Higher DNA Yield Research Articles

A deployable film method to enable replicable sampling of low-abundance environmental microbiomes

Urbanizing global populations spend over 90% of their time indoors where microbiome abundance and diversity are low. Chronic exposure to microbiomes with low abundance and diversity have demonstrated negative long-term impacts on human health. Sequencing-based analyses of environmental nucleic acids are critical to understanding the impact of the indoor microbiome on human health, however low DNA yields indoors, alongside sample collection and processing inconsistencies, currently challenge study replicability. This study presents a comparative assessment of a novel, passive, easily replicable sampling strategy using polydimethylsiloxane (PDMS) sheets alongside a representative swab-based collection protocol. Deployable, customizable PDMS films designed for whole-sample insertion into standardized extraction kits demonstrated 43% higher DNA yields per sample, and 76% higher yields per cm2 of sampler over swab-based protocols. These results indicate that this accessible, scalable method enables sufficient DNA collection to comprehensively evaluate indoor microbiome exposures and potential human health impacts using smaller, more space efficient samplers, representing an attractive alternative to swab-based collection. In addition, this process reduces the manual steps required for microbiome sampling which could address inter-study variability, transform the current microbiome sampling paradigm, and ultimately benefit the replicability and accessibility of microbiome exposure studies.

Comparison of Different DNA Sampling and Extraction Protocols for Bacterial and Archaeal Populations Analysis in Water Buffalo

Methane (CH4) is a potent greenhouse gas, and ruminants are a significant source of agricultural emissions. It has been hypothesized that the host's genome controls rumen microbial communities, but robust results require numerous samples. The feasibility of a research project will depend on the ease and representativeness of the sampling method, as well as the cost-efficiency of large-scale sequencing. This study aimed to compare different protocols to investigate whether non-invasive samples could serve as a substitute for ruminal digesta. DNA recovery was tested in various matrices (whole rumen content, feces, and buccal swabs) from five cannulated buffalo cows. Three types of buccal swabs were tested, as well as feces in different forms (as-is, pelleted, or in a glycerol solution) and the rumen content. The study compared different protocols for DNA extraction, including WUR protocol, Maxwell®, and Quick Extract™, and two sampling times. Saliva was a challenging matrix to process, obtaining unsatisfactory DNA yield. Feces showed higher yields when pelleted but lower than rumen. The highest amount of DNA was obtained from whole rumen content using all three DNA extraction methods. Quick Extract was the easiest method to perform, while WUR resulted in the highest yield of DNA, swabs excluded. The Maxwell® method gave satisfactory results with all three matrices. However, further metagenomic analysis is required to verify if the species composition is comparable.

Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance.

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has emerged as the source of choice to sample pathogen burdens in the environment. Wastewater from open drains and community water treatment plants is a reservoir of both pathogens and antimicrobial resistance (AMR) genes, and frequently comes in contact with humans. While there are many methods of tracking AMR from water,isolating good-quality DNA at high yields from heterogeneous samples remains a challenge. To compensate, sample volumes often need to be high, creating practical constraints. Additionally, environmental DNA is frequently fragmented, and the sources of AMR (plasmids, phages, linear DNA) consist of low-molecular-weight DNA. Yet, few extraction processes have focused on methods for high-yield extraction of linear and low-molecular-weight DNA. Here, a simple method for high-yield linear DNA extraction from small volumes of wastewater using the precipitation properties of polyethylene glycol (PEG) is reported. This study makes a case for increasing overall DNA yields from water samples collected for metagenomic analyses by enriching the proportion of linear DNA. In addition, enhancing low-molecular-weight DNA overcomes the current problem of under-sampling environmental AMR due to a focus on high-molecular-weight and intracellular DNA. This method is expected to be particularly useful when extracellular DNA exists but at low concentrations, such as with effluents from treatment plants. It should also enhance the environmental sampling of AMR gene fragments that spread through horizontal gene transfer.

Optimization of Total DNA Extraction from Dried Blood Samples.

