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OBJECTIVES: To investigate the clinical implication of quantitative polymerase chain reaction (PCR)-based high-sensitivity detection of hepatitis B virus (HBV)-DNA levels in patients with HBV-related liver cirrhosis (LC). METHODS: From January 2020 to December 2022, 100 fasting serum samples were collected and retrospectively analyzed from patients with treated HBV-related LC attending the Suzhou Hospital of Integrated Traditional Chinese and Western Medicine and Suzhou Guangci Cancer Hospital. Patients were divided into a negative group (HBV-DNA < 20 IU/mL) and a positive group (HBV-DNA ≥ 20 IU/mL) according to their high-sensitivity HBV-DNA test results. The clinical characteristics and serological indicators of the two groups were compared, mainly including gender, age, liver function [total protein (TP), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), total bilirubin (TBIL), direct bilirubin (DBIL), and indirect bilirubin (IBIL)], lipids [total cholesterol (TC) and triglycerides (TG)], platelets (PLT), five serum liver fibrosis markers [cholyglycine (CG), hyaluronic acid (HA), laminin (LN), precollagen type III (PCIII), and type IV collagen (IV-C)], serum gastrointestinal tumor markers [α-fetoprotein (AFP) and carcinoembryonic antigen (CEA)], and hepatitis B surface antigen (HBsAg). The differences between the two groups in terms of liver function Child-Pugh grades and the incidence of hepatocellular carcinoma (HCC) were also compared. RESULTS: There were 39 patients in the positive group, including 29 males and 10 females, and 61 patients in the negative group, including 38 males and 23 females, with no statistically significant differences in gender and age distribution between the two groups (P > 0.05). The levels of serological indicators (TP, ALB, AST, GGT, ALP, TBIL, DBIL, IBIL, TC, TG, PLT, CG, HA, LN, PCIII, IV-C, AFP, CEA, and HBsAg) in both groups showed no significant differences (P > 0.05), but the ALT level in the positive group was higher than that in the negative group (P < 0.0001). The positive group had worse Child-Pugh grades and higher HCC incidence compared to the negative group (P < 0.0001, P = 0.028). CONCLUSIONS: Patients with HBV-related LC and HBV-DNA ≥ 20 IU/mL have higher serum ALT levels, worse liver function Child-Pugh grades, and higher HCC incidence than those with HBV-DNA < 20 IU/mL. High-sensitivity HBV-DNA quantification can reflect the deterioration of liver function in patients with HBV-related LC to some extent.
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DNA quantification is a crucial step in the STR typing workflow for human identification purposes. Given the reaction's nature, qPCR assays may be subjected to the same stochastic effects of traditional PCR for low-input concentrations. The study aims to evaluate the precision of the PowerQuant® (Promega) kit assay measurements and the degree of variability for DNA templates falling below the optimal threshold of the PowerPlex® ESX-17 Fast STR typing kit (Promega). Five three-fold dilutions of the 2800 M control DNA (Promega) were set up. Each dilution (concentrations: 0.05, 0.0167, 0.0055, 0.00185, and 0.000617 ng/µL) was quantified and amplified in four replicates. Variability for qPCR results, STR profile completeness, and EPGs' peak height were evaluated. The qPCR-estimated concentration of casework samples was correlated with profile completeness and peak intensity, to assess the predictive value of qPCR results for the successful STR typing of scarce samples. qPCR was subjected to stochastic effects, of which the degree was inversely proportional to the initial input template. Quantitation results and the STR profile's characteristics were strongly correlated. Due to the intrinsic nature of real casework samples, a qPCR-derived DNA concentration threshold for correctly identifying probative STR profiles may be difficult to establish. Quantitation data may be useful in interpreting and corroborating STR typing results and for clearly illustrating them to the stakeholders.
