动物医学进展

为了建立同时检测奶牛临床型乳房炎中肺炎克雷伯菌(Kp)、产色葡萄球菌(Sc)和牛支原体(Mb),基于Kp ZKIR基因、Sc sodA基因和Mb oppD/F基因设计特异性引物，建立三重实时定量荧光PCR方法(real-time PCR)。试验采用在单一real-time PCR检测方法的基础上对三重real-time PCR检测方法进行优化，并确定退火条件为60℃,肺炎克雷伯菌、产色葡萄球菌以及牛支原体上、下游引物浓度为20μmol/L、荧光探针浓度为10μmol/L。结果表明，该方法对标准品pUC57-ZKIR-Kp、pUC57-sodA-Sc、pUC57-oppD/F-Mb最低检测限分别为1.55×10² copies/μL、1.44×10² copies/μL、1.34×10² copies/μL,灵敏度高；仅对Kp、Sc、Mb这3种病原产生荧光曲线，对其他病原无交叉反应，特异性强；其中组内、组间变异系数均小于2%,重复性良好。利用建立的多重real-time PCR对233份临床样品进行检测，Kp、Sc、Mb检出率分别为73.09%、21.97%、6.72%,与单一real-time PCR方法检测结果一致。说明建立的多重real-time PCR在实际应用中具有灵敏度高、特异性强、重复性良好、检测速度快等优点，可为奶牛临床型乳房炎病原的快速检测、临床诊断和流行病学调查提供有效检测手段。

In order to establish a method for simultaneous detection of Klebsiella pneumoniae(Kp),Staphylococcus chromogenes(Sc),and Mycoplasma bovis(Mb) in clinical mastitis of dairy cows, specific primers were designed based on the Kp ZKIR gene, Sc sodA gene, and Mb oppD/F gene, and a triplex real-time PCR method was established.The experiment optimized the triplex real-time PCR detection method based on single real-time PCR detection method and determined the annealing temperature to be 60 ℃ and the concentrations of the upstream and downstream primers for Kp, Sc, and Mb to be 20 μmol/L and the concentration of the fluorescent probe to be 10 μmol/L.The results showed that the method had the lowest detection limits of 1.55×10² copies/μL for pUC57-ZKIR-Kp, 1.44×10² copies/μL for pUC57-sodA-Sc, and 1.34×10² copies/μL for pUC57-oppD/F-Mb, with high sensitivity; it produced only fluorescence curves for Kp, Sc, and Mb and had no cross-reaction with other pathogens, with strong specificity; and the intra-group and inter-group coefficients of variation were less than 2%,indicating good repeatability.Using the multiplex real-time PCR method established in this study to detect 233 clinical samples, the detection rates of Kp, Sc, and Mb were 73.09%,21.97%,and 6.72%,respectively, which were consistent with the detection results of the single real-time PCR method.This indicates that the multiplex real-time PCR method established in this study has the advantages of high sensitivity, strong specificity, good repeatability, and fast detection in actual applications, and provides an effective detection method for the rapid detection, clinical diagnosis, and epidemiological investigation of clinical mastitis in dairy cows.