DNA Extraction Methods Research Articles

An improved DNA extraction method in okra for rapid PCR detection of Okra enation leaf curl virus from diverse Indian regions.

The extraction of DNA from okra (Abelmoschus esculentus) is challenging due to its high mucilage and polysaccharide content, which can hinder both the yield and quality of DNA.In this study, an improved DNA isolation method is described incorporating a key modification being the use of solution I (1 M NaCl and 2% Sarcosyl) as a pre-treatment before applying the CTAB buffer, resulting in high-purity genomic DNA in just 1h and 45min., making it suitable for handling large sample sizes due to its rapid processing capabilities. This enhanced DNA extraction method was crucial for the accurate and rapid molecular detection of Okra enation leaf curl virus (OELCuV), a monopartitebegomovirus that has spread across various regions of India. Transmitted by the whitefly (Bemisia tabaci), OELCuV causes leaf curling, enations, and stunted growth in okra, leading to significant yield losses. The surveys conducted during the 2020-21 and 2021-22 sowing seasons revealed disease incidence ranging from 14.03 to 67.57%. The extracted DNA via the improved DNA extraction method enhanced the speed of PCR based molecular identification of OELCuV, using virus-specific coat protein primers. The amplified CP genes were cloned and sequenced to study the CP gene based diversity among OELCuV isolates from different states of India. The CP gene nucleotide identity among the studied OELCuV isolates ranged from 95.57 to 99.27%, while comparison with previously reported Indian OELCuV CP sequences, the nucleotide identity ranged from 89.35 to 98.83%.The successful application of this optimized DNA extraction method sped up the detection process but also holds promise for broader use in the molecular study of okra and other mucilaginous crops, particularly in the rapid and reliable identification ofbegomoviruses. The optimized DNA extraction method significantly accelerated the detection of OELCuV, demonstrating its efficiency and reliability. This method shows strong potential for broader applications in the molecular study of okra and other mucilaginous crops, making it a valuable tool for future research and disease management.

Single Laboratory Evaluation of the Q20+ Nanopore Sequencing Kit for Bacterial Outbreak Investigations

Leafy greens are a significant source of produce-related Shiga toxin-producing Escherichia coli (STEC) outbreaks in the United States, with agricultural water often implicated as a potential source. Current FDA outbreak detection protocols are time-consuming and rely on sequencing methods performed in costly equipment. This study evaluated the potential of Oxford Nanopore Technologies (ONT) with Q20+ chemistry as a cost-effective, rapid, and accurate method for identifying and clustering foodborne pathogens. The study focuses on assessing whether ONT Q20+ technology could facilitate near real-time pathogen identification, including SNP differences, serotypes, and antimicrobial resistance genes. This pilot study evaluated different combinations of two DNA extraction methods (Maxwell RSC Cultured Cell DNA kit and Monarch high molecular weight extraction kits) and two ONT library preparation protocols (ligation and the rapid barcoding sequencing kit) using five well-characterized strains representing diverse foodborne pathogens. High-quality, closed bacterial genomes were obtained from all combinations of extraction and sequencing kits. However, variations in assembly length and genome completeness were observed, indicating the need for further optimization. In silico analyses demonstrated that Q20+ nanopore sequencing chemistry accurately identified species, genotype, and virulence factors, with comparable results to Illumina sequencing. Phylogenomic clustering showed that ONT assemblies clustered with reference genomes, though some indels and SNP differences were observed, likely due to sequencing and analysis methodologies rather than inherent genetic variation. Additionally, the study evaluated the impact of a change in the sampling rates from 4 kHz (260 bases pair second) to 5 kHz (400 bases pair second), finding no significant difference in sequencing accuracy. This evaluation workflow offers a framework for evaluating novel technologies for use in surveillance and foodborne outbreak investigations. Overall, the evaluation demonstrated the potential of ONT Q20+ nanopore sequencing chemistry to assist in identifying the correct strain during outbreak investigations. However, further research, validation studies, and optimization efforts are needed to address the observed limitations and fully realize the technology’s potential for improving public health outcomes and enabling more efficient responses to foodborne disease threats.

