Location: Horticultural Crops Disease and Pest Management Research Unit
Project Number: 2072-22000-045-054-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jun 1, 2025
End Date: Nov 30, 2026
Objective:
The objectives of this project are:
Objective 1: Enrich the near full genome library of BlShV and BlScV by high throughput sequencing (USDA-ARS and University of Minnesota)
Objective 2: Develop an inclusive duplex TaqMan probe real time RT-PCR test to inclusively detect BlScV and an internal endogenous control (University of Minnesota)
Objective 3. Develop an inclusive duplex TaqMan probe real time RT-PCR test to inclusively detect BlShV and an endogenous internal control (USDA-ARS)
Approach:
To fulfill the purpose of this project, USDA-ARS will first perform high throughput sequencing (HTS) of at least 40 samples, (20 infected with BlScV and 20 infected with BlShV) to increase the number full genomes available for the viruses. Tissue samples are already collected from previous surveys. Total nucleic acid (TNA) will be isolated as needed, checked for quality and integrity and sent for HTS in an outsourcing laboratory. Bioinformatics analysis will be performed at the University of Minnesota. University of Minnesota cooperator will generate and an in-house pipeline to analysis HTS data and will produce near full genome assembly of both viruses.
Primers and probes selection:
Primers/probe systems will be designed for best efficiency based on the sequence similarities from above and those in Gene Bank. USDA-ARS will develop the assay for the BlShV TaqMan real-time RT-PCR, where two primer/probe sets to target RNA-dependent RNA Polymerase (RdRp) in RNA2 and capsid protein (CP) in RNA3 will be selected and evaluated. For the BlScV, University of Minnesota will identify regions covering the RdRp and the CP in the single RNA and develop the TaqMan real-time RT-PCR. However, if other regions of the genome are found to be more conserved and less prone to mutations, these alternative regions will be prioritized for primers and probes design. An endogenous internal control gene targeting the Nad-5gene, known to work properly in RT-qPCR approaches, will be adapted in duplex reactions with the viruses of interest.
For assay optimization primers and probes will be tested independently to verify that each target is amplified with specificity and efficiency, without amplifying non-target viruses or host plant nucleic acids. To accomplish this a panel of non-infected plants and plants infected with other viruses will be tested with the new assays. Each primer and probe test will subsequently be evaluated in a duplex system with the endogenous gene test. As part of the optimization process, primer and probes concentration, reagents concentration and cycling parameters for best efficiency will be performed.
Analytical specificity will be evaluated against a panel of ilarviruses and carlaviruses and other blueberry infecting viruses and against RNA from healthy plant. Reference isolates of BlShV and BlScV, available in-house or provided by our collaborators will be confirmed by sequencing, and then used in an RNA serial dilution standard to develop standard curves and test parameters.
All the qPCR setting up and optimization will be run in a Quant Studio 3 real time thermocycler, then tested on an ABI StepOne before moving to the ring-test validation.