PENENTUAN PATOTIPE VIRUS NEWCASTLE DISEASE (ND) ISOLAT LOKAL PADA UNGGAS DENGAN METODE REVERSE TRANSCRIPTION POLYMERATION CHAIN REACTION DAN RESTRICTION ENDONUCLEASE ANALYSIS (RT-PCR DAN REA)

Medania Purwaningrum, Dr.drh. Aris Haryanto, M.Si

2014 | Tesis | S2 Sain Veteriner

Newcastle disease (ND) adalah penyakit infeksius pada unggas yang disebabkan oleh virus famili Paramyxoviridae, genus Avulavirus, spesies Avian paramyxovirus serogrup Avian paramyxovirus tipe 1 (APMV-1). Virus ND menyebabkan kerugian ekonomi yang sangat besar pada industri perunggasan di Indonesia. Newcastle disease di lapangan pada umumnya ditandai dengan beberapa gejala seperti gangguan pernapasan, gangguan pencernaan, gangguan saraf, penurunan produksi telur dan dapat berakhir dengan kematian. Penentuan patotipe virus ND untuk membedakan virulensi virus sulit dilakukan dan membutuhkan waktu yang lama serta biaya yang mahal. Oleh karena itu, diperlukan pengembangan metode untuk membedakan virus ND strain virulen dan avirulen secara sederhana, mudah dan cepat. Penelitian ini bertujuan untuk membedakan strain virus ND virulen dan avirulen dari gen F yang merupakan penentu patogenesitas virus ND dengan metode RT-PCR-REA menggunakan enzim restriksi Hin1l, Bam H1, dan Apa1. Sepuluh sampel virus ND diperoleh dari koleksi BBVet Wates periode 2012-2013 yang didapat dari kasus lapangan. Ekstraksi dilakukan untuk mendapatkan RNA virus ND. Hasil ekstraksi digunakan sebagai template untuk amplifikasi dengan RT-PCR menggunakan primer pada target gen F. Sepasang primer digunakan untuk mengamplifikasi gen F(forward 5’-GGAGCCAAACCGCGCACCTGCGG-3’ dan reverse 5’-GAGGATGTTGGCAGCAT-3’). Produk RT-PCR divisualisasi dengan elektroforesis gel agarose 1,5% pada UV Transilluminator. Hasil amplifikasi sampel ND dengan metode RT-PCR pada target gen F menunjukkan hasil positif dengan munculnya fragmen DNA sebesar 767 bp. Metode RT-PCR dan REA dengan enzim restriksi Hin 1l dan Bam H1 dapat digunakan untuk menentukan patotipe virus ND dari spesimen lapangan. Produk RT-PCR dan REA yang menunjukkan perbedaan pola pemotongan disekuensing untuk di analisis menggunakan software MEGA 5.10 dan analisis peta pola pemotongannya dengan CLC sequence viewer versi 6.8.1. Hasil penentuan patotipe virus ND dengan metode RT-PCR dan REA memiliki kecocokan dengan hasil sekuensing dalam menentukan patotipe virus ND.

Newcastle disease (ND) is a contagious viral disease caused by Avian Paramyxovirus Serotype-1 (APMV-1), species Avian paramyxovirus, genus Avulavirus, family Paramyxoviridae. This viral infection is responsible for devastating outbreak by attacking nerve, respiration, and also digestive system. This disease often followed with decreasing of eggs production and also responsible for economic loss in the poultry industries around the globe. Existing conventional method to identify the patotype of ND virus to differentiate between avirulent and virulent strain was time consuming and expensive. Those reason indicate the necessity to develop a new method to differentiate virulent or avirulent strain of ND virus. The main goal was to differentiate virulent or avirulent strain of ND virus from gene F, which is the virulent marker of ND virus, by RT-PCR and REA method, using 3 restriction enzymes, Hin1L, BamH1, and Apa1. Ten ND virus samples came from Animal Disease Investigation Center (ADIC) Wates virus collection, collected from field case in 2012-2013. RNA of ND virus was collected by extraction from the samples. The product of extraction were used as a template for amplification in RT-PCR. The target of RT-PCR amplification was gene F. In order to amplify this gene, a pair of primer were used (forward 5'-GGAGCCAAACCGCGCACCTGCGG-3' and reverse 5'-GAGGATGTTGGCAGCAT-3'). After the process of RT-PCR, the product then visualized by 1,5% gel agarose electrophoresis on UV transilluminator. The results indicated positive reaction due to existing of DNA fragment band in size of 767 bp. RT-PCR and REA with Hin1l and BamH1 can be used as tool to determine the patotype of ND virus from field specimen. RT-PCR product and REA that showed different restriction pattern were sequenced to get the analysis by using MEGA 5.10 and its restriction pattern map were analyzed by CLC squence viewer 6.8.1. The final result of patotype identification showed similarity between RT-PCR and REA method with sequencing method.

Kata Kunci : Virus Newcastle disease, gen penyandi protein F, RT-PCR dan REA, sekuensing