MALDI-TOF MS技术在侵袭性丝状真菌快速鉴定中的应用

MALDI-TOF MS in rapid identification of invasive Filamentous fungi

目的探索不同培养基、不同培养时间和不同蛋白质提取方法对侵袭性丝状真菌质谱鉴定准确率的影响,旨在提高基质辅助激光解析电离飞行时间质谱技术鉴定侵袭性丝状真菌的准确率。方法采用分子生物学方法为金标准,同时运用基质辅助激光解析电离飞行时间质谱技术对所收集临床丝状真菌进行鉴定。根据分子生物学的鉴定结果,去除VITEK-MS v3.0数据库中没有的菌株,其余菌株接种在沙氏葡萄糖琼脂(SDA)、马铃薯葡萄糖琼脂(PDA)和察氏培养基(CA)3种不同的培养基中,采用2种不同的蛋白质提取方法(甲酸乙腈法和磁珠法),获得了不同培养时间点(2、3、5、7和9d)的特异性质谱指纹图谱。结果不同丝状真菌蛋白质提取方法进行比较,甲酸乙腈法总鉴定准确率为79.8%,磁珠法总鉴定准确率为77.5%,2种丝状真菌蛋白质提取方法的质谱鉴定准确率差异无统计学意义(χ^(2)=1.040,P=0.308)。不同培养基进行比较,SDA培养基总鉴定准确率为90.7%,PDA培养基总鉴定准确率为81.4%,CA培养基总鉴定准确率为67.4%,3种不同培养基的丝状真菌质谱鉴定准确率差异有统计学意义(χ^(2)=36.609,P<0.001),其中使用SDA培养基鉴定准确率最高,使用CA培养基鉴定准确率最低(SDAvs PDA,χ^(2)=7.748,P=0.005;SDAvs CA,χ^(2)=35.131,P<0.001;PDAvs CA,χ^(2)=10.994,P=0.001)。不同培养时间进行比较,丝状真菌培养2、3、5、7和9d后的质谱总鉴定准确率分别为64.3%、88.4%、89.1%、79.1%和78.3%,不同培养时间的质谱鉴定准确率差异有统计学意义(χ^(2)=32.274,P<0.001)。培养3d和培养5d的质谱鉴定准确率优于培养7d和培养9d的质谱鉴定准确率,培养2d的质谱鉴定准确率明显低于其他时间(3dvs 5d,χ^(2)=0.039,P=0.844;7dvs 9d,χ^(2)=0.023,P=0.879;3dvs 7d,χ^(2)=4.095,P=0.043;2dvs 9d,χ^(2)=6.139,P=0.013)。结论侵袭性丝状真菌使用SDA培养基培养3d,运用甲酸乙腈法提取蛋白质进行质谱鉴定最理想。

Objective To investigate the influence of different culture media,culture time and protein extraction methods on the accuracy of mass spectrometry(MS)identification of invasive Filamentous fungi(IFF),and improve the accuracy of MALDI-TOF MS in the identification of IFF.Methods MALDI-TOF MS was used to identify clinical Filamentous fungi;molecular identification was used as the standard method.According to the identification results of molecular sequencing,the strains that were not included in VITEK-MS v3.0database were removed,and the remaining strains were inoculated in three different culture media(SDA,PDA and CA).The specific spectral fingerprints at different culture time points(2,3,5,7 and 9days)were obtained by using two different protein extraction methods(extraction with formic acid and acetonitrile;extraction with magnetic bead,formic acid and acetonitrile).Results Comparison of different protein extraction methods showed that the total identification accuracy for extraction with formic acid and acetonitrile was 79.8%,while that with magnetic bead,formic acid and acetonitrile was 77.5%;there was no significant difference between the two extraction methods(χ^(2)=1.040,P=0.308).Comparison of different media showed that the total identification accuracies for SDA medium,PDA medium and CA medium were 90.7%,81.4%and 67.4%,respectively.There were significant differences among the three culture media(χ^(2)=36.609,P<0.001).SDA medium resulted in the highest identification accuracy and CA medium did the lowest(SDAvs PDA,χ^(2)=7.748,P=0.005;SDAvs CA,χ^(2)=35.131,P<0.001;PDAvs CA,χ^(2)=10.994,P=0.001).Comparison of different culture time point showed that the total identification accuracies at Day 2,3,5,7and 9were 64.3%,88.4%,89.1%,79.1%and 78.3%,respectively;there were significant differences among the five culture3鎪time points(χ^(2)=32.274,P<0.001).The identification accuracies of MS on the third and fifth day of culturing were better than those on the seventh and ninth day.In addition,the identification accuracy of MS at Day 2of culturing was significantly lower than those at other days(3days vs 5days,χ^(2)=0.039,P=0.844;7days vs 9days,χ^(2)=0.023,P=0.879;3days vs 7days,χ^(2)=4.095,P=0.043;2days vs 9days,χ^(2)=6.139,P=0.013).Conclusion The best procedure to identify invasive Filamentous fungi with mass spectrometry is to culture them on SDA for 3days and then extract them with formic acid and acetonitrile.