噬菌体Chy2生物扩增法的建立及其检测效果的评价.docVIP

噬菌体Chy2生物扩增法的建立及其检测效果的评价 敖知，郭述良 (400016 重庆，重庆医科大学附属第一医院呼吸与危重症医学科) ?摘要? 目的 对分离鉴定的新分枝杆菌噬菌体Chy2，运用于噬菌体生物扩增法的方法学进行探讨，评价其检测临床痰标本结核分枝杆菌的应用价值。 方法 噬菌斑测试法观察噬菌体杀灭剂灭活Chy2的工作浓度和作用时间；噬菌斑计数法判断Chy2的最佳感染时间和感染滴度；分别用噬菌体Chy2和D29生物扩增法检测58份临床痰标本，以改良罗氏培养法作诊断标准，对比评价Chy2和D29的检测准确性。 结果 6％硫酸亚铁铵溶液作用5 min可彻底灭活游离Chy2；Chy2滴度为2.2×1011CFU/ml时，与结核分枝杆菌孵育4 h的感染效率最高；噬菌体Chy2生物扩增法对痰标本的检测灵敏度、特异性、Youden指数、阳性预测值和阴性预测值分别为94.7％、85.0％、0.797、0.923、0.895，D29的检测结果相应为76.3%、95.0%、0.713、0.967、0.678，Chy2扩增法的灵敏度、阴性预测值优于D29，两者特异性、Youden指数、阳性预测值无统计学差异。结论 成功建立了噬菌体Chy2生物扩增法，Chy2较D29检测特异性相当，灵敏度有所提高，可对结核病的早期发现、早期诊断提供参考依据，具有一定临床应用价值。 ?关键词? 分枝杆菌噬菌体Chy2；结核分枝杆菌；灵敏度；特异性 ?中图法分类号? R446.5,R373.9,R521 Establishment of Phage Amplified Biologically Assay Using Phage Chy2 and Evaluation of its Capacity of Detecting Mycobacterium Tuberculosis in Sputum Samples Ao Zhi, Guo Shuliang(Department of Respiratory and Critical Care Medicine, the First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, China) ?Abstract? Objective to explore the methodology of the phage amplified biologically assay (PhaB assay) using a newly isolated and identified mycobacteriophage which is named Chy2 and evaluate its clinical application value for detecting mycobacterium tuberculosis (MTB) in sputum samples. Methods The optimal concentration and time of phage-inactivation agents were observed by spot tests. The time and titer of phage to infect MTB most effectively were confirmed by counting plaques. 58 clinical sputum samples were collected from patients suspected of having TB with no previous history of TB treatment and were detected by PhaB assay using phage Chy2 and D29，respectively, with L?wenstein- Jensen (L-J) culture method as the diagnostic standard. Results 6％FAS mixed with Chy2 for 5 minutes was able to completely inactivate the extracellular phages. Chy2 of 2.2×1011CFU/ml incubating with MTB for 4 hours infected MTB most effectively. Compared to L-J culture method, PhaB assay using Chy2 to detect MTB in sputum samp