Development of a Loop-Mediated Isothermal Amplification Assay for Rapid and sensitive Detection of Hemileia vastatrix in Coffee

Abstract

Coffee leaf rust (CLR) caused by Hemileia vastatrix is a worldwide devastating disease. Early monitoring is crucial for controlling CLR quickly and efficiently. However, it is difficult to accurately identify CLR at the early stage via naked eye; and the detection of H. vastatrix using PCR-based methods were time-consuming, laborious, and sometimes low- sensitivity. LAMP technology is known for its speed, specificity, and sensitivity to accurately identify many different pathogens. Therefore, in this study, by comparing ITS sequences of H. vastatrix with other H. vastatrix and Uredinales strains using National Center for Biotec- hnology Information (NCBI) by BLASTn, we designed specific primers targeting the unique region and its flanking regions of ITS sequences for H. vastatrix. Using SYBR Green I dye, we established a LAMP technique for rapid and sensitive detection of H. vastatrix. Furthermore, we optimized the LAMP protocol was to enhance both sensitivity and specificity for H. vastatrix detection. Under the optimized condition, the establised LAMP protocol could detect as low as 1pg/μl of H. vastatrix DNA in 60min at 63℃, which is ~100 times more sensitive than conventional PCR. This method can successfully detect H. vastatrix in the early CLR symptom on the coffee leaves in the field.