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turboLysis is a novel mechanical cell lysis device that utilizes small beads to efficiently lyse tough cells like Mycobacterium, Saccharomyces, and Arabidopsis. We compared turboLysis to bead beating using the BeadBug 6 for several concentrations of Mycobacterium tuberculosis roughly correlated to the bacterial load commonly seen in patient samples. turboLysis performed similarly to the BeadBug at low bacterial concentrations and outperformed it at high concentrations above 2x105 CFU/ml (p

DNA- and RNA-based diagnostics play a pivotal role in accurately detecting and characterizing health-relevant bacteria, offering insights into bacterial presence, viability and treatment efficacy. Herein, we present the development of a novel extraction protocol for both DNA and RNA, designed to enable simple and rapid molecular diagnostics. The extraction method is based on the hydrophilic ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate and silica-coated magnetic beads. First, we developed an IL-based cell lysis protocol for bacteria that operates at room temperature. Subsequently, we established a magnetic bead purification procedure to efficiently and reproducibly extract DNA and RNA from the IL-lysates. The IL not only lyses the cells, but also facilitates the adsorption of nucleic acids (NAs) onto the surface of the magnetic beads, eliminating the need for a chaotropic binding buffer and allowing for purification of NAs without significant effort and materials required. Lastly, we combined the cell lysis step and the purification step and evaluated the novel IL-based extraction method on periopathogenic bacterial cultures, comparing it to commercial DNA and RNA extraction kits via (RT)-qPCR. In comparison to the reference methods, the IL-based extraction protocol yielded similar or superior results. Furthermore, costs are lower, required materials and equipment are minimal and the process is fast (30 min), simple and automatable. These characteristics favour the developed method for use in routine and high-throughput testing as well as in point-of-care, on-site and low-resource settings, thereby advancing the field of molecular diagnostics. [ABSTRACT FROM AUTHOR]

The Atacama Desert in Chile is one of the driest and most inhospitable places on Earth. To analyze the diversity and distribution of microbial communities in such an environment, one of the most important and challenging steps is DNA extraction. Using commercial environmental DNA extraction protocols, a mixture of living, dormant, and dead cells of microorganisms is extracted, but separation of the different DNA pools is almost impossible. To overcome this problem, we applied a novel method on soils across a west-east moisture transect in the Atacama Desert to distinguish between extracellular DNA (eDNA) and intracellular DNA (iDNA) at the cell extraction level. Here, we show that a large number of living and potentially active microorganisms, such as Acidimicrobiia, Geodermatophilaceae, Frankiales, and Burkholderiaceae, occur in the hyperarid areas. We observed viable microorganisms involved as pioneers in initial soil formation processes, such as carbon and nitrogen fixation, as well as mineral-weathering processes. In response to various environmental stressors, microbes coexist as generalists or specialists in the desert soil environment. Our results show that specialists compete in a limited range of niches, while generalists tolerate a wider range of environmental conditions. Use of the DNA separation approach can provide new insights into different roles within viable microbial communities, especially in low-biomass environments where RNA-based analyses often fail. [ABSTRACT FROM AUTHOR]

Genomic resources have yielded unprecedented insights into ecological and evolutionary processes, not to mention their importance in economic and conservation management of specific organisms. However, the field of macroalgal genomics is hampered by difficulties in the isolation of suitable DNA. Even when DNA that appears high quality by standard metrics has been isolated, such samples may not perform well during the sequencing process. We here have compared Oxford Nanopore long‐read sequencing results for three species of macroalgae to those of nonmacroalgal species and determined that when using macroalgal samples, sequencing activity declined rapidly, resulting in reduced sequencing yield. Chemical analysis of macroalgal DNA that would be considered suitable for sequencing revealed that DNA derived from dried macroalgae was enriched for polyphenol–DNA adducts (DNA with large polyphenols chemically attached to it), which may have led to sequencing inhibition. Of note, we observed the strongest evidence of sequencing inhibition and reduced sequence output when using samples dried using silica gel—suggesting that such storage approaches may not be appropriate for samples destined for Oxford Nanopore sequencing. Our findings have wide‐ranging implications for the generation of genomic resources from macroalgae and suggest a need to develop new storage methods that are more amenable to Oxford Nanopore sequencing or to use fresh flash‐frozen tissue wherever possible for genome sequencing. [ABSTRACT FROM AUTHOR]

Under current EU legislation, genetically modified organisms (GMOs) and derived food and feed products must be authorized as GM food, feed, or seed and appropriate detection methods must be made available for use in official controls. A Real-Time PCR method has recently been published by Chhalliyil et al. claiming to be specific for the detection and identification of genome-edited oilseed rape (OSR) lines commercialized in North America. In a previous study, we have independently assessed this method in three reference laboratories for sensitivity, specificity, and robustness. We found that the method does not meet all the minimum performance requirements (MPR) for GMO testing in the EU, which contradicts the claims of the method developer. Here we show, in addition to the previously published method assessment study that a modified DNA extraction is not the reason for the contradictory findings and does not affect the specificity of the method. We also discuss the procedures recently proposed by the method developers for interpreting PCR results with high Cq values. [ABSTRACT FROM AUTHOR]

