* Corresponding Author
Perbaikan Metode Ekstraksi dsRNA Virus secara Sederhana untuk RT-PCR Tiga Virus Tumbuhan
https://doi.org/10.22146/jpti.26026 Wiwik Endarsih(1*), Sedyo Hartono(2), Sri Sulandari(3)
(1) Balai Besar Karantina Pertanian Surabaya Jln. Raya Ir H. Juanda, Sidoarjo, Jawa Timur 61253
(2) Departemen Hama dan Penyakit Tumbuhan, Fakultas Pertanian, Universitas Gadjah Mada Jln. Flora 1, Bulaksumur, Sleman, Yogyakarta 55281
(3) Departemen Hama dan Penyakit Tumbuhan, Fakultas Pertanian, Universitas Gadjah Mada Jln. Flora 1, Bulaksumur, Sleman, Yogyakarta 55281
(*) Corresponding Author
Abstract
Replicative form (RF) of RNA viruses are dsRNA structured nucleic acid, always found in plants infected by RNA virus. The principle of dsRNA extraction is based on the different affinity of nucleic acids for the cellulose powder and the specific adsorption in 16.6% ethanol buffer. The study aims to develop the simple dsRNA extraction method for the preparation of RT-PCR detection for Rehmannia mosaic virus (ReMV), Cucumber mosaic virus (CMV), Tomato chlorosis virus (ToCV), and compared with commercial kit. The analysis was performed by quantification of nucleic acid with spectrophotometer, efficiency of method (level of complexity, time, cost per reaction) and sequencing. The RNA concentration with simple methode of dsRNA extraction was lower than kit extraction method but the both methods have same pure RNA result. The PCR and sequencing result showed that viral pathogen of pepper, tobacco, and tomato leaf was CMV, ReMV, and ToCV, respectively with amplicon size at 500, 568, and 360 bp. This method is quite cheap and the RNA quantity is proportional to the commercial kit. The simple method of dsRNA extraction can be proposed for the preparation of RT-PCR detection for CMV, ReMV, ToCV.
Intisari
Replicative form (RF) virus RNA merupakan asam nukleat berstruktur dsRNA, selalu ditemukan pada tumbuhan terinfeksi oleh virus RNA. Prinsip kerja ekstraksi dsRNA berdasarkan afinitas serbuk selulosa terhadap asam nukleat dan adsorbsi spesifik dsRNA pada konsentrasi etanol 16,6 %. Penelitian ini bertujuan untuk mengembangkan metode ekstraksi dsRNA secara sederhana untuk preparasi deteksi RT-PCR terhadap Rehmannia mosaic virus (ReMV), Cucumber mosaic virus (CMV), Tomato chlorosis virus (ToCV) dan dibandingkan dengan kit komersil. Data yang dibandingkan adalah kuantitas asam nukleat, analisa efisiensi metode (tingkat kerumitan, waktu, biaya per reaksi) serta sekuensing. Konsentrasi RNA hasil ekstraksi metode dsRNA secara sederhana lebih rendah dibanding dengan metode kit, namun kedua metode menghasilkan RNA yang murni. Berdasarkan hasil PCR dan sekuensing disimpulkan bahwa virus penyebab mosaik daun lada dan tembakau serta klorosis daun tomat berturut-turut adalah CMV, ReMV, dan ToCV dengan ukuran amplikon berturut turut 500, 568 dan 360 pb. Metode ini cukup murah dan kuantitas RNA yang dihasilkan sebanding dengan kit komersil. Ekstraksi RNA menggunakan metode dsRNA secara sederhana dapat dikembangkan untuk preparasi deteksi RT-PCR terhadap CMV, ReMV, ToCV.
Keywords
Full Text:
PDFReferences
Adiputra, J., S.H. Hidayat, & T.A. Damayanti, 2012. Evaluasi Tiga Metode Preparasi RNA Total untuk Deteksi Turnip mosaic potyvirus dari Benih Brassica rappa dengan Reverse Transcription-Polymerase Chain Reaction Jurnal Fitopatologi Indonesia 8: 44−49.
Azizi, P., M. Y. Rafii, M. Mahmood, S.N.A. Abdullah, M.M.Hanafi, M.A. Latif & S. Ashkani. 2017. Evaluation of RNA Extraction Methods in Rice and their Application in Expression Analysis of Resistance Genes against Magnaporthe oryzae. Biotechnology and Biotechnological Equipment 31: 75−84.
Balijja, A., A. Kvarnheden, & T. Turchetti, 2008. A Non-phenol-chloroform Extraction of Double-stranded RNA from Plant and Fungal Tissues. Journal of Virological Methods 152: 32–37.
Diningsih, E. 2016. Carnation mottle virus pada Tanaman Anyelir di Indonesia: Etiologi, Ekspresi Gen Protein Selubung, dan Eliminasinya. Tesis. Institut Pertanian Bogor, Bogor. 119 p.
Dodds, J.A., T.J. Morris & R.L. Jordan, 1984. Plant Viral Double-Stranded RNA. Annual Review of Phytopathology 22: 151−168.
Dovas, C.I., K. Efthimiou, & N.I. Katis, 2004. Generic Detection and Differentiation of Tobamoviruses by a Spot nested RT-PCR-RFLP Using dI-Containing Primers Along with Homologous dG-Containing Primers. Journal of Virological Methods 117: 137–144.
