荧光定量PCR检测发酵豆酱中黄曲霉毒素相关基因nor-1

Research of Quantification of the Copy Number of nor-1,a Gene of the Aflatoxin Biosynthetic Pathway,in Fermented Bean Sauce by Real-time PCR

建立了TaqMan荧光定量PCR快速检测产黄曲霉毒素曲霉菌的方法,并将其应用于发酵豆酱中。以黄曲霉毒素合成相关结构基因nor-1基因作为靶标基因,设计特异性引物及TaqMan探针,以含nor-1基因的质粒为模板建立标准曲线,将其应用于豆酱发酵过程中nor-1基因的动态监测,并与传统平板培养法测定产毒曲霉菌结果相比较。结果显示,两检测方法呈较好的相关性,Pearson等级相关系数为0.808,P=0.098。灵敏度及特异性检测结果显示,该方法灵敏度达10CFU/mL,特异性针对含nor-1基因的曲霉菌。所建立的方法检测时间短,仅6h即能完成整个过程,相比传统平板培养法,具有优势。该研究在国内首次建立了快速、灵敏、准确的荧光定量检测方法检测豆酱中黄曲霉毒素相关基因nor-1,能够在黄曲霉毒素产生前期,对产毒曲霉菌污染状况进行监测以更好控制食品中黄曲霉毒素,提高食品安全性。

This research developed a TaqMan real-time PCR method to detect aflatoxigenic Aspergilli in fermented bean sauce.We designed specific primers and TaqMan probe targeted nor-l gene,a structure gene related to aflatoxin synthetic.The standard curve was established using nor-1 gene plasmid as template and applied to monitoring nor-1 gene copies in the fermentation process of bean sauce.Then the copy numbers of the nor-l gene were compared to colony -forming unit data obtained from the same set of samples by conventional count（CC） method.A correlation between RT-PCR results and CC data was observed（Pearson,r = 0.808;P = 0.098 ）.Sensitivity and specificity detection results showed that the limited detection was 10 CFU/mL,and the system was specific for nor-l containing Aspergilli. The entire detection time was only six hours and had great advantages compared with the traditional CC method. This was the first time a quick,sensitive and accurate RT-PCR detection was established to detect the nor-1 gene in fermented bean sauce.This method can monitor poisoned aflatoxigenic Aspergilli before aflatoxins producing,give better control of aflatoxins and improve food security.