Abstract
Background Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics.Methodology/Principal FindingsThe objective of this work was to develop an alternative to conventional phage lysis tests – a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages ϕA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. ϕA1122-specific qPCR enabled the detection of an initial bacterial concentration of 103 CFU/ml (equivalent to as few as one Y. pestis cell per 1-µl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, ϕA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR.Conclusions/SignificanceThus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.
Highlights
Yersinia pestis is a Gram-negative nonsporulating bacterium belonging to the family Enterobacteriaceae
We have previously shown that P2 vir1 lyses Y. pestis at 37uC, has relatively low plaquing efficiency at 28uC, but is not active against a wild-type strain of Y. pseudotuberculosis at either temperature [50]
Since the speed of phage propagation is critical for quantitative real-time PCR (qPCR) efficiency, we first checked some parameters of amplification of QA1122, L-413C, and P2 vir1 on Y. pestis CO92 pgm2
Summary
Yersinia pestis is a Gram-negative nonsporulating bacterium belonging to the family Enterobacteriaceae. Y. pestis is the causative agent of bubonic and pneumonic plague, a primarily zoonotic infection. Pneumonic plague is a severe infection transmissible from person to person by respiratory droplets and thought to be responsible for about 200 million human deaths during three historic pandemics. Y. pestis is classified by the CDC as a category A biothreat agent due to its easy person-to-person dissemination via aerosol, high lethality, and wide recognition as a biowarfare agent that is likely to cause mass casualties [4]. The agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call