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Rapid and quantitative detection of F usarium oxysporum f. sp. cubense race 4 in soil by real‐time fluorescence loop‐mediated isothermal amplification
Author(s) -
Peng Jun,
Zhang He,
Chen Fengping,
Zhang Xin,
Xie Yixian,
Hou Xianwen,
Li Guangyi,
Pu Jinji
Publication year - 2014
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12645
Subject(s) - fusarium oxysporum f.sp. cubense , fusarium oxysporum , loop mediated isothermal amplification , biology , plasmid , primer (cosmetics) , detection limit , fusarium wilt , microbiology and biotechnology , chromatography , dna , botany , genetics , chemistry , organic chemistry
Aims In this study, a real‐time fluorescence loop‐mediated isothermal amplification (RealAmp) was developed and evaluated for the rapid and quantitative detection of F usarium 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 × 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 TR 4‐specific detection methods with the newly established R4‐specific RealAmp assay, an indirect approach to detect and differentiate ST 4 isolates was achieved by comparing the detection results of R4 and TR 4 simultaneously. The existence of ST 4 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 ST 4 isolates through the comparative analysis of detection results between TR 4 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.