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AbstractWe 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. [ABSTRACT FROM AUTHOR]

Background: Control 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. Result: The 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. Conclusion: Morphological 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. [ABSTRACT FROM AUTHOR]

Sugarcane white leaf (SCWL) disease, caused by Candidatus Phytoplasma sacchari, poses a significant threat to sugarcane cultivation. An obligate parasite, phytoplasma is difficult to culture in laboratory conditions, making the isolation of its DNA from the massive amount of plant host DNA extremely challenging. Yet, the appropriate amount and quality of plant microbiome-derived DNA are key for high-quality DNA sequencing data. Here, a simple, cost-effective, alternative method for DNA isolation was applied using a guanidine-HCl-hydroxylated silica (GuHCl-Silica)-based method and microbiome DNA enrichment based on size-selective low-molecular-weight (LMW) DNA by PEG/NaCl precipitation. qPCR analysis revealed a significant enrichment of phytoplasma DNA in the LMW fraction. Additionally, the NEBNext Microbiome DNA enrichment kit was utilized to further enrich microbial DNA, demonstrating a remarkable increase in the relative abundance of phytoplasma DNA to host DNA. Shotgun sequencing of the isolated DNA gave high-quality data on the metagenome assembly genome (MAG) of Ca. Phytoplasma sacchari SCWL with completeness at 95.85 and 215× coverage. The results indicate that this combined approach of PEG/NaCl size selection and microbiome enrichment is effective for obtaining high-quality genomic data from phytoplasma, surpassing previous methods in efficiency and resource utilization. This low-cost method not only enhances the recovery of microbiome DNA from plant hosts but also provides a robust framework for studying plant pathogens in complex plant models. [ABSTRACT FROM AUTHOR]

Fixation and demineralization protocols for bone marrow (BM) across diagnostic laboratories are not standardized. How different protocols affect histomorphology and DNA amplification is incompletely understood. In this study, 2 fixatives and 3 demineralization methods were tested on canine BM samples. Twenty replicate sternal samples obtained within 24 hours of death were fixed overnight in either acetic acid-zinc-formalin (AZF) or 10% neutral-buffered formalin (NBF) and demineralized with formic acid for 12 hours. Another 53 samples were fixed in AZF and demineralized with hydrochloric acid for 1-hour, formic acid for 12 hours, or ethylenediamine tetraacetic acid (EDTA) for 24 hours. Histologic sections were scored by 4 raters as of insufficient, marginal, good, or excellent quality. In addition, DNA samples extracted from sections treated with the different fixation and demineralization methods were amplified with 3 sets of primers to conserved regions of T cell receptor gamma and immunoglobulin heavy chain genes. Amplification efficiency was graded based on review of capillary electrophoretograms. There was no significant difference in the histomorphology scores of sections fixed in AZF or NBF. However, EDTA-based demineralization yielded higher histomorphology scores than demineralization with hydrochloric or formic acid, whereas formic acid resulted in higher scores than hydrochloric acid. Demineralization with EDTA yielded DNA amplification in 29 of 36 (81%) samples, whereas demineralization with either acid yielded amplification in only 2 of 72 (3%) samples. Although slightly more time-consuming and labor-intensive, tissue demineralization with EDTA results in superior morphology and is critical for polymerase chain reaction (PCR) amplification with the DNA extraction method described in this article. [ABSTRACT FROM AUTHOR]

The document titled "Supplementary Material" from the journal Frontiers in Microbiology provides detailed information on the composition of media used in a study, MIQE guidelines for experimental details, and the inactivation rate of IAV in different experimental conditions. The study explores discrepancies in virus inactivation rates between aerosol particles and microliter droplet experiments, speculating on factors such as initial virus titer, pH of the solution, and surface tension forces. The document emphasizes the need for further research to fully understand these discrepancies. [Extracted from the article]

Background: Toxocara canis and Toxocara cati are parasitic nematodes that occur worldwide. As embryonated Toxocara spp. eggs in the environment pose a zoonotic risk, especially for children, optimal diagnostic approaches are necessary for effective disease response and management, including surveillance. However, little is known about the performance of different diagnostic protocols for detecting Toxocara spp. in the faeces of cats and dogs, hampering movement towards an optimal diagnostic process. This study aimed to compare detection methods, including a newly developed sequential sieving protocol (SF-SSV) and a high-throughput multiplex qPCR-based method to facilitate epidemiological studies. Methods: Species-specific Toxocara spp. egg suspensions and canine and feline faecal samples from the field were used to estimate analytical and diagnostic sensitivity of the protocols. The performance of two automated DNA extraction protocols using enzymatic and mechanical lysis were compared by multiplex qPCR, targeting both T. canis and T. cati-specific genomic sequences. All samples were examined by microscopy-based techniques, the sedimentation flotation technique (SF) and a newly developed SF-SSV for the detection, enrichment and purification of parasite eggs. The costs and processing times necessary for all protocols were estimated and compared for both single samples and sets of 100 samples. Results: To detect Toxocara spp. eggs, SF-SSV showed the highest analytical sensitivity and a significantly higher diagnostic sensitivity than the DNA detection methods. Mechanical lysis performed better than enzymatic lysis for automated DNA extraction. In automated DNA extraction, 96-well plates performed better than 24-well plates. DNA detection and microscopy-based parasitological methods showed substantial agreement between the results generated by each method. Microscopy-based techniques required the lowest costs and least hands-on time for a single sample. However, when costs and labour were estimated for a set of 100 samples, the DNA detection protocol using 96-well plates for extraction revealed costs similar to SF-SSV and the fastest processing times. Conclusions: SF-SSV was superior in terms of analytical and diagnostic sensitivity for the detection of Toxocara spp. eggs. For larger sets of samples, multiplex qPCR-based DNA detection represents an alternative to microscopy-based methods, based on the possibility of faster sample processing at similar costs to SF-SSV, and the ability to provide species-specific diagnoses. [ABSTRACT FROM AUTHOR]

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 virusfocused methods for DNA extraction and specific sequencing aimed at understanding viral impact on the microbiome in the built environment. [ABSTRACT FROM AUTHOR]

In Australia, native possums are a major wildlife reservoir for Mycobacterium ulcerans, the causative agent of the neglected tropical skin disease Buruli ulcer (BU). Large-scale possum excreta surveys that use PCR to detect M. ulcerans in 100-1,000 s of excreta specimens are an important tool that can inform geospatial modeling and predict locations of future human BU risk. However, the significant expense of commercial kits used to extract DNA from specimens is a major barrier to routine implementation. Here, we developed a low-cost method for DNA extraction from possum excreta, possum tissue, and pure mycobacterial cultures, using a guanidinium isothiocyanate lysis solution and paramagnetic beads. In a 96-well plate format for high-throughput processing, the paramagnetic bead DNA extraction method was threefold less sensitive but only 1/6 the cost of a commonly used commercial kit. Applied to tissue swabs, the method was fourfold more sensitive and 1/5 the cost of a commercial kit. When used for preparing DNA from pure mycobacterial cultures, the method yielded purified genomic DNA with quality metrics comparable to more lengthy techniques. Our paramagnetic bead method is an economical means to undertake large-scale M. ulcerans environmental surveillance that will directly inform efforts to halt the spread of BU in Victoria, Australia, with potential for applicability in other endemic countries. IMPORTANCE Buruli ulcer (BU) is a neglected tropical skin disease, with an incidence that has dramatically increased in temperate southeastern Australia over the last decade. In southeastern Australia, BU is a zoonosis with native possums the major wildlife reservoir of the causative pathogen, Mycobacterium ulcerans. Infected possums shed M. ulcerans in their excreta, and excreta surveys using PCR to screen for the presence of pathogen DNA are a powerful means to predict future areas of Buruli ulcer risk for humans. However, excreta surveys across large geographic areas require testing of many thousands of samples. The cost of commercial DNA extraction reagents used for preparing samples for PCR testing can thus become prohibitive to effective surveillance. Here, we describe a simple, low-cost method for extracting DNA from possum excreta using paramagnetic beads. The method is versatile and adaptable to a variety of other sample types including swabs collected from possum tissues and pure cultures of mycobacteria. [ABSTRACT FROM AUTHOR]

