Improved RNA Extraction from Woody Plants for the Detection of Viral Pathogens by Reverse Transcription-Polymerase Chain Reaction

Abstract

An efficient procedure for the extraction of high-quality RNA from woody plants without the use of phenol, organic solvents, or alcohol precipitation is described. The method employs commercially available spin-column matrices and mitigates the inhibitory effects of plant polysaccharides and polyphenolic compounds commonly observed on subsequent polymerase chain reaction amplification when conventional extraction methods are applied to woody plant species. The method described has been successfully used in the development of highly sensitive reverse transcription-polymerase chain reaction (RT-PCR) techniques for the detection of a number of viruses in their woody hosts. The viruses detected included apple stem grooving capillovirus (ASGV), apple stem pitting virus, Prunus necrotic ringspot ilarvirus (PNRSV), grapevine fanleaf and Arabis mosaic nepoviruses, and grapevine leafroll-associated closterovirus type 3. The method described was equally effective for the extraction of viral RNA from either budwood, leaves, or flower blossoms as determined by the equivalent RT-PCR detection of ASGV and PNRSV from these tissues. Detection of viral RNA in samples of total plant RNA prepared using this method was found to be as sensitive as was previously described for the immunocapture RT-PCR technique.

Detection of Apple Stem Grooving Virus in Dormant Apple Trees with Crude Extracts as Templates for One-Step RT-PCR

“…Kinard et al (7) and MacKenzie et al (11) also reported the detection of ASGV, especially from bark and leaves of apple trees, by RT-PCR. Both used total RNA preparations as template in a classical twostep system.…”

“…Serological detection by enzyme-linked immunosorbent assay (ELISA) using commercially available antisera represents the first alternative for biological indexing, but its use is limited to a short time period in the growing season and appears inappropriate with dormant woody tissues (7,8,11).…”

“…ASGV detection methods based on reverse transcription-polymerase chain reaction (RT-PCR) have been reported by Kummert et al (8) using degenerate primers designed from conserved sequences of the putative viral RNA polymerase. Recently, Kinard et al (7) and MacKenzie et al (11) reported RT-PCR detection of ASGV from apple tissues using specific primers targeting the putative coat protein encoding region of the viral genome. These protocols rely on two-step RT-PCR procedures using reverse transcriptase with oligo(dT) primer for first strand cDNA synthesis, and Taq DNA polymerase with ASGV-specific primers for PCR amplification.…”

“…The standard sample extraction procedure for RT-PCR detection of ASGV is based on nucleic acid isolation (total RNA) (7,8,11), which is time-consuming and not amenable for processing large numbers of samples. Potentially improved sample processing procedures for plant virus detection by PCR have been reported and include immunocapture (24), direct binding (19), print capture (17), or direct application of clarified plant extracts in reaction tubes (25).…”

Evidence of Grapevine leafroll associated virus-1–3, Grapevine fleck virus and Grapevine virus B Occurring in Himachal Pradesh, India

“…Total RNA was extracted using RNeasy Plant Mini kit (Qiagen, Germany). For DNA extraction CTAB method was used [14,15]. Reverse transcription was carried out in a 25 ll reaction mixture containing 7 ll total RNA (1-1.5 lg), 1.0 ll of 0.2 lg/ll of random hexamer primers, 1.0 ll of 40 mM dNTP mix (Fermentas, Lithuania), 5 ll of 59 RT buffer and 100 Units of M-MuLV Reverse Transcriptase (USB Corporation, USA).…”