Search Results (25)

When conducting sequence-based analysis of microbiome samples, it is important to accurately represent the bacterial communities present. The aim of this study was to compare two commercially available DNA isolation and PCR amplification approaches to determine their impact on the taxonomic composition of microbiome samples following 16S rRNA gene sequencing. A well-established 16S rRNA gene profiling approach, which was widely used in the Human Microbiome Project (HMP), was compared with a novel alkaline degenerative technique that utilizes alkaline cell lysis in combination with a degenerate pool of primers for nucleic acid extraction and PCR amplification. When comparing these different approaches for the microbiome profiling of human and mouse fecal samples, we found that the alkaline-based method was able to detect greater taxonomic diversity. An in silico analysis of predicted primer binding against a curated 16S rRNA gene reference database further suggested that this novel approach had the potential to reduce population bias found with traditional methods, thereby offering opportunities for improved microbial community profiling. Full article

Background/Objectives: Candida auris is an emerging multidrug-resistant pathogen with the potential to cause invasive fungal infections and healthcare-associated outbreaks. Currently, there is no systematic review explicitly focusing on the up-to-date molecular diagnostics of this pathogen to cover the entire process, including sample pre-extraction procedures, nucleic acid extraction, and DNA-based detection. Sample pre-treatment and extraction are the prerequisites before molecular testing and have implications on the downstream detection but have not been reviewed elsewhere. This review aims to summarize a comprehensive update in the past 5 years. Methods: A systematic review was conducted to search for articles published in the period between 1 January 2020 and 20 November 2024 from various databases, including PubMed, Google Scholar, and Web of Science. The findings were produced through narrative synthesis, with quantitative analysis conducted where applicable. Results: Starting from 1115 records, 28 studies that met the inclusion criteria were included in the analysis. This review summarized the key updates on three categories, including (i) sample pre-extraction procedures and nucleic acid extraction, including magnetic, bead-beating, mechanical, chemical, thermal, and column-based protocols; (ii) commercial molecular assays; and (iii) laboratory-developed tests (LDTs). For real-time PCR, commercial molecular assays and LDTs showed sensitivity (ranging from 94.9% to 100% and 44% to 100%, respectively) and specificity (ranging from 98.2% to 100% and 92% to 100%, respectively). Conclusions: Here, we describe a useful summary to enlighten readers from clinical microbiology laboratories on the nucleic acid extraction protocols and performance of various molecular diagnostic assays used for the detection of C. auris. Full article

Rhus typhina, an invasive plant species, contains valuable compounds that can be utilized in various fields. The main aim of this paper was to find the optimal conditions for extracting high amounts of bioactive compounds from R. typhina fruits using ultrasound-assisted and bead-beating techniques under different parameters (solvent concentration, solvent/solid ratio, extraction time, bead size, and material). A Box–Behnken design was applied for ultrasound-assisted extraction. The following process parameters were found to be optimal: 20/1 solvent/solid ratio (v/w), 61.51% aqueous ethanol, 10 min extraction time, with a composite desirability of 0.7719. The HPLC profile indicates that p-coumaric acid was the most abundant phenolic compound found in the BBE extract. The BBE extract was subjected to in vitro biological tests. The results indicate a high antimicrobial activity on Streptococcus pyogenes (20 mm inhibition zone) and Salmonella enterica (12 mm inhibition zone). A hemolysis rate of 19.85% was found at an extract concentration of 1000 µg/mL on sheep erythrocytes. We report for the first time the protective role of the extract on cell viability of human gingival fibroblasts, but also a weak antiproliferative effect on the HepG2 human liver cancer cell line. Overall, we conclude that R. typhina fruits are rich in bioactive compounds that can be recovered using proper extraction conditions. Further research is required to understand and valorize their biological potential. Full article

