Ambient stable quantitative PCR reagents for the detection of Yersinia pestis.
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 degrees C.


RESULTS
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 degrees C for at least 49 days for a lower concentration of template DNA (10 copies/microl), and up to 79 days for higher concentrations (> or =10(2) copies/microl). The reagents were used subsequently to detect soil samples spiked with Y. pestis vaccine strain EV76, and 5x10(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
The vacuum-dried reagents for real-time PCR maintain accuracy and reproducibility for at least 49 days at 37 degrees 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.
Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.
Shi
S
Qu
Qinghai
Q
Shi
Lei
L
Zhou
Zhaobiao
Z
Guo
Dongsheng
D
Zhou
Junhui
J
Zhai
Ruifu
R
Yang
D001426:Bacterial Proteins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D002876:Chromosomes, Bacterial; D005612:Freeze Drying; D006801:Humans; D007202:Indicators and Reagents; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012988:Soil Microbiology; D013696:Temperature; D013997:Time Factors; D037521:Virulence Factors / Q000235:genetics; D015010:Yersinia pestis / Q000235:genetics / Q000302:isolation & purification*
D001426:Bacterial Proteins; D007202:Indicators and Reagents; D037521:Virulence Factors; C086725:caf1 protein, Yersinia pestis