地骨皮的多重位点特异性PCR鉴别

Identification of Original Plants of Lycii Cortex by Multiplex Allele-specific PCR

目的:使用多重位点特异性聚合酶链式反应(PCR)的方法,简单高效地鉴定地骨皮(Lycii Cortex)的2种基原植物枸杞(Lycium chinense)和宁夏枸杞(L.barbarum)。方法:在使用植物叶绿体基因组数据库(CGIR)获取枸杞和宁夏枸杞叶绿体基因组序列的基础上,利用IdenDSS软件筛选出两基原间特定的单核苷酸多态性(SNP)位点,并利用Primer 5.0软件设计获得特异性鉴别引物,包括鉴别枸杞的引物GQ-F/R、鉴别宁夏枸杞的引物NX-F/R。对引物浓度比、退火温度、循环次数、Taq酶进行优化,形成最适的PCR鉴别体系和条件,并对采集自不同地区的地骨皮进行适用性考察。结果:对影响PCR结果的各个条件进行考察优化后,最终确定枸杞和宁夏枸杞的引物浓度比为2∶1、退火温度为59℃、循环次数为30次时,来自不同产区地骨皮的2个基原枸杞和宁夏枸杞分别在183、295 bp的位置出现特异性鉴别条带。结论:通过一次PCR即可方便、快捷、高效地鉴定地骨皮的基原,为枸杞和宁夏枸杞的专属性鉴别提供新方法,同时对含有地骨皮成分的经典名方的研发提供一定的技术支撑。

Objective:To identify Lycium chinense and L.barbarum as the original plants of Lycii Cortex simply and efficiently by multiple allele-specific polymerase chain reaction(PCR).Method:The chloroplast genome sequences of L.chinense and L.barbarum were downloaded from the Chloroplast Genome Information Resource(CGIR),and then IdenDSS was employed to screen out the specific single nucleotide polymorphism(SNP)sites between the two plants.Primer 5.0 was used to design the specific primers,including primers GQ-F/R for identifying L.chinense and primers NX-F/R for identifying L.barbarum.Furthermore,the primer concentration ratio,annealing temperature,cycles,and Taq enzyme were optimized to establish the optimal PCR system and conditions for plant identification.Finally,the applicability of the established method was examined with the plant samples collected from different regions.Result:The PCR with GQ-F/R∶NX-F/R concentration ratio of 2∶1 at the annealing temperature at 59℃and for 30 cycles showed specific bands at 183 bp and 295 bp,respectively,for L.chinense and L.barbarum samples from different regions.Conclusion:The established PCR approach can simply,rapidly,and efficiently identify the original plants of Lycii Cortex,serving as a new method for the discrimination between L.chinense and L.barbarum.Moreover,the method provides technical support for the research and development of classic famous prescriptions containing Lycii Cortex.