DEVELOPMENT QPCR METHOD FOR QUANTIFICATION OF HOST RESIDUAL DNA OGATEA POLYMORPHA STRAIN R

• Main Author: Elfira Kurniati, Dwi
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/52079
• Institution: Institut Teknologi Bandung

Quantification of residual host DNA is necessary to ensure the safety and quality of vaccine products. WHO made a requirement that recombinant vaccines produced in yeast should contain less than 10 pg residual host DNA per dose. PT. B develops HBsAg subunit vaccine which are expressed in the yeast cell Ogatea polymorpha strain R. The development of qPCR method aims to generate a specific and validated self-quantification system that can be used for quantification of O. polymorpha strain R residual DNA. Primers targeting X, Y, and Z regions in the O. polymorpha strain R genome have been designed in a previous study and the primer specificity assay has been demonstrated by conventional PCR. This research aims to prepare DNA standard to be used in quantification and to determine primer specificity using qPCR SYBR Green-base and Taqman qPCR. DNA standard from O. polymorpha strain R were made with a concentration of 30ng/µL with SD+2, SD+3, SD-2 and SD-3 are 31.54, 32.25, 28.71, and 28.00 respectively after repetition in 3 different times. DNA standard 260/280 purity ratio is 1.966 + 0.058 which fulfills the acceptance criteria of ISO (1.8-2.0). On the other hand, 260/230 ratio purity were 1.655 + 0.052 below the acceptance criteria of ISO (2.0-2.2). Primer X, Y, and Z have high specificity with a Ct value 11.88, 11.85, and 11.93 when using 3000 pg O. polymorpha DNA as a template and have a single peak result from the melting curve, when tested using qPCR SYBR Green method. Taqman qPCR Specificity assay compared to non-target DNA (Pichia pastoris 3000 pg). The Ct value of primer X, Y and Z is 15.57, 16.14, and 15,29 respectively. While non-target sample has a value > 28. This result shows that all primers was able to amplified the target spesficically using qPCR SYBR Green-base and Taqman qPCR method.