College of Veterinary Medicine, Hebei Agricultural University, Baoding, 071001, China.
Center of Inspection and Quarantine Technology, Hebei Entry-Exit Inspection and Quarantine Bureau, Shijiazhuang, 050051, China.
Mol Cell Probes. 2019 Jun;45:14-18. doi: 10.1016/j.mcp.2019.03.007. Epub 2019 Mar 28.
Actinobacillus pleuropneumoniae is the etiological agent of swine contagious pleuropneumoniae, which is distributed globally and associated with severe economic losses in the pig rearing industry. In this study, a real-time recombinase polymerase amplification assay (real-time RPA) based on the apxIVA gene was developed to rapid detect A. pleuropneumoniae. Real-time RPA was performed successfully in Genie III at the constant temperature of 39 °C for 20 min. The developed assay was highly specific for A. pleuropneumoniae, and the sensitivity at 95% probability was 536 fg of A. pleuropneumoniae genomic DNA. The real-time RPA for A. pleuropneumoniae was further evaluated on the 112 clinical swine lung and tonsil samples, and 25 (22.3%), 27 (24.1%), and 12 (10.7%) samples were positive for A. pleuropneumoniae by the real-time RPA, real-time PCR and bacterial isolation, respectively. With a real-time PCR as the reference method, the real-time RPA showed a diagnostic specificity of 98.8%, a diagnostic sensitivity of 88.9%, a positive predicative value of 96.0%, a negative predictive value of 96.5%, and a kappa value of 0.900. The above data demonstrated the well potentiality and usefulness of the developed real-time RPA assay in the reliable detection of A. pleuropneumoniae, especially in resource limited settings.
副猪嗜血杆菌是猪传染性胸膜肺炎的病原体，广泛分布于全球各地，给养猪业造成严重的经济损失。本研究基于 apxIVA 基因，建立了一种实时重组酶聚合酶扩增检测方法（real-time RPA），用于快速检测副猪嗜血杆菌。实时 RPA 在 Genie III 恒温 39°C 下 20 分钟即可成功进行。该检测方法对副猪嗜血杆菌具有高度特异性，95%概率的检测灵敏度为 536 fg 副猪嗜血杆菌基因组 DNA。进一步用 112 份临床猪肺和扁桃体样本评估了实时 RPA 检测方法，实时 RPA、实时 PCR 和细菌分离分别检测到 25（22.3%）、27（24.1%）和 12（10.7%）份样本为副猪嗜血杆菌阳性。以实时 PCR 为参考方法，实时 RPA 检测副猪嗜血杆菌的诊断特异性为 98.8%，诊断敏感性为 88.9%，阳性预测值为 96.0%，阴性预测值为 96.5%，kappa 值为 0.900。上述数据表明，该实时 RPA 检测方法具有良好的潜力和实用性，可用于可靠检测副猪嗜血杆菌，特别是在资源有限的情况下。
