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


cellulose powder; dsRNA extraction; ekstraksi dsRNA; kuantitas RNA; Rehmannia mosaic virus; RNA quantity; RT-PCR; serbuk selulosa

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DOI: https://doi.org/10.22146/jpti.26026

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