title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. niveum in soil.
abstract: Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is one of the major limiting factors for watermelon production worldwide. Rapid and accurate detection of the causal pathogen is the cornerstone of integrated disease management. In this paper, a real-time fluorescence loop-mediated isothermal amplification (RealAmp) assay was developed for the rapid and quantitative detection of Fon in soil. Positive products were amplified only from Fon isolates and not from any other species or formae speciales of F. oxysporum tested, showing a high specificity of the primer sets. The detection limit of the RealAmp assay was 1.2 pg μL(-1) genomic DNA or 10(3) spores g(-1) of artificially inoculated soil, whereas real-time PCR could detect as low as 12 fg μL(-1) or 10(2) spores g(-1). The RealAmp assay was further applied to detect eight artificially inoculated and 85 field soil samples. No significant differences were found between the results tested by the RealAmp and real-time PCR assays. The RealAmp assay is a simple, rapid and effective technique for the quantitative detection and monitoring of Fon in soil under natural conditions.
authors.affiliation: Key Laboratory of the Ministry of Agriculture for Integrated Pest Management on Tropical Crops, Institute of Environmental and Plant Protection, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Peng
authors.forename: Yuanfeng
authors.initials: Y
authors.lastname: Zhan
authors.forename: Fanyun
authors.initials: F
authors.lastname: Zeng
authors.forename: Haibo
authors.initials: H
authors.lastname: Long
authors.forename: Yuelin
authors.initials: Y
authors.lastname: Pei
authors.forename: Jianrong
authors.initials: J
authors.lastname: Guo
mesh_terms: D004271:DNA, Fungal; D005670:Fusarium / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012988:Soil Microbiology
chemical_list: D004271:DNA, Fungal
keywords: Fusarium wilt of watermelon; RealAmp assay; detection of fungi; real-time PCR