鉴别蜂蜜真实属性实时荧光PCR法的建立

Establishment of Real-time Fluorescence PCR Method Toidentifying real Properties of Honey

通过建立能够鉴别完全掺假蜂蜜的实时荧光PCR检测方法,为打击掺假蜂蜜的不良市场风气提供技术储备。筛选显花基因NADH、油菜基因和龙眼基因的引物探针;通过验证这三对引物探针的特异性、灵敏度来确定其可行性;提取蜂蜜的DNA进行这三组实时荧光PCR法的测试,同时采用传统蜂蜜感官检测法辨别真假蜂蜜,比较两者方法。结果显示三组引物探针的特异性强,灵敏度分别为0.0001 ng/μL、0.01 ng/μL和0.1 ng/μL;蜂蜜的DNA浓度与花粉粒数目呈正相关;NADH基因对15份蜂蜜样品的检测结果与传统检测法结果一致,一份样品镜检未见到花粉粒,其实时荧光PCR结果为阴性,掺假率达6.67%;6份油菜蜂蜜均检出油菜源性成分;6份龙眼蜂蜜中只有有3份检出龙眼源性成分。结论认为相对传统方法,该方法安全性好、操作简单、灵敏度高、结果直观,能够鉴别完全掺假蜂蜜,为蜂蜜掺假检测提供技术支持。

To establish a real-time fluorescence PCR detection method to identify totally adulterated honey,and to provide technical reserve for combating the unhealthy market atmosphere of adulterated honey.The primer probes of NADH,Canola and Longan genes were screened.The feasibility was determined by verifying the specificity dnd sensitivity of the primers・The DNA of honey was extracted to test the three groups of real-time fluoresce nee PCR,and the traditional honey sensory detection method was used to distinguish the true and false honey,compared the two methods・The three primers showed high specificity dnd sensitivity was 0.0001 ng/μL,0.01 ng/μL and 0.1 ng/μL respectively.The DNA concentration of honey was positively correlated with the number of pollen grains.The detection results of NADH gene on 15 honey samples were consistent with the results of traditional detection method.No pollen grains were found in one sample under microscopic examination,dnd the fluorescence PCR results were negative in real time PCR,with the adulteration rate up to 6.67%.Cdnold comp on ents were detected in 6 samples of rapeseed honey.Only 3 out of 6 longan honey samples were found to be of longdn origin.Compared with traditiondl methods,this method has better safety,simple operation,high sensitivity and intuitive results.It can identify totally adulterated honey and provide technical support for the detection of adulterated honey.