Rapid and sensitive detection of <i>Fusarium oxysporum</i> f. sp. <i>cubense</i> tropical race 4 using a RPA-DETECTR assay

Abstract Early and prompt detection of banana wilt pathogen Fusarium oxysporum f. sp. cubense (Foc), tropical race 4 (Foc TR4), causing significant banana crop losses globally is crucial to curtail the disease spread, minimize damage and implement quarantine measures. We report the first successfully developed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) assay with high sensitivity and specificity for detecting Foc TR4, which has been validated across several isolates. Notably, specific CRISPR RNA (crRNA) spacer sequences and recombinant polymerase amplification (RPA) primers designed and evaluated for the DETECTR assay exhibited high sensitivity, detecting Foc TR4 with genomic DNA as low as 0.005 ng, and demonstrated a very high specificity and reproducibility. The RPA-DETECTR assay was validated on a diverse panel of banana samples, including both target and non-target pathogens confirming its robustness and reliability across different types of samples, ensuring its practical applicability. In terms of throughput, the RPA-DETECTR assays enabled faster detection of Foc TR4 than other molecular diagnostics including PCR, quantitative PCR (qPCR) and droplet digital PCR (ddPCR) analyses. This makes the assay suitable for point-of-care (POC) detection of Foc TR4, and facilitating rapid decision making and immediate response. This rapid detection capability is crucial in restricting any potential transboundary movement as part of ongoing disease management efforts.