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, that is '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 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.

Keywords: cotton leaf curl Kokhran virus; cotton leaf curl Multan betasatellite; cotton leaf curl disease (CLCuD); loop-mediated isothermal amplification (LAMP); βC1 gene.

FIGURE 1:

map of study districts in Punjab Province, Pakistan.

FIGURE 2:

LAMP assay optimization, colorimetric and real-time LAMP assay for βC1 gene. (A) Molecular optimization of LAMP assay; Lane 1: 1 kb DNA ladder, Lane 2: amplification from βC1 gene cloned in plasmid DNA. Lane 3–5: amplification from field-infected cotton DNA samples, Lanes 6 and 7: negative controls, Lane 8: 1 kb PLUS DNA ladder. (B) Colorimetric detection of LAMP. (C) Real-time amplification of LAMP products.

FIGURE 3:

Determination of LOD for optimized LAMP assay. (A) Agarose gel electrophoresis of the products from the LAMP assays of the serially diluted template DNA; Lane: 1 kb DNA ladder, Lanes 2–13: products from the 10-fold serial dilutions of template, 2.2 × 10¹⁰ virus DNA copies (100 ng/µl) to 2.2 × 10⁻¹ virus copies (1 × 10⁻⁹ng/µl), respectively. Lane 14: negative control, Lane 15: 1 kb DNA ladder. (B) A plot of the fluorescence intensity against the products of the assays containing different concentrations of plasmid template containing the βC1 gene as indicated in Figure 3A. (C) Colorimetric LAMP assay products from the 10-fold serial dilutions of template, 100 ng/µl (2.21 × 10¹⁰ virus DNA copies) to 1 × 10⁻¹⁰ng/µ; (2.21 × 10⁻² virus copies), respectively.

FIGURE 4:

detection of the CLCuMuB-βC1 gene in field-infected cotton samples using PCR. Lane 1: 50 bp DNA ladder, Lane 2: amplification from CLCuMuB-βC1 gene cloned in plasmid DNA, Lanes 3–17: an amplicon of 213 bp from field-infected DNA samples, Lanes 18 and 19: 1 kb DNA ladder, Lanes 20–27: amplification 213 bp from infected DNA samples, Lanes 28–32: negative controls of nonspecific DNA from non-infected cotton (Ct), Potato (Pt), Benthamiana (Ben), Tobacco (Tb) and Tomato (Tm) plants, Lane 33: negative control without any DNA sample, Lane 34: 50 bp DNA ladder.

FIGURE 5:

LAMP assays on infected cotton DNA collected from diverse locations of Punjab. (A) and (B) Agarose gel analysis of LAMP assay; Lane 1: 1 kb DNA ladder, lane 2: CLCuMuB-βC1 gene as positive control. Lanes 3–16: LAMP amplification from DNA of infected field plants, Lanes 17–18: 1 kb DNA ladder, Lanes 19–27: LAMP amplification of DNA collected from infected field plants, Lanes 29–33: negative controls of non-specific DNA from non-infected cotton (Ct), potato (Pt), benthamiana (Ben), tobacco (Tb) and tomato (Tm) plants, Lane 34: negative control with water, Lane 35: 1 kb DNA ladder. (C) Colorimetric detection of LAMP. (D) Detection through real-time amplification of LAMP products.