Eva Green Research Articles

Construction of PCR-SERS Method for Detection of Vibrio parahaemolyticus.

A paper-based surface enhancement of a Raman scattering substrate consisting of silver-nanowires stacked on glass-fiber filter paper was prepared. At the same time, the DNA-embedding molecule Eva Green was introduced as a signaling molecule for surface-enhanced Raman scattering (SERS) detection. Polymerase chain reaction (PCR) was used to amplify target genes and the method was developed into a rapid molecular diagnostic system. The total detection time of the developed detection method was 40 min, including 30 min of PCR amplification and 10 min of SERS measurement. After 30 PCR cycles, bacterial DNA with an initial concentration of 20 fg/μL and a bacterial suspension with an initial concentration of 7.2 × 101 CFUs/mL could be detected. When the enrichment culture time was 4 h, target bacteria with an initial contamination inoculation volume of 1.5 CFUs/mL could be detected in artificially contaminated samples. The method is fast and highly sensitive, and has not been applied to the detection of V. parahaemolyticus.

The homeodomain of the <i>Raphanus sativus</i> WOX4 binds to the promoter of the <i>LOG3</i> cytokinin biosynthesis gene

BACKGROUND: The WOX4 transcription factor plays a crucial role in maintaining the organisation of cambium meristem during secondary growth, but its direct targets are unknown. AIM: The aim of our work were to study the effect of WOX4 overexpression on the root development and gene expression in radish (Raphanus sativus L.), a root crop related to Arabidopsis thaliana, and to search for direct targets of the WOX4 in radish. MATERIALS AND METHODS: Radish line 19 of the St. Petersburg State University radish genetic collection was used. Plants were grown on Murashige–Skoog medium and then in soil at 23оС and 16 h of daylight. Total DNA was extracted from radish seedlings using the CTAB method. The PCR-amplified full-length RsWOX4-2 gene, gene fragments or homeobox sequence were cloned into the vectors for overexpression (pB7WG2D), RNA interference (pH7GWIWG2) and yeast one-hybrid assay (pDEST22), respectively, using the Gateway system. The vectors for overexpression and RNA interference of RsWOX4-2 were transformed into Escherichia coli DH10B and then into Agrobacteium rhizogenes Arqua chemically competent cells. Radish seedlings were transformed with A. rhizogenes containing vectors for overexpression and RNA interference of RsWOX4-2, and GUS-overexpressing A. rhizogenes was used as a control. Total RNA from transgenic radish roots was extracted with Trizol reagent. RNA reverse transcription was performed using dT-18 primers and RevertAid reverse transcriptase. qPCR was performed using the Eva Green reagent kit on a CFX96 thermocycler with fluorescence detection system. Results were processed using the 2–ΔΔCT method. Yeast transformation was performed using the competent Saccharomyces cerevisiae cells of Y2H Gold strain. For the yeast one-hybrid assay, the obtained yeast colonies transformed with plasmids containing TF homeodomain sequence and promoter regions of genes were grown on DDO and TDO selective media with different concentrations of 3-amino-1,2,4-triazole. Statistical processing based on Student’s t-test and graphing were performed using the ggplot2 package for the R programming language (v.4.0.2). RESULTS: Overexpression of the RsWOX4-2 gene affects the structure of the radish root stele and alters the number of vessels and cambium cells. Overexpression and RNA interference of the RsWOX4-2 causes changes in the expression levels of putative target genes with the WOX family transcription factor conserved binding sites in their promoters. Using the yeast one-hybrid assay, we have shown that the DNA-binding homeodomain of RsWOX4-2 interacts with the TAATCC site in the promoter of the RsLOG3 gene, which encodes the enzyme for cytokinin biosynthesis. CONCLUSIONS: We have demonstrated the effect of RsWOX4-2 overexpression on radish root stele and gene expression and identified the RsLOG3 as the putative direct target of the WOX4 transcription factor in radish.

Development of a Melting Curve-Based Triple Eva Green Real-Time PCR Assay for Simultaneous Detection of Three Shrimp Pathogens.

