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Background: Optimizing DNA extraction for the preservation of telomere length – as a biomarker for ageing and vari-ous diseases – is highly important. Long-term stored blood samples are considered essential resources for genetic studies. In this study, different lysis buffers were used along with CTAB extraction buffer to inves-tigate the quality of extracted DNA and the effect of its accompanying inhibitors on quantitative PCR (q-PCR) in telomere studies. Materials and Methods: DNA extraction was performed using six RBC lysis buffers and CTAB- based extraction buffer. DNA integrity was evaluated by gel electrophoresis, quantity by absorbance at 260 nm and its purity with expected values of 1.8 and 2-2.2 for the ratios of A260/A280 and A260/A230. The quantitative effect of each buffer in q-PCR and the repeatability of the results were assessed by calculating the reaction efficiency, coefficient of determination (R²), and percentage of coefficient of variation (%CV). Results: Buffer number 5 (10 mM Tris-HCl, pH 7.6, 50 mM NaCl) yielded the highest amount of DNA extraction (324.93 ng/ml, %CV 11.53). All the extracted DNA samples were pure, as indicated by the acceptable A260/A280 and A260/A230 ratios (p>0.05). Gel analysis revealed that the extracted DNA of all the buffers except one was intact. The DNA molecule extracted with buffer number 1 (155 mM NH4Cl, 10 mM KHCO3, and 5 mM EDTA) showed the best performance in q- PCR for HBG gene (efficiency=0.126, R²=0.97) and telomere (efficiency=0.99, R²=0.99). Conclusion: The DNA molecule extracted from frozen blood samples by buffer number 1 showed the least q-PCR inhibition for telomere study. [ABSTRACT FROM AUTHOR]

Puparia are commonly found in tsetse fly larviposition sites during studies on larval ecology. This chitinous shell is representative of past or ongoing exploitation of these sites by tsetse flies. The morphological characteristics of the puparium are not sufficiently distinctive to allow identification of the species. This study explores the applicability of biomolecular techniques on empty puparia for tsetse fly species identification. Five techniques were compared for DNA extraction from tsetse fly puparia, 1/Chelex® 100 Resin, 2/CTAB, 3/Livak's protocol, 4/DEB + proteinase K and 5/QIAamp® DNA Mini kit, using two homogenisation methods (manual and automated). Using a combination of two primer pairs, Chelex, CTAB, and DEB + K proved the most efficient on fresh puparia with 90, 85, and 70% samples identified, respectively. Shifting from fresh to one- to nine-month-old puparia, the Chelex method gave the best result allowing species identification on puparia up to seven months old. The subsequent testing of the Chelex extraction protocol identified 152 (60%) of 252 field-collected puparia samples at species level. The results show that reliable genetic identification of tsetse flies species can be performed from empty puparia, what can prove of great interest for future ecological studies on larviposition sites. The Chelex technique was the most efficient for DNA extraction, though the age-limit of the samples stood at seven months, beyond which DNA degradation probably compromises the genetic analysis. [ABSTRACT FROM AUTHOR]

Porcine adulteration in dairy products for economic purposes has been tackled mostly by protein- and lipid-based porcine detection methods. In this study, a DNA-based approach was used to determine the presence of porcine DNA in dairy products, especially butter and cheese. DNA was extracted by optimizing the conventional CTAB extraction method followed by an assessment of DNA fragmentation by real-time PCR assays targeting different sizes of amplicon. Butter and cheese DNA samples were analyzed using both endogenous and porcine-specific real-time PCR assays and the lard content was estimated based on the linear regression analysis of a series of different percentages of lard-adulterated butter and cheese DNA samples. Even though the optimization steps did not improve the DNA yield, the successful amplification of the 200 bp amplicon depicted high DNA quality. Approximately 1% lard was able to be quantified exhibiting high potential of the assay in quantifying porcine content in lard-adulterated highly processed food products. [ABSTRACT FROM AUTHOR]

Cronobacter sakazakii (Cz) infections linked with powdered milk/flour (PMF) are on the increase in recent times. The current study aimed at assessing worldwide and regional prevalence of Cz in PMF. Cz-PMF-directed data were conscientiously mined in four mega-databases via topic-field driven PRISMA protocol without any restriction. Bivariate analysis of datasets was conducted and then fitted to random-intercept logistic mixed-effects regressions with leave-one-study-out-cross-validation (LOSOCV). Small-study effects were assayed via Egger's regression tests. Contributing factors to Cz contamination/detection in PMF were determined using 1000-permutation-bootstrapped meta-regressions. A total of 3761 records were found out of which 68 studies were included. Sample-size showed considerable correlation with Cz positivity (r = 0.75, p = 2.5e−17), Milkprod2020 (r = 0.33, p = 1.820e−03), and SuDI (r = − 0.30, p = 4.11e−03). The global prevalence of Cz in PMF was 8.39% (95%CI 6.06–11.51, PI: 0.46–64.35) with LOSOCV value of 7.66% (6.39–9.15; PI: 3.10–17.70). Cz prevalence in PMF varies significantly (p < 0.05) with detection methods, DNA extraction method, across continents, WHO regions, and world bank regions. Nation, detection method, world bank region, WHO region, and sample size explained 53.88%, 19.62%, 19.03%, 15.63%, and 9.22% of the true differences in the Cz prevalence in PMF, respectively. In conclusion, the results indicated that national will power in the monitoring and surveillance of Cz in PMF matched with adequate sample size and appropriate detection methods will go a long way in preventing Cz contamination and infections. [ABSTRACT FROM AUTHOR]

