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

PeerJ

article

Kek Heng Chua¹  E. Wei Tan¹  Hwa Chia Chai¹  SD Puthucheary²  Ping Chin Lee³  Suat Moi Puah¹ 

[1] Department of Biomedical Science, Faculty of Medicine, University of Malaya;Faculty of Medicine, University of Malaya, University of Malaya;Faculty of Science and Natural Resources, Universiti Malaysia Sabah

关键词: bimA;    Burkholderia pseudomallei;    Insulated isothermal PCR;    On-site detection;    Real-time PCR;   

DOI  :  10.7717/peerj.9238

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

BackgroundBurkholderia 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.MethodIn 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.ResultsAll 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.ConclusionThis bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen.

【 授权许可】

CC BY