Enhancing DNA and RNA extraction quality for viral detection in horticultural seeds

Abstract

Imported seeds must meet Australian Biosecurity inspection and clearance requirements. Rapid, cost-effective diagnoses are needed to ensure imported seeds are free of seed-transmitted pathogens. Our project aims to develop an adaptable and reliable molecular diagnostics system to detect and mitigate the risk of introducing regulated pathogens into Australian horticultural production systems on seed, which is cost-effective and simple to implement. However, seeds are challenging for many molecular studies due to their high content of polyphenols, polysaccharides, and lipids, which can bind to nucleic acids, resulting in low yield and quality. Although there are published protocols claiming better seed nucleic acid extraction methods, none are applicable to high throughput analysis. We are attempting to create total nucleic acid extractions that are high quality, high yield, and high throughput to allow possible HTS analysis for infected seeds. To this end, investigations into the utility of SDS-based buffers were conducted extracting DNA and RNA from cucurbit seeds spiked with Cucumber Green Mottle Mosaic Virus. The improved protocols generated may contribute to rapid and sensitive diagnostics of viruses in horticultural seeds imported into Australia, reducing the risks of disease outbreaks in horticultural systems.