Immunocapture Loop Mediated Isothermal Amplification for Rapid Detection of Tomato Yellow Leaf curl Virus (TYLCV) without DNA Extraction

Abstract

To diminish the time required for some diagnostic assays including polymerase chain reaction (PCR), loopmediated isothermal amplification (LAMP; due to mainly DNA extraction step) and also DAS-ELISA into a minimum level, an innovative immunocapture LAMP (IC-LAMP) and immunocapture PCR (IC-PCR) protocol on the basis of Tomato Yellow Leaf curl Virus (TYLCV) genome were used and optimized. Even though DAS-ELISA, IC-PCR and IC-LAMP assays could successfully detect positive infected plant samples, considering the time, safety, sensitivity, cost, no need of DNA extraction and simplicity the last one was overall superior. The hydroxynaphthol blue could produce long stable colour change and brightness in a close tube-based approach to prevent cross-contamination risk. Altogether, as IC-LAMP is sensitive, cost effective, fairly user friendly and also can generate more accurate results than previous diagnostic procedures, we accordingly propose this assay as a highly reliable alternative viral recognition system regarding TYLCV recognition and probably other viral-based diseases.

Rapid real-time quantitative colorimetric LAMP methodology for field detection of Verticillium dahliae in crude olive-plant samples

“…For plant pathogens, LAMP assays have been developed for fungi (e.g., Aspergillus nomius, A. parasiticus, A. flavus, Fomitiporia torreyae, Fusarium oxysporrum f. sp., lycopersici, Fulviformes umbrinellus, Ceratocystis platani and Alternaria solani) (Luo et al, 2012;Ayukawa et al, 2017;Fukuta et al, 2017;Khan et al, 2018); oomycetes (e.g., Pythium myriotylum, Phytophthora infestans and P. ramorum) (Fukuta et al, 2014;Khan et al, 2017); bacteria (Xylella fastidiosa, Erwinia amylovora) (Harper et al, 2010;Bühlmann et al, 2013;Aglietti et al, 2019;Amoia et al, 2023) and viruses/viroids (e.g., ApNMV, ASPV, ASGV, ACLSV, ASSVd, TMV and TYLCV) (Liu et al, 2010;Dehabadi, 2013;Zhao et al, 2014;Lee et al, 2018;Lu et al, 2018;Nabi et al, 2020;Lee & Jeong, 2022). A common feature of the above methodologies is the use of a sample pretreatment purification step and mostly use of fluorescence probes for lab-based detection, with reported detection limits for plant pathogenic fungi and oomycetes in the range of 2 fg to 100.000 fg/μl reaction (Luo et al, 2012;Ayukawa et al, 2017;Fukuta et al, 2017;Khan et al, 2018).…”