DNA Extraction Efficiency Research Articles

Engineering the bacteriophage 80 alpha endolysin as a fast and ultrasensitive detection toolbox against Staphylococcus aureus

The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.

Improving DNA recovery and sample throughput using the PrepFiler™ Automated Forensic DNA Extraction Kit on two customised Tecan Fluent® 1080 Automated Workstations

One of the primary goals of forensic laboratories performing DNA recovery and profiling is obtaining a high-quality DNA extract from crime scene samples. This is largely dependent on the sensitivity and reliability of the DNA extraction chemistry utilised, as well as the liquid handling and contamination minimisation techniques employed. Automation of DNA extraction methods on large liquid handling platforms allows high-throughput laboratories to apply sensitive chemistries to reliably process a large number of samples, while minimising manual processes and cross-contamination.This study describes the first known implementation of the PrepFiler™ Automated Forensic DNA Extraction Kit on a Tecan Fluent® Gx 1080 Automation Workstation. Two Workstations were customised with the addition of novel “Safe Pipetting Modules” to eliminate sample crossover between wells, which is important in a forensic biology setting to reduce inadvertent DNA transfer. A comparison of DNA extraction efficiency between an optimised PrepFiler™ method and the DNA IQ™ System performed on a Perkin Elmer Janus® Integrator platform showed the optimised PrepFiler™ method consistently extracted a higher yield of DNA from a range of blood inputs, as well as blood and buccal swabs. The PrepFiler™ chemistry also more efficiently removed humic acid and haematin, reducing subsequent PCR inhibition. The subsequent implementation of the optimised PrepFiler™ method onto the Tecan Fluent® Gx workstations showed a further increase in sensitivity, with no evidence of DNA cross-contamination observed. However, the optimised PrepFiler™ method encountered difficulties extracting DNA from fabric substrates, with the PrepFiler Express™ chemistry extracting higher yields on the cartridge-based AutoMate Express™ System.Overall, this study demonstrated the Tecan Fluent® Gx 1080 Automation Workstation is a sensitive, reliable and robust method for DNA extraction using the PrepFiler™ Automated Forensic DNA Extraction Kit, and the addition of the novel Safe Pipetting Module makes this platform an attractive option for forensic biology laboratories where minimising inadvertent DNA transfer is of paramount importance.

Mobile lab: A novel pathogen assay using the nucleic acid automatic assay system assisted by a self-contained microfluidic cassette and chitosan decorating magnetic particles

Microfluidic pathogen detection cartridges with integrated switching valves have gained significant attention. Traditionally, these microfluidic cartridges utilized magnetic beads for a “bind-wash-elute-reaction” protocol to extract DNA but often lacked PCR-based aerosol contamination validation and focused primarily on isothermal applications. There is room to improve these systems, such as using novel DNA extraction methods and integrating PCR as the gold standard. In this study, we developed chitosan-decorated magnetic (CDM) particles and incorporated them into a mass-produced, self-contained microfluidic cartridge for the first time. Our results demonstrated that the DNA can be efficiently enriched from the lysis buffer (pH 5) using 75 % degree of deacetylation and 1 % of chitosan-modified CDM particles (20 μL). The DNA extraction efficiency exceeded 90 %. The CDM particles enabled “in-situ PCR”, simplifying the on-chip detection process to a “bind-wash-reaction” protocol. Additionally, we developed a nucleic acid automatic assay system (NA3 system) featuring a fluid thermocycler, four-channel optical detection, magnet, microcontroller, and software. The entire assay, from sample preparation to detection, can be completed within approximately 60 minutes, simultaneously detecting 16 targets. The performance of the NA3 system was better than that of the pristine magnetic microparticles-based assay and the commercial magnetic beads kit-based assay. The linearity, specificity, multi-pathogen detection, and user-friendliness of the NA3 system were tested. We validated the NA3 system's performance in detecting food-borne and serum infectious pathogens, demonstrating its potential for use in resource-limited settings.

