Rapid identification of melioidosis agent by an insulated isothermal PCR on a field-deployable device

PeerJ
Kek Heng ChuaSuat Moi Puah

Abstract

Burkholderia pseudomallei causes melioidosis, a serious illness that can be fatal if untreated or misdiagnosed. Culture from clinical specimens remains the gold standard but has low diagnostic sensitivity. In this study, we developed a rapid, sensitive and specific insulated isothermal Polymerase Chain Reaction (iiPCR) targeting bimA gene (Burkholderia Intracellular Motility A; BPSS1492) for the identification of B. pseudomallei. A pair of novel primers: BimA(F) and BimA(R) together with a probe were designed and 121 clinical B. pseudomallei strains obtained from numerous clinical sources and 10 ATCC non-targeted strains were tested with iiPCR and qPCR in parallel. All 121 B. pseudomallei isolates were positive for qPCR while 118 isolates were positive for iiPCR, demonstrating satisfactory agreement (97.71%; 95% CI [93.45-99.53%]; k = 0.87). Sensitivity of the bimA iiPCR/POCKIT assay was 97.52% with the lower detection limit of 14 ng/µL of B. pseudomallei DNA. The developed iiPCR assay did not cross-react with 10 types of non-targeted strains, indicating good specificity. This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen.

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Citations

Jan 11, 2022·Brazilian Journal of Microbiology : [publication of the Brazilian Society for Microbiology]·Apoorva SaxenaAjay Kumar Goel

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Datasets Mentioned

BETA
K96342

Methods Mentioned

BETA
PCR
nucleic acid amplification

Software Mentioned

BLASTN
Primer Express
LightCycler
GraphPad Prism
MedCalc

Related Concepts