小麦种子规模化DNA提取及检测体系的优化

以小麦种子分子检测农业行业标准(NY/T 2859-2015)为基础,开发小麦种子快速、规模化提取DNA方法,优化反应体系中各组分含量,为小麦真实性快速执法提供技术支撑。通过对SDS、CTAB、高盐低p H、快速提取和试剂盒5种方法提取的DNA质量、浓度和PCR扩增效果进行比较,发现改进的高盐低p H方法提取种子的DNA质量、浓度能够满足小麦真实性鉴定中42对SSR引物重复鉴定的需求,是利用96孔深孔板和自动化移液工作站规模化、高通量提取DNA的较优方法。进一步优化结果显示该方法在65℃温浴条件下比室温的浓度高出25%,但两种温度条件下提取的DNA质量及浓度均能够满足品种鉴定的需求;沉淀时用0.5倍提取液体积的预冷异丙醇沉淀浓度最高,用室温的异丙醇沉淀的最佳体积是提取液体积的0.6倍。对标准中42对引物的最佳引物浓度和模板浓度均进行了优化,综合所有42对引物的优化结果发现,反应体系为20μL时,引物终浓度为0.437 5μmol/L,模板终浓度为10 ng/μL时扩增效率相对较高,扩增产物稳定,能够满足多重电泳的需求。

Large-scale DNA Extraction and Optimization of Detection System for Wheat Seeds

Based on the industry standard of wheat authenticity SSR (NY/T 2859-2015), this study developed a rapid and large-scale DNA extraction method for wheat seeds, and optimized the content of components in the reaction system toprovide technical support for rapid enforcement of wheat authenticity law. By comparing the DNA quality, concentration and PCR amplification effect that extracted by five methods such as SDS, CTAB, high-salt low-pH, rapid extraction and kit, it was found that the improved method of the high-salt low-pH could meet the demand of 42 SSR primers for repeated identification in wheat authenticity identification. It was the better method for 96-well deep-hole plate and automated liquid transfer station. The results of further optimization showed that the concentration of this method at room temperature was 25% higher than that at at 65℃, but the quality and concentration of DNA extracted at both temperatures could meet the requirements of variety identification. The highest concentration of DNA was obtained with 0.5-fold volume of precooled isopropanol, and with 0.6-fold volume of isopropanol at room temperature. The optimal primer concentration and template concentration of 42 pairs of primers in the standard were optimized. The results showed that when the reaction system was 20 μL, the final primer concentration was 0.437 5 μmol/L, and the final template concentration was 10 ng/μL, the amplification efficiency was relatively high, and the amplification products were stable, which could meet the needs of multiple electrophoresis.