抗虫耐除草剂玉米瑞丰125 RPA快速检测方法的建立

Development of a recombinase polymerase amplification(RPA)method for rapid detection of transgenic maize Ruifeng 125

瑞丰125为农业农村部首批获得生产应用农业转基因生物安全证书的抗虫耐除草剂转基因玉米之一。为给监管转基因生物安全提供支撑,利用重组酶聚合酶扩增(recombinase polymerase amplification,RPA)技术,针对瑞丰125的转化体插入位点序列设计了引物并进行筛选,对反应的温度、时间和引物浓度进行了优化。结果显示,RPA体系在30~45℃范围内,20 min即可有效扩增,比PCR检测时间大大缩短。引物浓度会影响RPA的扩增效率,引物终浓度为0.35μmol/L时扩增效果最好。同时对RPA检测方法的特异性、灵敏度等进行考察,结果表明该检测方法的特异性良好,检测灵敏度可达到36拷贝。该恒温快速的检测方法为瑞丰125的快速检测提供依据,为商业化转基因作物的监管提供支撑,有望用于转基因成分的现场快速检测。

Transgenic maize Ruifeng 125 is one of the first series of transgenic maize varieties requesting safe⁃ty certificate in China.To improve the supervise efficiency by rapid detection for Ruifeng 125,recombinase poly⁃merase amplification(RPA)technology was developed.RPA primes were designed according to the junction border between insertion DNA and the host genome.Meanwhile,the screening procedure,reaction temperature,time and primer concentration were also optimized.Results showed that developed RPA method had a wide range of tempera⁃ture from 30℃to 45℃,and could be implemented within 20 min,showing less time cost than PCR.The best primer concentration of 0.35μmol/L was adopted for obtaining satisfactory assay performance and better amplification effi⁃ciency.The results demonstrated that the PRA method was capable for rapid amplification of Ruifeng 125 with high specificity and sensitivity.The detection limit of this method was 36 copies.This work provides a promising plat⁃form for the detection of Ruifeng 125 or other commercial transgenic crops,and expects to develop of point-of-care identification.