DETEKSI GEN env-TM VIRUS PENYAKIT JEMBRANA STRAIN TABANAN 1987 DENGAN METODE NUCLEIC ACID SEQUENCE BASED AMPLIFICATION

ATIK RATNAWATI, Dr. drh. Asmarani Kusumawati, MP. ; Prof. Dr. drh. Sri Hartati, SU.

2015 | Tesis | S2 Bioteknologi

Penyakit Jembrana merupakan penyakit viral pada sapi Bali yang diakibatkan oleh Lentivirus yang disebut VPJ, mengakibatkan sindrom penyakit yang bersifat berat dan akut hingga menyebabkan kematian setelah masa inkubasi yang pendek. Penyakit ini telah menyebar ke beberapa daerah di Indonesia, sehingga diperlukan metode deteksi dini penyakit Jembrana yang dapat diaplikasikan secara sederhana tetapi memberikan hasil yang spesifik dan sensitif. Metode Nucleic Acid Sequence Based Amplification (NASBA) dikembangkan untuk deteksi VPJ strain Tabanan 1987 dengan amplifikasi target gen env-TM pada kondisi isothermal dengan memanfaatkan aktivitas transkripsi oleh tiga enzim yaitu AMV-RT, RNAse H dan T7 RNA Polimerase. Optimasi kondisi reaksi NASBA dilakukan dengan variasi waktu dan konsentrasi KCl. Hasil optimasi digunakan untuk uji spesifitas dan sensitifitas. Sensitifitas hasil uji NASBA dibandingkan dengan hasil uji RT-PCR. Spesifisitas hasil uji virus VPJ dibandingkan dengan hasil uji sapi Bali dan sapi PO sehat serta pGEX-CA. Hasil penelitian menunjukkan kondisi optimum reaksi NASBA untuk deteksi gen env-TM VPJ strain Tabanan 1987 pada waktu 90 menit dengan konsentrasi KCl 70 mM. Reaksi NASBA hanya mampu mengamplifikasi RNA dengan konsentrasi 10-5 ng/ul, 10 kali kurang sensitif dari reaksi RT PCR yang mengamplifikasi RNA dengan konsentrasi 10-6 ng/ul dalam mendeteksi gen env-TM VPJ strain Tabanan 1987. Reaksi NASBA spesifik hanya mengamplifikasi gen env-TM VPJ. Teknik ini memberikan hasil positif adanya VPJ pada sampel organ berupa limpa, hati, paru, jantung, lgl. prescapularis dan lgl. prefemoralis yang berasal dari sapi yang diinfeksi VPJ strain Tabanan 1987. Kondisi optimal reaksi NASBA dapat mendeteksi keberadaan gen env-TM VPJ strain Tabanan 1987 pada sampel darah.

Jembrana disease is a viral disease in Bali cattle caused by a member of the Lentivirus called JDV, which lead to an acute and severe disease syndrome, and even death, short after the incubation period. Since it has spread to other areas of Indonesia, an early detection technique which can be applied simply but provides a spesific and sensitive result is highly required. Nucleic Acid Sequence Based Amplification (NASBA) was developed to detect JDV strain Tabanan 1987 with env-TM gene as the target of amplification using transcription activity at isothermal condition of three enzymes, i.e., AMV-RT, RNAse H and T7 RNA Polymerase. The optimization condition of NASBA reaction included time and KCl concentration. The optimization results obtained was then applied for specivicity and sensitivity tests. Sensitivity result of NASBA was compared with RT PCR method. Whereas specivicity result of JDV viruses was compared with a healthy Bali and PO cattles, and also pGEX-CA. Results of the study showed that the optimum condition for NASBA reaction for detection of env-TM gene on JDV strain Tabanan 1987 was 90 minutes, and 70mM KCl concentration. Nasba reaction was able to amplify RNA up to 10-5 ng/ul, 10 time less sensitive than RT PCR which was able to amplify up to 10-6 ng/ul for env-TM gene detection on JDV strain Tabanan 1987 samples. NASBA reaction was able to amplify env-TM gene on JDV. This technique gave a JDV positive results in organ samples of spleen, liver, lung, cor, lgl prefemoralis and lgl. prescapularis which were derived from experimental cattle inoculated with JDV strain Tabanan 1987. The optimum condition of NASBA reaction was able to detect the presence of env-TM gene on JDV strain Tabanan 1987 in blood sample.

Kata Kunci : NASBA, sapi Bali ,VPJ strain Tabanan 1987, gen env-TM