Genomic DNA extraction methods and phylogenetic analysis of Beauveria bassiana from Central Java, Indonesia, and its toxicity against the fall armyworm, Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae)

BackgroundControl techniques using biological control agents such as Beauveria bassiana (Balsamo) Vuillemin have the advantage of not showing any negative impacts on environmental health and safety issues. This study used isolates from B. bassiana collection from Laboratory of Pest Monitoring belonging to Indonesian Ministry of Agriculture (LPHP) in Central Java which showed potential in controlling target pest. The problems that still occur are the lack of facilities and infrastructure and the lack of quality testing of collection isolates in LPHP, so that the isolate identification process is still carried out in simple method, and bioassay testing on the fall armyworm (FAW), Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) as a target pest, is not commonly conducted. The results of bioassay testing can be used to determine the potential of a biological agent to control target pest.ResultThe two DNA extraction methods showed different results regarding DNA concentration and purity values, but both methods were good and could be used to amplify DNA using PCR. The DNA band was amplified at 500–600 bp using primers ITS 1 and ITS 4. The results of molecular analysis showed that the four isolates of B. bassiana from Central Java were found in the same clade as B. bassiana from South Sumatra, Dhaka, and Oromia, where these isolates showed similar similarities descended from a common ancestor. Genetically, B. bassiana isolates from Central Java show more genetic similarities to B. bassiana isolates from South Sumatra, Indonesia. Quality testing was carried out by calculating the density and germination ability values for LPHP isolates from Sukoharjo (Sukoharjo isolate), Temanggung (Purworejo isolate), and Banyumas (Banyumas and Cilacap isolate), which showed varying results. The bioassay test used three isolates, namely B. bassiana from Sukoharjo, Banyumas, and Cilacap, which were selected based on density values, germination ability, and molecular analysis. The ability to cause death of the three isolates against S. frugiperda showed different results where the isolate from Sukoharjo, Banyumas, and Cilacap caused mortality of 60, 40, and 60%, and the LC50 value of each isolate was 3.3 × 106, 1.3 × 107, and 3.5 × 107 conidia ml−1, respectively.ConclusionMorphological identification by macroscopic, microscopic, and molecular analysis showed that the isolate from the LPHP collection in Central Java, Indonesia, was B. bassiana. Genetically, the four isolates showed similar characteristics to isolates from South Sumatra, Indonesia. B. bassiana isolates from collections from Central Java showed potentials as a biological control agent against S. frugiperda.

Comparison of Four Different DNA Isolation Methods from MGIT Culture for Long-Read Whole Genome Sequencing of Mycobacterium tuberculosis

Background: Tuberculosis (TB) remains a global health challenge, particularly due to drug resistance and limitations in rapid diagnosis. Next-generation sequencing (NGS), especially long-read whole genome sequencing (WGS), shows promise for rapidly detecting TB and drug resistance, but it requires high-quality DNA, which is difficult to extract from Mycobacterium tuberculosis due to its complex cell wall. Objectives: This study evaluated four DNA isolation methods for extracting pure DNA from M. tuberculosis, aiming to standardize protocols for long-read WGS. Methods: Mycobacterium tuberculosis H37RV colonies were grown in BACTEC MGIT liquid medium. Two pellets were prepared as the initial material for the DNA extraction protocol: Pellets from 1 mL McFarland 2 suspensions and all growing colonies from two MGIT liquid cultures. Four DNA extraction methods were used: The cetyltrimethylammonium bromide (CTAB) method, GeneJET Genomic DNA Purification Kit, Quick-DNA Fecal/Soil Microbe Kit, and Genematrix Tissue/Bacterial DNA Purification Kit, with some modifications. DNA quality was assessed based on concentration, purity, and integrity. Results: Among the tested methods, the Quick-DNA Fecal/Soil Kit yielded approximately 85 ng/mL of DNA and a purity of 1.9 at 260/280 nm from the colonial pellet of two MGIT tubes. However, lower intact DNA [DNA integrity number (DIN) ~ 6.8] was obtained with this kit. The CTAB method provided the highest intact DNA (DIN ~ 9.5), although the purity of the DNA was not sufficient. Conclusions: Based on three repetitions of McF-2 and colonial pellet extractions, the Quick-DNA Fecal/Soil Kit yielded the highest DNA quantity and purity but showed lower integrity compared to other methods, indicating the need for adjustments. A pellet from two MGIT cultures (~ 100 µL) is suitable for long-read WGS with this kit. However, a larger sample size is required to generalize these findings. For effective long-read sequencing of M. tuberculosis, DNA extraction protocols must be optimized to balance yield, fragment size, and purity for accurate sequencing and drug resistance analysis.

