A sensitive & specific multiplex PCR assay for simultaneous detection of Bacillus anthracis, Yersinia pestis, Burkholderia pseudomallei & Brucella species

Abstract

Background & objectives: 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.

Interpretation & conclusion: The assay described in this study showed promise to be useful in application as a routine detection cum diagnostic method for these pathogens.

Fig

Cross-reactivity testing of the mPCR with closely related organisms. Lane 1: 100 bp DNA ladder, Lane 2: Burkholderia pseudomallei, Lane 3: Burkholderia mallei, Lane 4: Y. pestis, Lane 5: Y. pseudotuberculosis, Lane 6: Bacillus anthracis, Lane 7: Bacillus cereus, Lane 8: Brucella mellitus, Lane 9: Multiplex positive control. Lane 10: Y. enterocolitica.