Extraction of genomic DNA from wild boar (Sus scrofa) muscle tissue using hydrophobic magnetic deep eutectic solvents

AbstractExtraction of high‐quality genomic DNA from wild boar tissue is of great significance for the study of its genetic information, population quantity, and distribution. In this work, a hydrophobic magnetic deep eutectic solvent (HMDES) was synthesized. Based on the HMDES, a HMDES‐based vortexed extraction method was developed for extracting genomic DNA from wild boar muscle tissue. Compared to the traditional high salt‐extraction method, this approach is faster and embraces higher DNA extraction efficiency. Different pretreatment methods before extraction were evaluated. Single‐factor experiments were used to optimize the extraction condition, for example, extraction time, temperature, and HMDES volume. After extraction, the DNA can be quickly and easily recovered from the HMDES phase, and the HMDES can be reused. This work provides a simple and environmental‐friendly extraction method for DNA extraction from wild boar tissue.

Detection of transgenes in equine dried blood spots using digital PCR and qPCR for gene doping control.

Due to the ease of collection, transport and storage, the use of dried blood spots (DBS) offers an attractive alternative matrix for detection of the abuse of gene therapy, otherwise known as gene doping. This study evaluated the recovery, extraction efficiency and resulting detection capability of DNA from DBS by evaluating different target types, DNA extraction kits, the number of punches and blood tube preservatives. The long-term storage stability of low-copy-number transgene targets in DBS was not assessed in this study but would be noteworthy to investigate further. DNA was quantified using two detection methods: qPCR and digital PCR (dPCR). Using six punches with the Qiagen Investigator kit gave the best overall DNA yield compared with other extraction methods. Including three punches, however, gave better DNA extraction efficiency. Reference material could be detected using qPCR and dPCR in DBS spiked with 5000 copies/mL of blood (approximately 15 copies per 3 mm of punch). The optimal DNA extraction protocol was used on DBS samples from a custom recombinant adeno-associated virus administration study and showed successful detection of vector targets in DBS samples.

Using a GFP-labeled Stagonospora nodorum strain as a DNA extraction efficiency standard in plant disease diagnosis

Quantitative polymerase chain reaction (qPCR) is a commonly used technique for plant disease diagnosis, which generally requires DNA extraction from the plant samples. The efficiency of DNA extraction has a significant influence on the accuracy of qPCR. Thus, including a DNA extraction efficiency control is important in qPCR analyses. To develop and evaluate a DNA extraction efficiency control, a Stagonospora nodorum (syn. Phaeosphaeria nodorum, synonym and correct taxonomic name: Parastagonospora nodorum) strain named HUA-22 was created by transferring a green florescent protein (GFP) gene into the genome of the S. nodorum strain Sn15. HUA-22 showed a similar pathogenicity as Sn15 but carried a strong GFP activity. A qPCR primers/probe set named P-GFP, targeting the GFP sequence, was designed. Using P-GFP, qPCR analysis was conducted on DNA extracted from replicated samples of HUA-22 conidia, and confirmed that HUA-22 could be used to characterize the variation in replicated DNA extractions. Conidia of HUA-22 were used to spike soil samples inoculated with the canola clubroot pathogen Plasmodiophora brassicae, canola stem samples infected with the blackleg pathogens Leptosphaeria biglobosa and/or L. maculans and wheat/barley samples infected with Xanthomonas translucens pv. translucens (Xtt) or X. translucens pv. undulosa (Xtu). Duplex qPCR using P-GFP and a primers/probe set specific to P. brassicae, triplex qPCR using P-GFP and primers/probe sets specific to L. biglobosa and L. maculans, and triplex qPCR using P-GFP and primers/probe sets specific to Xtt and Xtu were conducted. The results indicated that HUA-22 could be used as a standard for DNA extraction efficiency in qPCR-based plant disease diagnosis. Adding HUA-22 conidia to plant or soil samples prior to DNA extraction, and subsequent use of the P-GFP detection control, provided an added control that could distinguish truly negative from false-negative qPCR results.