Magnetic Nanoparticle-Mediated Extraction and Electrochemical Detection of E. coli O157:H7 Genomic DNA

We report a rapid magnetic nanoparticle-mediated protocol to extract and detect DNA of E. coli O157:H7. Bare iron oxide nanoparticles (Fe3O4 NPs) were used as solid phase support to extract high purity DNA from concentrated and spiked water samples. Electrochemical detection of the DNA is achieved by employing polyaniline-functionalized magnetic nanoparticles (PANI-MNPs) which serve as DNA pre-concentrator and transducer. The DNA-based nanobiosensor shows good selectivity and sensitivity (detection limit of 0.10 ng/µL DNA) and offers faster and comparable results relative to conventional methods. This DNA extraction and detection method is expected to be applicable in food, water, and environmental samples.

Molecular and Clinical Spectrum of Hemoglobin D: An Experience at a Tertiary Care Diagnostic Center

Objective: To determine the clinical and molecular pattern of Hemoglobin D in patients presenting at tertiary care hospital. Study Design: Cross-sectional study. Place and Duration of study: Department of Hematology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Jul 2020 to Mar 2021. Methodology: An automated cell analyzer was used to determine the whole blood count and red cell indices. D-10 BIORAD HPLC was used for quantitative assessment of Hb A, Hb F, Hb D and Hb A2. Where D-10 BIORAD HPLC showed an abnormal Hb D, capillary electrophoresis was performed using Sebia Capillary 2 Flex Piercing. Chelex DNA Extraction method was used. Molecular analyses for the confirmation of homozygosity and heterozygosity were done using Polymerase Chain Reaction (PCR) amplification. The clinical symptoms of the patients such as anemia, jaundice, pallor, weakness, hepatomegaly and splenomegaly were also noted Results: Among 90 patients, Hemoglobin D trait was found in 78(80.4%) of the patients out of which HbD-Punjab was seen in 57(73%) and HbD-Iran was found in 21(27%). Compound Heterozygotes (HbD/β) was found in 17(17.5%) and Compound Homozygous HbD/D was found in 02 (2.1%). All HbD-Punjab and HbD-Iran traits patients were asymptomatic, and few clinical symptoms were found in Compound Heterozygotes (HbD/β) and Homozygous HbD/D. Conclusion: Collective clinical history records, full blood count and electrophoresis and molecular results allow for the conclusive detection of the variant of Hemoglobin D. More multicenter, organized and detailed studies with larger sample size are required to draw concrete conclusions.

Variation in the metagenomic analysis of fecal microbiome composition calls for a standardized operating approach.

The reproducibility in microbiome studies is limited due to the lack of one gold-standard operating procedure. The aim of this study was to examine the impact of protocol variations on microbiome composition using metagenomic data sets from a single center. We assessed the variation in a data set consisted of 2,722 subjects, including 9 subcohorts harboring healthy subjects and patients with various disorders, such as inflammatory bowel disease, colorectal cancer, and type 2 diabetes. Two different DNA extraction kits, with or without lyticase, and two sample storage methods were compared. Our results indicated that DNA extraction had the largest impact on gut microbiota diversity among all host factors and sample operating procedures. Healthy subjects matched by age, body mass index, and sample operating methods exhibited reduced, yet significant differences (PERMANOVA, P < 0.05) in gut microbiota composition across studies. The variations contributed by DNA extraction were primarily driven by different recovery efficiency of gram-positive bacteria, e.g., phyla Firmicutes and Actinobacteria. This was further confirmed by a parallel comparison of fecal samples from five healthy subjects and a standard mock community. In addition, the DNA extraction method influenced DNA biomass, quality, and the detection of specific lineage-associated diseases. Sample operating approach and batch effects should be considered for cohorts with large sample size or longitudinal cohorts to ensure that source data were appropriately generated and analyzed. Comparison between samples processed with inconsistent methods should be dealt with caution. This study will promote the establishment of a sample operating standard to enhance our understanding of microbiome and translating in clinical practice.IMPORTANCEThe reproducibility of human gut microbiome studies has been suboptimal across cohorts and study design choices. One possible reason for the disagreement is the introduction of systemic biases due to differences in methodologies. In our study, we utilized microbial metagenomic data sets from 2,722 fecal samples generated from a single research center to examine the extent to which sample storage and DNA extraction influence the quantification of microbial composition and compared this variable with other sources of technical and biological variation. Our research highlights the impact of DNA extraction methods when analyzing microbiome data and suggests that the microbiome profile may be influenced by differences in the extraction efficiency of bacterial species. With metagenomics sequencing being increasingly used in clinical biology, our findings provide insight into the challenges using metagenomics sequencing in clinical diagnostics, where the detection of certain species and its abundance relative to a "healthy reference" is key.

