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To date, studies on microbial forensics have focused mainly on sequence analysis and generally do not include information on the quantification of and comparison between the human and bacterial DNA present in forensic samples. Knowing the amount of each type of DNA can be important for determining when and how best to employ bacterial DNA analysis, especially when there is insufficient human DNA for successful short tandem repeat (STR) typing. The goal of this work was to develop a quantitative PCR (qPCR) assay that simultaneously quantifies human and bacterial DNA that would be simple and cost-effective for laboratories to implement. Through a reproducibility study and several small-scale experiments, the reliability of a custom qPCR assay was established. A reproducibility study illustrated that the multiplex assay produced data comparable to that of previously established bacterial DNA and human DNA qPCR assays. The small-scale experiments showed that common surfaces such as keyboards (6.76 pg/μL), elevator buttons (11.9 pg/μL), cleaning supplies (7.17 pg/μL), and dispensers (16.4 pg/μL) failed to produce human DNA quantities sufficient for quality STR analysis (≥250 pg). However, all tested surfaces produced bacterial DNA quantities suitable for reaching 1 ng of amplified bacterial targets necessary for sequence analysis. In fact, bacterial DNA concentrations down to 10−8 ng/uL produce enough amplified product for sequencing. The newly developed qPCR multiplex tool will allow scientists to make better decisions regarding whether human or bacterial DNA analysis methods can be pursued during forensic or other investigations. [ABSTRACT FROM AUTHOR]

Quantitative PCR is a molecular technique for DNA quantification that depends on reaction efficiency and the Ct value ("cycle threshold"). However, the results are dependent on laboratory conditions and mathematical approaches. Thus, the data of 16 genes from Pseudomonas aeruginosa strain AG1 were generated using qPCR to assess the effect of DNA concentration and three mathematical methods (a standard curve and two individual-curve-based approaches called exponential and sigmoidal models) on efficiency and DNA quantification. Differences in efficiency were revealed depending on the mathematical method used; the values were 100% in three out of the four standard curves, but estimations of the expected fold change in DNA serial dilutions were not achieved, indicating the possible overestimation of efficiency. Moreover, when efficiency was compared to DNA concentration, a decreasing trend in efficiency as DNA concentration increased in the reaction was observed in most cases, which is probably related to PCR inhibitors. For all 16 genes at a single DNA concentration, the efficiencies for the exponential model were found in the range of 1.5–2.79 (50–79%), and for the sigmoidal approach, the range was 1.52–1.75 (52–75%), with similar impact on normalized expression values, as indicated by the genes for standard curves. Jointly, DNA concentration and mathematical model choice were demonstrated to impact the estimation of reaction efficiency and, subsequently, DNA quantification when using qPCR. [ABSTRACT FROM AUTHOR]

Quantitative PCR is a molecular technique for DNA quantification that depends on reaction efficiency and the Ct value (“cycle threshold”). However, the results are dependent on laboratory conditions and mathematical approaches. Thus, the data of 16 genes from Pseudomonas aeruginosa strain AG1 were generated using qPCR to assess the effect of DNA concentration and three mathematical methods (a standard curve and two individual-curve-based approaches called exponential and sigmoidal models) on efficiency and DNA quantification. Differences in efficiency were revealed depending on the mathematical method used; the values were 100% in three out of the four standard curves, but estimations of the expected fold change in DNA serial dilutions were not achieved, indicating the possible overestimation of efficiency. Moreover, when efficiency was compared to DNA concentration, a decreasing trend in efficiency as DNA concentration increased in the reaction was observed in most cases, which is probably related to PCR inhibitors. For all 16 genes at a single DNA concentration, the efficiencies for the exponential model were found in the range of 1.5–2.79 (50–79%), and for the sigmoidal approach, the range was 1.52–1.75 (52–75%), with similar impact on normalized expression values, as indicated by the genes for standard curves. Jointly, DNA concentration and mathematical model choice were demonstrated to impact the estimation of reaction efficiency and, subsequently, DNA quantification when using qPCR.

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. [ABSTRACT FROM AUTHOR]

In this study, 241 mixed autosomal STR genetic profiles (swabs − 172, slides − 69) from processed casework samples were analysed. From these samples, DNA was extracted and quantified by qPCR; the results were analysed with the PowerQuant Analysis Software. The extracted DNA samples were amplified with the Investigator 24Plex QS and VeriFiler kits and separated and detected in an ABI 3500 Genetic Analyzer. A quantification correlation was observed between the total autosomal DNA targets [Auto] and the Y chromosome target [Y] in mixed samples in which the success of amplification of the male autosomal STRs markers was associated with high values of the quantification of the target of [Y] and low DNA ratio values [Auto]/[Y] (p = 0.00 < 0.05). Samples with [Auto]/[Y] ratio values much higher than the estimated value required analysis with Y-STR markers. The study herein, which assessed a relatively large number of sexual assault samples, demonstrates that quantitation ratio values of DNA mixtures effectively contribute to the decision-making process on whether and what genetic marker system should be selected for analysis of a male component in unknown casework samples. [ABSTRACT FROM AUTHOR]

