Development of a LAMP assay using a portable device for the real-time detection of cotton leaf curl disease in field conditions

Abstract

Cotton production is seriously affected by the prevalent cotton leaf curl disease (CLCuD) that originated from Nigeria (Africa) to various parts of Asia including Pakistan, India, China and Philippines. Due to CLCuD, Pakistan suffers heavy losses approximately 2 billion USD per annum. Numerous reports showed that CLCuD is associated with multiple species of begomoviruses, alphasatellites and a single species of betasatellite, i.e., Cotton leaf curl Multan betasatellite (CLCuMuB). The most prevalent form of CLCuD is the combination of Cotton leaf curl Kokhran virus-Burewala strain (CLCuKoV-Bur) and CLCuMuB. Thus, the availability of an in-field assay for the timely detection of CLCuD is important for the control and management of the disease. In this study, a robust method using the loop-mediated isothermal amplification (LAMP) assay was developed for the detection of CLCuD. Multiple sets of six primers were designed based on the conserved regions of CLCuKoV-Bur and CLCuMuB-βC1 genes. The results showed that the primer set targeting the CLCuMuB-βC1 gene performed best when the LAMP assay was performed at 58 °C using 100 ng of total plant tissue DNA as a template in a 25 µL reaction volume. The limit of detection (LOD) for the assay was as low as 22 copies of total purified DNA template per reaction. This assay was further adapted to perform as a colorimetric and real-time LAMP assay which proved to be advantageously applied for the rapid and early point-of-care detection of CLCuD in the field. Application of the assay could help to prevent the huge economic losses caused by the disease and contribute to the socio-economic development of underdeveloped countries.

RT-LAMP as Diagnostic Tool for Influenza—A Virus Detection in Swine

“…This current screening method is costly in terms of time and money. In contrast, RT-LAMP is a low-cost (USD 5 for extraction and LAMP reagents), low-overhead NAAT that can be easily performed with minimal lab equipment, and this makes it ideal to use as the basis for developing tests to detect the presence of viruses in agricultural or wild animals in field settings [ 14 , 45 , 46 ]. For influenza, the M gene is well conserved over the 825 sequences chosen, which included all clades and subtypes isolated from swine from 2017 to 2020.…”

Cotton Microbiome Profiling and Cotton Leaf Curl Disease (CLCuD) Suppression through Microbial Consortia associated withGossypium arboreum

“…CLCuD is an acute disease of cotton, with economic losses reaching approximately 2 billion USD per annum in Pakistan [31]. In comparison, the major losses happened in the 1990s, when cotton yields were reduced by 75% [32].…”

Improved visual detection of DNA amplification using pyridylazophenol metal sensing dyes

“…In this approach, LAMP is performed in a weakly buffered solution and the acidification of the reaction during DNA polymerization drives the color change of the pH sensitive dye. This method is particularly suitable for point-of-care settings and has been used in the detection of human pathogens 19 , 20 , field surveillance of mosquitos 21 and plant viral infections 22 – 24 . During the ongoing COVID-19 pandemic, pH-based LAMP detection has been applied to the detection of SARS-CoV-2 in numerous clinical diagnostics 25 – 33 , resulting in the first ever molecular test approved for at-home use 34 .…”