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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.

SUBMITTER: Liljander A

PROVIDER: S-EPMC4540935 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae.

Liljander Anne A Yu Mingyan M O'Brien Elizabeth E Heller Martin M Nepper Julia F JF Weibel Douglas B DB Gluecks Ilona I Younan Mario M Frey Joachim J Falquet Laurent L Jores Joerg J

Journal of clinical microbiology 20150617 9

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, ...[more]

PMID: 26085615

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Project description:BackgroundMycoplasmas are among the smallest prokaryotic microbes that can grow and proliferate on non-living media. They have reduced genomes, which may be associated with a concomitant reduction in their metabolic capacity. Mycoplasma capricolum subsp. capripneumoniae (Mccp) and Mycoplasma capricolum subsp. capricolum (Mcc), both belong to the Mycoplasma mycoides cluster, are significant important pathogenic Mycoplasma species in veterinary research field. They share high degree of genome homology but Mcc grows markedly faster and has higher growth titer than Mccp.MethodsThis study investigated the metabolites of these two pathogenic bacteria from the middle and late stages of the logarithmic growth phase through liquid chromatography-mass spectrometry-based metabolomics and targeted energy metabolomics. The multivariate analysis was conducted to identify significant differences between the two important Mycoplasma species.ResultsA total of 173 metabolites were identified. Of them, 33 and 34 metabolites involved in purine and pyrimidine, pyruvate metabolism, and amino acid synthesis were found to significantly differ in the middle and late stages, respectively. The abundance of fructose 1,6-bisphosphate, ADP, and pyruvate was higher in Mcc than in Mccp during the whole logarithmic period. Lactate was upregulated in slow-growing Mccp. The pH buffering agent N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] added to media effectively prevented pH reduction and increase bacterial viability and protein biomass. The multivariate analysis revealed that the two Mycoplasma species significantly differed in glucose metabolism, growth factor transport and metabolism, cholesterol utilization, and environmental regulation.ConclusionThe study data are beneficial for understanding the metabolomic characteristics of these two crucial Mycoplasma species and shedding more light on mycoplasma metabolism, and serve as a resource for the pathogenesis and development of related vaccines.

Dataset Information

Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae.

Publications

Field-Applicable Recombinase Polymerase Amplification Assay for Rapid Detection of Mycoplasma capricolum subsp. capripneumoniae.

Similar Datasets

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