The adulteration and mislabeling of food ingredients, particularly meat products, pose a significant concern for consumers. In order to address this issue, it is crucial to use precise and sensitive methods for species identification, mainly when dealing with samples that contain a limited number of copies of DNA. The primary objective of this study is to design a digital nanoplate polymerase chain reaction (PCR) technique for the purpose of quantifying species identification. The primer used in this study was cytochrome b gene. The results showed that target DNA can be amplified using the digital PCR nanoplate until the lowest sample concentration of 0.0013 ng/µL. This study can be applied to enhance species identification processes in food samples by utilizing digital PCR nanoplates. [ABSTRACT FROM AUTHOR]

The real-time quantitative PCR (qPCR) calibration curves are highly reproducible and allow the generation of specific, sensitive, and reproducible data that can be used for gene quantification. However, it is important to rigorously validate the external calibration curve model in qPCR since absolute quantification is dependent on the standards used. We present a method for standardising qPCR-based quantification of the β-parvalbumin (β-pvalb) gene of Lophius piscatorius, a major fish allergen, using a plasmid DNA (pDNA) calibrator. In parallel experiments, standard curves were generated and compared from the genomic DNA (gDNA) isolated from L. piscatorius and pDNA carrying the target, pvalb. The commutability of pDNA and gDNA calibrators for the quantification of β-pvalb was assessed by employing a TaqMan qPCR, targeting the second intron of the pvalb gene of L. piscatorius. Higher PCR efficiencies, good linearity, and lower standard deviation (S.D.) values were observed with pDNA instead of gDNA calibrants. pDNA calibrants exhibited a lower bias in terms of closeness to the expected value of unknown samples than their genomic counterparts. The assay was specific and sensitive, where the limit of detection (LOD) and limit of quantification (LOQ) were five copies and ten copies per reaction. The short-term stability study of the pDNA calibrants indicated its stability for 60 days at − 20 °C and 30 days at 4 °C. The efficient results indicated a plasmid calibrator as a potential tool for absolute quantification of the pvalb gene and an alternative to conventional gDNA standards. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Accurate DNA quantification is a highly important method within molecular biology. Methods widely used to quantify DNA are UV spectrometry and fluorometry. In this research, seven different DNA samples and one blank (MilliQ ultrapure water) were quantified by three analysts using one spectrophotometric (i.e., a NanoDrop instrument) and three fluorometric (i.e., the AccuGreen High Sensitivity kit, the AccuClear Ultra High Sensitivity kit, and the Qubit dsDNA HS Assay kit) methods. An analysis of variance (ANOVA) scheme was used to determine the influence of the analyst, the method, and the combination of analyst and method, on DNA quantification. For most samples, the measured DNA concentration was close to or slightly above the concentration of 10 ng/μL as specified by the supplier. Results obtained by the three analysts were equal. However, it was found that, compared to the fluorometric kits, the used spectrophotometric instrument in the case of fish DNA samples tends to overestimate the DNA concentration. Therefore, if sufficient sample volume is available, a combination of a spectrophotometric and a fluorometric method is recommended for obtaining data on the purity and the dsDNA concentration of a sample.

For microscopic investigation, archaeological bone samples are often embedded in Biodur® epoxy resin. This study wants to test whether it is possible to extract DNA suitable for PCR amplification from this sample type. For eight individuals a set of samples – each consisting of a Biodur-embedded femur sample, a native femur sample and a control sample of different anatomical origin – were submitted to organic DNA extraction. The extraction success was tested by autosomal short tandem repeat amplification. Seven out of eight Biodur-embedded femur samples revealed successful amplification results. If Biodur-embedded bone material exists from earlier microscopic investigations, our results encourage the use of this sample type as a source for genetic research.

