DNA Concentration Research Articles

High release of Candida albicans eDNA as protection for the scaffolding of polymicrobial biofilm formed with Staphylococcus aureus and Streptococcus mutans against the enzymatic activity of DNase I.

This study aimed to determine the protective role of the high release of C. albicans extracellular DNA (eDNA) in a polymicrobial biofilm formed by S. aureus and S. mutans in the course of DNase I treatment. A tube-flow biofilm bioreactor was developed to mimic biofilm formation in the oral cavity. eDNA release was quantified by real-time PCR (qPCR) and confocal microscopy analysis were used to determine the concentration and distribution of eDNA and intracellular DNA (iDNA). The mean amount of eDNA released by each species in the polymicrobial was higher than that in monospecies biofilms (S. aureus: 3.1 × 10-2ng/μl polymicrobial versus 5.1 × 10-4ng/μl monospecies; S. mutans: 3 × 10-1ng/μl polymicrobial versus 2.97 × 10-2ng/μl monospecies; C. albicans: 8.35ng/μl polymicrobial versus 4.85ng/μl monospecies). The large amounts of eDNA released by C. albicans (96%) in polymicrobial biofilms protects the S. aureus and S. mutans cells against the degradation by DNase I and dampens the effect of clindamycin.

Circulating nucleases degrade high-molecular-weight cell-free DNA in coronary vessels during acute myocardial infarction

Abstract Introduction Extracellular cell-free DNA (cfDNA) originates from various biological processes, including cell turnover, apoptosis, necrosis, NETosis, and other forms of cell death. In healthy individuals, highly fragmented cfDNA is elevated in the bloodstream due to infection, cancer, inflammation and thrombosis. In thrombotic vascular diseases, neutrophils release elongated, high-molecular-weight DNA with bioactive and prothrombotic properties in a process termed NETosis. Circulating nucleases are thought to counteract pathogenic properties of NETs. However, attempts to connect total cfDNA concentration to nuclease activity have failed. Purpose We aimed to elucidate the in vivo connection between extracellular DNA and circulating nucleases in thrombotic vascular diseases by investigating DNA length as a potential novel biomarker in cardiovascular disease. Methods Citrated plasma from healthy volunteers (n=363) and from ST-elevation myocardial infarction (MI) coronary aspirates (n=59) and peripheral arterial sites (n=96) were analyzed for DNA concentration, length, and nuclease activity. Total DNA concentration was measured with a fluorescent DNA binding dye, and elongated DNA assessed using qPCR and Agilent bioanalyzer high-sensitivity DNA chip. Nuclease activity was measured by single radial enzyme-diffusion (SRED) assay. Cardiac MRI assessed infarct size acutely and six months after the event. Results Coronary aspirates exhibited an elevation in total DNA, with a significant rise in high-molecular weight cell-free DNA in addition to an increase in sub-nucleosome fragmentation (Figure 1). Peripheral and coronary plasma samples from individuals with myocardial infarction (MI) showed elevated levels of total and elongated cfDNA. When compared to healthy individuals, the integrity of cfDNA was lower in peripheral samples and higher in coronary aspirates. During myocardial infarction, circulating nuclease activity was elevated systemically but low at the coronary culprit lesion site. Elongated high-molecular-weight cfDNA accumulates primarily in coronary vessels but is also elevated systemically. In coronary aspirates, nuclease activity negatively correlated with elongated DNA and DNA integrity (rs=0.6255, p<0.0001). Conclusions Elongated DNA accumulates during MI at the culprit lesion site. Its inverse correlation with nuclease activity underscores the role of nucleases in cleaving elongated DNA to shorter, soluble fragments. This study establishes DNA length and integrity as novel biomarkers in thrombotic cardiovascular disease.Figure 1:Coronary cfDNA lengthFigure 2:Nuclease activity and cfDNA

A Novel PCR-Free Ultrasensitive GQD-Based Label-Free Electrochemical DNA Sensor for Sensitive and Rapid Detection of Francisella tularensis

