利用SSR快速鉴定甜菜品种纯度和真实性的研究

Research of the Rapid Identification System for Purity and Authenticity of Sugarbeet Variety by Using SSR Markers

为建立一套快速鉴定甜菜品种纯度和真实性的实验体系,对DNA的提取、检测、PCR反应体系、程序、电泳以及显影方法等多个方面进行了优化,最终确立了一套简单、快速、节约、污染小的甜菜品种纯度和真实性的鉴定体系。该方法使用96孔PCR板,利用碱裂解法提取干种子(或者叶片干粉)DNA;使用无毒的Gelred替代致癌性的EB,利用λDNA检测提取样品DNA的浓度;PCR反应的体系为5μL,使用多重PCR替代单一PCR;PCR反应程序为94℃变性15 s,退火15 s,72℃延伸30 s,30个循环,最后72℃延伸5 min;PCR产物的分离使用8%非变性聚丙烯酰胺凝胶电泳,最后使用快速银染法对电泳后的聚丙烯酰胺凝胶进行显影。利用优化后的程序,一位成熟的实验室操作人员只需1天就可以完成192份样品的检测。

The study aims to establish an experimental system of rapid identification of beet variety purity and authenticity, to optimized extraction, detection of DNA, PCR reaction system, program, electrophoresis and the method of developing multiple aspects, and finally to set up a set of simple, rapid, economical and less polluted identification system for sugarbeet variety purity and authenticity. With 96- hole PCR plates, alkali decomposition method was used to extract DNA from dried seed（or leaf powder）. Using nontoxic Gelred substitute carcinogenic EB, DNA concentration was extracted with λDNA. PCR reaction system was 5 μL,using multiple PCR to replace a single PCR; PCR reaction procedure was 94℃, degeneration 15 s, annealing 15 s, extension 72℃ for 30 s, 30 cycles and extended at 72℃ for 5 min finally. Separation of the PCR products was conducted using 8% of Native-PAGE, and rapid argentation was used to develop polyacrylamide gel after electrophoresis. Using the optimized program, a skillful laboratory operator can complete 192 samples of detection within one day.