Rapid and Visual Detection of <i>Meloidogyne hapla</i> Using Recombinase Polymerase Amplification Combined with a Lateral Flow Dipstick Assay

Abstract

The northern root-knot nematode, <i>Meloidogyne hapla</i>, is a biotrophic parasite that infects many crops and causes severe economic losses worldwide. Rapid and accurate detection of <i>M. hapla</i> is crucial for disease forecasting and control. We developed a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay for rapid detection of <i>M. hapla</i>. The primers and probe were designed based on the effector gene <i>16D10</i> sequence and were highly specific to <i>M. hapla</i>. The RPA reaction was performed at a wide range of temperatures from 25 to 45°C within 5 to 25 min, and the amplicon was visualized directly on the LFD within 5 min. The detection limits of the RPA-LFD assay were 10^-3 females and 10^-2 second-stage juveniles/0.5 g of soil, which was 10 times more sensitive than the conventional PCR assay. In addition, the RPA-LFD assay can detect <i>M. hapla</i> from infested plant roots and soil samples, and the entire detection process can be completed within 1.5 h. These results indicate that the RPA-LFD assay is a simple, rapid, specific, sensitive, and visual method that can be used for rapid detection of <i>M. hapla</i> in the field and in resource-limited conditions.