Multi-amplicon nitrogen cycling gene standard: An innovative approach for quantifying N-transforming soil microbes in terrestrial ecosystems

Agriculture accounts for nearly three-fourths of all nitrous oxide (N2O) anthropogenic emissions, a potent greenhouse gas (GHG). Improvements in agricultural management practices are critical for improving plant nitrogen (N) use efficiency and reducing N2O emissions. While implementing “climate-smart” management can reduce N2O emissions, building a linkage between these emissions and the N-cycling microbes that drive them will require N-cycling gene assays that are cost-effective, practical, and accessible to climate-smart researchers. In a recent study, a group of researchers demonstrated the suitability of synthetic oligonucleotides to serve as real-time quantitative PCR (qPCR) standards for quantifying microbial genes in soils. While this study is not based on that study, our results complement their findings and offer fresh insights into this novel methodology. Here, we describe an innovative approach that provides precision in the absolute quantification of five N-transforming target genes (AOB amoA, AOA amoA, nirK, nirS, and nosZ) in soils. Similar to the aforementioned study, our method obviates the cost-intensive aspects of traditional qPCR methods for preparing standard curves in soil microbial ecology (e.g., gel purification of PCR products and in-vivo cloning of plasmids) and is capable of being extended to any biochemical system, or environment of interest. Additionally, our qPCR approach provides several advantages: it utilizes a single multi-amplicon synthetic oligonucleotide, requires only a single dilution series and one stock solution to quantify all gene targets, and contains a non-edaphic gene (ZIKV) that can serve as a soil spike to determine DNA extraction efficiency and DNA recovery estimates. Furthermore, our protocol mitigates PCR inhibition from soil-derived substances using a modified DNA extraction protocol and inhibitor-tolerant polymerase. Ultimately, our work aims to reduce the technical burden on non-biologists/biochemists when conducting soil microbiological assays, support the development of climate mediation practices, and advance climate-smart agriculture goals.

Prospects for use of genetic tagging technology in animal husbandry

The efficiency of DNA extraction with using different extraction methods: from the cartilage tissue of the outer ear and the leukocyte fraction of whole blood of cattle, followed by an assessment of the possible prospects for the use of “genetic tagging” technology in animal husbandry has been compared in the work. It was established that according to the results of the research the highest concentration of DNA (146,26±13,180 ng/μl at p < 0,001) was obtained in samples from ear tissues when using the method of precipitation with reagents compared to isolating DNA from the leukocyte fraction of whole blood using highly eff ective methods extraction of genomic deoxyribonucleic acid on magnetic particles (22,80±3,138 ng/μl) and on columns (39,46±8,941 ng/μl). It was found when assessing DNA contamination with polypeptides that despite the fact that when using diff erent extraction methods, the ratio of optical densities at the corresponding absorption wavelengths A260/A280 was close to the optimal ~1,8. The smallest scatter of measurement values was obtained in DNA samples from earmarks tissue 1,89±0,008. In a subsequent comparison of available technologies for taking ear pluck tissue samples used for genetic research (population genetic characterization, genomic analysis, etc.), the FlexoPlusGeno “genetic tagging” system turned out to be the most preferable. In this case, the resulting ear pluck tissue sample is correlated with corresponding ear visual or RFID tags, which creates a “link” of the biomaterial to a specific animal with the ability to track it throughout its life. The technology under consideration is applicable for collecting and storing samples of cartilage tissue obtained by ear plucking from cattle, small ruminants, pigs and deer.