Biological warfare agents are infectious microorganisms or toxins capable of harming or killing humans. Francisella tularensis is a potential bioterrorism agent that is highly infectious, even at very low doses. Biosensors for biological warfare agents are simple yet reliable point-of-care analytical tools. Developing highly sensitive, reliable, and cost-effective label-free DNA biosensors poses significant challenges, particularly when utilizing traditional techniques such as fluorescence, electrochemical methods, and others. These challenges arise primarily due to the need for labeling, enzymes, or complex modifications, which can complicate the design and implementation of biosensors. In this study, we fabricated Graphene Quantum dot (GQD)-functionalized biosensors for highly sensitive label-free DNA detection. GQDs were immobilized on the surface of screen-printed gold electrodes via mercaptoacetic acid with a thiol group. The single-stranded DNA (ssDNA) probe was also immobilized on GQDs through strong π−π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, leading to a decrease in the effect of GQD but a positive shift associated with the increase in DNA concentration. The specificity of the developed system was observed with different microorganism target DNAs and up to three-base mismatches in the target DNA, effectively distinguishing the target DNA. The response time for the target DNA molecule is approximately 1010 s (17 min). Experimental steps were monitored using UV/Vis spectroscopy, Atomic Force Microscopy (AFM), and electrochemical techniques to confirm the successful fabrication of the biosensor. The detection limit can reach 0.1 nM, which is two–five orders of magnitude lower than previously reported methods. The biosensor also exhibits a good linear range from 105 to 0.01 nM and has good specificity. The biosensor’s detection limit (LOD) was evaluated as 0.1 nM from the standard calibration curve, with a correlation coefficient of R2 = 0.9712, showing a good linear range and specificity. Here, we demonstrate a cost-effective, GQD-based SPGE/F. tularensis DNA test suitable for portable electrochemical devices. This application provides good perspectives for point-of-care portable electrochemical devices that integrate sample processing and detection into a single cartridge without requiring a PCR before detection. Based on these results, it can be concluded that this is the first enzyme-free electrochemical DNA biosensor developed for the rapid and sensitive detection of F. tularensis, leveraging the nanoenzyme and catalytic properties of GQDs.

Reentrant DNA shells tune polyphosphate condensate size

The inorganic biopolymer polyphosphate (polyP) occurs in all domains of life and affects myriad cellular processes. A longstanding observation is polyP’s frequent proximity to chromatin, and, in many bacteria, its occurrence as magnesium (Mg2+)-enriched condensates embedded in the nucleoid region, particularly in response to stress. The physical basis of the interaction between polyP, DNA and Mg2+, and the resulting effects on the organization of the nucleoid and polyP condensates, remain poorly understood. Here, using a minimal system of polyP, Mg2+, and DNA, we find that DNA can form shells around polyP-Mg2+ condensates. These shells show reentrant behavior, that is, they form within a window of Mg2+ concentrations, representing a tunable architecture with potential relevance in other multicomponent condensates. This surface association tunes condensate size and DNA morphology in a manner dependent on DNA length and concentration, even at DNA concentrations orders of magnitude lower than found in the cell. Our work also highlights the remarkable capacity of two primordial inorganic species to organize DNA.

Illumina Sequencing in Conjunction with Propidium Monoazide to Identify Live Bacteria After Antiseptic Treatment in a Complex Oral Biofilm: A Study Using an Ex Vivo Supragingival Biofilm Model

Background/Objectives: The evaluation of the efficacy of antibacterial treatments in complex oral ecosystems is limited by the inability to differentiate live from dead bacteria using omic techniques. The objective of this study was therefore to assess the ability of the combination of the 16S rRNA Illumina sequencing methodology and the action of propidium monoazide (PMA) to study viable bacterial profiles in oral biofilms after exposure to an antiseptic compound. Methods: Cariogenic supragingival biofilms were developed in an ex vivo model for 96 h, using saliva from healthy volunteers. The biofilms were treated with 0.12% chlorhexidine (CHX) combined with 0.05% cetylpyridinium chloride (CPC), for 60 s, using phosphate buffered saline as a control. After exposure, each biofilm was treated or not with PMA to then extract the bacterial DNA, quantify it by Qubit, quantify the bacterial population using qPCR, and perform the metataxonomic study of the samples using Illumina 16S rRNA sequencing. Results: A significantly lower DNA concentration in the PMA-treated biofilms (p < 0.05 compared with those not exposed to PMA) was observed. The viable bacterial count obtained by qPCR differed significantly from the total bacterial count in the biofilm samples exposed to the antiseptic (p < 0.05). The viable microbiome differed significantly from the total bacterial profile of the samples treated with CHX/CPC after exposure to PMA (p < 0.05 at the α- and β-diversity levels). Conclusions: The combination of Illumina 16S rRNA sequencing and PMA helps solve the inability to evaluate the efficacy of antibacterial treatments in the bacterial profile of complex ecosystems such as oral biofilms.