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Introduction: The increasing global pressure to explore alternative protein sources derived from animal by-products has opened-up opportunities, but it has also created the need to assess their compliance with labelling statements, to ensure consumer's trust in the composition of both feed and food products. Assessing the authenticity of highly processed animal by-products, particularly within the rapidly expanding Halal food market, presents a significant challenge due to the lack of robust and standardized methodologies. However, the success of DNA based authenticity system is highly dependent on the extracted DNA quantity, quality, and purity ratios from heterogeneous matrices. Material and methods: In this work, nine DNA extraction methods were tested on selected processed animal by-products with high-value and interest for the feed industry: meals from poultry meat, blood and feather, and hydrolysates from swine meat and bone, fish, and black soldier fly. The proposed DNA extraction methods are developed to specifically target swine-specific mitochondrial region, as a case study. Results and discussion: Both the conventional CTAB method and the commercial kits, specifically Invisorb® Spin Tissue Mini and NucleoSpin™ Food, demonstrated superior extraction efficiency and quality ratios. Nevertheless, commercial kits enabled faster detection in comparison to the conventional methods. The absence of swine DNA was successfully validated and confirmed in all animal meals and hydrolysates that did not contain swine in their composition beforehand, demonstrating their compliance with the Halal market requirements. [ABSTRACT FROM AUTHOR]

Aims Many countries are in the process of designing a deep geological repository (DGR) for long-term storage of used nuclear fuel. For several designs, used fuel containers will be placed belowground, with emplacement tunnels being backfilled using a combination of highly compacted powdered bentonite clay buffer boxes surrounded by a granulated "gapfill" bentonite. To limit the potential for microbiologically influenced corrosion of used fuel containers, identifying conditions that suppress microbial growth is critical for sustainable DGR design. This study investigated microbial communities in powdered and gapfill bentonite clay incubated in oxic pressure vessels at dry densities between 1.1 g cm−3 (i.e. below repository target) and 1.6 g cm−3 (i.e. at or above repository target) as a 1-year time series. Results Our results showed an initial (i.e. 1 month) increase in the abundance of culturable heterotrophs associated with all dry densities <1.6 g cm−3, which reveals growth during transient low-pressure conditions associated with the bentonite saturation process. Following saturation, culturable heterotroph abundances decreased to those of starting material by the 6-month time point for all 1.4 and 1.6 g cm−3 pressure vessels, and the most probable numbers of culturable sulfate-reducing bacteria (SRB) remained constant for all vessels and time points. The 16S rRNA gene sequencing results showed a change in microbial community composition from the starting material to the 1-month time point, after which time most samples were dominated by sequences associated with Pseudomonas, Bacillus, Cupriavidus , and Streptomyces. Similar taxa were identified as dominant members of the culture-based community composition, demonstrating that the dominant members of the clay microbial communities are viable. Members of the spore-forming Desulfosporosinus genus were the dominant SRB for both clay and culture profiles. Conclusions After initial microbial growth while bentonite was below target pressure in the early phases of saturation, microbial growth in pressure vessels with dry densities of at least 1.4 g cm−3 was eventually suppressed as bentonite neared saturation. [ABSTRACT FROM AUTHOR]

A hybrid material, consisting of commercially available nitrocellulose (NC) membrane non-covalently modified with amino-polyethylene glycol functionalized reduced graphene oxide (NH2-PEG-rGO) nanoparticles, was successfully synthesized for oligonucleotide extraction. Fourier Transform Infrared Spectroscopy (FTIR) confirmed the modification of the NC membrane, revealing characteristic peaks of both compounds, i.e., NC and NH2-PEG-rGO. Scanning Electron Microscopy (SEM) exhibited morphological changes in the NC/NH2-PEG-rGO hybrid membrane, marked by the introduction of NH2-PEG-rGO particles, resulting in a distinctly smothered surface compared to the porous surface of the NC control membrane. Wettability assays revealed hydrophobic behavior for the NC/NH2-PEG-rGO hybrid membrane, with a water contact angle exceeding 90°, contrasting with the hydrophilic behavior characterized by a 16.7° contact angle in the NC membrane. The performance of the NC/NH2-PEG-rGO hybrid membrane was evaluated for the extraction of ssDNA with fewer than 50 nucleotides from solutions containing various ionic species (MnCl2, MgCl2, and MnCl2/MgCl2). The NC/NH2-PEG-rGO hybrid membrane exhibited optimal performance when incubated in MgCl2, presenting the highest fluorescence emission at 525 relative fluorescence units (r.f.u.). This corresponds to the extraction of approximately 610 pg (≈13%) of the total oligo-DNA, underscoring the efficacy of the pristine material, which extracts 286 pg (≈6%) of oligo-DNA in complex solutions. [ABSTRACT FROM AUTHOR]

Central to the domain of molecular biology resides the foundational process of DNA extraction and purification, a cornerstone underpinning a myriad of pivotal applications. In this research, we introduce a DNA extraction and purification technique leveraging polypropylene (PP) threads. The process commences with robust cell lysis achieved through the vigorous agitation of interwoven PP threads. The friction between the threads facilitates cell lysis especially those microbes having tough cell wall. For purification of DNA, thread-based isotachophoresis was employed which makes the whole process swift and cost-effective. Lysed cell-laden threads were submerged in a trailing electrolyte which separated DNA from other cellular contents. The process was performed with a tailored ITP device. An electric field directs DNA, cell debris, trailing electrolyte, and leading electrolyte toward the anode. Distinct ion migration resulted in DNA concentrating on the PP thread's anode-proximal region. The SYBR green dye is used to visualize DNA as a prominent green zone under blue light. The purified DNA exhibits high purity levels of 1.82 ± 0.1 (A260/A280), making it suitable for various applications aiming at nucleic acid detection. [ABSTRACT FROM AUTHOR]

