Assistant Professor Oklahoma State University Stillwater, OK, USA
The ability to sensitively detect plant viruses is essential for effective surveillance and biosecurity. Building upon previous work on hybridization-based enrichment of viral RNA, we present a robust strategy leveraging customized dual-indexed oligonucleotides for reverse transcription and cDNA synthesis to enrich nucleotide sequences belonging to pepper mild mottle virus (PMMoV, species Tobamovirus capsici) and cucumber green mottle mosaic virus (CGMMV, species Tobamovirus viridimaculae). This approach utilizes indexed gene primers, designed from multiple sequence alignments of complete genomes, in combination with indexed template switching-primers, and subsequent amplification to generate double-stranded complementary DNA (dscDNA) libraries amenable to real-time PCR, high-throughput sequencing, and target enrichment using biotinylated baits complementary to the viral genomic sequences. Capture using streptavidin magnetic beads was based on the IDT protocol for long-read sequencing, which comprises hybridization and heated washes at 65°C. The targeted PMMoV and CGMMV fragments were consistently enriched while those from the host were depleted, as evidenced by comparing the PCR cycle thresholds relative to the viruses and the housekeeping gene NAD5 in postcapture amplicons versus precapture libraries (15.87 ≤ |ΔCt| ≤ 19.59 versus 10.70 ≤ |ΔCt| ≤ 12.29 for PMMoV). Sequencing by Oxford Nanopore MinION will confirm the enrichment of dscDNA libraries. This dual-indexed dscDNA platform refines the molecular selection of plant viruses and sets the stage for multiplex diagnostics applicable to other viral pathogens.
