title: Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae.
abstract: Contagious caprine pleuropneumonia (CCPP) is a highly contagious disease caused by Mycoplasma capricolum subsp. capripneumoniae that affects goats in Africa and Asia. Current available methods for the diagnosis of Mycoplasma infection, including cultivation, serological assays, and PCR, are time-consuming and require fully equipped stationary laboratories, which make 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 the detection of M. capricolum subsp. capripneumoniae. We developed the assay using a specific target sequence in M. capricolum subsp. capripneumoniae, 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. Detection limits corresponding to 5 × 10(3) and 5 × 10(4) cells/ml were obtained using genomic DNA and bacterial culture from M. capricolum subsp. capripneumoniae strain ILRI181, while no amplification was obtained from 71 related Mycoplasma isolates or from the Acholeplasma or the Pasteurella isolates, demonstrating a high degree of specificity. The assay produces a fluorescent signal within 15 to 20 min and worked well using pleural fluid obtained directly from CCPP-positive animals without prior DNA extraction. We demonstrate that the diagnosis of CCPP can be achieved, with a short sample preparation time and a simple read-out device that can be powered by a car battery, in <45 min in a simulated field setting.
authors.affiliation: International Livestock Research Institute, Nairobi, Kenya a.liljander@cgiar.org.
authors.forename: Anne
authors.initials: A
authors.lastname: Liljander
authors.affiliation: International Livestock Research Institute, Nairobi, Kenya.
authors.forename: Mingyan
authors.initials: M
authors.lastname: Yu
authors.affiliation: International Livestock Research Institute, Nairobi, Kenya.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: O'Brien
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Jena, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Heller
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Madison, Wisconsin, USA.
authors.forename: Julia F
authors.initials: JF
authors.lastname: Nepper
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, Madison, Wisconsin, USA.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Weibel
authors.affiliation: Vétérinaires Sans Frontières Suisse, Nairobi, Kenya.
authors.forename: Ilona
authors.initials: I
authors.lastname: Gluecks
authors.affiliation: Vétérinaires Sans Frontières Germany, Nairobi, Kenya.
authors.forename: Mario
authors.initials: M
authors.lastname: Younan
authors.affiliation: Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland.
authors.forename: Joachim
authors.initials: J
authors.lastname: Frey
authors.affiliation: Biochemistry Unit, University of Fribourg and Swiss Institute of Bioinformatics, Fribourg, Switzerland.
authors.forename: Laurent
authors.initials: L
authors.lastname: Falquet
authors.affiliation: International Livestock Research Institute, Nairobi, Kenya Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland.
authors.forename: Joerg
authors.initials: J
authors.lastname: Jores
mesh_terms: D000818:Animals; D006041:Goats; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D045623:Mycoplasma capricolum / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011002:Pleuropneumonia, Contagious / Q000175:diagnosis*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014730:Veterinary Medicine / Q000379:methods*