ABSTRACT Monitoring the levels of opportunistic pathogens in drinking water is important to plan interventions and understand the ecological niches that allow them to proliferate. Quantitative PCR is an established alternative to culture methods that can provide a faster, higher-throughput, and more precise enumeration of the bacteria in water samples. However, PCR-based methods are still not routinely applied for Legionella monitoring, and techniques, such as DNA extraction, differ notably between laboratories. Here, we quantify the impact that DNA extraction methods had on downstream PCR quantification and community sequencing. Through a community science campaign, we collected 50 water samples and corresponding shower hoses, and compared two commonly used DNA extraction methodologies to the same biofilm and water phase samples. The two methods showed clearly different extraction efficacies, which were reflected in both the quantity of DNA extracted and the concentrations of Legionella enumerated in both the matrices. Notably, one method resulted in higher enumeration in nearly all samples by about one order of magnitude and detected Legionella in 21 samples that remained undetected by the other method. 16S rRNA amplicon sequencing revealed that the relative abundance of individual taxa, including sequence variants of Legionella, significantly varied depending on the extraction method employed. Given the implications of these findings, we advocate for improvement in documentation of the performance of DNA extraction methods used in drinking water to detect and quantify Legionella, and characterize the associated microbial community.IMPORTANCEMonitoring for the presence of the waterborne opportunistic pathogen Legionella is important to assess the risk of infection and plan remediation actions. While monitoring is traditionally carried on through cultivation, there is an ever-increasing demand for rapid and high-throughput molecular-based approaches for Legionella detection. This paper provides valuable insights on how DNA extraction affects downstream molecular analysis such as the quantification of Legionella through droplet digital PCR and the characterization of natural microbial communities through sequencing analysis. We analyze the results from a risk-assessment, legislative, and ecological perspective, showing how initial DNA processing is an important step to take into account when shifting to molecular-based routine monitoring and discuss the central role of consistent and detailed reporting of the methods used.

BackgroundLeprosy, a chronic infectious disease caused by Mycobacterium leprae, continues to pose a public health challenge in many parts of the world. Early and accurate diagnosis is crucial for effective treatment and prevention of disabilities associated with the disease. Molecular techniques such as PCR have demonstrated great potential as a diagnostic tool for directly detecting M. leprae DNA in different clinical samples, providing better sensitivity and specificity than conventional diagnostic techniques. The objective of this study was to measure the amount of M. leprae DNA in leprosy patients’ urine samples using the Rlep gene target through qPCR.MethodsDifferent clinical samples such as smear, blood, and urine samples were collected from leprosy patients and healthy individuals. Leprosy patients were classified by the Ridley–Jopling classification. The Ziehl–Neelsen staining method was used for the slit skin smear (SSS) samples, and the bacteriological index (BI) was calculated for leprosy patients. DNA extraction and qPCR were performed for all three types of clinical samples using the Rlep gene target.ResultsThe Mycobacterial leprae DNA was successfully detected and quantified in all clinical samples across all types of leprosy among all the study groups using the Rlep gene (129 bp) target. The Rlep gene target was able to detect the presence of M. leprae DNA in 100% of urine, 96.1% of blood, and 92.2% of SSS samples of leprosy patients. Urine samples showed significant differences (p < 0.001) between the control and the different clinical forms and between borderline tuberculoid (BT) and pure neuritic leprosy (PNL) cases. There are significant differences in cycle threshold (Ct) values between control cases and clinical categories (p < 0.001), as well as specific differences within clinical categories (p < 0.001), reflecting the variability in bacterial load and detection sensitivity across different sample types and clinical manifestations of leprosy.ConclusionOverall, this study's findings suggest that the qPCR technique can be used to detect M. leprae DNA in urine samples of leprosy patients using the Rlep gene target. It can also be used for diagnosing the disease and monitoring the effectiveness of anti-leprosy drugs, including multi-drug therapy (MDT), across various leprosy disease groups.

The objective of this study was to develop a DNA-based method for the identification and tracking of edible oils, which is important for health management. Three different DNA extraction methods (CTAB, MBST kit, and manual hexane-based method) were used to obtain high-purity DNA from crude and refined soybean, maize, and canola oils. PCR was then conducted using specific primers to identify the presence of genes related to each oil type and to assess transgenicity. The results showed that DNA was present in crude and refined oils, but in very low amounts. However, using method 3 for DNA extraction provided sufficient quantity and quality of DNA for successful PCR amplification. The study concluded that the main challenge in DNA extraction from oils is the presence of PCR inhibitors, which can be overcome using the manual hexane-based method. Also, the examination of protein presence in the oils using SDS-PAGE did not indicate any protein bands.

ABSTRACT Microbiome studies are becoming larger in size to detect the potentially small effect that environmental factors have on our gut microbiomes, or that the microbiome has on our health. Therefore, fast and reproducible DNA isolation methods are needed to handle thousands of fecal samples. We used the Chemagic 360 chemistry and Magnetic Separation Module I (MSMI) instrument to compare two sample preservatives and four different pre-treatment protocols to find an optimal method for DNA isolation from thousands of fecal samples. The pre-treatments included bead beating, sample handling in tube and plate format, and proteinase K incubation. The optimal method offers a sufficient yield of high-quality DNA without contamination. Three human fecal samples (adult, senior, and infant) with technical replicates were extracted. The extraction included negative controls (OMNIgeneGUT, DNA/RNA shield fluid, and Chemagic Lysis Buffer 1) to detect cross-contamination and ZymoBIOMICS Gut Microbiome Standard as a positive control to mimic the human gut microbiome and assess sensitivity of the extraction method. All samples were extracted using Chemagic DNA Stool 200 H96 kit (PerkinElmer, Finland). The samples were collected in two preservatives, OMNIgeneGUT and DNA/RNA shield fluid. DNA quantity was measured using Qubit-fluorometer, DNA purity and quality using gel electrophoresis, and taxonomic signatures with 16S rRNA gene-based sequencing with V3V4 and V4 regions. Bead beating increased bacterial diversity. The largest increase was detected in gram-positive genera Blautia, Bifidobacterium, and Ruminococcus. Preservatives showed minor differences in bacterial abundances. The profiles between the V3V4 and V4 regions differed considerably with lower diversity samples. Negative controls showed signs from genera abundant in fecal samples. Technical replicates of the Gut Standard and stool samples showed low variation. The selected isolation protocol included recommended steps from manufacturer as well as bead beating. Bead beating was found to be necessary to detect hard-to-lyse bacteria. The protocol was reproducible in terms of DNA yield among different stool replicates and the ZymoBIOMICS Gut Microbiome Standard. The MSM1 instrument and pre-treatment in a 96-format offered the possibility of automation and handling of large sample collections. Both preservatives were feasible in terms of sample handling and had low variation in taxonomic signatures. The 16S rRNA target region had a high impact on the composition of the bacterial profile.IMPORTANCENext-generation sequencing (NGS) is a widely used method for determining the composition of the gut microbiota. Due to the differences in the gut microbiota composition between individuals, microbiome studies have expanded into large population studies to maximize detection of small effects on microbe–host interactions. Thus, the demand for a rapid and reliable microbial profiling is continuously increasing, making the optimization of high-throughput 96-format DNA extraction integral for NGS-based downstream applications. However, experimental protocols are prone to bias and errors from sample collection and storage, to DNA extraction, primer selection and sequencing, and bioinformatics analyses. Methodological bias can contribute to differences in microbiome profiles, causing variability across studies and laboratories using different protocols. To improve consistency and confidence of the measurements, the standardization of microbiome analysis methods has been recognized in many fields.

