Duan Yabing, Zhang Xiaoke, Ge Changyan, Wang Yong, Cao Junhong, Jia Xiaojing, Wang Jianxin, Zhou Mingguo
College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China.
Sci Rep. 2014 Nov 18;4:7094. doi: 10.1038/srep07094.
Resistance of Fusarium graminearum to carbendazim is caused by point mutations in the β2-tubulin gene. The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field resistant isolates in China. To establish a suitable method for rapid detection of the F167Y mutation in F. graminearum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed and optimized to specially distinguish the F167Y mutation genotype. The LAMP reaction was optimal at 63 °C for 60 min. When hydroxynaphthol blue dye (HNB) was added prior to amplification, samples with DNA of the F167Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. The developed LAMP had good specificity, stability and repeatability and was suitable for monitoring carbendazim-resistance populations of F. graminearum in agricultural production.
禾谷镰刀菌对多菌灵的抗性是由β2-微管蛋白基因突变引起的。在中国，超过90%的田间抗性分离株中存在密码子167处的点突变（TTT → TAT，F167Y）。为建立一种快速检测禾谷镰刀菌中F167Y突变的合适方法，基于环介导等温扩增（LAMP）开发了一种高效、简便且特异性高的方法。设计并优化了一组四条引物，以专门区分F167Y突变基因型。LAMP反应在63℃下进行60分钟效果最佳。在扩增前加入羟基萘酚蓝染料（HNB）时，含有F167Y突变DNA的样品在反应后呈现出特征性的天蓝色，而无DNA或含有不同DNA的样品则无此现象。通过凝胶电泳再次证实了HNB染色法的结果。所开发的LAMP具有良好的特异性、稳定性和重复性，适用于农业生产中监测禾谷镰刀菌对多菌灵的抗性群体。
