摘要
目的采用实时荧光PCR结合融解曲线分析的方法快速将临床常见曲霉菌鉴定到种的水平。方法①普通PCR扩增真菌ITS区后进行序列比对,准确鉴定菌种并设计种特异性引物和探针。②采用实时荧光PCR的方法及融解曲线分析,根据不同的解链温度将5种临床常见曲霉菌鉴定到种的水平。③特异性、敏感性、重复性试验。结果①解链曲线分析显示,不同种曲霉菌的种特异性探针有特异的Tm值,根据Tm值的不同可以将5种曲霉菌区分开来:烟曲霉Tm=61.4℃,黄曲霉Tm=57.4℃,黑曲霉Tm=67.7℃,土曲霉Tm=55.2℃和64.5℃,构巢曲霉Tm=65.8℃。其中小孢根霉和疣状瓶霉与烟曲霉探针发生交叉反应,阴性对照不出现特异性的解链曲线。②该方法对5种曲霉菌的检测下限分别为:烟曲霉56.8 fg,黄曲霉1 110 fg,黑曲霉13.7 fg,土曲霉123 fg,构巢曲霉780 fg。③重复性试验结果显示,同一种曲霉菌的Tm值波动范围不超过0.5℃。结论采用实时荧光PCR结合融解曲线分析的方法可以快速准确地将临床常见曲霉菌鉴定到种的水平,具有良好的敏感性、特异性和可重复性,有助于临床侵袭性曲霉感染的诊断和指导抗真菌药物使用。
Objective Real-time PCR combined with melting curve analysis were carried out for rapid identification of clinical important Aspergillus to the species level. Method （ 1 ） PCR amplification of fungal ITS region, and then sequence alignment were done to identificate Aspergillus isolates correctly and design species specfic primers and probes. （2） Five species of clinical impor- tant Aspergillus were identified to species level according to different melting temperatures （Tm） of species specific probes in a hi- probe real-time PCR. Results （ 1 ） The Tm value of A. terrus specific probe were 64.5℃ and 55.2℃ , and that of A.flavus,A. fumigatus ,A. nidulans and A. niger specific probe was 57.4℃ , 61.4℃, 65.8℃, and 67.7℃ respectively. Phialophora verrucosa and Rhizopus microspores had cross reactions with A. furnigatus specific probe. （2）The detection limit of A.fumigatus was 56.8 fg, and that of A. flavus 1.11 x 103 fg, A. niger 13.7 fg, A. terrus 1.23 x 102 fg and A. nidulans 7.80 x 102 fg. （ 3 ） The Tm value fluctua-tion was within 0.5 ℃ in repeated reactions. Conclusion The real-time PCR is time-saving and with high sensitivity, specificity and repeatablity. Therefore, it is helpful for diagnosis of invasive aspergillosis and the use of antifungal drugs.
出处
《中国真菌学杂志》
2012年第6期330-334,338,共6页
Chinese Journal of Mycology
