ANALISIS GEN PENYANDI PROTEIN VP2 VIRUS AVIAN ENCEPHALOMYELITIS (AE) ISOLAT LOKAL DENGAN METODE REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION (RT-PCR)

RATNA ERMAWATI, Dr. drh. Aris Haryanto, M.Si ; Dr. drh. A.E.T.H. Wahyuni, M.Si.

2013 | Tesis | S2 Sain Veteriner

Avian Encephalomyelitis (AE) merupakan penyakit infeksi virus yang dapat menginfeksi ayam, burung, dan kalkun. Kejadian AE di Indonesia telah dilaporkan sejak 2009 yaitu di Jawa Tengah, Yogyakarta, Serang, dan Kalimantan Timur. Gejala klinis yang muncul adalah ataksia, inkoordinasi, paralisis, dan rapid tremor pada kepala dan leher. Diagnosa AE terutama di lapangan masih cukup sulit karena banyak penyakit unggas dengan gejala klinis yang sangat mirip seperti Newcastle Disease (ND), Marek’s Disease (MD), Ricketsia, defisiensi vitamin B1 dan B2, Aspergillosis, Salmonellosis, Koksidiosis, Omphalitis, dan Mycoplasmosis. Oleh karena itu, diperlukan suatu pengujian untuk mendeteksi AE secara akurat, cepat, tepat, dan sensitif. Penelitian ini bertujuan melakukan analisis penggunaan metode diagnostik melalui teknik amplifikasi pada gen penyandi protein VP2 virus AE dari spesimen lapangan berbasis Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Tahapan penelitian ini meliputi koleksi sampel virus AE dari koleksi Balai Besar Veteriner (BBVet) Wates periode 2011-2012. Ekstraksi dilakukan untuk mendapatkan RNA virus AE dan diukur konsentrasinya dengan spektrofotometer. Hasil ekstraksi digunakan sebagai template untuk amplifikasi dengan RT-PCR menggunakan primer pada target gen penyandi protein VP2. Produk RT-PCR divisualisasi dengan elektroforesis gel agarose 1,5% pada UV Transilluminator. Hasil positif ditunjukkan dengan munculnya fragmen DNA sebesar 619 bp yang diinterpretasikan sebagai bagian gen virus AE. Produk RT-PCR disekuensing untuk mengetahui urutan basa nukleotidanya. Hasil sekuensing di-multiple alignment dengan gen penyandi protein VP2 virus AE dari data base Genbank menggunakan program MEGA versi 5.05. Sensitivitas amplifikasi dengan teknik RT-PCR ini hingga 127,75 ng/μl RNA dan spesivisitas primer mencapai 92% dianalisis dengan program BLAST NCBI. Hasil multiple alignment dengan program MEGA 5.05 ditemukan adanya perbedaan nukleotida sebanyak 46 nt dibandingkan dengan isolat AV1775/07 (Genbank) dan 93 nt dibandingkan dengan strain ZCHP2/0912695. Metode RT-PCR dengan target gen penyandi protein VP2 dapat mendeteksi dan mengidentifikasi virus AE dari spesimen lapangan sehingga dapat digunakan sebagai metode diagnostik rutin untuk deteksi penyakit AE pada unggas.

Avian Encephalomyelitis (AE) is a viral infectious disease which can infects chickens, birds, and turkeys. Avian Encephalomyelitis cases in Indonesia have been reported since 2009 in Central Java, Yogyakarta, Serang, and East Kalimantan province. The clinical symptoms of AE disease are ataxia, incoordination, paralysis, and rapid tremor of the head and the neck. Diagnosis of AE, especially in the field is quite difficult because many avian diseases have the similar clinical symptoms, such as Newcastle Disease (ND), Marek's Disease (MD), Rickets, deficiency of vitamin B1 and B2, Aspergillosis, Salmonellosis, Coccidiosis, Omphalitis, and Mycoplasmosis. Therefore, we need a test to detect AE accurately, rapidly, precisely, and sensitively. The aim of this study was to analyze the use of diagnostic method through amplification technique for VP2 protein encoding gene of AE virus from field specimens using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) method. Stages of this study was included the collection of AE virus samples from the collection of Animal Diseases Investigation Center (ADIC) in Wates during 2011-2012. The RNA extraction is conducted to isolate AE virus RNA and its concentration was measured with a spectrophotometer. Extraction product was used as a template for amplification by RT-PCR using a specific primers targeted to VP2 protein encoding gene. The RT-PCR product was visualized with 1.5% agarose gel electrophoresis on a UV Transilluminator. Positive results are shown with the appearance of 619 bp DNA fragment which interpreted as part of AE virus gene. The RT-PCR product was sequenced to determine the sequence of nucleotide bases. The sequencing result was aligned by multiple alignment with VP2 protein encoding gene of AE virus from Genbank data base using MEGA program version 5.05. The sensitivity of the amplification by RT-PCR method reached 127.75 ng/mL RNA. Based on the analysis method by NCBI BLAST program, the primer specificity reached untill 92%. The multiple alignment result by MEGA program version 5.05 also found there are 46 nucleotides differences found compared with AV1775/07 isolates and 93 nucleotides compared with ZCHP2/0912695 strain (Genbank). Amplification by RT-PCR method for VP2 protein encoding gene as a gene target can detect and identify AE virus from field specimens, so it can be used as a routine diagnostic method to detect AE disease in poultry.

Kata Kunci : Avian Encephalomyelitis, gen penyandi protein VP2, RT-PCR, sekuensing