DNA Extraction Methods

KATIE LONG and MARK FLOOD, Forensic Science Program, Fairmont State University, Fairmont, WV 26554. Evaluation of DNA Extraction Methods. Three forms of DNA extraction types include solution-based, column-based, and magnetic bead-based methodologies, with the latter being the accepted methodology used in forensic laboratories. Magnetic bead-based DNA extraction uses the negative charge of DNA and the positive charge of the magnetic beads to isolate the DNA strands and produce a purified sample. However, solution and column-based extraction methods use the same techniques of washing to purify DNA and typically have comparable DNA yields. Cheek swabs were obtained from ten individuals and the three extraction methods were performed to determine the quantitation of double-stranded DNA and the quality of the gel electrophoresis profile. Preliminary results indicate that solution-based extraction produces a slightly higher yield of double-stranded DNA having a 3.92% difference between the average quantitation of 26.29 ng/µL from solution-based and 25.28 ng/µL from magnetic bead-based methods. However, it is expected that the magnetic bead-based extraction will produce higher quality gel electrophoresis results. Overall, the methods examined produced similar DNA extraction efficiencies. This research was made possible by NASA West Virginia Space Grant Consortium, Training Grant #80NSSC20M0055 and the STaR Sure Grant.

Nanoparticle-Mediated Plasmid Curing in Combating Antibiotic Resistance in Pathogenic Bacteria

Background: Nanotechnology has emerged as a pivotal domain in material science research with extensive applications across various sectors including biotechnology and medicine. Nanoparticles offer unique properties facilitating advancements in nanobiotechnology, particularly in nanomedicine, to combat bacterial infections and antibiotic resistance. This study aimed to determine the application of nanoparticles, specifically nano-TiO2, in treating plasmid-mediated antibiotic resistance in both Gram-negative and Gram-positive bacteria. Method: We evaluated antibiotic and nanomaterial sensitivity through disc diffusion and broth microdilution assays. Plasmid curing experiments were conducted using varying concentrations of nano-TiO2 and SDS as curing agents, followed by plasmid isolation and DNA extraction. The efficacy of nano-TiO2 in plasmid curing and DNA extraction was assessed, alongside the impact on bacterial growth and antibiotic resistance. Results: Results showed successful plasmid elimination with nano-TiO2 treatment, evidenced by the loss of plasmid DNA bands. Additionally, nano-TiO2 substantially enhanced DNA extraction efficiency and quality. The study indicated nano-TiO2's potential in combating antibiotic resistance by targeting plasmids, thereby presenting a novel approach in molecular biology techniques. Conclusion: In conclusion, this study underscores the promising role of nanoparticles in addressing bacterial infections and combating antibiotic resistance. Nano-TiO2 emerges as a valuable tool in DNA purification and plasmid curing, offering new avenues in molecular biology and antibiotic resistance research. However, further investigations are warranted to elucidate the broader implications of nanoparticles across diverse bacterial species and strains. These findings represent a significant step towards harnessing the potential of nanotechnology in combating antimicrobial resistance and advancing healthcare paradigms.

Analytical evaluation of circulating tumor DNA sequencing assays

In China, circulating tumor DNA analysis is widely used and numerous assays are available. Systematic evaluation to help users make informed selections is needed. Nine circulating tumor DNA assays, including one benchmark assay, were evaluated using 23 contrived reference samples. There were two sample types (cell-free DNA and plasma samples), three circulating tumor DNA inputs (low, < 20 ng; medium, 20–50 ng; high, > 50 ng), two variant allele frequency ranges (low, 0.1–0.5%; intermediate, 0.5–2.5%), and four variant types (single nucleotide, insertion/deletion, structural, and copy number). Sensitivity, specificity, reproducibility, and all processes from cell-free DNA extraction to bioinformatics analysis were assessed. The test assays were generally comparable or superior to the benchmark assay, demonstrating high analytical sensitivity. Variations in circulating tumor DNA extraction and quantification efficiency, sensitivity, and reproducibility were observed, particularly at lower inputs. These findings will guide circulating tumor DNA assay choice for research and clinical studies, allowing consideration of multiple technical parameters.

