基于RPA-CRISPR-Cas12a和聚噻吩显色技术快速检测转基因植物外源基因CP4-EPSPS

Rapid detection of transgenic plant exogenous CP4-EPSPS gene based on RPA-CRISPR-Cas12a and polythiophene chromogenic technology

目的开发基于重组酶恒温扩增(recombinase polymerase amplification,RPA)结合簇状规则间隔短链重复序列及相关蛋白(clustered regularly interspaced short palindromic repeats-associated 12a protein,CRISPR-Cas12a)和聚噻吩显色技术快速检测转基因植物外源基因CP4-EPSPS的方法。方法利用Cas12a附属切割机制进行精准识别RPA产物中的靶标,结合阳离子水溶性共轭聚噻吩(cationic water-soluble conjugated polythiophene,PMNT)与体系中单链DNA(single stranded DNA,ssDNA)的颜色变化检测转基因产品中耐除草剂草甘膦CP4-EPSPS抗性基因。结果167 bp的RPA引物进行扩增时,扩增体系为20μL时条带最清晰;ssDNA的碱基数为15,Cas12a酶的浓度为0.28 U为最优组合。进行RPA-CRISPR-Cas12a反应后,加入PMNT溶液,裸眼观察是溶液从红色变为黄色,紫外下照射下则颜色为橘红色变为橙黄色,检出限为45拷贝。结论构建了一种基于PMNT颜色变化进行转基因产品的CRISPR-Cas12a的检测方法,30 min内完成整个检测过程,仅需要恒温加热可实现快速灵敏、可视化的检测。

Objective To develop a method for rapid detection of transgenic plant exogenous CP4-EPSPS gene based on recombinase polymerase amplification(RPA)combined with clustered regularly interspaced short palindromic repeats-associated 12a protein(CRISPR-Cas12a)and polythiophene chromogenic technology.Methods The Cas12a accessory cleavage mechanism was used to accurately identify the target in the RPA product,and the color change of cationic water-soluble conjugated polythiophene(PMNT)and single stranded DNA(ssDNA)in the system was combined to detect the transgenic plant exogenous CP4-EPSPS.Results The RPA primer of 167 bp was selected for amplification,and the band was the clearest when the amplification system was optimized to 20μL,the number of bases of ssDNA was 15,and the concentration of Cas12a enzyme was 0.28 U as the best combination.After RPA-CRISPR-Cas12a reaction,PMNT solution was added.The solution changed from red to yellow under naked eye observation,and then changed from orange-red to orange-yellow under ultraviolet irradiation.The limit of detection was 45 copies.Conclusion A CRISPR-Cas12a detection method for transgenic products based on PMNT color changes has been constructed.The whole detection process is completed within 30 min,and only constant temperature heating is needed to realize rapid,sensitive and visual detection.