Development of a recombinase polymerase amplification-lateral flow assay for rapid visual detection of Meloidogyne...

Root-knot nematodes (genus Meloidogyne) are among the most destructive agricultural pests globally, causing severe economic damage. Among these, Meloidogyne hapla is particularly problematic for many crops, including yams. Therefore, the rapid and accurate detection of M. hapla is essential for effective crop loss management and prevention. To establish a rapid, specific, and easy-to-use method for potential field-adaptable under semi-controlled conditions, we combined DNA extraction, recombinase polymerase amplification (RPA), and dipstick lateral flow (LF) assay. M. hapla DNA was extracted from a single nematode egg collected from infected yam tissue within 25 min using a lab-made DNA extraction buffer. To establish an efficient RPA process, the amplification temperature (45 °C), reaction time (20 min), and primer length (24 bp) were optimized, enabling rapid and selective amplification of M. hapla. The RPA-LF assay, using biotin- and fluorescein-labeled primers, enabled visual detection within 10 min and achieved a detection limit of 52.06 pM for RPA product under controlled conditions with high specificity. The developed RPA-LF assay provides a rapid, specific, and user-friendly approach for detecting M. hapla in agricultural samples. Its simplicity and speed make it highly suitable for field applications and future development as a portable diagnostic kit for effective management of M. hapla infestations.