Evaluation of intron containing potential reference gene-specific primers to validate grapevine nucleic acid samples prepared for conventional PCR and RT-PCR

Abstract

Previously we proved the usefulness of an intron containing reference gene, phosphoenolpyruvate carboxylase (PEP) to validate cDNA synthesis for detection of grapevine viruses by conventional RT-PCR from crude nucleic acid preparations. Thus amplicons derived from residual genomic DNA (gDNA) and cDNA can be clearly distinguished by their sizes. Here we designed novel sets of primers which encompass one or two intron containing sequences of grapevine housekeeping genes such as actin, tubulin and elongation factor 1-α. Using these primers the expected sequences were amplified from gDNAs of the tested 24 grapevine cultivars. Thereafter they were challenged using cDNAs prepared from total nucleic acid samples isolated from cambial scrapings of dormant canes, leaf laminas, petioles and in vitro leaves of 12 grapevine cultivars. All of these novel, and the previously published PEP gene-specific primers generated the amplification of the expected shorter DNA fragments without introns. Thus they are suitable to check the quality of nucleic acid preparations and to validate subsequent cDNA synthesis prior to pathogen detection assays.