Comparative evaluation of PCR and loop-mediated isothermal amplification (LAMP) assays for detecting Pasteurella multocida in poultry

ABSTRACT Aims To develop a colourimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Pasteurella multocida in clinical poultry samples and compare the performance of this assay with PCR. A secondary aim was to evaluate a simple DNA extraction method that could enable LAMP-based testing in the field without the need for specialised laboratory equipment. Methods Primer sets for both LAMP and PCR were designed to amplify the KMT1 gene of P. multocida. DNA was extracted from 12 P. multocida isolates using a commercial extraction kit, and subjected to analysis using both LAMP and PCR. The analytical specificity of the LAMP assay was evaluated by testing it against a panel of 12 unrelated bacterial species, and the analytical sensitivity (limit of detection) was determined through testing of serial dilutions of the target DNA and compared to that of PCR. Subsequently, cloacal swabs (n = 40) from a commercial turkey flock were subjected to analysis using both LAMP and PCR assays, using a rapid DNA extraction method and a commercial extraction kit. Clinical sensitivity and specificity of the LAMP assay were calculated in comparison to PCR. Results A single DNA fragment of the expected size (∼ 200 base pairs), was amplified by PCR from 12 P. multocida isolates, which were also all positive by the LAMP assay. The identity of all PCR amplicons was confirmed by sequencing. Both PCR and LAMP showed similar analytical sensitivity, with a LOD of 20 pg of target DNA. As neither PCR nor LAMP assays produced positive results with 12 non-related bacterial species, the analytical specificity was assessed as 100%. However, LAMP demonstrated lower clinical specificity (94.74%) compared to PCR (100%) when 40 clinical samples were tested. None of the DNA samples extracted using the simplified DNA extraction method were amplified by either LAMP or PCR. Conclusion The LAMP assay developed in this study exhibits comparable performance to PCR in detecting P. multocida. Clinical relevance The use of a rapid and portable DNA extraction method, in conjunction with LAMP assays, could create opportunities for point-of-care testing for fowl cholera in field settings.

Association between forensic DNA and odontology in human identification during mass disaster: a systematic review

BACKGROUND: According to the literature, forensic odontology is one of the most effective and affordable scientific techniques for identifying victims of mass disasters. This research methodically reviews the role and function of forensic odontologists in several global mass disasters. Forensic DNA in odontology is associated with the importance of its application in the identification of humans of mass disasters. As the crime rate continues to rise, the field of forensic medicine has evolved significantly. Forensic dentists play a pivotal role in various areas of crime scene investigations, thereby helping solve innumerable mysteries. AIM: The study aimed to increase the body of knowledge for future research on forensic odontology by conducting a systematic review search to investigate possible forensic DNA in odontology associated with the importance of its application in the identification of humans of mass disasters. MATERIALS AND METHODS: Six databases, including Google Scholar, PubMed, Scopus, Embase, Web of Science, and ScienceDirect, were analyzed using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) quality scale. The literature search, conducted until February 2024, informed important research choices. RESULTS: A total of 16 (100%) studies related to forensic DNA of mass disasters were identified. Of these, only seven (43.75%) articles related to dental DNA were included in this review. Of the 4,808 articles obtained, 138 duplicates or irrelevant articles were eliminated. Following a full-text review, seven studies were selected based on eligibility criteria. The highest percentage of victims was identified using dental DNA. In a few studies, some samples were insufficient for complete DNA profiling due to factors such as the method of DNA extraction. CONCLUSION: Forensic odontology has played a significant role in the identification of victims of several mass disasters around the world. Although teeth are an excellent source of DNA for humans, future studies with larger sample sizes, appropriate control groups, and standardized techniques of DNA extraction need to be conducted.