Infections with Enterocytozoon hepatopenaei (EHP), infectious hypodermal and hematopoietic necrosis virus (IHHNV), and Decapod iridescent virus 1 (DIV1) pose significant challenges to the shrimp industry. Here, a melting curve-based triple real-time PCR assay based on the fluorescent dye Eva Green was established for the simultaneous detection of EHP, IHHNV, and DIV1. The assay showed high specificity, sensitivity, and reproducibility. A total of 190 clinical samples from Shandong, Jiangsu, Sichuan, Guangdong, and Hainan provinces in China were evaluated by the triple Eva Green real-time PCR assay. The positive rates of EHP, IHHNV, and DIV1 were 10.5%, 18.9%, and 44.2%, respectively. The samples were also evaluated by TaqMan qPCR assays for EHP, DIV1, and IHHNV, and the concordance rate was 100%. This illustrated that the newly developed triple Eva Green real-time PCR assay can provide an accurate method for the simultaneous detection of three shrimp pathogens.

Nucleic acid-binding bis-acridine orange dyes with improved properties for bioimaging and PCR applications.

Understanding the intricate interactions of molecular dyes with nucleic acids is pivotal for advancing medical and biochemical applications. In this work, we present a comprehensive study of the interplay between a novel series of bis-acridine orange (BAO) dyes and double-stranded DNA (dsDNA). These BAO dyes were intentionally designed as two acridine orange units connected by neutral linkers featuring a 2,5-disubstituted thiophene moiety. Comparative analysis of BAO compounds with the widely utilized DNA-binding dye EvaGreen (EG) was carried out for fibroblast staining and qPCR analysis. The results show that BAO dyes outperform EG by supporting PCR amplification over a broader concentration range (0.5-5.0 μM). Furthermore, they exhibit an exceptional capability to generate consistent DNA melting curves regardless of DNA concentration fluctuations. Molecular dynamics simulations showed that BAO dyes when interacting with dsDNA unfold from the stacked conformation to the elongated one. The difference in the energy between the conformations is shown to be concomitant with fluorescence enhancement. This study enriches our understanding of the intricate interplay between innovative BAO dyes and dsDNA, fostering their applications in medical and biochemical research, particularly in qPCR methodologies and bioimaging techniques.

Assessing ST18 gene polymorphisms (rs17315309, rs2304365) in Iraqi patients with Pemphigus vulgaris.

Pemphigus vulgaris (PV) is a potentially fatal autoimmune disease characterized by blistering of the skin, mucous membranes, and oral cavity. Genetics are implicated in its etiology, with the ST18 gene identified as a potential risk factor for pemphigus in certain populations, suggesting its role as a novel molecular target for therapeutic intervention. This study aimed to detect single nucleotide polymorphisms (SNPs) rs17315309 A/G and rs2304365 C/G in the ST18 gene among Iraqi/Arabic patients with PV. A total of 90 Iraqi subjects participated in this study, including 45 patients diagnosed with PV and 45 healthy controls. SNP analysis was performed using High-Resolution Melt Analysis (HRMA) with Eva Green I Dye. For SNP rs17315309 A/G, the distribution of heterozygous genotypes showed highly significant differences between the patient and healthy groups (P = 0.005), with the mutant G-allele being significantly more prevalent in patients than in the healthy group (P = 0.001). In contrast, for SNP rs2304365 C/G, the distribution of heterozygous and mutant genotypes did not differ significantly between patients and healthy individuals (P = 0.8 and P = 0.3, respectively), with the mutant G-allele also showing no significant difference (P = 0.4). Our data indicate a significant association between PV and the rs17315309 A/G SNP in the ST18 gene among the Iraqi population of Arabic origin. However, no association was found between patients with PV and the rs2304365 C/G SNP in the same gene.

Diagnostic and prognostic value of c-MYC gene expression in hematological malignancies.

c-MYC plays vital role in regulation of cell proliferation and has been associated with tumorigenesis. This study is aimed at assessing diagnostic and prognostic value of plasma c-MYC expression to aid in early diagnosis and prognosis of hematological malignancies. Plasma c-MYC expression was determined by quantitative real time PCR using EVA Green chemistry and cluster of differentiation markers performed via immunocytochemistry. Plasma c-MYC was higher in subject with hematological malignancies (8.8 ± 1.1) when compared with apparently healthy controls (4.5 ± 0.5). A screening cut-off c-MYC ratio value of 9.42 with sensitivity and specificity of 65.5% and 100% respectively were obtained using receiver operator characteristic curve analysis. Plasma c-MYC was found to have no prognostic value using Kaplan-Meier analysis. Plasma c-MYC ratio showed promising screening/diagnostic value for hematological malignancies.