While dried blood spots are a convenient source of genetic material, they are usually associated with a lower DNA yield than from a native sample. The study evaluated the DNA yield from dried blood samples prepared on glass fibre and cellulose membranes and investigated the reasons for the yield reduction. The extraction of total DNA from membrane-dried blood samples was optimized by spin-column extraction method. It was shown that preliminary short-term (20min) solubilization of a dried matrix in an aqueous medium, followed by standard extraction protocols for the mixture of the eluate with membranes, provides the highest DNA yield. The yield of DNA from a glass fibre membrane was 40-50% lower compared to a native sample, but on average, two times higher than from a conventional cellulose membrane (filter paper). The reduction of DNA yield when using a dried sample was found to be due to partial retention of nucleic acids by the membrane material during the lysis stage.

Optimization of DNA Extraction for ITS/U4U3 analysis of Rhipsalis baccifera

Genetic analysis of plants relies on high yields of pure DNA. For Rhipsalis baccifera this represents a great challenge since its plant tissue can accumulate large amounts of mucilage, polysaccharides, polyphenols and secondary metabolites, which co-purify with amplifiable DNA. These contaminating compounds lead to a poor yield and prevent acces to PCR-based analysis. These contaminating compounds lead to a poor DNA yield and prevent access to PCR-based analysis. A number of factors, including choice of plant tissue, tissue preparation, and modifications of the extraction buffer, can impact on DNA extraction process. In this study, four different DNA extraction procedures were tested aiming to develop a simple protocol based on CTAB buffer. The obtained results showed that the use of the outer cuticle of old lyophilized tissue allowed reliable results in R. baccifera plants with a good purity ranging from 1.6 to 1.8 and high DNA yield yield ? 500 ng ?L-1.

DNA Isolation From Six Spotted Ladybird Beetle <i>Cheilomenes Sexmaculata</i> (F)

DNA extraction is a routine step in many insect molecular studies and extraction methods need to be evaluated for their efficiency, cost, and side effects, such as DNA degradation. Individuals of six spotted ladybird beetle, Cheilomenes sexmaculata (F.) were subjected to DNA extraction by the CTAB method and PureLink® Kit. The methods were compared in terms of DNA quantity and quality, cost of materials, and time consumed. The CTAB method resulted in higher DNA yield (ng DNA) at a much lower price and less degradation, as revealed on agarose gels. The PureLink® Kit was the most time-efficient but costliest, and degradation was observed on agarose gels. The DNA samples obtained were tested on agarose gel PCR for six SSRs located in various positions of the beetle’s genome. The results revealed that DNA isolated from the CTAB method showed successful amplifications, but the PureLink® Kit method did not show any amplification. These evaluations guide the choice of DNA extraction methods from C. sexmaculata beetles.

High Quality and Purified DNA Extraction Protocol from Transplastomic Potato Lines by Cetyltrimethylammonium Bromide (CTAB)-Based Method

For the advanced molecular techniques high quality and pure DNA is important requirement where a quick and cost-effective extraction method is a crucial factor. Commonly, prolong incubation period, multiple precipitations, and highly expensive commercial kits are remaining basic barrier to introduce swift and easiest method of DNA extraction from transgenic plants. Non-cellulose components present in the leaf and elevated level of starch content as well as protein content also makes this extraction procedure challenging and difficult. Present study was conducted to establish a unique DNA isolation method with some modification of existing CTAB method to ensure high-quality and purified genomic DNA from the potato plants. Through the implementation of solution based and selective DNA precipitation method, 100 mg of potato leaves from every transplastomic potato lines was applied to extract genomic DNA. Ethanol (70%) was used as elution buffer where maximum DNA yield was 3415.500 ng/µl and the ranges of DNA purity (A260/A280 nm) was recorded as 1.954-2.048, respectively. Compared to existing methods, this modified CTAB mediated procedure is swift (at least one hour can be saved ) and proficient enough for high quality DNA yield, free from the selective precipitation, regents and laboratory equipment’s are cost-effective and non-toxic to human health.