The article published in the Journal of Orthopaedic Research discusses the use of bead-beating DNA extraction during synovial fluid collection to improve the accuracy of PCR-based genetic diagnosis for periprosthetic joint infection (PJI). The study compared the detection rates between conventional and bead-beating DNA extraction methods, showing an improvement from 86.7% to 95.6% accuracy with the bead-beating method. The findings suggest that bead-beating DNA extraction can enhance the accuracy of PCR-based genetic diagnosis for PJI caused by Gram-positive bacteria. [Extracted from the article]

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. [ABSTRACT FROM AUTHOR]

Scrub typhus is caused by the Gram-negative obligate intracellular bacterium Orientia tsutsugamushi, and this tick-borne disease is difficult to distinguish from other acute febrile illnesses as it typically presents with symptoms such as rash, crusting at the bite site, headache, myalgia, lymphadenopathy, and elevated liver transaminases. It can often be diagnosed clinically, but not all patients present with characteristic symptoms, so serological diagnosis and molecular techniques may be required. However, existing diagnostic tests often have low sensitivity and specificity, making early detection difficult. This study presents a nucleic acid extraction method using large volumes of plasma and buffy coat to increase sensitivity, as well as an improved detection method using two target genes. Using the I-PULL device, nucleic acids can be extracted from up to 4 mL of sample in 30 min, avoiding contamination. The extracted DNA detects two genes of O. tsutsugamushi, increasing sensitivity compared to single-gene detection. Clinical validation in 38 patient samples showed 100% specificity and 95.24% sensitivity for the single target gene, with specificity and sensitivity rising to 100% when both genes are analyzed. This molecular diagnostic platform can be useful for distinguishing scrub typhus from similar diseases. [ABSTRACT FROM AUTHOR]

Introduction: Transcriptomics studies speed up the basic and applied research on the identification of genes involved in the biosynthesis of medicinally significant primary and secondary metabolites as well as plant responses to biotic and abiotic stresses. The adequate quality and quantity of RNA are essential for successful transcriptomics investigations such as RNA sequencing (RNA-seq) and microarrays. It is extremely difficult to isolate RNA from medicinal plants with high levels of polyphenols and polysaccharides, such as Borago officinalis. Moreover, isolating nucleic acids from tissues exposed to stressful conditions of heavy metal toxicity such as cadmium is challenging due to the increased accumulation of reactive oxygen species (ROS) and secondary metabolites. Any RNA-seq experiment requires high-quality RNA because the isolated RNA should meet stringent quality control requirements in order to be sequenced on the various platforms. In the present study, we evaluated different RNA extraction methods to obtain an efficient protocol for isolating high-quality total RNA from borage tissue exposed to cadmium stress. Materials and methods: The borage seedlings were grown in hydroponic containers containing half-strength Hoagland's nutrient solution in a growth chamber. Borage seedlings were exposed to 162 μM Cd using cadmium nitrate (Cd (NO3)2.4H2O) at 5-6 leaves stage and sampled at 48 h after treatment. The roots and leaves were subjected to five RNA isolation methods, including phenol/chloroform-based method, CTAB-based method, SDS-based method, RNX-plus protocol, and modified RNX-plus method to obtain an efficient protocol for isolating high-quality total RNA. The concentration and purity of the RNAs extracted using the abovementioned protocols were determined using gel electrophoresis and NanoDrop spectrophotometer. The quality and integrity of selected total RNA were approved with cDNA synthesis, RT-PCR, Bioanalyzer System, and transcriptome sequencing. After evaluating the extraction methods, a quick, simple and efficient instruction based on the modified RNX-Plus extraction method was afforded. Results and discussion: The results showed that the modified RNX-plus method was a fast and efficient protocol for the isolation of RNA from the borage leaf and root when compared with other methods. The method overcame the limitations posed by poor quality and low concentration of isolated RNA from borage samples exposed to cadmium stress. The A260/A280 and A260/A230 ratios of the RNA extracted using the modified RNX-plus method were 2.1 and 2.07, respectively, revealing its high purity. The key factors in the optimized protocol that resulted in removing the impurities were included the increasing ratio of extraction buffer to the amount of the powdered plant sample, using the optimized volume of chloroform, raising the RNA precipitation time at -20°C, washing RNA with lithium chloride and washing again with ethanol. Also, the yields of 333±15 and 463±43 ng μl-1 of RNA with RNA integrity (RIN) numbers of 8.6 and 9.05 were obtained from roots under cadmium stress and control conditions using the described optimized method, respectively. Conclusion: In general, the results of this study showed that the modified RNX-Plus method is convenient, fast, and effective for the isolation of total RNA from borage root and leaf tissues that contain different levels of polysaccharides, polyphenols, and secondary metabolites, and no solution is needed to be prepared before, except for ethanol and Lithium chloride. Since the RNA extracted from this procedure was successfully used for cDNA library construction, RT-PCR, and RNA sequencing, it can be considered as a simple and efficient method for the isolation of RNA from medicinal plants. [ABSTRACT FROM AUTHOR]

In response to the growing need for effective food safety protocols in game meat, this study introduces a novel DNase I treatment and heat kill method to detect live bacterial pathogens in meat, particularly in game. Focusing on male wild boar shoulder meat, we tested the method’s efficacy under a stringent condition of 59°C for 60 minutes. The results demonstrate that DNase I remains active, enabling the accurate differentiation between viable and non-viable bacterial cells for precise pathogen quantification. This method’s simplicity and potential for high-throughput processing suggest it could be an invaluable tool for enhancing the safety and quality of game meat. These findings offer a practical approach to game meat safety, and can permit large-scale food safety testing across various meat types. [ABSTRACT FROM AUTHOR]

[Objective] To establish a multiple fluorescence PCR detection method for common cattle, sheep and pig derived components in animal meat skewer products, and to achieve rapid identification of animal derived components in meat skewer products. [Method] Compared the extraction effect of DNA from meat mince tissue using magnetic bead method and column membrane method, optimized the real-time fluorescence PCR multiple detection method, and compared the amplification effect of cattle, sheep, and pig derived components at two annealing temperatures of 58 and 60 ℃. [Result] The two DNA extraction methods used in the experiment had similar effects, and the A260/A280 values of the DNA solution could meet the requirements of PCR detection. 58 ℃ was the optimal annealing temperature, at which point the cattle, sheep and pig derived DNA could be effectively amplified, and the lowest detected DNA concentration was 10-3 ng/µL. The primers and probes used in the method had good specificity and had not produced non-specific amplification. [Conclusion] The multiplex PCR detection method established by the application scope experiment for cattle, sheep and pig origin can provide a method reference for rapid qualitative analysis of animal origin components in meat products. [ABSTRACT FROM AUTHOR]

Background: Next-generation sequencing (NGS) approaches have revolutionized gut microbiome research and can provide strain-level resolution, but these techniques have limitations in that they are only semi-quantitative, suffer from high detection limits, and generate data that is compositional. The present study aimed to systematically compare quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for the absolute quantification of Limosilactobacillus reuteri strains in human fecal samples and to develop an optimized protocol for the absolute quantification of bacterial strains in fecal samples. Results: Using strain-specific PCR primers for L. reuteri 17938, ddPCR showed slightly better reproducibility, but qPCR was almost as reproducible and showed comparable sensitivity (limit of detection [LOD] around 104 cells/g feces) and linearity (R2 > 0.98) when kit-based DNA isolation methods were used. qPCR further had a wider dynamic range and is cheaper and faster. Based on these findings, we conclude that qPCR has advantages over ddPCR for the absolute quantification of bacterial strains in fecal samples. We provide an optimized and easy-to-follow step-by-step protocol for the design of strain-specific qPCR assays, starting from primer design from genome sequences to the calibration of the PCR system. Validation of this protocol to design PCR assays for two L. reuteri strains, PB-W1 and DSM 20016 T, resulted in a highly accurate qPCR with a detection limit in spiked fecal samples of around 103 cells/g feces. Applying our strain-specific qPCR assays to fecal samples collected from human subjects who received live L. reuteri PB-W1 or DSM 20016 T during a human trial demonstrated a highly accurate quantification and sensitive detection of these two strains, with a much lower LOD and a broader dynamic range compared to NGS approaches (16S rRNA gene sequencing and whole metagenome sequencing). Conclusions: Based on our analyses, we consider qPCR with kit-based DNA extraction approaches the best approach to accurately quantify gut bacteria at the strain level in fecal samples. The provided step-by-step protocol will allow scientists to design highly sensitive strain-specific PCR systems for the accurate quantification of bacterial strains of not only L. reuteri but also other bacterial taxa in a broad range of applications and sample types. -oGjUmj5e8MXZDxyDjzcm- Video Abstract [ABSTRACT FROM AUTHOR]

