Agronomy

PROJECT DESCRIPTION

An intensive agriculture and global market present a high risk for fast spreading of plant diseases, which can result in enormous economic losses, and ultimately in a starvation and worldwide crisis. Among various plant pathogens, viroids are the smallest and the simplest; however, they can cause severe and incurable diseases, which cannot be controlled by using plant protection products and their impact on susceptible host can be devastating. Discovery of the Citrus bark cracking viroid (CBCVd) on hop in Slovenia is a recent case of a severe viroid transmission to a new highly susceptible host and spreading to new regions. For efficient management of viroid disease, it is obvious that rapid, sensitive and selective on-site detection is an imperative.

Electrochemistry offers attractive features, such as favorable sensitivity, selectivity, inexpensive and portable instrumentation, and almost unlimited possibilities for electrode surface modifications aimed at designing powerful electrochemical (bio)sensors. Furthermore, electrochemical methods have been proven to be potent tools for a simple and sensitive analysis of nucleic acids (NAs). Apart from using a variety of labeling and amplification protocols, the intrinsic electrochemical activity of nucleobases offers also a label-free NA detection.

In this project, and in collaboration with our Czech partner from IBP, we will develop electrochemical NA biosensors (genosensors) for sensitive, selective and on-site detection of the Citrus bark cracking viroid (CBCVd). The genosensor for detecting viroid’s RNA (or cDNA as a product of reverse transcription) will be based on selected supporting screen-printed electrodes, which will be surface modified with sensing coatings assuring effective immobilization of biorecognition elements, adequate sensitivity, selectivity, and robustness. The selection, (pre)treatment and modification of supporting electrode surfaces and the application of sensing membrane will depend on the detection protocol. We will examine and combine different electrode materials with strategies for detecting the viroid using the principles such as:

(a) hybridization of CBCVd RNA/cDNA with an unlabeled capture probe;

(b) hybridization of CBCVd RNA/cDNA with a redox system- or enzyme-labeled capture probe;

(c) detection of proton reduction reaction as a function of the hybridization event in the presence of catalysts;

(d) tagging the probes with selected metal nanoparticles, which allow for highly sensitive detection using stripping voltammetry.

Special attention will be devoted to strategies involving incorporation of labelled nucleotides by DNA polymerases. Namely, we will investigate and select labelled deoxynucleoside triphosphates and DNA polymerases along with the optimization of corresponding protocols for:

(i) introduction of labels into the viroid cDNA during reverse transcription;

(ii) amplification of the labelled cDNA by PCR, and specifically by an isothermal amplification protocol such as LAMP, and

(iii) non-templated terminal tail labeling of RNA or DNA.

Research efforts will be focused on the biocatalytic (enzymatic) or electrocatalytic signal amplification techniques and on the development of simple detection approaches relying on preferential adsorption of labelled/amplified target cDNA at the electrode directly from reaction mixtures.

Finally, we will hyphenate the electrochemical genosensor with a portable, in this project developed and optimized NA amplification protocol, such as RT-LAMP, combined with NA labelling, which will significantly improve the sensitivity and selectivity of the genosensor. We will use a 3D printing technology to build a suitable and portable heating device for isothermal amplification step.

The resulting genosensing platform will be validated and tested on-site for early detection of CBCVd viroid in a real sample of hop.

PROJECT PHASES AND REALIZATION

WP 1. SELECTION OF THE SUPPORTING ELECTRODES AND THEIR PRETREATMENT

WP 2. MODIFICATION OF THE ELECTRODE SURFACE WITH A CAPTURE PROBE

WP 3. ENHANCING THE SENSITIVITY AND SELECTIVITY

WP 4. COUPLING OF ISOTHERMAL AMPLIFICATION WITH THE GENOSENSOR

WP 5. ADAPTATION TO THE ON-SITE GENOSENSING AND VALIDATION