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Rapid and sensitive detection of Mycobacterium ulcerans by use of a loop-mediated isothermal amplification test.

ABSTRACT: This work reports the design and evaluation of a rapid loop-mediated isothermal amplification test for detecting Mycobacterium ulcerans DNA based on the multicopy insertion sequence IS2404. The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome). The test has potential for the diagnosis of Buruli ulcer under field conditions.

SUBMITTER: Njiru ZK

PROVIDER: S-EPMC3347114 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Rapid and sensitive detection of Mycobacterium ulcerans by use of a loop-mediated isothermal amplification test.

Njiru Zablon K ZK Yeboah-Manu Dorothy D Stinear Timothy P TP Fyfe Janet A M JA

Journal of clinical microbiology 20120222 5

This work reports the design and evaluation of a rapid loop-mediated isothermal amplification test for detecting Mycobacterium ulcerans DNA based on the multicopy insertion sequence IS2404. The test is robust and specific with a detection limit equivalent to 20 copies of the target sequence (0.01 to 0.1 genome). The test has potential for the diagnosis of Buruli ulcer under field conditions. ...[more]

PMID: 22357495

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Rapid detection of Mycobacterium ulcerans with isothermal recombinase polymerase amplification assay.

Project description:BACKGROUND:Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease. METHODOLOGY AND PRINCIPAL FINDINGS:A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42°C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84-100), whiles the sensitivity was 88% (95% CI, 77-95). CONCLUSION:The Mu-RPA assay represents an alternative to PCR, especially in areas with limited infrastructure.

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| S-EPMC2446398 | biostudies-literature

Sensitive and rapid detection of Giardia lamblia infection in pet dogs using loop-mediated isothermal amplification.

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| S-EPMC3662070 | biostudies-literature

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