This study presents the development of a simple DNA extraction method and a novel PCR-RFLP protocol for genotyping the MALE STERILITY 4 (MS4) gene in Cryptomeria japonica as a proof of concept. Traditional CTAB-based DNA extraction methods, while effective, involve hazardous chemicals and require high-speed centrifugation, which are impractical in many field settings. Our approach utilizes a household dish detergent-based buffer, sodium chloride, and polyvinylpyrrolidone K-30 to extract DNA from C. japonica needle leaf tissues—without the need for liquid nitrogen. The simplicity of this method makes it more accessible and environmentally friendly. The extracted DNA was successfully used in PCR-RFLP analysis, targeting a single nucleotide polymorphism in the MS4 gene, demonstrating its efficacy for genotyping. The PCR-RFLP markers reliably discriminated between individual genotypes, confirming the practical application of our simple extraction method, even for conifers containing inhibitory substances. This technique is particularly advantageous for use in arboretums and field stations, where the use of hazardous chemicals, specialized equipment, and a draft chamber is limited. Our study contributes to genetic resource management by providing an easy, reliable, and safer method for DNA extraction and genetic analysis. [ABSTRACT FROM AUTHOR]

Culex pipiens is a major vector of pathogens, including West Nile and Usutu viruses, that poses a significant public health risk. Monitoring pyrethroid resistance in mosquito populations is essential for effective vector control. This study aims to evaluate four DNA extraction protocols—QIAsymphony, DNAzol® Direct reagent, PrepMan® Ultra Sample Preparation Reagent (USPR), and Chelex® 100—to identify an optimal method to extract DNA from individual Culex pipiens, as part of a high-throughput surveillance of pyrethroid resistance using Real-Time Genotyping PCR. The target is the L1014F mutation in the voltage-sensitive sodium channel (VSSC) gene, which confers knockdown (kdr) resistance to pyrethroids. Mosquitoes were collected from wintering and summer habitats in Lazio and Tuscany, Italy, and DNA was extracted using the four methods. The quality, quantity, extraction time, and cost of the DNA were compared among the various methods. The PrepMan® USPR protocol was the most efficient, providing high-quality DNA with a 260/280 purity ratio within the optimal range at the lowest cost and in a short time. This method also demonstrated the highest amplification success rate (77%) in subsequent real-time PCR assays, making it the preferred protocol for large-scale genotyping studies. [ABSTRACT FROM AUTHOR]

Background: Zygnematophyceae green algae represent the closest living relatives of land plants. Adaptions to hydro-terrestrial environments are evident through the production of mucilage carbohydrates, which are secreted outside algal cell walls to retain water. However, the mucilage poses significant challenges for the extraction of high molecular weight (HMW) DNA. Methods: To address this, we have developed an efficient protocol optimized for algae nuclei isolation and HMW DNA extraction with modified CTAB method, facilitating the use of third-generation long read sequencing technologies, e.g., PacBio or Oxford Nanopore. Furthermore, we have benchmarked the performance of our method against eight established DNA extraction methods or commercial kits. Results: Of the eight existing DNA extraction methods assessed, the PowerPlant DNeasy Kit, prominent for its use in plant DNA extraction, was the only protocol to successfully isolate DNA from Zygnema circumcarinatum algae. However, the DNA quality was insufficient for applications in PacBio or Oxford Nanopore sequencing. In contrast, our novel method not only yielded a high DNA concentration but also high purity with optimal A260/A230 and A260/A280 ratios suitable for long read DNA sequencing. Notably, the integrity of algal DNA obtained via our method surpassed that from commercial kits, demonstrating a significant increase in the length of extracted DNA, with a peak at 55.7 kb compared to 17.6 kb for the PowerPlant DNeasy Kit. Additionally, our method substantially reduced the organellar DNA, lowering it from 72.5% in the commercial kits to 9.6%, thereby enhancing the yield of nuclear genomic DNA. Discussion: Our method represents a significant advancement in the extraction of DNA from challenging plants and algae characterized by high extracellular mucilage contents. Our protocol removes most organellar DNA, and thus substantially increases the proportion of nuclear DNA reads in the sequencing data to lower the cost for nuclear genome sequencing. Our cost-efficient method will facilitate the whole genome sequencing of mucilage-rich algae and plants. [ABSTRACT FROM AUTHOR]

Background: The purity of the extracted DNA is critical for successful molecular testing. This study aimed to compare the effect of various DNA extraction methods, extraction processes, and sources of consumables, such as microtubes, on PCR results. Methods: DNA extraction from whole blood was performed using four different approaches utilizing four types of microtubes: chloroform-based, sodium perchlorate-based, heat-assisted salting out, and solid-phase extraction. Extracted DNA was evaluated by nanodrop spectrophotometry and used in PCR-based methods for human leukocyte antigens (HLA) typing. Results: The lowest and highest concentrations of extracted DNA were observed in the column-based and heat-assisted methods, respectively. The mean ratios of A260/A230, represents chemical contamination, were significantly lower in all extraction methods using all types of microtubes versus "A" microtube. We observed the highest mean ratio of A260/A230 (> 1.9) in the sodium perchlorate method by using the "A" microtube compared to other types of microtubes and extraction methods. Extracted DNA samples using microtube "A" showed acceptable results for HLA typing compared to other microtubes that represented uninterpretable results in the PCR using the sequence-specific oligonucleotide probe (PCR-SSOP) method. Conclusion: Our findings indicate that the chemical contaminations derived mainly from microtubes can decrease the quality of DNA and consequently interfere with the amplification or hybridization reactions in the PCR-SSOP method for HLA typing. Although, technical issues and extraction processes may also influence the quality of DNA. [ABSTRACT FROM AUTHOR]

