Quantitative DNA Detection Research Articles

Fluorescence microarrays for enzyme-free DNA detection based on web hybrid chain reaction

The diagnosis of pathogenic microorganism infection mainly relies on molecular detection of nucleic acids or proteins, and the nucleic acid detection is the gold standard for the pathogen detection. The viral genome mutates quickly, and the genome of the host cell usually binds to multiple sites in the viral genome, leading to patient infections. Therefore, rapid nucleic acid detection methods with high specificity are essential for controlling the spread of viruses and maintaining human health. In this study, Cy3-labeled DNA probes were obtained based on a convenient and enzyme-free web hybridization chain reaction (wHCR) and used for DNA diagnosis of hepatitis B virus (HBV) in an isothermal platform. In this microarray platform, the target DNA is directly captured on the microarray and identified by the aggregated DNA probes to amplify the fluorescence signal. After fluorescence scanning analysis, the detection limit of HBV DNA fragments on this DNA microarray is 84 fM. In addition, this method not only specifically distinguishes single-base mismatched sequences, but also obtains the quantitative detection of HBV DNA in serum samples. Compared with the enzymatic amplification reaction in which the target nucleic acids are used as the amplification template, this method effectively avoids cross-contamination. Furthermore, compared to enzymatic reactions, the requirements for reaction conditions such as temperature and pH are relatively lenient. This method is user-friendly and suitable for molecular detection of infectious diseases under limited resource conditions.

DNA-based qualitative and quantitative identification of bovine whey powder in goat dairy products

As one of the main ingredients in some milk powders, whey powder is sometimes added to pure goat milk products, which can cause health risks, economic fraud, and unfair competition of food industries. This study is the first to explore qualitative and quantitative methods to identify adulteration of bovine whey powder in goat dairy products based on DNA. We extracted DNA from whey powder using a modified DNA extraction method; this exhibited good quality and integrity, with purity of 1.53 to 1.75 and concentration of 122 to 179 ng/μL. Conventional PCR and real-time PCR were compared for qualitative detection of bovine whey powder; real-time PCR demonstrated sensitivity of 0.01 ng/μL, which was higher than the 0.05 ng/μL detected by the conventional PCR method. Furthermore, real-time PCR was conducted for DNA quantitative detection, with good linearity (R2 = 0.9858) obtained for bovine whey powder contents from 0.1% to 30%. Relative error decreased with increase of the mixing proportion of whey powder; the coefficient of variation above 0.1% of the mixing ratio was close to or less than 5%; and the relative standard deviation of repeatability results was less than 5%. Considering the economic costs of testing, conventional PCR could be performed first, and samples with obvious intentional adulteration detected can be further accurately quantified by real-time PCR. Overall, this research provides a realistic and effective method for qualitative and quantitative identification of bovine whey powder in goat dairy products, thus laying a good foundation for verification of goat dairy product label claims and industrial control.

Ultrasensitive DNA Methylation Ratio Detection Based on the Target-Induced Nanoparticle-Coupling and Site-Specific Base Oxidation Damage for Colorectal Cancer.

DNA methylation analysis holds great promise in the whole process management of cancer early screening, diagnosis, and prognosis monitoring. Nevertheless, accurate detection of target methylated DNA, especially its methylation ratio in the genome, remains challenging. Herein, we report for the first time an integrated strategy of target-induced nanoparticle-coupling and site-specific base oxidation damage for DNA methylation analysis with the assistance of well-designed nanosensors. The ultrahigh sensitivity for detecting target methylated DNA as low as 32 × 10-17 M and high specificity for distinguishing 0.001% methylation ratio are achieved by this proposed strategy without amplification operations. Notably, the precise quantification of target DNA methylation ratio has been achieved for the first time. Through quantitative detection of target methylated DNA and methylation ratio, this proposed strategy could reliably diagnose and monitor cancer progression and treatment responses for colorectal cancer, which is superior to the clinical Septin 9 kit. It is anticipated that the proposed strategy has attractive application prospects in early diagnosis and monitoring for colorectal cancer and other various diseases.

Boronic acid chemistry for fluorescence-based quantitative DNA sensing.

