PCR结合基因扫描分析技术对几种深部真菌病致病菌的快速检测及鉴定

目的 用PCR法快速鉴定22种(23株)深部真菌病致病菌种。方法 应用荧光标记的真菌通用引物ITS4与ITS86对22种(23株)深部真菌病致病菌种的菌悬液进行PCR扩增，扩增产物经ABI PRISM^TM377测序仪及基因扫描分析软件精确测定片段大小，与对照组——相应菌种采用传统方法抽提DNA后扩增片段的扫描分析结果相对照。结果 ①17种致病菌种菌悬液经ITS4、ITS86扩增出单一的片段(除了构巢曲霉和黑曲霉、白念珠菌和类星形念珠菌、裴氏着色真菌和皮炎外瓶霉片段长度相同)。②菌悬液扩增片段的扫描分析结果与其DNA扩增结果相近，差异无显著性。③整个检定过程仅需6h。结论 采用ITS通用引物及菌悬液PCR检测法，结合基因扫描分析技术可准确、特异、快速、敏感地检测并鉴定出22种深部真菌病致病菌种，这将有望成为快速诊断深部真菌病的一种方法。

Rapid Detection and Identification of Pathogenic Fungi of Some Deep Fungal Infections by PCR in Combination with Genescan Analysis

Objective To rapidly detect and identify pathogenic fungi of some deep fungal infections by PCR.Methods The suspensions of22pathogenic fungi(23strains)were amplified by PCR with fungal universal primers ITS86and ITS4which were labeled by FAM.The precise length of amplified fragments was determined by ABI PRISM TM 377Sequencer and Genescan analysis software,then compared with that of am-plicons of corresponding fungal DNA which were previously extracted.Results(1)Amplification of17pathogenic fungi with ITS4,ITS86resulted in a unique fragment length(except for A.nidulans and A.niger,C.albicans and C.stellatoidea,F.pedrosoi and E.dermatitidis).(2)No significant difference of the length of am-plicons was found between the fungal suspension and control organisms,based on the results of Genescan analysis.(3)The whole process took only6h to complete the detection.Conclusion The combination of fun-gal suspension PCR with ITS fungal universal primers and Genescan analysis might provide an accurate,spe-cific,sensitive,and rapid approach to detect and identify22pathogenic fungi causing deep fungal infections,and hold promise to be applied for the diagnosis of deep fungal infection.