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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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在基因编辑食品中定性和定量检测CRISPR相关的Cas基因
Lin Ding1, Xiaoli Xu1, Xiaofu Wang1
1State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
Foods (Basel, Switzerland)
|October 14, 2023
概括
开发精确的Cas12a (Cpf1) 检测方法对于调节基因编辑食品至关重要. 本研究建立了针对CPF1检测的特定,敏感和稳定的定性和定量PCR测定方法.
科学领域:
- 农业生物技术 农业生物技术
- 分子生物学分子生物学
- 食品科学 食品科学 食品科学
背景情况:
- 基因编辑作物的工业化需要有效的监管和公众的接受.
- 与CRISPR相关的 (Cas) 蛋白质是基因编辑系统的关键组成部分,需要强大的检测方法.
- 准确的定性和定量检测Cas基因对于监测基因编辑产品至关重要.
研究的目的:
- 开发和验证用于检测Cas12a (Cpf1) 基因的特定,敏感和稳定的定性和定量PCR分析.
- 为监测基因编辑产品和衍生食品提供可靠的方法.
主要方法:
- 为Cas12a (Cpf1) 设计和选原料和探针.
- 优化PCR系统参数,包括原料度和回火温度.
- 通过特异性,敏感性和稳定性测试建立和验证定性PCR和定量PCR (qPCR) 分析.
主要成果:
- 定性PCR和qPCR测定在检测Cas12a (Cpf1) DNA时显示出100%的特异性.
- 定性PCR检测的检测极限为0.1% (约. 44份),而对于qPCR,则是14份.
- 这两种测试都显示出高稳定性,在60次重复测试中,在最低检测极限时产生积极结果.
结论:
- 为Cas12a (Cpf1) 开发的定性和定量PCR分析具有特异性,敏感性和稳定性.
- 这些测试为有效监测基因编辑产品及其衍生食品提供了必要的技术支持.
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