结核分枝杆菌噬菌体试剂盒的研制与初步评价

A study and evaluation of bacteriophage's utilization in rapid detection of Mycobacterium tuberculosis

目的:研制能快速检测标本中结核分枝杆菌的噬菌体试剂盒,并对之在临床上的应用做出初步评价。方法:采用不同方法对噬菌体的生产和保存工艺进行探讨,并且观察该方法中所用试剂的稳定性。建立噬菌体快速检测结核分枝杆菌的方法,分别用噬菌体法与传统培养法检测34份临床标本中的结核分枝杆菌,以培养法作为金标准观察噬菌体法的敏感度、特异度和符合率。结果:①在37℃环境中采用琼脂双层法进行噬菌体扩增生产,48h后采用孵育-过滤法收集,可以获得较为理想的产量且简单易行。②噬菌体以液体形式4℃保存或以噬菌体宿主菌的无细胞菌苗作为保护剂进行真空冻干后4℃保存,28周后效价下降不足10的一个数量级。③噬菌体法中的重要试剂:辅助细菌和杀病毒剂,4℃保存40周均具有良好的稳定性。④34份痰标本培养4周,其中2份污染,29份阳性,3份阴性;噬菌体法检测标本中结核分枝杆菌的敏感度、特异度和符合率分别为86.2%(25/29)、66.7%(2/3)、84.4%(27/32)。结论:噬菌体法在48h内可以快速检测标本中的结核分枝杆菌活菌,并且具有以试剂盒形式进行商业化生产的可行性,将有助于临床结核病的快速诊断。

Objective：To study and evaluate the utilization of bacteriophage in rapid detection of Mycobacterium tuberculosis in clinical samples.Methods：The titer of replicated phage with different methods in 24 or 48 hours were compared,including centrifuge-filter method and incubate-filter method with 0.2μm filter.When the phages were preserved at different conditions with various preservers,the titer was tested and compared in 28 weeks.Other agents of the method,including virucide and help bacteria（Mycobacterium smegmatis） were investigated at different time when they were preserved at 4℃.After the phage assay was established,34 sputum samples were tested by both phage assay and culture method in L-J medium.Based on the results of culture method,phage assay in detection of mycobacterium tuberculosis were evaluated by the sensitivity,specificity and accordance.Results：①The titer of replicated phage was high,when it had been collected after infected for 48 hours.The collection through incubation-filter method was simple and effect.② The phage stored at 4℃ with liquid form was stable,and the declined titer was not more than 101.when the preserver was phage＇s host bacteria and it was stored at 4℃,it was as stable as that in dehydration form.③The important agents,including virucide and help bacteria,had the same character in 40 weeks,when they were stored at 4℃.④ Compared with the results of culture method,the sensitivity,specificity and accordance of phage assay in rapid detection of mycobacterium tuberculosis were 86.2%,66.7%,and 84.4% respectively.Conclusion：The phage assay could detect viable Mycobacterium tuberculosis of sputum samples in 48 hours.it is feasible to produce testing reagent box from the phage,and can be expected to be used for clinical.