Improving the efficiency of DNA extraction from iron incrustations and oilfield-produced water

The quantity and quality of DNA isolated from environmental samples are crucial for getting robust high-throughput sequencing data commonly used for microbial community analysis. The differences in the nature and physicochemical properties of environmental samples impact DNA yields, and therefore, an optimisation of the protocols is always recommended. For instance, samples collected from corroded areas contain high concentrations of metals, salts, and hydrocarbons that can interfere with several steps of the DNA extraction protocols, thereby reducing yield and quality. In this study, we compared the efficiency of commercially available DNA extraction kits and laboratory-adopted methods for microbial community analysis of iron incrustations and oilfield-produced water samples. Modifications to the kits manufacturers’ protocols were included to maximise the yield and quality. For iron incrustations, the modified protocol for FastDNA Spin Kit for Soil yielded higher DNA and resulted in higher diversity, including the recovery of low-abundant and rare taxa in the samples, compared to DNeasy PowerSoil Pro Kit. The DNA extracted with modified phenol–chloroform methods yielded higher DNA but failed to pass quality control PCR for 16S sequencing with and without purification. The protocols mentioned here can be used to maximise DNA recovery from iron incrustations and oilfield-produced water samples.

Assessment of the efficiency of DNA isolation and profiling applying a temperature-driven method in human remains.

The identification of human remains is of utmost importance in a variety of scenarios. One of the primary identification methods is DNA. DNA extraction from human remains could be difficult, particularly in situations where the remains have been exposed to environmental conditions and other insults. Several studies tried to improve extraction by applying different approaches. ForensicGEM Universal (MicroGem) is a single-tube approach to DNA extraction and a temperature-driven method that could have some advantages with respect to previous techniques, among them, reducing the risk of contamination, not requiring specialized equipment, or several steps to perform. The aim of this study was to assess, for the first time, the efficiency of DNA extraction and quality of STR profiles applying the MicroGem protocol and modifications of this protocol from tooth samples in comparison with automatic extraction (AE). Our results indicated that AE and MicroGem performed similar, though with variability depending on the MicroGem modifications, increasing the DNA yield and STR profile quality when DNA is concentrated with Microcon. These findings demonstrated the efficiency of this methodology for DNA extraction from human remains while also providing a simple and quick technique suitable to apply in a variety of forensic scenarios.

Preserving morphology while extracting DNA: a non-destructive field-to-museum protocol for slide-mounted specimens.

Our study aimed to develop an optimised laboratory protocol ensuring the preservation of morphological structures and extraction of high-quality DNA sequences from Psychodidae (Insecta, Diptera) specimens. With 310 analysed specimens, we investigated the impact of distinct laboratory treatments by employing two shaking categories (constant and interrupted) with five different incubation periods (16, 12, 8, 4 and 2 hours) during the DNA extraction process. Notably, 80.65% of the specimens exhibited morphological changes during DNA extraction. Our results indicated no statistical difference between constant and interrupted shaking for the total of morphological structures lost. However, within each shaking category, the loss of structures was influenced significantly by the incubation period. Prolonged incubation correlated with increased structural losses, whereas shorter incubation periods caused minor alterations in structures lost. In addition, our results showed a significant difference between constant and interrupted shaking treatments for DNA concentration. Likewise, the incubation period showed differences within each shaking category. Successful COI sequencing was achieved in 89.6% of specimens, with negligible differences in DNA fragment lengths across treatments. Our findings underscore the importance of an optimised protocol and its potential in systematic research involving nematoceran dipteran specimens by balancing morphological integrity and DNA extraction efficiency.

Spore-DNA localization and extraction efficiencies of Bacillus subtilis for accurate results in quantitative real-time polymerase chain reaction.

Mechanical bead disruption is an efficient DNA extraction method from spore cells for subsequent quantification of the spore population by quantitative polymerase chain reaction（qPCR）. In this study, to validate spore DNA localization and extraction efficiencies, the fractionated DNA included the total DNA（tDNA）extracted from spore cells and intracellular（iDNA）and extracellular DNA（eDNA）extracted from fractionated spores through chemical decoating and alkaline lysis buffers, each followed by bead disruption. Furthermore, alkaline lysis buffer-treated spore cells were intensively washed three and five times after each centrifugation to determine how the amount of DNA is affected by repeated centrifugation. This process was achieved through fractionated spore pellet and suspension treatments with propidium monoazide xx（PMAxx）before mechanical bead disruption. Three fractionated and extracted DNAs were assessed with qPCR. The amount of eDNA was higher than that of iDNA, and closer to tDNA levels in the qPCR assay. These results indicted the following: 1）amount of eDNA was more than iDNA and responsible for majority of amount of tDNA through the combination method involving alkaline lysis buffer and bead disruption, 2）lysis buffer partially eliminated the eDNA fragments through multiple washing steps, but it was not largely independent of the number of times centrifugation was performed.

