Center of Inspection and Quarantine Technology, Hebei Entry-Exit Inspection and Quarantine Bureau, Shijiazhuang 050051, China; Hebei Academy of Science and Technology for Inspection and Quarantine, Shijiazhuang 050051, China.
College of Veterinary Medicine, Agricultural University of Hebei, Baoding 071001, China.
J Microbiol Methods. 2019 Apr;159:56-61. doi: 10.1016/j.mimet.2019.02.015. Epub 2019 Feb 23.
Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, which is associated with high economic losses in swine production worldwide. In this study, recombinase polymerase amplification assays using real-time fluorescence detection (real-time RPA) and lateral flow strip detection (LFS RPA) were developed to detect M. hyopneumoniae based on the conserved region of the mhp165 gene. Real-time RPA was performed in Genie III at 39 °C for 20 min, while the LFS RPA was performed in an incubator block at 39 °C for 15 min, and the products were visible on the LFS inspected by the naked eyes within 2 min. Both assays were specific for M. hyopneumoniae, as there were no cross-reactions with other pathogens tested. The limit of detection of both RPA assay was 5.0 × 10 fg of M. hyopneumoniae DNA, which was the same as that of a real-time PCR assay. Of the 146 clinical samples, M. hyopneumoniae DNA was identified in 41, 42, and 47 samples by the real-time RPA, LFS RPA and real-time PCR, respectively. Compared to real-time PCR, the real-time RPA and LFS RPA assays showed diagnostic specificity of 100%, a diagnostic sensitivity of 87.23% and 89.36%, and a kappa value of 0.903 and 0.909, respectively. These results have demonstrated that the developed RPA assays are suitable for rapid and reliable detection of M. hyopneumoniae in diagnostic laboratory and at point-of-need facility.
猪肺炎支原体是猪地方性肺炎的病原体，它与全球范围内的养猪业的高经济损失有关。在本研究中，基于 mhp165 基因保守区，开发了使用实时荧光检测的重组酶聚合扩增检测（实时 RPA）和侧向流动条带检测（LFS RPA）来检测猪肺炎支原体。实时 RPA 在 Genie III 中于 39°C 下进行 20 分钟，而 LFS RPA 在孵育块中于 39°C 下进行 15 分钟，产物在 2 分钟内通过肉眼在 LFS 上可见。两种检测均特异性针对猪肺炎支原体，因为与测试的其他病原体没有交叉反应。两种 RPA 检测的检测限均为 5.0×10 fg 猪肺炎支原体 DNA，与实时 PCR 检测相同。在 146 个临床样本中，实时 RPA、LFS RPA 和实时 PCR 分别在 41、42 和 47 个样本中鉴定出猪肺炎支原体 DNA。与实时 PCR 相比，实时 RPA 和 LFS RPA 检测的诊断特异性均为 100%，诊断敏感性分别为 87.23%和 89.36%，kappa 值分别为 0.903 和 0.909。这些结果表明，所开发的 RPA 检测适用于诊断实验室和现场即时检测猪肺炎支原体的快速可靠检测。
