DEVELOPMENT OF ISOTHERMAL RNA-BASED AMPLIFICATION METHODS FOR ANTIRETROVIRAL THERAPY MONITORING IN HIV PATIENTS IN INDONESIA

• Main Author: IFFAT WIRUSANTI (nim : 10413032), NURUL
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29836
• Institution: Institut Teknologi Bandung

<p align="justify">Monitoring of antiretroviral therapy in HIV patients as an effort to manage HIV epidemic in Indonesia is still hampered by the need of sophisticated equipment for viral load testing which is only available at central hospitals. One alternative solution is to develop an RNA-based diagnostic kit that works on isothermal conditions. The advantage of this diagnostic kit is that it can be applied to point-of-care laboratory in primary healthcare services. The purpose of this study was to develop an isothermal amplification system that can detect HIV RNA. There were two methods tested, namely Transcription Mediated Amplification (TMA) method and Reverse Transcription-Recombinase Polymerase Amplification (RT-RPA) method. The sensitivity of these two methods was compared to the current standard method, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). In this experiment, we used RNA samples that were transcribed from pET-HIVMRNAseH plasmids. This synthetic RNA was then diluted serially so we obtained RNA with concentration ranging from 104 to109 copies/ml. This target RNA was then tested on TMA, RT-RPA and RT-PCR methods. The results showed that RT-PCR as a standard method can detect all concentrations of RNA, ie 104-109 copies/ml. Meanwhile, TMA and RT-RPA methods can successfully detect RNA, however only with high RNA concentrations of 107-109 copies/ml in TMA and 108-109 copies/ml in RT-RPA. Thus, it is concluded that both TMA and RT-RPA methods can be used as an alternative method to quantify HIV RNA, however with lower sensitivity if compared to RT-PCR. Therefore, to obtain more sensitive measurement, optimization of these two isothermal methods is necessary.<p align="justify">