Development of SYBR Green-based Real-Time Multiplex PCR Assay for Monitoring of HIV-1/HCV Co-infection

• Main Author: Puspadewi, Melati
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/42802
• Institution: Institut Teknologi Bandung

HIV/HCV confection exhibits high prevalence globally, including in Indonesia. Data shows that HIV/HCV coinfection prevalence in Indonesia reached 5–20%, even exceeded 50% in IDU users. Heart disease due to HCV co-infection in HIVinfected patients is observed to progress more rapidly and results in more severe damages; causing high risk of death if not immediately treated. However, HCV and HIV therapies are costly and present high health risks; denoting effective and efficient clinical monitoring is essential during treatment. Currently available commercial diagnostic assays are relatively unaffordable by many HIV and HCV patients due to expensive cost. Thus, this research aims to develop an affordable diagnostic assay for simultaneous detection of HIV-1 and HCV with high sensitivity and specificity. The method employed in this research is SYBR Greenbased multiplex real-time PCR assay. Primer pairs targeting a 95-bp sequence in the HIV-1 5’LTR and a 87-bp sequence in HCV 5'UTR were designed. Plasmids inserted with fragments from HIV-1 and HCV genes were used in this experiment as the positive controls. Detection limit and assay linearity were investigated through positive control assay with concentration ranging from 108 to 500 copy/mL. Results show that each amplicon produces specific melting temperature (Tm). HIV-1 specific Tm is 82.6 ± 0.4 oC, while that of HCV is 84.6 ± 0.4 oC. Analysis using standard solutions of known concentration shows that this assay displays high linearity with R2 value more than 0,99. Detection limits of this assay are 1000 copies/mL for singleplex reaction and 5000 copies/mL for multiplex reaction. In conclusion, an alternative diagnostic assay for monitoring of HIV- 1/HCV coinfection has been successfully developed. However, further modifications and optimizations are needed to increase its sensitivity.