Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for the detection ofTomato brown rugose fruit virus(ToBRFV)

Abstract

Tomato brown rugose fruit virus (ToBRFV) is a member of Tobamovirus infecting tomato and pepper. Within North America, both the United States and Mexico consider ToBRFV to be a regulated pest. In Canada, the presence of ToBRFV has been reported, but an efficient diagnostic system has not yet been established. Here, we describe the development and assessment of a loop-mediated isothermal amplification (LAMP)-based assay to detect ToBRFV. The LAMP test was efficient and robust, and results could be obtained within 35 min with an available RNA sample. Amplification was possible when either water bath or oven were used to maintain the temperature at isothermal conditions (65°C), and results could be read by visual observation of colour change. Detection limit of the LAMP was eight target RNA molecules. Under the experimental conditions tested, LAMP was as sensitive as qPCR and 100 times more sensitive than the currently used rt-PCR. We recommend this sensitive, efficient LAMP protocol to be used for routine lab testing of ToBRFV.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) method for detection of the potato wart pathogen Synchytrium endobioticum

“…Therefore, the LAMP sensitivity is comparable with both probebased and SYBR green-based qPCR assays. Similar sensitivities between LAMP and qPCR have been reported in previous studies (Okiro et al 2019;Sarkes et al 2020). In the conventional PCR test, a very weak band was observed at the molecule number of 60 per reaction, which indicated the LAMP was about 10 times more sensitive than the conventional PCR.…”

“…LAMP diagnostic protocols have been developed for different plant diseases (Huang et al 2017; Sarkes et al 2020; Yang et al 2021; Sun et al 2022). However, it has not been applied on the detection of S. endobioticum .…”

“…In Canada, a new record of potato production was reported with 6.9 million tons in 2021, rising up 18.2% from 2020 (Statistics Canada, 2021). In 2019/2020, Canada exported potatoes with a value of 1.93 billion CAD (Potato Market Information Review: 2019-2020. These statistics address the importance of potato as an edible crop in Canada and worldwide.…”

“…The results of LAMP can be visually detected and therefore LAMP can be applied with portable equipment. LAMP diagnostic protocols have been developed for different plant diseases (Huang et al 2017;Sarkes et al 2020;Yang et al 2021;Sun et al 2022). However, it has not been applied on the detection of S. endobioticum.…”

Toward a CRISPR-based point-of-care test for tomato brown rugose fruit virus detection

“…In contrast, isothermal nucleic acid amplification methods such as loop-mediated amplification (LAMP) (Notomi et al 2000) can be performed over crude plant extracts at a constant temperature by using a water bath or a thermoblock. Some colorimetric LAMP methods have also been developed to easily detect ToBRFV by the naked eye (Sarkes et al 2020, Rizzo et al 2021), but these rely on a type of amplification that can be nonspecific. On the other hand, the clustered regularly interspaced short palindromic repeats and its associated proteins (CRISPR/Cas) systems are trespassing the genome editing frontiers and revolutionizing diagnostics.…”