RELATIONSHIP BETWEEN THE SPECTROMETRIC VALUES OF DNA, RNA, AND THE PCR PRESENCE OF A PATHOGEN IN SINGLE TICK SAMPLES

Aim. Ticks are the vectors of many pathogens, which cause diseases with fatal consequences. Polymerase chain reaction (PCR) was used to detect the presence of these different pathogens in ticks, but there is a need of isolated nucleic acid to conduct the molecular assays. In our previous research, we found that some ticks give huge yield of isolated nucleic acid during spectrometric measurements, therefore aim of this study is to find whether there is any relation between spectrometric values of DNA, RNA and presence of Borrelia burgdorferi as example pathogen in single tick samples. Methods. DNA and RNA were isolated with mini column method from single tick samples. They were run in real time as well as conventional PCR tests for the presence of Borrelia burgdorferi. The nucleic acid yields of isolated nucleic acid samples were measured with a spectrophotometer. Results. It was found that there were 47 ticks positive for Borrelia burgdorferi and 40 were negative. Average isolated DNA and RNA quantity was higher in pathogen positive ticks than those of negative ticks. There was no correlation between the yield of nucleic acid and presence of pathogen in a single tick, but there was tendency that pathogen positive tick gave higher yield of DNA and RNA during the isolation. Conclusions. This study shows some of Borrelia burgdorferi positive ticks give very high yield of DNA and RNA during the isolation. There is no correlation between presence of pathogen and nucleic acid in a single tick, but there is tendency that pathogen positive tick may have higher nucleic acid yield. Therefore, our recommendation is that laboratory should always measure the nucleic acid yield along with conducting the PCR tests.

New polymer brush-coated monodisperse magnetic nanoparticles prepared via interface-mediated RAFT polymerization for high-throughput DNA extraction from pathogen bacteria

Polymerase chain reaction (PCR) is gold-standard method for molecular detection of pathogens. DNA extraction from a complex biological medium is a crucial process for PCR based detection. Recently, magnetic nanoparticles-based methods are of great interest for genetic material isolation. In our work, we offer new magnetic nanoparticles based high-throughput DNA extraction method for pathogen detection in biofluids. For this purpose, first time polymer brush coated monodisperse magnetic nanoparticles were prepared with interface-mediated RAFT polymerization to be used in DNA extraction process. After synthesizing Fe3O4 nanoparticles, silica shell was coated on the magnetic nanoparticle core. Polymer brushes was immobilized on the amine functionalized silica layer using RAFT agent. Subsequently, the resulting particles with poly(vinylbenzyltrimethylammonium chloride) (PVBTAC) grafted were finalized through interface-mediated RAFT polymerization. The functional groups on the nanoparticles were characterized using FTIR spectroscopy. Size distribution and zeta potential analysis of the particles were performed by TEM and Zeta-sizer respectively. Adsorption and recovery studies of plasmid DNA were carried out on the polymer-coated magnetic nanoparticles. The developed particle provided both the rapid extraction of DNA and high-yield DNA recovery without requiring extended incubation and elution time. The maximum adsorption capacity was determined as 238.1 μg/mg. About 90 % of the input DNA was recovered. The total time of DNA extraction took just 20 min. We have successfully demonstrated the extraction of dsDNA of Enterococcus faecalis spiked to reference blood sample using the developed magnetic particles. The extracted plasmid DNA and dsDNA were confirmed by gel electrophoresis and qPCR methods respectively. The results showed that the proposed magnetic iron oxide nanoparticles (MIONp) will be a strong candidate for DNA extraction process from biological matrices.

Saliva As a Non-Invasive Source to Monitor JAK2 V617F variant Allele Frequency (VAF) in Myeloproliferative Neoplasm (MPN): Implications for Dissecting the Mechanism of Interferon Alfa (IFNα) Response and Identification of Predictors of Response

