Location: Molecular Plant Pathology Laboratory
Project Number: 8042-22000-320-007-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jun 1, 2025
End Date: Nov 30, 2026
Objective:
Objective 1: Analyze and identify molecular targets in the genomes of three bacterial pathogen of citrus (Liberibacter spp., Spiroplasma citri and 'Ca. Phytoplasma' spp.) to design a multiplex TaqMan assay.
Objective 2: Develop and optimize a quadruplex TaqMan assay including a plant internal control
Objective 3: Validate the assay performance: Analitical sensitivity and specificity, efficiency, range, linearity, LOD, repeatability and reproducibility.
Approach:
Design and screen TaqMan qPCR assays targeting conserved region of Spiroplasma citri, Liberibacter asiaticus, L. africanus, L. americanus and 'Ca. Phytoplasma' spp. Genomes will be downloaded from NCBI GenBank genome database and will be evaluated for unique and conserved gene. Thermodynamically competent primers and probes will be designed for 4 qPCR reactions (3 pathogen + plant internal control) to achieve the highest sensitivity. The multiplex TaqMan qPCR protocol will be standardized for conditions and components. The qPCR assays will undergo performance characteristics verifications: analytical sensitivity and specificity, accuracy, precision, reportable range and LOD.