| Plague is one of the oldest and the most notorious deadly disease in the history of human beings. As it usually causes serious consequences to humans, the rapid detection and effective prevention of the Yersinia pestis infection are of critical importance. The methods used for detecting Yersinia pestis includes the traditional four-step protocol, serological techniques and nucleic acids-based methods.Aims: Although assays for detecting Yersinia pestis using real-time PCR based on TaqMan probe have been developed for years, until now 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: 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 pMT1 of Y. pestis, respectively. The concentration of primers, probes and magnesium ion were optimized, and the parameters of real-time PCR protocol were also tested.In order to evaluate the specificity of all the primers and probes designed, the genomic DNAs used in this study included 9 species of the Yersinia; 4 biovars of Yersinia pestis (Antiqua, Mediaevalis, Orientalis, Microtus); 16 serotypes of Yersinia pseudotuberculosis which is closely related to Yersinia pestis; Brucella spp., Francisella tularensis, Bacillus anthracis; and E. coli DH5a; and DNAs from human blood and mouse. In the evaluation of the sensitivity, Y. pestis live attenuated strain EV76 was cultivated in Luria-Bertani broth and then serially diluted by 10-fold. The number of viable cells was determined by counting colony forming unit (CFU) on the HBI (Heart Brain Infusion) agar plate. Five microliters of each sample were directly applied to PCR amplification for the evaluating test.In the study on the stability of real-time PCR reagents, all reagents needed for real-time PCR were mixed up at appropriate proportion and dispensed into 200μl microcentrifuge tubes with 15μl each for a single reaction. The dispensed reaction mixture was then vacuum-dried. For stability evaluation, the dry reagents were kept at 37°C in the dark. The reagents were reconstituted by adding 10μl of ddH2O, and then, 5μl of the corresponding templates. The real-time PCRs were performed using the reagents kept at -20°C and 37°C simultaneously. Only the system for 3a primers and probe was evaluated in this experiment.For the detection of the spiked soil samples, a culture of Y. pestis EV76 was serially diluted by 10-fold, and the number of viable cells was determined. Each of serial dilutions was added to soil samples, and the contaminated soil samples were used in the specificity, sensitivity and blind tests. A simple protocol for processing the soil samples was established.Finally, comparison of the homemade reagents vs. the imported reagents was carried out in the field, in which the contaminated water samples and the dilutions of Y. pestis were tested.Results: For both 3a and caf1 primers and probes, all four biovars of Y. pestis gave positive signals, and the other genomic DNAs gave negative results, indicating the good specificity of these reagents. The sensitivity of the reagents based on the 3a and the caf1 sequences is 1.5 and 15 CFU per reaction, respectively, using dilutions of EV76 as template.The 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 the lower concentration of template DNA (10 copies/μl), and up to 79 days for the higher concentrations (102 copies/μl). The reagents were used subsequently to detect soil samples spiked with Y. pestis vaccine strain EV76, and 5×104 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: Although study on room-temperature-stable PCR reagents was first reported in 1995, until now study on room-temperature-stable reagents for quantitative PCR based on TaqMan probe has never been reported. However, the need for the application of the detection methods based on PCR was quite practical in field epidemic surveillance of plague, immediate response to public health emergencies, counter-bioterrorism investigation, etc. In our work presented, 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.
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