A Novel Quantification Method for Gene-Edited Animal Detection Based on ddPCR
Kaili Wang,
Yi Ji,
Cheng Peng,
Xiaofu Wang,
Lei Yang,
Hangzhen Lan,
Junfeng Xu,
Xiaoyun Chen
Affiliations
Kaili Wang
School of Food Science and Engineering, Ningbo University, Ningbo 215211, China
Yi Ji
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, Hangzhou 310021, China
Cheng Peng
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, Hangzhou 310021, China
Xiaofu Wang
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, Hangzhou 310021, China
Lei Yang
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, Hangzhou 310021, China
Hangzhen Lan
School of Food Science and Engineering, Ningbo University, Ningbo 215211, China
Junfeng Xu
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, Hangzhou 310021, China
Xiaoyun Chen
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, Hangzhou 310021, China
As gene-editing technologies continue to evolve, gene-edited products are making significant strides. These products have already been commercialized in the United States and Japan, prompting global attention to their safety and regulatory oversight. However, the detection of gene editing still relies on qPCR, and there is a lack of quantitative detection methods to quantify gene-editing components in products. To ensure consumer safety and transparency, we developed a novel droplet digital PCR (ddPCR)-based detection method for gene-edited products. Primers and probes were designed targeting the editing sites of MSTN-edited cattle, and the method was evaluated for specificity, sensitivity, real sample testing, and detection thresholds. Our results demonstrate that this ddPCR method is highly specific, with a detection limit of 5 copies/µL, and it successfully detected MSTN edits in all 11 tested samples. Tests using both actual gene-edited cattle samples and plasmid DNA at concentrations of 5%, 1%, and 0.01% yielded consistent results, indicating the method’s suitability for real-world applications. This ddPCR assay provides a sensitive and specific tool for detecting MSTN gene-edited cattle and determining the presence of gene-edited products, offering crucial support for regulatory monitoring of gene-edited animal-derived foods.
