Deteksi Cepat Carnation mottle virus pada Tanaman Anyelir (Dianthus caryophyllus L.)

Abstract

ABSTRAK.Carnation mottle virus (CarMV) merupakan virus penting pada tanaman anyelir di dunia, termasuk di Indonesia. Deteksi virus yang mudah dan cepat, diperlukan untuk memantau tanaman induk anyelir bebas virus. Tujuan penelitian adalah mengevaluasi tiga metode preparasi RNA total yang mudah dan cepat dari tanaman anyelir sebagai template one step RT-PCR. Sumber RNA total berasal dari daun dan batang anyelir yang terinfeksi CarMV. Metode yang dievaluasi, yaitu simple direct method (SDT), simple extraction method (SEM), dan kit komersial sebagai pembanding. Optimasi dilakukan terhadap konsentrasi akhir primer (0,4 -1,0 µM) dan MgCl 2 (1,5 dan 2,0 mM). Metode SDT dan SEM berhasil mendapat RNA total dari sampel daun maupun batang. Hasil yang didapat dengan metode SDT dan SEM sebanding dengan kit komersial. One step RT-PCR RNA total yang digabungkan dengan metode SDT dan SEM menghasilkan intensitas DNA yang sebanding dengan kit komersial. RNA total dari daun sebagai sumber templat one step RT-PCR lebih baik dibandingkan batang. Preparasi RNA total dengan metode SDT dan SEM merupakan metode cepat, mudah, dan murah dalam menyediakan templat one step RT-PCR. Konsentrasi primer 0,4 µM dan MgCl 2 2 mM merupakan konsentrasi optimum dan mendapatkan hasil amplifikasi terbaik. Kata kunci: Carmovirus; Simple extraction method (SEM); Simple direct tube (SDT); One step RT-PCR ABSTRACT. Carnation mottle virus (CarMV) is an important virus on carnation plants in the world, including in Indonesia.A rapid and easy virus detection is necessary to monitor the source of virus free carnation mother plant. The aim of the research was to evaluate three methods of rapid and easy total RNA preparation from carnation plants as template of one step RT-PCR. The total RNA source is from the CarMV infected leaf and stem of carnations. The evaluated method namely simple direct method (SDT), simple extraction method (SEM), and commercial kit as comparison. Optimization was performed to a final concentration of premiere (0.4 -1.0 µM) and MgCl 2 (1.5 and 2.0 mM). SDT and SEM method successfully obtained a total RNA from both leaves and stems samples. The obtained success by the SDT and SEM methods were comparable with these of commercial kit. One step RT-PCR of total RNA combined with SDT and SEM methods produced DNA intensity comparable with commercial kits. Total RNA from leaves known to be the best source of one step RT-PCR template compared to these from stem. Total RNA preparation by SDT and SEM method is a method of quick, easy, and inexpensive to provide a template one step RT-PCR. Premiere and MgCl 2 concentration of 0.4 μM and 2 mM, respectively were optimum concentration and produced best amplification result.

Multiplex RT-PCR Assay for Crinivirus Detection Using RNA Prepared from Three Extraction Methods on Tomato Plant

“…A buffer containing guanidine thiocyanate is the preferred lysis buffer, yet this buffer can cause freezing of the sample depending on the number and type of secondary metabolites hence RNA extraction is difficult to be conducted, whereas for the extraction of plants containing many secondary metabolites is better to use a lysis buffer containing guanidine hydrochloride (Qiagen, 2012). Diningsih et al (2017). Therefore, based on the complexity level to extract the samples, the SDT is the easiest method compared to the other.…”

“…Whereas the highest RNA concentration extracted by Kit was from Kopeng (332.40 µg/ml) and the lowest was from Malang (23.20 µg/ml). To obtain total RNA or virus RNA with the high quality and quantity, certain chemicals in extraction buffers are needed to inhibit the RNase enzymes (Claros & Canovas, 1999;Diningsih et al, 2017). SDT method uses DEPC containing RNase inhibitors as RNA solvents to prevent RNA degradation by RNase (Amanda & Cartealy, 2015).…”

“…The simple dsRNA uses 2-mercaptoethanol in extraction buffers to eliminate ribonuclease (RNase) which is released during cell lysis. Likewise, RNase degrading agents in the buffer are present in commercial kits (Diningsih et al, 2017). In addition, sample selection also influences the concentration level resulted from the extraction as reported by Endarsih et al (2017) that a higher concentration of virus would be obtained in leaf samples with symptoms of early viral infections than in old tissue with advanced infection symptoms.…”

“…In some developed-extraction methods, RNA extraction requires quite long and complicated stages and uses hazardous chemicals, i.e. phenol and chloroform (Diningsih et al, 2017). The long and complicated extraction stages can allow contamination present between samples hence the results are not as expected (Suehiro et al, 2005).…”

“…Those methods are able to obtain RNA from plants and viruses. Simple RNA extraction using SDT method successfully detected several species of viruses, such as Turnip mosaic virus (TuMV), Green mottle mosaic virus (CGMMV), Cucumber mosaic virus (CMV), Sugarcane streak mosaic virus (SCSMV), TICV, ToCV, Chrysanthemum virus B (CVB), Chrysanthemum stunt viroid (CSVd), and Carnation mottle virus (CarMV) (Suehiro et al, 2005;Adiputra et al, 2012;Damayanti & Putra, 2012;Temaja et al, 2012;Diningsih et al, 2017). In addition, the simple dsRNA extraction method has succeeded in detecting several viruses, such as Rehmanian mosaic virus (ReMV), CMV, and ToCV (Fajarfika et al, 2015;Endarsih et al, 2017).…”