[关键词]
[摘要]
目的 探讨多重实时荧光PCR检测方法对川贝母及伪品鉴别的可行性。方法 通过对常见贝母属植物的ITS、psbA-trnH、rbcL和matK基因序列种间变异、遗传距离及系统发育关系分析,选择进化速率快、种间变异大、种内变异小的基因作为靶基因,设计川贝母及伪品贝母的特异性引物和Taqman探针,建立一种多重实时荧光PCR检测方法。通过特异性、灵敏度及混合样本检测与测序方法比较进行方法评估。结果 贝母属ITS和psbA-trnH变异位点高于rbcL和matK,通过遗传距离分析rbcL和matK基因显著低于ITS和psbA-trnH基因,以ITS作为靶基因,建立多重实时荧光PCR检测方法特异性强,灵敏度达0.01 ng,对18份样本抽查检出4份伪品贝母,检测结果与构建的NJ树聚类分析结果完全一致。结论 基于ITS区域序列为靶基因建立多重实时荧光PCR检测方法能够成功鉴定川贝母及其混伪品,为川贝母真伪鉴别提供依据。
[Key word]
[Abstract]
Objective To investigate the feasibility of multiple real-time PCR for the detection of Fritillariae Cirrhosae Bulbus and adulterants. Methods Based on the analysis of interspecies variation, genetic distance and phylogenetic relationship of ITS, psbA-trnH, rbcL and matK gene sequences, the genes with fast evolution rate, big interspecies variation and small intraspecies variation were selected as target genes. Fritillariae Cirrhosae Bulbus and adulterants specific primers and Taqman probes were designed to establish a multiplex real-time PCR assay. Methods were evaluated by comparison of specificity, sensitivity and mixed sample detection and sequencing. Results The ITS and psbA-trnH mutations were higher than rbcL and matK, and rbcL and matK were significantly lower than ITS and psbA-trnH genes by genetic distance analysis. And the sensitivity of the establish multiple real-time PCR using ITS as the target gene was 0.01 ng. Four samples of adulterants were detected in 18 samples, and the results were consistent with the results of NJ tree clustering analysis. Conclusion Based on the ⅡS region sequence as the target gene to establish multiple real-time fluorescence PCR detection method can successfully identify Fritillariae Cirrhosae Bulbus and its counterfeit goods, which provides a new basis for the authenticity of identification.
[中图分类号]
R282.12
[基金项目]
山东省创新公共服务平台:中药分子鉴定公共服务平台(2018JGX111);山东省农业科学院农业科技创新工程:山东道地药材产业关键技术创新与应用(CXGC2018D3);山东省高等学校对接产业类协同创新中心:中药质量控制与全产业链建设协同创新中心;山东省重点研发计划:30种柴胡SNP指纹图谱芯片的开发及利用(2018GSF11902)