A morphological and high throughput sequencing workflow to identify Australian ants (Hymenoptera, Formicidae): a new tool for biosecurity and biodiversity

Ants are often the most ubiquitous and ecologically influential components of terrestrial systems, exhibiting an exceptionally high level of endemic diversity. Simultaneously, exotic ants can be amongst the most environmentally and economically devastating biological invaders. Distinguishing between exotic and related natives is essential for early detection and to reduce the chance of establishment, but at the species-level there remains a great deal of uncertainty among several of the most diverse, widespread and ecologically dominant genera. This taxonomic impediment negatively impacts the use of molecular techniques relying on reference databases, due to the poor state of ant DNA sequences in publicly available repositories, with many species not represented at all and others incorrectly identified. As a result, biosecurity screening for targeted exotic ants and assigning ant species-level identifications for biodiversity remain two separate areas of focus in Australia. Here we propose a workflow for the identification of ants that enables simultaneous processing of large numbers of “bulk” ant samples each containing many individuals, increasing resolution for taxonomic assignment, and generating a curated database linked to voucher specimens. We use a non-destructive DNA extraction method and compare two high throughput sequencing (HTS) metabarcoding platforms – MiSeq (Illumina) and MinION (Oxford Nanopore Technologies) – processing up to 180 bulk mixed ant samples per run. This approach allowed for the acquisition of curated DNA sequences from voucher specimens morphologically examined by expert taxonomists. This work highlights the advantages and current limitations of DNA-based identifications, the needs for standardisation, as well as the importance of a taxonomy-based curation of DNA database for both biodiversity and biosecurity.

Applying nanopore sequencing technology in Paracoccidioides sp.: a high-quality DNA isolation method for next-generation genomic studies.

Paracoccidioidomycosis is a severe systemic endemic mycosis caused by Paracoccidioides spp. which mainly affects individuals in Latin America. Progress in Paracoccidioides genomics has been slow, as evidenced by the incomplete reference databases available. Next-generation sequencing is a valuable tool for epidemiological surveillance and genomic characterization. With the ability to sequence long reads without the need for prior amplification, Oxford Nanopore Technology (ONT) offers several advantages, but high-quality and high-quantity DNA samples are required to achieve satisfactory results. Due to the low concentration of Paracoccidioides DNA in clinical samples and inefficient culture isolation methods, DNA extraction can be a significant barrier to genomic studies of this genus. This study proposes a method to obtain a high-coverage de novo genome assembly for Paracoccidioides using an improved DNA extraction method suitable for sequencing with ONT. The assembly obtained was comparable in size to those constructed from available data from Illumina technology. To our knowledge, this is the first genome assembly of Paracoccidioides sp. of such a large size constructed using ONT.

Streamlined boiling lysis DNA extraction for Gram-positive aquaculture pathogen Streptococcus agalactiae.

Accurate genetic analysis is essential for the detection of pathogens as it necessitates suitable DNA extraction methods tailored to specific microorganisms such as Gram-positive bacteria. This study examined several commercial and simplified DNA extraction methods for their suitability in isothermal downstream applications. Extracted DNA was assessed using spectrophotometry, electrophoresis, polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) while its stability was inspected after five months of storage. The findings revealed variations in DNA yield, purity and integrity among the extraction methods. While extraction kits demonstrated high yield and purity, the in-house extraction techniques showed incoherent correlation between yield and purity, yet showed promise for a streamlined extraction process. The DNA acquired from all methods yielded positive amplification in PCR and LAMP. DNA extracted by kits exhibits prolonged stability than those obtained via boiling lysis. Both methods offer distinct advantages, with commercial kits providing longer stability and high-quality DNA while boiling lysis stands out for its simplicity, with shorter handling and processing periods. This study emphasizes selecting ideal extraction methods for Streptococcus agalactiae, in the prospect of aquaculture settings. In particular, successful LAMP reaction suggests that boiled extracts are feasible enough for detection, and suited for point-of-care (POC) testing where prompt detection of aquatic pathogens is often critical. Ultimately, the choice of method should be contemplated on a case-by-case basis such as the study goals, intended settings, and type of samples.

