Development of colorimetric and real time loop-mediated isothermal amplification (cr-LAMP) assay for rapid detection of Wheat dwarf virus (WDV)

Colorimetric and real-time loop-mediated isothermal amplification (cr-LAMP) assays were developed targeting the coat protein region of genomic DNA of Wheat dwarf virus (WDV). The method was optimized relating to primer selection, determination of isothermal temperature, and determination of incubation duration. The specificity of the assay was tested using potential hosts of WDV and Maize streak virus (MSV) from Mastrevirus genus. No cross-reaction took place with MSV or with the potential hosts tested. For the naked eye observation of positive LAMP reaction, WarmStart 2X Master Mix from NEB (New England Biolabs) was used, where the change in colour from pink to yellowish points out a positive result. The amplification was monitored in real-time by smart-DART platform (Diagenetix Inc, USA) that allows connecting to a tablet or smartphone via bluetooth. The detection sensitivity of the LAMP assay was 100-fold more sensitive than conventional PCR after optimization of the cr-LAMP reaction conditions. The novel LAMP assay showed a high specificity in distinction of WDV detection from potential hosts and MSV. The cr-LAMP is considered to be fast and effective assay since it only requires very basic equipment and the results can be evaluated easily by using colorimetric and real time approaches. The simple, low-power, handheld smart-DART platform and colorimetric detection both have a major powerful advantage of the LAMP reaction in that it can be enabled in agricultural industries, where laboratory capacity is often rudimentary.