Analisis komparative gen aroA pasteurella multocida isolat Indonesia dengan PCR-SSCP

MADUSARI, Lorensia Renni, Dr. Widya Asmara

2002 | Tesis | S2 Bioteknologi

I’usiezirellu tnzil~ocidus erotipe B 2 inenipakan bakteri patogen utama peiiyebab penyakit septikemia hemoragfika atau pasteurellosis primer, penyaht yang inenyerang sap1 dai kerbao. Keriigian ekonomi yang terbesar dilaporkan terjadi di Asia, tenrtama di Indonesia. Usaha pengendalian yang pernali dijalankan benipa vaksinasi, beripa peinbei-ran vaksin tnalstif dari bakteri 1). /rnil/ocidcr B:2 galiir Katha dari Myanmar dalam bentik oil c-~lJrrvnnHt.a sil vaksiiiasi tidak melindingi 100Y0 teiiiak Indonesia. Miitasi mxotrophic yang dilakitkan terhadap kemampiian bakteri I? t t i i i l l o c d r B:2 dalam mensiiitesis asain-asain amino aromatik tnenipakan iisalia lain intitk meinperoleli vaksin yang bersifat aktif. Gen crroA mutan yang akan diliasilkan inemberi liarapan ilntuk dapat memenuli kebuttulian vaksin yang dapat mengprangi kematiai hewan-liewan teniak akibat penyakit &lit hi. Penelitian ini berhditm inhik mernbandhingkan sekuen gen aroA dari enam isolat di Indonesia (dua isolat dari Bali, Madiira, Klaten dan dua isolat dari Kupang, NTT) yang dilaktkai sebagai penelitian pendahiduan imtilk inel ihat seberapa jaih perbedaan yang diiniliki dengan galur Katha dxi Myaiinar. Penelitian diawali dengan rekoiistitusi isolat-isolat bakteri koleksi FKH, UGM yang telah diuji secara biokimiawi. Isolasi DNA dilakukan dengan inetode blannur (1961) yang telali dimodifikasi, keinudian gen am A diamplifikasi dengm tehiik PCR (Polytnei.ase (‘hain I-kaction) inengguiiakan pniner spesifik FWPAl dai RWPAZ sehingga diperoleh satu fi-apmi gen aro A I’crstetrrdl(i rrrctltocrdu serotipe €3.2 benikiuan 1,2 kb. Hasil ainplifikasi gen trrriA dianalisis daigsui RFLP maiggiu~akm endonilklease restiiksi Hpdl inengliasilkan dua macam hcrncl benikiiran 740 dan 460 bp Selauljuttnya dilakikan analisis SSCP (Singlr-LStrcmdC ’rinjomution PulynioiThr.tm), iintuk melihat ada tidahnya variasi sekuen yang ditandai dengan mobilicv sh$. Hasil penelitian meniinjuk’kan bahwa analisis PCR-SSCP pada gen aroA I? inziI/oadu B:2 dari 6 isolat Indonesia dengan isolat Myaiinar, tidak menunjukkan adanya mobility . s h j dan ini mengiiidikasikan baliwa tidak ada perbedaan sekuen gen uroA diaiitara isolat-isolatl strain-strain yang diisolasi

I’u\teure//tr tnzi/locidu serotype B:2 is a pninary pathogen bacterium that cause haemorrhagic septicaeinia or primary pasteurellosis, the disease attacks the cattle, cows and buffaloes. The greatest economic losses sre recorded in Asia, especially in Indonesia. The controlling endeavor that had ever been nin was vaccination by giving inactive vaccine fTom B:2 f’.triii//oc/dcis train Katha from Myanmar in oil adjuvant fonn. The result was the vaccination did not protect 100% cattle in Indonesia. Auxotrophic mutation in the capability of P. t?ndtocidu serotype B:2 in synthesis aromatic amino acids is another attempt to generate live vaccine. The aroA mutant generated will fulfill the need of vaccine that could reduce the death of the cattle caused by this acute disease. The research aim is to compare aroA sequence from the six isolates in Indonesia (two isolates from Bali, one from Madura, one froin Klaten and two froin Kupang) arid is conducted as a prelimuiary research to find out their differences with Katlia strain from Myanmar. The research process began with the reconstitution of bacterium isolates, collection of the Faculty of, Veterinary Medical, Gadjah hlada University, that had been biochemical tested. The DNA was isolated with modified Marmir ( 196 1) method, whereas aroA by amplifying using PCR (Polymerase Chain Reaction) method with specific primer of FWPA-I and RWPA-2 so that one fi-agnent of a 1,2 kb aroA P. multocrda serotype B:2 was obtained. The amplified fragment was then analysed with RFLP (Restriction Fragment Length Polymorpl~isrn) using HpdI restriction endonuclease and producing two types of band sized 740 and 460 bp. SSCP (Single-Strand Conformation Polymorphism) analysis then conducted to observe if there was a sequence variant indicated by the mobility shift. DNA sequence variant in arhA was indicated by the mobility shift. The research resulted that PCR-SSCP analysis in crroA 1’. mii//ocicln B:2 did not demonstrate the occimence of mobility shift so it indicated that there were not any differences among the isolated isolates/ strains

Kata Kunci : Bioteknologi,Pasteurella Multocida Serotipe B2,PCR,SSCP