Contrary to the long-standing opinion of boronic acids being typically reactive with 1,2- and 1,3-diols and hence not suitable for quantitative sensing of DNA containing only a mono-ol unit, this proof-of-concept study has successfully shown the feasibility to quantitatively detect DNA in the concentration range of 5 to 50 nM plausibly through boronic acid-mediated bridging of two DNA double helices via the 3' hydroxy groups, which opens up new avenues in the realm of oligonucleotide biochemistry.

An enzyme-free three-dimensional DNA walker powered by catalytic hairpin assembly for H5N1 DNA ratiometric detection

The reliable and quantitative detection of virus DNA is highly desirable for the early diagnosis and clinical treatment of diseases. In this work, a three-dimensional (3D) DNA walker based on a non-enzyme-mediated nucleic acid cascade amplification reaction was constructed for the ratiometric detection of H5N1 DNA. When the DNA walker was developed by the surface modification of aminated silica microsphere (SiO2-NH2) with H1 hairpin structures were activated in the presence of target DNA, the fluorescence of the H1-conjugated Thioflavin T (ThT) fluorophore (495 nm) was turned on, while the fluorescence of 2-aminopurine (2-AP) (363 nm) conjugated to the H2 hairpin remained unchanged. This strategy combines DNA walker powered by catalyzed hairpin assembly (CHA) reaction with proportional fluorescence signal output methods, which can effectively reduce the risk of generating false-positive signals. The developed DNA walker demonstrated excellent analytical sensitivity and specificity, with a detection limit of 60 pM. The DNA walker was also capable of selectively detecting H5N1 DNA in clinical blood serum samples, which demonstrated its potential for use in various clinical applications.

Direct quantitative detection of host cell residual DNA in recombinant Filgrastim by qPCR

Host cell residual DNA is considered as an impurity in recombinant biopharmaceuticals. This study aimed to develop a direct qPCR method to quantify E. Coli residual DNA in recombinant Filgrastim. The specific primers were designed to amplify E. Coli's 16S-rDNA genomic region, which encodes the 16S-rRNA. The developed qPCR method showed that the designed primer has specifically amplified the target genome without any secondary reaction. The designed primer was also able to amplify the target gene as a representative of residual DNA in the drug matrix. Results show that the amount of residual DNA in Filgrastim is undetectable.

Jugular Vein Injection of High-Titer Lentiviral Vectors Does Not Transduce the Aorta-Brief Report.

Efficient gene transfer to the vascular wall via intravenous vector injection would be useful for experimental vascular biology and gene therapy. Initial studies of lentiviral vector tropism suggested that intravenously injected vectors do not transduce murine vascular tissue; however, there are also reports of highly efficient aortic transduction after jugular vein injection of high-titer lentiviral vectors. We sought to reproduce these results. Approach and Results: We injected high-titer preparations of GFP (green fluorescent protein)-expressing lentiviral vector into jugular veins of 8 mice; 6 mice received vehicle only. Four days later, samples of aorta (thoracic and abdominal), liver, spleen, and other tissues were harvested and processed for quantitative polymerase chain reaction detection of vector DNA and immunohistochemical detection of GFP. Our vector DNA assay did not detect transduction of any of the 16 aortic segments. This finding excludes an aortic transduction efficiency of >0.02 vector copies per cell. In contrast, vector DNA was detected in all 8 spleen and liver extracts (median, 0.8 and 0.1 vector copies per cell, respectively; P<0.001 versus vehicle controls). Quantitative polymerase chain reaction signals from DNA extracted from heart, lung, kidney, skeletal muscle, and femoral artery did not differ from background polymerase chain reaction signals from DNA extracted from tissues of vehicle-injected mice (P≥0.7 for all). Immunohistochemistry revealed GFP in scattered cells in spleen and liver, not in aorta. Injection of high-titer lentiviral vectors via the jugular vein transduces cells in the spleen and liver but does not efficiently transduce the aorta. Graphic Abstract: A graphic abstract is available for this article.