Dutch elm disease (DED) is a destructive tracheomycosis caused by Ophiostoma novo-ulmi, an ascomycete probably originating in East-Asia that is devastating natural elm populations throughout Europe, North America and Asia. The fungus is mainly spread by elm bark beetles that complete their life cycle between healthy and diseased elms. Recently, it has been highlighted that some fungi of the genus Geosmithia, which are similarly well associated with bark beetles, seem to also play a role in the DED pathosystem acting as mycoparasites of O. novo-ulmi. Although some relationship between the fungi is clear, the biological cycle of Geosmithia spp. within the DED cycle is still partly unclear, as is the role of Geosmithia spp. in association with the bark beetles. In this work, we tried to clarify these aspects by developing a qPCR duplex TaqMan assay to detect and quantify DNA of both fungi. The assay is extremely sensitive showing a limit of detection as low as 2 fg µl-1 for both fungi. We collected woody samples from healthy and infected elm trees throughout the beetle life cycle. All healthy elm samples were negative for both Geosmithia spp. and O. novo-ulmi DNA. Geosmithia spp. are never present in infected, but living trees, while they are present in frass of elm bark beetles (EBB - Scolytus spp.) and at each stage of the EBB life cycle in much higher quantities than O. novo-ulmi. This work provides a better understanding of the role and interactions occurring amongst the main players of the DED pathosystem. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Our study reports a reliable, cost-effective, and highly sensitive approach for the simultaneous extraction of both cir-mtDNA and cir-ncDNA using a single aliquot of 300 µL plasma sample. • This approach may help to avoid sample volume issues and the use of costly commercial kits, and kits and decreases the time and efforts in downstream diagnostic processes. • The cir-DNAs extracted using described approach are suitable for downstream analysis and provide a more precise and valuable tool to discriminate the significance of cir-mtDNA from cir-ncDNA in disease diagnosis. 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. [ABSTRACT FROM AUTHOR]

There is an increasing interest in the genetic quantification of bovine DNA. Analysis of genetic diversity using DNA extracted from the blood is affected by the quality and the quantity of the DNA extracts, which are critical factors that limit the accuracy and sensitivity of molecular studies. The purpose of this paper is to show the quantity and purity of DNA isolated from a number of 24 blood samples from Pinzgau cattle, using two basic techniques, the automatic DNA extraction technique, using Maxwell™ 16 and 16 MDx instruments and the spectrophotometric quantification technique, using Nanodrop ASP-3700 spectrophotometer. The obtained results are considered satisfactory in terms of the purity of the obtained DNA extracts, 13 samples having the DNA isolate contaminated with proteins based on the absorbance ratio A260/A280 lower than 1.7, the solution in this situation being the repetition of the protein precipitation process. [ABSTRACT FROM AUTHOR]

Context: Circulating free DNA (cfDNA) analysis has emerged as novel noninvasive diagnostic biomarker in several solid tumors. Raised levels have been reported in several malignancies and may correlate with clinicopathological and treatment response. The current study was designed to assess the diagnostics of cfDNA in different tumor types of malignancies correlating with tumor (T), nodes (N), and metastases (M) stage. Design: Serum samples were collected from treatment naïve cases with histologically diagnosed tumors including 23 brain tumors, 48 breasts, 50 gallbladder carcinoma (GBC), 13 lungs, 68 oral squamous cell carcinoma (OSCC), and 25 normal controls. CfDNA was quantified with real-time polymerase chain reaction (PCR), Invasive ductal carcinoma (IDC) using beta-globin gene amplification. Cut off values for diagnostics were calculated using receiver operating curve analysis. Results: Contrary to other cfDNA studies where it was postulated that cfDNA would not cross the blood–brain barrier and reach the systemic circulation, we found detectable cfDNA in glioma with median (Q1–Q3) of 349.22 ng/ml (19.87–1276.58). Median cfDNA concentration in breast, gallbladder, lung, oral and normal controls was 328.72 (128.38–624.44), 778.50 (589.88–1864.35), 348.73 (194.67–483.61), 386.27 (47.88–959.67), and 74.12 (49.66–120.00), respectively. Grades I and II glioma had significantly lower levels compared to Grades III and IV (P = 0.0001). Significant difference in median cfDNA values in IDC and GBC was observed with increasing tumor grades, stage, T stage, nodal stage and metastasis and with stage of OSCC cases. Conclusion: CfDNA levels showed good diagnostic discrimination in glioma, GBC, breast, lung carcinoma, and OSCC. Significant increase in titers was evident with increase in cancer stage from I to IV in breast, GBC and OSCC.

Accurate DNA quantification is a highly important method within molecular biology. Methods widely used to quantify DNA are UV spectrometry and fluorometry. In this research, seven different DNA samples and one blank (MilliQ ultrapure water) were quantified by three analysts using one spectrophotometric (i.e., a NanoDrop instrument) and three fluorometric (i.e., the AccuGreen High Sensitivity kit, the AccuClear Ultra High Sensitivity kit, and the Qubit dsDNA HS Assay kit) methods. An analysis of variance (ANOVA) scheme was used to determine the influence of the analyst, the method, and the combination of analyst and method, on DNA quantification. For most samples, the measured DNA concentration was close to or slightly above the concentration of 10 ng/ μ L as specified by the supplier. Results obtained by the three analysts were equal. However, it was found that, compared to the fluorometric kits, the used spectrophotometric instrument in the case of fish DNA samples tends to overestimate the DNA concentration. Therefore, if sufficient sample volume is available, a combination of a spectrophotometric and a fluorometric method is recommended for obtaining data on the purity and the dsDNA concentration of a sample. [ABSTRACT FROM AUTHOR]

