عنوان مقاله [English]
Introduction: To now, more than 70 viral diseases have been reported from grapevine. Serological methods are regular diagnostic tools of grapevine viruses, however, their sensitivity has affected by seasonal fluctuations of the virus. Reverse transcription polymerase chain reaction provides significant improvement in detection of grapevine viruses. Extraction of high-quality RNA is essential for the successful application of many molecular techniques, such as RT-PCR. Extraction of high-quality RNA from the leaves of woody plants, such as grapevine, is particularly challenging because of high concentrations of polysaccharides, polyphenols, and other secondary metabolites. Some RNA extraction methods yield pellets that are poorly soluble, indicating the presence of unknown contaminants, whereas others are gelatinous, indicating the presence of polysaccharides. RNA can make complexes with polysaccharides and phenolic compounds render the RNA unusable for applications such as reverse transcription. Grapevine fanleaf virus is a member of the genus Nepovirus in the family Secoviridae. The GFLV genome consists of two positive-sense single stranded RNAs. The genome has a poly (A) tail at the 3´ terminus and a covalently linked VPG protein at the 5´ terminus. Several extraction methods had been reported to be used for identification of GFLV in grapevine. Some of them require harmful chemical material; disadvantages of other are high costs. Immunocapture-RT-PCR requires preparation of specific antibody and direct binding RT-PCR (DB-RT-PCR) has a high contamination risk. In this study, four RNA extraction protocols were compared with a commercial isolation kit to explore the most efficient RNA isolation method for grapevines.
Material and Methods: 40 leaf samples were randomly collected during the growing season of 2011-2012. GFLV was detected in leaf samples by enzyme linked immunosorbent assay (ELISA) Using specific antibodies raised against Iranian isolate of the virus (Zakiaghl and Izadpanah 2003). The RNA isolation protocols of Triazol extraction, high salt phenol-chloroform extraction (Rowhani et al. 1993), RNA extraction using silicon dioxide, Silica (Boom et al 1990), CTAB-PVPP extraction and a commercial RNA isolation kit were used in the study. In all protocols, 50 mg leaf samples were used. The quality and purity of the extracted RNA were determined using spectrophotometry. For purity assessment, the absorbance for the A260/280 and A260/230 ratios was taking in water. RT-PCR was performed using DetF (5´-CGGCAGACTGGCAAGCTGT-3´) and DetR (5´-GGTCCAGTTTAATTGCCATCCA-3´) specific primer pair amplified 1000 bp of the coat protein gene of GFLV. PCR products were run on 1% agarose gel containing 0.5 µg/ml DNA Green Viewer, and visualized under UV irradiation.
Results: There were large differences in the amount of RNA extracted per gram of tissue depending on the protocol used. The commercial kit, CTAB-PVPP and TRIzol methods gave the highest yields in micrograms RNA per gram fresh weight (235-300 μg/g). In contrast, the application of Silica gave the lowest yield (11 μg/g). The high salt phenol-chloroform method gave a moderate yield of over 77 μg/g. In this respect, the CTAB-PVPP method provides the highest yield of RNA. RNA quality was assessed by three methods: A260/280, A260/230 and ability to produce RT-PCR products. A260/280 ratios indicate the level of protein contamination in the preparation. The commercial kit, CTAB-PVPP, TRIzol and high salt phenol-chloroform methods gave RNA with very low amounts of protein contamination. In contrast, RNA isolated by the Silica showed more protein contamination, as indicated by the lower A260/280 ratios. A260/230 ratios are used to assess the level of contamination by polysaccharides and polyphenols. The high salt phenol-chloroform method yielded RNA that was contaminated with polysaccharides. In contrast, the CTAB-PVPP, TRIzol methods yielded RNA with very little polysaccharide. RNA preparations were further tested for quality using RT-PCR reactions. In PCR analyses, whereas traditional methods yielded amplification ratios of 40-88% and the commercial isolation kits yielded amplification ratios of 16%. The RNA from Silica, high salt phenol-chloroform extraction, TRIzol and CTAB-PVPP extraction methods consistently resulted in amplification in 40, 56, 60 and 88% of the samples, respectively.
Discussion: The choice of extraction method depends upon the application in which the RNA will be used. Yield, A260/280, and A260/230 ratios are not good indicators of RT-PCR competent RNA. RNA should be checked for degradation. For RT-PCR, four extraction protocols of Silica, high salt phenol-chloroform extraction, TRIzol and CTAB-PVPP gave consistent product, and however, the last one was the most efficient method. The Silica extraction had significant RNA degradation, making problems in quantification with RT-PCR. Sodium perchlorate extractions had slight RNA degradation, low extraction efficiency, and cost substantially more than the Tris-LiCl method. The CTAB-PVPP method provided high yield and appropriate quality, consistent results for identification of the virus in RT-PCR. The high salt phenol-chloroform and TRIzol extraction were significantly cheaper than CTAB-PVPP methodical, but gave lower yields and were unsuitable for obtaining questionable results in RT-PCR.