Author ORCID Identifier:
Date of Graduation
12-2025
Document Type
Thesis
Degree Name
Master of Science in Plant Pathology (MS)
Degree Level
Graduate
Department
Entomology and Plant Pathology
Advisor/Mentor
Tzanetakis, Ioannis
Committee Member
Nelson, Christopher
Second Committee Member
Gibson, Kristen
Keywords
CRISPR-Cas 13a; Diagnostics; End-point PCR; Plant virus; TaqMan RT-PCR; ViMAPC
Abstract
Accurate and reliable detection of plant viruses is critical for protecting berries and other economically important crops. Yet diagnostic programs face persistent challenges with positive control availability, biosafety concerns, and specificity limitations. This thesis advances the diagnostic toolkit for berry viruses through three complementary innovations that address fundamental gaps in current detection methodologies. First, this research developed and validated virus-mimicking artificial positive controls (ViMAPCs) for diverse viruses affecting berry crops for endpoint PCR applications. ViMAPCs incorporate size differentiation, which enables immediate visual gel band discrimination between control and natural infection signals. Amplification efficiency analysis demonstrated close correspondence with true-positive controls (mean difference: 19.7% ± 7.3%), while concurrent synthesis in two independent laboratories achieved 100% success rates with 4–5-day turnaround times, establishing robust inter-laboratory reproducibility. Second, we extended the ViMAPC framework to TaqMan® quantitative real-time PCR through architectural innovations addressing probe-binding requirements. By incorporating complete probe-binding sequences into chimeric RT primers with strategically designed 5-nucleotide homopolymer spacers, we successfully developed ViMAPCs for seven economically significant viruses that achieved quantitative equivalence within ±4 Cq cycles of true-positive controls. Cross-laboratory validation using different operators, instruments, and reagent lots confirmed reproducibility, with observed ΔCq values ranging from -0.3 to +2 cycles, establishing TaqMan®-adapted ViMAPCs as viable biosafe alternatives to infectious positive controls while preserving quantitative fidelity that is essential for diagnostic applications. Lastly, we developed a CRISPR-Cas13a-based diagnostic assay specifically targeting episomal Rubus yellow net virus (RYNV) while avoiding false positives from ubiquitous endogenous viral sequences integrated into host genomes, a critical limitation plaguing current certification programs. Through systematic analysis of RYNV sequences, we designed a 29-crRNA panel targeting conserved regions across RYNV open reading frames. Integration of recombinase polymerase amplification (RPA) pre-amplification achieved a detection limit of 100 pg/µL in vitro transcribed RNA representing an at least 10-fold sensitivity enhancement over unamplified protocols. Collectively, these advances provide diagnostic laboratories, breeding programs, and quarantine facilities with validated, specific tools that overcome longstanding limitations in plant virus detection. These innovations represent significant methodological contributions toward sustainable management of viral diseases threatening berry production and broader agricultural biosecurity.
Citation
Atanda, H. Y. (2025). Advancing the virus diagnostic toolkit: Development of accurate and reliable artificial virus-mimicking positive controls and CRISPR-based detection. Graduate Theses and Dissertations Retrieved from https://scholarworks.uark.edu/etd/6090
