Development of Enzyme-Mediated Duplex Exponential Amplification Assay for Detection and Identification of Meloidogyne enterolobii in Field.

Abstract

The root-knot nematode Meloidogyne enterolobii has emerged as a devastating pathogen in global agricultural systems. Its geographic distribution is progressively expanding from tropical to temperate zones, leading to difficulties in discerning the symptoms it causes from those of congeners such as M. incognita. Currently, some molecular diagnostic technologies (e.g., qPCR) have been established for detecting M. enterolobii, but these methods fail to meet field-based detection demands due to their reliance on laboratory-grade thermocyclers. We thus developed a method for detecting M. enterolobii based on enzyme-mediated duplex exponential amplification (EmDEA) technologies to address this issue. The EmDEA detection method demonstrated strict specificity for the target species, showing no amplification in 13 non-target nematodes or host tissue samples. Sensitivity analyses revealed detection limits of 3.6 × 10^-4 ng/μL (purified DNA), 1/1000 of an individual nematode (single-organism detection), 8.97 nematodes/g sweet potato, and 4.08 nematodes/100 g soil, achieving equivalent performance to qPCR. Field validation confirmed successful on-site detection, with significantly higher nematode loads in root tissues (50.41-97.62 nematodes/g) than in rhizospheric soil (1.07-1.28 nematodes/g). The established detection method employs a 42 °C isothermal amplification technology paired with a palm-sized thermal module, enabling field-deployable detection. Its unique duplex exponential amplification mechanism achieves threshold determination 10 cycles (~10 min) faster than conventional qPCR. When integrated with rapid DNA extraction protocols, the entire workflow is completed within 40 min, improving detection efficiency. This study provides a molecular tool for the precise monitoring of M. enterolobii, offering critical support for formulating targeted control strategies.