The Chilean COVID-19 Genomics Network Biorepository: A Resource for Multi-Omics Studies of COVID-19 and Long COVID in a Latin American Population

Although a lack of diversity in genetic studies is an acknowledged obstacle for personalized medicine and precision public health, Latin American populations remain particularly understudied despite their heterogeneity and mixed ancestry. This gap extends to COVID-19 despite its variability in susceptibility and clinical course, where ethnic background appears to influence disease severity, with non-Europeans facing higher hospitalization rates. In addition, access to high-quality samples and data is a critical issue for personalized and precision medicine, and it has become clear that the solution lies in biobanks. The creation of the Chilean COVID-19 Biorepository reported here addresses these gaps, representing the first nationwide multicentric Chilean initiative. It operates under rigorous biobanking standards and serves as one of South America’s largest COVID cohorts. A centralized harmonization strategy was chosen and included unified standard operating procedures, a sampling coding system, and biobanking staff training. Adults with confirmed SARS-CoV-2 infection provided broad informed consent. Samples were collected to preserve blood, plasma, buffy coat, and DNA. Quality controls included adherence to the standard preanalytical code, incident reporting, and DNA concentration and absorbance ratio 260/280 assessments. Detailed sociodemographic, health, medication, and preexisting condition data were gathered. In five months, 2262 participants were enrolled, pseudonymized, and sorted by disease severity. The average Amerindian ancestry considering all participant was 44.0% [SD 15.5%], and this value increased to 61.2% [SD 19.5%] among those who self-identified as Native South Americans. Notably, 279 participants self-identified with one of 12 ethnic groups. High compliance (>90%) in all assessed quality controls was achieved. Looking ahead, our team founded the COVID-19 Genomics Network (C19-GenoNet) focused on identifying genetic factors influencing SARS-CoV-2 outcomes. In conclusion, this bottom-up collaborative effort aims to promote the integration of Latin American populations into global genetic research and welcomes collaborations supporting this endeavor. Interested parties are invited to explore collaboration opportunities through our catalog, accessible online.

Rapid Thermal Drying Synthesis of Nonthiolated Spherical Nucleic Acids with Stability Rivaling Thiolated DNA

AbstractAttaching DNA oligonucleotides to gold nanoparticles (AuNPs) to prepare spherical nucleic acids (SNAs) has offered tremendous insights into surface chemistry with resulting bioconjugates serving as critical reagents in biosensors and nanotechnology. While thiolated DNA is generally required to achieve stable conjugates, we herein communicate that using a thermal drying method, a high DNA density and excellent SNA stability was achieved using nonthiolated DNA, rivaling the performance of thiolated DNA such as surviving 1 M NaCl, 2 month stability in 0.3 M NaCl and working in 50 % serum. A poly‐adenine block with as few as two consecutive terminal adenine bases is sufficient for anchoring on AuNPs. By side‐by‐side comparison with the salt‐aging method, the conjugation mechanism was attributed to competitive adenine adsorption at high temperature along with an extremely high DNA concentration upon drying. Bioanalytical applications of nonthiolated SNAs were validated in both solution and paper‐based sensor platforms, facilitating cost‐effective applications for SNAs.

Applying nanopore sequencing technology in Paracoccidioides sp.: a high-quality DNA isolation method for next-generation genomic studies.

Paracoccidioidomycosis is a severe systemic endemic mycosis caused by Paracoccidioides spp. which mainly affects individuals in Latin America. Progress in Paracoccidioides genomics has been slow, as evidenced by the incomplete reference databases available. Next-generation sequencing is a valuable tool for epidemiological surveillance and genomic characterization. With the ability to sequence long reads without the need for prior amplification, Oxford Nanopore Technology (ONT) offers several advantages, but high-quality and high-quantity DNA samples are required to achieve satisfactory results. Due to the low concentration of Paracoccidioides DNA in clinical samples and inefficient culture isolation methods, DNA extraction can be a significant barrier to genomic studies of this genus. This study proposes a method to obtain a high-coverage de novo genome assembly for Paracoccidioides using an improved DNA extraction method suitable for sequencing with ONT. The assembly obtained was comparable in size to those constructed from available data from Illumina technology. To our knowledge, this is the first genome assembly of Paracoccidioides sp. of such a large size constructed using ONT.

