Implementation of loop-mediated isothermal amplification methods in lateral flow devices for the detection of Rhizoctonia solani

摘要

Rhizoctonia solani causes destructive diseases in many crops throughout the world, resulting in significant yield and quality losses. Early detection of R. solani would facilitate deployment of timely disease management strategies and prevention of spread of asymptomatic infected plant material. The aim of this study was to develop a simple, rapid and sensitive loop-mediated isothermal amplification (LAMP) method for the detection of R. solani in plants and soil samples. LAMP primers, based on the internal transcribed spacer DNA sequence, were designed for detecting most anastomosis groups of R. solani. Using these primers, a LAMP protocol was developed, which in sensitivity tests was shown to detect very low levels of DNA of R. solani and R. zeae, but not R. oryzae. This LAMP protocol successfully detected R. solani on inoculated plant tissues and soil samples. For onsite application in the field, the LAMP protocol was implemented in a generic anti-biotin and anti-fluorescein antibody-based LFD. LAMP reactions were performed using biotin-labelled primers, which were hybridized with a fluorescein amidite (FAM)-labelled hybridization probe and detected with the LFD. This LAMP-LFD successfully detected R. solani in infected plant tissues. The LAMP-LFD procedure presented here is simple and rapid, and is comparable to quantitative real-time PCR. It has potential for onsite diagnosis of R. solani in infected plant samples, seeds, vegetative cuttings and soil samples.