The advent of Oxford Nanopore Technologies has undergone significant improvements in terms of sequencing costs, accuracy, and sequencing read lengths, making it a cost-effective, and readily accessible approach for analyzing microbial genomes. A major challenge for bacterial whole genome sequencing by Nanopore technology is the requirement for a higher quality and quantity of high molecular weight DNA compared to short-read sequencing platforms. In this study, using eight pathogenic bacteria, we evaluated the quality, quantity, and fragmented size distribution of extracted DNA obtained from three different commercial DNA extraction kits, and one automated robotic platform. Our results demonstrated significant variation in DNA yield and purity among the extraction kits. The ZymoBIOMICS DNA Miniprep Kit (ZM) provided a higher purity of DNA compared to other kit-based extractions. All kit-based DNA extractions were successfully performed on all twenty-four samples using a single MinION flow cell, with the Nanobind CBB Big DNA kit (NB) yielding the longest raw reads. The Fire Monkey HMW-DNA Extraction Kit (FM) and the automated Roche MagNaPure 96 platform (RO) outperformed in genome assembly, particularly in gram-negative bacteria. Based on our finding, we recommend a minimum read coverage and raw read N50, obtained from the appropriate DNA extraction kit for each bacterial species, to optimize genome assembly and plasmid recovery. This approach will assist end-users in selecting the most effective kit-based extraction method for bacterial whole-genome assembly using only long-read nanopore sequences. [ABSTRACT FROM AUTHOR]

Grapevine (Vitis vinifera L.) varieties are particularly susceptible to the pathogens downy mildew Plasmopara viticola and powdery mildew Erysiphe necator. Conventional methods for identifying and classifying spores rely on time-consuming microscopic examinations susceptible to human error and requiring qualified personnel. The aim of the present work has focused on the establishment of a protocol for the rapid molecular detection of the fungal species P. viticola and E. necator from adhesive tapes used to trap spores in airborne inoculum collector cyclones. Four DNA extraction methods were tested. Subsequently, molecular detection of both pathogens was performed by validating some of the specific molecular markers available in the literature. PCR with the primers Nad9 cob-F/Nad9 cob-R and Uncin144/Uncin511 showed specific results for P. viticola and E. necator, respectively, and the best results were obtained with the T-CTAB method. The methodology developed in this work could be of great help for relating direct measurement of P. viticola and E. necator airborne inoculum to disease risk and detection of pathogens, which could be integrated into the early diagnosis of these grapevine pathogens, improving existing warning systems such as Decision Support Systems. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Leptospirosis is a worldwide zoonosis caused by Leptospira interrogans. Laboratory diagnosis of leptospirosis can be done through sample culture. However, microscopic observation of positive Leptospira spp. culture need to be supported by molecular detection to confirm the presence of pathogenic Leptospira spp. This study aimed to compare the effectiveness of two DNA extraction kits, from QIAGEN and Zymo Research, to extract bacterial DNA genome from positive Leptospira spp. culture for downstream molecular detection. This study is an analytical observational study with a cross-sectional design conducted in June-September 2023, and part of the PESTO-RITA 2023 study. Fourteen identified positive Leptospira spp. culture, with Leptospira spp. movement under dark field microscopy, was used for DNA extraction using both extraction kits. QIAGEN kit yielded higher mean DNA concentration (0.900±0.161 vs 0.790±0.167 log10 µg/mL, p>0.05) and better DNA purity (1.854 vs 1.632, p

Abstract During a bacterial infection or colonization, the detection of antimicrobial resistance (AMR) is critical, but slow due to culture-based approaches for clinical and screening samples. Culture-based phenotypic AMR detection and confirmation require up to 72 hours (h) or even weeks for slow-growing bacteria. Direct shotgun metagenomics by long-read sequencing using Oxford Nanopore Technologies (ONT) may reduce the time for bacterial species and AMR gene identification. However, screening swabs for metagenomics is complex due to the range of Gram-negative and -positive bacteria, diverse AMR genes, and host DNA present in the samples. Therefore, DNA extraction is a critical initial step. We aimed to compare the performance of different DNA extraction protocols for ONT applications to reliably identify species and AMR genes using a shotgun long-read metagenomic approach. We included three different sample types: ZymoBIOMICS Microbial Community Standard, an in-house mock community of ESKAPE pathogens including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli (ESKAPE Mock), and anonymized clinical swab samples. We processed all sample types with four different DNA extraction kits utilizing different lysis (enzymatic vs. mechanical) and purification (spin-column vs. magnetic beads) methods. We used kits from Qiagen (QIAamp DNA Mini and QIAamp PowerFecal Pro DNA) and Promega (Maxwell RSC Cultured Cells and Maxwell RSC Buccal Swab DNA). After extraction, samples were subject to the Rapid Barcoding Kit (RBK004) for library preparation followed by sequencing on the GridION with R9.4.1 flow cells. The fast5 files were base called to fastq files using Guppy in High Accuracy (HAC) mode with the inbuilt MinKNOW software. Raw read quality was assessed using NanoPlot and human reads were removed using Minimap2 alignment against the Hg38 genome. Taxonomy identification was performed on the raw reads using Kraken2 and on assembled contigs using Minimap2. The AMR genes were identified using Minimap2 with alignment against the CARD database on both the raw reads and assembled contigs. We identified all bacterial species present in the Zymo Mock Community (8/8) and ESKAPE Mock (6/6) with Qiagen PowerFecal Pro DNA kit (chemical and mechanical lysis) at read and assembly levels. Enzymatic lysis retrieved fewer aligned bases for the Gram-positive species (Staphylococcus aureus and Enterococcus faecium) from the ESKAPE Mock on the assembly level compared to the mechanical lysis. We detected the AMR genes from Gram-negative and -positive species in the ESKAPE Mock with the QIAamp PowerFecal Pro DNA kit on reads level with a maximum median time of 1.9 h of sequencing. Long-read metagenomics with ONT may reduce the turnaround time in screening for AMR genes. Currently, the QIAamp PowerFecal Pro DNA kit (chemical and mechanical lysis) for DNA extraction along with the Rapid Barcoding Kit for the ONT sequencing captured the best taxonomy and AMR identification for our specific use case.

