Skip to main content
ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » National Germplasm Resources Laboratory » Research » Publications at this Location » Publication #407734

Research Project: Characterization and Detection of Viruses that Impact the Exchange and Curation of Plant Germplasm

Location: National Germplasm Resources Laboratory

Title: A metagenomic sequencing spiked primer enrichment method for detection of tomato brown rugose fruit virus

item Abrahamian, Peter
item NUNZIATA, SCHYLER - Animal And Plant Health Inspection Service (APHIS)
item NAKHLA, MARK - Animal And Plant Health Inspection Service (APHIS)
item RIVERA, YAZMIN - Animal And Plant Health Inspection Service (APHIS)

Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/18/2023
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: High throughput sequencing (HTS) has proven useful for virus discovery, detection, and metagenomics. However, HTS for virus detection has some drawbacks, such as the lack of validated and optimized protocols that can accurately detect viruses. While HTS technologies have helped scientists detect new viruses, its sensitivity in some cases is comparable or less sensitive than real-time PCR. As a result, detection of low titer viruses can be problematic when using HTS, particularly in samples like seeds. Tomato brown rugose fruit virus (ToBRFV) is a regulated emerging plant virus under a USDA APHIS federal order. ToBRFV is a devastating seed-transmitted virus that can overcome Tm-22 resistance in tomatoes. In this study, we used ToBRFV as a model virus for designing a new and optimized method of HTS for targeted enrichment of a plant virus. This approach relies on both targeted and non-targeted enrichment by combining virus-specific primers and random hexamers. Total RNA libraries were prepared by spiking various primer concentrations (4, 2, 1, 0.5, 0.2, and 0.1 µM) and compared to non-spiked libraries. Libraries with spiked primer concentrations greater than 1 µM resulted in higher ToBRFV reads, genome coverage, and contig length compared to non-spiked libraries. Also, ToBRFV detection using HTS on serially diluted infected samples showed higher sensitivity when spiking libraries compared to non-spiked libraries. Overall, the specific spiking technique developed in this study showed significant improvement in HTS sensitivity.