PENGEMBANGAN TES DIAGNOSTIK BERBASIS REAL-TIME PCR UNTUK DETEKSI VIRUS HEPATITIS B (HBV) DI INDONESIA

• Main Author: Salsabilla Rizkita, Syifa
• Format: Final Project
• Language: Indonesia
• Subjects: Ilmu hayati ; Biologi
• Online Access: https://digilib.itb.ac.id/gdl/view/48535
• Institution: Institut Teknologi Bandung

Based on a serological test, the prevalence of hepatitis B in Indonesia is 7.1 % and categorizes Indonesia as a country with moderate endemicity of hepatitis B. The treatment of hepatitis B aims to suppress the replication of viral particles, and a diagnostic test based on real-time PCR is required to monitor the development of the treatment. But to date, there is no real-time PCR for HBV diagnostic test produced by Indonesia. Therefore, the goal of this research is to develop real-time PCR for HBV diagnostic test. A in silico study is conducted to determine the target gene through multiple sequence alignment. The primer is designed with SnapGeneTM software to create the desirable characteristics of primer, which is 15-30 bp in size, having 30-80% of GC Content, and Tm above 55oC. The control positive is also designed by inserting target sequence into the backbone of plasmid pUC57. The amplification performance of the primer is tested by real-time PCR. To get the best result of amplification performance, and the profile of an amplicon, the primer concentration used is the combination without dimer primer artifact. This combination is determined by conducting an experiment of real-time PCR without template and varies the primer concentration in range 100 to 300 nM. The target sequence is within the S region of the HBV genome, which is the 183rd to 269th nucleotide. The primer has good character and conserved in 60 sequences of HBV genome from different strain. From this study, there are four primer combination to minimize the formation of primer dimer artifact and lower the efficiency of amplification, those are 100-300 nM for forward primer, and 200 nM for reverse primer; 100 nM for forward primer and 300 nM for reverse primer. Real-time PCR results showed that the primer has succeeded to amplify a sequence which has melting temperature close to in silico prediction, 80oC. In conclusion, primer designed in this study could be a candidate for real-time PCR for HBV diagnostic test in Indonesia.