Automated nucleic acid extractors are useful instruments for the high-throughput processing of bio-samples and are expected to improve research throughput in addition to decreased inter-sample variability inherent to manual processing. We evaluated three commercial nucleic acid extractors Bioer GenePure Pro (Bioer Technology, Hangzhou, China), Maxwell RSC 16 (Promega Corporation, Madison, WI, USA), and KingFisher Apex (ThermoFisher Scientific, Waltham, MA, USA) based on their DNA yield, DNA purity, and 16S rRNA gene amplicon results using both human fecal samples and a mock community (ZymoBIOMICS Microbial Community Standard (Zymo Research Corp., Irvine, CA, USA)). Bead-beating provided incremental yield to effectively lyse and extract DNA from stool samples compared to lysis buffer alone. Differential abundance analysis and comparison of prevalent bacterial species revealed a greater representation of Gram-positive bacteria in samples subjected to mechanical lysis, regardless of sample type. All three commercial extractors had differences in terms of yield, inter-sample variability, and subsequent sequencing readouts, which we subsequently share in the paper and believe are significant considerations for all researchers undertaking human fecal microbiota research. Full article

Next-generation sequencing technology has driven the rapid advancement of human microbiome studies by enabling community-level sequence profiling of microbiomes. Although all microbiome sequencing methods depend on recovering the DNA from a sample as a first critical step, lysis methods can be a major determinant of microbiome profile bias. Gentle enzyme-based DNA preparation methods preserve DNA quality but can bias the results by failing to open difficult-to-lyse bacteria. Mechanical methods like bead beating can also bias DNA recovery because the mechanical energy required to break tougher cell walls may shear the DNA of the more easily lysed microbes, and shearing can vary depending on the time and intensity of beating, influencing reproducibility. We introduce a non-mechanical, non-enzymatic, novel rapid microbial DNA extraction procedure suitable for 16S rRNA gene-based microbiome profiling applications that eliminates bead beating. The simultaneous application of alkaline, heat, and detergent (‘Rapid’ protocol) to milligram quantity samples provided consistent representation across the population of difficult and easily lysed bacteria equal to or better than existing protocols, producing sufficient high-quality DNA for full-length 16S rRNA gene PCR. The novel ‘Rapid’ method was evaluated using mock bacterial communities containing both difficult and easily lysed bacteria. Human fecal sample testing compared the novel Rapid method with a standard Human Microbiome Project (HMP) protocol for samples from lung cancer patients and controls. DNA recovered from both methods was analyzed using 16S rRNA gene sequencing of the V1V3 and V4 regions on the Illumina platform and the V1V9 region on the PacBio platform. Our findings indicate that the ‘Rapid’ protocol consistently yielded higher levels of Firmicutes species, which reflected the profile of the bacterial community structure more accurately, which was confirmed by mock community evaluation. The novel ‘Rapid’ DNA lysis protocol reduces population bias common to bead beating and enzymatic lysis methods, presenting opportunities for improved microbial community profiling, combined with the reduction in sample input to 10 milligrams or less, and it enables rapid transfer and simultaneous lysis of 96 samples in a standard plate format. This results in a 20-fold reduction in sample handling time and an overall 2-fold time advantage when compared to widely used commercial methods. We conclude that the novel ‘Rapid’ DNA extraction protocol offers a reliable alternative for preparing fecal specimens for 16S rRNA gene amplicon sequencing. Full article

Microbiome analyses are essential for understanding microorganism composition and diversity, but interpretation is often challenging due to biological and technical variables. DNA extraction is a critical step that can significantly bias results, particularly in samples containing a high abundance of challenging-to-lyse microorganisms. Taking into consideration the distinctive microenvironments observed in different bodily locations, our study sought to assess the extent of bias introduced by suboptimal bead-beating during DNA extraction across diverse clinical sample types. The question was whether complex targeted extraction methods are always necessary for reliable taxonomic abundance estimation through amplicon sequencing or if simpler alternatives are effective for some sample types. Hence, for four different clinical sample types (stool, cervical swab, skin swab, and hospital surface swab samples), we compared the results achieved from extracting targeted manual protocols routinely used in our research lab for each sample type with automated protocols specifically not designed for that purpose. Unsurprisingly, we found that for the stool samples, manual extraction protocols with vigorous bead-beating were necessary in order to avoid erroneous taxa proportions on all investigated taxonomic levels and, in particular, false under- or overrepresentation of important genera such as Blautia, Faecalibacterium, and Parabacteroides. However, interestingly, we found that the skin and cervical swab samples had similar results with all tested protocols. Our results suggest that the level of practical automation largely depends on the expected microenvironment, with skin and cervical swabs being much easier to process than stool samples. Prudent consideration is necessary when extending the conclusions of this study to applications beyond rough estimations of taxonomic abundance. Full article

