基于特异性聚合酶链式反应的西红花快速分子鉴别研究

 目的 建立一种基于特异性聚合酶链式反应(PCR)及荧光染料检测快速鉴别西红花真伪品的方法。方法 通过对叶绿体基因片段测序和比对分析,找出西红花与其常见掺伪品序列差异,针对差异碱基设计特异性引物,构建并优化聚合酶链式反应扩增反应体系,使用荧光染料法检测聚合酶链式反应产物,实现西红花的快速鉴别。结果 建立了基于psbA-trnH序列的西红花鉴别体系,优化后的聚合酶链式反应反应条件为90 ℃预变性1 min,90 ℃变性5 s,58 ℃延伸5 s,26个循环。在聚合酶链式反应反应产物中加入染料SYBR Green I,西红花正品出现强烈绿色荧光,而伪品无荧光。结论 位点特异性聚合酶链式反应方法可以简单快速鉴别西红花及其掺伪品。

Rapid Molecular Identification of Saffron (Crocus sativus L.) Based on Specific PCR

OBJECTIVE To establish a rapid molecular method for identifying saffron (Crocus sativus L.) and its adulterants by PCR amplification using specific primers and fluorescent dyes detection. METHODS The chloroplast barcode was sequenced and analyzed to find the SNPs between saffron and its adulterants. Specific primers were designed for the SNPs, the PCR reaction systems were built and optimized, and fluorescent dyes method was used to identify PCR products. RESULTS A 421 bp saffron identification band based on the psbA-trnH barcode sequence was screened when 100× SYBR Green I was added into the optimized PCR product under the following condition: initial denaturation at 90 ℃ for 1 min, denaturation at 90 ℃ for 5 s, annealing at 58 ℃ for 5 s,26 cycles; the saffron (Crocus sativus L.) showed strong green fluorescence under 365 nm UV lamp whereas adulterants did not. CONCLUSION Fast site-specific PCR can rapidly identify saffron and its adulteration.