Comparison of low and high DNA Purity for quantitative detection of ratio Mitochondrial and Nucleus DNA among Drug-treated HIV Patients by Real-time PCR

3rd Annual Applied Science and Engineering Conference

工业技术;自然科学

Khairunisa, S.Q.^1 ; Masyeni, S.^2 ; Witaningrum, A.M.^1 ; Nasronudin, N.^1,3

Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia^1

Faculty of Medicine, Universitas Warmadewa, Bali, Indonesia^2

Airlangga Hospital, Universitas Airlangga, Surabaya, Indonesia^3

关键词: Annealing temperatures;    DNA quantification method;    Mitochondrial enzymes;    PCR amplification;    Peripheral blood mononuclear cells;    Quantitative detection;    Real time polymerase chain reactions;    Reverse transcriptases;   

Others  :  https://iopscience.iop.org/article/10.1088/1757-899X/434/1/012338/pdf
DOI  :  10.1088/1757-899X/434/1/012338

来源: IOP

【 摘 要 】

Antiretroviral in nucleoside reverse transcriptase inhibitors (NRTIs) can inhibit the mitochondrial enzyme polymerase γ, in-vitro and in-vivo to halt the extension of DNA known as the terminal chain terminator. The technique used is measuring the amount of mitochondrial and nucleus DNA in cells by real-time polymerase chain reaction (qPCR). Although the result is not consistent because of some factors. We optimized the mitochondrial and nucleus DNA quantification methods using high and low purity DNA by qPCR. 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 of DNA were measured by spectrofotometer. Quantification of mitochondrial and nucleus DNA using standard curve by qPCR. Optimizing primer and annealing temperature for quantification of mitochondrial DNA revealed that single peak in Tm 64,5?C for sets of primer CCOI and primer RNR1. In this study, standard curve was determined copy number of mitochondrial DNA. From standard curve, the high DNA purity was increased the PCR amplification efficiency. Otherwise, the low DNA purity was decreased the PCR amplification efficiency. Concentration and purity of DNA are influenced by PCR amplification efficiency. Therefore, we should be considered sample quality and technique of pipetting.