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. • Double emulsion (DE) droplets show enhanced stability in CRISPR/Cas12a-based DNA detection than single emulsion droplets. • The remarkable stability of DE droplets prevents content mixing, reducing contamination risks during incubation. • DE droplets significantly minimize sample loss during encapsulation and incubation, outperforming SE droplets. • DE droplets, compatible with flow cytometry, enable precise and high-throughput CRISPR/Cas12a-based DNA quantification. [ABSTRACT FROM AUTHOR]

In this present study, the preparation of chitosan functionalized gold‑nickel wire nanomachines (nanomotors) (CS@Au-Ni NMs) for motion-based double-stranded deoxyribonucleic acid (dsDNA) recognition and detection was described. Synthesis of the nanomachines was accomplished by Ni layer formation using direct current (DC) magnetron sputtering over electrochemically deposited Au wires. Subsequently, biopolymer chitosan was dispersed onto this bimetallic layer by drop casting which could provide a novel and functional surface for leading bio-applications. CS@Au-Ni NMs were characterized via scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and zeta potential analysis methods for the elucidation of structural morphology, elemental composition and electrophoretic mobility. On account of presenting the application of these magnetic nanomachines, they were interacted with different concentrations of dsDNA and the changes in their velocities were investigated. The speed CS@Au-Ni NMs were measured as 19 μm/s under 22 mT magnetic field. These magnetically guided nanomachines demonstrated a practical and good sensing ability by recognizing dsDNA between 0.01 mg/L and 10 mg/L. Electrochemical characterization was also performed to identify the surface characteristics. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments presented the interaction of the NMs with dsDNA by indicating the convenient recognition. [Display omitted] • Chitosan functionalized gold‑nickel nanowires were facilely synthesized. • The CS@Au-Ni nanowires as nanomotors were successfully moved under magnetic field in a controllable manner. • Motion-based dsDNA recognition was achieved efficiently. • Chitosan functionalization improved the sensing property of the gold‑nickel nanomotors. • SEM, XPS and XRD characterization studies showed the uniform distribution and effective functionalization. [ABSTRACT FROM AUTHOR]

Context: Dental tissue remains are the toughest, and chemically, the most stable tissue in the body. Its high resilience in the events of fire and bacterial decomposition makes them vital for DNA analysis by PCR method. Aims: Determination of sex of children through molecular analysis of pulp tissue of exfoliated deciduous teeth stored in different media and analyzed after a different time period. Settings and Design: Sixty samples of deciduous teeth were divided into three groups. Group IA and Group IIA were stored in natural soil and wet clay for 1 month, respectively. Group IB and Group IIB were stored in natural soil and wet clay for 6 months, respectively. Group III was analyzed immediately after extraction. Methods and Material: Sex determination was carried out in five steps: Pulp tissue removal, DNA isolation, DNA quantification, PCR amplification, Sex determination. X and Y specific chromosomes from each sample were amplified and compared. Statistical Analysis Used: Kruskal-Wallis test, Dunn's test, and Wilcoxon signed rank test. Results: Group III revealed the highest amount of DNA quantified. Amount of DNA quantified after 6 months of storage in natural soil and wet clay decreased in both the groups with the samples stored in wet clay showing a maximum decrease. Results of the PCR analysis also showed 100% accuracy rate in the samples of Group III. Conclusions: Sex determination from pulp tissue depends a lot on the quality and quantity of DNA extracted. Sex could be effectively determined among the samples evaluated immediately after extraction. This ability decreases as the storage condition changes and the time period increases. Samples stored in wet clay were found to show the least sex identification ability than dry soil.

Nanopore sensing is a promising tool well suited to capture and detect DNA and other single molecules. DNA is a negatively charged biomolecule that can be captured and translocated through a constricted nanopore aperture under an applied electric field. Precise assessment of DNA concentration is of crucial importance in many analytical processes and medical diagnostic applications. Recently, we found that hydrodynamic forces can lead to DNA motion against the electrophoretic force (EPF) at low ionic strength. This study utilized glass nanopores to investigate the DNA capture mechanism and detect DNA molecules due to volumetric flow at these low ionic strength conditions. We measured the DNA capture rate at five different pico-molar concentrations. Our findings indicated that the translocation rate is proportional to the concentration of DNA molecules and requires no calibration due to the volumetric flow rate and DNA counting directly correlates with concentration. Using finite element analysis, we calculated the volumetric flow and proposed a simple, straightforward approach for accurate DNA quantification. Furthermore, these experiments explore a unique transport mechanism where one of the most highly charged molecules enters a pore against electric field forces. This quantitative technique has the potential to provide distinct insight into nanopore-based biosensing and further enhance the nanopore’s capability as a biomolecule concentration sensor.

