A sensitive & specific multiplex PCR assay for simultaneous detection of Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei & Brucella species.
OBJECTIVE
Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei and Brucella species are potential biowarfare agents. Classical bacteriological methods for their identification are cumbersome, time consuming and of potential risk to the handler.


METHODS
We describe a sensitive and specific multiplex polymerase chain reaction (mPCR) assay involving novel primers sets for the simultaneous detection of B. anthracis, Y. pestis, B. pseudomallei and Brucella species. An additional non-competitive internal amplification control (IAC) was also included.


RESULTS
The mPCR was found to be specific when tested against closely related organisms. The sensitivity of the assay in spiked blood samples was 50 colony forming units (cfus)/25 μl reaction, for the detection of B. anthracis, Y. pestis and Brucella species; and 150 cfus/25 μl reaction, for B. pseudomallei. The assay proved useful in correctly and promptly identifing the clinical isolates of the targeted agents recovered from patients, compared to the gold standard culture methods.


CONCLUSIONS
The assay described in this study showed promise to be useful in application as a routine detection cum diagnostic method for these pathogens.
Division of Microbiology, Defence Research & Development Establishment (DRDO), Gwalior, India.
Sai Arun
SA
Batra
S
S
Krupanidhi
Urmil
U
Tuteja
D001408:Bacillus anthracis / Q000302:isolation & purification*; D001483:Base Sequence; D002002:Brucella / Q000302:isolation & purification*; D016957:Burkholderia pseudomallei / Q000302:isolation & purification*; D017931:DNA Primers; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D015010:Yersinia pestis / Q000302:isolation & purification*
D017931:DNA Primers