玉米种子不同部位基因组提取效率对CRISPR/Cas12a检测体系的影响

Effects of genomic extraction efficiency from different parts of maize seeds on CRISPR/Cas12a-based detection system

中文关键词:  转基因玉米种子  基因组提取  核酸检测  成簇规律间隔短回文重复序列及相关蛋白12a

英文关键词:genetically modified maize  genomic extraction  nucleic acid testing  clustered regularly interspaced short palindromic repeats/associated protein 12a

中文摘要:

      目的 探究玉米种子不同部位在基因组提取和核酸检测中的表现差异, 明确最优选择和对成簇规律间隔短回文重复序列及相关蛋白12a (clustered regularly interspaced short palindromic repeats/associated protein 12a, CRISPR/Cas12a)检测体系结果的影响。方法 本研究比较了玉米种子胚、胚乳和全籽粒的基因组提取效率; 通过常规聚合酶链式反应(polymerase chain reaction, PCR)级联CRISPR/Cas12a系统, 对各部位提取产物进行靶标扩增和识别, 评估所提取基因组对后续核酸检测的影响; 通过对比DNA提取效率、扩增效果和荧光信号强度, 验证各部位样本在检测中的适应性。结果 玉米种子不同组织部位的DNA提取效率存在差异, 胚部基因组含量最高、全籽粒次之, 胚乳相对较低, 符合玉米种子生物学结构特征; 各部位基因组均能成功扩增, 级联CRISPR/Cas12a系统检测产生明确阳性信号; 本研究构建的方法定性检出限为0.1 wt%, 符合大多数国家和地区对转基因成分的检测要求。结论 玉米种子不同部位基因组提取效率存在差异, 但均具备良好的核酸扩增和CRISPR/Cas12a检测适应性, 在实际检测中全籽粒取样即可满足检测灵敏度且操作更便捷。本研究开发的方法无需大型仪器设备, 能够满足对转基因玉米种子现场检测的需求, 具备转基因食品安全筛查的应用价值。

英文摘要:

      Objective To explore the performance differences of various maize seed tissues in genome extraction and nucleic acid testing to clarify the optimal choice and its impact on clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a)-based detection system. Methods In this study, the genome extraction efficiencies of maize seed embryo, endosperm and whole kernel were compared. Through the conventional polymerase chain reaction (PCR) cascade CRISPR/Cas12a system, the extracted products from each part were amplified and identified, and the impact of the extracted genome on subsequent nucleic acid detection was evaluated. By comparing the DNA extraction efficiency, amplification effect and fluorescence signal intensity, the adaptability of samples from various parts in the test was verified. Results There were differences in the efficiency of DNA extraction from different tissues of corn seeds. The genome concentration in the embryo was the highest, followed by the whole kernel, and the endosperm was relatively low, which was in line with the biological structural characteristics of corn seeds. The genomes in each part could be successfully amplified, and the cascade CRISPR/Cas12a system produced a clear positive signal. The qualitative detection limit of the method constructed in this study was 0.1 wt%, which met the detection requirements of genetically modified ingredients in most countries and regions. Conclusion Differences in extraction efficiency are found among maize seed tissues, but all parts supports effective nucleic acid amplification and CRISPR/Cas12a detection. In practical applications, tissue separation is not required; efficient and specific detection can be achieved using whole seeds or randomly selected samples. The method developed in this study does not require large-scale instrumentation and is suitable for on-site detection of genetically modified maize seeds, demonstrating potential for application in the safety screening of genetically modified food.