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

Han Yih Lau,Eugene J H Wee,Matt Trau,Yuling Wang,Haoqi Wu,Jose R Botella

doi:10.1038/srep38896

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.

Highlights

Develop simpler recombinase polymerase amplification (RPA) assays for plant pathogen detection in the field comprising all steps, from DNA isolation to visualization of results
We describe a rapid and highly sensitive diagnostic method coupling RPA with AuNPs as electrochemical probes to detect the presence of plant pathogen DNA by differential pulse voltammetry (DPV) on disposable screen printed carbon electrodes (SPCE)
RPA was used to amplify the target sequence using a biotin labeled reverse primer and a forward primer containing a 10 nt barcode covalently linked to the 5′end via a carbon spacer

Summary

Introduction

Develop simpler RPA assays for plant pathogen detection in the field comprising all steps, from DNA isolation to visualization of results. AuNPs-based DNA biosensors can provide a promising platform for the development of rapid, sensitive, specific and portable diagnostic tools for detecting DNA21–23. We describe a rapid and highly sensitive diagnostic method coupling RPA with AuNPs as electrochemical probes to detect the presence of plant pathogen DNA by differential pulse voltammetry (DPV) on disposable screen printed carbon electrodes (SPCE).

Results

Conclusion