Genetic analysis of the honey bee spermathecal content can be particularly useful to provide an estimate of the genetic diversity and purity of the surrounding populations. Here we compared the concentration and quality of DNA extracted from queen spermatheca using four commercial kits to determine the best method to obtain DNA suitable for single nucleotide polymorphism genotyping by next-generation sequencing. The four kits were tested with different adjustments in the lysis incubation time, use of RNA-carrier, elution conditions and number of re-elutions. Only the use of QIAamp DNA Microkit with 3 h of lysis incubation, the addition of RNA-carrier and multiple re-elutions produced a DNA concentration over the required threshold. [ABSTRACT FROM AUTHOR]

Background: Optimizing DNA extraction for the preservation of telomere length – as a biomarker for ageing and vari-ous diseases – is highly important. Long-term stored blood samples are considered essential resources for genetic studies. In this study, different lysis buffers were used along with CTAB extraction buffer to inves-tigate the quality of extracted DNA and the effect of its accompanying inhibitors on quantitative PCR (q-PCR) in telomere studies. Materials and Methods: DNA extraction was performed using six RBC lysis buffers and CTAB-based extraction buffer. DNA integrity was evaluated by gel electrophoresis, quantity by absorbance at 260 nm and its purity with expected values of 1.8 and 2-2.2 for the ratios of A260/A280 and A260/A230. The quantitative effect of each buffer in q-PCR and the repeatability of the results were assessed by calculating the reaction efficiency, coefficient of determination (R2), and percentage of coefficient of variation (%CV). Results: Buffer number 5 (10 mM Tris-HCl, pH 7.6, 50 mM NaCl) yielded the highest amount of DNA extraction (324.93 ng/ml, %CV 11.53). All the extracted DNA samples were pure, as indicated by the acceptable A260/A280 and A260/A230ratios (p>0.05). Gel analysis revealed that the extracted DNA of all the buffers ex-cept one was intact. The DNA molecule extracted with buffer number 1 (155 mM NH4Cl, 10 mM KHCO3, and 5 mM EDTA) showed the best performance in q-PCR for HBG gene (efficiency=0.126, R2=0.97) and telomere (efficiency=0.99, R2=0.99). Conclusion: The DNA molecule extracted from frozen blood samples by buffer number 1 showed the least q-PCR inhibition for telomere study.

Abstract 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. Graphical Abstract

Background. High content of organic compounds that worsen nucleic acid purification in aromatic plants, as well as the use of such toxic substances as phenol and mercaptoethanol in many protocols for plant DNA isolation, make it advisable to take the above disadvantages into account when optimizing the DNA extraction technique for the work with essential oil rose plants. Material and methods. Rose accessions from the Crimean Federal University’s Botanical Garden, and those from the collection held by the Research Institute of Agriculture of Crimea were included in the study. DNA extraction was done according to a modified CTAB protocol. Effectiveness of the technique was assessed using spectrophotometry, horizontal electrophoresis, and ISSR-PCR. Results. DNA preparations extracted with the modified technique were well visualized on the electropherogram and demonstrated high spectrophotometric values. DNA content was twice as high in preparations isolated with an extraction buffer with PVP, compared to a PVP-free buffer. The concentration was also higher in DNA extracts from stems than that from leaves. Purity parameters expressed by the absorption ratios at wavelengths A260/280 and A260/230 were again higher for DNA extracts from stems isolated with an enriched buffer, the A260/230 ratio falling within the normal range only in DNA extracted from stems in the presence of PVP. Besides, DNA extracts were effectively purified from proteins without phenol or mercaptoethanol, due to double rinsing with a chloroform/isoamyl alcohol (24 : 1) mixture. Conclusion. Using rose stem tissues as the research material, adding 2% PVP to the extraction buffer, and twofold rinsing with a chloroform/isoamyl alcohol mixture made it possible to obtain DNA extracts with high concentrations and purity indices within normal ranges suitable for the ISSR analysis of essential oil rose genetic diversity.

Abstract 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 (R 2 > 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. Video Abstract

Yellow rust (YR), caused by Puccinia striiformis f.sp. tritici (Pst), is a global threat to wheat production. In this study the response of 46 wheat and triticale cultivars to Pst at the adult plant stage (APS) was evaluated during two successive growing seasons at Sulaimania, Iraq. Also, we used a molecular analysis to find the yellow rust resistance (Yr) genes present in the individual cultivars. The results revealed large differences in the response to Pst between the cultivars. Most of the cultivars were susceptible to YR; the mean coefficients of infection (CI) varied from 0.23 in cv. Sarah to 83.33 in Hsad. High resistance levels were found in Al-Wand, Kalar 1, Rezan, and Sarahat APS, while Al-Rashid, Charmo, Faris 1, Maaroof, Rabiea, and Iratom displayed moderate resistance. The level of Yellow rust infection was higher in 2023 than in 2022 in most tested cultivars. Molecular analysis revealed the highest number of Yr genes (Yr2, Yr5, Yr7, Yr9, Yrvav, Yr15, Yr24, Yr26, and Yr32) in the cv. Al-Wand, followed by Sulaimani 2 with eight Yr genes (Yr2, Yr5, Yr7, Yr9, Yr15, Yr24, Yr26, and Yr32). Only one Yr gene was found in Iratom and Tamuz 3. Yr2 was the most frequently identified gene, present in the majority of tested cultivars (87%), followed by Yr7 (76%) and Yr9 (74%), respectively.

