摘要
为建立同时鉴别霍山石斛、铁皮石斛及齿瓣石斛的多重位点特异性PCR法,解决上述石斛属药材真伪掺杂的难题。通过比对石斛药材ITS及trn L-trn F序列,设计引物,建立多重PCR鉴别法,并对不同来源的霍山石斛、铁皮石斛及齿瓣石斛样品进行特异性扩增,根据特异性条带大小进行鉴别。结果在退火温度为61℃、循环数为35时,齿瓣石斛、铁皮石斛和霍山石斛分别可扩出584、397和211 bp的特异性条带。本文所建立的方法对齿瓣石斛和霍山石斛的最低检测限为1.2 ng,铁皮石斛低于0.24 ng,其对铁皮石斛、齿瓣石斛和霍山石斛的混伪检出限分别为1%、1%和5%。表明多重位点特异性PCR法能特异性鉴别霍山石斛、铁皮石斛及齿瓣石斛。
This study was designed to establish a multiplex allele-specific polymerase chain reaction method for simultaneous identification of Dendrobium huoshanense, D. officinale and D. devonianurn, which may resolve identification problems of caulis dendrobii. Internal transcribed spacer sequences and trnL-trnF sequences of the Dendrobium species were aligned by BioEdit software, then specific SNPs of the three species were analyzed for designing allele-specific primers and the multiplex allele-specific PCR reaction system was established. The different origin of Dendrobium huoshanense, D. officinale and D. devonianum was amplified and identified by the sizes of respective band. The results showed that 584 bp, 397 bp and 211 bp bands could be amplified by D. devonianum, Dendrobium officinale and Dendrobium huoshanense respectively, when the annealing temperature was 61 ℃ and the number of cycles was 35. The limit of detection （LOD） of D. devonianum and D. huoshanense were both 1.2 ng, while D. officinale was low than 0.24 ng. The detection limit of adulterates in D. devonianum, D. devonianurn and D. huoshanense mixture sample was 1%, 1% and 5% respectively. This result suggests that the method of multiplex allele-specific PCR is useful to identify D. huoshanense, D. officinale and D. devonianum is accurate and specific.
出处
《药学学报》
CAS
CSCD
北大核心
2017年第6期998-1006,共9页
Acta Pharmaceutica Sinica
基金
中医药行业科研专项“常用大宗中药材质量现场快速检测技术研究”(201407003)
国家杰出青年科学基金项目(81325023)
北京市科委-中药品质评价关键技术体系与平台建设
关键词
霍山石斛
铁皮石斛
齿瓣石斛
多重位点特异性PCR
掺伪鉴别
Dendrobium huoshanense
Dendrobium officinale
Dendrobium devonianum
multiplex allele-specific polymerase chain reaction
admixture identification
