丝状真菌MALDI-TOF MS鉴定中三种样品前处理方法比较  

Comparison of three sample pretreatment methods in MALDI-TOF MS identification of filamentous fungi

作　　者：邓穗燕[1,2] 郭旭光 林丽英[1,2] 唐晓华 夏勇[1,2] DENG Sui-yan;GUO Xu-guang;LIN Li-ying;TANG Xiao-hua;XIA Yong(Department of clinical laboratory,Third Affiliated Hospital of Guangzhou Medical University,Guangdong,Guangzhou 510150,China;不详)

机构地区：[1]广州医科大学附属第三医院检验科,广东广州510150 [2]广州医科大学金域检验学院,广东广州510180

出　　处：《现代预防医学》2020年第22期4144-4147,4159,共5页Modern Preventive Medicine

基　　金：国家自然科学基金青年项目(81700004);广州市卫生健康科技一般引导项目(20201A011091)。

摘　　要：目的探讨适用于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)鉴定丝状真菌的样本预处理方法。方法分别使用甲酸萃取法、液氮研磨法、液氮研磨和甲酸萃取相结合三种方法对35株丝状真菌进行破壁,用牛鲍计数板进行破壁率计算,采用单因素方差分析(F检验)比较三种方法的破壁率;MALDI-TOF MS分别测定三种方法预处理的样本,采用卡方检验(χ^2检验)分析比较三种方法的鉴定效果。结果在普通光学显微镜下,经液氮研磨的丝状真菌可见大量被碾碎的孢子和菌丝渣子,背景浑浊不清,偶见完整孢子;液氮研磨法和(液氮研磨+甲酸萃取法)的破壁率分别为(0.86±0.06)和(0.84±0.06),均高于甲酸萃取法(均P<0.001)。以(形态学+分子生物学鉴定)为金标准,经质谱鉴定,将35株丝状真菌鉴定到种和属的鉴定率:液氮研磨法为71%和14%,甲酸萃取法为54%和23%,(液氮研磨+甲酸萃)取法为86%和8%;(液氮研磨+甲酸萃取法种的鉴定效果优于甲酸萃取法,差异有统计学意义(χ^2=8.231,P=0.016),而液氮研磨法与甲酸萃取法和(液氮研磨+甲酸萃取法)在种的鉴定率上均无统计学差异(其χ^2=2.203和χ^2=2.121,P=0.214和P=0.192。)。结论液氮研磨破壁效果好,(液氮研磨+甲酸萃取法)是质谱鉴定丝状真菌良好的样本预处理方法,值得临床推广应用。Objective A sample pretreatment method for identification of filamentous fungi by matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)was studied.Methods The walls of 35 filamentous fungi were broken by three methods:formic acid extraction,liquid nitrogen grinding,liquid nitrogen grinding and formic acid extraction.The breaking rate was calculated by using the Neubauer hemocytometer.One-way ANOVA(F test)was used to compare the wall breakage rates of the three methods.MALDI-TOF MS was used to determine the samples pretreated by the three methods,and Chi-square test(χ^2 test)was used to compare the identification results of the three methods.Results Under the light microscope,a large number of crushed spores and mycelium residue can be seen in the filamentous fungi ground with liquid nitrogen,and the background is cloudy with occasional intact spores.The wall breaking rates of liquid nitrogen grinding and liquid nitrogen grinding combined formic acid extraction were 0.86±0.06 and 0.84±0.06,respectively,which were higher than that of formic acid extraction(P<0.001).The rate of identification of 35 filamentous fungi to species and genus was 71%and 14%by liquid nitrogen grinding method,54%and 23%by formic acid extraction method,86%and 8%by liquid nitrogen grinding and formic acid extraction method.The rate of species identification by liquid nitrogen grinding combined with formic acid extraction was higher than that by formic acid extraction,and the difference was statistically significant(χ^2=8.231,P=0.016),while there was no statistical difference between liquid nitrogen grinding and the other two methods(χ^2=2.203,P=0.214 andχ^2=2.121,P=0.192).Conclusion Liquid nitrogen grinding combined with formic acid extraction is a good sample pretreatment method for mass spectrometry identification of filamentous fungi.

关 键 词：液氮 破壁 丝状真菌 质谱 

分 类 号：R115[医药卫生—公共卫生与预防医学]