建立MALDI-TOF MS鉴定的海洋致病性弧菌数据库样本前处理的适宜条件

目的探讨建立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)鉴定海洋致病性弧菌数据库前样本处理的适宜条件，为完善MALDI-TOF MS鉴定该菌入数据库提供参考。 方法将32株海洋致病性弧菌培养至24、48、72 h时，分别用直接转移法、扩展的直接转移法、甲酸提取法得到上机样品，然后用MALDI-TOF MS进行采图鉴定，用SPSS20.0软件进行统计学分析，对样本不同培养时间及3种处理方法的鉴定结果进行比较评价。 结果培养24 h鉴定分数平均值为2.321，属水平鉴定正确率为96.88%，种水平鉴定正确率为90.63%；培养48 h鉴定分数平均值为2.315，属水平鉴定正确率为93.75%，种水平鉴定正确率为90.63%；培养72 h鉴定分数平均值为2.316，属水平鉴定正确率为93.75%，种水平鉴定正确率为87.50%，3种不同培养时间的前处理方法鉴定至属(P=0.129)、鉴定到种(P=0.078)，差异均无统计学意义。直接转移法鉴定分数平均值为1.986，属水平鉴定正确率为78.13%，种水平鉴定分数为59.38%；扩展的直接转移法鉴定分数平均值为2.135，属水平鉴定正确率为90.63%，种水平鉴定正确率为78.13%；甲酸提取法鉴定分数平均值为2.306，属水平鉴定正确率为96.88%，种水平鉴定分数为90.63%，3种不同上机前样本处理方法鉴定到属时差异有统计学意义(P=0.006)；鉴定到种时差异有统计学意义(χ²=8.691，P=0.013)。 结论建立MALDI-TOF MS海洋致病性弧菌数据库时，选择培养时间24 h的甲酸法提取菌体蛋白的前处理方法较适宜，可得到高质量的蛋白指纹图谱和鉴定正确率。

Research on the suitable sample processing conditions for establishing marine pathogenic vibrio database based on MALDI-TOF MS

ObjectiveTo explore the suitable sample processing conditions of establishing marine pathogenic vibrio database based on matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). MethodsThirty-two strains of marine pathogenic vibrio were cultured for 24 h, 48 h, 72 h, and then identified respectively by direct transfer method, extensional direct transfer method and formic acid extraction method. The results were statistically analyzed with SPSS20.0 statistical analysis software to find the suitable conditions for establishing MALDI-TOF MS marine pathogenic vibrio database. ResultsThe average value of 24 h culture identification score was 2.321 with the accuracy rate of genus identification was 96.88% and species identification was 90.63%. The average value of 48 h identification score was 2.315 with the accuracy rate of genus identification was 93.75% and the species identification was 90.63%. The average value of 72 h culture identification score was 2.316 with the accuracy rate of genus identification was 93.75% and the species identification was 87.50%. There was no statistical difference among these three pretreatment methods (P=0.129、P=0.078). In contrast, the average value of identification score by direct transfer method was 1.986, with the accuracy rate of genus identification was 78.13%, and the species identification was 59.38%; the average value of the extended direct transfer method was 2.135 with the accuracy rate of genus identification was 90.63% and the species identification was 78.13%; the average value of formic acid extraction was 2.306 with the accuracy rate of genus identification was 96.88% and species identification was 90.63%. There were significant differences among the three kinds of pretreatment methods on genus level (P=0.006) and species level(χ²=8.691, P=0.013). ConclusionsIn the establishment of MALDI-TOF MS marine pathogenic vibrio database, the culture time should be set at 24h and the pretreatment method should be formic acid extraction method, which can get high quality protein fingerprints.