马达加斯加龙树实时荧光定量PCR鉴定方法的研究.docxVIP

? ? 马达加斯加龙树实时荧光定量PCR鉴定方法的研究 ? ? 李井干 吴晶 伏建国 杨晓军 张明哲 Summary [目的]通过设计特异性引物与荧光探针，建立马达加斯加龙树（Didierea madagascariensis）实时荧光定量PCR鉴定方法。[方法]提取26份龙树科植物基因组，并用内源基因检测DNA质量;根据GenBank中已发表的马达加斯加龙树的PEPC基因序列，设计特异性引物与荧光探针，通过检测26份供试样本，检测该方法的特异性;将样品DNA进行10倍梯度稀释，以检测实时荧光定量PCR方法的灵敏度。[结果]只有3份马达加斯加龙树样本出现典型的扩增曲线，其他23份龙树科植物无典型的扩增曲线;核酸原液与10-1～10-4倍稀释液样本均能够得到典型扩增曲线，标准曲线的决定系数（R2）为0.995，检测限量为5.2×10-3 ng/μL。[结论]该方法特异性强、灵敏度高，可有效地应用于马达加斯加龙树的鉴定。 Key 马达加斯加龙树;实时荧光定量PCR;鉴定 Q 943? A 0517-6611（2022）12-0168-03 doi：10.3969/j.issn.0517-6611.2022.12.043 开放科学（资源服务）标识码（OSID）： Research on Identification of Didierea madagascariensis with Real-time PCR Method LI Jing-gan，WU Jing，FU Jian-guo et al （Animal，Plant and Food Inspection Center of Nanjing Customs，Nanjing，Jiangsu 210019） Abstract [Objective]A real-time fluorescent quantitative PCR detection method for Didierea madagascariensis was established by designing specific primers and fluorescent probes.[Method]26 samples of didiereaceae plants were extracted，and DNA quality was detected with endogenous genes.Specific primers and probe were designed according to the published PEPC genes of Didierea madagascariensis and were used to detecting 26 testing samples，the specificity and sensitivity were detected by screening 26 testing samples.The sample DNA was serially diluted 10 times to test the sensitivity of real-time PCR method.[Result]Typical amplification curves could be obtained for 3 Didierea madagascariensis samples，but not for other 23 samples.The nucleic acid stoste and its diluents from 10-1-10-4 times could also be deteced the signals，the determination coefficient （R2）of the standard curve was 0.995 and the detection limit was 5.2×10-3 ng/μL.[Conclusion]The method has strong specificity and high sensitivity，and can be effectively applied to the identification of Didierea madagascariensis. Key words Didierea madagascariensis;Real-time PCR;Identification 马达加斯加龙树（Didierea madagascariensis）属龙树科（Didiereaceae）刺棘木属（Didierea）[1]，马达加斯加特有种，因当地人类生活、农业生产、养牛场建设而变得濒