Myeloproliferative Neoplasm (MPN) is a group of chronic blood cancers characterized by the somatic acquisition of mutations, in particular Janus Activated Kinase 2 (JAK2V617F), which lead to constitutive activation of myeloid cytokine receptors. Interferon alfa (IFNα) is the only known therapy that induces a molecular response by decreasing the Variant Allele Frequency (VAF) of JAK2V617F (Mullally et al, Blood 2013; Mosca et al, Blood, 2021). However, not all MPN patients on IFNα enjoy a molecular response, and why some patients respond and others do not remains unclear. Our long-term goal is to identify early biomarkers that predict for a molecular response to IFNα by following in depth MPN patients who are beginning IFNα dosing for one year with daily sampling of leukocyte DNA to monitor day-to-day changes in the driver mutation VAF using digital PCR (dPCR) (Cordua et al, Blood, 2019). Biosample collection and processing: peripheral blood was drawn into an EDTA tube and aliquoted into 500 µL stocks. Saliva was collected via passive drool and Zymo DNA/RNA Shield was added in a 1:1 dilution. DNA purification: DNA was purified using the Zymo Research Quick-DNA Miniprep Plus Kit and DNA yields (ng/µL) were quantified using the Qubit Flurometer. dPCR: DNA was added to master mix, JAK2WT and JAK2V617F specific primers and fluorescent probes, and sterile water. dPCR protocol: 10-minute pre-heat at 96°C, followed by 45 cycles of denature at 96°C for 30 seconds/anneal-extend at 61°C for 60 seconds, analyzing for JAK2WT and JAK2V617F fluorescent signals to quantify the amount of DNA (cp/µL) per allele. VAF was calculated using the amounts of DNA (JAK2V617F)/(JAK2WT+JAK2V617F). First, using stored samples from patients received before and during IFNα treatment, we identified variable responses of the JAK2V617F VAF to IFNα. We observed that patients with a positive clinical response to IFNα had an increase in JAK2V617F VAF immediately after initiating this therapy, followed by an eventual decrease. Then, we showed the feasibility for a study using daily saliva sampling in an IRB-approved observational trial to monitor daily VAF trends via saliva sampling for one year. Participants provided daily sampling starting from ~1 month before initiating IFNα therapy. So far, we have recruited eight patients. For most patients, high DNA yields (> 5 ng/µL) can be consistently obtained (Additional Content A). For one patient (IFN01) with initial low yields, we were able to improve those yields with positive reinforcement (e.g., smelling lemon-scented stickers). Next, we view that most patients can maintain consistent daily sampling times and food/drink conditions. Using the daily sampling, we have developed JAK2V617F change curves in response to IFNα (Additional Content B). There is daily variation in VAFs (hypothesized to be from variable buccal epithelial contamination), however clear trends can be seen. One patient (IFN01) displays a steep increase in JAK2V617F VAF, another (IFN07) displays a more gradual increase, and two patients (IFN03, IFN04) display no noticeable change. These trends will be interesting for continued observation to determine if they will correlate with long-term outcome IFNα. From the above graphs, after viewing that weekly sampling (which for most patients are symbolized by the black circles for weekly IFNα injections) preserves the same VAF trends as daily sampling, we simulated 1000 weekly, bi-weekly and monthly data distributions by randomly selecting a data point from each consecutive time frame over the first 150-day period, respectively. We will then conduct a generalized linear model to construct a plot of predicted mean values across time and its 95% confidence intervals (CIs). We determined that moving forward, we can decrease the sampling frequency to weekly samples, since this will preserve fine detailed trends, as well as have the combination of ease for patient and frequent enough for patients to not forget sampling habits. Here, we explore the novel use of daily saliva for comprehensive and high resolution VAF tracking of MPN patients in a remote setting. We will continue to follow the day-to-day variation of VAF in response to IFNα and plan to expand the use of saliva to gene expression profiling. Overall, the results of this trial will be used to generate a preliminary model of IFNα response, which can be used to predict how likely a MPN patient will benefit from IFNα therapy.

