DEVELOPMENT OF REAL-TIME PCR METHOD TO DETECT VARIOUS SUBTYPES OF HEPATITIS C VIRUS COMMONLY FOUND IN INDONESIA

• Main Author: PUTRI NINGSIH (NIM: 21114018), AYU
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/21356
• Institution: Institut Teknologi Bandung

Data in Indonesia show that around 7 million Indonesians are infected with <br /> <br /> Hepatitis C virus. Therefore the development of a quantitative diagnostic test to <br /> <br /> detect the disease is needed. Until now several commercial assays had been <br /> <br /> developed to quantify the number of virus RNA in a person’s body as well as <br /> <br /> monitor the succes of antiviral therapy. But some of the existing comercial assays <br /> <br /> could not be fully implemented in Indonesia, especially in areas far from the city <br /> <br /> centre, due to their high operational costs. Given the very diverse HCV genome, <br /> <br /> some of these viral infections could not be detected with existing commercial <br /> <br /> assays. Based on this, in this study had developed an alternative test using Real- <br /> <br /> Time PCR method to detect and quantify the viral HCV RNA genotype <br /> <br /> commonly found in Indonesia (subtype 1a, 1b, 1c, 2a, 2e, 2f, 3a, 3k, and 4a) . In <br /> <br /> this study candidate pairs of primers obtained from the NCBI database were <br /> <br /> selected based on the HCV genomic conserved region that amplified the 5’UTR <br /> <br /> HCV fragment with a156 bp amplicon length(base sequence position: 74-229). <br /> <br /> The positive control used was a pUC57 plasmid containing a synthetic gene of <br /> <br /> 5’UTR HCV fragment designed using the Bioedit Sequence Alignment Editor <br /> <br /> software. The sensitivity and linearity of the developed assays determined by <br /> <br /> using the standard plasmid as a positive control. To know about the linearity of <br /> <br /> the standard curve, serial dilution of the positives control is made of <br /> <br /> aconcentration of 1011 copies/mL up to 101 copies/mL. The developed assay had <br /> <br /> linearity at the limit of 1011-106 copies/mL. Efficiency of assay developed was <br /> <br /> 67% with r value = 0,9912. Based on this, this Real-Time PCR method can be <br /> <br /> used as an easy and efficiet alternative diagnostic test to detect and quantify viral <br /> <br /> load of HCV with the lowest quantifiable limit 106 copies/mL.