Development of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Hemileia vastatrix in coffee plantations

Abstract

Coffee leaf rust (CLR) caused by Hemileia vastatrix is a devastating worldwide disease. Early monitoring is crucial for controlling CLR quickly and efficiently. However, accurately identifying CLR in its early stages via the naked eye is challenging. Moreover, detecting H. vastatrix using PCR-based methods is time-consuming, labour-intensive, and occasionally exhibits low sensitivity. Loop-Mediated Isothermal Amplification (LAMP) technology is known for its speed, specificity, and sensitivity to identifying many pathogens accurately. Therefore, in this study, we conducted a comparative analysis of ITS sequences from H. vastatrix and other H. vastatrix and Uredinales strains available in the National Center for Biotechnology Information (NCBI) database using the BLASTn tool. Based on this analysis, we designed specific primers that target the unique region and its flanking regions within the ITS sequences of H. vastatrix. Using SYBR Green I dye, we established a LAMP technique for rapid and sensitive detection of H. vastatrix. Moreover, we optimised the LAMP protocol to enhance sensitivity and specificity for H. vastatrix detection. Under the optimised conditions, the established LAMP protocol detected as little as 1pg/μL of H. vastatrix DNA within 60min at 63°C. This sensitivity is approximately 100 times higher than that achieved using conventional PCR. Our method proved effective in detecting H. vastatrix at the early stages of CLR symptom development on the coffee leaves in field conditions.