Cationic and anionic detergent buffers in sequence yield high-quality genomic DNA from diverse plant species

Because of the heterogeneity among seedlings of outbreeding species, the use of seedling tissues as a source of DNA is unsuitable for the genomic characterization of elite germplasms. High-quality DNA, free of RNA, proteins, polysaccharides, secondary metabolites, and shearing, is mandatory for downstream molecular biology applications, especially for next-generation genome sequencing and pangenome analysis aiming to capture the complete genetic diversity within a species. The study aimed to accomplish an efficient protocol for the extraction of high-quality DNA suitable for diverse plant species/tissues. We describe a reliable, and consistent protocol suitable for the extraction of DNA from 42 difficult-to-extract plant species belonging to 33 angiosperm (monocot and dicot) families, including tissues such as seeds, roots, endosperm, and flower/fruit tissues. The protocol was first optimized for the outbreeding recalcitrant trees viz., Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera, which are rich in proteins, polysaccharides, and secondary metabolites, and the quality of the extracted DNA was confirmed by downstream applications. Nine procedures were attempted to extract high-quality, impurities-free DNA from these three plant species. Extraction of the ethanol-precipitated DNA from cetyltrimethylammonium bromide (CTAB) protocol using sodium dodecyl sulfate (SDS) buffer, i.e., the extraction using a cationic (CTAB) detergent followed by an anionic (SDS) detergent was the key for high yield and high purity (1.75–1.85 against A260/280 and an A260/230 ratio of >2) DNA. A vice versa extraction procedure, i.e., SDS buffer followed by CTAB buffer, and also CTAB buffer followed by CTAB, did not yield good-quality DNA. PCR (using different primers) and restriction endonuclease digestion of the DNA extracted from these three plants validated the protocol. The accomplishment of the genome of P. cineraria using the DNA extracted using the modified protocol confirmed its applicability to genomic studies. The optimized protocol successful in extracting high-quality DNA from diverse plant species/tissues extends its applicability and is useful for accomplishing genome sequences of elite germplasm of recalcitrant plant species with quality reads.

Evaluation of commercial kits for isolation and bisulfite conversion of circulating cell-free tumor DNA from blood

BackgroundDNA methylation biomarkers in circulating cell-free DNA (cfDNA) have great clinical potential for cancer management. Most methods for DNA methylation analysis require bisulfite conversion, causing DNA degradation and loss. This is particularly challenging for cfDNA, which is naturally fragmented and normally present in low amounts. The aim of the present study was to identify an optimal combination of cfDNA isolation and bisulfite conversion kits for downstream analysis of DNA methylation biomarkers in plasma.ResultsOf the five tested bisulfite conversion kits (EpiJET Bisulfite Conversion Kit, EpiTect Plus DNA Bisulfite Kit (EpiTect), EZ DNA Methylation-Direct Kit, Imprint DNA Modification Kit (Imprint) and Premium Bisulfite Kit), the highest and lowest DNA yield and recovery were achieved using the EpiTect kit and the Imprint kit, respectively, with more than double the amount of DNA for the EpiTect kit. Of the three tested cfDNA isolation kits (Maxwell RSC ccfDNA Plasma Kit, QIAamp Circulating Nucleic Acid Kit (CNA) and QIAamp MinElute ccfDNA Mini Kit), the CNA kit yielded around twice as much cfDNA compared to the two others kits, although with more high molecular weight DNA present. When comparing various combinations of cfDNA isolation kits and bisulfite conversion kits, the CNA kit and the EpiTect kit were identified as the best-performing combination, resulting in the highest yield of bisulfite converted cfDNA from normal plasma, as measured by droplet digital PCR (ddPCR). As a proof of principle, this kit combination was used to process plasma samples from 13 colorectal cancer patients for subsequent ddPCR methylation analysis of BCAT1 and IKZF1. Methylation of BCAT1 and/or IKZF1 was identified in 6/10 (60%) stage IV patients and 1/3 (33%) stage III patients.ConclusionsBased on a thorough evaluation of five bisulfite conversion kits and three cfDNA isolation kits, both individually and in combination, the CNA kit and the EpiTect kit were identified as the best-performing kit combination, with highest DNA yield and recovery across a range of DNA input amounts. The combination was successfully used for detection of clinically relevant DNA methylation biomarkers in plasma from cancer patients.

An Efficient Extraction Method Allowing the Genetic Evaluation of Host DNA from Samples Collected for Virus Infection Diagnosis in Viral Transport Medium.

