Development of recombinase polymerase amplification assay for rapid detection of Meloidogyne incognita, M. javanica, M. arenaria, and M. enterolobii.
Recombinase polymerase amplification (RPA) is a novel isothermal DNA amplification approach that has been used to detect a variety of animal and plant pathogens. However, the RPA assay is rarely used for the molecular diagnosis of plant parasitic nematodes. In this study, we developed RPA assays for the detection of four Meloidogyne spp.; Meloidogyne incognita, M. javanica, M. arenaria, and M. enterolobii. The RPA amplification step could be completed at 38°C in 20 min without a thermal cycling instrument. The RPA assays were able to distinguish these four Meloidogyne spp. from closely related Meloidogyne species and other plant parasitic nematodes. The detection limits of the RPA assays were 10-2, 10-2, 10-1, and 10-1 dilutions of DNA from a single J2 nematode of M. incognita, M. javanica, M. arenaria, and M. enterolobii, which were less sensitive than polymerase chain reaction (PCR) detection methods. In addition, the RPA assays could detect these four Meloidogyne spp. directly from infested tomato roots. The simplicity, rapidity and practicability all indicated that the RPA assay will be an effective tool for molecular diagnosis of plant parasitic nematodes.