Development and application of loop-mediated isothermal amplification for detecting the highly benzimidazole-resistant isolates in Sclerotinia sclerotiorum

作者：Duan, Ya Bing; Yang, Ying; Wang, Jian Xin; Liu, Cong Chao; He, Ling Ling; Zhou, Ming Guo^*

来源：Scientific Reports, 2015, 5(1): 17278.

DOI：10.1038/srep17278

• RAPID DETECTION
• FUSARIUM-GRAMINEARUM
• REVERSE-TRANSCRIPTION
• RT-LAMP
• ASSAY
• DISEASES
• DNA
• PRODUCTS
• PRIMERS
• VIRUS

Resistance of benzimidazole fungicides is related to the point mutation of the beta-tubulin gene in Sclerotinia sclerotiorum. The point mutation at codon 198 (GAG -> GCG, E198A) occurs in more than 90% of field resistant populations in China. Traditional detection methods of benzimidazole-resistant mutants of S. sclerotiorum are time-consuming, tedious and inefficient. To establish a suitable and rapid detection of benzimidazole-resistant mutants of S. sclerotiorum, an efficient and simple method with high specificity was developed based on loop-mediated isothermal amplification (LAMP). Eight sets of LAMP primers were designed and four sets were optimized to specially distinguish benzimidazole-resistant mutants of S. sclerotiorum. With the optimal LAMP primers, the concentration of LAMP components was optimized and the reaction conditions were set as 60-64 degrees C for 60 min. This method had a good specificity, sensitivity, stability and repeatability. In the 1491 sclerotia, 614 (41.18%) were positive by LAMP, and 629 (42.19%) positive by MIC. Therefore, the LAMP assay is more feasible to detect benzimidazole-resistant mutants of S. sclerotiorum than traditional detection methods.

• 出版日期2015-11-26
• 单位南京农业大学