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ARS Home » Pacific West Area » Riverside, California » National Clonal Germplasm Repository for Citrus » Research » Research Project #444270

Research Project: Developing a Diagnostic Procedure for Candidatus Phytoplasma brasiliense

Location: National Clonal Germplasm Repository for Citrus

Project Number: 2036-21000-012-004-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: May 1, 2023
End Date: Oct 31, 2024

Develop a qPCR diagnostic assay for ‘Candidatus Phytoplasma brasiliense’.

To create a qPCR diagnostic assay, pre-existing isolates of ‘Ca. P. brasiliense’ obtained in Florida will be used as templates. Samples will be tested by PCR for secA, dnaK, tuf, and secY genes. Amplicons of expected sizes will be cloned using a cloning kit with chemically competent E. coli cells and sent for sequencing. Sequences will be aligned with all other available phytoplasma sequences in GenBank and primer/probe regions will be selected that will not allow cross amplification with other phytoplasmas. New primers will subsequently be tested on existing isolates by standard PCR. Amplicons of expected size will be cloned using same approach as above and sequenced to confirm identity. Plasmids containing the appropriate insert will be diluted so target is present in 109 copies per microliter then serially diluted to 101 copies per microliter. Each concentration will be tested in triplicate on a qPCR system to generate a standard curve for quantification of phytoplasma in tissue and to assess efficiency among different loci.