DEVELOPMENT OF ADVENTITIOUS AGENTS DETECTION IN ORAL POLIO VACCINE BASED ON SISPA-REVERSE HYBRIDIZATION METHOD

• Main Author: Rizaldy, Defri
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45239
• Institution: Institut Teknologi Bandung

Vaccine is a safe biological preparation to improves the body's immunity for certain diseases. One of the WHO requirements to ensure oral polio vaccine (OPV) safety is that OPVs are free from adventitious agents, which are microorganism contaminants that are unintentionally introduced to biological preparation in the production process. This study was aimed to develop a detection method of adventitious agents from sample of OPV products in one of vaccine producer in Indonesia. The method consists of two stages: (1) Random amplification of DNA sample suspected of containing adventitious agents using Sequence Independent Single Primer Amplification (SISPA) technique; and (2) Detection of adventitious agents DNA in the amplification product using reverse hybridization technique. Vero cells culture containing Simian Foamy Virus (SFV) was used as positive control and neutral vero cell culture was used as negative control. Total DNA in the cell culture was amplified using an infrared labelled random primer. The SISPA product was hybridized to SFV specific probes that were immobilized on the surface of a nylon membrane. Membrane was scanned using a LI-COR Odyssey instrument. Optimum condition of SISPA were: final concentration of 20 nM [PCR_454_A_IRDye700], final concentration of 3 µM MgCl2 annealing temperature at 55 °C, and 20 seconds of primer elongation. Hybridization signal from the detection of SISPA product appeared only in [Probe_Mac] probe. To obtain better hybridization signal, further concentration optimization on three other probes is needed.