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

作　　者：徐嵩月 赵金凤 赵锦琦 李梦瑶 张玉荣[1] 张咚咚 XU Songyue;ZHAO Jinfeng;ZHAO Jinqi;LI Mengyao;ZHANG Yurong;ZHANG Dongdong(School of Food and Strategic Reserves,Grain Storage and Security Engineering Research Center of Education Ministry,Henan University of Technology,Zhengzhou 450001,China;Hangzhou Grain Storage Co.,Ltd.,Hangzhou 311100,China)

机构地区：[1]河南工业大学粮食和物资储备学院粮食储藏与安全教育部工程研究中心,河南郑州450001 [2]杭州市粮食收储有限公司,浙江杭州311100

出　　处：《河南工业大学学报（自然科学版）》2024年第3期110-117,共8页Journal of Henan University of Technology：Natural Science Edition

基　　金：国家自然科学基金项目(32001745);中国科协第五届青年人才托举工程项目(2019QNRC001);国家小麦产业技术体系项目(CARS-03);河南省级科技研发计划联合基金(应用攻关类)项目(222103810081)。

摘　　要：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.

关 键 词：实时荧光定量 黄曲霉 检测 玉米 

分 类 号：TS210.1[轻工技术与工程—粮食、油脂及植物蛋白工程]