Ding Lin, Xu Xiaoli, Wang Xiaofu, Chen Xiaoyun, Lu Yuwen, Xu Junfeng, Peng Cheng
State 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.
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of MARA and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, China.
Foods. 2023 Oct 7;12(19):3681. doi: 10.3390/foods12193681.
Effective regulation of gene-edited products and resolution of public concerns are the prerequisites for the industrialization of gene-edited crops and their derived foods. CRISPR-associated protein, the core element of the CRISPR system, requires to be regulated. Thus, there is an urgent need to establish qualitative and quantitative detection methods for the gene. In the present study, the primers and probes were designed and screened for (), which is the most commonly used target site in gene editing; we performed PCR system optimization, determined the optimal primer concentration and annealing temperature, and established qualitative PCR and quantitative PCR (qPCR) assays for detecting in gene editing by specificity and sensitivity tests. In specificity testing, qualitative PCR and qPCR methods could 100% detect samples containing DNA, while the detection rate of other samples without was 0%. In the assay sensitivity test, the limit of detection of qualitative PCR was 0.1% (approximately 44 copies), and the limit of detection of the qPCR method was 14 copies. In the stability test, both the qualitative PCR and qPCR methods were repeated 60 times at their corresponding lowest detection limit concentrations, and the results were positive. Thus, the qualitative and quantitative assays for are specific, sensitive, and stable. The method provides technical support for the effective monitoring of gene-edited products and their derived foods in the future.
对基因编辑产品进行有效监管并解决公众关切问题是基因编辑作物及其衍生食品产业化的前提条件。CRISPR相关蛋白作为CRISPR系统的核心元件，需要进行监管。因此，迫切需要建立针对该基因的定性和定量检测方法。在本研究中，针对基因编辑中最常用的靶位点（）设计并筛选了引物和探针；我们对PCR体系进行了优化，确定了最佳引物浓度和退火温度，并通过特异性和灵敏度测试建立了用于检测基因编辑中（）的定性PCR和定量PCR（qPCR）检测方法。在特异性检测中，定性PCR和qPCR方法能够100%检测到含有（）DNA的样本，而其他不含（）的样本检测率为0%。在检测灵敏度测试中，定性PCR的检测限为0.1%（约44个拷贝），qPCR方法的检测限为14个拷贝。在稳定性测试中，定性PCR和qPCR方法均在其相应的最低检测限浓度下重复进行60次，结果均为阳性。因此，针对（）的定性和定量检测方法具有特异性、灵敏性和稳定性。该方法为未来有效监测基因编辑产品及其衍生食品提供了技术支持。
