快速检测马铃薯干腐病病原接骨木镰孢的环介导等温扩增技术

Rapid diagnosis of potato dry rot caused by Fusarium sambucinum using a loopmediated isothermal amplification assay

本研究基于环介导等温扩增技术(loop-mediated isothermal amplification,LAMP),建立了一种快速、准确和灵敏的接骨木镰孢的检测技术。通过比对接骨木镰孢与其近源种之间的候选靶标序列,选取TEF1-α(translation elongation factor1-α,翻译延伸因子)基因作为靶标,设计并筛选出一套对该病原菌具有种特异性的LAMP引物,建立了检测接骨木镰孢的LAMP体系。该体系在反应前加入染料羟基萘酚蓝(hydroxynaphthol blue,HNB),经62℃恒温反应70 min之后,可根据肉眼观察到的反应物的颜色判定结果。特异性分析结果表明,仅接骨木镰孢的DNA经检测后呈天蓝色的阳性反应,而其他供试菌株的DNA均呈紫色的阴性反应。该方法对DNA的最低检测限为100 pg·μL^(-1)。在采自内蒙古和黑龙江的28份马铃薯干腐病疑似病害样本中,检测到14份阳性样品。该方法的建立为接骨木镰孢的检测及其所致病害的诊断提供了快捷准确的技术。

In this study,we developed a rapid,accurate,and sensitive assay for detection of F. sambucinum based on loop-mediated isothermal amplification（ LAMP） technology. By comparing different candidate gene sequences among F. sambucinum and its relative species,TEF1-α（ translation elongation factor 1-α） was selected as a target to design the LAMP primers for specific detection of F. sambucinum. The developed LAMP assay can efficiently amplify the target gene in 70 min at 62℃. By adding HNB（ hydroxynaphthol blue） prior to the assay,the result can be directly visualized through eyes. A positive reaction exhibited sky blue color,while purple color corresponds to a negative reaction. The results showed specificity for F. sambucinum rather than the other assayed fungal isolates. The lowest detectable limit of the LAMP assay for F. sambucinum DNA was 100 pg·μL^（-1）. Among 28 suspected diseased samples of potato dry rot collected from Inner Mongolia and Heilongjiang provinces,F. sambucinum was detected in 14 samples. This technology can be used for fast and accurate detection of F. sambucinum and diagnosis of F. sambucinum-caused potato dry rot.