Rapid detection of plant diseases before they escalate can improve disease control. Our team has developed rapid nucleic acid extraction methods with microneedles and combined these with loop-mediated amplification (LAMP) assays for pathogen detection in the field. In this work, we developed LAMP assays for early blight (Alternaria linariae, A. alternata, and A. solani) and bacterial spot of tomato (Xanthomonas perforans) and validated these LAMP assays and two previously developed LAMP assays for tomato spotted wilt virus and late blight. Tomato plants were inoculated, and disease severity was measured. Extractions were performed using microneedles, and LAMP assays were run in tubes (with hydroxynaphthol blue) on a heat block or on a newly designed microfluidic slide chip on a heat block or a slide heater. Fluorescence on the microfluidic chip slides was visualized using EvaGreen and photographed on a smartphone. Plants inoculated with X. perforans or tomato spotted wilt virus tested positive prior to visible disease symptoms, whereas Phytophthora infestans and A. linariae were detected at the time of visual disease symptoms. LAMP assays were more sensitive than PCR, and the limit of detection was 1 pg of DNA for both A. linariae and X. perforans. The LAMP assay designed for early blight detected all three species of Alternaria that infect tomato and is thus an Alternaria spp. assay. This study demonstrates the utility of rapid microneedle extraction followed by LAMP on a microfluidic chip for rapid diagnosis of four important tomato pathogens. [ABSTRACT FROM AUTHOR]

The Ujung Genteng Beach, located in Sukabumi, is renowned for its extensive seagrass beds and functions as an intertidal area. However, there has been limited scientific investigation into the diversity of microalgae in this ecosystem. This research aimed to identify the diversity of microalgae sourced from the seawater of Ujung Genteng Beach based on morphological characteristics and optimize DNA isolation and amplification techniques. Sampling was conducted three times at predetermined research locations. Morphological identification was performed using a binocular microscope, while DNA isolation and amplification were performed using CTAB methods with modification. The number of species found at Ujung Genteng Beach, Sukabumi is 47 species, 12 of which are often found at the three research locations both morning and afternoon, namely Navicula sp., Frustulia sp., Diploneis parma, Nitzschia sp., Achnanthidium sp., Amphora sp., Oscillatoria teneuis, Achanthes sp., Planothidium sp., Uronema sp., Zygnema sp., and Flagillaria sp.Based on the gel image, genomic DNA of seawater microalgae was successfully isolated, despite the relatively low purity and concentration. Analysis of diversity index and species diversity revealed variations in species abundance and diversity among different locations, particularly in seawater microalgae found within distinct zones: 0-2 meters from the low tide line (location I), seawater within seagrass areas located 2-5 meters away (location II), and seawater near the open sea, situated 5-10 meters away (location III).Therefore, the diversity of microalgae at all sampling locations was in the medium category and the uniformity index between species was low. [ABSTRACT FROM AUTHOR]

To tease apart differences between conventional cage (CC) and cage-free (CF) housing systems, this study focuses on the effects of excreta exposure and age by comparing microbial communities, intestinal permeability, and corticosterone in hens in enriched colonies (EC) and CF housing systems during early- and late-lay. Hens were randomly selected from two rooms of CF (n = 20) and EC (n = 20) at 35 and 76 weeks of age. One hour following an oral gavage of fluorescein isothiocyanate dextran (FITC-D), hens were euthanized, and ileal contents and blood were collected. Serum FITC-D using a fluorescent spectrophotometer and corticosterone using a commercial competitive ELISA kit were analyzed. Following DNA isolation from the ileum contents, the V4 region of the 16S rRNA gene was sequenced. Sequence data were filtered in Mothur v1.43.0, followed by de novo operational taxonomic unit (OTU) clustering and classifying with the SILVA SSU v138 reference database. Serum FITC-D was altered by housing type, age of hens, and the interaction between housing type and age of hens (p < 0.001), with 76-week-old hens housed in EC having the highest FITC-D. Corticosterone increased with age (p = 0.023). Microbial community diversity measurements favored hens housed in the CF housing system as ileal contents tended to have increased species evenness (p = 0.008) and greater alpha diversity (p = 0.006). The majority of the over-representation of OTUs were associated with peak lay. [ABSTRACT FROM AUTHOR]

Studies in evolution, ecology and conservation are increasingly based on genetic and genomic data. With increased focus on molecular approaches, ethical concerns about destructive or more invasive techniques need to be considered, with a push for minimally invasive sampling to be optimised. Buccal swabs have been increasingly used to collect DNA in a number of taxa, including amphibians. However, DNA yield and purity from swabs are often low, limiting its use. In this study, we compare different types of swabs, preservation method and storage, and DNA extraction techniques in three case studies to assess the optimal approach for recovering DNA in anurans. Out of the five different types of swabs that we tested, Isohelix MS‐02 and Rapidry swabs generated higher DNA yields than other swabs. When comparing storage buffers, ethanol is a better preservative than a non‐alcoholic alternative. Dried samples resulted in similar or better final DNA yields compared to ethanol‐fixed samples if kept cool. DNA extraction via a Qiagen™ DNeasy Blood and Tissue Kit and McHale's salting‐out extraction method resulted in similar DNA yields but the Qiagen™ kit extracts contained less contamination. We also found that samples have better DNA recovery if they are frozen as soon as possible after collection. We provide recommendations for sample collection and extraction under different conditions, including budgetary considerations, size of individual animal sampled, access to cold storage facilities and DNA extraction methodology. Maximising efficacy of all of these factors for better DNA recovery will allow buccal swabs to be used for genetic and genomic studies in a range of vertebrates. [ABSTRACT FROM AUTHOR]

As DNA sequencing technology continues to rapidly improve, studies investigating the microbial communities of host organisms (i.e., microbiota) are becoming not only more popular but also more financially accessible. Across many taxa, microbiomes can have important impacts on organismal health and fitness. To evaluate the microbial community composition of a particular microbiome, microbial DNA must be successfully extracted. Fecal samples are often easy to collect and are a good source of gut microbial DNA. Additionally, interest in the avian preen gland microbiome is rapidly growing, due to the importance of preen oil for many aspects of avian life. Microbial DNA extractions from avian fecal and preen oil samples present multiple challenges, however. Here, we describe a modified PrepMan Ultra Sample Preparation Reagent microbial DNA extraction method that is less expensive than other commonly used methodologies and is highly effective for both fecal and preen oil samples collected from a broad range of avian species. We expect our method will facilitate microbial DNA extractions from multiple avian microbiome reservoirs, which have previously proved difficult and expensive. Our method therefore increases the feasibility of future studies of avian host microbiomes. [ABSTRACT FROM AUTHOR]

Aspergillus species are known to be very important in human and domestic animal health. Aspergillus species commonly cause severe systemic and skin infections, as well as allergic lung diseases. With the development of PCR techniques, these methods are used to identify and diagnose fungi. DNA extraction from Aspergillus species is difficult because the fungal cell wall structure is very durable and complex. Fungal DNA extraction methods containing proteinase K and liquid nitrogen are widely used to break down the cell wall. However, these methods cause DNA loss during the extraction in Aspergillus species. In this study, on the contrary, the commonly used DNA extraction by means of ammonium hydroxide, which is generally used to break down chitin in DNA extraction of ticks and plants, is used. The efficiency of the cell wall lysis method from A. flavus with ammonium hydroxide was compared with methods containing proteinase K and liquid nitrogen. For this purpose, DNA extraction of A. flavus was tried using three different methods. As a result, the cell wall of A. flavus was lysed using ammonium hydroxide in this study. The obtained DNA's quality, concentration, and PCR performance were sufficient. This method has been evaluated as a faster, more straightforward, and more economical alternative. [ABSTRACT FROM AUTHOR]

In samples of harmful algal blooms (HABs), seawater can contain a high abundance of microorganisms and elemental ions. Along with the hardness of the walls of key HAB dinoflagellates such as Prorocentrum triestinum, this makes RNA extraction very difficult. These components interfere with RNA isolation, causing its degradation, in addition to the complex seawater properties of HABs that could hinder RNA isolation for effective RNA sequencing and transcriptome profiling. In this study, an RNA isolation technique was established through the modification of the Trizol method by applying the Micropestle System on cell pellets of P. triestinum frozen at −20 °C, obtained from 400 mL of culture with a total of 107 cells/mL. The results of the modified Trizol protocol generated quality RNA samples for transcriptomics sequencing, as determined by their measurement in Analyzer Agilent 4150. [ABSTRACT FROM AUTHOR]