Fish species are commonly used as experimental models in the laboratory. DNA is routinely collected from these animals to permit identification of their genotype. The current standard procedure to sample DNA is fin clipping, which involves anaesthetising individuals and removing a portion of the caudal fin. While fin clipping reliably generates good quality DNA samples for downstream applications, there is evidence that it can alter health and welfare, and impact the fish’s behaviour. This in turn can result in greater variation in the data collected. In a recent study we adapted a skin swabbing protocol to collect DNA from small-bodied fish, including sticklebacks and zebrafish, without the use of analgesics, anaesthetics or sharp instruments. A rayon-tipped swab was used to collect mucus from the flank of the fish, which was then used for DNA extraction. We subsequently demonstrated that compared to fin clipping, skin swabbing triggered fewer changes in stress axis activation and behaviour. We also found that gene expression and behaviour data collected from swabbed fish were less variable than similar data collected from fish that had been fin clipped. This potentially allows smaller sample sizes in experimental groups to be used after skin swabbing, thereby reducing animal use. Here we provide a detailed protocol explaining how to collect DNA samples from small laboratory fish using skin swabs.

The aim is to evaluate the effect of viral inactivation methods on the quality and quantity of compounds in dried blood spots (DBS).

Abstract Objective Roller swabbing of surfaces is an effective way to obtain environmental DNA, but the current DNA extraction method for these samples is equipment heavy, time consuming, and increases potential contamination through multiple handling. Here, we used rollers to swab a dog kennel and compared three DNA extraction approaches (water filtration, roller trimming and direct buffer) using two different platforms (Qiacube, Kingfisher). DNA extraction methods were evaluated based on cost, effort, DNA concentration and PCR result. Results The roller trim method emerged as the optimal method with the best PCR results, DNA concentration and cost efficiency, while the buffer-based methods were the least labour intensive but produced mediocre PCR results and DNA concentrations. Additionally, the Kingfisher magnetic bead extractions generally ranked higher in all categories over the Qiacube column-based DNA extractions. Ultimately, the ideal DNA extraction method for a particular study is influenced by logistical constraints in the field such as the size of the roller, the availability of cold storage, and time constraints on the project. Our results demonstrate the strengths and weaknesses of each approach, allowing for informed decision making by researchers.

Background: Formalin-fixed paraffin-embedded (FFPE) specimen archives are a valuable source of sample material for molecular biological analysis. However, the DNA isolated from FFPE samples is usually low in concentration and fragmented. Thus, it is necessary to optimize the FFPE DNA extraction protocol to obtain the best results. Proteinase K incubation is undoubtedly crucial in DNA extraction procedures, but this step is often not well explained in the manufacturer’s manual. Purpose: This study aimed to find the optimal duration for proteinase K incubation protocols to achieve the highest DNA yields. Methods: Fifteen paraffin blocks of Oral Squamous Cell Carcinoma (OSCC) specimens were obtained, and the cancerous areas were microdissected into smaller cuts for DNA extraction. The samples were randomly divided into three groups (n=5) and subjected to three different proteinase K incubation protocols: one-hour incubation at 56ºC as per the manufacturer’s instructions (Group I), 24-hour incubation at 56ºC (Group II), and 48 hours at room temperature with an additional four hours at 56ºC (Group III). The extracted DNA was then quantified using a Nanodrop spectrophotometer. The recorded data were analyzed using ANOVA-LSD. Results: The highest DNA concentration was found in Group III (107.74 ± 41.92), which was significantly higher compared to Group II (59.46 ± 30.32) and Group I (6.46 ± 1.97) (p

Technological advancements in genome sequencing, assembly and annotation platforms and algorithms that resulted in several genomic studies have created an opportunity to further our understanding of the biology of phytopathogens, including Armillaria species. Most Armillaria species are facultative necrotrophs that cause root- and stem-rot, usually on woody plants, significantly impacting agriculture and forestry worldwide. Genome sequencing, assembly and annotation in terms of samples used and methods applied in Armillaria genome projects are evaluated in this review. Infographic guidelines and a database of resources to facilitate future Armillaria genome projects were developed. Knowledge gained from genomic studies of Armillaria species is summarized and prospects for further research are provided. This guide can be applied to other diploid and dikaryotic fungal genomics.