Introduction: During the COVID-19 pandemic, an extraordinary number of nasopharyngeal secretion samples inoculated in viral transport medium (VTM) were collected and analyzed to detect SARS-CoV-2 infection. In addition to viral detection, those samples can also be a source of host genomic material, providing excellent opportunities for biobanking and research. Objective: To describe a simple, in-house-developed DNA extraction method to obtain high yield and quality genomic DNA from VTM samples for host genetic analysis and assess its relative efficiency by comparing its yield and suitability to downstream applications to two different commercial DNA extraction kits. Methods: In this study, 13 VTM samples were processed by two commercial silica-based kits and compared with an in-House-developed protocol for host DNA extraction. An additional 452 samples were processed by the in-House method. The quantity and quality of the differentially extracted DNA samples were assessed by Qubit and spectrophotometric measurements. The suitability of extracted samples for downstream applications was tested by polymerase chain reaction (PCR) amplification followed by amplicon sequencing and allelic discrimination in real-time PCR. Results: The in-House method provided greater median DNA yield (0.81 μg), being significantly different from the PureLink® method (0.14 μg, p < 0.001), but not from the QIAamp® method (0.47 μg, p = 0.980). Overall satisfactory results in DNA concentrations and purity, in addition to cost, were observed using the in-House method, whose samples were able to produce clear amplification in PCR and sequencing reads, as well as effective allelic discrimination in real-time PCR TaqMan® assay. Conclusion: The described in-House method proved to be suitable and economically viable for genomic DNA extraction from VTM samples for biobanking purposes. These results are extremely valuable for the study of the COVID-19 pandemic and other emergent infectious diseases, allowing host genetic studies to be performed in samples initially collected for diagnosis.

AirDNA sampler: An efficient and simple device enabling high-yield, high-quality airborne environment DNA for metagenomic applications.

Analyzing temporal and spatial distributions of airborne particles of biological origins is vital for the assessment and monitoring of air quality, especially with regard to public health, environmental ecology, and atmospheric chemistry. However, the analysis is frequently impeded by the low levels of biomass in the air, especially with metagenomic DNA analysis to explore diversity and composition of living organisms and their components in the air. To obtain sufficient amounts of metagenomic DNA from bioaerosols, researchers usually need a long sampling time with an expensive high-volume air sampler. This work shows the utilization of an air sampling device containing an economical, high-volume portable ventilation fan in combination with customized multi-sheet filter holders to effectively obtain high yields of genomic DNA in a relatively short time. The device, named 'AirDNA' sampler, performed better than other commercial air samplers, including MD8 Airport and Coriolis compact air samplers. Using the AirDNA sampler, an average DNA yield of 40.49 ng (12.47-23.24 ng at 95% CI) was obtained in only 1 hour of air sampling with a 0.85 probability of obtaining ≥10 ng of genomic DNA. The genomic DNA obtained by the AirDNA system is of suitable quantity and quality to be further used for amplicon metabarcoding sequencing of 16S, 18S, and cytochrome c oxidase I (COI) regions, indicating that it can be used to detect various prokaryotes and eukaryotes. Our results showed the effectiveness of our AirDNA sampling apparatus with a simple setup and affordable devices to obtain metagenomic DNA for short-term or long-term spatiotemporal analysis. The technique is well suited for monitoring air in built environments, especially monitoring bioaerosols for health purposes and for fine-scale spatiotemporal environmental studies.

DNA extraction protocol impacts ocular surface microbiome profile.

