Recombinase polymerase amplification assay combined with a dipstick-readout for rapid detection of Mycoplasma ovipneumoniae infections

PLoS One. 2021 Feb 4;16(2):e0246573. doi: 10.1371/journal.pone.0246573. eCollection 2021.

Abstract

Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Mycoplasma ovipneumoniae / genetics
  • Mycoplasma ovipneumoniae / isolation & purification
  • Mycoplasma ovipneumoniae / pathogenicity*
  • Nucleic Acid Amplification Techniques
  • Plasmids / genetics
  • Pneumonia, Mycoplasma / genetics
  • Pneumonia, Mycoplasma / microbiology*
  • Real-Time Polymerase Chain Reaction
  • Recombinases / metabolism*

Substances

  • Recombinases

Grants and funding

This work was supported by an AgResearch Curiosity Fund awarded to SKG, funded through the Strategic Science Investment Fund supported by the Ministry of Business, Innovation and Employment (Wellington, New Zealand). The funder provided support in the form of salaries for authors SKG and QD as well as research reagents and consumables, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section'.