Cohesion policy and metropolitan governance: innovation in stakeholders collaboration practices in Lisbon

This study examines the impact of Cohesion Policy on metropolitan governance and stakeholders’ collaboration practices in the Lisbon region. The financial crisis and subsequent austerity policies have underscored the importance of institutional arrangements and governance processes, particularly at the local and regional levels. Focusing on the Lisbon metropolitan area, which faces fragmented institutional structures and governance challenges, this research highlights the role of Cohesion Policy through Integrated Territorial Investments (ITI) in fostering innovation and collaboration among stakeholders. The EVA (Green and Blue Corridor) project in the Lisbon Metropolitan Area exemplifies the role of EU funding in promoting cooperation and governance innovation. The introduction of ITI under the Cohesion Policy has facilitated the establishment of collaborative networks and innovative practices in the metropolitan area. These initiatives have broken down planning silos, promoting "soft spaces of governance" that encouraged knowledge exchange and institutional learning among territorial stakeholders. However, challenges persist in the absence of a coherent regional strategy and the prioritization of broad EU objectives over tailored regional needs. This hampers the potential for integrated and proactive territorial management. Nevertheless, Cohesion Policy has stimulated stakeholder collaboration and innovation in metropolitan governance, paving the way for sustainable and inclusive development in the Lisbon region.

An Eva Green Real-Time PCR Assay for Porcine DNA Analysis in Raw and Processed Foods

Abstract The prevalence of the inclusion or substitution of porcine and its derivatives in foods by imprudent food manufacturers accentuated the need for a sensitive and specific method to detect porcine DNA. This work aimed to develop and validate an EvaGreen dye-based quantitative real-time (qPCR) assay for porcine DNA analysis. The primer set used in this work targets a 95-bp fragment of the porcine cytochrome b gene. Application of the assay to dilutions of porcine genomic DNA showed that the assay is sensitive down to 10 pg/μL of porcine DNA and the detection limit, under binary admixture conditions, was 0.001%. A correlation coefficient (R2) of 0.998 and PCR efficiency of 101.6% over the dynamic range from 10 to 10000 pg/μL indicated that the assay has high linearity and efficiency. The method’s specificity towards porcine was revealed by the amplification profile, which only displayed an amplification signal for porcine DNA. These findings suggest that the developed assay is sensitive, specific, and rapid. In conclusion, the assay can be used as an alternative method to detect and quantify porcine DNA in raw and processed food products.

The Female Burden Visualized: Cinematic Representation of Women during Epidemics

The Female Burden Visualized: Cinematic Representation of Women during Epidemics

The curse and the chora

Referring to the work of Julia Kristeva, this article seeks to perform a comparative analysis between Cordelia Chase (Charisma Carpenter), as she appears within the remit of Joss Whedon and David Greenwalts’s Angel (1999–2004), and Vanessa Ives (Eva Green) in John Logan’s Penny Dreadful (2014–2016). Taking each character as a case study, this article seeks to elucidate the precarious subject positions of central female leads in a team/ensemble horror television series in order to assess whether or not and how the portrayal and characterisation thereof has changed over two decades. To do so, this article employs a theoretical framing that examines the question of agency and power by assessing both characters as what I will call “Choraic conduits”. As such, both characters’ relation, manipulation of/by, and mediation of the supernatural as envisaged and presented in their respective diegetic worlds are analysed in themselves and comparatively against one another. Key concerns here are the questions and problems surrounding each character’s agency over her powers and the supernatural/spiritual realm(s) from which they emerge, as well as the psycho-physical and symbolic consequences of not only the possession of their respective powers, but the micro and macroscopic consequences of how they are used in their respective diegetic worlds.

A DNA molecular diagnostic technology with LAMP-like sensitivity based on one pair of hairpin primers-mediated isothermal polymerization amplification

With the rapid development of isothermal amplification technology, DNA molecular diagnosis has become an important reference for clinical treatment. In this work, we have designed a DNA molecular diagnostic technology with LAMP-like sensitivity for nucleic acid analysis and detection based on only one pair of hairpin primers. This DNA molecular diagnostic technology consists of Bst DNA polymerase and one pair of hairpin primers, which are designed easily by adding a stem-loop structure to a target binding domain. When the target is present, the polymerization reaction between the hairpin primers and the target generates a specific dumbbell DNA similar to LAMP, which triggers cyclic amplification reactions to extend a series of long dsDNA products with repeated sequences by inserting fluorescent dye Eva Green observed the increase in fluorescence signal. In our method, using the hairpin primers-mediated isothermal polymerization amplification, we can specifically monitor 3–5 copies of the target nucleic acid in the system without labeling and temperature cycling in the reaction. In addition, serum samples from 13 patients with suspected schistosomiasis were targeted; we further demonstrated the ability of the technology to detect complex clinic samples, and its potentially inestimable applicability in clinic early molecular diagnostic research.

