Evaluating the utilization of synthetic oligonucleotides as a positive control in the detection of ToBRFV in pepper seeds

Abstract

A constant challenge experienced by plant diagnostic laboratories is having reliable and readily available controls. Often, requests must be made from colleagues to obtain materials for use as controls. This can be problematic if the pathogen is not present in the country and/or is subject to regulations. gBlocks™ are synthetic oligonucleotides that are widely used in genomic-based applications and studies. We evaluated the use of synthesized gBlocks™ as a positive control for tomato brown rugose fruit virus (ToBRFV) in a seed extraction PCR (SE-PCR) assay. ToBRFV is a highly virulent Tobamovirus that expresses symptoms of mosaic patterns, browning, and undersized wrinkled fruits in tomatoes, peppers, and other solanaceous plants. Transmission occurs in the seeds and can spread via mechanical contamination of equipment, humans, and other infected plants. Regulations with ToBRFV in the US makes it difficult to obtain a positive biological control for use in diagnostic clinics. Therefore, we wanted to assess if this synthetic oligonucleotide could serve as a process control. In our study, pepper (Capsicum annuum) seeds were “spiked” with synthesized oligonucleotides, which were then used as a point of comparison to biologically positive seeds and negative controls by way of quantifying viral titer. The stability of these synthesized oligonucleotides was evaluated over several temperatures and temporal parameters. Our results suggest that the oligonucleotides are suitable for use in the production of synthetically contaminated seeds that are to be used as a positive control in the validation of the diagnostic process for ToBRFV.