关键词: 分枝杆菌噬菌体, 牛分枝杆菌, 检测方法, 牛乳

Abstract: A technique for detection of Mycobacterium bovis by PhaB assay was developed, and the method was applied to detect 22 clinical isolates of Mycobacterium bovis,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 Lǒwenstein-Jensen culture. It was demonstrated that the optimal working concentration of mycobacteriophage was 1×10⁹PFU/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 mycobacteriophage was selected as the working concentration. Under these conditions, the optimal results could be obtained, and the heatinactivated or indicated bacteria were not infected with mycobacteriophage. The positive results could be obtained by using this method of detection for control strain of M.bovis，M.smegmatis and 22 clinical isolates of M. bovis,but the negative results were demonstrated in 16 reference strains of nonMtb strains of Mycobacteria and 5 other bacteria. The 4 strains of NTM（M.fortuitum, M.intrcellulare, M.aurum, M.phlei）can be positive reaction at concentration of high level (>10⁵ CFU/mL). By using this method 60-120 CFU/mL of M.bovis 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 Lǒwenstein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 bovis.

Key words: Mycobacteriophage, Mycobacterium bovis, detect method, milk