Comprehensive Analysis of Complex Copy Number Variations in the Plasmodium falciparum Genome via Long-Read Sequencing: Implications for Antimalarial Resistance and Broad Genomic Insights

Abstract Introduction/Objective The genome of Plasmodium falciparum, characterized by a high AT content and a propensity for copy number variations (CNVs), presents a model for understanding how genomes adapt to stress. Our research focused on the prevalence and distribution of CNVs highlights long-read sequencing as a tool for detecting complex structural variants in other models. Methods/Case Report P. falciparum parasites were cultured and treated with DSM1 and aphidicolin that induce replication stress. Long-read sequencing was conducted using the Oxford Nanopore Technology Minion, with an emphasis on an optimizing DNA extraction method for preserving intact chromosomes. Visualization of CNVs, including tandem and inverted duplications, was achieved through a custom R Shiny application that allowed both quantitative and qualitative assessments. Results (if a Case Study enter NA) Long read sequencing identified an enrichment of multiple CNV types in treated samples, with a significant enrichment of inverted duplications as well as other types of structural variants. Aphidicolin and DSM1-treated samples demonstrated significant accumulation of these variations compared to controls (2.0 and 5.6-fold, respectively with a p value of &amp;lt; 0.0001 in both treatment groups). These occurred mainly in core genomic regions that do not contain variable gene copies. An increase in the number of inverted duplications may be related to stress response to the treatment and may be a potential adaptive mechanism of the parasite. Our manual single-read visualization technique made it possible to visualize the structure of various CNV types, which provides unique insight on CNVs as they arise in single genomes. Conclusion We hypothesize that inverted duplications represent stalled replication forks, and we are currently assessing their frequency in replicating and non-replicating cells. This study highlights the role of long-read sequencing in detecting complex genomic structures in P. falciparum, which may influence the parasite’s resistance to drug treatment. These findings not only advance our understanding of malaria pathology but also aid our comprehension of how cancer cells might react to drug-induced stress and the mechanisms behind CNV formation in neoplastic cells.

Oxygen exposure decreases the yield of high-molecular-weight DNA from some anaerobic bacteria and bacterial communities during DNA extraction

ObjectivesThe central challenge in third-generation sequencing lies in meeting the requirements for DNA quality (integrity and purity) and quantity. Therefore, novel improvements in DNA extraction methods are needed to satisfy these requirements. We reasoned that in anaerobic microbial communities, the presence of certain strict anaerobes containing oxygen-activated DNase activity might contribute substantially to the poor integrity of extracted metagenomic DNA (or genomic DNA from some pure cultures) if exposed to air. MethodsTo test this hypothesis, we developed an enhanced genomic and metagenomic DNA isolation technique that we applied to a specifically chosen set of both strict and aerotolerant anaerobes, as well as to the hindgut microbiota of a herbivorous marine fish. ResultsConsidering the quality (or degradation) of extracted DNA obtained under anaerobic versus aerobic conditions, we found that DNA extracted aerobically from cells of some strict anaerobes showed more degradation of high molecular weight DNA than analogous preparations under anaerobic conditions. In contrast, with the selected aerotolerant anaerobes, no discernible difference was found between the molecular sizes of DNA extracted aerobically and anaerobically. Metagenomic DNA extracted from the fish hindgut microbiota showed higher yields and better quality under anaerobic conditions compared to aerobic conditions. ConclusionOur study effectively demonstrates the advantages of our improved extraction protocol in anaerobic conditions. This is evident through the improved quality of extracted DNA. Such findings may be valuable for studies, especially metagenomic studies, where the quality and quantity of DNA are crucial for downstream analysis.

