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Testing 21 different fecal DNA extraction protocols in multiple laboratories results in a standardized protocol with the potential to improve comparability across human gut microbiome studies. Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

In this study, we have demonstrated for the first time the performance of LDR real-time PCR assay in amplifying the genomic DNA of W. bancrofti extracted by a simple TE based method in comparison with Qiagen (standard) method for detecting filarial infection in large number of field collected mosquito pools from an endemic area in a cost-effective manner. Cost comparison in our laboratory showed that the TE based extraction of genomic DNA saves the cost (12 times) and the time (75%)8. [...]TE based extraction of DNA could be used as an alternative method as it could be less expensive and time saving while processing large number of mosquito samples by real-time PCR during post-MDA surveillance and monitoring.

Interactions between the diet and intestinal microbiota play a role in health and disease, including obesity and related metabolic complications. There is great interest to use dietary means to manipulate the microbiota to promote health. Currently, the impact of dietary change on the microbiota and the host metabolism is poorly predictable and highly individual. We propose that the responsiveness of the gut microbiota may depend on its composition, and associate with metabolic changes in the host. Our study involved three independent cohorts of obese adults (n = 78) from Belgium, Finland, and Britain, participating in different dietary interventions aiming to improve metabolic health. We used a phylogenetic microarray for comprehensive fecal microbiota analysis at baseline and after the intervention. Blood cholesterol, insulin and inflammation markers were analyzed as indicators of host response. The data were divided into four training set - test set pairs; each intervention acted both as a part of a training set and as an independent test set. We used linear models to predict the responsiveness of the microbiota and the host, and logistic regression to predict responder vs. non-responder status, or increase vs. decrease of the health parameters. Our models, based on the abundance of several, mainly Firmicute species at baseline, predicted the responsiveness of the microbiota (AUC  =  0.77-1; predicted vs. observed correlation  =  0.67-0.88). Many of the predictive taxa showed a non-linear relationship with the responsiveness. The microbiota response associated with the change in serum cholesterol levels with an AUC of 0.96, highlighting the involvement of the intestinal microbiota in metabolic health. This proof-of-principle study introduces the first potential microbial biomarkers for dietary responsiveness in obese individuals with impaired metabolic health, and reveals the potential of microbiota signatures for personalized nutrition.

Current freshwater biomonitoring with diatoms is based on microscopic examination of the morphology of their silica skeleton. This standardized approach is time consuming and requires a high degree of taxonomic expertise. Metabarcoding combined with high-throughput sequencing (HTS) has great potential for next-generation biomonitoring applications but requires standardization. Molecular inventories are strongly influenced by the DNA extraction method used, but the effect of extraction protocols has not been tested to enable selection of the best DNA extraction method for HTS metabarcoding. We used 5 DNA extraction methods combining various types of cell lysis and DNA purification to extract DNA from 8 pure diatom cultures and 8 samples from streams and lakes with differing water quality. We compared the methods based on: 1) quality and purity of the extracted DNA, 2) community inventories obtained from HTS targeting the ribulose-1, 5-bisphosphate carboxylase (rbcL) barcode, and 3) similarity between molecular and microscopy-based inventories of community composition and the Specific Pollution-sensitivity Index [SPI]. A method based on GenElute™-LPA had higher extraction efficiency than the 4 commercial kits but had the highest polymerase chain reaction inhibition level. All 5 methods were efficient for HTS, and method did not affect operational taxonomic unit richness. We observed variations in the relative abundance of some taxa within Nitzschia, Amphora, Encyonema, Gomphonema, and Navicula between 2 of the 5 methods, but method did not affect global diatom community composition or SPI values. SPI values calculated from microscopy-based inventories and molecular inventories based on all 5 extraction methods were strongly correlated. For convenience purposes (high DNA quantity and low cost), we encourage standardization of HTS diatom biomonitoring based on the SA-Gen method.

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

Correct mosquito species identification is essential for mosquito and disease control programs. However, this is complicated by the difficulties in morphologically identifying some mosquito species. In this study, variation of a partial sequence of the cytochrome c oxidase unit I ( COI ) gene was used for the molecular identification of British mosquito species and to facilitate the discovery of cryptic diversity, and monitoring invasive species. Three DNA extraction methods were compared to obtain DNA barcodes from adult specimens. In total, we analyzed 42 species belonging to the genera Aedes Meigen, 1818 (21 species), Anopheles Meigen, 1818 (7 species), Coquillettidia Theobald, 1904 (1 species), Culex Linnaeus, 1758 (6 species), Culiseta Felt, 1904 (7 species), and Orthopodomyia Theobald, 1904 (1 species). Intraspecific genetic divergence ranged from 0% to 5.4%, while higher interspecific divergences were identified between Aedesgeminus Peus, 1971/ Culisetalitorea (Shute, 1928) (24.6%) and Ae.geminus / An.plumbeus Stephens, 1828 (22.5%). Taxonomic discrepancy was shown between An.daciae Linton, Nicolescu & Harbach, 2004 and An.messeae Falleroni, 1828 indicating the poor resolution of the COI DNA barcoding region in separating these taxa. Other species such as Ae.cantans (Meigen, 1818)/ Ae.annulipes (Meigen, 1830) showed similar discrepancies indicating some limitation of this genetic marker to identify certain mosquito species. The combination of morphology and DNA barcoding is an effective approach for the identification of British mosquitoes, for invasive mosquitoes posing a threat to the UK, and for the detection of hidden diversity within species groups.

