实时荧光定量PCR用于玉米中黄曲霉的快速检测

Development of a specific qPCR method for the rapid detection of Aspergillus flavus in maize

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0.015μg/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=-3.5192x+55.905(R^(2)=0.9973),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(R^(2)=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。

Rapid and accurate detection of Aspergillus flavus is crucial for preventing fungal contamination-induced deterioration of grain,controlling aflatoxin production contamination 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.A linear relationship between the copy number of the OMT-1 gene in A.flavus producing aflatoxin and the CT value was determined,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 Aspergillus flavus.This method demonstrated high specificity and sensitivity,with a detection limit of 0.015μg/mL.The logarithm of copy number of OMT-1 gene is linearly correlated with CT value,with a standard curve equation of y=-3.5192x+55.905(R^(2)=0.9973)and an amplification efficiency of 107.0%.This method demonstrated high specificity and sensitivity,with a detection limit of 0.015μg/mL.Additionally,the copy number of the OMT-1 gene showed a strong correlation with the colony count data obtained from the same set of samples(R^(2)=0.97).These results indicate that the qPCR absolute quantification method established in this study could be used to monitor the expression of the A.flavus OMT-1 gene in maize.