Isolation of high quality and quantity genomic DNA is essential for molecular studies. It is crucial to select a non-invasive and straightforward technique to ensure the efficient collection of DNA, particularly at the farm level. The aim of this study was to determine if nasal swabs are an appropriate biological matrix to obtain good quality genomic DNA suitable for SNP genotyping. In this study, two biological matrices (blood and nasal swabs) were evaluated and compared for the isolation of genomic DNA obtained from 15 female Texel sheep. DNA quality and quantity were assessed using spectrophotometry and gel electrophoreses. Genotype concordance rates were used for comparison. Results showed that the highest concentration mean was obtained from blood samples (159.14 ng/µl), while from nasal swab samples the concentration mean was lower (130.12 ng/µl), but the difference was non-significant. Regarding purity, DNA obtained from nasal swabs presented a higher A260/A280 ratio (1.96), while the one obtained from blood samples was 1.90. Total DNA yield obtained from blood samples (15.91 µg) was significantly higher than the one obtained from nasal swabs (6.51). Blood and nasal swab genotyping concordance rates were high (mean = 0.984). In conclusion, our results indicate that nasal swabs can yield good quality DNA; however, the DNA extraction protocol should be optimized.

ABSTRACTThis manuscript describes the development of a streamlined, cost-effective laboratory workflow to meet the demands of increased whole genome sequence (WGS) capacity while achieving mandated quality metrics. From 2020 to 2021, the Wadsworth Center Bacteriology Laboratory (WCBL) used a streamlined workflow to sequence 5,743 genomes that contributed sequence data to nine different projects. The combined use of the QIAcube HT, Illumina DNA Prep using quarter volume reactions, and the NextSeq allowed the WCBL to process all samples that required WGS while also achieving a median turn-around time of 7 days (range 4 to 10 days) and meeting minimum sequence quality requirements. Public Health Laboratories should consider implementing these methods to aid in meeting testing requirements within budgetary restrictions.IMPORTANCEPublic Health Laboratories that implement whole genome sequencing (WGS) technologies may struggle to find the balance between sample volume and cost effectiveness. We present a method that allows for sequencing of a variety of bacterial isolates in a cost-effective manner. This report provides specific strategies to implement high-volume WGS, including an innovative, low-cost solution utilizing a novel quarter volume sequencing library preparation. The methods described support the use of high-throughput DNA extraction and WGS within budgetary constraints, strengthening public health responses to outbreaks and disease surveillance.

Abstract Marine sediments as excellent climate archives, contain among other biomolecules substantial amounts of extracellular DNA. Through mineral binding, some of the DNA remains protected from degradation which aids its preservation. While this pool of DNA represents genomic ecosystem fingerprints spanning over millions of years, the capability of current DNA extraction methods in recovering mineral‐bound DNA remains poorly understood. We evaluated current sedimentary DNA extraction approaches and their ability to recover short DNA fragments from artificially created DNA‐mineral complexes involving pure clay minerals or quartz, as well as from different types of natural marine sediments. We separately investigated lysis (DNA release) and isolation steps (purification of DNA) comparing five different lysis buffers across two commonly used DNA isolation approaches: silica magnetic beads and liquid‐phase organic extraction and purification. The choice of lysis buffer significantly impacted the amount of recovered mineral‐bound DNA and facilitated selective desorption of DNA fragments. High molarity EDTA and phosphate lysis buffers recovered on average an order of magnitude more DNA from clay minerals than other tested buffers, while both isolation approaches recovered comparable amounts of DNA. In marine sediments, however, liquid‐phase organic extraction caused inhibitory effects in subsequent downstream applications (e.g., PCR), across all assessed DNA extracts, while silica magnetic beads induced inhibition only in half of the tested DNA extracts. Thus, the isolation approach, together with the lysis buffer, played a decisive role in successful library preparation with lysis buffer choice ultimately impacting final library fragment distribution. With this study, we underscore the critical importance of lysis buffer selection to maximize the recovery of mineral‐bound DNA and show its profound impact on recovered fragment lengths in sedimentary DNA extractions, a crucial factor alongside existing isolation approaches in facilitating high‐quality DNA extracts for downstream analysis related to ancient environmental DNA research.

In this study, the ability of the Quick-DNA™ Tissue/Insect Miniprep Kit and Chelex® methods to extract DNA from O. niloticus skin, muscle, and gill tissue was compared. The quantity and purity of the DNA were measured with a NanoDrop spectrophotometer. Based on the results obtained, it appears that the DNA extracted using the Kit has good quality based on A260/280 (1.67–1.98), and the Chelex method (1.52-1.81) was acceptable. ANOVA for the amount of nucleic acid revealed a significant difference between muscle and skin with gill tissue (P < 0.05). However, the skin of O. niloticus subjected to both methods was the best at extracting DNA (1.89-1.81). The extracted DNA was also studied by 28S ribosomal DNA and COI of mitochondrial DNA genes. Phylogenetic analysis based on 28S rDNA and COI of mtDNA placed the South African population of O. niloticus in a clade with other related species with a posterior probability value of 1.00. Finally, the molecular results showed that 28S ribosomal DNA is a suitable marker for the identification of O. niloticus. In conclusion, precise identification of O. niloticus is critical for breeding for farmers and commercial sectors.