Genetic assessment of highly incinerated and/or degraded human skeletal material is a persistent challenge in forensic DNA analysis, including identifying victims of mass disasters. Few studies have investigated the impact of thermal degradation on whole‐genome single‐nucleotide polymorphism (SNP) quality and quantity using next‐generation sequencing (NGS). We present whole‐genome SNP data obtained from the bones and teeth of 27 fire victims using two DNA extraction techniques. Extracts were converted to double‐stranded DNA libraries then enriched for whole‐genome SNPs using unpublished biotinylated RNA baits and sequenced on an Illumina NextSeq 550 platform. Raw reads were processed using the EAGER (Efficient Ancient Genome Reconstruction) pipeline, and the SNPs filtered and called using FreeBayes and GATK (v. 3.8). Mixed‐effects modeling of the data suggest that SNP variability and preservation is predominantly determined by skeletal element and burn category, and not by extraction type. Whole‐genome SNP data suggest that selecting long bones, hand and foot bones, and teeth subjected to temperatures <350°C are the most likely sources for higher genomic DNA yields. Furthermore, we observed an inverse correlation between the number of captured SNPs and the extent to which samples were burned, as well as a significant decrease in the total number of SNPs measured for samples subjected to temperatures >350°C. Our data complement previous analyses of burned human remains that compare extraction methods for downstream forensic applications and support the idea of adopting a modified Dabney extraction technique when traditional forensic methods fail to produce DNA yields sufficient for genetic identification. [ABSTRACT FROM AUTHOR]

Background: Extracting high-quality total RNA is pivotal for advanced RNA molecular studies, such as Next-generation sequencing and expression microarrays where RNA is hybridized. Despite the development of numerous extraction methods in recent decades, like the cetyl-trimethyl ammonium bromide (CTAB) and the traditional TRIzol reagent methods, their complexity and high costs often impede their application in small-scale laboratories. Therefore, a practical and economical method for RNA extraction that maintains high standards of efficiency and quality needs to be provided to optimize RNA extraction from human and mice tissues. Method: This study proposes enhancements to the TRIzol method by incorporating guanidine isothiocyanate (GITC-T method) and sodium dodecyl sulfate (SDS-T method). We evaluated the effectiveness of these modified methods compared to the TRIzol method using a micro-volume UV-visible spectrophotometer, electrophoresis, q-PCR, RNA-Seq, and whole transcriptome sequencing. Result: The micro-volume UV-visible spectrophotometer, electrophoresis, and RNA-Seq demonstrated that the GITC-T method yielded RNA with higher yields, integrity, and purity, while the consistency in RNA quality between the two methods was confirmed. Taking mouse cerebral cortex tissue as a sample, the yield of total RNA extracted by the GITC-T method was 1,959.06 ± 49.68 ng/mg, while the yield of total RNA extracted by the TRIzol method was 1,673.08 ± 86.39 ng/mg. At the same time, the OD260/280 of the total RNA samples extracted by the GITC-T method was 2.03 ± 0.012, and the OD260/230 was 2.17 ± 0.031, while the OD260/280 of the total RNA samples extracted by the TRIzol method was 2.013 ± 0.041 and the OD260/230 was 2.11 ± 0.062. Furthermore, q-PCR indicated that the GITC-T method achieved higher yields, purity, and greater transcript abundance of total RNA from the same types of animal samples than the TRIzol method. Conclusion: The GITC-T method not only yields higher purity and quantity of RNA but also reduces reagent consumption and overall costs, thereby presenting a more feasible option for small-scale laboratory settings. [ABSTRACT FROM AUTHOR]

Stick-tight fleas, Echidnophaga gallinacea (Westwood), are major ectoparasites of domesticated chickens and can cause serious diseases and even death if left untreated. In the present study the flea samples were collected from three traditional chicken-raising farms in the Al-Baha region. The samples were examined under a stereomicroscope and identified using classification keys. After extracting DNA from the insects, the polymerase chain reaction technique was used to identify the hereditary gene, cytochrome oxidase, present in the insect biopics. The gene was purified, its nucleotide sequence was obtained, and the accession number (OR161051) has been assigned in GenBank. After determining its nucleotide sequence, it was compared with other insects in GenBank, where it was found to be identical (99.82%) to the E. gallinacea isolate from Thailand recorded in GenBank [OQ291364 and MW492259 (https://www.ncbi.nlm.nih.gov/nucleotide/MW492259.1?report=genbank&log$=nucltop&blast%5frank=1&RID=VJR9ZAEE013)]. The match was 94.33% with Echidnophaga iberica from Spain (KF479239) and 93.97% with Echidnophaga oschanini from China (KU880666). The phylogenetic tree also showed similarities between the Saudi Arabian isolates and other isolates. This genetic study of sticktight fleas is the first of its kind in the Kingdom of Saudi Arabia, therefore will be valuable for assessing the prevalence and geographical distribution of this parasite. [ABSTRACT FROM AUTHOR]

The study aims to enhance students' system thinking skills through phylogenetic analysis based on DNA sequencing results. The study is carried out through three steps: (i) pretest: evaluate students' knowledge, (ii) treatment: analysis of affinity relationships through phylogenetics using DNA sequence data, and (iii) postest: measurement of improvement in systems thinking skills after implementation. Evaluations are conducted using written tests as well as worksheets and supported by surveys, questionnaires, and interviews for qualitative analysis. The experiment began with the isolation of citrus DNA, determination of concentration and purity, amplification, reading of sequencing results, and phylogenetics. The results of the teaching analysis show that there is a significant improvement in students' mastery of conceptual and system thinking skills, and there is a high correlation between the pretest and posttest. The problem analysis of interviews and questionnaires is also done to see the enthusiasm for activities as well as the effectiveness of the worksheets used. The study comes with new information on determining the relationship between local citrus in Bengkulu and improving the students' system thinking skills. [ABSTRACT FROM AUTHOR]

BACKGROUND: Streptococcus mutans is an important pathogen of dental caries, and timely detection of its levels is of great significance for early detection and treatment of dental caries. OBJECTIVE: To evaluate the effect of loop-mediated isothermal amplification (FTA-LAMP) direct extraction of Streptococcus mutans DNA. METHODS: (1) Bacterial suspensions containing ATCC standard strains (Streptococcus mutans) were prepared and inoculated into the brain-heart leachate medium. After mixed thoroughly, the mixture was then diluted in a 10-fold gradient into seven concentrations (4.2×107, 4.2×106, 4.2×105, 4.2×104, 4.2×10³, 4.2×10², 4.2×10 CFU/ mL), two parallel controls were made for each dilution level, and sterile water was used as a blank control. (2) The DNA of Streptococcus mutans was extracted using FTA Elute card, boiling method, kit extraction and lysate extraction methods separately and then amplified using LAMP technology was amplified. A specificity test was also performed to compare the differences between the four DNA extraction methods. RESULTS AND CONCLUSION: The DNA extracted by all four methods met the requirements for LAMP amplification. Specificity test results showed that only Streptococcus mutans could specifically amplify the target gene. The detection limit value of the DNA concentration was 4.2×10³ CFU/mL for the lysate method, 4.2×104 CFU/mL for the FTA Elute card extraction method, 4.2×106 CFU/mL for the kit extraction method, and 4.2×107 CFU/mL for the boiling method. In the other aspects of the four extraction methods, the kit extraction method had the highest experimental cost, number of steps and time; the other three methods had the same number of steps, with the FTA Elute card method requiring the least amount of instruments, the boiling method having the lowest single cost, and the lysate extraction method taking the least amount of time. Only a small amount of bacteria were needed for successful extraction using both the FTA Elute card and lysate extraction methods. Compared with the FTA Elute card method, the lysate extraction method was superior in terms of time, but it had a high single cost and required more equipment. To conclude, the FTA-LAMP technology established in this study has the advantages of ease of operation, high specificity, high sensitivity, and visualization, which is expected to be a new way for efficient extraction and detection of Streptococcus mutans. [ABSTRACT FROM AUTHOR]

