牛支原体、多杀性巴氏杆菌、溶血性曼氏杆菌三重qPCR方法的建立与流行病学调查

Establishment of a triplex qPCR detection method for Mycoplasma bovis, Pasteurella multocida, Mannheimia haemolytica and an epidemiological investigation

摘要:

目的 建立牛支原体(Mycoplasma bovis)、多杀性巴氏杆菌(Pasteurella multocida, P.m)和溶血性曼氏杆菌(Mannheimia haemolytica, M.h)的三重qPCR检测方法，并开展规模化奶牛养殖场牛呼吸道疾病综合征(bovine respiratory disease complex, BRDC)的流行病学调查。方法 以实验室分离鉴定后纯化的菌株制备敏感性质控样品；选取国内主要流行株的保守基因，分别以牛支原体的uvrC基因、多杀性巴氏杆菌的Kmt1基因和溶血性曼氏杆菌的lktD基因作为靶标，建立多重探针和引物体系，优化反应体系参数，构建三重qPCR方法。验证该方法的敏感性、重复性和特异性，并与细菌分离鉴定方法进行符合率验证。利用该方法对1 252份临床疑似BRDC的样本进行病原学检测，分析病原的流行特征和时空分布特征。结果 三重qPCR方法的标准曲线R²值分别为0.998 6、0.994 6和0.998 6；牛支原体、多杀性巴氏杆菌和溶血性曼氏杆菌的最低检出量分别为2.50×10³、1.26×10³和7.50×10² CFU/mL。批内和批间变异系数均小于2%，仅3种菌的敏感性质控样品出现特异性扩增曲线。符合率试验结果显示，牛支原体、多杀性巴氏杆菌和溶血性曼氏杆菌与细菌分离鉴定方法的符合率分别为100.00%、98.40%和97.60%。流行病学调查显示，BRDC细菌性病原总阳性率为75.9% [95% confidence interval (CI), 73.4%-78.3%]，其中混合感染占比48.8%，冬季为高发期，北方地区阳性率显著高于南方(P<0.001)。结论 本研究建立的三重qPCR方法具有良好的特异性、稳定性和重复性，为我国BRDC主要病原的一步法检测提供了候选方案。本研究还分析了BRDC细菌病原的协同感染特征及地理-季节分布规律，为精准防控策略的制定提供了参考。

Abstract:

Objective To establish a triple qPCR detection method for Mycoplasma bovis, Pasteurella multocida (P.m) and Mannheimia haemolytica (M.h), and to conduct an epidemiological investigation of bovine respiratory disease complex (BRDC) in large-scale dairy farms with it.Methods Sensitive quality control samples were prepared using the purified strains isolated and identified in our laboratory. The conserved genes of the major prevalent strains in China were used as targets, including the uvrC of M. bovis, Kmt1 of P.m, and lktD of M.h, to establish a multiplex qPCR containing multiple pairs of primers and probes. By optimizing the parameters of the reaction system, a triple qPCR method was established and its sensitivity, repeatability and specificity were verified. The coincidence rate with the bacterial isolation and identification method was verified. A total of 1 252 clinical samples suspected of BRDC were tested using the established method, and the prevalence and spatiotemporal distribution characteristics of three pathogens were analyzed based on the test results.Results The R² values of the standard curves for the triple qPCR for detecting M. bovis, P.m, and M.h were 0.998 6, 0.994 6 and 0.998 6 respectively; the minimum detectable quantities were 2.50×10³, 1.26×10³ and 7.50×10² CFU/mL, respectively. The intra-batch and inter-batch coefficients of variation of the reaction system were both less than 2%, and only the specific amplification curves were observed for the three sensitive quality control samples established. The results of the test showed that the concordance rates of M. bovis, P.m, and M.h with bacterial isolation and identification methods were 100.00%, 98.40% and 97.60%, respectively. The epidemiological survey indicated that the total positive rate of bacterial pathogens in BRDC was 75.9% (95% confidence interval (CI), 73.4%-78.3%), among which the proportion of mixed infections was 48.8%. The incidence was higher in winter, and the positive rate in the northern region was significantly higher than that in the southern region (P<0.001).Conclusion The multiple qPCR method established in this study demonstrated excellent specificity, stability and repeatability, which provided a candidate solution for the detection of major pathogens of BRDC in China. The analysis of the co-infection characteristics and geographical-seasonal distribution patterns of BRDC bacterial pathogens offered meaningful references for the formulation of precise prevention and control strategies.