Abstract
Detection of viruses in grapevine plants is important to prevent their spreading with propagating stocks. For this purpose PCR based protocols are commonly and routinely used. Since most grapevine viruses are RNA viruses, the detection procedure starts with RNA purification followed by cDNA synthesis prior to PCR. The cDNAs should be validated by an internal control for which usually constitutively expressed housekeeping genes are used. Here we publish a new set of primers designed in the Vitis vinifera phosphoenolpyruvate carboxylase gene to encompass two or three introns. PCR products amplified from remaining genomic DNA in the RNA samples and from cDNA can be clearly distinguished since cDNA-derived products are smaller in size compared to those amplified from genomic DNA. Thus these primers may be useful reference markers in RT-PCR-based virus detection assays both for the validation of cDNA synthesis and the detection of trace amounts of genomic DNA.
References
Al Rwahnih, M., Dave, A., Anderson, M. M., Rowhani, A., Uyemoto, J. K., & Sudarshana, M. R. (2013). Association of a DNA virus with grapevines affected by red blotch disease in California. Phytopathology, 103, 1069–1076.
Basso, M. F., da Silva, J. C. F., Fajardo, T. V. M., Fontes, E. P. B., & Zerbini, F. M. (2015). A novel, highly divergent ssDNA virus identified in Brasil infecting apple, pear and grapevine. Virus Research, 210, 27–33.
Bertolini, E., García, J., Yuste, A., & Olmos, A. (2010). High prevalence of viruses in table grape from Spain detected by real-time RT-PCR. European Journal of Plant Pathology, 128, 283–287.
Borges, A. F., Fonseca, C., Ferreira, R. B., Lourenco, A. M., & Monteiro, S. (2014). Reference gene validation for quantitative RT-PCR during biotic and abiotic stresses in Vitis vinifera. PloS One, 9(10), e111399.
Gambino, G., Cuozzo, D., Fasoli, M., Pagliarani, C., Vitali, M., Boccacci, P., Pezzotti, M., & Mannini, F. (2012). Co-evolution between Grapevine rupestris stem pitting-associated virus and Vitis vinifera L. leads to decreased defence responses and increased transcription of genes related to photosynthesis. Journal of Experimental Botany, 63, 5919–5933.
Gambino, G., & Gribaudo, I. (2006). Simultaneous detection of nine grapevine viruses by multiplex reverse transcription-polymerase chain reaction with coamplification of a plant RNA as internal control. Phytopathology, 96, 1223–1229.
Gambino, G., Perrone, I., & Gribaudo, I. (2008). A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochemical Analysis, 19, 520–525.
Jelly, N. S., Schellenbaum, P., Walter, B., & Maillot, P. (2012). Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in grapevine somatic embryos. Transgenic Research, 21, 1319–1327.
Jooste, A. E. C., Molenaar, N., Maree, H. J., Bester, R., Morey, L., de Koker, W. C., & Burger, J. T. (2015). Identification and distribution of multiple virus infections in grapevine leafroll diseased vineyards. European Journal of Plant Pathology, 142, 363–375.
Komorowska, B., Berniak, H., & Golis, T. (2014). Detection of grapevine viruses in Poland. Journal of Phytopathology, 162, 326–332.
Lima, M. F., Alkowni, R., Uyemoto, J. K., Golino, D., Osman, F., & Rowhani, A. (2006). Molecular analysis of a California strain of Rupestris stem pitting-associated virus isolated from declining Syrah grapevines. Archives of Virology, 151, 1889–1894.
Maliogka, V. I., Martelli, G. P., Fuchs, M., & Katis, N. I. (2015). Control of viruses infecting grapevine. Advances in Virus Research, 91, 175–227.
Martelli, G. P. (2014). Directory of virus and virus-like diseases of the grapevine and their agents. Journal of Plant Pathology, 96(1Suppl), S1–S136.
Nakaune, R., & Nakano, M. (2006). Efficient methods for sample processing and cDNA synthesis by RT-PCR for the detection of grapevine viruses and viroids. Journal of Virological Methods, 134, 244–249.
OEEP/EPPO. (2008). Pathogen-tested material of grapevine varieties and rootstocks. OEEP/EPPO Bulletin, 38, 422–429.
Oliver, J. E., & Fuchs, M. (2011). Tolerance and resistance to viruses and their vectors in Vitis sp.: A virologist’s perspective of the literature. American Journal of Enology and Viticulture, 62, 438–451.
Osman, F., Leutenegger, C., Golino, D., & Rowhani, A. (2008). Comparison of low-density arrays, RT-PCR and real-time TaqMan® RT-PCR in detection of grapevine viruses. Journal of Virological Methods, 149, 292–299.
Thompson, J. R., Fuchs, M., McLane, H., Celebi-Toprak, F., Fischer, K. F., Potter, J. L., & Perry, K. L. (2014). Profiling viral infections in grapevine using a randomly primed reverse transcription-polymerase chain reaction/macroarray multiplex platform. Phytopathology, 104, 211–219.
Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M., & Rozen, S. G. (2012). Primer3-new capabilities and interfaces. Nucleic Acids Research, 40(15), e115.
White, E. J., Venter, M., Hiten, N. F., & Burger, J. T. (2008). Modified cetyltrimethylammonium bromide method improves robustness and versatility: The benchmark for plant RNA extraction. Biotechnology Journal, 3, 1424–1428.
Xu, Q., Wen, X., & Deng, X. (2004). A simple protocol for isolating genomic DNA from chestnut rose (Rosa roxburghii Tratt) for RFLP and PCR analyses. Plant Molecular Biology Reporter, 22, 301a–301g.
Zhang, Y., Singh, K., Kaur, R., & Qiu, W. (2011). Association of a novel DNA virus with the grapevine vein-clearing and vine decline syndrome. Phytopathology, 101, 1081–1090.
Acknowledgements
This work was supported by National Research, Development and Innovation Office (NKFIH) grants no. K-119783 (to R. O.) and K-115403 (to. E. Sz.) and by Hungarian Ministry of Agriculture (to Á. B.). The authors are grateful to Dr. Éva Várallyay (ABC, Gödöllő, Hungary) for critical reading of manuscript prior to submission.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Oláh, R., Deák, T., Turcsán, M. et al. Use of an intron containing grapevine gene as internal control for validation of cDNA synthesis in virus detection by RT-PCR. Eur J Plant Pathol 149, 765–770 (2017). https://doi.org/10.1007/s10658-017-1218-5
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10658-017-1218-5