Mycoplasma cynos 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 M. cynos that performs well under standard rtPCR conditions. Primers and probes were designed to target the M. cynos tuf gene. Reaction efficiencies for the M. cynos tuf gene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3-97.9% (r2 ≥ 0.9935); QuantStudio OpenArray platform, 119.1-122.5% (r2 = 0.9784). The assay performed very well over a range of template input, from 109 copies to the lower limit of quantification at 4 copies of the M. cynos 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 Mycoplasma species. To substantiate the high specificity of the M. cynos tuf gene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealed M. mucosicanis rather than M. cynos. The complete protocol of the newly developed M. cynos tuf assay is provided to facilitate assay harmonization.

中文翻译：

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新型支原体实时PCR检测的表征。

食蟹支原体是世界范围内犬传染性呼吸道疾病（CIRD）的一种新兴病原体。我们为食蟹猴开发了一种新的开源实时PCR（rtPCR）分析方法，该方法在标准rtPCR条件下表现良好。设计引物和探针以靶向食蟹猴tuf基因。在2个平台上进行食蟹猴Tuf基因测定的反应效率基于8个数量级的标准曲线的扩增：ABI 7500平台，94.3-97.9％（r2≥0.9935）；QuantStudio OpenArray平台，119.1-122.5％（r2 = 0.9784）。在模板输入的范围内，从109个拷贝到在ABI 7500平台上的4个拷贝的食蟹猴基因组的定量下限，该测定法均表现出色。通过与临床标本上的内部传统检测方法以及以前通过基因间隔区（ISR）测序表征的分离株进行比较，评估了诊断性能。通过测试其他12种支原体来确定排他性。为了证实食蟹猴tuf基因测定的高特异性，对从临床标本中获得的ISR PCR扩增子进行了序列确认。一个ISR扩增子序列揭示了粘膜分枝杆菌而不是食蟹猴。提供了新开发的猕猴tuf测定的完整方案，以促进测定的协调。对从临床标本中获得的ISR PCR扩增子进行序列确认。一个ISR扩增子序列揭示了粘膜分枝杆菌而不是猕猴。提供了新开发的猕猴tuf测定的完整方案，以促进测定的协调。对从临床标本中获得的ISR PCR扩增子进行序列确认。一个ISR扩增子序列揭示了粘膜分枝杆菌而不是猕猴。提供了新开发的猕猴tuf测定的完整方案，以促进测定的协调。