基于便携式和CFX96实时荧光PCR仪的冬虫夏草及其混伪品鉴定研究

Identification of Ophiocordyceps sinensis and its adulterants based on portable and CFX96 real-time fluorescent PCR systems

采用TaqMan探针实时荧光PCR方法对冬虫夏草及其混伪品进行分子鉴定研究。从100份冬虫夏草及其混伪品中提取总DNA,依据核糖体(rDNA)内部转录间隔区(internal transcribed spacer, ITS)序列,利用MEGA7.0软件进行比较分析,找出冬虫夏草及其混伪品的变异位点,通过Primer Premier 6.0软件设计一对特异性引物和TaqMan探针,分别在两种实时荧光PCR仪(Genesig q16和Bio-Rad CFX96)上进行灵敏度和特异性鉴定研究。灵敏度研究表明,在Bio-Rad CFX96系统中,该方法对冬虫夏草DNA模版的检测下限为0.016 ng·μL^(-1);在Genesig q16系统中,该方法对冬虫夏草DNA模版的检测下限为15.527 ng·μL^(-1)。特异性鉴定研究表明,在Genesig q16和Bio-Rad CFX96系统上,该方法对冬虫夏草均具有良好的特异性,能与混伪品下垂虫草、古尼虫草、蛹虫草、蝉花、凉山虫草、新疆虫草明显区分。TaqMan探针实时荧光PCR方法可实现对冬虫夏草及其混伪品的准确鉴定,为药材市场的管理提供技术支撑,对名贵中药材的鉴别具有较好应用前景。

The molecular identification of Ophiocordyceps sinensis and its adulterants was carried out by real-time fluorescent PCR with TaqMan probe. Genomic DNA was extracted from 100 samples of Ophiocordyceps sinensis and its adulterants. MEGA 7.0 software was used for comparative analysis to define the variable sites between Ophiocordyceps sinensis and its adulterants according to the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). A set of specific primers and TaqMan probe were designed using Primer Premier 6.0 software, and sensitivity and specificity studies were performed on two different real-time fluorescent PCR systems (Genesig q16 and Bio-Rad CFX96). The sensitivity study showed that the detectable DNA template concentration of Ophiocordyceps sinensis for the real-time fluorescent PCR was 0.016 ng·μL^-1 in the Bio-Rad CFX96 system and 15.527 ng·μL^-1 in the Genesig q16 system, respectively. Meanwhile, this method had good specificity for Ophiocordyceps sinensis on Genesig q16 and Bio-Rad CFX96 systems, so Ophiocordyceps sinensis could be clearly distinguished from Ophiocordyceps nutans, Cordyceps gunnii, Cordyceps militaris, Cordyceps cicadae, Cordyceps liangshanensis, Cordyceps gracilis. Our results indicate that real-time fluorescent PCR with TaqMan probe can be used to accurately identify Ophiocordyceps sinensis from its adulterants. This provides a technical method that has wide applications for market management and quality control of Chinese materia medica.