In molecular genetics analysis, isolation and quantification of DNA represents a step extremely important. DNA isolation methods are based on purity, integrity and the amount of DNA obtained. The degree of DNA purity is one of the most important factors in the reproducibility of the PCR method. DNA is considered pure if the ratio of the two spectrophotometric readings, A260/A280, shows values between 1.7 - 2.0. Values lower than 1.7 indicate impurities with proteins, and a ratio higher than 2.0 indicates impurities with other contaminants. The aim of this research was to evaluate the effectiveness of the automatic method of DNA extraction from a number of 24 blood samples collected from Pinzgauer cattle breed, for the analysis of genetic diversity. The amounts of DNA extracted from the 24 blood samples ranged from 13-61.9 ng/µl and the absorbance ratio A260/A280 showed values between 1.61-2.13. The results obtained demonstrated the effectiveness of the automatic method of DNA extraction, and thus, isolated and quantified genetic material can be used further in the next stages of genomic analysis of this breed. [ABSTRACT FROM AUTHOR]

In their 2019 study, Molina et al. evaluate a cell proliferation assay that fluorescently quantifies DNA for assessing ballast water treatment efficacy of organisms = 10 to 50 µm. Because of concerns with the overall experimental design, procedures, and authors' interpretations, their conclusions do not appear to be justified and the assay used to arrive at those conclusions is not proven appropriate. Specific concerns we highlight include UV and chlorine dose calculations and exposure conditions, bacterial contamination issues, poor agreement between controls, high detection limit of the assay, and mismatch between conclusions in the abstract and statistical significance of the data. Their conclusions that "population maintenance or growth was evident ... after UV treatment" and "photoreactivation could have been attributed to increased mean DNA concentrations" are therefore not scientifically defensible without further experimentation and verification. These concerns call into serious question the use of this study by the US Coast Guard or other governing bodies to (1) determine the applicability of this cell proliferation assay for assessing ballast water disinfection or (2) make conclusions regarding the suitability of UV treatment for inactivating organisms in ballast water. [ABSTRACT FROM AUTHOR]

• Species specificity of commercial human DNA assay kits was examined. • Cross-species amplifications of non-human primate DNAs is observed. • Species specificity differed among the examined kits. • Cross-species data will aid human identification in forensic cases involving non-human primates. 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. [ABSTRACT FROM AUTHOR]

Establishing the age of a bloodstain provides forensic investigators with critical information on the time that a crime occurred. Our work presents a time since deposition (TSD) analysis that integrates visible absorbance spectroscopy and high-resolution automated electrophoresis data to quantify light absorbance and DNA degradation of whole blood over time. Passive bloodstains were created and treated on either FTA cards without anticoagulant or in microcentrifuge tubes with anticoagulant and tested over 11 different timepoints across 15 days at controlled temperature and relative humidity. A total of 41 variables were analyzed using linear regression, with six variables showing statistical independence and a relationship to time. A general negative trend was noticeable for the visible absorbance of α and β bands and concentration of high molecular weight DNA of the samples over time. We then conducted a principle component analysis (PCA) of all variables; the principal components integrated both DNA and absorbance data and generally improved the model fit (i.e. increased R2). Importantly, our PCA findings demonstrated that the experimental effect of treatment and donor was largely accounted for in PC1, with PC2 reflecting the true relationship of the integrated metrics to time. Our data set and models are publicly available in an effort to build upon this study by incorporating environmental variables. [ABSTRACT FROM AUTHOR]

Pooling individual samples prior to DNA extraction can mitigate the cost of DNA extraction and genotyping; however, these methods need to accurately generate equal representation of individuals within pools. The objective of this study was to determine accuracy of pool construction of blood samples based on white blood cell counts compared to two common DNA quantification methods. Fifty individual bovine blood samples were collected, and then pooled with all individuals represented in each pool. Pools were constructed with the target of equal representation of each individual animal based on number of white blood cells, spectrophotometric readings, spectrofluorometric readings, and whole blood volume with 9 pools per method and a total of 36 pools. Pools and individual samples that comprised the pools were genotyped using a commercially available genotyping array. ASReml was used to estimate variance components for individual animal contribution to pools. The correlation between animal contributions between two pools was estimated using bivariate analysis with starting values set to the result of a univariate analysis. Adonis test on distance matrix from the animal correlation showed clustering with method, and higher correlations between methods than within (P < 1 × 10–6). White blood cell count was predictive of sample representation when compared to pooling based on DNA concentration. Therefore, constructing pools using white blood cell counts prior to DNA extraction may reduce cost associated with DNA extraction and genotyping and improve representation of individuals in a pool.