Background: The rapid transmission and high pathogenicity of respiratory viruses significantly impact the health of both children and adults. Extracting and detecting their nucleic acid is crucial for disease prevention and treatment strategies. However, current extraction methods are laborious and time-consuming and show significant variations in nucleic acid content and purity among different kits, affecting detection sensitivity and efficiency. Our aim is to develop a novel method that reduces extraction time, simplifies operational steps, and ensures high-quality acquisition of respiratory viral nucleic acid. Methods: We extracted respiratory syncytial virus (RSV) nucleic acid using reagents with different components and analyzed cycle threshold (Ct) values via quantitative real-time polymerase chain reaction (qRT-PCR) to optimize and validate the novel lysis and washing solution. The performance of this method was compared against magnetic bead, spin column, and precipitation methods for extracting nucleic acid from various respiratory viruses. The clinical utility of this method was confirmed by comparing it to the standard magnetic bead method for extracting clinical specimens of influenza A virus (IAV). Results: The solution, composed of equal parts glycerin and ethanol (50% each), offers an innovative washing approach that achieved comparable efficacy to conventional methods in a single abbreviated cycle. When combined with our A Plus lysis solution, our novel five-minute nucleic acid extraction (FME) method for respiratory viruses yielded superior RNA concentrations and purity compared to traditional methods. FME, when used with a universal automatic nucleic acid extractor, demonstrated similar efficiency as various conventional methods in analyzing diverse concentrations of respiratory viruses. In detecting respiratory specimens from 525 patients suspected of IAV infection, the FME method showed an equivalent detection rate to the standard magnetic bead method, with a total coincidence rate of 95.43% and a kappa statistic of 0.901 (P < 0.001). Conclusions: The FME developed in this study enables the rapid and efficient extraction of nucleic acid from respiratory samples, laying a crucial foundation for the implementation of expedited molecular diagnosis. [ABSTRACT FROM AUTHOR]

Introduction: Buruli ulcer (BU) caused by Mycobacterium ulcerans (MU) is a devastating necrotic skin disease. PCR, recommended for confirmation of BU by WHO, requires an adequately equipped laboratory, therefore often delaying timely diagnosis and treatment of BU patients in remote settings. Loop-mediated isothermal amplification (LAMP) is a PCR-based protocol for isothermal amplification of DNA that has been suggested for diagnosis of BU in low-resource settings. Study aims and methods: This is an exploratory diagnostic test evaluation study, with an embedded qualitative sub-study. Its aims are two-fold: First, to evaluate a simple rapid syringe-based DNA extraction method (SM) in comparison with a more elaborate conventional DNA extraction method (CM), followed by a LAMP assay targeting IS2404 for the detection of MU, either using a commercially available pocket warmer (pw) or a heat block (hb) for incubation. Second, to complement this by exploring the diagnostic workflow for BU at a community-based health centre in an endemic area in rural Ghana as an example of a potential target setting, using interviews with researchers and health care workers (HCWs). Diagnostic test evaluation results are discussed in relation to the requirements of a target product profile (TPP) for BU diagnosis and the target setting. Results: A protocol using SM for DNA extraction followed by IS2404 PCR (IS2404 PCRSM) was able to identify MU DNA in 73 out of 83 BU clinical specimens submitted for diagnosis. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IS2404 PCRSM were 90.12%, 100%, 100% and 65.21% respectively, as compared to the reference standard IS2404 PCR in combination with a standard extraction protocol for mycobacterial DNA. Evaluation of the LAMP assay on 64 SM DNA extracts showed a sensitivity, specificity, PPV and NPV of 83.6%, 100%, 100% and 50%, respectively, using either pocket warmer (pwLAMPSM) or heat block (hbLAMPSM) for incubation of the reaction, as compared to the same reference standard. The limit of detection of pwLAMPSM was found to be 30 copies of the IS2404 target. Interview findings explored barriers to BU diagnosis and treatment, including perceptions of the disease, costs, and availability of transport. Participants confirmed that a diagnosis at the PoC, in addition to screening based on clinical criteria, would be advantageous in order to prevent delays and loss to follow-up. Discussion and conclusions: The high diagnostic and analytic accuracy of the pwLAMP, evaluated by us in combination with a syringe-based DNA extraction method, supports its potential use for the rapid detection of MU in suspected BU samples at the community or primary health care level without reliable electricity supply. Further optimization needs include a lysis buffer, evaluation directly at the PoC and/or other sites, assessing staff training requirements and quality control. Author summary: Buruli ulcer (BU) is a severe bacterial skin disease. Molecular diagnostic methods, as recommended by the World Health Organization (WHO), require a well-equipped laboratory with reliable electricity supply, which is challenging in low-resource settings. We aimed to evaluate a protocol for loop-mediated isothermal amplification (LAMP), independent of power supply, as an alternative DNA-detection based diagnostic tool for peripheral health care facilities at the point-of-care (PoC). Our protocol included a rapid syringe-based DNA extraction method, followed by a LAMP assay using a commercial pocket warmer for incubation. It showed promising results, with around 84% sensitivity and 100% specificity, as compared to standard molecular methods. Through interviews and discussions with researchers, healthcare workers and community members, we characterized the diagnostic and treatment workflow at a potential target site in an endemic area in Ghana's Eastern Region. Reported barriers to treatment seeking included perceptions of BU and price and availability of transport, and confirmed potential advantages of diagnosis at the PoC in preventing delays and loss to follow-up. Our study suggests that this LAMP protocol could hold promise for rapid and accurate diagnosis of BU in settings with unreliable electricity; further research will need to include further optimization and evaluation. [ABSTRACT FROM AUTHOR]

Tongue swabs hold promise as a non-invasive sample for diagnosing tuberculosis (TB). However, their utility as replacements for sputum has been limited by their varied diagnostic performance in PCR assays compared to sputum. The use of silica-based DNA extraction methods may limit sensitivity due to incomplete lysis of Mycobacterium tuberculosis (MTB) cells and co-extraction of non-target nucleic acid, which may inhibit PCR. Specificity may also be compromised because these methods are labor-intensive and prone to cross-contamination. To address these limitations, we developed a sample preparation method that combines sonication for MTB lysis and a sequence-specific MTB DNA capture method using hybridization probes immobilized on magnetic beads. In spiked tongue swabs, our hybridization capture method demonstrated a 100-fold increase in MTB DNA yield over silica-based Qiagen DNA extraction and ethanol precipitation. In a study conducted on clinical samples from South Africa, our protocol had 74% (70/94) sensitivity and 98% (41/42) specificity for detecting active pulmonary TB with sputum Xpert MTB/RIF Ultra as the reference standard. While hybridization capture did not show improved sensitivity over Qiagen DNA extraction and ethanol precipitation, it demonstrated better specificity than previously reported methods and was easier to perform. With integration into point-of-care platforms, these strategies have the potential to help enable rapid non-sputum-based TB diagnosis across key underserved patient populations. [ABSTRACT FROM AUTHOR]

Purpose: Metagenomic next-generation sequencing(mNGS) is a novel molecular diagnostic technique. For nucleic acid extraction methods, both whole-cell DNA (wcDNA) and cell-free DNA (cfDNA) are widely applied with the sample of bronchoalveolar lavage fluid (BALF). We aim to evaluate the clinical value of mNGS with cfDNA and mNGS with wcDNA for the detection of BALF pathogens in non-neutropenic pulmonary aspergillosis. Methods: mNGS with BALF-cfDNA, BALF-wcDNA and conventional microbiological tests (CMTs) were performed in suspected non-neutropenic pulmonary aspergillosis. The diagnostic value of different assays for pulmonary aspergillosis was compared. Results: BALF-mNGS (cfDNA, wcDNA) outperformed CMTs in terms of microorganisms detection. Receiver operating characteristic (ROC) analysis indicated BALF-mNGS (cfDNA, wcDNA) was superior to culture and BALF-GM. Combination diagnosis of either positive for BALF-mNGS (cfDNA, wcDNA) or CMTs is more sensitive than CMTs alone in the diagnosis of pulmonary aspergillosis (BALF-cfDNA+CMTs/BALF-wcDNA+CMTs vs. CMTs: ROC analysis: 0.813 vs.0.66, P=0.0142/0.796 vs.0.66, P=0.0244; Sensitivity: 89.47% vs. 47.37%, P=0.008/84.21% vs. 47.37%, P=0.016). BALF-cfDNA showed a significantly greater reads per million (RPM) than BALF-wcDNA. The area under the ROC curve (AUC) for RPM of Aspergillus detected by BALF-cfDNA, used to predict "True positive" pulmonary aspergillosis patients, was 0.779, with a cut-off value greater than 4.5. Conclusion: We propose that the incorporation of BALF-mNGS (cfDNA, wcDNA) with CMTs improves diagnostic precision in the identification of nonneutropenic pulmonary aspergillosis when compared to CMTs alone. BALFcfDNA outperforms BALF-wcDNA in clinical value. [ABSTRACT FROM AUTHOR]