Background: Nails are one of the objects that are more durable than other witness objects. This study was interested in whether the Spin column-based nucleic acid purification technique could extract DNA from nails to give satisfactory results. Aims and Objectives: This study aimed to evaluate the yield and quality of DNA extracted from the nail. MATERIALS AND METHODS: Nail samples from 15 males and 15 females over 18 were extracted using GeneAll® Exgene™ Cell SV Mini Kit. The DNA concentration and purity were measured using a NanoDrop Spectrophotometer, and the quality of DNA was assessed by polymerase chain reaction (PCR) using primers specific for human growth hormone (HGH). Results: The average yield of DNA was 0.816 μg (0.141–2.706 μg) obtained from the average nail weight of 30.2 mg (22.2–40.0 mg). The average DNA concentration was 27.2 ng/μL (4.7–90.2 ng/μL), corresponding to 30 μL in volume. Almost all DNA samples (96.7%) had high purity (A260/A280 ≥1.80) and gave a band of PCR product of the HGH gene on agarose gel electrophoresis. Conclusion: Spin column-based nucleic acid purification is recommended for nail DNA extraction due to its simplicity and high quality.

The collection and preservation of biological material before DNA analysis is critical for inter alia biomedical research, medical diagnostics, forensics and biodiversity conservation. In this study, we evaluate an in-house formulated buffer called the Forensic DNA Laboratory-buffer (FDL-buffer) for preservation of biological material for long term at room temperature. Human saliva stored in the buffer for 8 years, human blood stored for 3 years and delicate animal tissues from the jellyfish Pelagia noctiluca comb jelly Beroe sp., stored for 4 and 6 years respectively consistently produced high-quality DNA. FDL-buffer exhibited compatibility with standard organic, salting out and spin-column extraction methods, making it versatile and applicable to a wide range of applications, including automation.

BACKGROUND RESULTS CONCLUSION The identification of adulterated sweet potato vermicelli faces significant challenges, seriously hindering the development of the vermicelli industry. Herein, we investigate effects of energy‐gathered ultrasound (EGU) and energy‐divergent ultrasound (EDU) (30, 40 and 50 W  L−1) on structure, DNA extraction and adulterated quantification of sweet potato vermicelli and its starch, thereby exploring their potential in adulteration of sweet potato vermicelli.EGU‐assisted modified cetyltrimethylammonium bromide (CTAB) protocol with β‐mercaptoethanol significantly improved DNA extraction from sweet potato vermicelli (223.7–249.2 ng μL−1) and its starch (133.4–186.4 ng μL−1), followed by EDU‐assisted DNA extraction from sweet potato vermicelli (115.1–209.3 ng μL−1) and its starch (33.4–61.0 ng μL−1). Both EGU and EDU treatments resulted in the destruction of microstructure and crystalline structure, as well as changes in pasting and thermal properties of sweet potato vermicelli and its starch. Real‐time polymerase chain reaction (PCR) amplification results revealed that EGU and EDU enhanced the efficiency of DNA amplification, and EDU showed smaller cycle threshold (Ct) values than EGU. In addition, EDU‐assisted CTAB protocol combined with real‐time PCR could detect levels of less than 1% of cassava and maize starches in sweet potato vermicelli.EDU‐assisted CTAB protocol combined with real‐time PCR shows promise as a sensitive and reliable analytical tool for vermicelli adulteration. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

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]

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]

Water samples for bacterial microbiome studies undergo biomass concentration, DNA extraction, and taxonomic identification steps. Through benchmarking, we studied the applicability of skimmed milk flocculation (SMF) for bacterial enrichment, an adapted in-house DNA extraction protocol, and six 16S rRNA databases (16S-DBs). Surface water samples from two rivers were treated with SMF and vacuum filtration (VF) and subjected to amplicon or shotgun metagenomics. A microbial community standard underwent five DNA extraction protocols, taxonomical identification with six different 16S-DBs, and evaluation by the Measurement Integrity Quotient (MIQ) score. In SMF samples, the skimmed milk was metabolized by members of lactic acid bacteria or genera such as Polaromonas, Macrococcus, and Agitococcus, resulting in increased relative abundance (p < 0.5) up to 5.0 log fold change compared to VF, rendering SMF inapplicable for bacterial microbiome studies. The best-performing DNA extraction protocols were FastSpin Soil, the in-house method, and EurX. All 16S-DBs yielded comparable MIQ scores within each DNA extraction kit, ranging from 61–66 (ZymoBIOMICs) up to 80–82 (FastSpin). DNA extraction kits exert more bias toward the composition than 16S-DBs. This benchmarking study provided valuable information to inform future water metagenomic study designs. [ABSTRACT FROM AUTHOR]

