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

ABSTRACT: 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 non-targeted 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.

SUBMITTER: Chua KH

PROVIDER: S-EPMC7261116 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Chua Kek Heng KH Tan E Wei EW Chai Hwa Chia HC Puthucheary S D SD Lee Ping Chin PC Puah Suat Moi SM

PeerJ 20200527

<h4>Background</h4><i>Burkholderia pseudomallei</i> 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.<h4>Method</h4>In this study, we developed a rapid, sensitive and specific insulated isothermal Polymerase Chain Reaction (iiPCR) targeting <i>bimA</i> gene (<i>Burkholderia</i> Intracellular Motility A; BPSS1492) for the identification of <i>B. pseudomallei.</i> A pai ...[more]

PMID: 32518734

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Project description:BackgroundSeneca Valley virus (SVV) has emerged in multiple countries in recent years. SVV infection can cause vesicular lesions clinically indistinguishable from those caused by other vesicular disease viruses, such as foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and vesicular exanthema of swine virus (VESV). Sensitive and specific RT-PCR assays for the SVV detection is necessary for differential diagnosis. Real-time RT-PCR (rRT-PCR) has been used for the detection of many RNA viruses. The insulated isothermal PCR (iiPCR) on a portable POCKIT™ device is user friendly for on-site pathogen detection. In the present study, SVV rRT-PCR and RT-iiPCR were developed and validated.ResultsNeither the SVV rRT-PCR nor the RT-iiPCR cross-reacted with any of the vesicular disease viruses (20 FMDV, two SVDV, six VSV, and two VESV strains), classical swine fever virus (four strains), and 15 other common swine viruses. Analytical sensitivities of the SVV rRT-PCR and RT-iiPCR were determined using serial dilutions of in vitro transcribed RNA as well as viral RNA extracted from a historical SVV isolate and a contemporary SVV isolate. Diagnostic performances were further evaluated using 125 swine samples by two approaches. First, nucleic acids were extracted from the 125 samples using the MagMAX™ kit and then tested by both RT-PCR methods. One sample was negative by the rRT-PCR but positive by the RT-iiPCR, resulting in a 99.20% agreement (124/125; 95% CI: 96.59-100%, κ = 0.98). Second, the 125 samples were tested by the taco™ mini extraction/RT-iiPCR and by the MagMAX™ extraction/rRT-PCR system in parallel. Two samples were positive by the MagMAX™/rRT-PCR system but negative by the taco™ mini/RT-iiPCR system, resulting in a 98.40% agreement (123/125; 95% CI: 95.39-100%, κ = 0.97). The two samples with discrepant results had relatively high CT values.ConclusionsThe SVV rRT-PCR and RT-iiPCR developed in this study are very sensitive and specific and have comparable diagnostic performances for SVV RNA detection. The SVV rRT-PCR can be adopted for SVV detection in laboratories. The SVV RT-iiPCR in a simple field-deployable system could serve as a tool to help diagnose vesicular diseases in swine at points of need.

Project description:BackgroundDetection of Plasmodium spp. is sometimes inconvenient especially in rural areas that are distant from a laboratory. In this study a portable diagnostic test of Plasmodium spp. was developed using insulated isothermal polymerase chain reaction (iiPCR) as an alternative approach to improve this situation.MethodsA pair of universal primers and probe were designed to amplify and detect gene encoding 18S small sub-unit rRNA of Plasmodium spp using iiPCR method in a portable device, POCKIT™. The efficiency and detection limit of the assay were evaluated using quantitative real-time polymerase chain reaction (qPCR) approach before being subjected to testing in POCKIT™. Detection results of POCKIT™ were displayed as '+', '-' or '?' based on the fluorescence ratio after/before reaction. A total of 55 and 35 samples from malaria patients and healthy subjects, respectively, were screened to evaluate the feasibility of this newly designed iiPCR assay.ResultsThe iiPCR assay allowed the detection of various species of Plasmodium, including those infecting humans (Plasmodium falciparum, P. vivax, P. knowlesi, P. malariae, P. ovale), monkeys, birds, and rodents. Efficiency of the assay achieved 96.9 % while the lower detection limit was ≥100 copies of plasmodial DNA. Specificity of the assay was assured as it could not detect human, bacterial and other parasitic DNA. Among the 55 clinical samples tested, 47 (85.4 %) of them were detected as positive by POCKIT™. Four (7.3 %) samples with fluorescence ratio after/before reaction of <1.2 were reported as negative while another four (7.3 %) were ambiguously detected as they had fluorescence ratios between 1.2 and 1.3. The fluorescence ratio was not found to be associated with the copy number of plasmodial DNA. This approach can only be considered as a qualitative method.ConclusionsThe portable iiPCR system may serve as an alternative approach for preliminary screening of malaria in endemic rural areas. The system may also be useful for detecting animal malaria in the field. Although it is not as quantitative as qPCR method, it is comparatively fast and easy to handle. It is believed that the POCKIT-iiPCR assay is able to achieve 100 % sensitivity if increased amount of DNA from each sample is used. The iiPCR assay can also be upgraded in future to detect multiple Plasmodium spp. at the same time by designing the specific primers and probes.

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

Publications

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

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