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Repetições de Microssatélites , Reação em Cadeia da Polimerase em Tempo Real , Humanos , Repetições de Microssatélites/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Impressões Digitais de DNA/métodos , Genética Forense/métodos , DNA/genéticaRESUMO
The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process, ensuring maximum efficiency and avoiding repeated analyses, over-amplified samples, or unnecessary examinations. In our laboratory, we use the Quantifiler® Trio system to quantify DNA extracts from a wide range of samples extracted from traces (bloodstains, saliva, semen, tissues, etc.), including swabs from touched objects, which are very numerous in the forensic context. This method has been extensively used continuously for nine years, following an initial validation process, and is part of the ISO/IEC 17025 accredited method. In routine practice, based on the quantitative values determined from the extracts of each trace, we use a standard method or a low-copy-number method that involves repeating the amplification with the generation of a consensus genetic profile. Nowadays, when the quantification results are less than 0.003 ng/µL in the minimum extraction volume (40 µL), we do not proceed with the DNA extract analysis. By verifying the limits of the method, we make a conscious cost-benefit choice, in particular by using the least amount of DNA needed to obtain sufficiently robust genetic profiles appropriate for submission to the Italian DNA Forensic Database. In this work, we present a critical re-evaluation of this phase of the method, which is based on the use of standard curves obtained from the average values of the control DNA analysed in duplicate. Considering the various contributions to uncertainty that are difficult to measure, such as manual pipetting or analytical phases carried out by different operators, we have decided to thoroughly investigate the contribution of variability in the preparation of calibration curves to the final results. Thus, 757 samples from 20 independent experiments were re-evaluated using two different standards for the construction of curves, determining the quantitative differences between the two methods. The experiments also determined the parameters of the slope, Y-intercept, R2, and the values of the synthetic control probe to verify how these parameters can provide information on the final outcome of each analysis. The outcome of this revalidation demonstrated that it is preferable to use quantification ranges rather than exact quantitative limits before deciding how to analyse the extracts via PCR or forgoing the determination of profiles. Additionally, we present some preliminary data related to the analysis of samples that would not have been analysed based on the initial validation, from which genetic profiles were obtained after applying a concentration method to the extracts. Our goal is to improve the accredited analytical method, with a careful risk assessment as indicated by accreditation standards, ensuring that no source of evidence is lost in the reconstruction of a criminal event.
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DNA , Genética Forense , Reação em Cadeia da Polimerase em Tempo Real , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Genética Forense/métodos , Genética Forense/normas , DNA/análise , DNA/genética , Impressões Digitais de DNA/métodos , Repetições de Microssatélites , Sêmen/químicaRESUMO
Environmental DNA (eDNA) workflows contain many familiar molecular-lab techniques, but also employ several unique methodologies. When working with eDNA, it is essential to avoid contamination from the point of collection through preservation and select a meaningful negative control. As eDNA can be obtained from a variety of samples and habitats (e.g., soil, water, air, or tissue), protocols will vary depending on usage. Samples may require additional steps to dilute, block, or remove inhibitors or physically break up samples or filters. Thereafter, standard DNA isolation techniques (kit-based or phenol:chloroform:isoamyl [PCI]) are employed. Once DNA is extracted, it is typically quantified using a fluorometer. Yields vary greatly, but are important to know prior to amplification of the gene(s) of interest. Long-term storage of both the sampled material and the extracted DNA is encouraged, as it provides a backup for spilled/contaminated samples, lost data, reanalysis, and future studies using newer technology. Storage in a freezer is often ideal; however, some storage buffers (e.g., Longmires) require that filters or swabs are kept at room temperature to prevent precipitation of buffer-related solutes. These baseline methods for eDNA isolation, validation, and preservation are detailed in this protocol chapter. In addition, we outline a cost-effective, homebrew extraction protocol optimized to extract eDNA.
