Li Kaili, Zhang Yu, Luo Tingyu, Li Changwen, Yu Haibo, Wang Wei, Zhang He, Chen Hongyan, Xia Changyou, Gao Caixia
State Key Laboratory for Animal Disease Control and Prevention, Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine, National Poultry Laboratory Animal Resource Center, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China.
Microorganisms. 2024 Oct 5;12(10):2017. doi: 10.3390/microorganisms12102017.
Porcine respiratory disease is a significant economic problem for the global swine industry. (), (), and () are three important pathogenic bacteria of the swine respiratory tract. Notably, the three pathogens not only frequently manifest as mixed infections, but their striking clinical similarities also present difficulties for pig populations in terms of disease prevention and treatment. Thus, we developed a triplex real-time quantitative polymerase chain reaction (qPCR) assay based on a TaqMan probe for the detection of , serotype 2, and . Primers and probes were designed to target the conserved regions of the gene, the serotype 2 gene, and the gene. By optimizing the reaction system and conditions, a triplex qPCR method for simultaneous detection of , serotype 2, and was successfully established. The amplification efficiencies of the standard curves for all three pathogens were found to be highly similar, with values of 102.105% for , 105.297% for serotype 2, and 104.829% for , and all R values achieving 0.999. The specificity analysis results showed that the triplex qPCR method had a strong specificity. The sensitivity test results indicated that the limit of detection can reach 50 copies/μL for all three pathogens. Both intra- and inter-assay coefficients of variation for repeatability were below 1%. This triplex qPCR method was shown to have good specificity, sensitivity, and reproducibility. Finally, the triplex qPCR method established in this study was compared with the nested PCR as recommended by the Chinese national standard (GB/T34750-2017) for , the PCR as recommended by the Chinese national standard (GB/T 19915.9-2005) for serotype 2, and the PCR as recommended by the Chinese agricultural industry standard (NY/T 564-2016) for by detecting the same clinical samples. Both methods are reasonably consistent, while the triplex qPCR assay was more sensitive. In summary, triplex qPCR serves not only as a rapid and accurate detection and early prevention method for these pathogens but also constitutes a robust tool for microbial quality control in specific pathogen-free pigs.
猪呼吸道疾病是全球养猪业面临的一个重大经济问题。（）、（）和（）是猪呼吸道的三种重要病原菌。值得注意的是，这三种病原体不仅经常表现为混合感染，而且它们显著的临床相似性也给猪群的疾病预防和治疗带来了困难。因此，我们基于TaqMan探针开发了一种三重实时定量聚合酶链反应（qPCR）检测方法，用于检测（）、2型（）和（）。引物和探针被设计用于靶向（）基因、2型（）基因和（）基因的保守区域。通过优化反应体系和条件，成功建立了一种同时检测（）、2型（）和（）的三重qPCR方法。发现所有三种病原体标准曲线的扩增效率高度相似，（）的值为102.105%，2型（）为105.297%，（）为104.829%，所有R值均达到0.999。特异性分析结果表明，三重qPCR方法具有很强的特异性。灵敏度测试结果表明，所有三种病原体的检测限均可达到50拷贝/μL。重复性的批内和批间变异系数均低于1%。该三重qPCR方法具有良好的特异性、灵敏度和重现性。最后，将本研究建立的三重qPCR方法与中国国家标准（GB/T34750-2017）推荐的用于（）的巢式PCR、中国国家标准（GB/T 19915.9-2005）推荐的用于2型（）的PCR以及中国农业行业标准（NY/T 564-2016）推荐的用于（）的PCR进行比较，通过检测相同的临床样本。两种方法结果基本一致，而三重qPCR检测更灵敏。总之，三重qPCR不仅是这些病原体的快速准确检测和早期预防方法，也是无特定病原体猪微生物质量控制的有力工具。
