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 103 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.

Shining a Light on LAMP Assays — A Comparison of LAMP Visualization Methods Including the Novel use of Berberine

“…Both dyes emit a weak fluorescence signal in the presence of single-stranded DNA (ssDNA) but emit strongly upon binding to double-stranded DNA (dsDNA) and are therefore ideal candidates for the detection of DNA in LAMP reactions. Because of its inhibitory effect on DNA polymerization at higher concentrations, SYBR Green I is mainly used in LAMP for end point detection by adding it to the reaction mixture after the amplification reaction (13, 14) or by depositing the dye into the lid of the reaction vessel (15). EvaGreen is more applicable for the monitoring of LAMP reactions in real time because it does not affect DNA polymerization and therefore may be present throughout the entire reaction (16, 17).…”

Rapid detection of Fusarium wilt in basil (Ocimum sp.) leaves by desorption electrospray ionization mass spectrometry (DESI MS) imaging

“…22 Therefore, customized methods suitable for rapid detection of contaminated leaves within hours is focused for this study. are well known, 21 but its susceptibility to Fusarium wilt has to be understood in detail to prevent outbreak of the disease.…”

Rapid detection of Colletotrichum gloeosporioides in infected strawberry plants using loop-mediated isothermal amplification

“…The LAMP assay has been used to detect pathogenic plant viruses (Fukuta et al 2003, 2004, 2013b), viroids (Boubourakas et al 2009), fungi (Duan et al 2014a, b;Fukuta et al 2014;Lenarčič et al 2014;Moradi et al 2014;Takahashi et al 2014;Tomlinson et al 2010;Zhang et al 2013), bacteria (Okuda et al 2005;Rigano et al 2010Rigano et al , 2014, oomycete pathogens (Dai et al 2012;Fukuta et al 2013a;Tomlinson et al 2007), and nematodes (Kikuchi et al 2009). The LAMP assay requires 4 different primers that recognize 6 distinct regions of a target gene.…”