Size Effects of Gold Nanoparticles on Surface Plasmon Resonance Assays for DNA Hybridization.

Recent advancements in signal amplifiers, such as biofunctionalized gold nanoparticles (AuNPs) have improved the surface plasmon resonance (SPR) performance. However, the correlation between the sizes of DNA-Au conjugates and the SPR chips remains elusive. We investigated how the size of AuNPs functioned with DNA detection probes (D-AuNPs) affect SPR signals in sandwich DNA hybridization assays. The effects of three sizes (5, 13, and 29 nm) of D-AuNPs with an equal surface probe density were systematically compared to delineate the relationship between signal amplification and steric hindrance. Sporadically adsorbed target DNA on sparse capture probe-coated chips led to a growth of signal amplification with larger D-AuNPs. In contrast, on dense capture probe-coated SPR chips, when the target DNA concentration was above 1.5 nM, the medium-sized 13-nm AuNPs displayed 1.7- and 1.3-fold enhancement factors than 5-nm and 29-nm ones, respectively. Our results indicate the steric hindrance disturbs the capture of D-AuNPs on dense target DNA-modified chips, rendering the surface density of captured D-AuNPs a determining factor of the sensor response. Alternatively, the sensor sensitivity to D-AuNP surface density is crucial on chips with sparse target DNA. These insights should stimulate and guide future research on surface functionalization toward SPR sensors and AuNPs.

Application of Genetic Origin Analysis of Copy Number Variations in Non-Invasive Prenatal Testing.

This study aimed to assess the application of origin analysis of copy number variations (CNVs) in non-invasive prenatal testing (NIPT) and provide a basis for expanding the clinical application of NIPT. We enrolled 35,317 patients who underwent NIPT between January 2019 and March 2023. Genome sequencing of copy number variation (CNV-Seq) analysis was performed using the CNV calling pipeline to identify subchromosomal abnormalities in maternal plasma. Genetic origin was determined by comparing the chimaerism ratio of CNV and the concentration of cell-free foetal DNA (cffDNA). All pregnant women with a high risk of CNV, as indicated by the NIPT, were informed of their genetic origins. Amniocentesis was recommended for detecting the CNVs in foetal chromosomes, and pregnancy outcomes were tracked. A total of 109 pregnancies showed clinically significant positive results for CNV after NIPT, including 65 cases of maternal/foetal (M/F)-CNVs and 44 cases of F-CNVs. The occurrence of M/F-CNVs was independent of age, screening (serological or ultrasound) indications for abnormalities, and mode of pregnancy. The incidence of pathogenic/likely pathogenic (P/LP)-F-CNVs was high in cases where serological screening indicated intermediate, high-risk, or abnormal US findings (p<0.05). In the M/F-CNV group, most of the P/LP-CNVs were small fragments with low penetrance; 55 (84.62%) were less than 5Mb in size, and nine (13.85%) were between 5 and 10Mb. In the F-CNV group, foetal P/LP-CNV was detected in 36 of 42 cases undergoing prenatal diagnosis, and no significant bias was noted in the size distribution of P/LP-F-CNV fragments. The prenatal diagnostic rate and positive predictive value in the F-CNV group were 95.45% and 85.71%, respectively, which were significantly different from those in the M/F group (26.15% and 52.95%), respectively (p<0.05). Genetic origin analysis of CNV can effectively improve adherence to prenatal diagnosis in pregnant women and the accuracy of prenatal diagnosis.

The Impact of Heavy Metal Ions on Arginase Produced from Pseudomonas aeruginosa Isolated from Sewage

