Deteksi Cepat Carnation mottle virus pada Tanaman Anyelir (Dianthus caryophyllus L.) [Rapid Detection of Carnation mottle virus on Carnation Plant (Dianthus caryophyllus L.)]

Erniawati Diningsih, Gede Suastika, Tri Asmira Damayanti, Slamet Susanto

Abstract


Carnation mottle virus (CarMV) merupakan virus penting pada tanaman anyelir di Indonesia maupun di dunia.  Deteksi virus yang mudah dan cepat, diperlukan untuk memantau sumber induk anyelir bebas virus. Tujuan penelitian adalah mengevaluasi tiga metode preparasi RNA total yang mudah dan cepat dari tanaman anyelir sebagai templat one step RT-PCR.  Sumber RNA total adalah dari daun dan batang anyelir terinfeksi CarMV. Metode yang dievaluasi yaitu SDT, SEM, dan kit komersial sebagai pembanding. Optimasi dilakukan terhadap konsentrasi akhir primer (0.4 – 1.0 µM) dan MgCl2 (1.5 dan 2.0 mM).  Metode SDT dan SEM berhasil mendapat RNA total dari tanaman anyelir baik dari sampel daun maupun batang.  Keberhasilan 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 terbaik dibandingkan batang. Preparasi RNA total dengan metode SDT dan SEM adalah metode cepat, mudah, dan murah dalam menyediakan templat one step RT-PCR. Konsentrasi primer 0.4 µM dan MgCl2 2 mM merupakan konsentrasi optimum dan menghasilkan hasil amplifikasi terbaik

Keywords

Carmovirus; Simple extraction method (SEM); Simple direct tube (SDT); One step RT-PCR

Abstrack

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 MgCl2 (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 MgCl2 concentration of 0.4 μM and 2 mM, respectively were optimum concentration and produced best amplification result.


Keywords


Carmovirus; Simple extraction method (SEM); Simple direct tube (SDT); One step RT-PCR

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References


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DOI: http://dx.doi.org/10.21082/jhort.v27n1.2017.p95-104

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