用重组噬菌体检测结核分枝杆菌耐药性

Specific mycobacteriophage for detecting mycobacterium tuberculosis and drug susceptibility

目的 建立一种特异性强 ,灵敏度高的结核分枝杆菌 (结核菌 )的检测及药敏试验方法。方法 采用生物发光技术检测可表达荧光素酶的分枝杆菌噬菌体phage 40 ,对不同细菌的发光反应和药敏试验进行测定 ,探讨噬菌体生物发光检测方法的灵敏度和特异性。结果 phage 40对各种分枝杆菌均有特异性发光 ,对非分枝杆菌发光值很低 ,两者差异有显著性意义 ;不同的分枝杆菌发光值有差异 :卡介苗的发光值最高 ,在 4 2× 10 6 ml时最大发光值为 2 84 7mV ,结核菌的发光值最低 ,H37Ra在 6 4× 10 6 ml时最大发光值为 72 4mV ,H37Rv在 7 6× 10 6 ml时的最大发光值是 74 3mV ;在含抗结核药物的培养基中 ,耐药结核菌的发光强度比非耐药结核菌强 ,其强度差异有显著性意义。phage40的药敏试验结果与常规罗氏培养基法符合率一致 ,可在 72h内得到结果。结论 生物发光技术可快速、敏感地检测结核菌 ,通过发光分析 ,建立了一种简便。

Objective To find a fast, sensitive and correct way to detect mycobacterium and drug sensitivity of mycobacterium by recombinant mycobacteriophages and bioluminescent method.Method The luciferase activity in different bacteria and in different mycobacterium strains was detected by recombinant mycobacteriophage (phage 40), and the drug susceptibilities of different mycobacterium were assessed. Finally, the sensitive and corrective rate between luciferase assay and L J medium culture were compared.Result phage 40 showed specifically a high light production in mycobacterium and low light production in E.coli. Among different mycobacterium, BCG showed the highest light production. At the concentration of 4.2×10 6/ml, the highest light production was 284.7 mV and mycobacterium tuberculosis, the lowest light production. In H37Ra, the concentration of 6.4×10 6/ml showed the highest light production of 72.4 mV, and in H37Rv, the concentration of 7.6×10 6/ml has the highest light production of 74.3 mV. The light production of durg resistant mycobacterium strains was much higher than that of drug sensitive strains in culture with antituberculosis durgs, including rifampicin, isoniazid and streptomycin. The time for drug resistant test was 72 h, which is much less than that of the normal L J method.Conclusion Luciferase reporter phage can detect mycobacterium specifically and can be used in drug resistant test. This assay is fast, sensitive and corrective in detecting mycobacterium stains and their drug resistance.