Trichomonas gallinae is a widespread protozoan parasite that primarily affects birds, causing a disease known as avian trichomonosis. The present study aimed to investigate the prevalence and genetic diversity of T. gallinae, a parasite causing avian trichomoniasis in feral pigeons, budgerigars, and finches in Tehran, Iran. The 5.8S ribosomal RNA locus, along with the internal transcribed spacer 2 (ITS2) region, has been extensively utilized for genotype identification and for determining inter- and intra-specific diversity. More recently, the Fe-hydrogenase (Fe-Hyd) gene has been suggested as an additional genetic marker to enhance the accuracy of strain subtyping discrimination. In the present study, a total of 12% (12/100) birds examined were infected with T. gallinae using microscopy and PCR methods. Infection was found in seven of 30 (23.3%) feral pigeons, three of 40 (7.5%) budgerigars, and two of 30 (6.66%) finches. Analysis of the ITS2 region of T. gallinae isolates revealed two highly similar sequences. The first sequence (GenBank: OQ689964-OQ689970) was found in five feral pigeons and two budgerigars, whereas the second sequence (GenBank: OQ689971-OQ689975) was identified in two feral pigeons, one budgerigar, and two finches. Phylogenetic analysis confirmed the presence of two distinct clusters (cluster I and cluster II) within the trichomonads based on the ITS2 region. However, further analysis using Fe-Hyd revealed greater diversity, with three subtypes identified (A1, A2, and C1). One isolate identified in the present study (GenBank accession number: OQ694508.1) belonged to subtype A1. Combining ITS2 and Fe-Hyd markers holds promise for a more comprehensive understanding of the population structure of T. gallinae and the potential role of ITS2 in host adaptation. [ABSTRACT FROM AUTHOR]

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Yellow rust (YR), caused by Puccinia striiformis f.sp. tritici (Pst), is a global threat to wheat production. In this study the response of 46 wheat and triticale cultivars to Pst at the adult plant stage (APS) was evaluated during two successive growing seasons at Sulaimania, Iraq. Also, we used a molecular analysis to find the yellow rust resistance (Yr) genes present in the individual cultivars. The results revealed large differences in the response to Pst between the cultivars. Most of the cultivars were susceptible to YR; the mean coefficients of infection (CI) varied from 0.23 in cv. Sarah to 83.33 in Hsad. High resistance levels were found in Al-Wand, Kalar 1, Rezan, and Sarahat APS, while Al-Rashid, Charmo, Faris 1, Maaroof, Rabiea, and Iratom displayed moderate resistance. The level of Yellow rust infection was higher in 2023 than in 2022 in most tested cultivars. Molecular analysis revealed the highest number of Yr genes (Yr2, Yr5, Yr7, Yr9, Yrvav, Yr15, Yr24, Yr26, and Yr32) in the cv. Al-Wand, followed by Sulaimani 2 with eight Yr genes (Yr2, Yr5, Yr7, Yr9, Yr15, Yr24, Yr26, and Yr32). Only one Yr gene was found in Iratom and Tamuz 3. Yr2 was the most frequently identified gene, present in the majority of tested cultivars (87%), followed by Yr7 (76%) and Yr9 (74%), respectively. [ABSTRACT FROM AUTHOR]

The extraction of DNA from Cryptosporidium oocysts is challenging due to the robust oocyst wall. Nanoparticles have been applied to disinfect Cryptosporidium oocysts; here, we demonstrate the utilisation of nanoparticles to disrupt the oocyst wall to enable sporozoite lysis and detection via PCR. Both silver and zinc oxide nanoparticles are investigated under different conditions and compared to existing techniques. Zinc oxide nanoparticles are shown to be as effective as freeze–thaw methods, suggesting that a nanoparticle lysis approach offers a viable alternative to existing methods. [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]

At many undergraduate institutions it is not possible for every student to participate in one-on-one student-faculty research experiences. However, large numbers of undergraduates could gain research experience through the use of course-based undergraduate research (CURE) in laboratory courses. Here we present a CURE using a viral emerging infection in amphibians that is suitable for undergraduate-level students and will permit them to develop an understanding of how to calculate epidemiologically relevant sample sizes, genomic DNA extraction procedures, traditional or quantitative PCR use, and associated techniques. [ABSTRACT FROM AUTHOR]

Background: Symbiotic relationships with diverse microorganisms are crucial for many aspects of insect biology. However, while our understanding of insect taxonomic diversity and the distribution of insect species in natural communities is limited, we know much less about their microbiota. In the era of rapid biodiversity declines, as researchers increasingly turn towards DNA-based monitoring, developing and broadly implementing approaches for high-throughput and cost-effective characterization of both insect and insect-associated microbial diversity is essential. We need to verify whether approaches such as high-throughput barcoding, a powerful tool for identifying wild insects, would permit subsequent microbiota reconstruction in these specimens. Methods: High-throughput barcoding ("megabarcoding") methods often rely on non-destructive approaches for obtaining template DNA for PCR amplification by leaching DNA out of insect specimens using alkaline buffers such as HotSHOT. This study investigated the impact of HotSHOT on microbial abundance estimates and the reconstructed bacterial community profiles. We addressed this question by comparing quantitative 16S rRNA amplicon sequencing data for HotSHOT-treated or untreated specimens of 16 insect species representing six orders and selected based on the expectation of limited variation among individuals. Results: We find that in 13 species, the treatment significantly reduced microbial abundance estimates, corresponding to an estimated 15-fold decrease in amplifiable 16S rRNA template on average. On the other hand, HotSHOT pre-treatment had a limited effect on microbial community composition. The reconstructed presence of abundant bacteria with known significant effects was not affected. On the other hand, we observed changes in the presence of low-abundance microbes, those close to the reliable detection threshold. Alpha and beta diversity analyses showed compositional differences in only a few species. Conclusion: Our results indicate that HotSHOT pre-treated specimens remain suitable for microbial community composition reconstruction, even if abundance may be hard to estimate. These results indicate that we can cost-effectively combine barcoding with the study of microbiota across wild insect communities. Thus, the voucher specimens obtained using megabarcoding studies targeted at characterizing insect communities can be used for microbiome characterizations. This can substantially aid in speeding up the accumulation of knowledge on the microbiomes of abundant and hyperdiverse insect species. [ABSTRACT FROM AUTHOR]