Pooling individual samples prior to DNA extraction can mitigate the cost of DNA extraction and genotyping; however, these methods need to accurately generate equal representation of individuals within pools. The objective of this study was to determine accuracy of pool construction of blood samples based on white blood cell counts compared to two common DNA quantification methods. Fifty individual bovine blood samples were collected, and then pooled with all individuals represented in each pool. Pools were constructed with the target of equal representation of each individual animal based on number of white blood cells, spectrophotometric readings, spectrofluorometric readings, and whole blood volume with 9 pools per method and a total of 36 pools. Pools and individual samples that comprised the pools were genotyped using a commercially available genotyping array. ASReml was used to estimate variance components for individual animal contribution to pools. The correlation between animal contributions between two pools was estimated using bivariate analysis with starting values set to the result of a univariate analysis. Adonis test on distance matrix from the animal correlation showed clustering with method, and higher correlations between methods than within (P < 1 × 10–6). White blood cell count was predictive of sample representation when compared to pooling based on DNA concentration. Therefore, constructing pools using white blood cell counts prior to DNA extraction may reduce cost associated with DNA extraction and genotyping and improve representation of individuals in a pool. [ABSTRACT FROM AUTHOR]

Decellularization of animal tissues is a novel route to obtain biomaterials for use in tissue engineering and organ transplantation. Successful decellularization is required as animal DNA causes inflammatory reactions and contains endogenous retroviruses, which could be transmitted to the patient. One of the criteria for successful decellularization is digestion (fragmentation) and elimination (residual quantity) of DNA from the tissue. Quantification of DNA can be done in many ways, but it has recently been shown that silica‐based solid‐phase extraction methods often do not completely purify in particular small DNA fragments. In the context of decellularization, this means that the measured DNA amount is underestimated, which could compromise safety of the processed tissue for in‐patient use. In this article, we review DNA quantification methods used by researchers and assess their influence on the reported DNA contents after decellularization. We find that underestimation of residual DNA amount after silica‐based solid‐phase extraction may be as large as a factor of ten. We therefore recommend a direct assessment of DNA amount in tissue lysate using dsDNA‐specific binding dyes, such as Picogreen, due to their higher accuracy for small fragment detection as well as ease of use and widespread availability. [ABSTRACT FROM AUTHOR]

A wet‐vacuum‐based collection method with the M‐Vac® was compared to a wet‐swabbing collection method by examining the recovery of diluted blood on 22 substrates of varying porosity. The wet‐vacuum method yielded more total nuclear DNA than wet‐swabbing on 18 porous substrates, recovering on average 12 times more DNA. However, both methods yielded comparable amounts of total DNA on two porous and two nonporous substrates. In no instance did wet‐swabbing significantly recover more DNA. The wet‐vacuum method also successfully collected additional DNA on previously swabbed substrates. Mitochondrial DNA yields were assessed, and outcomes were generally similar to the nuclear DNA outcomes described above. Results demonstrate that wet‐vacuuming may serve as an alternative collection method to swabbing on difficult porous substrates and could potentially recover additional DNA on previously swabbed substrates. However, swabbing remains the preferred collection method on substrates with visible stains and/or nonporous surfaces for reasons of convenience, simplicity, and lower cost relative to the wet‐vacuum method. [ABSTRACT FROM AUTHOR]

A wet‐vacuum‐based collection method with the M‐Vac® was compared to a wet‐swabbing collection method by examining the recovery of diluted blood on 22 substrates of varying porosity. The wet‐vacuum method yielded more total nuclear DNA than wet‐swabbing on 18 porous substrates, recovering on average 12 times more DNA. However, both methods yielded comparable amounts of total DNA on two porous and two nonporous substrates. In no instance did wet‐swabbing significantly recover more DNA. The wet‐vacuum method also successfully collected additional DNA on previously swabbed substrates. Mitochondrial DNA yields were assessed, and outcomes were generally similar to the nuclear DNA outcomes described above. Results demonstrate that wet‐vacuuming may serve as an alternative collection method to swabbing on difficult porous substrates and could potentially recover additional DNA on previously swabbed substrates. However, swabbing remains the preferred collection method on substrates with visible stains and/or nonporous surfaces for reasons of convenience, simplicity, and lower cost relative to the wet‐vacuum method. [ABSTRACT FROM AUTHOR]

Background: Caprine skin is a promising biomaterial for tissue‐engineering applications. However, tissue processing is required before its xenogenic use. Aims: Therefore, the purpose of this study was to evaluate the structural integrity and biocompatibility of the caprine skin after de‐epithelialization, using sodium chloride (NaCl) and trypsin solutions, followed by de‐cellularization using sodium dodecyl sulfate (SDS) solution. Materials & Methods: The caprine skin was de‐epithelialized using NaCl (2‐4 mol/L) and trypsin (0.25%‐0.5%) followed by the treatment of SDS (1%‐4%) solution over a period of time. Acellularity of the prepared matrix was confirmed histologically and characterized by appropriate staining, scanning electron microscopy (SEM), DNA quantification, and Fourier‐transform infrared (FTIR) spectroscopy. The caprine acellular dermal matrix (CADM) was used for the repair of spontaneously occurring abdominal hernia in ten buffaloes. The biocompatibility of the CADM was evaluated using clinical, hematological, biochemical, and anti‐oxidant parameters. Results: Histologically, the skin treated with 0.25% trypsin in 4 mol/L NaCl for 8 hours resulted in complete de‐epithelialization. Further treatment with 2% SDS for 48 hours demonstrated complete acellularity and orderly arranged collagen fibers. The SEM confirmed a preservation of collagen arrangement within CADM. The DNA content was significantly (P <.05) lower in CADM (46.20 ± 7.94 ng/mg) as compared to fresh skin (662.56 ± 156.11 ng/mg) indicating effective acellularity. The FTIR spectra showed characteristic collagen peaks of amide A, amide B, amide I, amide II, and amide III in CADM. All the 10 animals recovered uneventfully and remained sound. Hematological, biochemical, and anti‐oxidants findings were unremarkable. Conclusion: Results indicated the acceptance and biocompatibility of the xenogenic caprine acellular dermal matrix for abdominal hernia repair in buffaloes without complications. [ABSTRACT FROM AUTHOR]

