MALDI-TOF MS鉴定马尔尼菲篮状菌的实验条件优化

Study on the optimized pretreatment conditions for identification of Talaromyces marneffei by MALDI-TOF MS

目的构建用于基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF MS)鉴定马尔尼菲篮状菌的数据库,并探索用于MALDI-TOF MS鉴定马尔尼菲篮状菌的最佳培养条件和前处理方法。方法利用8株马尔尼菲篮状菌构建自建数据库,比较不同培养基[沙保弱葡萄糖琼脂培养基(SDA)、沙氏葡萄糖肉汤培养基(SDB)]、培养温度(28、35℃)、培养天数、前处理方法(甲酸乙腈法、直涂法)对质谱谱图质量及鉴定正确率的影响。结果在VITEK MS科研模式(RUO模式)中成功构建马尔尼菲篮状菌质谱自建数据库。SDA较SDB更适合MALDI-TOF MS鉴定马尔尼菲篮状菌,后者采集的质谱谱图中干扰峰较多。两种温度培养采集的质谱谱图的特征峰相似,但35℃菌落的鉴定正确率略高。培养3~5 d时的菌落鉴定正确率均高于71.4%,其中培养5 d时鉴定正确率最高,可达100.0%。甲酸乙腈法的提取效果优于直涂法。结论马尔尼菲篮状菌在SDA上35℃培养3~5 d,并使用甲酸乙腈法进行前处理可得到最佳的鉴定正确率。这对于形态不典型的马尔尼菲篮状菌同样适用。

Objective To construct the database of Talaromyces marneffei for Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF MS)identification,and to explore the best culture conditions and pretreatment method for MALDI-TOF MS identification of Talaromyces marneffei.Methods A self-built mass spectrometry database was constructed by using 8 strains of Talaromyces marneffei.The effects of different culture media[Sabouraud Dextrose Agar(SDA),Sabouraud Dextrose Broth(SDB)],culture temperature(28,35℃),culture day and pretreatment methods(formic acid acetonitrile method and direct coating method)on the quality of spectrum and the accuracy of identification were compared.Results In VITEK MS research use only mode(RUO mode),the self-built mass spectrometry database of Talaromyces marneffei was successfully constructed.SDA was more suitable for MALDI-TOF MS identification of Talaromyces marneffei than SDB,and the latter had more interference peaks in the acquisition spectrum.The characteristic peaks of the spectra collected at the two temperatures were similar,but the identification accuracy of colonies at 35℃was slightly higher.The accuracy rate of colony identification was higher than 71.4%when cultured for 3-5 days,and the highest rate could reach 100.0%when cultured for 5 days.The extraction effect of formic acid acetonitrile method was better than that of direct coating method.Conclusion The best identification accuracy could be obtained by culturing Talaromyces marneffei on SDA at 35℃for 3-5 days and pretreated with formic acid acetonitrile method.This is also applicable to the Talaromyces marneffei with atypical morphology.