| [Objective] The aim of our research was to establish a rapid technique to detect Mycobacterium bovis by mycobacteriophage and studied the optimal test conditions. At the same time studied the value of this method in detecting of Mycobacterium bovis in simulative clinical sample and 203 clinical cow milk samples.[Methods] To set up a rapid technique for detecting Mycobacterium bovis by mycobacteriophage, the optimal test conditions were investigated. The stability of main agents were also studied in our research.The control strain of Mycobacterium bovis,21 clinical isolates of Mycobacterium bovis,20 non-Mtb strains of Mycobacteria,other bacteria and simulative clinical samples were detected by this assay. 203 cow milk samples were detected by PhaB Assay and compared the results with skin test,smear method and Lǒwenstein-Jensen culture.[Results] It was demonstrated the optimal working concentration of mycobacteriophage was 1×10~9PFU/ml and the optimal condition for detection was the infection at 37℃for 2 hours. By using this method 60-120CFU/ml of M.bovis could be detected. Meanwhile, the concentration of virucidal agent to inactivate the mycobacteriophage completely was 100 mmol/L at room temperature for 10min, and 4mg/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. 16 reference strains of non-Mtb strains of Mycobacteria and 6 other bacteria were detected negative by PhaB assay. The 4 strains of NTM(M.fortuitum, M.intrcellulare, M.aurum, M.phlei) can be positive reaction at concentration of high level(>10~5/ml),while it can be negative reaction at concentration of low level(<10~5/ml).The intra-and inter-batch variation coefficies were all below 15%, as demonstrated by repeated testing, indicating a good repeatability.The stability of main agents were studied in our experiment,four percent FAS could inactive 1.0×l0~9PFU/ml after it preservation 40 weeks.The title of replicated phage was higher when it preservation 28 weeks ago. The viable organism of M.smegmatis drop to 1.0×l0~9PFU/ml when it stored at 4℃after 40 weeks, which did not influence the plaques. The method was used to detect Mycobacterium bovis directive and indirect. It can get positive result directive.4 strains of Mycobacterium bovis was detected negative while add in milk. It can get good results by centrifugation. In all 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 this method compared to other three methods were all above 96%, the sensitivity was between 71%-100%.[Conclusions] The optimal test conditions were investigated and a technique for detecting Mycobacterium bovis by phage amplified biologically assay was established. The control strain of Mycobacterium bovis,21 clinical isolates of Mycobacterium bovis,20 non-Mtb strains of Mycobacteria,other bacteria and simulative clinical samples were detected by this assay and get satisfied results.The stability of main agents were studied, it can get good result stored at 4℃. Simulative clinical samples were detected by this assay and get satisfied results.The result of the PhaB assay compared smear method,skin test and Lowenstein-Jensen culture respectively.The correspondence rate and specificity of this method compared to other three methods were all above 96%, the sensitivity was between 71%-100%. The result shows that it can get high Coincidence and specificity of PhaB assay compared to routine techniques. This method had good value in detection and generalization, which had good use prospect. |
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