Sperm limitation is a concern for a number of heavily fished decapods; however, work to assess this concern is sometimes hampered by a lack of simple techniques to quantify sperm transferred during reproduction. Our primary goal was to determine if DNA measurements could be used to quantify the sperm content of spermatophores and thus facilitate investigations of sperm limitation in American lobsters (Homarus americanus H. Milne Edwards, 1837). This was achieved by measuring the amount of DNA in a sample and then calibrating those values by using flow cytometry to count the number of individual sperm present in the sample. Our results show that the DNA quantification technique provides a fast and accurate way to quantify sperm. We then demonstrated the utility of the method by using it to examine the rate at which males can produce sperm under simulated conditions of repeated mating events, a situation that might lead to a reduction in the number of sperm per spermatophore. While spermatophores obtained from male lobsters at three-day intervals varied substantially in the number of sperm they contained (range 427,090–5,028,996; mean 2,306,473), there was no clear decline in sperm count over time. These results suggest that male lobsters replenish their sperm supplies rapidly, and that sperm recharge rate is unlikely to be a factor that could lead to sperm limitation in American lobster populations. [ABSTRACT FROM AUTHOR]

Context: Dental tissue remains are the toughest, and chemically, the most stable tissue in the body. Its high resilience in the events of fire and bacterial decomposition makes them vital for DNA analysis by PCR method.Aims: Determination of sex of children through molecular analysis of pulp tissue of exfoliated deciduous teeth stored in different media and analyzed after a different time period.Settings and Design: Sixty samples of deciduous teeth were divided into three groups. Group IA and Group IIA were stored in natural soil and wet clay for 1 month, respectively. Group IB and Group IIB were stored in natural soil and wet clay for 6 months, respectively. Group III was analyzed immediately after extraction.Methods and Material: Sex determination was carried out in five steps: Pulp tissue removal, DNA isolation, DNA quantification, PCR amplification, Sex determination. X and Y specific chromosomes from each sample were amplified and compared.Statistical Analysis Used: Kruskal-Wallis test, Dunn's test, and Wilcoxon signed rank test.Results: Group III revealed the highest amount of DNA quantified. Amount of DNA quantified after 6 months of storage in natural soil and wet clay decreased in both the groups with the samples stored in wet clay showing a maximum decrease. Results of the PCR analysis also showed 100% accuracy rate in the samples of Group III.Conclusions: Sex determination from pulp tissue depends a lot on the quality and quantity of DNA extracted. Sex could be effectively determined among the samples evaluated immediately after extraction. This ability decreases as the storage condition changes and the time period increases. Samples stored in wet clay were found to show the least sex identification ability than dry soil. [ABSTRACT FROM AUTHOR]

