丝状真菌质谱鉴定前处理方法及应用  

The research and application of pretreatment method for matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of filamentous fungi

作　　者：黄艳飞[1] 常峥[2] 白婕[1] 朱敏[1] 张明新[1] 王玫[1] 张桂[1] 李修远[1] 童贻刚[3] 王景林[3] 鲁辛辛[1] Huang Yanfei Chang Zheng Bai Jie Zhu Min Zhang Mingxin Wang Mei Zhang Gui Li Xiuyuan Tong Yigang Wang Jinglin Lu Xinxin(Department of Laboratory Medicine, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China)

机构地区：[1]首都医科大学附属北京同仁医院检验科,100730 [2]北京市海淀医院检验科 [3]解放军军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室

出　　处：《中华医学杂志》2017年第30期2379-2383,共5页National Medical Journal of China

基　　金：国家高技术研究发展计划（863计划）（2014AA021402）;国家科技重大专项（2014ZX10004005）

摘　　要：目的 建立并评价实验室自行研发的丝状真菌质谱鉴定前处理方法的可行性.方法复苏2014年1月至2016年12月北京同仁医院菌库中380株丝状真菌菌株,沙保弱葡萄糖琼脂平板（SDA）28 ℃培养至成熟状态,同时使用Bruker仪器推荐的垂直旋转法培养和本实验室研发的＂水平震荡法＂培养至足量菌落.3种方法培养菌株均采用＂改良磁珠提取法＂提取蛋白行质谱鉴定.结果380株真菌菌株,使用SDA培养时间需要3~10 d,鉴定到种和属的比率分别是47%和81%;使用Bruker仪器推荐方法培养时间需要5~7 d,鉴定到种和属的比率分别是76%和94%;＂水平震荡法＂培养时间只需1~2 d,鉴定到种和属的比率分别是96%和99%.＂水平震荡法＂和SDA培养法比较,差异有统计学意义（χ2=39.026,P〈0.01）,和仪器推荐的方法比较,差异有统计学意义（χ2=11.310,P〈0.01）.结论 本实验室研发的＂水平震荡法＂和＂改良磁珠提取法＂优于仪器推荐的方法和SDA培养鉴定方法,可以应用到临床.Objective To establish and evaluate the feasibility of a pretreatment method for matrix-assisted laser desorption ionization-time of flight mass spectrometry identification of filamentous fungi developed by the laboratory. Methods Three hundred and eighty strains of filamentous fungi from January 2014 to December 2016 were recovered and cultured on sabouraud dextrose agar （SDA） plate at 28 ℃ to mature state. Meanwhile, the fungi were cultured in liquid sabouraud medium with a vertical rotation method recommended by Bruker and a horizontal vibration method developed by the laboratory until adequate amount of colonies were observed. For the strains cultured with the three methods, protein was extracted with modified magnetic bead-based extraction method for mass spectrum identification.Results For 380 fungi strains, it took 3-10 d to culture with SDA culture method, and the ratio of identification of the species and genus was 47% and 81%, respectively; it took 5-7 d to culture with vertical rotation method, and the ratio of identification of the species and genus was 76% and 94%, respectively; it took 1-2 d to culture with horizontal vibration method, and the ratio of identification of the species and genus was 96% and 99%, respectively. For the comparison between horizontal vibration method and SDA culture method comparison, the difference was statistically significant （χ2=39.026,P〈0.01）; for the comparison between horizontal vibration method and vertical rotation method recommended by Bruker, the difference was statistically significant（χ2=11.310,P〈0.01）.Conclusion The horizontal vibration method and modified magnetic bead-based extraction method developed by the laboratory is superior to the method recommended by Bruker and SDA culture method in terms of the identification capacity for filamentous fungi, which can be applied in clinic.

关 键 词：真菌 水平震荡法 改良磁珠提取法 

分 类 号：R446.5[医药卫生—诊断学]