噬菌体生物扩增法快速检测结核分枝杆菌标准化研究

A study of standardization to the rapid detection of Mycobacterium tuberculosis based on phage amplified biologically assay

目的 对噬菌体生物扩增法快速检测和鉴别标本中分枝杆菌的方法学进行探讨。方法 对噬菌体生物扩增法的条件 :感染时间、杀灭胞外噬菌体的药物浓度和时间进行探讨和比较 ;观察不同分枝杆菌应用此法的检测灵敏度和特异性 ,以及对呼吸道常见细菌的特异度 ;观察实验中所用噬菌体、杀菌剂、辅助细菌在 2 8～ 4 0周内的稳定性。结果 (1)结核分枝杆菌与噬菌体感染 3～ 4h时感染效率较高 ,4 %硫酸亚铁胺 10 0 μl作用 5min能全部杀灭 1 1ml中 1× 10 9噬菌斑形成单位(PFU)的噬菌体 ;(2 ) 80～ 2 0 0集落形成单位 (CFU) /ml的结核分枝杆菌活菌能用此法检测出阳性结果 ,非结核分枝杆菌≤ 1× 10 3 CFU/ml时仅耻垢分枝杆菌出现阳性结果 ,特异性为 95 % ,而≥ 1× 10 4CFU/ml时结果依据不同的菌株而不同 ,呼吸道常见的非分枝杆菌细菌用此法的检测特异性为 10 0 % ;(3)液体 4℃保存实验中的主要成分 ,稳定性均较好。结论 应用噬菌体方法可以快速检测和鉴别结核分枝杆菌 ,所需试剂的稳定性较好 ,为广泛应用于临床检测提供了依据。

Objective To evaluate the phage amplified biologically (PhaB) assay in the rapid detection of Mycobacteria tuberculosis in samples Methods The conditions of the PhaB assay, including various infection times prior to addition of virucide and the effect of the inactivation agents which could inactive the extracellular phages, were investigated and compared The sensitivity, specificity and accuracy of PhaB assay were tested when it was used in rapid detection of Mycobacterium tuberculosis Some agents of the method, including Mycobacteriophage , virucide and help cells( Mycobacterium smegmatis ) were investigated at different times when they were preserved at 4℃ Results (1) The optimal infection time prior to addition of virucide was between 3 and 4 hours. Four percent FAS(ferrous ammonium sulphate) could inactive 1×10 9 PFU( plaque forming unit) in five minutes.(2) The samples were positive when 80 200 CFU of Mycobacterium tuberculosis were present However, the positive rate of non Tuberculosis Mycobateria (NTM) was varied All bacteria lived in the respiratory tract were negative (3)The important agents used in this test showed optical effect when they were preserved at 4℃ Conclusions The method based on mycobacteriophage amplified boiologically assay could rapidly detect Mycobacterium tuberculosis , and it was effective, accurate, and simple to perform It was appropriate for using in developing countries, compared with a variety of molecular techniques