[Objective]To determine efficient method for extracting genomic DNA from D. sinensis. [Method]Four methods were used to extract genomic DNA from young leaves of D. sinensis. The efficiency of genomic DNA extraction was assessed by ultraviolet spectrophotometer, agarose gel electrophoresis, restriction enzyme digestion, and amplification of conserved gene fragments. [Result]The results showed that the high-salt, low-pH method led to more DNA loss due to the extended operation time, resulting in low concentration of DNA with low impurity content. The SDS method had a poor extraction efficiency, yielding DNA with low content and poor integrity. The urea method produced the worst DNA extraction, with severe degradation that renders it unsuitable for molecular biology research. The improved CTAB method successfully extracted a large amount of DNA suitable for enzyme digestion and PCR amplification. [Conclusion] The determination of an efficient DNA extraction method will provide scientific basis and technical references for related molecular biology research of D. sinensis. [ABSTRACT FROM AUTHOR]

The bacterium Erwinia amylovora causes fire blight and continues to threaten global commercial apple and pear production. Conventional microbiology techniques cannot accurately determine the presence of live pathogen cells in fire blight cankers. Several factors may prevent E. amylovora from growing on solid culture media, including competing microbiota and the release of bacterial-growth-inhibitory compounds by plant material during sample processing. We previously developed a canker processing methodology and a chip-based viability digital PCR (v-dPCR) assay using propidium monoazide (PMA) to bypass these obstacles. However, sample analysis was still time-consuming and physically demanding. In this work, we improved the previous protocol using an automatic tissue homogenizer and transferred the chip-based v-dPCR to the BioRad QX200 droplet dPCR (ddPCR) platform. The improved sample processing method allowed the simultaneous, fast, and effortless processing of up to six samples. Moreover, the transferred v-ddPCR protocol was compatible with the same PMA treatment and showed a similar dynamic range, from 7.2 × 102 to 7.6 × 107 cells mL−1, as the previous v-dPCR. Finally, the improved protocol allowed, for the first time, the detection of E. amylovora viable but nonculturable (VBNC) cells in cankers and bark tissues surrounding cankers. Our v-ddPCR assay will enable new ways to evaluate resistant pome fruit tree germplasm, further dissect the E. amylovora life cycle, and elucidate E. amylovora physiology, epidemiology, and new options for canker management. [ABSTRACT FROM AUTHOR]

Multiple methods for collecting genetic samples from amphibians exist, each with their own implications for study design, animal welfare, and costs. Toe clipping is one common method, but there is ongoing debate regarding its potential detriment. Less invasive methods should be implemented, if efficacious, as amphibians are a particularly vulnerable vertebrate group. Skin and buccal swabbing are less invasive methods for genetic sampling, but the potential for contamination and a lower yield of DNA may exist. To compare these methods, we gathered skin swabs, buccal swabs, and toe clips from the same individuals of a relatively small anuran species, Blanchard's Cricket Frog (Acris blanchardi). We then compared DNA yield, DNA purity, amplification success rate, and genotypic data quality among sample types. We found toe clips and buccal swabs generated similar DNA yield and purity, with skin swabs yielding significantly less DNA of significantly lower purity than the other sample types. Amplification success rate was significantly higher using toe clips compared to the other sample types, though buccal swab samples amplified more readily than skin swabs. Genotypic data from toe clips and buccal swabs did not differ significantly in quality, but skin swab data quality was significantly lowest among sample types. Thus, skin swabbing could produce erroneous data in some situations, but buccal swabbing is likely an effective substitute to toe clipping, even for small species. Our results can help future researchers select which genetic sampling method might best suit their research needs. [ABSTRACT FROM AUTHOR]

This document summarizes a study on the gene sequencing of the EGFR gene in patients with hepatocellular carcinoma (HCC) in Egypt. The study found correlations between HCC and factors such as viral infections, smoking, diabetes, hypertension, and familial predisposition. It also identified specific genetic variations in the EGFR gene in HCC patients. The findings contribute to a better understanding of HCC and its risk factors in Egypt, but further research with larger patient cohorts is needed. [Extracted from the article]

This article discusses a study conducted in Egypt on the genetic variations and mutations found in hepatocellular carcinoma (HCC), a type of liver cancer. The study used next-generation sequencing to analyze the BRAF gene in HCC patients. The results showed that almost half of the patients had BRAF gene mutations, suggesting that targeting these mutations could be a potential treatment for HCC. The study also explored other risk factors and characteristics associated with HCC. However, more research is needed to fully understand the genetic changes in HCC. [Extracted from the article]

The butterfly genus of Teinopalpus , endemic to Asia, embodies a distinct species of mountain-dwelling butterflies with specific habitat requirements. These species are rare in the wild and hold high conservation and research value. Similar to other protected species, the genetic analysis of the rare Teinopalpus aureus poses challenges due to the complexity of sampling. In this study, we successfully extracted DNA and amplified mitochondrial genomic DNA from various noninvasive sources such as larval feces, larval exuviae, larval head capsules, pupal exuviaes, and filamentous gland secretions, all integral parts of butterfly metamorphosis. This was conducted as part of a research initiative focused on the artificial conservation of T. aureus population in Jinggang Shan Nature Reserve. Our findings illustrated the successful extraction of DNA from multiple noninvasive sources, achieved through modified DNA extraction methodologies. Although the DNA concentration obtained from noninvasive samples was lower than that from muscle tissues of newly dead larvae during rearing, all samples met the requirements for PCR amplification and sequencing, yielding complete circular sequences. These sequences are pivotal for both interspecific and intraspecific genetic relationship analysis. Our methods can be extended to other insects, especially scarce species. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) are pervasive environmental contaminants, with benzo(a)pyrene (B(a)P) standing out as a prototypical example. B(a)P is a known mutagen and carcinogen that, upon metabolic activation stimulated by cytochromes P450 (CYP1A) with microsomal epoxide hydrolase, reacts with DNA. In this study, common carp (Cyprinus carpio) were subjected to three different doses of B(a)P (1µg/kg and 10µg/kg) to investigate the mutagenic effects on B(a)Pderived DNA in vivo. Blood samples were collected, followed by thorough DNA extraction and polymerase chain reaction analysis. The findings revealed a pronounced mutational impact of B(a)P on the amplified segment of the CYP1A gene. Specifically, at a concentration of 1µg/kg, B(a)P induced 8 mutations, 3 amino acid alterations, and the emergence of 5 new haplotype patterns. In contrast, at a concentration of 10µg/kg, B(a)P resulted in 21 mutations, 10 changes in amino acids, and the generation of 7 new haplotype patterns. Additionally, alterations in the three-dimensional protein composition were observed in both dosage groups. This study underscores the significant mutagenic potential of B(a)P, shedding light on its capacity to induce genetic changes and protein structure modifications, thereby emphasizing the importance of monitoring and addressing the environmental presence of PAHs for both ecological and human health considerations. [ABSTRACT FROM AUTHOR]

