噬菌体快速检测结核分支杆菌方法的改良研究

The improvement of the rapid detection method for Mycobacterium tuberculosis with phage splitting assay

目的改良噬菌体裂解法检测结核分支杆菌试验的步骤,提高该试验的阳性率和敏感性。方法通过采用2%～5%不同浓度NaOH和0～24h不同时长的孵育时间等对结核患者痰液标本和结核菌株进行对比试验,分析不同试验条件对结果的影响,总结该方法的最佳试验条件。结果单因素分析结果显示,3%NaOH用于痰液标本的消化处理可以在杀灭痰液标本中的杂菌的同时更好地保持结核分支杆菌的活力。消化后的标本是否用磷酸盐缓冲液清洗及是否进行24h孵育对结果中的噬菌斑数量无显著影响。同时对临床标本的检测结果表明,未经过夜孵育标本污染率小于过夜孵育标本。结论采用3%NaOH消化处理痰液标本后不进行缓冲液清洗和24h孵育步骤,直接用噬菌体感染标本中的结核分支杆菌较原方法能更好地保存了结核分支杆菌活力,避免了杂菌过度的繁殖,提高了噬菌体裂解法快速检测结核分支杆菌试验(FASTTPlaque TBTM)检测结果的阳性率和敏感性,同时简化了试验步骤,缩短了试验时间。

Objective To improve the sensitivity and positive rate of rapid detection for Mycobacterium tuberculosis with phage splitting assay. Methods Tests with different concentration of NaOH solution （2%-5%） and different incubation time （0-24 h） were carried out to acquire the optimal experimental condition. Results The univariate analysis showed that 3 %NaOH preprocess could kill the mixed bacteria in sputum and keep the activity of Mycobacterium tuberculosis. Ablution with phosphate buffer and 24 hour＇ incubation of digested samples didn＇t affect the number of negative colonies. The detection result showed the contaminative rate of overnight incubated clinical samples was lower than that of non-overnight incubated ones. Conclusion The test of 3% NaOH preprocess with out buffer ablution and 24 hour＇ incubation may preferably keep the activity of Mycobacterium tuber culosis, avoid excessive multiplication of mixed bacteria, and improve the sensitivity and positive rate of rapid detection for Mycobacterium tuberculosis with phage splitting assay. Meanwhile, it still simplifies the test procedure and shortens the test duration.