Prevalence and geographical distribution of Nosema apis and Nosema ceranae in apiaries of Northwest Mexico using a duplex real-time PCR with melting-curve analysis

In many rural regions of Mexico, beekeeping is an important socioeconomic activity which is currently facing various health issues, being the main one the presence of infectious diseases. The aim of our study was to optimize a duplex Eva Green RT-PCR assay along with melting-curve analysis to detect and differentiate Nosema apis from Nosema ceranae infection in Apis mellifera. At four locations in Northwest Mexico 765 samples from 76 apiaries were tested using a RT-PCR and melting-curve analysis. Infection was detected in all the four geographical regions under study. Overall, 87.32% samples resulted positive for N. apis, 12.68% for N. ceranae and 10.20% resulted co-infected with both microsporidians. The Mexicali valley had the highest prevalence of N. apis with 56.68% positive apiaries, while in Ensenada the lowest prevalence was found with 7.89% positive apiaries. The highest prevalence of N. ceranae was detected in Mexicali (15.79%), while Tijuana and San Luis Rio Colorado showed the lowest prevalence with 1.32%, respectively. Our work indicates a wide spread of nosemosis in all the four geographical regions with a marked dominion of N. apis. The RT-PCR assay resulted highly specific and accurate for the detection of N. apis and N. ceranae and could provide a rapid and accurate diagnostic tool for the simultaneous detection and discrimination of both microsporidians. It can also be used for clinical diagnostics and disease surveillance and to evaluate the effectiveness of the measures for control and prevention of nosemosis in the Northwest corner of Mexico.

Development of an EvaGreen-based real-time PCR assay for detection of Aleutian mink disease virus

The objective of this study was to develop a rapid, sensitive and specific EvaGreen (EG)-based real-time PCR assay capable of detecting Aleutian mink disease virus (AMDV) and to evaluate the reliability of the assay for analysis of blood or tissue samples. For this assay, a pair of primers was designed based on a nonstructural protein (NS)-encoding gene of AMDV, and the identity of PCR products was identified based on a melting temperature of 82.8°C. The EG-based real-time PCR assay did not detect canine distemper virus or mink enteritis virus, and the assay could be used to detect Chinese and American AMDV strains, in contrast to a commercial TaqMan kit that could only detect American AMDV strains. The amplification efficiencies of the EG assay were 104.8% for the Chinese strain and 94.4% for the American strain, and the detection limit was 1 copy/μL of AMDV plasmid or 3 pg/μL of viral DNA (Chinese strain). The intra- and inter-assay variation coefficients of melting temperature were all lower than 0.15%, confirming the high reproducibility of the assay. Forty-five clinical blood samples were simultaneously analyzed using the EG real-time PCR, TaqMan kit and conventional PCR, and the detection rates were 91.1%, 0.0% and 86.7%, respectively. Serum samples were also collected from the corresponding blood samples and tested using the counterimmunoelectrophoresis (CIEP) assay, where positive samples accounted for 24.4% of the 45 samples. In conclusion, EG-based real-time PCR is a rapid, sensitive, universal assay that can be effectively utilized as a reliable and specific tool for detection and quantitation of AMDV.

INFLUENCE OF APPROACHES TO ISOLATION OF ANIMAL BIOACTIVE SUBSTANCES ON ANTIMICROBIAL ACTION

This article highlights the problem of the use of chemical preservatives in the food industry. The prospects of implementation natural substances with an antimicrobial effect for prolonging shelf life and improving the quality of food products are also discussed. Methods for isolating of antimicrobial substances from epithelial and mucous tissues of animal origin are proposed, taking into account the minimization of losses of their biological activity in accordance with the structure of protein-peptide molecules. Based on the results of the determination of antimicrobial activity against Listeria monocytogenes, Staphylococcus aureus and Pseudomonas aeruginosa using flow cytometry, stains Eva Green and PI, the most promising raw materials were determined and the feasibility of using weakly acid extraction followed by trypsinolysis for releasing antimicrobial substances from preprotein molecules and ultrafiltration for purification from high-molecular compounds were investigated.