The aim of this study is to provide a reference frame to allow the comparison and interpretation of currently published studies on 16S ribosomal ribonucleic acid amplicon sequencing of ocular microbiome samples using different DNA extraction protocols. Alongside, the quantitative and qualitative yield and the reproducibility of different protocols has been assessed. Both eyes of 7 eligible volunteers were sampled. Five commercially available DNA extraction protocols were selected based on previous publications in the field of the ocular surface microbiome and 2 host DNA depletion protocols were added based on their reported effective host DNA depletion without significant reduction in bacterial DNA concentration. The V3-V4 region of the 16S rRNA gene was targeted using Illumina MiSeq sequencing. The DADA2 pipeline in R was used to perform the bio-informatic processing and taxonomical assignment was done using the SILVA v132 database. The Vegdist function was used to calculate Bray-Curtis distances and the Galaxy web application was used to identify potential metagenomic biomarkers via linear discriminant analysis Effect Size (LEfSe). The R package Decontam was applied to control for potential contaminants. Samples analysed with PowerSoil, RNeasy and NucleoSpin had the highest DNA yield. The host DNA depletion kits showed a very low microbial DNA yield; and these samples were pooled per kit before sequencing. Despite pooling, 1 of both failed to construct a library.Looking at the beta-diversity, clear microbial compositional differences - dependent on the extraction protocol used - were observed and remained present after decontamination. Eighteen genera were consistently retrieved from the ocular surface of every volunteer by all non-pooled extraction kits and a comprehensive list of differentially abundant bacteria per extraction method was generated using LefSe analysis. High-quality papers have been published in the field of the ocular surface microbiome but consensus on the importance of the extraction protocol used are lacking. Potential contaminants and discriminative genera per extraction protocol used, were introduced and a reference frame was built to facilitate both the interpretation of currently published papers and to ease future choice - making based on the research question at hand.

Evaluation of DNA Extraction Methods for Reliable Quantification of Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa

Detection and quantification of DNA biomarkers relies heavily on the yield and quality of DNA obtained by extraction from different matrices. Although a large number of studies have compared the yields of different extraction methods, the repeatability and intermediate precision of these methods have been largely overlooked. In the present study, five extraction methods were evaluated, using digital PCR, to determine their efficiency in extracting DNA from three different Gram-negative bacteria in sputum samples. The performance of two automated methods (GXT NA and QuickPick genomic DNA extraction kit, using Arrow and KingFisher Duo automated systems, respectively), two manual kit-based methods (QIAamp DNA mini kit; DNeasy UltraClean microbial kit), and one manual non-kit method (CTAB), was assessed. While GXT NA extraction kit and the CTAB method have the highest DNA yield, they did not meet the strict criteria for repeatability, intermediate precision, and measurement uncertainty for all three studied bacteria. However, due to limited clinical samples, a compromise is necessary, and the GXT NA extraction kit was found to be the method of choice. The study also showed that dPCR allowed for accurate determination of extraction method repeatability, which can help standardize molecular diagnostic approaches. Additionally, the determination of absolute copy numbers facilitated the calculation of measurement uncertainty, which was found to be influenced by the DNA extraction method used.

Validation of the InnoXtract™ DNA extraction method for bone, teeth, and rootless hair

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.

High Yield of Pleural Cell-Free DNA for Diagnosis of Oncogenic Mutations in Lung Adenocarcinoma

Pleural cytology is currently used to assess targetable mutations in patients with advanced lung adenocarcinoma. However, it is fraught with low diagnostic yield. Can pleural cell-free DNA (cfDNA) be used to assess targetable mutations in lung adenocarcinoma patients with malignant pleural effusions (MPE)? Patients with lung adenocarcinoma MPE were recruited prospectively between January 2017 and September 2021. Oncogenic mutations were assessed by treating providers using pleural fluid cytology or lung cancer biopsies. Pleural and plasma cfDNA were used to assess the mutations using next-generation sequencing (NGS). Fifty-four pleural fluid samples were collected from 42 patients. The diagnostic yield to detect oncogenic mutations for pleural cfDNA, pleural cytology, biopsy, and plasma cfDNA was 49/54 (90.7%), 16/33 (48.5%), 22/25 (88%), and 24/32 (75%), respectively, P< .001. The agreement of mutations in positive samples between pleural cfDNA and pleural cytology was 100%, whereas the agreement of pleural cfDNA with biopsies was 89.4%. The median concentration (interquartile range) of pleural cfDNA was higher than plasma: 28,444 (4,957-67,051) vs2,966.5 (2,167-5,025) copies of amplifiable DNA per mL, P< .01. Median of 5mL (interquartile range, 4.5-5) of pleural fluid supernatant was adequate for cfDNA testing. The diagnostic yield of pleural cfDNA NGS for oncogenic mutations in lung adenocarcinoma patients is comparable to tumor biopsies and higher than pleural cytology and plasma cfDNA. The pleural cfDNA can be longitudinally collected, can be readily incorporated in clinical workflow, and may decrease the need for additional biopsies.