Whole blood as an alternative to peripheral blood mononuclear cell for detection of total HIV-1 DNA

BackgroundA more time saving, convenient, reproducible, and scalable method is needed to assess total HIV-1 DNA levels.MethodsFrozen whole blood and peripheral blood mononuclear cell (PBMC) samples both 200 μl at the same point were used to detect total HIV-1 DNA. Automatic extraction of total HIV-1 DNA was used to ensure the consistency of sample extraction efficiency. The detection reagent was HIV-1 DNA quantitative detection kit and real-time quantitative PCR was utilized.ResultsOf the 44 included patients, 42 were male and 2 were female, with a median age of 33 years. Thirty-three cases were collected after receiving antiviral treatment, with a median duration of treatment of 3 months, and the other 11 cases were collected before antiviral treatment. The median viral load was 1.83 log10 copies/mL, the median CD4 and CD8 count were 94 and 680 cells/μL, and the median CD4/CD8 ratio was 0.18. The results of the two samples were 3.02 ± 0.39 log10 copies/106 PBMCs in PBMC samples and 3.05 ± 0.40 log10 copies/106 PBMCs in whole blood samples. The detection results of the two methods were highly correlated and consistent by using paired t test (P = 0.370), pearson correlation (r = 0.887, P < 0.0001) and intra-group correlation coefficient (ICC = 0.887, P < 0.0001) and bland-altman [4.55% points were outside the 95% limits of agreement (− 0.340 ~ 0.390)].ConclusionsThe results of the whole blood sample test for total HIV-1 DNA are consistent with those of PBMC samples. In a clinical setting it is recommended to use whole blood samples directly for the evaluation of the HIV reservoir.

Humoral Immune Memory to Hepatitis B Vaccine after Primary Vaccination of Children and Adolescents in Assiut, Egypt.

ObjectivesWe sought to assess the prevalence of hepatitis B virus (HBV) seroprotection among vaccinated children in the Assiut governorate, Egypt, and assess a booster dose immune memory response among non-seroprotected children.MethodsUsing a multistage cluster sample, 566 children were recruited from three clusters: one urban and two rural. Children were aged from nine months to 16 years old. All participants received the full three doses of the compulsory HBV vaccine during infancy. Serum hepatitis B surface antigen (HBsAg), total anti-hepatitis B core (anti-HBc) antibodies, and quantitative detection of anti-HBs were measured using enzyme-linked immunosorbent assay. Repeatedly positive samples for HBsAg/anti-HBc were submitted for quantitative HBV DNA detection using real-time polymerase chain reaction. Non-seroprotective participants (anti-HBs < 10 IU/L) were given a booster dose of HBV vaccine. Two weeks later, a blood sample was taken from each child to assess an anamnestic response.ResultsThe seroprotection rate was 53.2%, and only two children had HBV breakthrough infection (0.4%) with positive serum anti-HBc and HBV DNA. Age was the only significant predictor for non-seroprotection with an adjusted odds ratio (OR) of 3.2, 9.4, and 9.9 among children aged 5–10, 11–15, and > 15 years, respectively, compared to younger children (p < 0.001). About 85% of non-seroprotected children developed an anamnestic response after receiving the booster dose, and 84.3% of responders had a good response (3 100 IU/L). Undetectable pre-booster titer was found to be the only risk factor for non-response to booster with OR = 3.2 (p < 0.010). About 95.7% of children who were not responding to booster dose developed immune response after receiving the three doses of HBV vaccine.ConclusionsOlder age of children was the only significant predictor for HBV non-seroprotection. High anamnestic response rate signifies the presence of immune memory with long-term protection despite the waning of anti-HBs over time. However, some children with pre-booster undetectable anti-HBs titers may be unable to develop anamnestic response, and a second vaccination series might be necessary for HBV protection for these children.

Quantitative Detection of G-Quadruplex DNA in Live Cells Based on Photon Counts and Complex Structure Discrimination.

G-quadruplex DNA show structural polymorphism, leading to challenges in the use of selective recognition probes for the accurate detection of G-quadruplexes in vivo. Herein, we present a tripodal cationic fluorescent probe, NBTE, which showed distinguishable fluorescence lifetime responses between G-quadruplexes and other DNA topologies, and fluorescence quantum yield (Φf ) enhancement upon G-quadruplex binding. We determined two NBTE-G-quadruplex complex structures with high Φf values by NMR spectroscopy. The structures indicated NBTE interacted with G-quadruplexes using three arms through π-π stacking, differing from that with duplex DNA using two arms, which rationalized the higher Φf values and lifetime response of NBTE upon G-quadruplex binding. Based on photon counts of FLIM, we detected the percentage of G-quadruplex DNA in live cells with NBTE and found G-quadruplex DNA content in cancer cells is 4-fold that in normal cells, suggesting the potential applications of this probe in cancer cell detection.

