OPTIMASI METODE QUANTITATIVE POLYMERASE CHAIN REACTION (qPCR) UNTUK PENENTUAN KANDUNGAN DNA VIRUS HEPATITIS B DENGAN TARGET GEN X
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 the...
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| Format: | Final Project |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/45561 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | 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.
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