Mycobacterium tuberculosis is the microorganism that causes tuberculosis, a disease affecting millions of people worldwide. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast, reliable, and cost-effective method for microorganism identification which has been used for the identification of Mycobacterium spp. isolates. However, the mycobacteria cell wall is rich in lipids, which makes it difficult to obtain proteins for MALDI-TOF MS analysis. In this study, two cell preparation protocols were compared: the MycoEx, recommended by MALDI-TOF instrument manufacturer Bruker Daltonics, and the MycoLyser protocol described herein, which used the MagNA Lyser instrument to enhance cell disruption with ethanol. Cell disruption and protein extraction steps with the two protocols were performed using the Mycobacterium tuberculosis H37Rv strain, and the MALDI-TOF MS results were compared. The MycoLyser protocol allowed for improved Biotyper identification of M. tuberculosis since the log(score) values obtained with this protocol were mostly ≥ 1.800 and significantly higher than that underwent MycoEx processing. The identification reliability was increased as well, considering the Bruker criteria. In view of these results, it is concluded that the MycoLyser protocol for mycobacterial cell disruption and protein extraction improves the MALDI-TOF MS method’s efficacy for M. tuberculosis identification. Full article

One of the main challenges for the mass introduction of the molecular diagnostics of soil-transmitted helminths (STHs) into clinical practice is the lack of a generally recognized effective method for isolating parasitic DNA from fecal samples. In the present study, we assessed the effects of various pretreatment procedures on the efficiency of removing PCR inhibitors and extracting Toxocara canis DNA from feces. We evaluated the effectiveness of four destructive methods (bead beating, the action of temperature-dependent enzymes, freeze-heat cycles, and incubation in a lysis buffer) on the integrity of T. canis eggs and the efficiency of DNA extraction. Also, we evaluated the effects of prewashes and the use of commercial concentrators on DNA extraction from fecal samples contaminated with T. canis eggs. A bead beating procedure was sufficient to destroy the T. canis eggs, while the effects of enzymes and freeze-heat cycles did not lead to a significant destruction of the eggs or the release of Toxocara DNA. Helminth DNA isolation protocols that do not include a bead beating step are not preferred. The preconcentration of STH eggs from feces using a commercial concentrator and subsequent washing can significantly increase the yield of DNA from STHs and reduce PCR inhibition. Full article

Diatoms are unicellular algae and occur ubiquitously in almost every marine and freshwater habitat on earth. They produce intricately structured cell walls, which mainly consist of amorphous silica. To synthesize their cell walls, diatoms take up monosilicic acid from the environment and store it. These silicon storage pools (SSPs) can exceed the solubility of silicic acid by one to two orders of magnitude, as observed in various diatom species. However, their chemical composition and cellular localization has not yet been elucidated. It is suggested that SSPs may consist of stabilized aggregates such as pre-condensed silica particles or silica-containing vesicles. Isolation protocols for SSPs without significant chemical modification are required to prove such hypotheses. A critical issue is the efficient separation of components of the SSPs from cell wall fragments or artefacts, which may interfere with analytical methods targeting silicon. To this end, a comparative study was performed on exponentially grown cells and extracted, purified cell walls (biosilica) to observe the sedimentation behavior after lysis. Cell cultures were lysed by bead beating and then fractionated by differential centrifugation. The obtained fractions were analyzed for total silicon content (tSi) using molybdenum blue assay (MBA) after alkaline treatment. It was revealed that cell wall fragments are almost absent in fractions above 1000 × g. Compared with biosilica, a significantly higher silicon concentration is found in lysed cell pellets after centrifugation at moderately high forces. The differences correspond to a few percent of total cellular silicon, which are assumed to be part of SSPs. Only relatively low amounts of silica/silicic acid remain in the supernatant at high centrifugal forces. This indicates that SSPs are mainly present in larger aggregates that sediment at lower centrifugal forces. According to Stokes’ law, only silica particles below ca. 25 nm radius would remain in the final supernatant. This leads to the conclusion that SSPs must mainly consist of larger silica particles and/or are associated with larger compartments/aggregates. Full article

