OPTIMASI METODE QUANTITATIVE POLYMERASE CHAIN REACTION (qPCR) UNTUK PENENTUAN KANDUNGAN DNA VIRUS HEPATITIS B DENGAN TARGET GEN X

• Main Author: Astriani, Nadia
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45561
• Institution: Institut Teknologi Bandung

Hepatitis B Virus (HBV) infection has infected an estimated 2 billion people in the world. It is divided into two types of infections, acute and chronic. Chronic HBV infection can develop into liver cirrhosis or hepatocellular carcinoma. One of the methods used in diagnostic and monitoring of therapy efficacy for chronic HBV infection is by quantitative Polymerase Chain Reaction (qPCR) to determine viral titer based on HBV DNA level. The c (core) gene is usually used as the target gene in this method. This study aimed to optimize the qPCR method so it can be used to determine HBV DNA level with x gene as the target. Three primer pairs were designed based on journal and Primer3 application. Primer pairs were then analyzed with PRaTo program. The results showed that each primer pair has the score of -6, -2 and -4, which is still in the range of acceptable score. Conventional PCR was then performed to confirm whether its amplicon size fit its theoretical size. Primer pair that showed single amplicon and correct amplicon size was then used in qPCR to determine its efficiency. From three primers that were designed, only one primer pair showed good efficiency score, which is 2.02. Primer pair with good efficiency was then used to analyze various HBV DNA samples with determined level. The log 10 HBV DNA level was then plotted against Ct value and it showed that DNA sample with 109 IU/ml titer and its serial dilutions produced a linear curve for samples within the 105 - 109 IU/ml HBV DNA level range. However, several of the undiluted HBV DNA samples with various HBV DNA levels showed Ct value that did not match its actual level. The conclusion of this study is that qPCR method is not optimum yet for HBV DNA level determination using x gene, nevertheless it still can be developed to quantify HBV DNA samples with high titer.