植物病理学および微生物学ジャーナル

概要

Development and Application of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Fusarium Oxysporum f. Sp. lycopersici

Mohammad Amin Almasi, Seyed Mohammad Hosseini Dehabadi, Aboubakr Moradi, Zahra Eftekhari, Mehdi Aghapour Ojaghkandi and Saeedeh Aghaei

A reliable and rapid pathogen detection protocol, for the first time, that utilizes colorimetric loop-mediated isothermal amplification (LAMP) was developed for detection of Fusarium oxysporum f. sp. lycopersici. In this regard, all six LAMP primers (i.e. F3, B3, FIP, BIP, LF and LB), together with PCR primers (F and R) were designed on the basis of the 28s ribosomal RNA gene (GenBank accession number: HM057281.1) of the fungi genome. Even though PCR and LAMP assays could successfully detect positive infected samples, considering the time, safety, cost and simplicity, the latter was overall superior. Furthermore, the results demonstrated that the LAMP assay was 100-fold sensitive and 4-fold faster compared to PCR. Interestingly, LAMP reaction could successfully detect Fusarium oxysporum f. sp. lycopersici without DNA purification (direct-LAMP). Meanwhile, among six visual dyes used to detect LAMP products, hydroxynaphthol blue, GeneFinderTM and SYBR Green I could produce long stable color change and brightness in a close tube-based approach, to prevent cross-contamination risk. Altogether, as LAMP is sensitive, cost effective, fairly user friendly, and also can generate more accurate results than previous diagnostic procedures, such as serological methods, PCR and other molecular methods, we accordingly propose this colorimetric assay as a highly reliable alternative fungi recognition system regarding Fusarium oxysporum f. sp. lycopersici recognition, and probably other fungi-based diseases.