Characterization of a novel <i>Mycoplasma cynos</i> real-time PCR assay

Published on 2019-11-23T13:10:16Z (GMT) by
<div><p><i>Mycoplasma cynos</i> is recognized as an emerging causative pathogen of canine infectious respiratory disease (CIRD) worldwide. We developed a new open-source real-time PCR (rtPCR) assay for <i>M. cynos</i> that performs well under standard rtPCR conditions. Primers and probes were designed to target the <i>M. cynos tuf</i> gene. Reaction efficiencies for the <i>M. cynos tuf</i> gene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3–97.9% (<i>r</i><sup>2</sup> ≥ 0.9935); QuantStudio OpenArray platform, 119.1–122.5% (<i>r</i><sup>2</sup> = 0.9784). The assay performed very well over a range of template input, from 10<sup>9</sup> copies to the lower limit of quantification at 4 copies of the <i>M. cynos</i> genome on the ABI 7500 platform. Diagnostic performance was estimated by comparison with an in-house legacy assay on clinical specimens as well as testing isolates that were characterized previously by intergenic spacer region (ISR) sequencing. Exclusivity was established by testing 12 other <i>Mycoplasma</i> species. To substantiate the high specificity of the <i>M. cynos tuf</i> gene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealed <i>M. mucosicanis</i> rather than <i>M. cynos</i>. The complete protocol of the newly developed <i>M. cynos tuf</i> assay is provided to facilitate assay harmonization.</p></div>

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Tallmadge, Rebecca L.; Anderson, Renee; Mitchell, Patrick K.; Forbes, Zachary C.; Werner, Brenda; Gioia, Gloria; et al. (2019): Characterization of a novel Mycoplasma cynos real-time PCR assay. SAGE Journals. Collection. https://doi.org/10.25384/SAGE.c.4751840.v1