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 × 103 and 5 × 104 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.

Development of a Novel Cocktail Enzyme-Linked Immunosorbent Assay and a Field-Applicable Lateral-Flow Rapid Test for Diagnosis of Contagious Bovine Pleuropneumonia

“…Thus, culture of the causative organism from specimens such as pleural fluid or infected lung tissues is still the gold standard for confirmation of CBPP. However, due to the fastidious nature of Mycoplasma, culturing is time-consuming, and the isolation is often hindered by sample contamination and reduced bacterial viability as a result of long times of transportation of specimens from the sampling site to the laboratory in Africa (9). Furthermore, culture findings still require molecular confirmation using nucleic acid-based hybridization or amplification techniques.…”

“…Thus, in the absence of better vaccines, improved diagnostic assays for recurrent testing combined with isolation and treatment of positive animals represent an option for CBPP control in Africa (8). Of particular importance as an integrated part of a CBPP outbreak response would be rapid and easy-to-use diagnostic tests that are applicable for field use, such as the recently described assay for diagnosis of contagious caprine pleuropneumonia (9). The different disease outcomes (acute, subacute, or chronic disease) associated with different or a total absence of clinical signs hamper the diagnosis of CBPP, and the use of more than one serological test is currently recommended (10)(11)(12).…”

Mycoplasmosis in Small Ruminants in India: A Review

“…This particular study is indicative of the fact that as far as investigation of contagious agalactia is concerned the use of p80 protein (conserved) aids to the specificity of diagnosis. It is important to note that some assays require further assessment of clinical specimens while others are very promising for large scale diagnosis and have field applicability Liljander et al 2015). Mycoplasma mycoides subsp.…”

A Novel Isothermal Assay of Borrelia burgdorferi by Recombinase Polymerase Amplification with Lateral Flow Detection

“…A series of RPA detection kits produced by the British company TwistDx can be used to detect both DNA and RNA targets. RPA is widely used in many fields [6,7,8,9] and many improved applications were shown in recent reports [10,11,12]. …”