Salmon sperm DNA increases sample recovery from cotton swabs

Forensic samples with low DNA template amounts are difficult to analyze and interpret. There is a large body of research demonstrating that adding carrier nucleic acid to storage tubes, solid phase extractions, or filtering devices can improve yields of target DNA. However, the addition of carrier nucleic acid to sampling substrates, like cotton swabs, has not yet been attempted. In this proof-of-concept study, carrier nucleic acids in the form of either Poly (A) RNA or salmon sperm DNA were spotted onto cotton swabs, followed by human genomic DNA, to determine if introducing the carrier prior to sample collection would increase recovery from the swabs post-extraction. Extracts were also evaluated to determine whether adding the carrier nucleic acids to human DNA would interfere with downstream forensic DNA analysis processes such as real-time PCR quantitation, PCR amplification of STR loci, or capillary electrophoresis. The RNA carrier did not improve human sample recovery from cotton swabs. The extraction efficiency of human DNA from cotton swabs was increased when the DNA carrier was applied to the swabs prior to sample deposition, and the scale of the increase depended on the amount of carrier DNA used. When applying the salmon sperm DNA carrier to cotton swabs, with each increase from no carrier to 0.001–1–10 µg, human DNA recovery went from ∼29 % to ∼50 % to ∼75 % to ∼100 %. Additionally, no inhibitory effects from the carrier DNA were observed post-extraction with quantitation or in the DNA profile after amplification. Therefore, salmon sperm DNA carrier will increase human DNA yield from cotton swabs without negative effects on downstream forensic DNA profiling methods, with the optimal carrier amount being 10 µg.

Recovery of microbial DNA by agar-containing solution from extremely low-biomass specimens including skin

Recovering a sufficient amount of microbial DNA from extremely low-biomass specimens, such as human skin, to investigate the community structure of the microbiome remains challenging. We developed a sampling solution containing agar to increase the abundance of recovered microbial DNA. Quantitative PCR targeting the 16S rRNA gene revealed a significant increase in the amount of microbial DNA recovered from the developed sampling solution compared with conventional solutions from extremely low-biomass skin sites such as the volar forearm and antecubital fossa. In addition, we confirmed that the developed sampling solution reduces the contamination rate of probable non-skin microbes compared to the conventional solutions, indicating that the enhanced recovery of microbial DNA was accompanied by a reduced relative abundance of contaminating microbes in the 16S rRNA gene amplicon sequencing data. In addition, agar was added to each step of the DNA extraction process, which improved the DNA extraction efficiency as a co-precipitant. Enzymatic lysis with agar yielded more microbial DNA than conventional kits, indicating that this method is effective for analyzing microbiomes of low-biomass specimens.

Physical Pretreatments Applied in Three Commercial Kits for the Extraction of High-Quality DNA from Activated Sewage Sludge.

