黄曲霉的快速精确检测,既有利于预防真菌对粮食造成污染,也有利于控制粮食中真菌毒素的产生,确保粮食储藏安全.依据特异性邻甲基转移酶基因(OMT-1)设计了黄曲霉特异性引物,构建基于SYBR Green I实时荧光定量PCR(quantitative real-time PCR,qPCR)的检测体系,建立了黄曲霉毒素产生菌OMT-1基因拷贝数与循环阈值(cycle threshold,CT)之间的线性关系,对黄曲霉qPCR绝对定量方法进行验证.研究结果表明:该方法特异性强,检测灵敏度高,检测限为0.015 μg/mL,OMT-1基因拷贝数的对数与CT线性相关,标准曲线方程为y=-3.519 2x+55.905(R2=0.997 3),扩增效率为107.0％.OMT-1基因的拷贝数与同一组样本获取的菌落总数相关性高(R2=0.97).所建立的qPCR绝对定量方法可用于监测玉米中黄曲霉基因的表达.
Development of a specific qPCR method for the rapid detection of Aspergillus flavus in maize
Rapid and accurate detection of Aspergillus flavus is crucial for preventing fungal contamination of grain,controlling aflatoxin production in grains,and ensuring safety of grain storage.In this study,specific primers targeting the OMT-1 gene of A.flavus were designed and a SYBR Green I qPCR-based detection system was established.We determined a relationship between the copy number of the OMT-1 gene in A.flavus producing aflatoxin and the CT value,and verify the absolute quantitative method of qPCR for Asper-gillus flavus.This method demonstrated high specificity and sensitivity,with a detection limit of 0.015 µg/mL.The logarithm of copy number of OMT-1 gene is linearly correlated with CT value,with a standard curve equation of y=-3.519 2x+55.905(R2=0.997 3)and an amplification efficiency of 107.0％.Additionally,the copy number of the OMT-1 gene showed a strong correlation with the colony count data ob-tained from the same set of samples(R2=0.97).These results indicate that the qPCR absolute quantification method could be used to monitor the expression of the A.flavus OMT-1 gene in maize.
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