临床常见镰刀菌rDNA ITS序列分析

Sequence analysis of ribosomal DNA ITS of clinical common Fusarium species.

目的探讨rDNA ITS序列分析用于快速鉴定临床常见镰刀菌的方法,为临床早期诊断镰刀菌感染提供依据。方法选择临床常见的茄病镰刀菌、串珠镰刀菌、尖孢镰刀菌和半裸镰刀菌,提取基因组DNA,用真菌通用引物ITS1、ITS4、PCR扩增rDNA ITS,纯化后测定其序列,所得序列在GenBank核酸序列数据库中进行同源序列搜索,并和其中镰刀菌标准株的rDNA ITS序列进行序列比对。利用C lustalX软件将序列匹配排列,进行1000次bootstrap统计学检验,用TreeV iew软件显示系统树。结果rDNA ITS序列分析的结果与表型基本一致,以前经形态学鉴定的2株串珠镰刀菌经过rDNA ITS序列分析鉴定为层生镰刀菌。系统树显示茄病镰刀菌和标准株聚在一起,串珠镰刀菌大部分也和标准株聚在一起。结论形态学鉴定镰刀菌有一定的局限性,rDNA ITS序列分析可用于快速鉴定临床常见的镰刀菌。

Objective To study rapid identification method of clinically isolated common Fusarium spp, providing the basis fur early diagnosis of Fusarium infection. Methods Cliniccaly isolated con,mon Fusarium spp, such as Fusrium solani ,Fusrium monififorme ,Fusrium oxysporum and Fusrium semitectum were selected. Their genumic DNAs were extracted. Fungal general primers of ITSI, ITS4 were used to amplify the internal transcribed spacer of rDNA of the Fusarium spp tested. After purification, the PCR products were sequenced directly. For the sequences acquired, homologous sequences were searched in Genebank, the nucleic acid sequence database ; the sequences acquired were paired with sequences of rD- NA rFS of standard strain of Fusarium found in Genebank. Phylogentic tree were displayed by using TreeView software after these sequences were matched and arranged by means of Clustal X software and verified by bootstrap statistical test one thousand times. Results ＇lhe sequence analysis results of rDNA UUS were basically coincident with their phenotype. Two strains of F. moniliforme identified morphologically were actually F. proliferatum confirmed by their ITS sequence analysis. All F. solani strains and almost all F. monilifomle stains were allied with their standard strains in phylogentic tree. Conclusion To identify Fusarium spp by their morphological characters has some limitations. Sequence analysis of rDNA ITS can identify clinical common Fusarium spp rapidly.