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DNA Ambiental , DNA Ambiental/isolamento & purificação , DNA Ambiental/análise , DNA Ambiental/genética , Preservação Biológica/métodos , Manejo de Espécimes/métodosRESUMO
Pairing droplet microfluidics and CRISPR/Cas12a techniques creates a powerful solution for the detection and quantification of nucleic acids at the single-molecule level, due to its specificity, sensitivity, and simplicity. However, traditional water-in-oil (W/O) single emulsion (SE) droplets often present stability issues, affecting the accuracy and reproducibility of assay results. As an alternative, water-in-oil-in-water (W/O/W) double emulsion (DE) droplets offer superior stability and uniformity for droplet digital assays. Moreover, unlike SE droplets, DE droplets are compatible with commercially available flow cytometry instruments for high-throughput analysis. Despite these advantages, no study has demonstrated the use of DE droplets for CRISPR-based nucleic acid detection. In our study, we conducted a comparative analysis to assess the performance of SE and DE droplets in quantitative detection of human papillomavirus type 18 (HPV18) DNA based on CRISPR/Cas12a. We evaluated the stability of SEs and DEs by examining size variation, merging extent, and content interaction before and after incubation at different temperatures and time points. By integrating DE droplets with flow cytometry, we achieved high-throughput and high-accuracy CRISPR/Cas12a-based quantification of target HPV18 DNA. The DE platform, when paired with CRISPR/Cas12a and flow cytometry techniques, emerges as a reliable tool for absolute quantification of nucleic acid biomarkers.
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Técnicas Biossensoriais , Sistemas CRISPR-Cas , Emulsões , Emulsões/química , Humanos , Técnicas Biossensoriais/métodos , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/isolamento & purificação , Citometria de Fluxo , DNA Viral/análise , DNA Viral/genética , Ácidos Nucleicos/química , Ácidos Nucleicos/análiseRESUMO
Decellularized human-adipose tissue (hDAT) can serve as an alternative to two-dimensional monolayer culture and current ECM hydrogels due to its unlimited availability and cytocompatibility. A major hurdle in the clinical translation and integration of hDAT and other hydrogels into current in vitro culture processes is adherence to current good manufacturing practices (cGMP). Transferring of innovative technologies, including hydrogels, requires the establishing standardized protocols for quality assurance and quality control (QA/QC) of the material.Integration of basic characterization techniques, including physiochemical characterization, structural/morphological characterization, thermal and mechanical characterization, and biological characterization, in addition to the reduction of batch-to-batch variability and establishment of proper sterilization, storage, and fabrication processes verifies the integrity of the hydrogel. Obatala Sciences has established a characterization protocol that involves a series of assays including the evaluation of gelation properties, protein content, glycosaminoglycan content, soluble collagen content, and DNA content of hDAT.
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Matriz Extracelular , Hidrogéis , Humanos , Hidrogéis/química , Matriz Extracelular/metabolismo , Colágeno/metabolismo , Glicosaminoglicanos/metabolismo , Controle de Qualidade , Engenharia Tecidual/métodosRESUMO
Species specificity of commercial human DNA quantification kits and short tandem repeat (STR) profiling kits was examined using primate DNA samples. These samples comprised 33 individuals from eight primate species, each with gender and kinship data, including human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), and orangutan (Pongo pygmaeus) of Hominidae family, and Japanese macaque (Macaca fuscata), long-tailed macaque (Macaca fascicularis), hamadryas baboon (Papio hamadryas), and savannah monkey (Chlorocebus sp.) of Cercopithecidae family. The findings revealed varying levels of cross-species amplifications in all non-human DNA samples that correlated with their evolutionary proximity to humans, both kit types. Moreover, cross-species amplification, including female DNA samples, was observed in a Y-chromosomal STR profiling kit. Additionally, species specificity differed among the commercial kits examined. The cross-species amplification data presented in this study offer valuable assistance in interpreting the results of individual human identification in forensic cases involving non-human primates.