Quantitative analysis of hepatitis B virus DNA based on raman spectroscopy combined with multivariate statistical methods

Quantitation of hepatitis B virus deoxyribonucleic acid (HBV DNA) is the most accurate method for determining the infection level of hepatitis B. It is a very meaningful means of detecting the different replication states of HBV infection. In this study, HBV DNA was detected and quantified in 499 patients with hepatitis B by confocal Raman spectroscopy combined with multivariate analysis. The effective information related to HBV DNA in spectral data was extracted by different pretreatments according to the vibrational modes of different functional groups. Quantitative analysis of HBV DNA was carried out by using the spectrum in the characteristic range of 807.027–1700.29 cm−1 for spectral peak assignment analysis and using partial least squares to construct a quantitative model. The results show that the regression model is established by combining normalization pretreatment and multivariate analysis methods. The correlation coefficient (Rc) and root mean square error of calibration from the calibration set of the selected feature range are 0.9751 and 0.3881, and the correlation coefficient (Rp) of the verification set and the root mean square error of prediction from the prediction set are 0.9673 and 0.4314. respectively. In clinical medical applications, Raman spectroscopy combined with diversified analysis methods has great research potential for the quantitative detection of HBV DNA in HBV serum.

Comparison of benzothiazole-based dyes for sensitive DNA detection

For efficient and quantitative DNA detection, fluorescence staining is the most often explored approach, which relies on non-covalent binding of dyes with double stranded DNA (dsDNA). Ethidium bromide (EB) is the most classic DNA stain, but suffers from its high carcinogenicity. A series of less toxic alternatives were developed, many of which contain the core structure of the benzothiazole ring. However, the relationship between the structure and the DNA detection performance was not illustrated. Herein, five benzothiazole dyes, namely thiazole orange, SYBR Green I, PicoGreen, SYBR Safe, and thioflavine-T, were compared for DNA detection through direct fluorescence and gel electrophoresis, with particular focus on the structure-performance relationship. It turned out that SYBR Green I is currently the best choice for DNA detection. The results in this work may be useful for future DNA-staining dye developments.

Dual platform based sandwich assay surface-enhanced Raman scattering DNA biosensor for the sensitive detection of food adulteration.

Surface enhanced Raman scattering (SERS) DNA biosensing is an ultrasensitive, selective, and rapid detection technique with the ability to produce molecule-specific distinct fingerprint spectra. It supersedes the long amplicon based PCR assays, the fluorescence and spectroscopic techniques with their quenching and narrow spectral bandwidth, and the electrochemical detection techniques using multiplexing. However, the performance of the SERS DNA biosensor relies on the DNA probe length, platform composition, both the presence and position of Raman tags and the chosen sensing strategy. In this context, we herein report a SERS biosensor based on dual nanoplatforms with a uniquely designed Raman tag (ATTO Rho6G) intercalated short-length DNA probe for the sensitive detection of the pig species Sus scrofa. In the design of the signal probe (SP), a Raman tag was incorporated adjacent to the spacer arm, followed by a terminal thiol modifier, which consequently had a strong influence on the SERS signal enhancement. The detection strategy involves the probe-target DNA hybridization mediated coupling of the two platforms, i.e., the graphene oxide-gold nanorod (GO-AuNR) functionalized capture probe (CP) and SP-conjugated gold nanoparticles (AuNPs), consequently enhancing the SERS intensity by both the electromagnetic hot spots generated at the junctions or interstices of the two platforms and the chemical enhancement between the AuNPs and the adsorbed intercalated Raman tag. This dual platform based SERS DNA biosensor exhibited outstanding sensitivity in detecting pork DNA with a limit of detection (LOD) of 100 aM validated with DNA extracted from a pork sample (LOD 1 fM). Moreover, the fabricated SERS biosensor showed outstanding selectivity and specificity for differentiating the DNA sequences of six closely related non-target species from the target DNA sequences with single and three nucleotide base-mismatches. Therefore, the developed short-length DNA linked dual platform based SERS biosensor could replace the less sensitive traditional methods of pork DNA detection and be adopted as a universal detection approach for the qualitative and quantitative detection of DNA from any source.