Obtaining sufficient and high-quality genomic DNA from sludge samples is a fundamental issue of feasibility and comparability in genomic studies of microbial diversity. Commercial kits for soil are often used for the extraction of gDNA from sludge samples due to the lack of specific kits. However, the evaluation of the performance of commercial kits for sludge DNA extraction is scarce and optimization of these methods to obtain a high quantity and quality of DNA is necessary, especially for downstream genomic sequencing. Sequential batch reactors (SBRs) loaded with lignocellulosic biomass are used for the synthesis of renewable resources such as levulinic acid (LA), adipic acid (AA), and polyhydroxyalkanoates (PHAs), and the biochemical synthesis of these compounds is conducted through the inoculation of microbes present in the residual activated sludge (AS) obtained from a municipal wastewater treatment plant. To characterize these microbes, the extraction of DNA from residual sewage sludge was conducted with three different commercial kits: Nucleospin® Soil from Macherey-Nagel, DNEasy® PowerSoil® from Qiagen, and E.Z.N.A.® Plant DNA Kit from Omega BIO-TEK. Nevertheless, to obtain the highest load and quality of DNA for next-generation sequencing (NGS) analysis, different pretreatments and different combinations of these pretreatments were used. The pretreatments considered were an ultrasonic bath and a temperature of 80 °C, together and separately with different incubation time periods of 30, 60, and 90 min. The results obtained suggest a significant improvement in the efficiency and quality of DNA extraction with the three commercial extraction kits when used together with the ultrasonic bath and 80 °C for 60 min. Here, we were able to prove that physical pretreatments are a viable alternative to chemical lysis for DNA extraction from complex samples such as sludge.

Design of a Protocol for Soil-Transmitted Helminths (in Light of the Nematode Toxocara canis) DNA Extraction from Feces by Combining Commercially Available Solutions.

One of the main challenges for the mass introduction of the molecular diagnostics of soil-transmitted helminths (STHs) into clinical practice is the lack of a generally recognized effective method for isolating parasitic DNA from fecal samples. In the present study, we assessed the effects of various pretreatment procedures on the efficiency of removing PCR inhibitors and extracting Toxocara canis DNA from feces. We evaluated the effectiveness of four destructive methods (bead beating, the action of temperature-dependent enzymes, freeze-heat cycles, and incubation in a lysis buffer) on the integrity of T. canis eggs and the efficiency of DNA extraction. Also, we evaluated the effects of prewashes and the use of commercial concentrators on DNA extraction from fecal samples contaminated with T. canis eggs. A bead beating procedure was sufficient to destroy the T. canis eggs, while the effects of enzymes and freeze-heat cycles did not lead to a significant destruction of the eggs or the release of Toxocara DNA. Helminth DNA isolation protocols that do not include a bead beating step are not preferred. The preconcentration of STH eggs from feces using a commercial concentrator and subsequent washing can significantly increase the yield of DNA from STHs and reduce PCR inhibition.

Importance of sample pre-treatments for the DNA-based characterization of microbiomes in cropland and forest soils

Soil-extracted DNA is widely used to characterise the composition of microbiomes. However, there is no commonly accepted protocol or understanding of how sample preservation and laboratory pre-incubation affect the results obtained. Here, we evaluated six different pre-treatments of sieved cropland and forest topsoils from five sites in Northern Germany, including freezing, drying, fresh analyses, and pre-incubations for 1 or 14 day prior to DNA extraction. Based on PCR-amplified 16S rRNA genes and ITS sequences, we characterized the abundance and diversity of bacteria, archaea and fungi, and compared it to a reference community composition obtained from samples immediately frozen with liquid nitrogen and without further manipulation. Irrespective of the pre-treatments, soil microbiomes could be differentiated according to their land use and sampling site. For prokaryotes, Acidobacteriota, Actinobacteriota or Thermoproteota, were more affected by pre-treatments than Bacteriodota, Firmicutes or Planctomycetota. For fungi, Basidiomycota and Mucoromycota, which predominated in forest soils, were more susceptible than the cropland predominant Ascomycota, indicating that different pre-treatments have a stronger impact on assessing the composition of fungal microbiomes from forest than from cropland. Differences between the reference and pre-treatments suggest that generally soil sieving enhanced the DNA extraction efficiency, except for dried soils, but it shifted the proportionality of detected taxa, as shown by reduced Shannon diversity. Overall, oven-drying had a more detrimental effect than air drying, freezing treatments or fresh analyses. For large scale monitoring projects, where fresh analysis is not possible, the gentle freezing approach as applied in this study would be the best choice.