mRNA作为结核分支杆菌活菌检测标志的可行性研究

目的 探讨mRNA作为结核分支杆菌活菌检测标志的可行性。方法 采用定量聚合酶链反应(PCR)方法测定结核分支杆菌H37Rv在利福平、异烟肼、乙胺丁醇、链霉素、氧氟沙星处理后24、48、72h 85B mRNA表达水平的变化，并与活菌计数方法对比。结果 在异烟肼、利福平和链霉素处理24h时，结核分支杆菌H37Rv活菌数下降明显；在异烟肼、链霉素、乙胺丁醇及氧氟沙星各药各浓度处理72h时，活菌数相似，但利福平处理后活菌数较它们低。利福平处理24h后85B mRNA下降到无药对照的0．02％，其他药物处理分别下降到无药对照的1％～10％；各药处理72h后85B mRNA均下降到1％以下。结论 mRNA表达水平可以在短时间内反映出用药与无药的区别，并与菌落形成单位(cfu)几乎呈平行关系。mRNA是检测和判定结核分支杆菌“死”“活”的分子标志物。

Quantitative analysis of mRNA as a marker for viability of Mycobacterium tuberculosis

OBJECTIVE: To study whether quantitative analysis of M. tuberculosis mRNA could be used to assess bacterial viability. METHODS: The levels of M. tuberculosis mRNA were compared 24, 48, and 72 hours after M. tuberculosis H(37)R(V) was treated with no drug, 1.0 micro g/ml and 10.0 micro g/ml rifampicin, 0.2 micro g/ml and 1.0 micro g/ml isoniazid, 2 micro g/ml and 4 micro g/ml ethambutol, 2 micro g/ml and 4 micro g/ml streptomycin, 1.25 micro g/ml and 5.00 micro g/ml ofloxacin. The levels of mRNA were determined by quantitative PCR. RESULTS: After exposure to isoniazid, rifampicin, and streptomycin for 24 h, the level of M. tuberculosis H(37)R(V) was reduced markedly. Similarly, after exposure to isoniazid, streptomycin, ethambutol, and ofloxacin for 72 h, the level of M. tuberculosis H(37)R(V) was identical, but higher than that after exposure to rifampicin. Exposure of M. tuberculosis H(37)R(V) to rifampicin for 24 h reduced the levels of 85B mRNA to 0.02% of the control; exposure to other drugs for 24 h reduced the levels to 1% approximately 10% of the control, and for 72 h to less than 1% of the control. CONCLUSIONS: The levels of mRNA can show the difference between M. tuberculosis H(37)R(V) exposed and unexposed to drugs, and are consistent with colony forming unit. mRNA could be used as a marker for assessing the viability of M. tuberculosis.