摘要
目的利用特异性引物、DNA条形码技术对松花粉、蒲黄进行鉴定。方法利用内源转录间隔区(ITS)通用引物对松花粉、蒲黄进行扩增并测序,获得ITS碱基序列。基于ITS碱基序列设计松花粉、蒲黄特异性引物,并对特异性引物、退火温度、模板量、特异性引物量、循环次数、检测限及混合样品检测能力进行考察。利用协同结合试剂(SYBR)荧光定量聚合酶链反应(PCR)对松花粉、蒲黄的特异性引物进行验证并做熔解曲线,获得松花粉、蒲黄的Tm值。利用MEGA软件对测序获得的碱基序列构建邻接(NJ)系统发育树。结果基于松花粉、蒲黄ITS设计的引物只能对松花粉、蒲黄进行扩增,分别在230,193 bp处出现特异性条带。最佳扩增体系和条件为:退火温度62℃,特异性引物量1.0μL,模板量0.1μL(110 ng/μL),循环次数28次,最低检测限0.1 pg/μL。当松花粉、蒲黄相互掺伪比例达2%时,依然能够对二者进行鉴别。松花粉、蒲黄的特异性引物熔解曲线对应的Tm值分别为88.5,77.5℃;通过松花粉、蒲黄ITS2碱基序列构建的NJ系统发育树显示,二者分别独立聚为一支。结论设计的松花粉、蒲黄特异性引物能够准确地对松花粉、蒲黄进行鉴定,SYBR荧光定量PCR技术能够根据其熔解曲线Tm值对松花粉、蒲黄进行鉴定,DNA条形码NJ系统发育树能够准确鉴定松花粉、蒲黄。
Objective To identify pine pollen and typha pollen using specific primers and DNA barcoding technology.Methods The internal transcribed spacer(ITS)universal primers were used to amplify and sequence pine pollen and typha pollen,obtaining their ITS base sequences.Specific primers for pine pollen and typha pollen were designed based on the ITS sequences,and their specificity,annealing temperature,template quantity,primer concentration,cycle number,detection limit,and ability to detect mixed samples were evaluated.SYBR fluorescent quantitative PCR was employed to validate the specific primers,with melting curves analyzed to determine the Tm values.A neighbor-joining(NJ)phylogenetic tree was constructed using MEGA software based on the ITS2 sequences.Results Primers designed for pine pollen and typha pollen exclusively amplified their respective targets,producing specific bands at 230 bp and 193 bp.The optimal amplification system and conditions were as follows:annealing temperature of 62℃,1.0μL of specific primers,0.1μL of template DNA(110 ng/μL),and 28 cycles,with a minimum detection limit of 0.1 pg/μL.The method reliably distinguished between pine pollen and typha pollen even when adulterated at 2%.The Tm values from the melting curves of the specific primers were 88.5℃and 77.5℃for pine pollen and typha pollen,respectively.The NJ phylogenetic tree based on ITS2 sequences showed that pine pollen and typha pollen clustered into distinct branches.Conclusion The designed specific primers enable accurate identification of pine pollen and typha pollen.SYBR fluorescent quantitative PCR,combined with Tm values from melting curves,and the DNA barcoding-based NJ phylogenetic tree provide reliable methods for authenticating these pollens.
作者
王孟虎
徐义菁
郭雯雅
吴亚楠
张圣阳
钟明月
陈晨昱
孟祥松
WANG Menghu;XU Yijing;GUO Wenya;WU Yanan;ZHANG Shengyang;ZHONG Mingyue;CHEN Chenyu;MENG Xiangsong(School of Chinese Medicine,Bozhou University,Bozhou,Anhui,236800,China)
出处
《甘肃中医药大学学报》
2025年第4期44-51,共8页
Journal of Gansu University of Chinese Medicine
基金
安徽省优秀青年培育项目(YQYB2024085)
亳州市重点研发项目(bzzc2023030,bzzc2024057)
安徽省高校科学研究项目(KJ2021A1143)
安徽高校协同创新项目(GXXT-2023-073)。
