铁皮石斛灰霉病多菌灵高抗菌株的LAMP快速检测体系的建立

Establishment of Loop-mediated Isothermal Amplification Assay to Rapidly Detect Carbendazim High Resistant Strain of Botrytis cinerea

为了建立一种新型的能够快速、准确、高效地检测铁皮石斛灰霉病菌对多菌灵抗药性的检测技术,利用环介导的等温扩增技术(Loop-mediated isothermal amplification)通过对临安3个铁皮石斛基地的灰霉病菌进行抗性分析发现,本地铁皮石斛灰霉病菌对多菌灵表现出高水平抗药性的菌株编码β-微管蛋白的198位氨基酸由E(GAG)突变为V(GTG)。根据高抗菌株第198位密码子的突变位点设计了特异性检测引物,对高抗灰霉菌株E198V基因型进行分子检测。结果表明:使用钙黄绿素染料作为反应指示剂,在等温条件下(65℃)进行核酸扩增反应40 min后,以抗药性菌株DNA为模板的反应液呈阳性(变为亮绿色),而敏感菌株的为阴性(仍为橙色),灵敏度试验发现最低检测限为10 pg/μL,琼脂糖凝胶电泳结果与LAMP检测结果一致。该方法的建立为快速检测铁皮石斛灰霉病菌抗多菌灵菌株及科学合理的防治铁皮石斛灰霉病奠定了基础。

This study uses the loop-mediated isothermal amplification to set up a fast,accurate and efficient way to detect Botrytis cinerea that confered high resistant to carbendazim. Carbendazim resistance analysis of B. cinerea was conducted in three produce bases of Dendrobium candidumin in Lin'an county. We found that local high-level carbendazim resistance of B. cinerea was due to 198 amino acids mutantion of β-tubulin encoding protein from E( G AG) to V( GTG). In this study,point mutant was designed in primer F2 to detect the B. cinerea E198 V genotype that confered high resistant to carbendazim. The LAMP assay efficiently amplified the target DNA in 40 min under isothermal conditions at 65 ℃ and was evaluated for specificity and sensitivity. A positive colour( bright green) was only observed in the presence of B. cinerea E198 V strain by addition of calcein dye as an indicator reaction prior to amplification,whereas none of other isolates showed a colour change( still orange). The detection limit of the LAMP assay for B. cinerea E198 V was 10 pg/μL of genomic DNA per reaction. Agarose gel electrophoresis results were consistent with the LAMP test results. Our establishment of LAMP assay provides a new alternative method for rapid identification of carbendazim resistant strains of B. cinerea E198 V strain of Dendrobium candidumin.