Background: Researchers remain captivated by the impact of heavy metal ions, commonly found in sewage, on arginase synthesis. Objective: The study examines the presence and characteristics of Pseudomonas aeruginosa in 24 sewage samples, identifying 18 isolates (75%). It assesses DNA concentration and purity, utilizes PCR-based methods for subtype analysis and investigates the inhibitory effects of heavy metals on purified enzymes. Methods: Between November 2022 and April 2023, 52 samples were collected from various sewage sites in Baghdad, Iraq. These samples were cultured in brain heart infusion broth and on nutrient agar plates. The isolates were then subjected to laboratory procedures, including biochemical tests, analysis with the Vitek2 system and PCR to identify Pseudomonas aeruginosa. Biochemical testing specifically focused on Pseudomonas aeruginosa. DNA extraction was performed on P.aeruginosa isolates from sewage, which had already been identified through morphological and biochemical tests as P.aeruginosa. This was accomplished by screening for the presence of the 16SrRNA gene using PCR. Result: Metal ion effects on L-arginase were tested at various concentrations (100mM, 50mM, 25mM, 12.5 mM). Metal ions were obtained via the cutting method and added 15 minutes before the substrate. Enzyme activity was measured and the activity ratio (enzyme activity with and without metal ions) calculated. Activity was assessed in the absence of 100% metal ions.Arginase isolated and purified from Pseudomonas aeruginosa from sewage, heavy metals (mercury, cadmium, cobalt) has effect on arginase, mercuric chloride and cadmium chloride inhibit and decrease enzyme activity, while Cobalt chloride increases enzyme activity.

Development and evaluation of a panel of newly screened Y chromosome InDels for inferring paternal ancestry information in Southwest China.

Y-InDels (insertions/deletions) are genetic markers which are extremely understudied. It is unknown whether this type of markers can be utilized for genetic ancestry inference. We have developed an innovative Y chromosome ancestry inference system tailored for forensic applications. This panel amplifies 21 Y chromosome loci, encompassing Y-InDels and Y-SNPs (Single Nucleotide Polymorphism), utilizing the capillary electrophoresis (CE) platform. The system performed well at DNA concentrations greater than 0.125 ng/ul and produced accurate results at a 1:100 mixing ratio of male and female DNA. The Cumulative probability of matching (CPM) was between 0.95 and 0.97 in the experimental population. The system's efficacy in inferring ancestral origins was demonstrated through intercontinental population discrimination, revealing high discrimination power between African and East Asian populations. Population genetic analyses conducted on Han, Qiang and Hui populations in Southwest China, where the smallest FST value was 0.0002 between Han Chinese in Beijing (from 1000 Genomes Project) and Qiang Chinese from Sichuan (CQSC). Phylogenetic tree construction further illuminated distinct haplotypes among populations, with ethnically unique haplotypes observed in 34.6% of Hui and 7.1% of Qiang populations. K-fold cross-validation show the system's inference abilities at the intercontinental level. In addition, our investigations identified potential associations between the Y-InDel locus Y: 15,385,547 (GRCh37) and haplogroup R1a1a1b2a2- Z2124, as well as locus Y: 13,990,180 (GRCh37) and haplogroup F-M89. In conclusion, we have established a Y-chromosome inference system tailored for grassroots-level application, underscoring the value of incorporating Y-InDel markers in forensic analyses.

Circulating tumour DNA in patients with hepatocellular carcinoma across tumour stages and treatments

ObjectiveCirculating tumour DNA (ctDNA) is a promising non-invasive biomarker in cancer. We aim to assess the dynamic of ctDNA in patients with hepatocellular carcinoma (HCC).DesignWe analysed 772 plasmas from 173...

Validation of the performance of a point of care molecular test for leprosy: From a simplified DNA extraction protocol to a portable qPCR.

The study aimed to optimize qPCR reactions using oligonucleotides from the first Brazilian molecular diagnostic kit for leprosy on a portable platform (Q3-Plus). In addition, we sought to develop a simplified protocol for DNA extraction that met point-of-care criteria. During optimization on the Q3-Plus, optical parameters, thresholds, and cutoffs for the 16S rRNA and RLEP targets of M. leprae were established using synthetic DNA, purified DNA from M. leprae, and pre-characterized clinical samples. For the simplified extraction protocol, different lysis solutions were evaluated using chaotropic agents, and purification was carried out by transferring the lysed material to FTA cards. The complete protocol (simplified extraction + qPCR on the portable platform) was then evaluated with pre-characterized clinical skin biopsy samples and compared with standard equipment (QuantStudio-5). LOD95% for the optimized reactions was 113.31 genome-equivalents/μL for 16S rRNA and 17.70 genome-equivalents/μL for RLEP. Among the lysis solutions, the best-performing was composed of urea (2 M), which provided good dissolution of the skin fragment and a lower Ct value, indicating higher concentrations of DNA. The complete technological solution showed a sensitivity of 52% in reactions. Our results highlight the need for additional optimization to deal with paucibacillary samples, but also demonstrate the feasibility of the portable platform for the qPCR detection of M. leprae DNA in low infrastructure settings.

