Megalocytivirus merupakan salah satu patogen merugikan dalam industri akuakultur, menyebabkan mortalitas benih ikan hingga 100%. Megalocytivirus terbagi dalam tiga klaster yakni Infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), dan turbot reddish body iridovirus (TRBIV). Oleh karena gejala klinis dan patologi akibat infeksi Megalocytivirus adalah identik antar klaster, maka diperlukan metode deteksi molekular. Metode polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) diharapkan dapat mendeteksi Megalocytivirus, serta membedakan antara klaster ISKNV dan RSIV yang menyerang ikan budidaya di Indonesia. Obyek dalam penelitian ini adalah sekuen konservatif dalam ORF 120R121L pada ISKNV dan sekuen homolog pada Megalocytivirus lainnya. Koleksi jaringan dan sampel ikan dikumpulkan kemudian diekstraksi DNA genomnya, dianalisis bersama koleksi isolat DNA genom lainnya. Sekuen DNA target diamplifikasi dengan PCR menggunakan primer yang telah ditentukan. DNA amplikon dipotong menggunakan enzim EcoRV dan disekuensing. Analisis hasil amplifikasi DNA target sepanjang 729 bp, diperoleh 13 sampel terindikasi positif Megalocytivirus dari 51 sampel pada ikan air laut dan air tawar, yang berasal dari daerah Purwokerto, Gondol, Karimunjawa, dan Situbondo. Pemotongan menggunakan enzim EcoRV, menunjukkan seluruh isolat tersebut berada dalam klaster ISKNV. Berdasarkan sekuensing DNA amplikon isolat PW14, KJ01, GN11 dan ST06 menunjukkan positif terinfeksi Megalocystivirus dan memiliki similaritas level nukleotida 99,45-99,86% dengan ISKNV.

Megalocytivirus was known as agents that caused serious systemic disease in freshwater and marine fishes. The mortality up to 100% and been reported in Indonesia. Megalocytivirus can be divided into three clusters i.e. infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), and turbot reddish body iridovirus (TRBIV). Because of clinical symptoms and pathology of those Megalocytiviruses were similar, the molecular detection methods were needed to distinguish the clusters. In this research we used polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) to detect Megalocytivirus and distinguish between clusters ISKNV and RSIV from cultured fishes in Indonesia. A primer pair was designed base on the conserved sequences of ORF 120R-121L from ISKNV and the homologous sequences from other Megalocytivirus. The DNA collection and DNA which extracted from tissue and fish samples were used as templates to amplify a fragment DNA of Megalocytivirus. The amplified DNA sequences were digested using EcoRV to determine the cluster. The four DNA amplicon of Megalocytivirus represent the sampling area were sequenced. In this research we successfully designed primers which able to detect Megalocytivirus by producing 729 bp DNA fragment. Among 51 samples, we found that 13 isolates from marine and freshwater fishes which originally from Purwokerto, Gondol, Karimunjawa and Situbondo were positive infected by Megalocytivirus. Digestion using EcoRV enzyme showed that all isolates were belonged into ISKNV cluster. Sequences analysis showed that those isolates shared 99,45-99,86% similarity on nucleotide level with ISKNV reference.

Kata Kunci : Megalocytivirus, ikan, amplifikasi, enzim EcoRV, ISKNV, RSIV