临床常见镰刀菌的鉴别

Identification of clinically important Fusarium spp.

目的从分子生物学角度寻找一种快速准确鉴定临床常见镰刀菌的方法。方法将受试镰刀菌接种于PDA培养基,观察其菌落及镜下形态,在此基础上PCR扩增受试镰刀菌的rDNA ITS并测其序列,在GenBank核酸序列数据库进行同源序列搜索及分析。选择限制性内切酶Dra Ⅱ和Cfr13 Ⅰ进行RFLP。设计了茄病镰刀菌的种特异性引物Sol1、Sol2,初步验证其特异性。结果形态学鉴定结果显示,茄病镰刀菌所占比例最高,除2株串珠镰刀菌外,其余镰刀菌ITS序列分析的结果与形态学鉴定结果一致。茄病、层生和串珠镰刀菌的Dra Ⅱ、Cfr13 I酶切带形互不相同。用Sol1、Sol2扩增受试菌的rDNA ITS,只有茄病镰刀菌为阳性。结论rDNA ITS序列测定及其PCR-RFLP可用于初步鉴别几种临床常见镰刀菌,合适的种特异性引物可以初步快速鉴定茄病镰刀菌。

Objective To find rapid and reliable molecular methods to identify medically important Fusarium spp.. Methods The Fusarium spp. tested were cultured on PDA. Their ITS of rDNA were amplified using PCR and their PCR products were sequenced. BLAST and sequences analysis were performed through the nucleic acid bank of the GenBank. Endonuclease including Dra II and Cfr13 I were selected to perform RFLP analysis. A pair of species-specific primers-Sol1 and Sol, ofF. solani were designed and their specificity were tested. Results F. solani were the most predominant species based on morphological characters. The sequences of ITS analysis results were coincident with their morphologies except two F. moniliforme . The RFLP patterns of F. solani, F. moniliforme and F. proliferatum digested by Dra II and Cfr13 I were different from each other. The species-specific primers can only amplify rDNA of F. solani. Conclusions PCR- RFLP and sequencing of rDNA ITS can identify some Fusarium spp.. Appro- priate species-specific primers can be used to identify Fusarium solani rapidly.