Nucleic Acid Purification From Plant Tissues for Viral Detection

The transmission of virus and virus-like diseases through plants has implications throughout modern agriculture practices, not only in localized crop production, but in the trafficking of seeds and plant propagules between countries and continents.  Molecular screening for these infectious particles requires high quality nucleic acid extraction methods and robust amplification reagents.  Nucleic Acid purification from many economically important crops for pathogen detection is complicated by the presence of inhibitors within seed or leaf materials and oftentimes tedious protocols have been required to recover sufficient amounts of amplifiable material for screening assays. We explore the use of purification chemistries for extraction of high quality plant nucleic acid that is readily amplifiable without the need for organics or multi-step precipitation techniques.  We tested a variety of plant leaf and seed models including corn, soybean, and Arabidopsis leaves, and pepper, tomato and watermelon seeds.  Amplifiability in all cases was evaluated by delta Cq assessment of serially diluted inputs into qPCR and RT-qPCR.  Using a Tobacco Mosaic virus model system we further examined viral RNA isolation from ground plant material.  On average, viral percent recoveries were greater than 90% using a particle-based method easily amenable to automated medium and high throughput purification formats.