Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.
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.


RESULTS
The 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 phiA1122 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. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl 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, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR.


CONCLUSIONS
Thus, 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.
Sergueev
Kirill V
KV
Division of Bacterial and Rickettsial Diseases, Department of Emerging Bacterial Infections, Walter Reed Army Institute of Research, Silver Spring, Maryland, United States of America. kirill.sergueev@amedd.army.mil
He
Yunxiu
Y
Borschel
Richard H
RH
Nikolich
Mikeljon P
MP
Filippov
Andrey A
AA
D001435:Bacteriophages / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D006801:Humans; D010930:Plague / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D015010:Yersinia pestis / Q000235:genetics / Q000302:isolation & purification* / Q000821:virology
D017931:DNA Primers; D004279:DNA, Viral