Possibilities of Immunogenetic Methods in Testing the Activity of Tuberculosis Infection in Children

The objective: to test the TREC/KREC DNA levels in children with various manifestations of tuberculosis infection.Subjects and Methods. A prospective, cross-sectional study was conducted in 2022-2024. Observation Group consisted of 115 children aged 6 (5; 7) years, of them 25 were diagnosed with tuberculosis, 63 were diagnosed with latent tuberculosis infection, 27 were not infected with Mycobacterium tuberculosis (conditionally healthy). Results. Findings of the study were as follows: among children with LTBI and a positive response to TRA, only 18.2% had the high TREC DNA concentration which corresponded to the level in the children ill with tuberculosis (t = 0.971; p = 0.342), there was a direct relationship between the TREC DNA and KREC DNA levels in children with LTBI; in 21.2% of children the level was low and indicative of immunodeficiency. If the positive response to TRA persisted in children for two years or more, there was a decrease in the average TREC DNA level, which was statistically significant compared to that in children with TRA+ for less than 2 years. Taking into account the results obtained, the TREC DNA level in children with LTBI can be considered as an additional criterion to assess activity of tuberculosis infection and can be an evidence to justify preventive anti-tuberculosis therapy if studied further. We also found a direct correlation between the TREC and KREC DNA levels in children with LTBI, r = 0.48 (by Spearman). When the positive response to TRA persisted for more than two years, a statistically significant decrease in the TREC DNA level was noted versus the results in children with the first positive response to TRA (t = 2.965; p = 0.005).Conclusion. Taking into account the results obtained, the TREC DNA level in patients with various manifestations of tuberculosis infection can be considered as an additional criterion of infection activity, which will allow differentiated approach to preventive treatment being an important evidence to justify anti-tuberculosis therapy.

Optimization of loop-mediated isothermal amplification assay for sunflower mildew disease detection

Loop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops. Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P. halstedii genomic DNA, targeting the ribosomal Large Subunit (LSU). These primers were verified by in silico analysis and experimental validation using both target and non-target species' DNAs. Optimizations encompassing reaction conditions (temperature, time) and component concentrations (magnesium, Bst DNA polymerase, primers, and dNTP) were determined. Validation of these optimizations was performed by agarose gel electrophoresis. Furthermore, various colorimetric chemicals (Neutral Red, Hydroxynaphthol Blue, SYBR Safe, Thiazole Green) were evaluated to facilitate method analysis, and the real-time analysis has been optimized, presenting multiple approaches for detecting sunflower downy mildew using the LAMP technique. The analytical sensitivity of the method was confirmed by detecting P. halstedii DNA concentrations as low as 0.5 pg/μl. This pioneering study, establishing P. halstedii detection through the LAMP method, stands as unique in its field. The precision, robustness, and practicality of the LAMP protocol make it an ideal choice for studies focusing on sunflower mildew, emphasizing its recommended use due to its operational ease and reliability.

B-025 Revolutionizing NGS-driven Laboratory Developed Tests: The impact of automated liquid handling on plate-based bead clean-up

