Real-time Loop-mediated Isothermal Amplification Assay for Rapid Detection of Fusarium Circinatum

Abstract

Fusarium circinatum is the causal agent of pitch canker, a lethal disease of pine and other conifers. Since F. circinatum is a quarantine organism, its timely detection could efficiently prevent its introduction into new areas or facilitate spread management in already infected sites. In this study, we developed a sequence-specific probe loop-mediated isothermal amplification (LAMP) assay for F. circinatum using a field-deployable portable instrument. The assay was able to recognize the pathogen in host tissues in just 30 min, and the sensitivity of the assay made it possible to detect even small amounts of F. circinatum DNA (as low as 0.5 pg/μl). The high efficiency of this method suggests its use as a standard diagnostic tool during phytosanitary controls.

Rapid Detection of Pine Pathogens Lecanosticta acicola, Dothistroma pini and D. septosporum on Needles by Probe-Based LAMP Assays

“…The LAMP-based assays developed in this work were capable of rapidly identifying these three pathogens in less than 30 min (Figure 1); a substantial improvement compared to the currently available DNA-based diagnostics for these species [45][46][47][48]. These assays also have the potential to be deployable in-field, directly at point-of-care with the use of portable devices developed for supporting LAMP reactions (e.g., Genie ® II and III by OptiGene Limited, Horsham, UK) [55,56,59]. To the best of our knowledge, there are no published LAMP-based assays targeting L. acicola and D. pini, while for D. septosporum a LAMP-based assay has been recently published [60].…”

“…Efforts should also be directed toward validating the developed assays for use at point-of-care on portable devices and using crude DNA extracts, as this has already been successfully performed in numerous pathosystems, including forest pests and pathogen species such as Heterobasidion irregulare Garbel and Otrosina, Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz and Hosoya, Phytophthora spp, Raffaelea lauricola Harrington, Fraedrich and Aghayeva, Xylella fastidiosa Wells, Raju, Hung, Weisburg, Parl and Beemer, Ceratocystis spp., and Fusarium spp. [54,[56][57][58][59].…”

“…All these features make it an ideal solution for in-field point-of-care molecular diagnostics [55]. In addition, the technology has already proved successful for the detection of pathogens in forest systems [54,[56][57][58][59][60]. The aim of this study was to develop three LAMP-based diagnostic assays for the rapid and early detection of the pine pathogens L. acicola, D. pini and D. septosporum.…”

Recent Advances in Molecular Diagnostics of Fungal Plant Pathogens: A Mini Review

“…Conventional LAMP (cLAMP) and a quantitative LAMP (qLAMP) assays were performed to diagnose a quarantine fungal pathogen Fusarium circinatum that causes pitch canker in pine and other conifers. LAMP probes targeting TEF-1α revealed that qLAMP tests had higher specificity than cLAMP for the detection of F. circinatum ( Stehliková et al., 2020 ). A widespread pathogen, Sclerotinia sclerotiorum , with a broad host range including rape seed, was detected using a LAMP assay targeting Ssos5 and a visualizing indicator hydroxynaphthol blue (HNB) for detection.…”

Rapid Detection of Pityophthorus juglandis (Blackman) (Coleoptera, Curculionidae) with the Loop-Mediated Isothermal Amplification (LAMP) Method

“…Molecular assays based on LAMP have been developed to diagnose a range of parasitic infections in both humans and animals (e.g., malaria, leishmaniasis, and cysticercosis) [ 24 ]. Recently, several LAMP tests have also been devised for the identification of invasive plant pathogens [ 25 , 26 , 27 , 28 ] and insect pests [ 29 , 30 , 31 ], both for field applications and in the laboratory. The LAMP technique is a robust and suitable technique for in situ application owing to its low infrastructure requirements and minimal operator training [ 32 ].…”