Wang Yuling, Peng Cheng, Ding Lin, Su Zhixun, Chen Xiaoyun, Wang Xiaofu, Sun Meihao, Xu Junfeng
College of Life Sciences, Zhejiang Normal University, Jinhua 321004, China.
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
Foods. 2023 Feb 1;12(3):615. doi: 10.3390/foods12030615.
CRISPR/Cas12a technology is used for nucleic acid detection due to its specific recognition function and non-specific single-stranded DNA cleavage activity. Here, we developed a fluorescence visualisation detection method based on PCR and CRISPR/Cas12a approaches. The method was used to detect the nopaline synthase terminator (T-nos) of genetically modified (GM) crops, circumventing the need for expensive instruments and technicians. For enhanced sensitivity and stability of PCR-CRISPR/Cas12a detection, we separately optimised the reaction systems for PCR amplification and CRISPR/Cas12a detection. Eleven samples of soybean samples were assessed to determine the applicability of the PCR-CRISPR/Cas12a method. The method could specifically detect target gene levels as low as 60 copies in the reaction within 50 min. In addition, accurate detection of all 11 samples confirmed the applicability. The method is not limited by large-scale instruments, making it suitable for mass detection of transgenic components in plants in the field. In conclusion, we developed a new, accurate, rapid, and cost-effective method for GM detection.
CRISPR/Cas12a技术因其特异性识别功能和非特异性单链DNA切割活性而被用于核酸检测。在此，我们基于PCR和CRISPR/Cas12a方法开发了一种荧光可视化检测方法。该方法用于检测转基因作物的胭脂碱合成酶终止子（T-nos），无需昂贵的仪器和技术人员。为提高PCR-CRISPR/Cas12a检测的灵敏度和稳定性，我们分别优化了PCR扩增和CRISPR/Cas12a检测的反应体系。对11份大豆样品进行评估，以确定PCR-CRISPR/Cas12a方法的适用性。该方法能够在50分钟内特异性检测反应中低至60拷贝的目标基因水平。此外，对所有11份样品的准确检测证实了该方法的适用性。该方法不受大型仪器的限制，适用于田间植物中转基因成分的大规模检测。总之，我们开发了一种新的、准确、快速且经济高效的转基因检测方法。
