Immuno-Dipstick for Colletotrichum gloeosporioides Detection: Towards On-Farm Application

Abstract

Early and quick detection of pathogens are crucial for managing the spread of infections in the biomedical, biosafety, food, and agricultural fields. While molecular diagnostics can offer the specificity and reliability in acute infectious diseases, detection of pathogens is often slowed down by the current benchtop molecular diagnoses, which are time consuming, labor intensive, and lack the mobility for application at the point-of-need. In this work, we developed a complete on-farm use detection protocol for the plant-devastating anthracnose agent: Colletotrichum gloeosporioides. Our methods combined a simplified DNA extraction on paper that is compatible with loop-mediated isothermal amplification (LAMP), coupled with paper-based immunoassay lateral flow sensing. Our results offer simple, quick, easy, and a minimally instrumented toolkit for Colletotrichum gloeosporioides detection. This scalable and adaptable platform is a valuable alternative to traditional sensing systems towards on-the-go pathogen detection in food and agriculture, biomedical, and other fields.

Keywords: Colletotrichum gloeosporioides; anthracnose; immuno-dipstick; loop-mediated amplification (LAMP); point-of-need molecular diagnostic; simplified DNA extraction.

Figure 1

Schematic diagram of the fabricated immuno-dipstick.

Figure 2

Electropherogram of PCR-amplified fungal DNA: (a) lane M—100 bp DNA marker; lane 1, clear visualization of C. gloeosp; lanes 10, 100, and 1000, C. gloeosporioides DNA diluted 10, 100, and 1000 times, respectively; lane NTC, no-template control; (b) lane M—100 bp DNA marker; lane A.f = Aspergillus fumigatus; lane C.g = C. gloeosporioides; lane C.c = C. candidum; lane C.t = C. theobromicola.

Figure 3

Images of the immuno-dipstick for detection of C. gloeosporioides DNA without dilution (1); diluted 10-fold (10); diluted 100-fold (100); 1000-fold (1000); no template control (NTC). Test and control dots are indicated by arrows.

Figure 4

Images of the immuno-dipstick specificity test against Aspergillus fumigatus (A.f), C. theobromicola (C.t), and C. candidum (C.c). Test and control dots are indicated by arrows.

Figure 5

Absolute quantification of C. gloeosporioides DNA purified using four in-house developed simplified DNA extraction methods (MA, MB, MC, and MD; details in Table S1). Paper strips were used to purify the DNA from the samples and elute it into 25 µL Milli-Q. Purification was performed in triplicate (n = 3), and qPCR quantification values were used to calculate the initial concentration of ITS sequence in each amplification reaction. All bar graphs represent the mean of three replicates, and error bars represent the STD.

Figure 6

Loop-mediated isothermal amplification (LAMP) of C. gloeosporioides DNA extracted using (a) a conventional extraction kit: white tube had C. gloeosporioides DNA extract, clear tube had no DNA (negative control); (b) in-house simplified method C for C. gloeosporioides (n = 3). Fluorescence (milky white) indicated successful amplification.

Figure 7

Summary diagram of a complete detection of C. gloeosporioides for on-farm use. The proposed methodology involving the solid-phase of nucleic acid extraction, an isothermal amplification, and the visual detection of the pathogen in just 5 quick steps. This protocol can be adaptable to different point-of-need uses and scalable to other pathogens.