Comparison of two affordable DNA extraction methods for molecular detection of Salmonella isolates from broiler farm’s boot swabs

There are many extraction methods available to purify bacterial DNA. PCR efficiency can vary depending on the quantity and quality of the template DNA used. This study evaluated the efficiency, quality, cost and rapidness of two in-house protocols, silica particles and Chelex-100 resin, for the extraction of twenty Salmonella isolates from boot swabs. The aim of this experiment was to compare the extraction methods for Salmonella spp. detection. DNA extraction was performed for each method and subjected to real-time PCR amplification. The amounts and integrity of DNA were determined using spectrophotometry and agarose gel electrophoresis, and the efficiency measured with real-time PCR. Limit of Detection (LOD) was determined with serial dilutions of a S. Typhimurium reference strain resulting in linear regression coefficients of R 2=0.992 (efficiency 119.31) for silica and R 2=0.958 (efficiency 93.33) for Chelex, with LOD of 10 -4 for both. Silica particles method resulted in higher DNA yield, 85.01 ± 59.11 compared to 50.74 ± 12.95 and 260/280 ratio, 1.77 ± 0.02 versus 1.63 ± 0.13. DNA integrity was superior using silica, gel showed a unique band higher than 2.000 bp, while Chelex-100 was imperceptive or degraded. Regarding PCR results, the mean quantification cycle (Cq) for silica was 17.08 ± 0.73 and Chelex-100, 17.64 ±0.56 (suggesting lower DNA values). Results showed that both methods were effective for the DNA extraction of Salmonella, once PCR resulted positive for all samples, efficiency was higher for silica. Chelex-100 resin was performed in less time at a lower cost.

Protocol for the High-quality Plasmid Isolation from Different Recalcitrant Bacterial Species: Agrobacterium spp., Rhizobium sp., and Bacillus thuringiensis.

High yield of good quality plasmid DNA from gram -ve bacteria (Agrobacterium tumefaciens, A. rhizogenes, and Rhizobium sp.) and gram +ve bacterium (Bacillus thuringiensis) is difficult. The widely used plasmid extraction kits for Escherichia coli yield a low quantity of poor-quality plasmid DNA from these species. We have optimized an in-house modification of the QIAprep Spin Miniprep kit protocol of Qiagen, consisting of two extraction steps. In the first, the centrifugation after adding neutralization buffer is followed by ethanol (absolute) precipitation of plasmid DNA. In the second extraction step, the precipitated DNA is dissolved in Tris-EDTA (TE) buffer, followed by an addition of 0.5 volumes of 5 M sodium chloride and 0.1 volumes of 20% (w/v) sodium dodecyl sulfate. After incubation at 65 °C for 15 min, the plasmid DNA is extracted with an equal volume of chloroform:isoamyl alcohol (CIA). RNase (20 mg/mL) is added to the upper phase retrieved after centrifugation and is incubated at 37 °C for 15 min. The extraction of the plasmid DNA with an equal volume of CIA is followed by centrifugation and is precipitated from the retrieved upper phase by adding an equal volume of absolute ethanol. The pellet obtained after centrifugation is washed twice with 70% (v/v) ethanol, air dried, dissolved in TE buffer, and quantified. This easy-to-perform protocol is free from phenol extraction, density gradient steps, and DNA binding columns, and yields high-quality plasmid DNA. The protocol opens an easy scale up to yield a large amount of high-quality plasmid DNA, useful for high-throughput downstream applications. Key features The protocol is free from density gradient steps and use of phenol. The protocol is an extension of the QIAprep Spin Miniprep kit (Qiagen) and is applicable for plasmid DNA isolation from difficult-to-extract bacterial species. The protocol facilitates the direct transformation of the ligation product into Agrobacterium by skipping the step of E. coli transformation. The plasmids isolated are of sequencing grade and the method is useful for extracting plasmids for metagenomic studies. Graphical overview Overview of the plasmid isolation protocol (modified QIAprep Spin Miniprep kit) of the present study.