OPTIMASI METODE MAPREC UNTUK DETEKSI MUTASI 480 G-A DAN 525 U--+C PADA VAKSIN POLIO ORAL SABIN TIPE 1

• Main Author: Kusumawardhani, Hadiastri
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/64329
• Institution: Institut Teknologi Bandung

Oral polio vaccine (OPV) containing live attenuated virus potentially causes neurovirulence if back mutation or reversion occurs in the virus. This event occasionally occurs during the process of vaccine production. WHO recommends a method to detect the reversion i.e. Mutant Analysis by PCR and Restriction Enzyme Cleavage (MAPREC). The first step of this method is RNA extraction from vaccine sample, reverse transcription, amplification of cDNA, labeling of cDNA using fluorophore¬labeled primer and cleavage of amplified DNA fragments using restriction enzymes. The restriction result is analyzed by electrophoresis and visualization is done by infrared fluorescence or ultra violet using SYBR Green dye to quantify the reversion. In previous work, MAPREC method has been optimized and validated for type 2 and 3 of OPV. This research was done to optimize MAPREC method to he used to quantify reversion of type OPV at 0-480-A and U-525-C. The result showed that the optimum condition was achieved when the final concentration of MgCl2 was 4 mM, the addition of Proteinase K after digestion of DNA sample with restriction enzyme, the amount of each of restriction enzyme Neil and Data was 10 unit, the addition of both enzymes was simultaneous, and the visualization of DNA fragments was done using ultraviolet light. The optimum condition gave linear quantification of reversion percentage. MAPREC method is specific to type 1 OPV. For the application of MAPREC method using infrared fluorescence detection for type 1 OPV further optimization is required.