title: Rapid and quantitative detection of Fusarium oxysporum f. sp. cubense race 4 in soil by real-time fluorescence loop-mediated isothermal amplification.
abstract: OBJECTIVE
  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.
  
  
  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 × 10(3) copies of plasmid DNA or 10(3) 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.
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  The technique is an alternative quantitative detection method, which will be used for a routine detection service for the soil-borne pathogen in 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, Hainan, China; Ministry of Agriculture Key Laboratory for Rubber Biology, Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou, Hainan, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Peng
authors.forename: He
authors.initials: H
authors.lastname: Zhang
authors.forename: Fengping
authors.initials: F
authors.lastname: Chen
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.forename: Yixian
authors.initials: Y
authors.lastname: Xie
authors.forename: Xianwen
authors.initials: X
authors.lastname: Hou
authors.forename: Guangyi
authors.initials: G
authors.lastname: Li
authors.forename: Jinji
authors.initials: J
authors.lastname: Pu
mesh_terms: D005453:Fluorescence; D005670:Fusarium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D019105:Random Amplified Polymorphic DNA Technique; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012988:Soil Microbiology
keywords: Fusarium oxysporum f. sp. cubense (Foc) race 4; detection; loop-mediated isothermal amplification; real-time fluorescence loop-mediated isothermal amplification; soil,subtropical race 4 (STR4),tropical race 4 (TR4)