The standard procedure for the detection of candidemia is blood culture, a method that might require 3–5 days for a positive result. Compared with culturing, molecular diagnosis techniques can provide faster diagnosis. The current paper aimed to present the main strengths and constraints of current molecular techniques for Candida spp. DNA extraction, analyzing their efficiency from a time, price, and ease of usage point of view. A comprehensive search was conducted using the PubMed NIH database for peer-reviewed full-text articles published before October 2022. The studies provided adequate data on the diagnosis of the infection with the Candida spp. DNA extraction is a relevant step in yielding pure qualitative DNA to be amplified in molecular diagnostic techniques. The most used fungal DNA extraction strategies are: mechanical (bead beating, ultrasonication, steel-bullet beating), enzymatic (proteinase K, lysozyme, lyticase), and chemical extraction (formic acid, liquid nitrogen, ammonium chloride). More clinical studies are needed to formulate adequate guidelines for fungal DNA extraction as the current paper highlighted discrepancies in the reported outcome. Full article

Wastewater monitoring became a promising solution in the early detection of outbreaks. Despite the achievements in the identification of pathogens in wastewater using real-time PCR, there is still a lack of reliable rapid nucleic acid extraction protocols. Therefore, in this study, samples were subjected to alkali, proteinase K and/or bead-beating followed by reverse purification magnetic beads-based separation. Wastewater samples spiked with S. aureus, E. coli and C. parvum were used as examples for Gram-positive and -negative bacteria and protozoa, respectively. All results were compared with a spin column technology as a reference method. Proteinase K with bead beating (vortexing with 0.1 mm glass beads for three minutes) was particularly successful for bacterial DNA extraction (three- to five-fold increase). The most useful extraction protocol for protozoa was pre-treatment with proteinase K (eight-fold increase). The selected methods were sensitive as far as detecting one bacterial cell per reaction for S. aureus, ten bacterial cells for E. coli and two oocysts for C. parvum. The extraction reagents are cold chain independent and no centrifuge or other large laboratory equipment is required to perform DNA extraction. A controlled validation trial is needed to test the effectiveness at field levels. Full article

Liquid chromatography coupled with mass spectrometry (LC-MS) metabolomic approaches are widely used to investigate underlying pathogenesis of gastrointestinal disease and mechanism of action of treatments. However, there is an unmet requirement to assess faecal metabolite extraction methods for large-scale metabolomics studies. Current methods often rely on biphasic extractions using harmful halogenated solvents, making automation and large-scale studies challenging. The present study reports an optimised monophasic faecal extraction protocol that is suitable for untargeted and targeted LC-MS analyses. The impact of several experimental parameters, including sample weight, extraction solvent, cellular disruption method, and sample-to-solvent ratio, were investigated. It is suggested that a 50 mg freeze-dried faecal sample should be used in a methanol extraction (1:20) using bead beating as the means of cell disruption. This is revealed by a significant increase in number of metabolites detected, improved signal intensity, and wide metabolic coverage given by each of the above extraction parameters. Finally, we addressed the applicability of the method on faecal samples from patients with Crohn’s disease (CD) and coeliac disease (CoD), two distinct chronic gastrointestinal diseases involving metabolic perturbations. Untargeted and targeted metabolomic analysis demonstrated the ability of the developed method to detect and stratify metabolites extracted from patient groups and healthy controls (HC), highlighting characteristic changes in the faecal metabolome according to disease. The method developed is, therefore, suitable for the analysis of patients with gastrointestinal disease and can be used to detect and distinguish differences in the metabolomes of CD, CoD, and HC. Full article