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DNA , Repetições de Microssatélites , Especificidade da Espécie , Animais , Humanos , Repetições de Microssatélites/genética , DNA/genética , DNA/análise , Feminino , Masculino , Impressões Digitais de DNA/métodos , Primatas/genética , Reação em Cadeia da Polimerase/métodos , Genética Forense/métodosRESUMO
DNA quantification prior to STR amplification is a crucial step in forensic casework. Obtaining good-quality genetic STR profiles depends mainly on the amount and integrity of the DNA input in the PCR. In addition, the detection of male trace DNA provides key information for forensic investigation. AIM: To evaluate the correlation between the quantification results obtained with the previously developed Amel-Y system, and its ability to detect Y-chromosome DNA by HRM, with the resulting STR profiles, and to ultimately show that Amel-Y can be routinely used in forensic casework to improve STR and Y-STR results. MATERIAL & METHODS: Biological samples derived from forensic casework (85 reference and 391 evidence samples) were quantified by the Amel-Y system (a duplex qPCR/HRM based on SYTO™ 9 chemistry) using Rotor-Gene 6000. STRs were amplified and analyzed with GeneAmp™ PCR System 9700 or Veriti™ Thermal Cyclers and ABI 3500 Genetic Analyzer, respectively. RESULTS: After DNA normalization, a total of 386 STR profiles were obtained (305 full and 81 partial). Sex typing by HRM was 100% successful in reference samples. Male DNA was detected by HRM in 210 evidence samples. 80/201 were mixed with an excess of female DNA. In addition, Amel-Y was able to detect Y-chromosome DNA in mixed samples that did not amplify the Y-variant of Amelogenin marker with commercial STR kits. The reproducibility and precision of the Amel-Y system were demonstrated (CVCt% ≤ 9.55) within the dynamic range analyzed (0.016-50 ng/µL; 41 independent runs). Amel-Y also proved to be compatible with other real-time PCR platforms. CONCLUSION: We demonstrated that Amel-Y is a robust quantification system that can be routinely used in forensic casework to obtain reliable autosomal STR profiles and can be suitable as a predictor for Y-STR typing success when male DNA is detected. HRM can be used as a rapid screening tool for male DNA detection in mixed samples. Alternative designs like Amel-Y offer independence from commercial quantification kits in forensic labs.
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Impressões Digitais de DNA , Repetições de Microssatélites , Masculino , Humanos , Feminino , Impressões Digitais de DNA/métodos , Reprodutibilidade dos Testes , DNA/análise , Cromossomos Humanos YRESUMO
We used droplet digital PCR (ddPCR) assays to detect/quantify DNA from Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus spp. in blood samples. Bacterial DNA from clinical strains (4 < n < 12) was extracted, quantified and diluted (10-0.0001 ng/µL) and ddPCR assays were performed in triplicate. These ddPCR assays showed low replication variability, low detection limit (1-0.1 pg/µL), and genus/species specificity. ddPCR assays were also used to quantify bacterial DNA obtained from spiked blood (1 × 104-1 CFU/mL) of each bacterial genus/species. Comparison between ddPCR assays and bacterial culture was performed by Pearson correlation. There was an almost perfect correlation (r ≥ 0.997, P ≤ 0.001) between the number of CFU/mL from bacterial culture and the number of gene copies/mL detected by ddPCR. The time from sample preparation to results was determined to be 3.5 to 4 hours. The results demonstrated the quantification capacity and specificity of the ddPCR assays to detect/quantify 4 of the most important bloodstream infection (BSI) bacterial pathogens directly from blood. SIGNIFICANCE AND IMPACT: This pilot study results support the potential of ddPCR for the diagnosis and/or severity stratification of BSI. Applied to patients' blood samples it can improve diagnosis and diminish sample-to-results time, improving patient care.
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Escherichia coli , Sepse , Humanos , DNA Bacteriano/genética , Projetos Piloto , Reação em Cadeia da Polimerase/métodos , Escherichia coli/genética , Staphylococcus aureus/genéticaRESUMO
This research evaluates the current DNA quantification (Quantifiler™ Trio, PowerQuant®, Investigator® Quantiplex® Pro and InnoQuant® HY Fast) and autosomal STRs amplification kits (GlobalFiler™, PowerPlex® Fusion 6 C, Investigator® 24Plex QS) using 62 degraded skeletal remains from armed conflicts (petrous bone, femur, tibia, and tooth) with several parameters (autosomal small, large, and male target, degradation index, probability of degradation, number of alleles above analytical threshold, number of alleles above stochastic threshold, RFU, peak height ratio, number of reportable loci). The best qPCR/autosomal STRs amplification tandem was determined by comparing quantification results by a DNA quantity estimation based on sample average RFU. InnoQuant® HY Fast was the most sensitive kit, and no significative differences were observed among amplification kits; however, Investigator® 24 Plex QS was found to be the most sensitive in our samples. That is why InnoQuant™ and Investigator® 24Plex QS were determined to be the best tandem.