Recent reports have disclosed the occurrence of Wolbachia in Aedes aegypti. Our study detected Wolbachia infection in Ae. aegypti by screening wild adult mosquitoes using two Wolbachia‐specific molecular markers. Overall, 444 adult Ae. aegypti mosquitoes were collected from April 2022 to October 2022 in Jeddah, Saudi Arabia. Each individual sample was processed and screened for the presence of Wolbachia using selected markers, the Wolbachia‐specific 16S rDNA and the Wolbachia surface protein gene (wsp), under optimized polymerase chain reaction (PCR) conditions, and sequenced. In total, 39 (8.78%) and 48 (10.81%) individual mosquito samples were determined to be infected with Wolbachia using the wsp and 16S rDNA markers, respectively. By utilizing two Wolbachia‐specific molecular markers, our study demonstrated the presence of Wolbachia from individual Ae. aegypti samples. Our results showed a low rate of Wolbachia infection and inferred that the detected strain belongs to supergroup B. [ABSTRACT FROM AUTHOR]

The virome, composed of viruses inhabiting diverse ecosystems, significantly influences microbial community dynamics and host health. The phenol-chloroform DNA extraction protocol for viromes, though effective, is time-intensive and requires the use of multiple toxic chemicals. This study introduces a streamlined, scalable protocol for DNA extraction using a commercially-available kit as an alternative, assessing its performance against the phenol-chloroform method across human fecal, mouse fecal, and soil samples. No significant differences in virome diversity or community composition were seen between methods. Most viral operational taxonomic units (vOTUs) were common to both methods, with only a small percentage unique to either approach. Alpha- and beta-diversity analyses showed no significant impact of the extraction method on virome composition, confirming the kit’s efficacy and versatility on sample types beyond those officially supported by the manufacturer. While the kit approach offers benefits like reduced toxicity and increased throughput, it has limitations such as higher costs and potential issues reliably capturing low-abundance taxa. This protocol provides a viable option for large-scale virome studies, although the phenol-chloroform approach may still be preferable for specific sample types.

ABSTRACT Understanding transmission pathways of important opportunistic, drug-resistant pathogens, such as extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli, is essential to implementing targeted prevention strategies to interrupt transmission and reduce the number of infections. To link transmission of ESBL-producing E. coli (ESBL-EC) between two sources, single-nucleotide resolution of E. coli strains, as well as E. coli diversity within and between samples, is required. However, the microbiological methods to best track these pathogens are unclear. Here, we compared different steps in the microbiological workflow to determine the impact different pre-enrichment broths, pre-enrichment incubation times, selection in pre-enrichment, selective plating, and DNA extraction methods had on recovering ESBL-EC from human stool samples, with the aim to acquire high-quality DNA for sequencing and genomic epidemiology. We demonstrate that using a 4-h pre-enrichment in Buffered Peptone Water, plating on cefotaxime-supplemented MacConkey agar and extracting DNA using Lucigen MasterPure DNA Purification kit improves the recovery of ESBL-EC from human stool and produced high-quality DNA for whole-genome sequencing. We conclude that our optimized workflow can be applied for single-nucleotide variant analysis of an ESBL-EC from stool.IMPORTANCEDrug-resistant infections are increasingly difficult to treat with antibiotics. Preventing infections is thus highly beneficial. To do this, we need to understand how drug-resistant bacteria spread to take action to stop infection and transmission. This requires us to accurately trace these bacteria between different sources. In this study, we compared different laboratory methods to see which worked best for detecting extended-spectrum beta-lactamase (ESBL)-producing E. coli, a common cause of urinary tract or bloodstream infections, from human stool samples. We found that enriching stool in a nutrient broth for 4 h, then plating the bacterial suspension on antibiotic-selective MacConkey agar, and finally extracting DNA from the bacteria using a specific DNA purification kit resulted in improved recovery of ESBL E. coli and high-quality DNA. Sequencing multiple isolates from stool allowed us to distinguish unambiguously and at high resolution between different variants of ESBL E. coli present in stool.

The health of the terrestrial ecosystems is directly dependent on the microbial composition that fulfills essential functions, such as sustaining plant growth, nutrient cycling and carbon sequestration. The study of the soil microbiome has gained popularity in the last decades due to its significant impact on the health of the environment and its inhabitants. This review explores the diversity and functions of soil microbial communities, with a particular focus on microbial dark matter, a subset of organisms that cannot be cultured through classical microbiological techniques. The evolution of DNA extraction methods and sequencing technologies coupled with the transition from amplicon sequencing to metagenome-assembled genomes (MAGs) and continuously developing bioinformatic pipelines has led to the discovery of novel microbial taxa, community networks, metabolic pathways and potentially useful molecules. Soil microbiome research is gaining momentum in Romania, as a big part of studies try to assess the impact of agricultural practices on the environment. Designing sustainable agricultural practices and implementing them with the goal of preserving the heterogeneity of the microbiome contributes significantly to the resilience of ecosystems, preserving the health of the environment, as well as the well-being of its residents.

