Development of a Polymerase Chain Reaction Assay for Detection of Burkholderia mallei, a Potent Biological Warfare Agent

Abstract

Burkholderia mallei is the etiological agent of glanders, primarily a disease of equines. B. mallei is closely related to B. pseudomallei, the causative agent of melioidosis. Therefore, detection of B. mallei and its differentiation from B. pseudomallei, has always been troublesome. In present investigation, a B. mallei specific DNA sequence was identified by performing BLASTn search using ~3000 ORFs of B. mallei NCTC 10229. A polymerase chain reaction (PCR) assay with internal amplification control (IAC) was developed for detection of B. mallei and its differentiation from B. pseudomallei. The PCR assay could amplify a specific 224-bp fragment from all the six B. mallei strains used in the study, whereas other closely related organisms were tested negative. The detection limit of the assay was found to be 10 pg of purified DNA of B. mallei. Incorporation of IAC in the assay makes the results reliable as false negative results which may arise due to presence of PCR inhibitors, can be avoided. For validation, the assay was tested on tap water, Bengal gram and grass artificially spiked with B. mallei. The developed assay can be used as a simple and rapid tool for detection of B. mallei.

Development of a real-time loop-mediated isothermal amplification assay for detection ofBurkholderia mallei

“…Burkholderia mallei ‐specific target DNA sequence was selected as described earlier (Pal, Kumar, Malik, & Rai, ; Pal, Singh, Tiwari, Jaiswal, & Rai, ). A subregion (89 bp) of gene BMA10229_0375 along with 145‐bp downstream sequence of BMA10229_0375 being specific for B. mallei was targeted for development of LAMP assay.…”

“…To the best of our knowledge, this is the first report of a specific and sensitive real-time LAMP assay for detection of B. mallei. 2.2 | Selection of B. mallei-specific target DNA sequence Burkholderia mallei-specific target DNA sequence was selected as described earlier (Pal, Kumar, Malik, & Rai, 2012;Pal, Singh, Tiwari, Jaiswal, & Rai, 2016). A subregion (89 bp…”

Current Trends in the Biosensors for Biological Warfare Agents Assay

“…The PCR is dependent on used primers, which are responsible for the specificity of the method. Various configurations of the method are available from which real time PCR is likely the most widely used in the current praxis [16,17]. There are also commercially available PCR devices suitable for field assay.…”