云南山茶TaqMan探针实时荧光PCR快速鉴定方法的建立

Development of Real-time Fluorescence PCR Assay Using TaqMan Probes for Rapid Detection of Camellia reticulata Lindl

为建立云南山茶(Camellia reticulata Lindl.)TaqMan探针实时荧光PCR鉴定方法,根据GenBank数据库中及本实验室所测的15条云南山茶ITS可变序列片段设计特异引物和相应的TaqMan探针.试验结果证明,该引物探针组合可对云南山茶"大理茶""蒲门茶""凤山茶""麻叶银红""泽河"5个品系分别进行特异性检测鉴定,而对山茶属华东山茶(Camellia japonica L.)、金花茶(Camellia petelotii(Merrill)Sealy)、普洱茶(Camellia sinensis var.assamica(J.W.Masters)Kitamura)以及非山茶属的番茄均未检出荧光信号.本研究建立的云南山茶TaqMan探针实时荧光PCR快速鉴定方法特异性强、敏感性高、重复性好,非常适合基层和口岸在物种资源查验工作中推广应用,解决对云南山茶实施口岸查验的关键技术问题,提高查验效率.

In order to establish a TaqMan probes based real-time fluorescence PCR identification method for Camellia reticulata Lindl.,specific primers and corresponding TaqMan probes were designed according to 15 ITS variable sequence fragments measured in the GenBank database and our laboratory.After experiments,the results confirmed that the primer probe combination could specifically detect five strains of Camellia reticulata Lindl.,i.e.,“Dali Camellia”,“Pumen Camellia”,“Fengshan Camellia”,“Reticulate Leaf Spinel Pink”and“Zehe Camellia”,while no fluorescence signal could be detected for Camellia japonica L.,Camellia petelotii(Merrill)Sealy,Camellia sinensis var.assamica(J.W.Masters)Kitamura and non-camellia tomato.The real-time fluorescence PCR assay using TaqMan probes for rapid detection of Camellia reticulata Lindl.established in this study has strong specificity,high sensitivity and good repeatability,which is perfect for field and port inspection of species resources.It can solve the key technical problems of port inspection of Camellia reticulata Lindl.,and improves the inspection efficiency.