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

• Published in: PLoS Neglected Tropical Diseases
• Main Authors: Shi Qu, Qinghai Shi, Lei Zhou, Zhaobiao Guo, Dongsheng Zhou, Junhui Zhai, Ruifu Yang
• Format: Article in Journal/Newspaper
• Language: English
• Published: Public Library of Science (PLoS) 2010
• Subjects: Arctic medicine. Tropical medicine; RC955-962; Public aspects of medicine; RA1-1270; Arctic
• Online Access: https://doi.org/10.1371/journal.pntd.0000629; https://doaj.org/article/fc449ad2c7684bec8b6162405d038b45

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. 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 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/SIGNIFICANCE: 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.