Abstract Background Automated liquid handling systems are proving to be a transformative technology in the landscape of Laboratory Developed Tests (LDTs), especially those utilizing Next-Generation Sequencing (NGS). By enhancing accuracy, boosting efficiency, and offering scalable solutions, these systems enable labs to meet current and future challenges more effectively. The right system not only improves the quality of diagnostics but also positions labs to be agile and adaptive in a rapidly advancing healthcare environment. firefly® is a well-suited liquid handling solution for diagnostics labs developing LDTs due to its compact footprint, user-friendly software and exceptional versatility. Validated protocol templates that support NGS library preparation are readily available from the firefly cloud and can be adapted to meet specific lab needs. For example, automated bead-based clean-ups designed for 96- and 384- NGS applications are available on firefly. These remove DNA fragments of undesirable sizes, enzymes or primers rom the amplified DNA after a PCR reaction. Automating this process facilitates higher throughput and scaling. firefly has a unique combination of 6 positive displacement dispense heads and a 96/384-well air-displacement pipetting head, making it ideal to distribute the reagents required for bead clean-ups. Methods firefly protocols were developed and optimized to be compatible with a range of elution volumes and bead ratios. Protocol performance was tested using library DNA (∼300bp). The same input DNA was plated out and quantified using the QuantiFluor dsDNA system (Promega) before and after running the protocol. This data was used to assess any reduction in DNA concentration and uniformity across the plate. The protocol's ability to successfully modulate the fragment size distribution was demonstrated using a 50bp DNA ladder and different bead ratios. Data was obtained using a Fragment Analyzer (Agilent, DNF-474 HS NGS Fragment Kit). Well-to-well contamination was assessed by running no-template-controls (NTCs) in wells adjacent to library DNA, then analyzed by qPCR using a LightCycler 480 System (Roche, KAPA Library Quantification kit). Results 96 format has a setup time of 10 minutes, run time of 43 and %CV library concentration of &amp;lt;6%. 384 format has a setup time of 10 minutes, run time of 47 and %CV library concentration of &amp;lt;8%. Uniformity of concentration and fragment sizes was seen across the plates. Comparison of the library concentration before and after the clean-up showed a 5% reduction in DNA concentration after the 96-well plate clean-up and an 11% reduction in library concentration when using the 384-well plate clean-up. Fragment size analysis showed that fragment sizes can be successfully modulated on firefly by changing the bead ratio of the firefly run and no detectable well-to-well contamination was detected by qPCR. Conclusions We have demonstrated that automated bead clean-ups on firefly are uniform show uniformity across both 96- and 384-well plates, with no detectable well-to-well contamination, to deliver the consistent and robust results required for an LDT. Bead-based cleanups on firefly offer a fast, 100% walkaway solution. By running 96-samples at once on firefly, we see approximately a 6-fold increase in throughput than when performed manually by a single user.

A-302 Evaluation of real-time PCR assays for detection of sexually-transmitted infections

Abstract Background Sexually transmitted infections (STIs) have serious negative consequences for reproductive health worldwide. They are associated with infertility, premature birth and neonatal infections. Five STI pathogens, namely Trichomonas vaginalis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma urealyticum were selected for the evaluation of three potential real-time PCR assays that can be adopted for diagnostic testing. Methods A total of 36 samples were included, consisting of genital swabs, urine, abscesses and samples provided by quality assurance programmes. DNA extraction was performed using an automated nucleic acid extraction system, abGenixTM. Commercial genomic controls and DNA extracts donated by external laboratories were used as well. The extracted nucleic acids were tested using three different assays, namely Seegene Anyplex II STI-5 Detection, Thermo Fisher Scientific customized STI Panel assay, and TIB MOLBIOL STI LightMix Modular kits, according to the manufacturers’ instructions. Results A total of 67% and 11% of the samples showed equivalent positive and negative results, respectively. However, 22% of the samples had non-concordant results. TIB MOLBIOL STI LightMix Modular kit was unable to differentiate between Ureaplasma parvum and Ureaplasma urealyticum in 9 samples. Genomic controls were tested, and Seegene Anyplex II STI-5 Detection was unable to detect lower concentrations of DNA for Trichomonas vaginalis, Mycoplasma genitalium and Mycoplasma hominis. All assays were able to detect lower concentrations of Ureaplasma parvum DNA, but detectable concentrations were higher for Ureaplasma urealyticum. Conclusions In conclusion, the performance of each assay differed according to pathogen. None of the assays offered equal performance for all pathogens. We have adopted Thermo Fisher Scientific customized STI Panel assay in our diagnostic testing due to its advantage of being user friendly and cost effective.

A-296 Streck Urine Preserve Provides Sample Stability for Cepheid Xpert CT/NG test

