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

Background. 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. Method. In this study, we developed a rapid, sensitive and specific insulated isot...

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Main Authors: Kek Heng Chua, E. Wei Tan, Hwa Chia Chai, SD Puthucheary, Lee, Ping Chin, Suat Moi Puah
Format: Article
Language:English
English
Published: 2020
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Online Access:https://eprints.ums.edu.my/id/eprint/26629/1/Rapid%20identification%20of%20melioidosis%20agent%20by%20an%20insulated%20isothermal%20PCR%20on%20a%20field%E2%80%93deployable%20device.pdf
https://eprints.ums.edu.my/id/eprint/26629/2/Rapid%20identification%20of%20melioidosis%20agent%20by%20an%20insulated%20isothermal%20PCR%20on%20a%20field%E2%80%93deployable%20device2.pdf
https://eprints.ums.edu.my/id/eprint/26629/
http://dx.doi.org/10.7717/peerj.9238
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Institution: Universiti Malaysia Sabah
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spelling my.ums.eprints.266292021-01-14T08:34:21Z https://eprints.ums.edu.my/id/eprint/26629/ Rapid identification of melioidosis agent by an insulated isothermal PCR on a field–deployable device Kek Heng Chua E. Wei Tan Hwa Chia Chai SD Puthucheary Lee, Ping Chin Suat Moi Puah Q Science (General) QR Microbiology Background. 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. Method. 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 nontargeted strains were tested with iiPCR and qPCR in parallel. Results. 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. Conclusion. This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen. 2020 Article PeerReviewed text en https://eprints.ums.edu.my/id/eprint/26629/1/Rapid%20identification%20of%20melioidosis%20agent%20by%20an%20insulated%20isothermal%20PCR%20on%20a%20field%E2%80%93deployable%20device.pdf text en https://eprints.ums.edu.my/id/eprint/26629/2/Rapid%20identification%20of%20melioidosis%20agent%20by%20an%20insulated%20isothermal%20PCR%20on%20a%20field%E2%80%93deployable%20device2.pdf Kek Heng Chua and E. Wei Tan and Hwa Chia Chai and SD Puthucheary and Lee, Ping Chin and Suat Moi Puah (2020) Rapid identification of melioidosis agent by an insulated isothermal PCR on a field–deployable device. PeerJ, 8. pp. 1-16. http://dx.doi.org/10.7717/peerj.9238
institution Universiti Malaysia Sabah
building UMS Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaysia Sabah
content_source UMS Institutional Repository
url_provider http://eprints.ums.edu.my/
language English
English
topic Q Science (General)
QR Microbiology
spellingShingle Q Science (General)
QR Microbiology
Kek Heng Chua
E. Wei Tan
Hwa Chia Chai
SD Puthucheary
Lee, Ping Chin
Suat Moi Puah
Rapid identification of melioidosis agent by an insulated isothermal PCR on a field–deployable device
description Background. 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. Method. 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 nontargeted strains were tested with iiPCR and qPCR in parallel. Results. 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. Conclusion. This bimA iiPCR/POCKIT assay will undoubtedly complement other methodologies used in the clinical laboratory for the rapid identification of this pathogen.
format Article
author Kek Heng Chua
E. Wei Tan
Hwa Chia Chai
SD Puthucheary
Lee, Ping Chin
Suat Moi Puah
author_facet Kek Heng Chua
E. Wei Tan
Hwa Chia Chai
SD Puthucheary
Lee, Ping Chin
Suat Moi Puah
author_sort Kek Heng Chua
title Rapid identification of melioidosis agent by an insulated isothermal PCR on a field–deployable device
title_short Rapid identification of melioidosis agent by an insulated isothermal PCR on a field–deployable device
title_full Rapid identification of melioidosis agent by an insulated isothermal PCR on a field–deployable device
title_fullStr Rapid identification of melioidosis agent by an insulated isothermal PCR on a field–deployable device
title_full_unstemmed Rapid identification of melioidosis agent by an insulated isothermal PCR on a field–deployable device
title_sort rapid identification of melioidosis agent by an insulated isothermal pcr on a field–deployable device
publishDate 2020
url https://eprints.ums.edu.my/id/eprint/26629/1/Rapid%20identification%20of%20melioidosis%20agent%20by%20an%20insulated%20isothermal%20PCR%20on%20a%20field%E2%80%93deployable%20device.pdf
https://eprints.ums.edu.my/id/eprint/26629/2/Rapid%20identification%20of%20melioidosis%20agent%20by%20an%20insulated%20isothermal%20PCR%20on%20a%20field%E2%80%93deployable%20device2.pdf
https://eprints.ums.edu.my/id/eprint/26629/
http://dx.doi.org/10.7717/peerj.9238
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