Specific colorimetric LAMP assay for the detection of maize bushy stunt phytoplasma in corn through comparative genomics

Abstract

Brazil stands out for being the third largest corn producer in the world, in addition to having self-sufficiency in national supply. Even with the high production, the country has, throughout its extension, climatic conditions that favor the attack of several pathogens. Maize red stunt, caused by maize bushy stunt phytoplasma (MBSP), is one of the most harmful diseases to the crop, therefore, there is a demand for detection methods that are fast and accurate. Loop-mediated isothermal amplification (LAMP) is one of these methods, being fast, sensitive, with high specificity and can be used in field analysis. The goal of the present work was the development of a LAMP protocol, through comparative genomics, for MBSP in maize. To design the sets of primers, MBSP genome sequence and other sequences of other pathogens were used in the RUCS software to select only core unique sequences. Three sets of primers presented the desired criteria and were synthetized. The most promising primer set, MBSP-LP, was used to test the LAMP assay together with the Warmstart colorimetric LAMP 2X master mix (NEB) Kit. The collection of 51 samples of corn with and without symptoms was tested with a typical nested-PCR and compared with the proposed LAMP assay. Considering the presence and absence of symptoms, there was confirmation that the symptomatic plants were positive for LAMP in a greater proportion than for nested-PCR. The proposed LAMP assay proved to be sensitive, detecting up to 0.1 fg µL-1 of DNA. The use of plant material directly in the reaction was evaluated for changing the color of the master mix and also for inhibiting the reaction. It was identified that there are no inhibitors in the maize plant tissue, being possible to use this LAMP with plant tissue without the DNA extraction step.