应用实时荧光PCR技术检测构巢曲霉的初步研究

目的 根据构巢曲霉（Aspergillus nidulans）3-磷酸甘油醛脱氢酶（Glyceraldehyde-3-phosphate dehydrogenase，GAPDH）基因特异位点设计并合成Taqman探针及引物，建立构巢曲霉实时荧光 PCR检测方法。方法 应用lasergene7.1软件对构巢曲霉与13种常见曲霉主要包括黑曲霉（A.niger）、烟曲霉（A.fumigatus）、杂色曲霉（A.versicolor）、土曲霉（A.terrus）、黄曲霉（A. flavus）、温特曲霉（A.wentii）、寄生曲霉（A. parasiticus）、泡盛曲霉（A.awamori）、米曲霉（A. oryzae ）、棒曲霉（A.cavatus）、赤曲霉（A.ruber ）、亮白曲霉（A.ochraceus）及赭曲霉（A.ochraceus）GAPDH基因序列比对分析，在特异位点设计引物和探针，建立构巢曲霉实时荧光 PCR检测方法，并对该方法进行特异性及敏感性分析。结果 用曲霉属22种41株不同曲霉及其他属的12株病原真菌验证实验表明，所建立的荧光PCR方法特异性强；检测灵敏度可达4.03×10－12μg/ml的模板DNA。 结论 应用实时荧光PCR技术能够有效检测构巢曲霉，该方法具有特异、灵敏、快速等特点，可在实际工作中应用。

Development of a Real-time PCR Method to Detect Aspergillus nidulans

Objective A real-time PCR method for the detection of Aspergillus nidulans was developed. Methods Multiple nucleotide sequence alignment of 13 Aspergillus species shows high Variation in partial sequence of Glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH gene). Base on the Variation region the primers and TaqMan probes were designed for specific detecting Aspergillus nidulans specie. The specific and sensitivity of the real-time PCR was tested. Results 41 Aspergillus isolates and 12 others genus fungi were used，No cross-amplification was observed. The lowest detection limits of the real-time PCR are proved by the sensitivity testing ，4.03×10－12μg/ml of the A. nidulans DNA template can be amplified successfully. Conclusion Real-time PCR is a time-efficient，specifically method，it is a novel tool for identifying and determining the strain of A. nidulans. This method may be useful for detecting and identifying Aspergillus nidulans specie.