多重PCR对真菌性角膜炎主要致病菌的菌属鉴定

目的:建立多重PCR体系对真菌性角膜炎主要致病真菌进行快速诊断并同时进行菌属鉴定的方法。方法:建立两个多重PCR体系(体系1和体系2),对真菌性角膜炎九种主要致病真菌DNA进行检测,观察该体系对真菌临床菌株、人类基因组及其他眼部常见致病微生物DNA的检测结果。结果:体系1对镰孢菌属扩增均产生约360bp的特异产物,对曲霉菌属、牵连青霉菌和新月弯孢菌扩增均产生约470bp的特异产物。体系2对镰孢菌属、曲霉菌属均无特异产物,而对牵连青霉菌产生了360bp的特异产物,对新月弯孢霉产生了300bp的特异产物。根据DNA模板在两个多重PCR体系中扩增出的不同特异条带可将九种真菌分为四个菌属。57株真菌临床菌株中55株的鉴定结果与常规鉴定结果一致。两体系对人类基因组及其他眼部常见致病微生物DNA的扩增结果均为阴性。结论:通过两个多重PCR体系检测可将真菌性角膜炎在菌属水平进行诊断及鉴定。该方法具有快速、简便、特异、灵敏的特点,具有较好的临床应用前景。

Genus Identification of Pathogens in Fungal Keratitis with Multiplex Polymerase Chain Reation

Objective:To develop a rapid,simple,sensitive and specific molecular biological method for diagnosis and genus identification of fungal pathogens in fungal keratitis.Methods:Two multi-PGR systems were established.System 1 contains sequence-specific primers for Fusarium spp.,Aspergillus spp.,Penicillum spp.and Curvularia spp..System 2 contains sequence- specific primers for Penicillum spp.and Curvularia spp..Fusarium solani,Fusarium moniliform,Fusarium equiseti,Fusarium graminum,Aspergillus fumigatus,Aspergillus flavus,Aspergillus niger,Penicillum implicatum and Curvularia lunata were tested by the two systems.57 clinical fungi strains,human genomic DNA and DNAs of other microorganism in human ocular infections were also tested using the same method.Results:The fungi could be distinguisbed according to the amplification products by the two multi-PCR systems:In system 1,size of Fusarium species amplificate products was about 360 bp;Aspergillus,Penicillum and Curvularia species amplificate products were about 470 bp.In system 2,size of Penicillum implicatum amplificate product was about 360 bp;Curvularia lunata amplification product was about 300 bp;and no amplificate product was obtained from Fusarium and Aspergillus species.55 out of the 57 straius of clinical microoganisms were identificated using this method.No cross-reaction with human DNA,DNAs of Chlamydia trachomatis,Herpes simplex and several other bacterial pathogens so far tested was observed. Conclusions:The combination of two multi-PGR systems is a rapid,simple,reliable method for diagnosis and genus identification of fungal pathogens in fungal keratitis.And it has a good prospect for clinical application.