Phage communities in household-related biofilms correlate with bacterial hosts

The average American spends 93% of their time in built environments, almost 70% of that is in their place of residence. Human health and well-being are intrinsically tied to the quality of our personal environments and the microbiomes that populate them. Conversely, the built environment microbiome is seeded, formed, and re-shaped by occupant behavior, cleaning, personal hygiene and food choices, as well as geographic location and variability in infrastructure. Here, we focus on the presence of viruses in household biofilms, specifically in showerheads and on toothbrushes. Bacteriophage, viruses that infect bacteria with high host specificity, have been shown to drive microbial community structure and function through host infection and horizontal gene transfer in environmental systems. Due to the dynamic environment, with extreme temperature changes, periods of wetting/drying and exposure to hygiene/cleaning products, in addition to low biomass and transient nature of indoor microbiomes, we hypothesize that phage host infection in these unique built environments are different from environmental biofilm interactions. We approach the hypothesis using metagenomics, querying 34 toothbrush and 92 showerhead metagenomes. Representative of biofilms in the built environment, these interfaces demonstrate distinct levels of occupant interaction. We identified 22 complete, 232 high quality, and 362 medium quality viral OTUs. Viral community richness correlated with bacterial richness but not Shannon or Simpson indices. Of quality viral OTUs with sufficient coverage (614), 532 were connected with 32 bacterial families, of which only Sphingomonadaceae, Burkholderiaceae, and Caulobacteraceae are found in both toothbrushes and showerheads. Low average nucleotide identity to reference sequences and a high proportion of open reading frames annotated as hypothetical or unknown indicate that these environments harbor many novel and uncharacterized phage. The results of this study reveal the paucity of information available on bacteriophage in indoor environments and indicate a need for more virus-focused methods for DNA extraction and specific sequencing aimed at understanding viral impact on the microbiome in the built environment.

New Refined Experimental Analysis of Fungal Growth in Degraded Bio-Based Materials

When exposed to different building environmental conditions, bio-composite materials, such as hemp mortars, represent a risk of mold proliferation. This later plays a critical role in the biodeterioration of the materials when their physical properties are locally modified by the natural aging process. The primary objectives of the present work are first to assess the evolution of the surface of contaminated mortar; second, to investigate an accurate DNA extraction method that could be used for both bio-composite mortars and their fiber sources collected in situ; then, to understand the process of the proliferation of mold strains on both hemp shives and hemp mortar; and finally, to compare mold strains present in these phases to show their relationship to mold contamination and their impact on human health. In situ hemp mortar contamination behavior was investigated in the region of Pau (France) two months after hemp mortar application in extreme conditions (high humidity, low temperature, no aeration), which did not match the standard conditions under which hemp mortar must be used. The SEM observations and FTIR and pH analyses highlighted the decrease in pH level and the presence of organic matter on the mortar surface. DNA sequencing results showed that hemp shives were the main source of fungal contamination of hemp mortar. A mold population analysis showed that the most dominant phylum was Ophistokonta, which represented 83.6% in hemp shives and 99.97% in hemp mortar. The Acrostalagmus genus representatives were the most abundant, with 42% in hemp shives and 96% in hemp mortar. The interconnection between the mold strain characteristics (particularly the ability to grow in extreme environments) and the presence of hemp mortar was emphasized.

B-229 Extraction-free Nucleic Acid Sample Preparation from Inhibitor Rich Samples via Hyperbaric Heating

Abstract Background Extraction-based nucleic acid sample preparation for PCR is limited by long processing times, liquid transfers, cost, and storage requirements. Extraction-free approaches enable streamlined sample prep but are severely impacted by inhibitors present in biological sample matrices. We previously reported the use of Hyperbaric Heating (HBH) for effective organism lysis and demonstrate here its effectiveness to prepare nucleic acids for PCR from various inhibitor-rich sample matrix types. Methods HBH exposes the sample to high temperature/high pressure environment for a short duration. This process effectively lyses organisms and denatures inhibitors within seconds. Samples are sealed within a pressure-tight metallic vessel, then rapidly heated using magnetic induction. Within the sealed metallic vessel, vaporization of the aqueous sample raises the internal pressure, enabling an internal temperature higher than 100°C. Proprietary sample processing beads containing chelating agents and other proteins protect nucleic acids from fragmentation during heating. Results The efficacy of HBH for PCR amplification was tested against commercial kits and extraction-free methods. HBH treatment of nasal swab samples spiked with inactivated SARS-CoV-2 elicited detection down to 125 viral copies/mL. Viruses such as RSV and Influenza spiked in nasal swab samples demonstrated comparable PCR results as extraction prepped samples. HBH treatment of microbe samples, including Candida albicans spiked in urine and Bacillus subtilis spiked in serum, had comparable PCR threshold cycles as microbe specific DNA extraction methods (Table 1). Conclusions HBH is a generalizable extraction-free nucleic acid sample prep technology affording detection capabilities comparable to commercial sample prep methods. We demonstrated the use of HBH sample prep for detection of several infectious microorganisms in common biological matrices (nasal swab, serum, urine) without additional sample purification. With its ease of use, speed, and lower cost, HBH will be a key tool in applications where nucleic acid amplification is pivotal for diagnostic and research use.

