Rapid and quantitative detection of Fusarium oxysporum f. sp. cubense race 4 in soil by real-time fluorescence loop-mediated isothermal amplification

Abstract

Aims: In this study, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) was developed and evaluated for the rapid and quantitative detection of Fusarium oxysporum f. sp. cubense race 4 (R4) in soil. Methods and Results: The LAMP primer set was designed based on previously verified RAPD marker sequences, and the RealAmp assay could specifically detect and distinguish R4 isolates from other related species. The detection sensitivity of the RealAmp assay was approx. 3·82 × 103 copies of plasmid DNA or 103 of spores per gram in artificially infested soil, indicating that the method is highly tolerant to inhibitor substances in soil compared to real-time PCR. Combining previously published TR4-specific detection methods with the newly established R4-specific RealAmp assay, an indirect approach to detect and differentiate ST4 isolates was achieved by comparing the detection results of R4 and TR4 simultaneously. The existence of ST4 isolates in China was subsequently confirmed through the developed approach. Conclusion: The developed RealAmp assay has been confirmed to be a simple, rapid and effective method to detect R4 in soil, which facilitates to further identify and distinguish ST4 isolates through the comparative analysis of detection results between TR4 and R4 simultaneously. Significance and Impact of the Study: The technique is an alternative quantitative detection method, which will be used for a routine detection service for the soil-borne pathogen in China.