检测牛分枝杆菌的噬菌体生物扩增法的建立及初步应用  

Establishment and Preliminary Application of Detection of Mycobacterium bovis by Phage Amplified Biologically Assay

作　　者：钟志军[1] 彭广能[1] 唐明霞[2] 白永平[1] 石梅[1] 马晓平[1] 胡忠义[3] 王洁[3] 秦莲花[3] 杨华[3] 

机构地区：[1]四川农业大学动物医学院,雅安625014 [2]西南大学荣昌校区动物医学系,重庆400715 [3]上海市结核重点实验室,上海200433

出　　处：《畜牧兽医学报》2007年第11期1224-1229,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：上海市科学技术委员会科研计划资助项目(044119638)

摘　　要：建立噬菌体生物扩增法（PhaB）检测牛分枝杆菌,将建立的方法用于22株牛分枝杆菌临床分离株及20种常见非结核分枝杆菌（NTM）及5种非分枝杆菌,同时对203头疑患牛结核病奶牛的奶样进行检测,其结果与皮内变态试验、涂片法、罗氏培养进行比较。结果表明,噬菌体工作浓度为1×10^9 PFU/mL、37℃感染2 h为最佳检测条件;杀毒剂浓度为100 mmol/L,室温作用10 min即可完全杀灭受试噬菌体;加热灭活的细菌和指示细菌不被噬菌体感染;牛分枝杆菌、耻垢分枝杆菌和22株牛分枝杆菌临床分离株检测结果均为阳性,16种NTM和5种非分枝杆菌为阴性,4种NTM（偶然、胞内、金色、草分枝杆菌）在高浓度时（〉10^5CFU/mL）检测结果为阳性;该法可检测出60-120 CFU/mL牛分枝杆菌;批内、批间变异系数均小于15%,重复性良好;203头检测奶牛中,PhaB法、涂片法、皮内变态试验和罗氏培养结果分别有14头（6.9%）、17头（8.4%）、21头（10.3%）和12头（6.0%）为阳性,PhaB法与其他3种方法比较,阳性准确性、特异性都在96%以上,敏感性在71%-100%。该法检测牛分枝杆菌具有快速、简便、灵敏、特异性高等特点。A technique for detection of Mycobacterium boris by PhaB assay was developed, and the method was applied to detect 22 clinical isolates of Mycobacterium boris,20 non-Mtb strains of Mycobacteria and 5 other bacteria. 203 cow milk samples were detected by PhaB assay and the results were compared with skin test,smear method and Lowenstein-Jensen culture. It was demonstrated that the optimal working concentration of mycobacteriophage was 1 × 10^9 PFU/mL and the optimal condition for detection was infection at 37℃ for 2 hours. Meanwhile, the concentration of virucidal agent to inactivate the mycobacteriophage completely was 100 mmol/L at room temperature for 10 min,and 4 mg/mL concentration of the logarithmic phage of mycobacteriophon. Under these conditions, the optimal results could icated bacteria were not infected with mycobacteriophby using this method of detection for control strain of tes of M. boris,but the negative results were demonstrains of Mycobacteria and 5 other bacteria. The 4 age was selected as the working concentrati be obtained, and the heat-inactivated or ind age. The positive results could be obtained M. bovis,M, smegmatis and 22 clinical isola strated in 16 reference strains of non-Mtb strains of NTM（M. fortuitum, M. intrcellulare, M. aurum, M. phlei）can be positive reaction at concentration of high level （〉10^5 CFU/mL）. By using this method 60-120 CFU/mL of M. boris could be detected. The intra- and inter-batch variation coefficies were all below 15%, indicating a good repeatability. In all milk samples from 203 cows, 14,17,21 and 12 were detected positive by PhaB assay,smear method,skin test and Lowenstein-Jensen culture respectively. The correspondence rate and specificity of phaB method compared to other three methods were all above 96%, the sensitivity was between 71%-100%. Rapidity, simplicity, sensitivity and high specificity enable PhaB assay can be used for detecting Mycobacterium boris.

关 键 词：分枝杆菌噬菌体 牛分枝杆菌 检测方法 牛乳 

分 类 号：S852.618[农业科学—基础兽医学] R446.5[农业科学—兽医学]