Background: Infections with soil-transmitted helminths (STH) and schistosomiasis (SCH) result in a significant global health burden, particularly in rural communities in low and middle-income countries. While microscopy remains the primary diagnostic method for STH and SCH in resource-limited settings, nucleic acid amplification tests (NAATs) are gaining prominence as tools for evaluation of public health control programs in endemic countries, and individual diagnosis in high-income countries. Despite the high sensitivity and specificity of NAATs, previous research has highlighted inter-laboratory variations, both in technical and clinical performance, justifying the need for continuous proficiency testing. Methodology: Results from 5 rounds over a 5-year period of the so far only longitudinal international Helminth External Molecular Quality Assessment Scheme (HEMQAS), coordinated by the Dutch Foundation for Quality Assessment in Medical Laboratories (SKML), were examined in order to (i) assess the diagnostic proficiency of laboratories in detecting helminths in stool and (ii) identify potential factors contributing to variations in performance. Outcome and conclusions: Thirty-six laboratories, from 18 countries and 5 continents, participated in HEMQAS. The overall diagnostic performances were satisfying, with remarkably low numbers (<2%) of false-positive results. False-negative results were more often reported for stool (15%) than for DNA (5%) samples. False-negative results varied largely between targets (the highest number (29%) for Trichuris trichiura). Twenty-five laboratories provided a sufficient number of results for a robust comparison between participating laboratories, which confirmed substantial inter-laboratory variability in quantitative NAAT results (Cq-values). This variability likely arises from differences in pre-treatment, DNA isolation and DNA-target amplification procedures. This study emphasizes the complexity of molecular diagnosis for STH and SCH, highlighting the critical role of proper stool preparation and DNA isolation methods. The results underscore the necessity for laboratory professionals and public health decision-makers to recognize these complexities and continuously undertake external quality assessment schemes to ensure accurate and reliable performance in molecular diagnosis. Author summary: Parasitic worms cause significant global health challenges, particularly in rural communities in low and middle-income countries. Laboratory tests that detect parasite DNA in human stool are increasingly being used for the diagnosis of parasitic worm infections. While parasite DNA detection can be an excellent diagnostic tool, its performance in clinical practice varies considerably, as demonstrated by previous research. To ensure the quality in diagnostic testing of these parasitic worms, it is important that laboratories evaluate the diagnostic performance of their DNA-based tests by participating in a so-called external quality assessment scheme (EQAS). This study, focusing on the first five-yearly rounds of the so far only longitudinal international EQAS to date, reveals that quantitative DNA detection results are not directly comparable across laboratories due to substantial inter-laboratory variability. This variability mainly stems from differences in pre-treatment and DNA isolation protocols, and to a lesser extent from differences in DNA target amplification procedures. Furthermore, the study underscores the complexity of molecular diagnosis for parasitic worms in clinical practice and the necessity for laboratories to engage in EQAs to guarantee the proper functioning of these diagnostic tests. [ABSTRACT FROM AUTHOR]

Obtaining high-quality DNA suitable for long-read sequencing can be difficult for many types of tissues and cells, and it is a key step in current genomic studies. The challenge is even greater when it comes to isolating genomic DNA from mammalian spermatozoa, as DNA is tightly packed into a cell with a robust membrane rich in disulfide bonds. Here we describe a method for isolating high molecular weight DNA from Bovine commercial semen straws. This protocol includes a cleaning step to remove diluents and preservatives used for the long-term storage of the semen, which may affect long read sequencing. It is based on a simple salting-out method and avoid the use of spin columns, strong mixing or intensive centrifugation, in order to limit DNA fragmentation. However, we have adapted this protocol to facilitate the disruption of cell membranes and disulfide bonds with strong chaotropic and reducing agents. The average size of the fragments produced was approximately 49 kb, ranging from 25 to 85 kb, according to the femto pulse profiles.This method was used to isolate DNA from semen straws, more than 80 of them were successfully sequenced using the Continuous Long-Read (CLR) sequencing mode on the PacBio SequelII platform to study genome diversity and notably to detect large structural variations within genomes. [ABSTRACT FROM AUTHOR]

This document is a diagnostic protocol for Anisogramma anomala, a pathogen that causes eastern filbert blight on European hazelnut trees. It provides information on identifying and detecting the pathogen, including its symptoms and characteristics. The document includes molecular tests for confirmation in critical cases and emphasizes the importance of early detection. It also provides guidance on reporting and documentation, as well as contact information for further information. The appendices provide instructions for isolating the organism and describe the media used for its growth. The document also includes instructions for conducting a real-time PCR test to detect A. anomala, with information on controls, interpretation of results, and performance characteristics. The test has been validated and shown to have high sensitivity and specificity. [Extracted from the article]

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

Insoluble phosphorous compounds solubilization by soil bacteria is of great relevance since it puts available the phosphorus to be used by plants. The production of organic acids is the main microbiological mechanism by which insoluble inorganic phosphorus compounds are solubilized. In Gram negative bacteria, gluconic acid is synthesized by the activity of the holoenzyme glucose dehydrogenase-pyrroloquinoline quinine named GDH-PQQ. The use of marker genes is a very useful tool to evaluate the persistence of the introduced bacteria and allow to follow-up the effect of biotic and abiotic factors on these beneficial microorganisms in the soil. In previous studies we detected the presence of the pqqE gene in a great percentage of both non-culturable and culturable native soil bacteria. The objective of this study was to analyze the phylogeny of the sequence of pqqE gene and its potential for the study of phosphate solubilizing bacteria from pure and mixed bacterial cultures and rhizospheric soil samples. For this, the presence of the pqqE gene in the genome of phosphate solubilizing bacteria that belong to several bacteria was determined by PCR. Also, this gene was analyzed from mixed bacterial cultures and rhizospheric soil associated to peanut plants inoculated or not with phosphate solubilizing bacteria. For this, degenerate primers designed from several bacterial genera and specific primers for the genus Pseudomonas spp., designed in this study, were used. DNA template used from simple or mixed bacterial cultures and from rhizospheric soil samples was obtained using two different DNA extraction techniques. Results indicated that pqqE gene amplification product was found in the genome of all Gram negative phosphate solubilizing bacteria analyzed. It was possible to detect this gene in the DNA obtained from mixed cultures where these bacteria grew in interaction with other microorganisms and in that obtained from rhizospheric soil samples inoculated or not with these bacteria. The phylogenetic analysis indicated that pqqE gene is a conserved gene within related genera. In conclusion, pqqE gene could be a potential marker for the study of phosphate solubilizing bacterial populations. [ABSTRACT FROM AUTHOR]

Simple Summary: Old World bollworm is a moth species that causes significant damage to a wide range of agricultural crops. This species was once confined to the eastern hemisphere but has recently spread throughout South America and the Caribbean and has the potential to establish in North America. Molecular detection methods for Old World bollworm have been developed to track its spread and rapidly differentiate it from the native sibling species, corn earworm. Currently, droplet digital PCR (ddPCR) is a preferred method for bulk screening as it is highly accurate and tolerant of PCR inhibitors; however, real-time PCR is less expensive and more widely available in molecular labs. Improvements to DNA extraction yield and purity are crucial for real-time PCR assay optimization, but these improvements must be time- and cost-efficient. In this study, we improve upon previously published bulk DNA extraction methods by reducing bench time and costly materials. Our results indicate that the addition of caffeine and RNase A improves DNA extraction, resulting in higher amounts of target DNA in real-time PCR. Such improvements will enable the use of high throughput screening methods across multiple platforms to improve the probability of detection of Old World bollworm. Helicoverpa armigera is among the most problematic agricultural pests worldwide due to its polyphagy and ability to evolve pesticide resistance. Molecular detection methods for H. armigera have been developed to track its spread, as such methods allow for rapid and accurate differentiation from the native sibling species H. zea. Droplet digital PCR (ddPCR) is a preferred method for bulk screening due to its accuracy and tolerance to PCR inhibitors; however, real-time PCR is less expensive and more widely available in molecular labs. Improvements to DNA extraction yield, purity, and throughput are crucial for real-time PCR assay optimization. Bulk DNA extractions have recently been improved to where real-time PCR sensitivity can equal that of ddPCR, but these new methods require significant time and specialized equipment. In this study, we improve upon previously published bulk DNA extraction methods by reducing bench time and materials. Our results indicate that the addition of caffeine and RNase A improves DNA extraction, resulting in lower Cq values during real-time PCR while reducing the processing time and cost per specimen. Such improvements will enable the use of high throughput screening methods across multiple platforms to improve the probability of detection of H. armigera. [ABSTRACT FROM AUTHOR]

