USU Conference Systems, International Conference on Tropical Medicine and Infectious Diseases (ICTROMI) 2017

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Comparison of Low and High DNA Purity for Quantitative Detection of Rasio Mitochondrial and Nucleus DNA among Drug-Treated HIV Patients by Realtime PCR
siti qamariyah khairunisa, DAP Sri Masyeni, nasronudin nasronudin, Brian Eka Rachman

Last modified: 2017-11-11



Background: Antiretroviral in nucleoside reverse transcriptase inhibitors (NRTIs) can inhibit the mitochondrial enzyme polymerase γ, in vitro and in vivo so as to halt the extension of DNA known as the terminase chain terminator. The technique used is measuring the amount of mithocondrial and nucleus DNA in cells by realtime polymerase chain reaction (qPCR). Although the result is not consistent because some factors. This study we optimized the mithocondrial and nucleus DNA quantification methods using high and low purity DNA by qPCR.

Methods: HIV-1-infected individuals were recruited from Gianyar and Denpasar, Bali.. HIV-1 DNA was extracted from peripheral blood mononuclear cells (PBMCs). Quality and quantity and DNA were measured by spectrofometer. Quantification of Mitochondrial and Nucleus DNA using standart curve by (qPCR).

Results and Discussions: Optimizing primer and annealing temperatur for quantification of mithocondrial DNA revealed that single peak in Tm 64,5 °C for sets of primer CCOI and sets of primer RNR1. It means the primers spesific for this target. In this study, standart curve were determine copy number of  mithocondrial DNA. From standart curve, the high DNA purity were increased the PCR amplification efficiency. Otherwise the low DNA purity were decreased the PCR amplification efficiency.

Conclusions: Concentration and purity of DNA is influenced to PCR amplification efficiency, the high DNA purity were increased the amplification product and  PCR amplification efficiency. Therefore, we should be considered sample quality and technique of pipetting.

Key words: mitochondrial DNA, HIV-1, purity, qPCR