三七染料法实时荧光聚合酶链式反应鉴别方法的建立  

Establishment of a real-time fluorescent polymerase chain reaction method for identification of Panax notoginseng based on fluorescent dye

作　　者：张芹 季超 汪星宇 李娟 伍志强[1,2] 郭俊利 马荥 郑文杰 曹际娟 ZHANG Qin;JI Chao;WANG Xing-Yu;LI Juan;WU Zhi-Qiang;GUO Jun-Li;MA Ying;ZHENG Wen-Jie;CAO Ji-Juan(College of Life Sciences,Tianjin Normal University,Tianjin 300387,China;Tianjin Key Laboratory of Conservation and Utilization of Animal Diversity,Tianjin Normal University,Tianjin 300387,China;State Key Laboratory for Conservation and Utilization of Yunnan Biological Resources,Yunnan Agricultural University,Kunming 650201,China;Zheng Wenjie Expert Workstation of Yunnan Province,Southwest Forestry University,Kunming,650224,China;Key Laboratory of Biotechnology and Resource Utilization of Ministry of Education,Dalian Minzu University,Dalian 116000,China)

机构地区：[1]天津师范大学,生命科学学院,天津300387 [2]天津师范大学,天津市动物多样性保护与利用重点实验室,天津300387 [3]云南农业大学,云南生物资源保护与利用国家重点实验室,昆明650201 [4]西南林业大学,云南省郑文杰专家工作站,昆明650224 [5]大连民族大学,生物技术与资源利用教育部重点实验室,大连116000

出　　处：《食品安全质量检测学报》2022年第3期894-901,共8页Journal of Food Safety and Quality

基　　金：云南省重大科技专项(202102AE090042);云南绿色食品国际合作研究中心项目子课题项目(2019ZG00901-04);生物技术与资源利用教育部重点实验室项目(KF2021006)。

摘　　要：目的采用TB Green染料法实时荧光聚合酶链式反应(polymerase chain reaction,PCR)技术,基于叶绿体基因psbA-trnH序列建立鉴别三七的实时荧光PCR方法。方法通过比较三七及其同属的人参、西洋参和竹节参的psbA-trnH序列,设计出一对三七特异性引物,优化退火温度,进行灵敏度实验,并验证方法的特异性、适用性和抗干扰性,最后对市售三七样品进行检测。同时以18SrDNA引物为内参质控,对DNA提取及反应体系进行质控。结果所建立的三七染料法实时荧光PCR方法灵敏度为0.001 ng/μL;特异性较好,与18种近缘及常见中药材均无交叉反应;适用性和抗干扰性也较好;对28份不同加工方式的市售三七样品检测的结果与测序结果一致。结论建立的三七染料法实时荧光PCR方法具有快速、准确、灵敏的特点,可用于三七产品中三七成分的鉴别。Objective To establish a real-time fluorescent polymerase chain reaction(PCR)method for the identification of Panax notoginseng based on the psb A-trnH sequence of chloroplast gene using fluorescent PCR technique by TB Green dye method.Methods By comparing the psbA-trnH sequences of Panax notoginseng and Panax ginseng,Panax quinquefolius and Panax japonicus,a pair of Panax notoginseng-specific primers was designed,the annealing temperature was optimized,sensitivity experiments were performed,and the specificity,applicability and anti-interference of the method were verified,and finally,commercial samples of Panax notoginseng were detected.The 18 S rDNA primer was used as the internal reference for quality control of DNA extraction and reaction system.Results The sensitivity of the established real-time fluorescence PCR method with Panax notoginseng dye method was 0.001 ng/μL;the specificity was good,and there was no cross-reactivity with 18 kinds of closely related and common Chinese herbal medicinal materials;the applicability and anti-interference were also good;the results of the detection of 28 commercially available Panax notoginseng samples with different processing methods were consistent with the sequencing results.Conclusion The established real-time fluorescence PCR method of Panax notoginseng dye method is rapid,accurate and sensitive,and can be used to identify Panax notoginseng components in Panax notoginseng products.

关 键 词：三七 实时荧光聚合酶链式反应 染料法 鉴别 

分 类 号：R282.5[医药卫生—中药学]