Probing DNA damage induced by common antiviral agents using multiple analytical techniques

Hepatocellular carcinoma is one of the most common malignant tumors in the world. Chronic hepatitis B and C infections are the most common etiologies of hepatocellular carcinoma worldwide. In this study, we explore the potential DNA damaging effect of some FDA-approved antiviral drugs which may be able to serve as anticancer agents for hepatocellular carcinoma, in order to better elucidate their mode of action. Five antiviral drugs were selected; ribavirin, sofosbuvir, tenofovir disoproxil fumerate, daclatasvir and ledipasvir. Several methods, including absorption spectroscopy, MALDI-TOF mass spectrometry and fluorimetric analysis using the EvaGreen (EG) intercalating dye, were used to probe the drug-induced DNA damage. Results show that only daclatasvir and ledipasvir induced DNA damage. Absorption spectroscopy showed hyperchromicity in the 260-nm DNA absorption band of DNA samples incubated with each drug, indicating disruption of the double-strand structure. Mass spectra for DNA samples incubated with each of the two drugs showed a disappearance of the DNA molecular ion peak with a concomitant appearance of peaks with smaller m/z, indicating DNA strand breaks. EG fluorescence was observed to decrease with increasing incubation time of daclatasvir and ledipasvir with DNA, indicating that the EG detaches from the DNA, likely due to DNA damage. All of these results are consistent with DNA damage, proposed as oxidative damage to both nucleobase and deoxyribose moieties of DNA as the mode of action for these two drugs. Moreover, these results are dependent on the antiviral drug concentration and show that DNA regions rich in guanine are affected more than other regions by these two drugs. Therefore, such antiviral drugs may present a promising therapeutic alternative to the currently used anticancer agents, especially for hepatitis B and C patients with hepatocellular carcinoma resistant to conventional treatment approaches.

Characterization of the binding interactions between EvaGreen dye and dsDNA.

Understanding the dsDNA·EG binding interaction is important because the EvaGreen (EG) dye is increasingly used in real-time quantitative polymerase chain reaction, high resolution melting analysis, and routine quantification of DNA. In this work, a binding isotherm for the interactions of EG with duplex DNA (poly-dA17·poly-dT17) has been determined from the absorption and fluorescence spectra of the EG and dsDNA·EG complex. The isotherm has a sigmoidal shape and can be modeled with the Hill equation, indicating positive cooperativity for the binding interaction. A Scatchard plot of the binding data yields a concave-down curve in agreement with the Hill analysis of the binding isotherm for a positive cooperative binding interaction. Analysis of the Scatchard plot with the modified McGhee and von Hippel model for a finite one-dimensional homogeneous lattice and nonspecific binding of ligands to duplex DNA yields the intrinsic binding constant, the number of lattice sites occluded by a bound ligand, and the cooperativity parameter of 3.6 × 105 M-1, 4.0, and 8.1, respectively. The occluded site size of 4 indicates that moieties of the EG intercalate into the adjacent base pairs of the duplex DNA with a gap of 1 intercalation site between EG binding sites, as expected for a bifunctional molecule. Interestingly, at high [EG]/[base pair], the intercalation is disrupted. A model is proposed based on the fluorescence spectrum where the formation of anti-parallel stacked chains of EGs bound externally to the duplex DNA occur at these high ratios.

The presence of residual gold nanoparticles in samples interferes with the RT-qPCR assay used for gene expression profiling

