Comparison of Three Conventional Extraction Methods for the Detection of Plant Virus/Viroid Rnas From Heat Dried High-Phenolic Host Leaves

Abstract

The presence of virus/viroid infections can go unnoticed since symptoms appear only if additional viruses are present. Detection of Plum bark necrosis stem pitting associated virus (PBNSPaV), Apricot latent virus (ApLV), Apple scar skin viroid (ASSVd), Prunus necrotic ringspot virus (PNRSV) and Potato virus Y (PVY) by reverse transcriptase polymerase chain reaction (RT-PCR) or nested-RT-PCR is possible; however, these assays could be unreliable if the tissue contains interfering compounds. This study reports on use of three extraction procedures in recently developed heat-dried virus/viroid preserved plant tissues. The methods tested were lithium chloride, silica-capture and citric buffer. The results showed that (1) the silica-capture RNA extraction method appears to be superior for total RNA extraction; (2) the increase in volume of silica improves the efficiency of RNA extraction from dried infected leaves and (3) the use of silica method minimizes the fragmentation of PCR products and improves the PCR detection of tested pathogens. The results of study indicate that the use of appropriate RNA extraction method is crucial for a successful PCR and an appreciable yield of PCR product from heat-dried infected leaves. © 2007 Asian Network for Scientific Information.