Location: National Clonal Germplasm Repository for Citrus
Project Number: 2036-21000-012-024-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Sep 1, 2024
End Date: Oct 31, 2025
Objective:
(1) Develop a qPCR assay that utilizes HRMA to distinguish strains of 16SrV phytoplasmas and (2) Develop a multiplex dPCR assay to amplify strains of 16SrV phytoplasmas.
Approach:
1) Generate a qPCR/HRMA assay for amplifying and differentiating strains of 16SrV phytoplasma, isolates of ‘Ca. P. ulmi’, ‘Ca. P. vitis’, ‘Ca. P. rubi’ and the un-named strain from virginia creeper held at the University of Florida’s Fort Lauderdale Research and Education Center (FLREC) will be used as template. Universal secA primers will be used to generate sequences for strains. Sequences will be aligned and primers will be designed around conserved regions that contain internal variability allowing for amplification of a 80 to 100 bp region for all strains that will be distinguishable by HRMA. General PCR will be performed using the new primers on each strain. Products will be cloned to generate plasmids with inserts for corresponding strains. Isolated plasmids will be screened using the designed primers by qPCR in triplicate reactions for seral dilutions ranging from 109 copies/µl to 101 copies/µl. Additionally, isolates of each strain will be screened with same primers. All reactions will be analyzed using the HRM App (Life Technologies) to evaluate variance of Tm product relative to concentration (plasmid-focused assays) as well as among strains. 2) Develop a multiplex dPCR for the same strains listed in objective 1, secA sequences will analyzed to find variable regions to design strain-specific TaqMan assays. Assay design will be accomplished using OligoArchitech (Sigma-Aldrich). dPCR reactions will be run on the Absolut Q dPCR system (Life Technologies) which has four open channels, allowing for up to four strain specific assays in a multiplex assay (tetraplex), allowing for all four strains listed in objective 1 to be contained within the same multiplex reactions. Should secA not possess sufficient variability to accommodate four strain specific assays, other loci (groEL, secY) will be evaluated. Regardless of the loci selected, each strain-specific assay will be purchased separetly with a 5' FAM labeled to determine optimal PCR parameters and verify there is no cross amplification among strains and assays. Once verified, the multiplex assay will be purchased and evaluated against plasmids with inserts for corresponding strains and actual samples/isolates of the corresponding strains. Each strain will have their region amplified by standard PCR, the product cloned and plasmids isolated and serially diluted (109 copies/µl to 101 copies/µl). Validation of functionality of the multiplex assay will be accomplished against plasmids at a concentration of 103 copies/µl and to establish flouresence thresholds. Isolates of each strain will be diluted to 25 ng/µl then serially diluted to 0.025 ng/µl. All dilutions for each strain will be screened with the multiplex assay to validate functionality on eluate from total DNA extractions and to establish optimal concentration (due to unknown titer of phytoplasma in samples).
