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Projects / Programmes source: ARIS

Development of a sensitive and selective electrochemical genosensor for on-site detection of Citrus bark cracking viroid (CBCVd)

Research activity

Code Science Field Subfield
1.04.00  Natural sciences and mathematics  Chemistry   

Code Science Field
1.04  Natural Sciences  Chemical sciences 
Keywords
Citrus bark cracking viroid (CBCVd), electrochemical genosensor, on-site detection, voltammetry, DNA/RNA sensor
Evaluation (rules)
source: COBISS
Points
5,960.3
A''
1,284.7
A'
3,199.02
A1/2
4,263.84
CI10
9,457
CImax
464
h10
46
A1
21.78
A3
10.97
Data for the last 5 years (citations for the last 10 years) on April 20, 2024; A3 for period 2018-2022
Data for ARIS tenders ( 04.04.2019 – Programme tender, archive )
Database Linked records Citations Pure citations Average pure citations
WoS  409  13,425  12,029  29.41 
Scopus  426  14,459  13,016  30.55 
Researchers (15)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  14360  PhD Mojca Benčina  Biotechnology  Researcher  2021 - 2024  392 
2.  51925  Maja Dobrajc  Plant production  Junior researcher  2021 - 2022  22 
3.  36371  PhD Tanja Guček  Plant production  Researcher  2021 - 2024  63 
4.  18555  PhD Samo Hočevar  Chemistry  Head  2021 - 2024  245 
5.  52020  PhD Jelena Isailović  Chemistry  Junior researcher  2021 - 2024  17 
6.  16379  PhD Jernej Jakše  Plant production  Researcher  2021 - 2024  688 
7.  37427  PhD Taja Jeseničnik  Plant production  Researcher  2022 - 2023  36 
8.  50528  PhD Urban Kunej  Biotechnical sciences  Researcher  2021 - 2023  56 
9.  50616  Arne Praznik  Biochemistry and molecular biology  Technical associate  2021 - 2022  35 
10.  20162  PhD Sebastjan Radišek  Plant production  Researcher  2021 - 2024  621 
11.  35123  PhD Tea Romih  Chemistry  Researcher  2021 - 2022  115 
12.  19184  PhD Nataša Štajner  Plant production  Researcher  2021 - 2024  324 
13.  56038  PhD Nikola Tasić  Chemistry  Researcher  2022 - 2024  76 
14.  37428  PhD Helena Volk  Biotechnical sciences  Researcher  2021  59 
15.  31990  PhD Lea Žibret  Civil engineering  Researcher  2021  57 
Organisations (3)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0104  National Institute of Chemistry  Ljubljana  5051592000  20,958 
2.  0416  Slovenian Institute of Hop Research and Brewing  Žalec  5051762000  4,259 
3.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  66,295 
Abstract
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 redox 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.
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