Collecting and preserving biological samples in the field, particularly in remote areas in tropical forests, prior to laboratory analysis is challenging. Blood samples in many cases are used for nucleic acid‐based species determination, genomics or pathogen research. In most cases, maintaining a cold chain is impossible and samples remain at ambient temperature for extended periods of time before controlled storage conditions become available. Dried blood spot (DBS) storage, blood stored on cellulose‐based paper, has been widely applied to facilitate sample collection and preservation in the field for decades. However, it is unclear how long‐term storage on this substrate affects nucleic acid concentration and integrity. We analysed nucleic acid quality from DBS stored on Whatman filter paper no. 3 and FTA cards for up to 15 years in comparison to cold‐chain stored samples using four nucleic acid extraction methods. We examined the ability to identify viral sequences from samples of 12 free‐ranging primates in the Amazon forest, using targeted hybridization capture, and determined if mitochondrial genomes could be retrieved. The results suggest that even after extended periods of storage, DBS will be suitable for some genomic applications but may be of limited use for viral pathogen research, particularly RNA viruses. [ABSTRACT FROM AUTHOR]

The article presents a study which evaluated the effects of acetyl-L-carnitine (ALC) in attenuating depressive-like symptoms in the cuprizone intoxication mouse model. Topics discussed include categories of C57BL/6 mice, RNA extraction, reverse transcription, and quantitative R-PCR, and effects of acetyl-L-carnitine on demyelination.

Most Pamphagidae species are at risk of being endangered due to their flightlessness, low mobility, and small habitat range. For the same reason, Haplotropis brunneriana (Orthoptera: Pamphagidae) has been designated an endangered species in South Korea. Endangered species with small populations are difficult to observe and investigate because any damage or disturbance to them are prohibited. To address these problems, we first performed non-invasive genomic DNA (gDNA) extraction using exuviae to identify H. brunneriana. Second, we tested the limit of detection of gDNA in the exuviae and how long it could persist when exposed to artificial environmental conditions. Using exuviae, we identified cytochrome b as a species-specific marker of H. brunneriana that could distinguish them from other grasshoppers with sufficient specificity. In the limit of detection test, gDNA could persist and be amplified from exuviae for up to 40 days. Our results demonstrated that fewer than 10 days is recommended for amplifying gDNA from exuviae to obtain reproducible results. In conclusion, this non-invasive identification method using exuviae can be used as an alternative to species identification when it is difficult to find H. brunneriana in the habitat and has the advantage of allowing genetic research to be conducted without harming the endangered species. Implications for insect conservation: Our study used a non-invasive genomic DNA extraction method to conserve endangered grasshoppers. Using exuviae, endangered grasshopper species could be identified, thus confirming their presence or absence in the habitat. This identification method can be an alternative method that can be used when conducting genetic analyses accompanied by physical harm is difficult due to small populations, such as endangered and rare species. [ABSTRACT FROM AUTHOR]

Natural history museums house the largest biodiversity collections in the world and represent an enormous repository of genetic information. Much of this information, however, has remained inaccessible until recently. Emerging technologies, such as techniques for isolation of historical DNA (hDNA) and target enrichment sequencing of ultraconserved elements (UCEs) that can utilize degraded DNA as input material, have the potential to unlock museum collections for genomics research. Here, we demonstrate that hDNA extracted from dried Pocillopora coral specimens, collected up to 90 yrs ago, can be used as input for UCE target enrichment sequencing. The resulting sequence data can be used in phylogenetic studies to resolve questions about taxonomic species identities, biogeographic distributions, and evolutionary histories. Our results provide a blueprint for research groups seeking to take advantage of untapped genetic information stored in natural history museum collections. [ABSTRACT FROM AUTHOR]

Objective To explore the genetic diversity of Aedes albopictus populations in plain, hilly, and mountainous areas of Hainan Province, and to analyze the genetic structure of Aedes albopictus populations in different terrain regions of Hainan Province. Methods Aedes albopictus were collected from the plain areas of Sanya and Haikou City, the hills of Danzhou and Tunchang City, and the mountainous areas of Baisha City in Hainan Province. DNA was extracted from a single mosquito and stored in a -80 °C refrigerator for use. Mitochondrial cytochrome C oxidase subunit I (mtDNA-CO I) was amplified by PCR and sequenced. The results were compared on the National Center for Biotechnology Information (NCBI) website, sequence peaks were observed using BioEdit 7.0, genetic diversity parameters were calculated using DnaSP v6, haplotype networks were constructed using PopART 1.7, and analysis of molecular variance (AMOVA) values were calculated using Arlequin to analyze differences between populations. Results A total of 414 mtDNA-CO I sequences were obtained from Aedes albopictus populations in 5 regions, with lengths of 663 bp. All sequences had five mutation sites, with a G+C content of 32.86% and an A+T content of 67.14%, consistent with mitochondrial DNA characteristics. Compared with other populations, the nucleotide diversity of the Danzhou and Tunchang populations in the plain and hilly areas was higher. The average nucleotide differences were higher in the Sanya, Tunchang, and Danzhou populations. Haploid analysis revealed 6 haplotypes, with H02 being the dominant haplotype. The Sanya and Tunchang populations had the highest number of haplotypes, while the Haikou and Sanya populations had exclusive haplotypes. Only the plains populations had unique haplotypes, while the hill and mountainous populations had relatively fewer haplotypes. The neutral results and mismatch distribution map indicated that the population of Aedes albopictus has recently expanded in all regions. The AMOVA value showed that the intra-population differences were greater than the inter-population differences. Conclusions The mtDNA-CO I gene can serve as a molecular marker for studying the genetic diversity of Aedes albopictus populations. The Sanya population in the plain region shows higher genetic diversity, and the two populations in the hilly region also have higher genetic diversity. However, the number of haplotypes is only higher in the Sanya population in the plains and the Danzhou population in the hills. [ABSTRACT FROM AUTHOR]

Background: Urogenital schistosomiasis is caused by the parasitic trematode Schistosoma haematobium. Sensitive and specific point-of-care diagnostics are needed for elimination of this disease. Recombinase polymerase amplification (RPA) assays meet these criteria, and an assay to diagnose S. haematobium has been developed (Sh-RPA). However, false-positive results can occur, and optimisation of reaction conditions to mitigate these is needed. Ease of use and compatibility of DNA extraction methods must also be considered. Methods: Using synthetic DNA, S. haematobium genomic DNA (gDNA), and urine samples from clinical cases, Sh-RPA reactions incorporating different betaine concentrations (0 M, 1 M, 2.5 M, 12.5 M) and the sample-to-water ratios were tested to determine effects on assay specificity and sensitivity. In addition, five commercial DNA extraction kits suitable for use in resource-limited settings were used to obtain gDNA from single S. haematobium eggs and evaluated in terms of DNA quality, quantity, and compatibility with the Sh-RPA assay. All samples were also evaluated by quantitative polymerase chain reaction (qPCR) to confirm DNA acquisition. Results: The analytical sensitivity of the Sh-RPA with all betaine concentrations was ≥ 10 copies of the synthetic Dra1 standard and 0.1 pg of S. haematobium gDNA. The addition of betaine improved Sh-RPA assay specificity in all reaction conditions, and the addition of 2.5 M of betaine together with the maximal possible sample volume of 12.7 µl proved to be the optimum reaction conditions. DNA was successfully isolated from a single S. haematobium egg using all five commercial DNA extraction kits, but the Sh-RPA performance of these kits varied, with one proving to be incompatible with RPA reactions. Conclusions: The addition of 2.5 M of betaine to Sh-RPA reactions improved reaction specificity whilst having no detrimental effect on sensitivity. This increases the robustness of the assay, advancing the feasibility of using the Sh-RPA assay in resource-limited settings. The testing of commercial extraction kits proved that crude, rapid, and simple methods are sufficient for obtaining DNA from single S. haematobium eggs, and that these extracts can be used with Sh-RPA in most cases. However, the observed incompatibility of specific kits with Sh-RPA highlights the need for each stage of a molecular diagnostic platform to be robustly tested prior to implementation. [ABSTRACT FROM AUTHOR]