ABSTRACT As technologies advance alongside metabarcoding and metagenomic resources, particularly for larger fungal genomes, DNA extraction methods must be optimized to meet higher thresholds, especially from complex environmental substrates. This study focused on extracting fungal genomic compounds from woody substrates, a challenge due to the embedment of endophytic and saprotrophic fungi within wood cells, the physical recalcitrance of wood, the adsorption of nucleic acids to wood polymers, and the release of downstream inhibitors. Hypothesizing that cetyltrimethylammonium bromide would be the best option, we compared prominent methods by extracting and sequencing microbial DNA from sound and decayed birch (Betula papyrifera) and pine (Pinus resinosa). DNA quantities varied significantly depending on extraction methods and decay stage. The quality of DNA, in terms of purity and integrity, significantly impacted whether the samples could be amplified and sequenced. However, amplicon sequencing of bacterial and fungal communities revealed no significant extraction bias. This, along with the sequencing effectiveness and cost/time efficiency, indicates that Qiagen is the gold standard for woody substrates. This study increases confidence in published amplicon data sets regardless of the extraction methods, provides a cost‐benefit table for making protocol decisions, and offers guidance on fungal DNA extractions from complex organic substrates (sound and decayed wood) that would best suit future metagenomic efforts.

ABSTRACT 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.

Large-scale genotyping platforms are currently being developed for several wild species. By querying thousands of polymorphic loci, genomics can be a useful ecological tool for describing and monitoring populations. Genomics is becoming increasingly useful as a forensic tool because of its ability to identify population of origin for purposes of enforcing anti-poaching laws. Our aim was to test the new SNP chip for caribou/reindeer (Rangifer tarandus) (Illumina iSelect caribou 60 K) under recommended and non-optimal sample conditions. Impact on signal detection (call rate) and error rate were assessed using reference samples. The SNP chip was shown to be robust, highly sensitive, reliable, and accurate at more than 10-fold below the recommended DNA input. Biological source of DNA had minor impact, even with fecal pellets given sufficient amount of host DNA. Hybridization of non-Rangifer samples as well as samples bearing DNA from two Rangifer samples both showed a drop in call rate and shifted levels of heterozygosity. Based on a population-targeted subset of SNPs included in the chip design, reassignment of 981 samples to a functional group (here to a caribou ecotype) was highly accurate (99.59 %) and the relative probability of reassignment error was estimated using the logarithm of odds score. Overall, the SNP chip is suitable for analysis of caribou/reindeer genomes even with suboptimal sampling and hence useful for population management and forensics.

Molecular diagnosis is a gold standard method for identifying an infectious disease. DNA extraction from a target pathogen is one of the most important procedures for accurate analysis of the disease-causative pathogen. In this study, a novel 3D-printed electrophoretic DNA extraction microdevice (3D-EDEM) was developed using a digital light processing-stereolithography (DLP-SL) for point-of-care analysis. The 3D-EDEM consists of a source chamber for a bacteria lysate reservoir, a sink chamber for an elution solution container, a hydrogel channel embracing capillary channels that act as a sieving matrix for size-based separation, and two electrode holders for supplying electrical current. Prior to fabricating the 3D-EDEM, UV-curable resin was prepared by using a poly(ethylene glycol) diacrylate (PEG-DA), Irgarcure 819 (IRG), and 2-isopropyl thioxanthone (ITX) as a monomer, a photoinitiator, and a photosensitizer, respectively. The 3D-printed 3D-EDEM provides numerous merits of being inexpensive, reproducible, and convenient, making it more suitable for on-site DNA extraction microdevices than soft-lithographic procedures. For DNA extraction on the 3D-EDEM, Escherichia coli O157:H7 (E. coli) lysate and elution buffer were loaded into the source chamber and the sink chamber, respectively. The optimum DNA extraction time and limit of the DNA extraction test of 3D-EDEM were carried out to evaluate DNA extraction performance, especially using a portable battery. Additionally, the successful DNA extraction test from artificially infected food samples confirms the applicability of the 3D-EDEM to real fields. The proposed 3D-EDEM is adequate for on-site DNA extraction in the field of clinical diagnosis, food safety, environmental monitoring, and forensic analysis.

Abstract Objective Bartonella are emerging bacterial zoonotic pathogens. Utilization of clotted blood samples for surveillance of these bacteria in wildlife has begun to supersede the use of tissues; however, the efficacy of these samples has not been fully investigated. Our objective was to compare the efficacy of spleen and blood samples for DNA extraction and direct detection of Bartonella spp. via qPCR. In addition, we present a protocol for improved DNA extraction from clotted, pelleted (i.e., centrifuged) blood samples obtained from wild small mammals. Results DNA concentrations from kit-extracted blood clot samples were low and A260/A280 absorbance ratios indicated high impurity. Kit-based DNA extraction of spleen samples was efficient and produced ample DNA concentrations of good quality. We developed an in-house extraction method for the blood clots which resulted in apposite DNA quality when compared to spleen samples extracted via MagMAX DNA Ultra 2.0 kit. We detected Bartonella in 9/30 (30.0%) kit-extracted spleen DNA samples and 11/30 (36.7%) in-house-extracted blood clot samples using PCR. Our results suggest that kit-based methods may be less suitable for DNA extraction from blood clots, and that blood clot samples may be superior to tissues for Bartonella detection.