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

Abstract

Brazil stands out as the third largest corn producer in the world, showing self-sufficiency to supply its entire national demand. However, maize red stunt, caused by maize bushy stunt phytoplasma (MBSP), is one of the most harmful diseases to this crop, requiring fast and accurate detection methods to deal with this threat. One such method is loop-mediated isothermal amplification (LAMP), a fast, sensitive, and highly specificity tool that can be used in field analysis. From this perspective, this study aimed to develop a LAMP protocol through comparative genomics for MBSP in maize. To design the sets of primers, the MBSP genome sequence and sequences of other pathogens were used in the RUCS software (Rapid identification of PCR primers for Unique Core Sequences) to select only core unique sequences. Three sets of primers had 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 reaction optimization uses a 4:1 proportion of primers and a temperature of 65 °C for 60 minutes. 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 the presence of any inhibitors of the reaction. It was identified that there are no inhibitors in the maize plant tissue, and this LAMP assay can be used without the DNA extraction step.