培养条件对克柔念珠菌MALDI-TOF MS鉴定结果的影响分析  

Impact of different culture conditions on the identification of Candida krusei clinical isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry system

作　　者：黄晶晶 范欣[1,2,3] 肖盟 徐志鹏[1] 张戈 徐英春[1,2] HUANG Jing-jing;FAN Xin;XIAO Meng;XU Zhi-peng;ZHANG Ge;XU Ying-chun(Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730,China;Beijing Key Laboratory for Mechanisms Research and Precision Diagnosis of Invasive Fungal Diseases, Beijing 100730,China;Beijing Chao-Yang hospital, Capital Medical University, Beijing 100020,China)

机构地区：[1]中国医学科学院北京协和医院检验科,北京100730 [2]侵袭性真菌病机制研究与精准诊断北京市重点实验室,北京100730 [3]首都医科大学附属北京朝阳医院感染和临床微生物科,北京100020

出　　处：《中国真菌学杂志》2020年第5期262-267,共6页Chinese Journal of Mycology

基　　金：北京协和医学院研究生创新基金(2019-1002-02);国家科技重大专项子课题“基于飞行质谱技术的真菌鉴定研究”(2018ZX10712001-008);北京市科技计划“飞行时间质谱的示范推广研究”(Z181100001618015)。

摘　　要：目的探究不同的培养时间、培养温度、培养基种类及培养气体环境对克柔念珠菌基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS)鉴定结果的影响。方法收集中国侵袭性真菌耐药监测网(CHIF-NET)2012-2013年中17所医院克柔念珠菌25株,每株菌平行接种在沙堡弱氯霉素琼脂平板(SDA-C)、血琼脂平板等6种培养基,28℃和35℃2种孵育温度,空气和5%CO22种气体环境中孵育,以真菌rDNA ITS测序为金标准,孵育24 h和48 h后,使用Vitek MALDI-TOF质谱仪(简称Vitek MS)和Bruker Autoflex Speed型MALDI-TOF质谱仪(简称Bruker MS)分别进行菌种鉴定,并对其鉴定正确率进行分析。结果克柔念珠菌孵育24 h和48 h时Vitek MS鉴定正确率均为100%,Bruker MS鉴定正确率分别是98.86%和87.43%。28℃SDA-C上生长的克柔念珠菌种水平鉴定准确率较35℃生长的菌株高。比较各种培养基,孵育48 h时SDA-C上生长的克柔念珠菌Bruker MS鉴定正确率(100%)最高。结论克柔念珠菌接种在SDA-C上,28℃空气培养24 h质谱鉴定效果最佳。常规细菌培养基上生长的克柔念珠菌,Bruker MS鉴定分值≥1.700即可认为菌种鉴定可靠。Objective To investigate the effects of different culture media,incubation time,culture temperature and gas environment on the identification of Candida krusei by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS).Methods Twenty-five strains of Candida krusei were collected from 17 hospitals of China Hospital Invasive Fungal Surveillance Net(CHIF-NET)program.Six culture media were tested,including Sabouraud dextrose agar supplemented with chloramphenicol(SDA-C),CHROMagar(CCP),trypticase soy agar supplemented with 5%sheep blood(BAP),chocolate agar supplemented with vancomycin(CAP-VA),China blue lactose rosolic acid agar(CBA/R)and MacConkey agar(MAC).All isolates were identified by sequencing of the rDNA internal transcribed spacer(ITS)region as“gold standard”.Isolates were incubated at 28℃and 35℃for 24 or 48 hours,with/without 5%CO2 gas,and then identification was carried out by two MALDI-TOF MS systems.Results All(175/175)isolates incubated for 24 hours and 48 hours were identified correctly by Vitek MS.In comparison,Bruker Biotyper correctly identified 98.86%(173/175)isolates incubated for 24 hours and 87.43%(153/175)isolates incubated for 48 hours.All strains inoculated on SDA-C medium were correctly identified by both MALDI-TOF MS systems,but the accuracy of the species level of identification of which grown on SDA-C at 28℃is higher than those grown at 35℃.With 48 hours’incubation,the correct identification rates by Bruker Biotyper were higher for strains grown on SDA than other media.Conclusion The correct identification rate of 24-hour incubated strains was higher than the rate of 48-hour incubated strains by Bruker MS.Compared with cultured at 35℃,the identification of Candida krusei strains grown at 28℃was more accurate.SDA-C was the most suitable media for MALDI-TOF MS identification in our study.The results were reliable for Bruker Biotyper MALDI-TOF MS system with score values of≥1.700.

关 键 词：克柔念珠菌 鉴定 培养条件 MALDI-TOF MS 

分 类 号：R379.4[医药卫生—病原生物学]