Ambient Stable Quantitative PCR Reagents for the Detection of Yersinia pestis

Citation: Qu S, Shi Q, Zhou L, Guo Z, Zhou D, et al. ( Ambient Stable Quantitative PCR Reagents for the Detection of Yersinia pestis Shi Qu. 0 Qinghai Shi. 0 Lei Zhou 0 Zhaobiao Guo 0 Dongsheng Zhou 0 Junhui Zhai 0 Ruifu Yang 0 Kirsten E. Lyke, University of Maryland School of Medicine, United States of America 0 Laboratory of Analytical Microbiology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology , Beijing , China 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 37uC. 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 37uC for at least 49 days for a lower concentration of template DNA (10 copies/ml), and up to 79 days for higher concentrations ($102 copies/ml). The reagents were used subsequently to detect soil samples spiked with Y. pestis vaccine strain EV76, and 56104 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 37uC, 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. - Funding: This work was supported by the National Key Program for Major Infectious Diseases of China (2009ZX10004-103). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Yersinia pestis, the causative pathogen of the plague, mainly resides in rodents and can be transmitted to humans by infected fleas [1]. As Y. pestis is highly virulent and infectious, it has always been recognized as one of the classical biological warfare agents [2] and was classified as a Category A pathogen by the U. S. Center for Disease Control and Prevention (http://www.bt.cdc. gov/agent/agentlist-category.asp) [3]. Y. pestis was traditionally identified by bacterial isolation and microscopy observation [4], the phage lysis assay [5] and animal experiments, which was termed as a four-step protocol in China. Although it is time-consuming and laborious, this protocol is still a gold standard for laboratory confirmation of Y. pestis infections. Immunological methods were also developed for the detection of F1 antigen and antibodies against Y. pestis [6,7,8]. Immunological biosensors, based on fiber optics, magnetic and upconverting phosphor technology, were recently applied in detection of antigen and antibodies of Y. pestis as well [9,10,11]. These methods have played important roles in fighting plague, however, nucleic acid-based detection techniques could be an even powerful alternative for detecting Y. pestis. Conventional PCR-gel electrophoresis method has been developed for detecting Y. pestis in fleas and other specimens [12,13,14]. A handful of real-time quantitative PCR assays in various formats were also established for detecting and identifying Y. pestis [15,16,17,18,19,20,21,22,23]. Real-time PCR assays provide greater specificity, and they require less time and labor to complete than conventional PCRs. The techniques applied include SYBR Green [24], molecular beacon [25], TaqMan probes [18,20] and minor groove binding (MGB) probes [22] ect., targeting specific sequences on the chromosome and (or) plasmids. Although real-time PCR has been successfully used in detecting and identifying Y. pestis, the relevant reagents need to be transported under low temperature in dry ice in order to keep the activities of enzymes and labeled probes. In this report we developed a room-temperature stable reagent for real-time PCRs which targeted the 3a sequence [26] in chromosome and the caf1 gene [14] in the plasmid pMT1. This reagent could be stable during transportation at room temperature and thus be reliably applied for on site detection of target microorganisms if the thermal cycler is available. Materials and Methods Genomic DNAs Genomic DNAs of four biovars (Microtus, Orientalis, Antiqua and Mediaevalis) of Y. pestis were stored in our lab. Closely related or Yersinia pseudotuberculosis Biovar or Serotype Author Summary Plague, caused by Yersinia pestis, is one of the oldest and most dangerous diseases in human history, and has claimed millions of lives in the three major historical pandemics. Although panic caused by the Black Death is fading, the threat of the reemergence of plague pandemics still exists, with the additional potential of misuse in biowarfare or bioterrorism. Rapid on-site detection and identification of the pathogen is of paramount significance for timely implementation of effective countermeasures. TaqMan probe-based real-time PCR assays can give quick and accurate identification; however, the need for cold delivery and storage prevents its potential on-site application. The objective of this study was to develop a stable PCR system for easy delivery and storage under room temperature, which is vital for conventional plague surveillance and for preparedness in public health emergencies. We present a solution to this particular issue, hoping that it is helpful to future applications. other genomic DNAs used in this study include 9 species of Yersinia (Y. enterocolitica, Y. intermedia, Y. aldovae, Y. bercovieri, Y. frederiksenii, Y. kristensenii, Y. mollaretii, Y. rohdei, Y. ruckeri), 16 different serotypes of Y. pseudotuberculosis, Brucella, Francisella tularensis, Bacillus anthracis, and E. coli DH5a; and DNAs from human blood and mouse. All bacterial strains used in this study were listed in Table 1. Primers and probes The primers and probes for real-time PCR were designed based on the 3a sequence in the chromosome [26] and caf1 in plasmid pMT1 using Primer Express 2.0 (PE Corporation,USA). Other primers were also designed for cloning the target 3a and calf1 sequences into pGEM-T Easy Vector (Promega, USA). The primers and probes used in this study were listed in Table 2. Real-time (...truncated)