三种前处理在基质辅助激光解析电离飞行时间质谱鉴定假丝酵母菌属中的应用比较

Three pretreatments in the identification of Candida species by matrix-assisted laser desorption ionization time-of-flight mass spectrometry:a comparison

目的比较3种前处理方法在基质辅助激光解析电离飞行时间质谱(MALDI TOF-MS)鉴定假丝酵母菌属中的结果可靠性。方法以ITS测序鉴定结果为金标准,对临床分离的66株假丝酵母分别采用传统直涂法、改良直涂法和甲酸-乙腈蛋白提取法进行前处理,MALDI TOF-MS鉴定,比较3种方法的Biotyper Log值,分析质谱图的差异。结果传统直涂法、改良直涂法和甲酸-乙腈提取法对66株假丝酵母的属水平鉴定率分别为48.5%、50.0%和97.0%,Biotyper Log均值分别为1.628、1.674和2.010,其中甲酸-乙腈提取法对66株假丝酵母的种水平鉴定率为53.0%。甲酸-乙腈提取法得到的质谱图比另2种方法的质谱图离子峰更加密集,图像更复杂,鉴定结果可信度更高。结论甲酸-乙腈蛋白提取法对假丝酵母菌属的鉴定成功率和可靠性明显高于传统直涂法和改良直涂法,对临床假丝酵母菌病的准确诊断具有重要价值。

Objective To compare the reliability of three pretreatment methods in the identification of Candida by matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI TOF-MS).Methods With the results of ITS sequencing as the gold standard,66 clinical isolates of Candida were pretreated with conventional direct smear method,modified direct smear method and formic acid-acetonitrile protein extraction method,respectively,and identified by using MALDI TOF-MS.The Biotyper Logs of the three methods were compared and the differences in mass spectra were analyzed.Results The identification rates of conventional direct smear method,modified direct smear method and formic acid-acetonitrile extraction method for the 66 strains were 48.5%,50.0%and 97.0%,respectively,and the average values of Biotyper Log were 1.628,1.674 and 2.010,respectively.The species-level identification rate by formic acid-acetonitrile extraction method was 53.0%.The ion peaks of the mass spectra obtained by formic acid-acetonitrile extraction method were denser,the images were more complex,and the identification results were more reliable than those of the other two methods.Conclusion The success rate and reliability of formic acid-acetonitrile protein extraction method in the identification of Candida is significantly higher than conventional direct smear method and modified direct smear method,which is of great value for the accurate diagnosis of clinical candidiasis.