APLIKASI MULTIPLEX POLYMERASE CHAIN REACTION (m-PCR) UNTUK DETEKSI Mycobacterium tuberculosis DAN Mycobacterium bovis PADA DOMBA

• Main Authors: , Yohanes Timbun Raja Mangihut Ronael Simarmata, , Prof. Dr. drh. Ida Tjahajati, MP.
• Format: Theses and Dissertations NonPeerReviewed
• Published: [Yogyakarta] : Universitas Gadjah Mada 2013
• Subjects: ETD
• Online Access: https://repository.ugm.ac.id/123166/; http://etd.ugm.ac.id/index.php?mod=penelitian_detail&sub=PenelitianDetail&act=view&typ=html&buku_id=63277
• Institution: Universitas Gadjah Mada

Tuberculosis (TB) is an important zoonotic disease which until now is a major problem in both humans and animals. Early diagnosis of TB is a very important issue considering TB disease can be transmitted through the air, so that rapid and accurate diagnosis is a key in preventing the spread of this disease. The fact of TB in cattle in the field as a potential source of transmission to humans, encouraging the development of methods for rapid and accurate diagnosis. The m-PCR method which can detect more than one type of bacteria at a time, enabling the construction of TB diagnosis kit which can simultaneously detect the presence of M. tuberculosis and M.bovis which is the cause of tuberculosis that is harmful to animals and humans. The purpose of this research is the application of m-PCR method for detection of M. tuberculosis and M.bovis in sheep with the specific primers that can differentiate the DNA of M. tuberculosis and M. bovis. The samples used were obtained from pulmonary and limfoglandula organs of sheep (ovis aries) with suspect TB from Yogyakarta. Isolation of DNA were conducted using PureLink TM The m-PCR results from samples of lung and limfoglandula of the suspect sheep showed 4 sheep infected by M tuberculosis and M.bovis. Two isolated DNA showed at position of 168 bp (positive M.bovis), and 2 isolates were at 297 bp (positive M. tuberculosis). Sequences analysis results showed a highly similarity (90% -100%) with others nucleotide sequences of Genomic DNA kit Invitrogen production by following the manufacturer's procedure. Isolated DNA then were used as template in the m-PCR amplification process using forward primer 5'-TTCCGAATCCCTTGTGA-3', coded CSB1, reverse primer 5'- GGAGAGCGCCGTTGTA-3', coded CSB2 (M.bovis) and reverse primer 5'- AGTCGCGTGGCTTCTCTTTTA-3', coded CSB3 (M.tuberculosis). The PCR amplification product then were reconfirmed by agarose gel electrophoresis as positive result showed at the band of 297 bp (M.tuberculosis) and 168 bp (M.bovis). M.bovis BCG str. Korea and M. tuberculosis H37Rv complete genomes in GenBank. The result were reinforced by the growth of bacterial colonies on ogawa media. From the whole analysis, this study conclude that the m-PCR method using specific primers (CSB1, CSB2, CSB3) can be applied for detection of M. tuberculosis and M. bovis on sheep. The TB disease in sheep can be caused by M.tuberculosis, besides M.bovis and M.caprae.