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Impressões Digitais de DNA , Dente , Masculino , Humanos , Impressões Digitais de DNA/métodos , Restos Mortais , Repetições de Microssatélites , DNA/análise , Dente/químicaRESUMO
The accurate quantification of DNA in forensic samples is of utmost importance. These samples are often present in limited amounts; therefore, it is indicated to use the appropriate analysis route with the optimum DNA amount (when possible). Also, DNA quantification can inform about the degradation stage and therefore support the decision on which downstream genotyping method to use. Consequently, DNA quantification aids in getting the best possible results from a forensic sample, considering both its DNA quantity and quality limitations. Here, we introduce NuMY, a new quantitative real-time PCR (qPCR) method for the parallel quantification of human nuclear (n) and mitochondrial (mt) DNA, assessing the male portion in mixtures of both sexes and testing for possible PCR inhibition. NuMY is based on previous work and follows the MIQE guidelines whenever applicable. Although quantification of nuclear (n)DNA by simultaneously analyzing autosomal and male-specific targets is available in commercial qPCR kits, tools that include the quantification of mtDNA are sparse. The quantification of mtDNA has proven relevant for samples with low nDNA content when conventional DNA fingerprinting techniques cannot be followed. Furthermore, the development and use of new massively parallel sequencing assays that combine multiple marker types, i.e., autosomal, Y-chromosomal, and mtDNA, can be optimized when precisely knowing the amount of each DNA component present in the input sample. For high-quality DNA extracts, NuMY provided nDNA results comparable to those of another quantification technique and has also proven to be a reliable tool for challenging, forensically relevant samples such as mixtures, inhibited, and naturally degraded samples.
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DNA Mitocondrial , Mitocôndrias , Feminino , Humanos , Masculino , DNA Mitocondrial/genética , Cromossomos Humanos Y/genética , Bioensaio , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Quantitative gel electrophoresis, also referred to as yield gel via gel electrophoresis, is an early quantification method that was developed to provide an estimate of the quality and the quantity of DNA extracted from evidence or reference samples. To conduct quantitative gel electrophoresis, an agarose gel that is combined with a nucleic acid gel stain is prepared. The gel stain intercalates between double-stranded DNA and can be visualized using UV light. DNA extract samples, along with DNA standards (ranging from 250 to 5 ng), and a 1 KB ladder are combined with a 6X loading dye and loaded on the agarose gel. Voltage is applied to facilitate DNA migration through the gel from the negative to the positive electrode, separating DNA fragments by size. After electrophoresis is complete, the results are visualized using UV light, and an image is captured for analysis. High-quality and -quantity DNA should contain a compact band comparable to that of the high molecular weight standards and ladder, with some smearing down the sample well. If a DNA extract sample does not produce a compact band and presents with only a smear, this is an indication that DNA degradation has occurred. This chapter provides instructions on how to successfully prepare an agarose gel, load DNA extract samples and corresponding controls, appropriately set up and run quantitative gel electrophoresis, interpret the results, and ensure comprehension of the method so troubleshooting can be performed if needed.
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Corantes , DNA , Eletroforese em Gel de Ágar/métodos , Sefarose , Eletroforese , DNA/análise , Peso MolecularRESUMO
Metals can pose challenges while conducting forensic DNA analysis. The presence of metal ions in evidence-related DNA extracts can degrade DNA or inhibit PCR as applied to DNA quantification (real-time PCR or qPCR) and/or STR amplification, leading to low success in STR profiling. Different metal ions were spiked into 0.2 and 0.5 ng of human genomic DNA in an "inhibition study" and the impact was evaluated by qPCR using the Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) and an in-house SYBR Green assay. This study reports on a contradictory finding specific to tin (Sn) ions, which caused at least a 38,000-fold overestimation of DNA concentration when utilizing Quantifiler Trio. This was explained by the raw and multicomponent spectral plots, which indicated that Sn suppresses the Quantifiler Trio passive reference dye (Mustang Purple™, MP) at ion concentrations above 0.1 mM. This effect was not observed when DNA was quantified using SYBR Green with ROX™ as the passive reference, nor when DNA was extracted and purified prior to Quantifiler Trio. The results show that metal contaminants can interfere with qPCR-based DNA quantification in unexpected ways and may be assay dependent. The results also highlight the importance of qPCR as a quality check to determine steps for sample cleanup prior to STR amplification that may be similarly impacted by metal ions. Forensic workflows should recognize the risk of inaccurate DNA quantification of samples that are collected from substrates containing tin.
