title: Development of a real-time fluorescence loop-mediated isothermal amplification assay for rapid and quantitative detection of Fusarium oxysporum f. sp. cubense tropical race 4 in soil.
abstract: Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10(3) spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.
authors.affiliation: Ministry of Agriculture Key Laboratory of Integrated Pest Management on Tropical Crops, Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Ministry of Agriculture Key Laboratory of Integrated Pest Management on Tropical Crops, Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China.
authors.forename: He
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Ministry of Agriculture Key Laboratory of Integrated Pest Management on Tropical Crops, Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China.
authors.forename: Jinji
authors.initials: J
authors.lastname: Pu
authors.affiliation: Ministry of Agriculture Key Laboratory of Integrated Pest Management on Tropical Crops, Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China.
authors.forename: Yanxiang
authors.initials: Y
authors.lastname: Qi
authors.affiliation: Ministry of Agriculture Key Laboratory of Integrated Pest Management on Tropical Crops, Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China.
authors.forename: Qunfang
authors.initials: Q
authors.lastname: Yu
authors.affiliation: Ministry of Agriculture Key Laboratory of Integrated Pest Management on Tropical Crops, Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China.
authors.forename: Yixian
authors.initials: Y
authors.lastname: Xie
authors.affiliation: Ministry of Agriculture Key Laboratory of Integrated Pest Management on Tropical Crops, Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, China ; State Key Laboratory of Agro-biotechnology and Ministry of Agriculture Key Laboratory for Plant Pathology, China Agricultural University, Beijing, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Peng
mesh_terms: D001483:Base Sequence; D005453:Fluorescence; D005670:Fusarium / Q000235:genetics / Q000302:isolation & purification*; D057230:Limit of Detection; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D012988:Soil Microbiology; D013172:Spores, Fungal / Q000302:isolation & purification