  Y. pestis  vaccine strain EV76, and 5×10 4  CFU per gram of soil could be detected by both 3a- and  caf1 -based PCR reagents. In addition, a simple and efficient method for soil sample processing is presented here.    Conclusions/Significance  The vacuum-dried reagents for real-time PCR maintain accuracy and reproducibility for at least 49 days at 37°C, indicating that they can be easily transported at room temperature for field application if the machine for performing real-time PCR is available. This dry reagent is of great significance for routine plague surveillance.

Ambient Stable Quantitative PCR Reagents for the Detection of Yersinia pestis