基于ITS2序列和特定引物区分铁皮石斛和伪品的PCR鉴别方法

Distinguishment Method of Tiepi Shihu(Dendrobium Officinale) from Counterfeit Species Based on DNA ITS2 Sequences and Specific Primer

目的:应用ITS2序列差异对铁皮石斛(Dendrobiumofficinale)和类似石斛品种进行归类分析,并建立快速分析鉴别特定铁皮石斛的方法。方法:提取9批石斛药材品种的DNA,采用ITS2通用引物进行PCR扩展,测序。对ITS2序列进行BLAST序列比对,Bio Edit软件进行Clustal W多重对比,针对差异序列设计特异引物,PCR扩增,通过琼脂糖电泳进行铁皮石斛特定品种的鉴别。结果:碱基序列配对BLAST未能将#1~4样品准确归类,#5~9确定为铁皮石斛(Dendrobiumofficinale)。9批样品进行ITS2序列多重对比,发现#1~4样品与#5~9样品的ITS2序列在23~49 bp位置存在显著差异,采用软件设计引物ITS2-specific R和ITS2-specific F,在退火温度为60℃PCR扩增,#5~9样品在136 bp出现条带,而#1~4在相应位置样品未见条带。结论:特定铁皮石斛品种的ITS2序列与伪品存在局部差异,能够通过PCR方法与伪品进行区分。该PCR鉴别方法将为区分特定铁皮石斛和伪品提供快速、有效的鉴别方法。

Objective： This study aims to distinguish Tiepi Shihu （dendrobium officinale）from related coun- terfeit species, and to establish quick identifieation method for Tiepi Shihu （dendrobium officinale）based on PCR. Methods： DNA of 9 batches of dendrobium species was extracted by using the CTAB method, and their ITS2 was amplified by using common primers before ITS2 sequencing. Alignment was performed by BLAST, and ITS2 sequence was analyzed by ClustalW of BioEdit software. Afterward, specific primer for distinguishing Tiepi Shihu（dendrobium officinale） from counterfeit was designed by Primer and employed for PCR. Then, PCR prod- ucts were condueted with an annealing temperature of 60 ℃ and put on electrophoresis for validation. Results： As our results show, there was a significant difference between Tiepi Shihu （dendrobium officinale）and related counterfeit species from 23 to 49 bp in ITS2 sequence, but ITS2 sequence can satisfy identification of concise species by BLAST in database. Therefore, based on the difference in ITS2 sequence, the specific primer for Tiepi Shihu （dendrobium offieinale）was designed for PCR amplification, and 60℃ was selected as annealing temperature. Bands of Tiepi Shihu （dendrobium officinale）（batch #5-9）appeared at 136 bp, while no bang was observed #1-4 in that same place in batch. Conclusion： Tiepi Shihu （dendrobium officinale）is possible to be distinguished from related counterfeit species based on DNA ITS2 sequences and specific primer, which serves as a fast and efficient method for identification of Tiepi Shihu （dendrobium officinale）.