1773. Development and Evaluation of an Automated Adenovirus Quantitative Assay Using the Luminex ARIES® System

BackgroundThe quantification of circulating human adenovirus (HAdV) DNA is the recommended diagnostic method to predict disseminated disease. Most current HAdV quantitative assays are manually performed and lack the flexibility needed to provide rapid answers when required.The purpose of this study was to evaluate the application of MultiCode® HAdV PCR assay for use with the Luminex ARIES® system (Luminex Corporation, Austin, TX) as an automated and random access test for the quantitative detection of HAdV DNA in plasma.MethodsAnalytical performance characteristics including assay limit of detection/quantitation (LoD/LoQ), accuracy, and inter-, intra-reproducibility were studied using commercial panels (Exact Diagnostics, Fort Worth, TX). Assay specificity was determined usingHAdV reference strains obtained from the American Type Culture Collection (Manassas, VA) and plasma spiked with related Herpes viruses and pathogens commonly found in the blood. Assay accuracy was verified with analysis of 30 plasma samples spiked with different concentrations of control material that covered the full range of HAdV DNA levels. We also prospectively analyzed 180 plasma samples collected from 102 patients. DNA from all samples were extracted, amplified and detected on a single automated Luminex ARIES® system.ResultsThe assay has a wide linear range from 2.55 to 9.4 log10 HAdV DNA copies/mL (coefficient of determination; R2 = 0.995) with a detection limit of 1.82 log10 (95% positivity rate), and a limit of quantification of 2.55 log10 copies/mL. The assay detected HAdV DNA from Adenovirus groups A-F, although slight shifts in Tm peaks were observed. Inter- and intra-assay reproducibility was evaluated using 6 panels of commercial standards, producing variation coefficients of 5% and 2%, respectively. Assay accuracy results reflected a good correlation with a mean difference of 0.10 log10 copies/mL. The results of the prospective study showed 6/102 (5.8%) patients had HAdV viremia, including 4 (3.9%) patients with a viral load ≥4 log10/mL, which might necessitate therapy.ConclusionThe HAdV quantitative assay using the Luminex ARIES® system provides excellent performance for routine testing with the additional advantage of random access capabilities for urgent testing to identify patients at risk for disseminated disease.Disclosures All authors: No reported disclosures.

Quantitative detection of cell-free fetal DNA in peripheral blood of pregnant women during early pregnancy

Objective: This study aimed for the quantitative detection of cell-free fetal DNA (cffDNA) in peripheral plasma of pregnant women, which provides basic data for clinical non-invasive prenatal screening in early pregnancy. Materials and Methods: A total of 243 individuals with gestational age of 5-10+6 weeks were selected for this study, who required abortion. mDNA was extracted from villi, and Y-chromosome specific SRY gene was detected by nested PCR, which was regarded as standard for quantitative detection of accuracy. CffDNA was extracted from peripheral blood and SRY gene’s expression was detected by real-time fluorescent quantitative PCR. Results: Among 243 cases, nested PCR detected 163 individuals with SRY gene positive, and 80 were negative. Out of 163, in 140 cases SRY was detected in cffDNA, but 23 cases with gestational age 5-6+6 weeks were negative. However at 7th week of pregnancy, it was also detected in those 23 cases. The average concentration of cff DNA in 10th week of pregnancy was found significantly higher than in 7th, 8th, and 9th week of pregnancy. Conclusion: Thus, this study indicates the efficiency and reliability of cffDNA in peripheral blood of 7-10th week of gestational period for the detection of early pregnancy.

The potential of sedimentary ancient DNA for reconstructing past sea ice evolution

Sea ice is a crucial component of the Arctic climate system, yet the tools to document the evolution of sea ice conditions on historical and geological time scales are few and have limitations. Such records are essential for documenting and understanding the natural variations in Arctic sea ice extent. Here we explore sedimentary ancient DNA (aDNA), as a novel tool that unlocks and exploits the genetic (eukaryote) biodiversity preserved in marine sediments specifically for past sea ice reconstructions. Although use of sedimentary aDNA in paleoceanographic and paleoclimatic studies is still in its infancy, we use here metabarcoding and single-species quantitative DNA detection methods to document the sea ice conditions in a Greenland Sea marine sediment core. Metabarcoding has allowed identifying biodiversity changes in the geological record back to almost ~100,000 years ago that were related to changing sea ice conditions. Detailed bioinformatic analyses on the metabarcoding data revealed several sea-ice-associated taxa, most of which previously unknown from the fossil record. Finally, we quantitatively traced one known sea ice dinoflagellate in the sediment core. We show that aDNA can be recovered from deep-ocean sediments with generally oxic bottom waters and that past sea ice conditions can be documented beyond instrumental time scales. Our results corroborate sea ice reconstructions made by traditional tools, and thus demonstrate the potential of sedimentary aDNA, focusing primarily on microbial eukaryotes, as a new tool to better understand sea ice evolution in the climate system.

