Objective Fusarium wilt of banana, caused by the fungus Fusarium oxysporum f.sp. cubense race 4 (Foc4), poses a severe threat to global banana production. This study aimed to develop a rapid and accurate detection method suitable for field and port quarantine use to facilitate early disease warning.

Method A enzyme-mediated duplex exponential amplification (EmDEA) assay was developed for Foc4 detection. A Foc4-specific genomic region was identified through comparative genomics and used to design six DNA primer pairs and six RNA probes. Systematic screening and evaluation then yielded the optimal primer–probe combination (F4R1N1). The assay was performed at 42 ℃ for 30 min with real-time fluorescence monitoring. Its specificity was assessed using genomic DNA from 31 fungal strains, including Foc4 isolates from different origins, other Fusarium oxysporum formae speciales, and non-Fusarium species. Sensitivity was evaluated using serially diluted Foc4 genomic DNA and further validated using DNA from naturally infected banana plants.

Result The assay showed high Foc4 specificity, with no cross-reactivity observed against any tested non-target strain. The detection limit was 0.5 pg/μL. The detection results of banana diseased plant samples showed that this method could effectively identify samples infected with Foc4, while all healthy samples yielded negative results.

Conclusion This EmDEA-based method combines precise target selection with a dual-signal amplification mechanism, achieving high sensitivity and specificity. Its rapidity, portability, and closed-tube operation make it particularly well-suited for on-site Foc4 detection, providing a practical tool for early disease management and quarantine efforts.