Stool samples typically contain PCR inhibitors; however, helminths are difficult to lyse and can cause false-negative PCR results. We assessed the effective methods for extracting DNA from different kinds of intestinal parasites. We compared the most common DNA extraction methods from stool samples, including the phenol-chloroform technique with or without a bead-beating step (P and PB), a QIAamp Fast DNA Stool Mini Kit (Q), and a QIAamp PowerFecal Pro DNA Kit (QB). Genomic DNA was extracted from 85 stool samples collected from patients infected with Blastocystis sp., Ascaris lumbricoides, Trichuris trichiura, hookworm, and Strongyloides stercoralis. DNA quantity and DNA quality were evaluated via spectrophotometry, and DNA integrity was assessed by PCR. We found that P and PB provided higher DNA yields (~4 times) than when using Q and QB. However, P showed the lowest detection rate of PCR (8.2%), wherein only S. stercoralis (7 out of 20 samples) was detected. QB showed the highest detection rate of PCR (61.2%). After plasmid spikes, only 5 samples by QB were negative while 60 samples by P were still negative. Remarkably, QB could extract DNA from all the groups of parasites that we tested. These results indicate that QB is the most effective DNA extraction method for the diagnosis and monitoring of intestinal parasites via PCR. Full article

The comprehension of the pathophysiological mechanisms, the identification of druggable targets, and putative biomarkers for aortic valve stenosis can be pursued through holistic approaches such as proteomics. However, tissue homogenization and protein extraction are made difficult by tissue calcification. The reproducibility of proteome studies is key in clinical translation of the findings. Thus, we aimed to optimize a protocol for aortic valve homogenization and protein extraction and to develop a standard operating procedure (SOP), which researchers can use to maximize protein yield while reducing inter-laboratory variability. We have compared the protein yield between conventional tissue grinding in nitrogen followed by homogenization with a Potter apparatus with a more advanced bead-beating system. Once we confirmed the superiority of the latter, we further optimized it by testing the effect of beads size, the number of homogenization cycles, tube capacity, lysis buffer/tissue mass ratio, and two different lysis buffers. Optimal protein extraction was achieved with 2.8 mm zirconium dioxide beads, in two homogenization cycles, in the presence of 20 µL RIPA buffer/mg tissue, using 2 mL O-ring cryotubes. As a proof of concept of the usefulness of this SOP for proteomics, the AV proteome of men and women with aortic stenosis was characterized, resulting in the quantification of proteins across six orders of magnitude and uncovering some putative proteins dysregulated by sex. Full article

Introduction: There are numerous confounding variables in the pre-analytical steps in the analysis of gut microbial composition that affect data consistency and reproducibility. This study compared two DNA extraction methods from the same faecal samples to analyse differences in microbial composition. Methods: DNA was extracted from 20 faecal samples using either (A) chemical/enzymatic heat lysis (lysis buffer, proteinase K, 95 °C + 70 °C) or (B) mechanical and chemical/enzymatic heat lysis (bead-beating, lysis buffer, proteinase K, 65 °C). Gut microbiota was mapped through the 16S rRNA gene (V3–V9) using a set of pre-selected DNA probes targeting >300 bacteria on different taxonomic levels. Apart from the pre-analytical DNA extraction technique, all other parameters including microbial analysis remained the same. Bacterial abundance and deviations in the microbiome were compared between the two methods. Results: Significant variation in bacterial abundance was seen between the different DNA extraction techniques, with a higher yield of species noted in the combined mechanical and heat lysis technique (B). The five predominant bacteria seen in both (A) and (B) were Bacteroidota spp. and Prevotella spp. (p = NS), followed by Bacillota (p = 0.005), Lachhnospiraceae (p = 0.0001), Veillonella spp. (p < 0.0001) and Clostridioides (p < 0.0001). Conclusion: As microbial testing becomes more easily and commercially accessible, a unified international consensus for optimal sampling and DNA isolation procedures must be implemented for robustness and reproducibility of the results. Full article