Hierarchic Interfacial Nanocube Assembly for Sensitive, Selective, and Quantitative DNA Detection with Surface-Enhanced Raman Scattering.

Surface-enhanced Raman scattering (SERS)-based sensing is promising in that it has potential to allow for highly sensitive, selective, and multiplexed detection and imaging. However, the controlled assembly and gap formation between plasmonic particles for generating strong SERS signals in a quantitative manner is highly challenging, especially on biodetection platforms, and particle-to-particle variation in the signal enhancement can vary by several orders of magnitude in a single batch, largely limiting the reliable use of SERS for practical sensing applications. Here, a hierarchic-nanocube-assembly based SERS (H-Cube-SERS) bioassay to controllably amplify the electromagnetic field between gold nanocubes (AuNCs) is developed. Based on this strategy, H-Cube-SERS assay allows for detecting target DNA with a wide dynamic range from 100 aM to 10 pM concentrations in a stable and reproducible manner. It is also found that the uniformly formed AuNCs with flat surfaces are much more suitable for highly sensitive, reliable, and quantitative biodetection assays due to faster DNA binding kinetics, sharper DNA melting transition, wider hot spot regions, and less dependence on light polarization direction than spherical Au nanoparticles with curved interfaces. This work paves the pathways to the quantitative and sensitive biodetection on a SERS platform and can be extended to other particle assembly systems.

A self-digitization chip integrated with hydration layer for low-cost and robust digital PCR

We present a self-discretization and zero-water-loss microfluidic digital PCR (dPCR) device to enable low-cost and robust quantitative nucleic acid assays. In this device, a thin void is integrated beneath the reaction chamber array. By utilizing the permeability of polydimethylsiloxane (PDMS) film, the integrated void serves a dual function: vacuum “accumulator” and hydration “reservoir”. The combination of pre-stored pumping energy and water compensation for evaporation loss enables simple, robust and reliable single-DNA-molecule amplification and detection in 10,000 reactors of picoliter volume. Compared to the conventional degassing PDMS pumps, the vacuum accumulator exhibits superior performance due to more vacuum storage and shorter diffusion distance. We also evaluated the performance of the embedded hydration layer in suppressing evaporation loss at elevated temperatures, and verified that zero-water-loss could be achieved for all reaction chambers in our dPCR chip during thermal cycling. By performing quantitative detection of T790M DNA from 10 to 104 copies/μL, the proposed dPCR chip demonstrated high accuracy and excellent performance for the absolute quantification of the target gene with a dynamic range of 104. The simplicity and robustness of our dPCR chip make it well suited for low-cost molecular diagnostic assays under resource-limited settings.

Quantitative and selective DNA detection with portable personal glucose meter using loop-based DNA competitive hybridization strategy

A portable, quantitative and selective DNA detection biosensor requiring only one step for target recognition and signal reporter generation using a loop-based DNA competitive hybridization assay and personal glucose meter (PGM) was developed. In the presence of target DNA, due to its competitive binding, the invertase-DNA conjugate was released which could be collected with the help of a magnet subsequently. The released invertase-DNA was used to catalyze the hydrolysis of sucrose into glucose with millions of turnovers which was target concentration dependent. We found that there was a linear relationship between the PGM signals and the target DNA concentration in the range of 100 pM to 100 nM with a detection limit of 100 pM. In addition, the sensor exhibits excellent sequence selectivity, being able to differentiate a single mismatch in the target DNA, and excellent anti-interference ability, having almost no effect in serum on the detection performance. The biosensor reported here is easier to operate, owning great potential in point of care testing in environments with limited resources and skilled personnel for rapid and sensitive detection of specific DNA sequence in real biological samples.

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

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.