Abstract Background Sexually transmitted infections (STIs) are one of the most common communicable diseases worldwide and are associated with significant morbidity and mortality. Data indicates that the four curable STIs - chlamydia, gonorrhea, trichomoniasis, and syphilis - cause over 375 million infections annually. Further, the infection rate for gonorrhea has increased by 63% in the United States and the emergence of resistant gonorrhea is an urgent global public health concern. To prevent transmission of gonorrhea and other curable STIs and minimize the escalation of untreated infections, early and effective prevention and control strategies are paramount. Rapid diagnostic testing and nucleic acid amplification tests (NAATs) have become the gold standard for STI detection. However, limited global access to these technologies often requires that samples be shipped to central laboratories for analysis. Current protocols recommend that neat urine be analyzed within 24 hours of collection if stored at 2 °C to 8 °C or within four days if frozen, as prolonged storage and exposure to freeze-thaws can induce cell lysis and release of nucleases, leading to degradation of DNA and false negative test results. Without sample stabilization, laboratories are left with a small window for accurate analysis. Here, we demonstrate that Streck Urine Preserve (SUP) maintains target and donor DNA concentrations during prolonged storage and that these stabilized samples are compatible with Cepheid’s automated GeneXpert System and associated Xpert CT/NG test. Methods Fresh human urine, with or without SUP, was tested neat or spiked with Neisseria gonorrhoeae (N. gonorrhoeae). Samples were stored at room temperature for up to 18 days before being analyzed on the Cepheid GeneXpert System using the Xpert CT/NG test per manufacturer’s instructions. Differences in average Ct values were compared for the sample processing control, sample adequacy control, and N. gonorrhoeae targets. DNA stability was assessed by comparing the changes between fresh and SUP-treated samples throughout the study period. Results Following prolonged storage, Ct values for neat, untreated urine increased by 3-7 cycles, correlating to a 10- to 100-fold decrease in detectable copies of N. gonorrhoeae and the sample adequacy control compared to urine samples stabilized with SUP. The average Ct value for samples stabilized with SUP did not change by more than 1.5 cycles throughout the study period. Additionally, Ct values for the sample processing control remained constant throughout the testing period, indicating that SUP did not impact sample processing. Conclusions When limited point-of-care testing options require storage and shipping of samples to central laboratories, urine stabilizers are imperative to obtain results representative of the patient sample at the time of collection. SUP effectively stabilized N. gonorrhoeae DNA and donor DNA prior to analysis with the Cepheid Xpert CT/NG test. This contrasts with the marked decrease in DNA copy number in non-stabilized urine over the study duration, which may lead to false negative results for low-positive urine samples. Taken together, SUP provides clinical researchers and assay developers with added flexibility when handling, shipping, and processing urine specimens and supports compatibility with automated NAAT systems and tests.

A-251 Simplification of Molecular Diagnostics for Sexually Transmitted Infection Point-of-Care Testing

Abstract Background HPV-caused cancers contribute to a large disease burden, accounting for 5% of all cancers. It is estimated that over 700,000 people develop HPV-related cancers each year with the majority being cervical cancer. The burden of this disease is disproportionately carried by low-income countries with the highest rates of HPV cervical infections primarily found in sub-Saharan Africa, Latin America, Eastern Europe, and South-East Asia. Many regions in low-resource countries lack adequate access to sensitive point-of-care screening tools, preventing timely diagnosis and treatment. Current low-cost methods, such as Pap smears and visual inspection using acetic acid suffer from low sensitivity, specificity, and reproducibility which limit their effectiveness as a screening modality. Many countries use nucleic acid amplification tests (NAATs) to identify key HPV infections which may progress towards cervical cancer. However, the expenditure for NAAT-capable equipment and reagents are cost-prohibitive for rural and underserved communities. To reduce screening barriers, we developed an isothermal HPV molecular test that can detect all 14 cancer-causing HPV strains using a novel amplification methodology combined with G-quadruplex DNAzyme colorimetric detection. Methods We developed a novel isothermal amplification technique that is related to loop-mediated isothermal amplification (LAMP) but uses modified looped primers to enable exponential amplification at 60°C for 1 hour. The modified primers only require two binding sites compared to 4-6 in LAMP. To detect the amplified products, we used G-quadruplex peroxidase-like DNAzyme probes which reduce ABTS to produce a visually interpreted result. Analytical validation evaluated the method’s limit of detection, specificity, and accuracy. As proof of concept in a relevant matrix, clinical cervical brushings collected ThinPrep Pap media were tested with the new method and compared to an FDA-approved reference qPCR test. Results Repeated experiments demonstrate that the lowest detectable HPV DNA concentration was approximately 1000 genomic copies/µL with no amplification with other pathogens. As proof of concept, the comparison to the reference standard demonstrated high accuracy. Conclusions The application of our assay is intended as a near-patient screening tool with further evaluation by a clinician for confirmation. With an accurate and rapid screening tool, we envision low-resource regions across the world will have the tools to rapidly detect and treat cervical cancers in their early stages.