Oral: Pathogen Detection
Fluorogenic Detection of Plant Viruses by Helicase Dependent Amplification with Self-Quenched Primers
S. MOLINA CÁRDENAS (1), A. Salazar Aguirre (1), F. Ochoa-Corona (1), J. Olson (1) (1) Oklahoma State University, U.S.A.
Thermophilic helicase dependent amplification (tHDA) is an isothermal DNA amplification that does not require temperature cycling. A study of primers sharing similar thermodynamics, but different GC content in their targeted amplification products, was made to assess fluorescence using self-quenched primers (SqP) reacting in tHDA standard temperature and chemistry. Model viruses used were Rose rosette virus (RRV), High plains wheat mosaic virus (HPWMV) formerly High plains virus (HPV) and Hosta virus X (HVX). RT-PCR was used as the reference method. Temperature gradients (60 to 70°C) were performed to each set of primers. tHDA reactions with SqP were optimized assessing combinations of two primer concentrations (0.15 and 0.2 µM) with two MgSO4 concentrations (4 and 4.5 mM). The sensitivity of each primer set was determined by quantitative tHDA (qtHDA) in real time. Subsequently the products amplified by qtHDA with SqP were visualized in 2% agarose gel electrophoresis. Detection limits using a plasmid DNA carrying the target sequences for RRV and HPWMV are 0.0001 ng. There was no amplification of the HVX targeted region due to its putative high GC content. This study explored primer design criteria for tHDA with SqP for sensitive detection of plant viruses. The use of SqP reduces the cost of qPCR and qtHDA and has acceptable levels of sensitivity. tHDA with SqP bring new possibilities for field deployment primer design in biosecurity and microbial forensics.