BackgroundRT-qPCR is routinely used in expression profiling of toxicity pathway genes. However, genetic and molecular level studies used to determine, understand and clarify potential risks of engineered nanomaterials (ENMs) are still incomplete. Concerns regarding possible interference caused by intracellular ENMs during analyses have been raised. The aim of this study was to verify a qPCR procedure for gene expression assays, which can be used in toxicity and exposure assessments.ResultsAmplification of ten reference genes was performed to test the expression stability. A preliminary study was performed on RNA from BEAS-2B cells that had been treated with AuNPs. Also, a reference total RNA standard from ten cell lines was spiked with various amounts of the same AuNP. This treatment mimics exposure assessment studies, where assay-interference may be caused by intracellular residual ENMs still being present in the biological samples (during and after isolation/purification procedures). Both types of RNA samples were reverse transcribed and then amplified by qPCR. The qPCR-related software and statistical programs used included BestKeeper, NormFinder, REST and qBase+. These results proved that using standard qPCR analysis and statistical programs should not be the only procedure applied to verify the assay for gene expression assessment related to ENMs. A comparison of SYBR Green to EVA Green was discussed, in addition to a comparison to the latest reports regarding the influence of ENM thermal conductivity, surface interactions with ENMs, effects of ENM size and charge, as well as, the limit of detection in a qPCR assay.ConclusionsAuNPs have the potential to interfere with the assay mechanism of RT-qPCR, thus, assay verification is required for AuNP-related gene expression studies used to evaluate toxicity. It is recommended to use HSP90 and YWHAZ as reference genes, i.e. these were the most stable in our study, irrespective of the source of the RNA, or, the point at which the AuNPs interacted with the assay. This report describes steps that can be utilised to generate a suitable method for gene expression studies associated with toxicity testing of various ENMs. For example, RNA standards that have been spiked with known amounts of ENMs should be run in conjunction with the unknown samples, in order to verify any RT-qPCR assay and determine the degree of error.

Identification and differentiation of Campylobacter species by high-resolution melting curve analysis.

Campylobacter jejuni and Campylobacter coli are the important food-born zoonotic pathogen, also are leading causes of human food borne illnesses worldwide. cadF gene is expressed in all C.jejuni and C.coli strains and mediates cell binding to the cell matrix protein, Fibronectin. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The goal of this study was to apply HRM analysis to identification of C.jejuni and C.coli. A total of 100 samples were obtained from chicken in Kermanshah, Iran. HRM analysis based on cadF gene and Eva Green was developed to the identification of Campylobacter to the species level. Fifty-five of 100 samples were positive as campylobacter (7 C.jejuni, 29 C.coli, 16 mixes and 3 unknown). Minor variations were observed in melting point temperatures of C.coli or C.jejuni isolates and this variation can be used to the differentiation between C.coli or C.jejuni isolates. The results of this study indicated that HRM curve analysis can be a suitable technique and rapid technique for distinguishing between C.jejuni and C.coli isolates.

Optimization of Eva Green real-time mPCR for differentiating C. jejuni/coli directly from feces.

To develop and optimize a rapid molecular method for diagnosing campylobacteriosis directly from a clinical fecal sample and at the same time for determining the most common causing agents - C. jejuni/coli. 38 clinical fecal samples from hospitalized patients with diarrheal syndrome were tested using a rapid immunochromatographic test. All positive samples were tested for confirmation by culturing in a microaerophilic atmosphere. The Eva Green real-time mPCR reaction of a direct fecal sample was conducted using the "IQ5TM Real-Time PCR System" apparatus. Out of 38 clinical fecal samples which were ICT positive, 18 strains were isolated by culture, namely, 17 of C. jejuni and 1 of C. coli. The Eva Green real-time mPCR reaction also reported 18 positive samples for Campylobacter, out of which 17 were of C. jejuni and only one of C.coli. We developed and optimized the Eva Green real-time mPCR for the detection and species differentiation of C. jejuni/coli directly from a clinical fecal sample. The molecular analysis we described has a 100% sensitivity and specificity when comparing the results obtained by it to those of the culture method, which is currently the "gold standard" in the diagnosis of campylobacteriosis (Tab. 2, Fig. 1, Ref. 6).

Optimization of Diamond Nucleic Acid Dye for quantitative PCR.

Here, we evaluate Diamond Nucleic Acid Dye (DD) for use in quantitative PCR (qPCR) applications. Although DD is a commercially available stain for detection of DNA separated by gel electrophoresis, its use as a detection dye in qPCR has yet to be described. To determine if DD can be used in qPCR, we investigated its inhibitory effects on qPCR at concentrations ranging 0.1-2.5×. Serial dilution of DNA was used to determine the efficiency, sensitivity, and linearity of DD-generated qPCR data in comparison to other commonly used fluorescent dyes such as SYBR Green (SG), EvaGreen (EG), and BRYT Green (BG). DD was found to be comparable with other dyes for qPCR applications, with an R2 value >0.9 and an efficiency of 0.83. Mitochondrial DNA (mtDNA) target signals were successfully produced by DD over a DNA dilution range of ~28 ng- 0.28 pg, demonstrating comparable sensitivity to the other dyes investigated. Cq values obtained using DD were lower than those using EG by almost 7 cycles. We conclude that Diamond Nucleic Acid Dye is a cheaper, less toxic alternative for qPCR applications.