In present study, 50 dairy cows of the local and Holstein breeds were used (34 local cows and 16 Holsteins). Milk samples were collected from 50 cows once every two weeks from the morning milking (to estimate the daily amount of milk, multiply the amount by 2). One-day test to calculate the daily and monthly milk yield. Samples were taken at a rate of 50 ml for each cow, and the proportions of the main milk components (fat, protein, lactose and non-fat solids) were estimated. Blood samples were drawn from cows for the purpose of DNA extraction. Then the DGAT1 gene was amplified using the primers of the studied piece, which was 411 bp in size. The gene was cut using Taq1 restriction enzyme. The results showed the presence of three genotypes: AA, AK, and KK. In terms of the quantity of daily, weekly, monthly, and first stage milk production, the findings also demonstrated that the genotypes AK and KK of the Holstein breed were superior (P< 0.05) than the two genotypes AA and AK of the local breed. While the genotype AA of the local breed was superior to the genotype AK in terms of daily, weekly, and monthly milk production as well as the first stage, it was also discovered that there is a correlation between the genotypes and the chemical components of milk, as the results showed that the genotype AA was significantly superior (P<0.05) by the percentage of fat in the local breed on the AK and KK genotypes of both breeds. [ABSTRACT FROM AUTHOR]

Copyright of Egyptian Journal of Botany is the property of Egyptian National Agricultural Library (ENAL) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

DNA extraction for downstream molecular diagnostic applications can be an expensive, time-consuming process. We devised a method to quickly extract total bacterial DNA from environmental samples based on the sodium hydroxide lysis of cells with or without capture by magnetic beads for subsequent PCR or quantitative PCR. The final DNA extraction method using NaOH is extremely low-cost and can be completed in as little as 10 min at room temperature with dilution, or the DNA can be further purified using silica-coated paramagnetic beads. NaOH extraction was effective for Gram-negative and Gram-positive bacteria in samples from air, soil, sewage, food, laboratory surfaces, and chicken cloacal swabs. The NaOH extraction method was comparable to commercial kits for extraction of DNA from pig fecal samples for 16S amplicon sequencing analyses. We demonstrated that an impinger and portable pump can efficiently capture bacteria from the air in poultry facilities for rapid NaOH extraction to quantify total bacteria and for detection of specific species using qPCR. The air sampling and NaOH extraction procedures are well suited for routine, high-throughput screening and for metagenomic analyses for specific pathogens, even in resource-limited situations. [ABSTRACT FROM AUTHOR]

Objective: Roller swabbing of surfaces is an effective way to obtain environmental DNA, but the current DNA extraction method for these samples is equipment heavy, time consuming, and increases potential contamination through multiple handling. Here, we used rollers to swab a dog kennel and compared three DNA extraction approaches (water filtration, roller trimming and direct buffer) using two different platforms (Qiacube, Kingfisher). DNA extraction methods were evaluated based on cost, effort, DNA concentration and PCR result. Results: The roller trim method emerged as the optimal method with the best PCR results, DNA concentration and cost efficiency, while the buffer-based methods were the least labour intensive but produced mediocre PCR results and DNA concentrations. Additionally, the Kingfisher magnetic bead extractions generally ranked higher in all categories over the Qiacube column-based DNA extractions. Ultimately, the ideal DNA extraction method for a particular study is influenced by logistical constraints in the field such as the size of the roller, the availability of cold storage, and time constraints on the project. Our results demonstrate the strengths and weaknesses of each approach, allowing for informed decision making by researchers. [ABSTRACT FROM AUTHOR]

The simple and rapid identification of meat via DNA-based methods is still subject to the conditions of complex and time-consuming preprocessing. It usually takes 3-4 h to obtain DNA sample using conventionally available DNA extraction kits. The existing approaches are cumbersome, require large equipment, and are not suitable for field operation. This study developed a swelling microneedle patch, constituted by micron-sized copolymers of methyl vinyl ether and maleic acid (PMVE/MA). The microneedle patch was applied to the meat sample for 1 min before being pulled out and rinsed with Tris-EDTA (TE) buffer to obtain DNA, which could be used for polymerase chain reaction (PCR) without purification. The DNA extracted by the microneedle patch could meet the needs for amplification and identification of meat samples. The developed approach which combines the patchbased extraction method and conventional PCR amplification is quick, simple, and reliable for DNA extraction and identification of meat samples. [ABSTRACT FROM AUTHOR]

Species-abundance distribution (SAD) is essential in understanding the formation and maintenance mechanisms of species diversity. In alpine meadows, aspect affects almost all biological and ecological processes. Therefore, the soil ciliate data obtained by Illumina MiSeq sequencing were used to explore the characteristics of SADs to the soil ciliate communities on different aspects in alpine meadows (south aspect, southwest aspect, west aspect, northwest aspect, north aspect; SA, SWA, WA, NWA, NA, respectively). Statistical models: Log-series model (LSM) and Log-normal model (LNORM), Niche models: Broken stick model (BSM) and Geometric series model (GSM), and a neutral theory model: Meta-community zero-sum multinomial distribution model (MZSM) were selected to fit the soil ciliate data for analysis. The results showed that: (1) the number of common species increased and then decreased from SA to NA, the number of rare species changed in the opposite trend; (2) BSM and GSM were able to fit the SAD of overall soil ciliates species on different aspects, while MZSM was rejected on all aspects, indicating that the niche process dominated the formation of soil ciliate communities in the alpine meadows, and the resource allocation pattern was dominated by fixed allocation; (3) best-fit models for common species were GSM and BSM which were consistent with best-fit models for all species, while the best-fit model for rare species was LNORM, common species dominated the formation of SAD on different aspects and had an irreplaceable role in maintaining community productivity and stability. [ABSTRACT FROM AUTHOR]