Vermicomposting reduces the antimicrobial resistance in livestock waste

Vermicomposting, a process in which housefly larvae are used to decompose organic waste, has attracted attention as a method for managing antimicrobial-resistant bacteria (ARB) in livestock manure. Vermicomposting effectively reduces antimicrobial resistance genes (ARGs) and residual antimicrobials. However, the evaluation of live bacteria, including ARB, remains scarce. Additionally, conventional DNA extraction methods include DNA from dead bacteria, impeding the accurate evaluation of ARG-associated risk in compost and the microbiome. This study assesses the effectiveness of vermicomposting pig manure against antimicrobial resistance (AMR) by evaluating the ARB, ARGs (focusing on DNA from live bacteria), and microbiome associated with vermicomposting processes. Vermicomposting significantly reduces the abundance of bacteria, including ARB, and decreases the ARG (tetA, tetB, blaTEM, and blaCTX−M) copy number in live bacteria. Bacterial community analysis revealed an increase in the abundance of Gammaproteobacteria. Moreover, the vermicomposted samples effectively cultivated myriad plants. Overall, vermicomposting effectively reduces the ARB and ARGs in pig manure, with potential benefits for plant growth and sustainable waste management. Hence, it can be widely applied to treat livestock manure and other organic wastes to combat AMR.

DNARS: A safe, environmentally friendly and high-quality DNA extraction method suitable for various biological samples

The functional and interactional relationships between nucleic acids and proteins form the foundational framework for molecular biology studies. In this context, obtaining high-quality nucleic acids is a crucial step for successful downstream applications. This study aimed to develop a cost-effective, eco-friendly, and versatile DNA extraction method that yields high-quality DNA from diverse biological samples. We utilized three types of borosilicate-based recycled laboratory glass as a silica source, combined with sodium iodide as a chaotropic agent, creating an efficient system for cell lysis and DNA extraction. Quality control was performed by assessing the concentration and purity of the extracted DNA using spectrophotometry, and the results were compared to those obtained with a commercial kit. DNA integrity was evaluated via agarose gel electrophoresis. To verify the suitability of the extracted DNA for downstream applications, we conducted 16S rRNA and ITS PCR amplifications. Our findings demonstrated that our DNA extraction method produced significantly higher yields, better purity, and greater integrity compared to the commercial kit. Moreover, the extracted DNA was readily applicable in PCR-based procedures, confirming the method's effectiveness for molecular biology applications.

Efficiency comparison of DNA extraction kits for analysing the cockle gut bacteriome

Cockles play a vital ecological role and provide valuable ecosystem services globally. However, the performance, production, and safe consumption of cockles are significantly influenced by their gut-associated bacteriome. Accurate understanding of gut-bacteriome interactions, and surveillance of pathogenic bacteria loads in cockles, rely on efficient DNA extraction methods that yield high-quality and representative bacterial DNA. Despite this importance, reliable extraction methods for cockles are currently overlooked. Therefore, we evaluated the performance of five DNA extraction kits (E.Z.N.A.® Soil DNA; FastDNA® Spin; DNeasy PowerSoil Pro; QIAamp PowerFecal DNA; ZymoBIOMICS™DNA Miniprep) in terms of DNA quality, yield, bacterial community structure (analysed by using denaturating gradient gel electrophoresis; DGGE), and bacteriome composition (analysed by 16S rRNA gene sequencing) in Cerastoderma edule gut. The DNeasy kit provided the highest purity and quantity of bacterial DNA, while the PowerFecal and Zymo kits exhibited reduced extraction efficiency. DGGE profiles revealed significant variability between the tested kits (R = 0.512; mean P = 0.011), but the FastDNA kit under-represented the bacterial community in cockles’ gut. Based on alpha diversity, the DNeasy kit outperformed the others and successfully detected all abundant genera found with the alternative kits. Our findings indicate that the DNeasy kit is an efficient DNA extraction method, enabling a molecular representation of the gut-associated bacteriome in C. edule. These results contribute to the development of effective techniques for studying the cockle gut bacteriome and its ecological implications.