DNA Extraction‐Free Loop‐Mediated Isothermal Amplification for Rapid and Sensitive Detection of Pratylenchus zeae

ABSTRACT Pratylenchus zeae is a globally distributed plant‐parasitic nematode with substantial economic impact on agriculture. To enable sensitive, accurate and rapid detection of P. zeae , a loop‐mediated isothermal amplification (LAMP) assay targeting the 28S rDNA sequence was developed. This assay, using the Pzeae primer set in a 4:1 ratio of inner to outer pairs and the WarmStart Colorimetric LAMP 2× Master Mix, successfully detected P. zeae collected in different geographic regions of Brazil, a country characterised by its large area and ecological diversity. The specificity of the LAMP assay was validated using DNA from 11 other species, including species within the genera Pratylenchus , Meloidogyne , Aphelenchoides , Heterodera and Helicotylenchus . The optimal assay conditions were established at 60°C for 60 min, achieving a detection sensitivity of up to 200 fg μL −1 (~2 × 10 −7 of a nematode) of DNA. Thus, this assay demonstrated greater sensitivity than conventional and quantitative PCR. Moreover, it effectively detected P. zeae directly from infected roots, eliminating the need for toxic compounds such as phenol and chloroform. This study has developed the first tool for direct detection of a plant‐parasitic nematode without DNA extraction and offers a practical and rapid solution for detecting P. zeae even in remote locations. The LAMP detection method will assist the development of effective strategies for management of P. zeae in the field.