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. 2017 Jan 17:7:38896.
doi: 10.1038/srep38896.

Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes

Han Yih Lau  1   2 , Haoqi Wu  1   3 , Eugene J H Wee  1 , Matt Trau  1   4 , Yuling Wang  1 , Jose R Botella  2
Affiliations

Specific and Sensitive Isothermal Electrochemical Biosensor for Plant Pathogen DNA Detection with Colloidal Gold Nanoparticles as Probes

Han Yih Lau et al. Sci Rep. .

Abstract

Developing quick and sensitive molecular diagnostics for plant pathogen detection is challenging. Herein, a nanoparticle based electrochemical biosensor was developed for rapid and sensitive detection of plant pathogen DNA on disposable screen-printed carbon electrodes. This 60 min assay relied on the rapid isothermal amplification of target pathogen DNA sequences by recombinase polymerase amplification (RPA) followed by gold nanoparticle-based electrochemical assessment with differential pulse voltammetry (DPV). Our method was 10,000 times more sensitive than conventional polymerase chain reaction (PCR)/gel electrophoresis and could readily identify P. syringae infected plant samples even before the disease symptoms were visible. On the basis of the speed, sensitivity, simplicity and portability of the approach, we believe the method has potential as a rapid disease management solution for applications in agriculture diagnostics.

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Figures

Figure 1
Figure 1. Schematic illustration of the electrochemical bioassay for plant pathogen DNA detection.
Figure 2
Figure 2. Specificity study for plant pathogen DNA detection.
(A) DPV curve and (B) Current-response to P. syringae (Psy), Botrytis cinerea (Bot) and Fusarium oxysporum f.sp. conglutinans (Foc) as well as a no template control (NTC). Error bars represent ±SD, n = 3. (C) Electrophoresis gel image of RPA products.
Figure 3
Figure 3. Electrophoresis gel image for the sensitivity comparison between RPA and PCR over a range of gDNA inputs.
Figure 4
Figure 4. Sensitivity study for plant pathogen DNA detection via RPA/electrochemistry.
(A) DPV curve and (B) Current-response to different amounts of amplification products, the error bars represent ±SD, n = 3. (C) Electrophoresis gel image of the RPA products.
Figure 5
Figure 5. RPA/electrochemistry detection on healthy and P. syringae infected A. thaliana plant tissue samples (Stage 1 and Stage 2).
(A) DPV curve and (B) Current-response of healthy and infected samples. Error bars represent ±SD, n = 3. (C) Images of healthy and diseased A. thaliana leaves (Stage 1 and Stage 2) with the electrophoresis gel image of RPA products.
See this image and copyright information in PMC

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