PERANCANGAN PRIMER DAN PROBE UNTUK DETEKSI RESIDUAL DNA SEL INANG DENGAN METODE TAQMAN QPCR PADA VAKSIN HEPATITIS B

• Main Author: Nur Hasan, Azmi
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/50302
• Institution: Institut Teknologi Bandung

Hepatitis B caused by Hepatitis B Virus (HBV) can be categorized as serious disease that affects liver function. Vaccination can be performed to prevent liver diseases caused by HBV. PT Bio Farma produces Hepatitis B vaccine with HBsAg expression using yeast cell host Ogataea polymorpha strain BF, Pichia genus. In the production and purification step of a recombinant Hepatitis B vaccine antigen possibly produced impurity in the form of host cell residual DNA. One of recombinant vaccine requirement from WHO for licensed vaccine is one vaccine dose has to contain less than 10 pg host cell residual DNA. resDNASEQ™Quantitative Pichia DNA kit from Applied Biosystems with Taqman qPCR method commercially available for that assay with Pichia pastoris DNA as a template. From preliminary test, the kit did not give PCR product using O. polymorpha BF DNA as a template. The aim of this research is to design primer and probe for detection of host cell residual DNA O. polymorpha BF for qPCR Taqman based detection system. Three sets of primer and probe were designed using https://idtdna.com, SnapGene, and DNASTAR PrimerSelection in accordance with recommended criteria. Primer annealing was analysed by conventional PCR and DNA agarose electrophoresis was used for visualization. Electrophoresis result showed that single band PCR product was formed in the size that corresponded with in silico prediction, i.e., 203, 147 and 157 bp. The DNA sequence in the target area is determined by the sequencing process using the BigDyeTM Terminator v 3.1 Cycle Sequencing Kit. The result of the sequence already corresponds to the targeted area and the designed probe can be recognized the sequence in silico.