DNA quantification is an essential step for successful multiplex short tandem repeat (STR) polymerase chain reactions (PCR), which are used for confirming identities using human genomic DNA. The new DNA quantification kit, named the National Forensic Service Quantification (NFSQ) kit, simultaneously provides total human DNA concentration, human male DNA concentration, and a DNA degradation index (DI) using multiplex TaqMan fluorescent probes. The NFSQ was validated according to developmental validation guidelines from the SWGDAM and MIQE. NFSQ detected up to 0.00128 ng/μL and could detect male DNA up to a 1:8000 ratio of male to female DNA. In PCR inhibitor tests, NFSQ could measure DNA at a concentration of 200 ng/μL of humic acid and 600 μM of hematin. The NFSQ kit showed a DI value trend similar to other qPCR kits. In the reproducibility study, the coefficient of variation of the NFSQ kit was within 10%. The quantitative results of the casework samples obtained using the NFSQ kit were consistent with the STR interpretation results. The NFSQ kit can be useful in the human identification process, as it has detection capabilities similar to those of other comparable quantification kits. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA (mtDNA) exposed to the extracellular space due to cell death has immunostimulatory properties. Case-control studies reported a positive association between odds of developing preeclampsia and circulating mtDNA. These findings are based on relative quantification protocols that do not allow determination of absolute concentrations of mtDNA and are highly sensitive to nuclear DNA contamination. Furthermore, circulating mtDNA concentrations in response to normal pregnancy, which is an inflammatory state characterized by continuous placental cell apoptosis, have not been established. The main objective of this study was to determine longitudinal changes in circulating mtDNA from preconception to first trimester, third trimester, and postpartum in healthy pregnant women. Absolute real-time PCR quantification of mtDNA and nuclear DNA (nDNA) was performed on whole genomic extracts from serum using TaqMan probes and chemistry. Serum cell-free mtDNA and nDNA concentrations were greater in late pregnancy as compared with early pregnancy and postpartum. Pregnant women carrying neonates at the upper quartile of birth length distribution had higher concentrations of mtDNA in late pregnancy compared with pregnancies carrying neonates at the lower quartile. The correlation between circulating mtDNA and nDNA concentrations varied by sex (i.e., pregnancies carrying female vs. male fetuses). This study is the first to establish temporal patterns of circulating cell-free mtDNA concentrations in normal human pregnancy using absolute DNA quantification techniques. Concentrations of circulating mtDNA in normal pregnancy may be used as reference values for the development of clinical prognostic or diagnostic tests in pregnant women with, or at risk of developing, gestational complications. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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 (CV Ct % ≤ 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. 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. • qPCR and HRM can be combined in a single reaction for forensic casework. • Amel-Y quantification results correlated well with STR typing. • HRM can be used as a rapid screening tool for male DNA detection and may be useful as a predictor for Y-STR typing success. [ABSTRACT FROM AUTHOR]

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. • Four qPCR and three autosomal STRs commercial kits performance is compared with 62 critical skeletal remains. • InnoQuant® HY Fast was found as the most sensitive kit. • The three autosomal STRs kits tested achieved similar results. • InnoQuant® HY Fast and Investigator® 24Plex were found as the best duo. [ABSTRACT FROM AUTHOR]

Abstract The purpose of the study was to develop a xenogenic bubaline diaphragm matrix (BDM) for abdominal hernia repair. A fresh diaphragm was decellularized using aqueous sodium dodecyl sulfate (SDS) solutions (0.5-4% w/v) over a period. Acellularity was confirmed histologically and characterized by Masson’s trichrome staining, scanning electron microscopy (SEM), DNA quantification, agarose gel electrophoresis, and Fourier-transform infrared spectroscopy. The BDM was used for clinical abdominal hernia repair in six cattle. Clinical, hemato-biochemical and antioxidant parameters were evaluated to assess biocompatibility of xenogenic BDM. Histologically, the diaphragm treated with 2% SDS for 48 h showed complete acellularity and orderly arranged collagen fibers. The SEM confirmed preservation of collagen structure and integrity. The DNA content was significantly (P < 0.05) reduced in BDM (33.12 ± 5.40 ng/mg) as compared to the native diaphragm (443.96 ± 162.60 ng/mg). DNA extracts from BDM show considerable removal of DNA material, with absence of DNA band in agarose gel. The FTIR spectrum of BDM has shown all characteristic transmittance peaks of bovine skin collagen indicating preserved collagen structure. Six cattle with BDM implant recovered uneventfully and remained sound at least upto 6 months. Hemato-biochemical and antioxidant findings were unremarkable. Bubaline diaphragm matrix shows excellent repair efficiency and biocompatibility for abdominal hernia repair in cattle without complications.

Background The application of an appropriate extraction method is a relevant factor for the success of all molecular studies. Methods Seven different DNA extraction methods suitable for high-throughput DNA sequencing with very small arthropods were compared by applying nine different protocols: three silica gel based spin methods, two cetyltrimethyl ammonium bromide (CTAB) based ones (one with an additional silica membrane), a protein precipitation method and a method based on a chelating resin (applying different protocols). The quantity (concentration) and quality (degradation, contamination, polymerase chain reaction (PCR) and sequencing success) of the extracted DNA as well as the costs, preparation times, user friendliness, and required supplies were compared across these methods. To assess the DNA quantity, two different DNA concentration measurements were applied. Additionally, the effect of varying amounts of starting material (different body sizes), variable lysis temperatures and mixing during DNA extraction was evaluated. Results Although low DNA concentrations were measured for all methods, the results showed that—with the exception of two methods—the PCR success was 100%. However, other parameters show vast differences. The time taken to perform DNA extraction varied from 20 min to 2.5 h (Chelex vs. CTAB) and the costs from 0.02 to 3.46 € (Chelex vs. QIAamp kit) per sample. High quality genomic DNA was only gained from four methods. Results of DNA quantity measurements further indicated that some devices cannot deal with small amounts of DNA and show variant results. Discussion In conclusion, using Chelex (chelating resin) turned out as a rapid, low-cost method which can provide high quality DNA for different kinds of molecular investigations.

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., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)