Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

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.

Application of bacteriophages for detection of foodborne pathogens

“…This way, the authors were able to correlate the fluorescence signal indicating the proportion of dead helper cells to the initial number of target cells present in the original sample, allowing detection of 10 CFU/ml within 4 h without prior enrichment. Further endpoint detection methods that have been used to replace the traditional plaque formation and enumeration include quantitative real-time PCR (qPCR) 53 and competitive ELISA (phage replication-competitive enzymelinked immunosorbent assay, PR-cELISA). 54…”

Bacteriophage-based tools: recent advances and novel applications

“…If the bacterial target is present and viable, detectable phage numbers will increase through amplification on the pathogen. Modifications of this method can generate results more rapidly, and in the case of Yersinia pestis , Sergueev et al ., for example, developed a quantitative real-time PCR to detect the increase in phage DNA instead of traditional plaque assays 10 . Reporter phages can also detect bacteria through infection without needing cell lysis and progeny phages.…”

Pathogen detection using engineered bacteriophages

“…Rather than relying on the production of visual plaques after phage amplification, end points such as optical density [40], live/dead fluorochromic staining [41], quantitative realtime PCR (qPCR) [42], matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry [43,44], and competitive ELISA [45] have all been applied for the detection of host-released phages or phage components, thus showcasing the versatility of this assay format.…”

“…Sergueev et al [42] used phage amplification of the Yersinia pestis specific phages ΦA1122 and L-413 C followed by qPCR to quantify resulting progeny phage genomes. Phage ΦA1122 was more sensitive than phage L-413 C, maintaining a detection limit of 1,000 CFU mL -1 in pure culture, but was less specific owing to cross-reactivity with Yersinia pseudotuberculosis, although this can be mediated by lowering the incubation temperature.…”