尖孢镰刀菌TaqMan探针荧光定量PCR快速检测方法的建立及应用

Establishing and Applying of TaqMan Probe Real-time PCR Detection of Fusarium Oxysporum

目的建立TaqMan探针荧光定量PCR检测方法，用于临床标本中尖孢镰刀菌的快速诊断。方法针对尖抱镰刀菌的B—tubulin基N。设计特异性引物和探针并构建该基N的重组质粒标准品；优化PCR反应条件，建立尖孢镰刀菌的荧光定量PCR检测方法；通过lO种标准菌株和33株，临床分离株的应用检测，验证该方法的可行性。结果该检测方法具有较好的特异度、敏感度和重复性，6袜尖孢镰刀茵均有明显扩增，而其它37株临床常见的致角膜炎真菌标准株或分离株无明显扩增；最低检出限为408copies／μl，且变异系数低于5％。结论研究成功建立了尖孢镰刀菌的TaqMan探针荧光定量PCR检测方法，对临床上真菌性角膜炎的快速诊断及指导用药具有实际应用性和重要意义。

Objective To establish TaqMan probe real-time PCR method for rapid detection of Fusarium oxysporum in clini- cal samples. Methods A set of specific primer pair and probe were designed and synthesized based on the 13-tubulin gene of Fusarium ozysporum. The sensitivity and specificity of primers and probe were assessed by constructing plasmid standard substance. The detection methods were built up through optimizing the real-time fluorescent quantitative PeR reaction sys- tem. The feasibility verification of methods was carried out by detecting 10 standard fungal strains and 33 clinical isolates. Results ]＇be method which detecting Fusarium oacysporum with high sensitivity,specificity and reproducibility was estab- lished. 6 strains of Fusarium oocysporum were amp^ificated obviously,while ~he amplifications of the o^her 37 standard fun- gal strains and clinical isolates which were common in fungal keratitis were nearly unmeasurable. The minimum detectability was 408 copies/μl. The coefficient of variation was lower than 5 %. Conclusion TaqMan probe real-time PCR method which was build up in this study has great importance and practical application in early diagnosis of Fusarium o^cysporum in clini- cal.