10株荚膜组织胞浆菌和7株球孢子菌的分型鉴定及质谱自建库建立

Molecular typing and establishment of MALDI-TOF MS in-house database of ten Histoplasma capsulatum and seven Coccidioides sp.strains

目的利用分子分型及基质辅助激光解析串联飞行时间质谱(MALDI-TOF MS)技术对现有的荚膜组织胞浆菌(Histoplasma capsulatum)和球孢子菌属(Coccidioides sp.)复核鉴定并聚类分析。方法通过多位点序列分型(multilocus sequence typing,MLST)对10株荚膜组织胞浆菌进行系统发育分析;利用特异性片段(Coi区)明确7株球孢子菌的种归属;MALDI-TOF MS技术建立荚膜组织胞浆菌和球孢子菌的MALDI-TOF MS自建数据库,并从蛋白质指纹图谱角度对两类真菌进行同源性分析。结果通过MLST分析,可以将10株荚膜组织胞浆菌分为3个进化支:欧洲型(Eurasia clade)、澳洲型(Australia clade)和北美2型(North American class 2 clade),且质谱聚类分析亦将10株荚膜组织胞浆菌分为3个簇;利用特异性片段(Coi区)鉴定7株球孢子菌为波萨达斯球孢子菌,同时MALDI-TOF-MS蛋白质指纹图谱同源性分析将其归为两个簇。两类真菌MALDI-TOF MS建库后验证符合率为100%。结论分子鉴定后所有球孢子菌明确为波萨达斯球孢子菌,与美国流行情况一致;国内分离的荚膜组织胞浆菌归于欧洲型、北美2型和澳洲型这3个型别;MALDI-TOF MS建库数据可靠且质谱同源性分析可作为分子分型的补充,成为荚膜组织胞浆菌和波萨达斯球孢子菌鉴定和溯源的另一手段。

ObjectiveTo recheck the Histoplasma capsulatum and Coccidioides sp.strains of the past by molecular identification and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS).MethodsThe phylogenetic relationships among the ten Histoplasma capsulatum isolates and reference strains were analyzed by multilocus sequence typing(MLST).Based on the Coi region,Coccidioides posadasii was distinguished from Coccidioides immitis accurately.MALDI-TOF MS was used to set up the MALDI-TOF MS database of Histoplasma capsulatum and Coccidioides sp.for rapid identification.In addition,hierarchical clustering of spectra was compared with MLST.ResultsAn unrooted dendrogram constructed with MLST showed that ten individuals of Histoplasma capsulatum were divided into three clades:Eurasia clade,Australia clade and North American class 2 clade,in agreement with the establishment by MALDI-TOF MS cluster analysis.All individuals of Coccidioides sp.were identified as Coccidioides posadasii with Coi region primers.The in-house MALDI-TOF MS database of Histoplasma capsulatum and Coccidioides posadasii was expanded and reached an identified accuracy of 100%.ConclusionsWe improve the recognition of Histoplasma capsulatum and Coccidioides posadasii by molecular pathways which shows the major species or clades in Chinese mainland.The in-house MALDI-TOF MS database of Histoplasma capsulatum and Coccidioides posadasii can provide a new efficient way to identify those pathogens rapidly.