Melon (Cucumis melo L.) is a global commercial crop that is sensitive to seed-borne wilt infections caused by Fusarium oxysporum f. sp. melonis (Fom). To address the challenge of detecting Fom contamination, we designed a probe-based real-time PCR method, TDCP2, in combination with rapid or column-based DNA extraction protocols to develop reliable molecular detection methods. Utilizing TDCP2, the detection rate reached 100% for both artificially Fom-inoculated (0.25–25%) and pod-inoculated melon seeds in conjunction with DNA samples from either the rapid or column-based extraction protocol. We performed analyses of precision, recall, and F1 scores, achieving a maximum F1 score of 1 with TDCP2, which highlights the robustness of the method. Additionally, intraday and interday assays were performed, which revealed the high reproducibility and stability of column-based DNA extraction protocols combined with TDCP2. These metrics confirm the reliability of our developed protocols, setting a foundation for future enhancements in seed pathology diagnostics and potentially broadening their applicability across various Fom infection levels. In the future, we hope that these methods will reduce food loss by improving the control and management of melon diseases.

We present a rapid high-throughput DNA extraction method for use with EDTA-anticoagulated blood using silicon dioxide (SiO2) powder in a guanidine–HCl solution, hereinafter referred to as “Glassmilk.” The method utilizes a 96-well deep-well plate, enabling DNA extraction from 96 samples in under 3 h. The method integrates cell lysis, washing, elution, and DNA storage within the same well, eliminating the need for DNA transfer. The Glassmilk extraction method is cost-effective and fast, and it avoids expensive or toxic reagents by using only basic lab equipment. The method yielded approximately 40 μg of high-quality DNA from 200 μl of blood. The DNA yield of the Glassmilk method was about 50% higher, and the purity of the DNA was comparable to those obtained using two commercial column-based extraction kits that were used for comparison. The cost per sample was around $1, with the most expensive item being the filter pipette tips, which account for about $0.80 per sample. As we show, the extracted DNA is suitable for downstream applications such as polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism analysis, and qPCR. The method can be adapted for various sample types, including biopsies, fecal samples, cultured cells, and bacteria (see “subprotocols” section), and can also be applied in individual Eppendorf tubes. Our protocol may be useful for basic molecular research in laboratories having limited funds.

Plant-based meat alternatives (PMAs), as an emerging food category gaining increasing popularity, face potential food safety risks and ethical concerns for vegetarians due to the illegal adulteration of animal-derived components. To address these challenges and enhance regulatory oversight, the development of a rapid, sensitive, and highly specific detection method is essential. In this study, five DNA extraction methods were evaluated and optimized to identify the most effective approach for PMA products. The optimal conditions were determined to be 60 mmol/L NaCl, 10 mmol/L Tris HCl, and a centrifugation speed of 12,000× g. Additionally, specific primers targeting four common animal-derived adulterants, namely pork, chicken, duck, and beef, were designed and screened for targeted amplification. To establish a rapid and visually interpretable detection system, the recombinant polymerase amplification conditions were optimized. The final protocol used 0.4 µmol/L primer and isothermal amplification at 39 °C for 25 min, with the incorporation of SYBR Green I dye enabling the rapid and specific visualization of animal-derived DNA. This optimized method is characterized by its simplicity, sensitivity (capable of detecting beef-derived components as low as 0.0514% w/w), and rapidity, significantly reducing detection time and providing a reliable tool for the identification of animal-derived adulteration in PMA products.

Research background. Sourdough is a spontaneously formed, complex microbial ecosystem of various lactic acid bacteria (LAB) and yeast which, by producing specific metabolites, determines the quality of the baked products. In order to design and control the sourdough with preferred nutritional characteristics, it is crucial that the LAB diversity ofthe product of interest be elucidated. Experimental approach. Using the opportunities of next-generation sequencing (NGS) of the V1–V3 hypervariable gene region of 16S rRNA, we studied the microbial ecosystem of a whole grain sourdough made of Triticum monococcum, originating from Southwestern Bulgaria. Since the DNA extraction method is considered crucial for the accuracy of the sequencing results, as it can introduce significant differences in the examined microbiota, we used three different commercial kits for DNA isolation and analyzed their impact on the observed bacterial diversity. Results and conclusions. All three DNA extraction kits provided bacterial DNA which passed quality control and was successfully sequenced on Illumina MiSeq platform. The results received from the different DNA protocols showed variations in the microbial profiles. Alpha diversity indices (ACE, Chao1, Shannon, and Simpson) were also different among the three groups of results. Nevertheless, a strong dominance of phylum Firmicutes, class Bacilli, order Lactobacillales, represented mostly by family Lactobacillaceae, genus Lactobacillus (relative abundance of 63.11–82.28 %) and family Leuconostocaceae, genus Weissella (relative abundance of 3.67–36.31 %) was observed. Lactiplantibacillus plantarum and Levilactobacillus brevis with relative abundance of 16.15–31.24 % and 6.21−16.29 % respectively, were the two dominant species identified in all three DNA isolates. Novelty and scientific contribution. The presented results give insight into the taxonomic composition of bacterial community of a specific Bulgarian sourdough. Having in mind that the sourdough is a difficult matrix for DNA isolation on the one hand, and that there is no standardized DNA extraction protocol for this matrix on the other hand, this pilot study aims to give a small contribution to the future establishment and validation of such a protocol, which will allow accurate assessment of the specific microbiota of sourdough samples.