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Impressões Digitais de DNA , Estanho , Humanos , Impressões Digitais de DNA/métodos , Repetições de Microssatélites , Reação em Cadeia da Polimerase em Tempo Real , DNA/análise , MetaisRESUMO
Trace DNA is a significant type of evidence for its ability to be collected from touched items or surfaces at crime scenes to link suspects to their crimes. In cases of violent crimes like assault, sexual offences, or even homicide, often touch DNA is collected from the victim's skin. However, the collection of touch DNA from the victim's skin can be complex because of the mixture of DNA present, as there is likely to be a small quantity of the offender's DNA compared to the victim's DNA. Validating different collection methods or techniques can improve touch DNA sampling; therefore, this study investigated three collection techniques involving cotton and nylon swabs to test their efficiency for the collection of touch DNA from the human neck. There was a significant difference between the three recovery techniques used to recover touch DNA with a cotton swab (CS) (p < 0.05) and nylon swab (NS) (p < 0.05), with more alleles observed when the neck skin was moistened with 100 µL of distilled water using a spray bottle before collection with both swabs.
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Nylons , Tato , Humanos , Impressões Digitais de DNA/métodos , Homicídio , DNA , Manejo de Espécimes , AsfixiaRESUMO
Submerged items are often thought to lack evidentiary value. However, previous studies have shown the ability to recover DNA from submerged porous items for upwards of six weeks. The crevices or interweaving fibers in porous items are thought to protect DNA from being washed away. It is hypothesized that, because non-porous surfaces do not have the same traits that might aid in DNA retention, then DNA quantities and the number of donor alleles recovered would decrease over longer submersion periods. Additionally, it is hypothesized that DNA quantity and the number of alleles would be negatively affected by flow conditions. Neat saliva of known DNA quantity was applied to glass slides and exposed to stagnant and flowing spring water to observe the effects on both DNA quantity and STR detection. Results supported that DNA deposited onto glass and subsequently submerged in water experienced a decrease in DNA quantity over time, yet submersion did not have as strong of a negative effect on the detected amplification product. Additionally, an increase in DNA quantity and detected amplification product from designated blank slides (no initial DNA added) could indicate the possibility of DNA transfer.
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Impressões Digitais de DNA , Água , DNA/genética , Alelos , SalivaRESUMO
The persistence of touch DNA deposited after realistic handling of items typically encountered in forensic investigations has been the subject of few studies. Understanding the long-term persistence of touch DNA on different substrates in varying conditions can be central to the effective triage of samples for further processing. As the time between an alleged incident and collection of evidence may vary from a few days to years after an alleged event, this study assessed three different common substrates for the persistence of touch DNA over a time span up to 9 months. These substrates included fabric, steel, and rubber, each of which were handled in a way to imitate what may happen during a criminal act. The three substrates were exposed to two different environments for up to 9 months: inside a dark cupboard with no traffic to act as a control and an outside semi-exposed environment. Ten replicates from each of the 3 substrates were tested at 5 time points to create 300 samples. All samples were processed using a standard operating workflow to provide genotype data after exposure to different environments. It was found that the fabric samples produced informative STR profiles (defined here as 12 or more alleles) up to the 9 month timepoint for either environment. The rubber and steel substrates for the inside condition produced informative STR profiles up to the 9 month timepoint, but only generated informative STR profiles for the outside condition up to 3 and 6 months, respectively. These data add to our understanding of the external factors that affect DNA persistence.