Next-generation sequencing technologies, such as Nanopore MinION, Illumina Hiseq and Novaseq, and PacBio Sequel II, hold immense potential for advancing genomic research on non-model organisms, including the vast majority of marine species. However, application of these technologies to marine invertebrate species is often impeded by challenges in extracting and purifying their genomic DNA due to high polysaccharide content and other secondary metabolites. In this study, we help resolve this issue by developing and testing DNA extraction protocols for Kellet's whelk (Kelletia kelletii), a subtidal gastropod with ecological and commercial importance, by comparing four DNA extraction methods commonly used in marine invertebrate studies. In our comparison of extraction methods, the Salting Out protocol was the least expensive, produced the highest DNA yields, produced consistent high DNA quality, and had low toxicity. We validated the protocol using an independent set of tissue samples, then applied it to extract high-molecular-weight (HMW) DNA from over three thousand Kellet's whelk tissue samples. The protocol demonstrated scalability and, with added clean-up, suitability for RAD-seq, GT-seq, as well as whole genome sequencing using both long read (ONT MinION) and short read (Illumina NovaSeq) sequencing platforms. Our findings offer a robust and versatile DNA extraction and clean-up protocol for supporting genomic research on non-model marine organisms, to help mediate the under-representation of invertebrates in genomic studies. [ABSTRACT FROM AUTHOR]

Aims: The inefficient lysis of recalcitrant bacterial cell wall and subsequent isolation of DNA from environmental samples can lead to a bias in the qualitative and quantitative assessment of bacteria present in the sample. Thus, the selection of an optimum DNA isolation method is the important first step for biosurveillance and metagenomic analyses. This study aims to determine the optimal DNA isolation method out of four commercial DNA isolation kits (A, B, C and D) and two conventional methods (E and F), for rodent faecal droppings. The key selection criterion is the general bacterial diversity contained in the isolated DNA, as evaluated by the Shannon-Weaver index based on the maximal number of PCRamplicons of partial 16S rRNA gene, derived from each method. The amplicons were separated in accordance with their difference in nucleotide sequences via DGGE. Methodology and results: Five faecal samples of wild rodents were collected from different sites and preserved in DNA/RNA shield reagent (Zymo Research). Each sample was extracted, and the DNA extracts were then subjected to amplification of the bacterial 16S rRNA and DGGE separation of the amplicons. Method E showed a higher yield of DNA (average 324.22 ng/µL) as compared to the other methods. However, the majority of the DNA extracts showed partial degradation. The DGGE profiles showed the highest number of amplicons were generated from DNA extracted from Method A and B with a total of 168 and 167 respectively. This is indicated by the Shannon-Weaver index which were 0.306 and 0.305, respectively. Conclusion, significance and impact of study: Method A is the optimum DNA isolation method for rodent faecal samples as its isolated DNA contains the most diverse bacteria. The isolated DNA can then be used for PCRbiosurveillance or metagenomic sequencing and analyses. [ABSTRACT FROM AUTHOR]

Taxus baccata L. is a tertiary relict, long-lived, wind-pollinated dioecious tree species found throughout Europe. In the rocky mountains of Thasos island, monumental old trees create a unique area of natural beauty. In recent times, the need to implement conservation measures for key endangered species such as Taxus baccata has intensified. Exploring the genetic diversity of the species is a prerequisite for successful forest management decisions aimed at conservation. In this study, 28 monumental trees from two natural populations of Thasos were investigated using eight Simple Sequence Repeat markers in order to assess the levels of genetic diversity and genetic differentiation within the individuals, to estimate the degree of inbreeding and the effective population size of each population, and to discuss the impact this study has on conservation efforts for the species. Although the population size was small (14 individuals per population), the results showed moderate to high genetic diversity parameters. The mean expected heterozygosity was He = 0.649 and the number of effective alleles was Ne = 3.270 for both populations. Moreover, allelic richness (AR = 3.395) was high, indicating a variable genetic pool which is probably a result of a past established expansion of the species in the area. The results of the present study present a unique genetic pool harbored by specific trees, which is an important advantage for ensuring their conservation and resistance against biotic and abiotic threats. Our study paves the way towards conservation measures, which can be prioritized as follows: (a) in situ conservation, (b) seed bank establishment, and (c) in vitro propagation in order to secure future resilience and sustainability of the species. [ABSTRACT FROM AUTHOR]

Most Dalbergia species are economically valuable and have been over-exploited, which has raised concerns. The regulation and protection of this genus require accurate and rapid authentication and identification processes. To address the issue of high residual inhibitors in extracted DNA from the Dalbergia xylem, an optimized DNA extraction experiment was performed on 10 species of Dalbergia wood stored for 1–5 years; in particular, no gene sequence for D. tsoi can be found in the NCBI database. Additionally, universal primers ITS2 were used for PCR amplification and sequencing to confirm the effectiveness of DNA extraction. The results revealed that rinsing the wood with 0.25 M ammonium acetate buffer produced DNA with a high purity, without a significant decrease in the DNA yield. To achieve an optimal DNA yield, the wood DNA should be rinsed with ammonium acetate fewer than three times. All the wood DNA obtained using the kit method and treated with the ammonium acetate buffer rinsing solution one to four times was successfully amplified. The NJ phylogenetic tree constructed based on ITS2 can distinguish D. tsoi from other Dalbergia spp., and the predicted ITS2 secondary structure showed the difference between species. This experiment extracted high-quality DNA from wood, without the need for purification kits, thereby improving the efficiency of the extraction process. The extracted DNA was directly used for follow-up molecular experiments. [ABSTRACT FROM AUTHOR]

Copyright of Arab Journal of Plant Protection is the property of Arab Society for Plant Protection and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)