Precision-cut liver slices (PCLS) are increasingly used as a model to investigate anti-fibrotic therapies. However, many studies use PCLS from healthy animals treated with pro-fibrotic stimuli in culture, which reflects only the early stages of fibrosis. The effects of different culture conditions on PCLS from cirrhotic animals has not been well characterized and there is no consensus on optimal methods. In this study, we report a method for the collection and culture of cirrhotic PCLS and compare the effect of common culture conditions on viability, function, and gene expression. Additionally, we compared three methods of RNA isolation and identified a protocol with high yield and purity. We observed significantly increased albumin production when cultured with insulin-transferrin-selenium and dexamethasone, and when incubated on a rocking platform. Culturing with insulin-transferrin-selenium and dexamethasone maintained gene expression closer to the levels in fresh slices. However, despite stable viability and function up to 4 days, we found significant changes in expression of key genes by day 2. Interestingly, we also observed that cirrhotic PCLS maintain viability in culture longer than slices from healthy animals. Due to the influence of matrix stiffness on fibrosis and hepatocellular function, it is important to evaluate prospective anti-fibrotic therapies in a platform that preserves tissue biomechanics. PCLS from cirrhotic animals represent a promising tool for the development of treatments for chronic liver disease. [ABSTRACT FROM AUTHOR]

Given extensive variability in feed composition, the absence of a dedicated DNA extraction kit for poultry feed underscores the need for an optimized extraction technique for reliable downstream sequencing analyses. This study investigates the impact of five DNA extraction techniques: Qiagen QIAamp DNA Stool Mini Kit (Qiagen), modified Qiagen with Lysing Matrix B (MQ), modified Qiagen with celite purification (MQC), polyethylene glycol (PEG), and 1-Day Direct. Genomic DNA amplification and Illumina MiSeq sequencing were conducted. QIIME2-2021.4 facilitated data analysis, revealing significant diversity and compositional differences influenced by extraction methods. Qiagen exhibited lower evenness and richness compared to other methods. 1-Day Direct and PEG enhanced bacterial diversities by employing bead beating and lysozyme. Despite similar taxonomic resolution, the Qiagen kit provides a rapid, consistent method for assessing poultry feed microbiomes. Modified techniques (MQ and MQC) improve DNA purification, reducing bias in commercial poultry feed samples. PEG and 1-Day Direct methods were effective but may require standardization. Overall, this study underscores the importance of optimized extraction techniques in poultry feed analysis, with potential implications for future standardization of effective methods. [ABSTRACT FROM AUTHOR]

Purpose: The study aimed to analyse the relationship of the rs4986790 locus of the TLR4 gene with the overall risk of preeclampsia, including both its early and late forms. Methods: The study used standard genetic analysis methods such as DNA extraction, PCR amplification, and genotyping of the rs4986790 locus of the TLR4 gene to assess the association with the development of preeclampsia and peripartal stroke in 207 pregnant women from the southern regions of Kazakhstan from 2016 to 2022, of whom 103 had peripartal stroke on the background of preeclampsia (the main group) and 104 preeclampsia (comparative group). Results: The results of the study demonstrate that the AG and AG + GG genotypes at the rs4986790 locus of the TLR4 gene are significantly associated with an increased risk of developing an early form of preeclampsia. This opens up a new perspective in the identification of genetic markers that can serve as indicators of a tendency to develop preeclampsia in earlier periods of pregnancy. Conclusion: It was noted that the rs4986790 locus did not show a statistically significant association with the risk of late preeclampsia. An important aspect of the study revealed the relationship of genotypes with the development of peripartal stroke on the background of preeclampsia. This study offers practical insights for creating targeted genetic screening and personalised treatments for preeclampsia, aiming to improve patient outcomes. To fully understand the molecular mechanisms underlying the identified association, additional research is required to identify deeper molecular pathways and relationships, and to develop new strategies for the prevention and treatment of preeclampsia. [ABSTRACT FROM AUTHOR]

Background: The Pacific white shrimp is one of the world's most economically significant aquatic species, being one of the top three species cultured globally. However, the increasing incidence of diseases such as acute hepatopancreatic necrosis disease and hepatopancreatic microsporidia has led to a serious decline in shrimp production and severe economic losses. With the increasing demand for pathogen detection in shrimp farms, rapid DNA extraction technology has become more sophisticated. In this study, a rapid and crude method of extracting genomic DNA from shrimp muscle and hepatopancreas using Chelex-100 was established. Results: DNA was successfully extracted from muscle and hepatopancreatic tissues using both the Chelex-100 method and commercial kits. The internal reference genes of shrimp were successfully amplified via PCR and real-time PCR using the obtained DNA samples. Moreover, a field assay was successfully conducted using real-time PCR and real-time enzymatic recombinase amplification (real-time ERA), indicating that the quality of the DNA extracted using Chelex-100 is sufficient for use in conjunction with nucleic acid amplification to detect pathogens in shrimps. Conclusions: Chelex-100 is an efficient method for extracting DNA from shrimp muscle or hepatopancreas tissues, with a short extraction time, high extraction efficiency, and simple operation, making it appropriate for use in the detection of pathogens in shrimp. [ABSTRACT FROM AUTHOR]

• Analyses of river microbiomes have major limitations;. • The method of water sample preparation is important;. • The search for microbiological indicators makes sense;. • Comparing results from different methods is problematic. A common method of preparing water samples for environmental DNA isolation is to vacuum filter the water sample through membrane filters. The aim of research was to test the performance of five methods of preparing river water samples for DNA isolation. DNA was isolated using a commercial kit and next-generation sequencing was performed on an Illumina Miseq platform. Pseudomonas spp. was the dominant bacterial genus in all samples. However, its relative abundance varied between samples. Depending on the volume of water filtered, the eluates yielded 62–63 % (V = 100 mL), 59–63.9 % (V = 50 mL), 17.8–19.4 % (V = 500 mL) of the relative abundance of Pseudomonas sp. In contrast, DNA isolation from the membrane filter (V = 100 mL) yielded 38 % and from the sediment after centrifugation 27 %. Differences were observed for all taxa obtained. The results indicate that even the sample volume used for filtration influences the results obtained from next-generation sequencing. [ABSTRACT FROM AUTHOR]

The present study, conducted from August to November 2022 in district Okara, Pakistan, focused on assessing the bacterial characterization in oral saliva swabs of black rats (Rattus rattus). DNA extraction was performed using the QIAamp DNA Microbiome kit, and the 16S rRNA gene was amplified using universal primers to amplify variable regions (V) V1 to V8 of 1380 bp. The identified bacterial phyla were as follows: Proteobacteria 98%, Firmicutes 1%, Actinobacteria 0.4%, and Bacteroidetes 0.05%. The bacterial classes included Gammaproteobacteria 95%, Alpha Enterobacterales 3%, and Bacilli 1%. The relative abundance of different bacterial orders was Pseudomonadales 40%, Enterobacterales 30%, Xanthomonadales 25%, Sphingomonadales 3%, Lactobacillales 1%, Micrococcales 0.4%, and Bacteroidales 0.05%. The identified families followed the order of Pseudomonadaceae 40%, Enterobacteriaceae 30%, Xanthomonadaceae 25%, and Sphingomonadaceae 3%. The percentage distribution of Pseudomonas was 40%, Stenotrophomonas 25%, Sphingomonas 3%, Pantoea 2%, and Porphyromonas 0.05%. This knowledge enhances our understanding of bacterial infections in rodents, serving as crucial baseline data for bacterial species. The high prevalence of potentially pathogenic bacteria like Pseudomonas suggests a significant risk of zoonotic diseases that could affect both local wildlife and human populations. It is crucial that future studies should focus on identified bacterial communities in other rodent species and small mammals to compare their roles in disease ecology. This ongoing research could identify specific species that are particularly significant in zoonotic transmission, thereby guiding future studies and public health measures. [ABSTRACT FROM AUTHOR]