黑曲霉实时荧光PCR检测方法的建立

Development of Real-Time Fluorescent PCR for Determination of Aspergillus niger

目的建立黑曲霉实时荧光PCR检测方法。方法对6种主要病原曲霉(黑曲霉、烟曲霉、杂色曲霉、构巢曲霉、土曲霉及黄曲霉)的GAPDH基因序列进行比对分析,选择黑曲霉特异位点设计引物和探针,对黑曲霉进行实时荧光PCR扩增,并检测该方法的灵敏度及特异性。结果该方法可检出2.78×10-10μg/ml的黑曲霉基因组DNA;对亲源关系较近的11株不同种曲霉及4株其他属临床常见的病原真菌进行实时荧光PCR检测,未发现有交叉反应。结论已建立了灵敏度高、特异性好的快速检测黑曲霉的实时荧光PCR方法。

Objective To develop a real-time fluorescent PCR for determination of Aspergillus niger. Methods The GAPDH gene sequences of 6 kinds of pathogenic Aspergillus, i.e. Aspergillus niger, Aspergillus fumigatus, Aspergillus versicolor, As- pergiUus nidulans, Aspergillus terreus and Aspergillus flavus, were compared, based on which primers and probes for real-time fluo- rescent PCR were designed according to the specific site of Aspergillus niger. The developed real-time fluorescent PCR was analyzed for sensitivity and specificity. Results By using the developed real-time fluorescent PCR, 2.78×10^-10μg/ml of genomic DNA of Aspergillus niger was detected. Eleven AspergiUus niger isolates with close genetic relationship and 4 isolates of other common pathogenic fungi in clinic were determined by the developed method, and no cross reactions were observed. Conclusion A real-time fluorescent PCR for rapid determination of Aspergillus niger, with high specificity and sensitivity, was developed.