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Impressões Digitais de DNA , Tato , Borracha , Repetições de Microssatélites , DNA , AçoRESUMO
A ring trial among five European laboratories was organized to reach consistency in microsatellite (MS) typing of the zoonotic parasite Toxoplasma gondii. Three sample sets were circulated and analyzed by each laboratory following a previously published method that is based on fragment length polymorphism of 15 MS markers. The first sample set compared typing results in general and focused on effects of DNA concentration; the second sample set focused on the polymorphic fingerprinting markers that can differentiate T. gondii strains within the same archetypal lineage; and the third set focused on non-archetypal genotypes. Methodological variations between laboratories, including the software programs used to determine MS fragment length, were collated using a questionnaire. Overall, lineage-level typing results reached a high level of agreement, especially in samples with the highest DNA concentrations. However, laboratory-specific differences were observed for particular markers. Major median differences in fragment length, of up to 6 base pairs, were related to the fluorophore used to label fragment-specific primers. In addition, primer pairs with identical sequences obtained from different suppliers resulted in fragments of differing length. Furthermore, differences in the way the sequencing profiles were assessed and interpreted may have led to deviating results in fragment length determination. Harmonization of MS typing, for example, by using the same fluorophores or by numerical adjustments applied to the fragment-lengths determined, could improve the uniformity of the results across laboratories. This is the first interlaboratory comparison, providing guidelines (added as a supplement) for the optimization of this technique.
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Toxoplasma , Toxoplasmose Animal , Humanos , Animais , Toxoplasma/genética , Variação Genética , Polimorfismo de Fragmento de Restrição , DNA de Protozoário/genética , Repetições de Microssatélites , GenótipoRESUMO
Recognition and repair of DNA lesions are critical for cell survival. Herein, we highlight recent advances in the sequencing, repair mechanisms, and biological consequences of DNA lesions presented at the 2022 Fall American Chemical Society meeting.
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Dano ao DNA , Reparo do DNA , DNARESUMO
The magnitude of variations in the level of circulating mitochondrial (cir-mtDNA) and nuclear DNA (cir-ncDNA) in different diseases has indicated the need for investigating a discriminative approach for evaluating their diagnostic significance. This study reports a typical in-house process for extracting both types of cir-DNAs from a single plasma sample and assessed their usefulness in discriminating type 2 diabetes mellitus patients from healthy individuals to eliminate the prevailing dispute about their discriminative role and improve their diagnostic value. This approach offers a more precise and valuable tool for distinguishing the impact of cir-mtDNA from cir-ncDNA in diagnostic implications.
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Diabetes Mellitus Tipo 2 , Humanos , Diabetes Mellitus Tipo 2/diagnóstico , Patologia Molecular , Mitocôndrias/genética , DNA Mitocondrial/genéticaRESUMO
The rapid development of biologics and vaccines in response to the current pandemic has highlighted the need for robust platform assays to characterize diverse biopharmaceuticals. A critical aspect of biopharmaceutical development is achieving a highly pure product, especially with respect to residual host cell material. Specifically, two important host cell impurities of focus within biopharmaceuticals are residual DNA and protein. In this work, a novel high-throughput host cell DNA quantitation assay was developed for rapid screening of complex vaccine drug substance samples. The developed assay utilizes the commercially available, fluorescent-sensitive Picogreen dye within a 96-well plate configuration to allow for a cost effective and rapid analysis. The assay was applied to in-process biopharmaceutical samples with known interferences to the dye, including RNA and protein. An enzymatic digestion pre-treatment was found to overcome these interferences and thus allow this method to be applied to wide-ranging, diverse analyses. In addition, the use of deoxycholate in the digestion treatment allowed for disruption of interactions in a given sample matrix in order to more accurately and selectively quantitate DNA. Critical analytical figures of merit for assay performance, such as precision and spike recovery, were evaluated and successfully demonstrated. This new analytical method can thus be successfully applied to both upstream and downstream process analysis for biologics and vaccines using an innovative and automated high-throughput approach.