Development and validation of the isothermal recombinase polymerase amplification assays for rapid detection of Mycoplasma ovipneumoniae in sheep

Abstract

Background: Mycoplasmal pneumonia is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae is one of major etiological agent causing mycoplasmal pneumonia. Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technique, and RPA-based diagnostic assays have been described for the detection of different types of pathogens. Results: The RPA assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. ovipneumoniae targeting a conserved region of the 16S rRNA gene. Real-time RPA was performed in a portable florescence scanner at 39°C for 20 min. LFS RPA was performed in a portable metal bath incubator at 39°C for 15 min, and the amplicons were visualized with the naked eyes within 5 min on the lateral flow strip. Both assays were highly specific for M. ovipneumoniae, as there were no cross-reactions with other microorganisms tested, especially the pathogens involved in respiratory complex and other mycoplasmas frequently identified in ruminants. The limit of detection of LFS RPA assay was 1.0 × 10 1 copies per reaction using a recombinant plasmid containing target gene as template, which is 10 times lower than the limit of detection of the real-time RPA and real-time PCR assays. The RPA assays were further validated on 111 clinical sheep nasal swab and fresh lung samples, and M. ovipneumoniae DNA was detected in 29 samples in the real-time RPA, 31 samples in the LFS RPA and 32 samples in the real-time PCR assay. Compared to real-time PCR, the real-time RPA and LFS RPA showed diagnostic specificity of 100 and 98.73%, diagnostic sensitivity of 90.63 and 93.75%, and a kappa coefficient of 0.932 and 0.934, respectively. Conclusions: The developed real-time RPA and LFS RPA assays provide the attractive and promising tools for rapid, convenient and reliable detection of M. ovipneumoniae in sheep, especially in resource-limited settings. However, the effectiveness of the developed RPA assays in the detection of M. ovipneumoniae in goats needs to be further validated.

Development and Evaluation of the Rapid and Sensitive RPA Assays for Specific Detection of Salmonella spp. in Food Samples

“…RPAs have successfully been practiced in the discovery of pathogenic bacteria ( Hong et al, 2020 ), fungus ( Ahmed et al, 2014 ), and viruses ( Boyle et al, 2013 ). The reagents in RPA are available in lyophilized form for long-term storage and are conveniently transported even without cold-chain ( Wang et al, 2020 ). Moreover, under the prerequisite that the testing results were visible, the real-time RPA assay was accomplished on the user-friendly PON (point of need) detection platform (Genie III) with battery power.…”

“…RPA has the merit of amplification at a relatively low temperature (37–42°C) within 10–20 min ( Piepenburg et al, 2006 ; Geng et al, 2018 ). The use of RPA-based methods has been proved to be a success in detecting pathogenic bacteria and viruses in clinical and food samples ( Euler et al, 2012 ; Abd El Wahed et al, 2015 ; Wang et al, 2020 ). RPA-based methods have been designed to be a miniaturized diagnostic device that includes all the components for the RPA assay ( Asiello and Baeumner, 2011 ).…”

Nucleic Acid Amplification Techniques in Immunoassay: An Integrated Approach with Hybrid Performance

“…The reaction can be carried out at 60–65 °C, so the target DNA can be amplified with high efficiency within 1 h at a constant temperature . After that, various ITA technologies gradually emerged and showed a good application prospect in on-site rapid detection. − …”

Genomic regions associated withMycoplasma ovipneumoniaepresence in nasal secretions of domestic sheep

“…Previous exposure to M. ovipneumoniae can be assessed by the presence of antibodies [11], though this does not enable quantification of bacteria equivalents present. There are several molecular amplification assays used to detect this bacterium [25–27]. In order to eliminate false positives and ensure bacterium-specific quantification of M. ovipneumoniae , a species-specific quantitative PCR was developed for use with DNA from nasal secretions.…”