Edy, V. G., M. Szekely, T. Loviny, & C. Dreyer, 1976. Action of Nucleases on Double Stranded RNA. European Journal of Biochemistry 61: 563–572.
Hartono, S., T. Natsuaki, H. Sayama, H. Atarashi, & S. Okuda, 2003. Yellowing Disease of Tomatoes Caused by Tomato infectious chlorosis virus Newly Recognized in Japan, Journal of General Plant Pathology 69: 61–64.
John, M. E., 1992. An Efficient Method for Isolation of RNA and DNA from Plants Containing Polyphenolics. Nucleic Acids Research 20: 2381.
Karan, M., S.Hicks, R.M. Harding & D.S.Teakle, 1991. Stability and Extractability of Double-stranded RNA of Pangola Stunt and Sugarcane Fiji Disease Viruses in Dried Plant Tissues. Journal of Virological Methods 33: 211–216.
Karp, A., P.G. Isaac, & D.S. Ingram, 1998. Isolation of Nucleic Acids Using Silica-Gel Based Membranes: Methods Based on the Use of QIAamp Spin Columns. p. 59−63. In A. Karp, D.S. Ingram, P.G. Isaac (eds.) Molecular Tools for Screening Biodiversity. Chapman & Hall Springer, Netherlands.
Li, R., R. Mock, Q. Huang, J. Abad, J. Hartung, & G. Kinard, 2008. A Reliable and Inexpensive Method of Nucleic Acid Extraction for the PCR-based Detection of Diverse Plant Pathogens. Journal of Virological Methods 154: 48–55.
Maréchal-Drouard, L & P. Guillemaut, 1995. A Powerful but Simple Technique to Prepare Polysaccharide-free DNA Quickly and without Phenol Extraction. Plant Molecular Biology Reporter13: 26−30.
Morris, T.J., & J.A. Dodds. 1979. Isolation and Analysis of Double-stranded RNA from Virus-infected Plant and Fungal Tissue. Journal of Phytopathology 69: 854−858.
Nassuth, A., E. Pollari, K. Helmeczy, S. Stewart, & S.A. Kofalvi. 2000. Improved RNA Extraction and One-tube RT-PCR Assay for Simultaneous Detection of Control Plant RNA Plus Several Viruses in Plant Extracts. Journal of Virological Methods 90: 37–49.
Newbury, H. J., & J.V. Possingham. 1977. Factors Affecting the Extraction of Intact Ribonucleic Acid from Plant Tissues Containing Interfering Phenolic Compounds. Plant Physiology 60: 543–547.
Okada, R., E. Kiyota, H. Moriyama, T. Fukuhara, & T. Natsuaki. 2015. A Simple and Rapid Method to Purify Viral dsRNA from Plant and Fungal Tissue. Journal of General Plant Pathology 81: 103–107.
Porebskim, S., L.G. Bailey & B.R. Baum. 1997. Modification of a CTAB DNA Extraction Protocol for Plants Containing High Polysaccharide and Polyphenol Components. Plant Molecular Biology Reporter Journal 15: 8−15.
Rapley, R. & J. Heptinstall. 1998. Protocol RNA Isolation and Characterization Protocols Volume 86 of the Series Methods in Molecular Biology. p. 65−68. In R. Rapley & D.L. Manning (eds.) Methods in Molecular Biology. Humana Press Inc., New Jersey.
Sahu, S.K., M. Thangaraj, & K. Kathiresan. 2012. DNA Extraction Protocol for Plants with High Levels of Secondary Metabolites and Poly-saccharides without Using Liquid Nitrogen and Phenol. Molecular Biology 1: 1−6.
Scholthof, K-B. G. 2008. Tobacco Mosaic Virus: The Beginning of Plant Pathology. APSnet Features. http://www.apsnet.org/publications/apsnetfeatures/ Pages/TMV.aspx, modified 13/6/17.
Tan, S. C. & B.C.Yiap. 2009. DNA, RNA, and Protein Extraction: The Past and The Present. Journal of Biomedicine and Biotechnology 2009: 1−10.
Tzanetakis, I.E. & R.R. Martin. 2008. A New Method for Extraction of Double-stranded RNA from Plants. Journal of Virological Methods 149: 167–170.
Wyley, S., C. R. Wilson, R. A. C. Jones & M. G. K. Jones. 1993. A Polymerase Chain Reaction Assay for Cucumber Mosaic Virus in Lupin Seeds. Australian Journal Agriculture Research 44: 41–51.
Valverde, R.A, 1990. Analysis of Double-stranded RNA for Plant Virus Diagnosis. Plant Disease 74: 255−258.
DOI: https://doi.org/10.22146/jpti.26026
Article Metrics
Abstract views : 6468
| 
views : 4309
Refbacks
- There are currently no refbacks.
Copyright (c) 2017 Jurnal Perlindungan Tanaman Indonesia
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
Jurnal Perlindungan Tanaman Indonesia ISSN 1410-1637 (print), ISSN 2548-4788 (online) is published by the Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada, in collaboration with Indonesian Entomological Society (Perhimpunan Entomologi Indonesia, PEI) and Indonesian Phytopathological Society (Perhimpunan Fitopatologi Indonesia, PFI). The content of this website is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
View website statistics