Location: Foreign Disease-Weed Science Research
Project Number: 8044-22000-052-016-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jul 1, 2024
End Date: Sep 30, 2025
Objective:
Goal: To formally validate standardized internal controls for molecular pathogen diagnostic assays in multiple host plant matrices and develop a central database of validated internal controls that can be used as a resource when developing diagnostic assays.
Objective 1: Select and Validate Endogenous Internal Controls.
Objective 2: Select and Validate Exogenous Internal Controls.
Objective 3: Conduct a Blind Ring Trial with All Suggestion Members.
Objective 4: Communicate Results with the Plant Pathogen Diagnostic Community.
Approach:
Objective 1: ARS will use a broad range of plant taxa to evaluate the following known endogenous internal controls: 18S rDNA, 16S rRNA, 28S rDNA, 5.8S rRNA, elongation factor 1a, NS1&2 rDNA, rbcl, nad5, and iterations of the plant mitochondrial cytochrome c oxidase.
Objective 2: ARS will validate the Applied Biosystems TaqMan Exogenous Internal Positive Control, the Haudenshield and Hartman Internal Control, and the exogenous control developed by Ramachandran et al 2021, all of which are general purpose and not limited to a specific primary target. Each will be tested on DNA extracted from soil, water, and spore trap samples to determine utility in a range of sample types.
Objective 3: Internal control assays will be validated by ring tests performed by multiple operators in four laboratories. Assays will be compared across at least two fluorescent thermocycler platforms (i.e. ABI QuantStudio 5 and Biorad CFX96). The internal controls will be evaluated for replicated consistency across operators, locations, and platforms.
Objective 4: The resulting database of validated internal controls will be available as a resource on the Diagnostic Assay Validation Network (DAVN) website. Additionally, we will host webinars with the National Plant Diagnostic Network affiliated diagnostic labs to inform and train the diagnosticians of this new valuable resource.
