A field-applicable recombinase polymerase amplification assay for rapid detection of Mycoplasma capricolum subsp. capripneumoniae

Liljander, A.; Yu, M.; O'Brien, E.; Heller, Martin GND; Nepper, J.F.; Weibel, D.B.; Gluecks, I.; Younan, M.; Frey, J.; Falquet, L.; Jores, J.

Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused by Mycoplasma capricolum subsp. capripneumoniae (Mccp) that affects goats in Africa and Asia. Current available methods for diagnosis of Mycoplasma, including cultivation, serological assays and PCR, are time-consuming and require fully equipped, stationary laboratories, which makes them incompatible with testing in the resource poor settings that are most relevant to this disease. We report a rapid, specific and sensitive assay employing isothermal DNA amplification using recombinase polymerase amplification (RPA) for detection of Mccp. We developed an assay using a specific target sequence in Mccp as found in the genome sequence of the field strain ILRI181 and the type strain F38 and that was further evidenced in 10 field strains from different geographical regions. A detection limit corresponding to 5×103 and 5×104 cells/mL was obtained using genomic DNA and bacterial culture from Mccp strain ILRI181 while no amplification was obtained from 71 related Mycoplasma, Acholeplasma as well as Pastaurella isolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15-20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. With a short sample preparation time and a simple read-out device that can be powered by a car battery, we demonstrate that the diagnosis of CCPP can be achieved in < 45 min in a simulated field setting.

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Liljander, A. / Yu, M. / O'Brien, E. / et al: A field-applicable recombinase polymerase amplification assay for rapid detection of Mycoplasma capricolum subsp. capripneumoniae. 2015.

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