Phylogenetic relationship analysis of common medicinal submetals and multiple real-time fluorescence PCR identification of Fritillariae Cirrhosae Bulbus / 中草药

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.