Efficient Enrichment Research Articles

A new and efficient enrichment method for metagenomic sequencing of Monkeypox virus

BackgroundThe methodology described in previous literature for Monkeypox virus (MPXV) sequencing shows low efficiency when using metagenomic approaches. The aim of the present study was to evaluate a new fine-tuned method for extraction and enrichment of genomic MPXV DNA using clinical samples and to compare it to a non-enrichment metagenomic approach.ResultsA new procedure that allows sample enrichment in MPXV DNA, avoiding wasting the sequencing capacity in human DNA, was designed. This procedure consisted of host DNA depletion using a saponin/NaCl combination treatment and DNase, together with high g-force centrifugations. After typical quality control, samples using the enrichment method contained around 96% of reads not classified as human DNA, while the non-enrichment protocol showed around 5-10%. When reads not belonging to Orthopoxvirus were removed, enriched samples kept about 50% of the original read counts, while non-enriched ones kept only 2-7%.ConclusionsResults showed a very significant improvement in sequencing efficiency, increasing the number of reads belonging to MPXV, the depth of coverage and the trustworthiness of the consensus sequences. This, in turn, allows for more samples to be included in a single cartridge, reducing costs and time to diagnosis, which can be very important factors when dealing with a contagious disease.

Acidification of blood plasma facilitates the separation and analysis of extracellular vesicles

BackgroundBlood plasma is available with minimal invasive sampling, it has significant diagnostic utility, and it is a valuable source of extracellular vesicles (EVs). Nevertheless, rich protein content, the presence of lipoproteins (LPs) that share similar biophysical properties, and relatively low abundance of EVs, especially those of rare subpopulations, make any downstream application a very challenging task. The growing evidence of the intricate surface interactome of EVs, and the association of EVs with LPs, impose further challenges during EV purification, detection, and biomarker analyses. ObjectivesIn this study, we tackled the fundamental issues of plasma EV yield and LP co-isolation and their implications in the subsequent marker analyses. MethodsModerate acidification of plasma was combined with size exclusion chromatography (SEC) and/or differential centrifugation (DC) to disrupt LPs and improve recovery of EVs and their subsequent detection by immunoassays and single-particle analysis methods. ResultsOur results demonstrate a surprisingly efficient enrichment of EVs (up to 3.3-fold higher than at pH 7) and partial depletion of LPs (up to 61.2%). Acidification of blood plasma samples enabled a quick single-step isoelectric precipitation of up to 20.4% of EVs directly from plasma, upon short low-speed centrifugation. ConclusionThus, acidification holds potential as a simple and inexpensive methodological step, which improves the efficacy of plasma EV enrichment and may have implications in future biomarker discoveries.

Efficient Enrichment Method for N-Phosphorylation Peptides in Mouse Brain Tissue.

In addition to O-phosphorylation, protein N-phosphorylation was proven to play important roles in multiple biological processes. Although affinity methods were developed for the enrichment of N-phosphorylation peptides in cells, it was still difficult to enrich N-phosphorylation peptides in tissue due to its complexity. In this study, we established a workflow for the identification of N-phosphorylation peptides in mouse brain tissue by direct enrichment in high concentration urea. In total, 989 N-phosphorylation sites were obtained using 0.5 M urea as enrichment buffer. Among all identified N-phosphorylation sites, the localization probability over 0.75 was as high as 80%, suggesting the reliability of the method. Furthermore, the sequence motif analysis and gene ontology analysis results showed a good match to previous studies. The method was successfully used for N-phosphorylation analysis of mouse hippocampus from Alzheimer's disease model, and 533 N-phosphorylation sites were identified in 5 × FAD mouse hippocampus tissue. Biological process analysis results showed that "brain development", "cellular response to reactive oxygen species", "microtubule cytoskeleton organization", and "peptidyl-serine phosphorylation" were especially enriched in 5 × FAD mouse. It is suggested that N-phosphorylation may be related to Alzheimer's disease in these aspects.

Cell-Free DNA Fragmentomics: The Novel Promising Biomarker.

Cell-free DNA molecules are released into the plasma via apoptotic or necrotic events and active release mechanisms, which carry the genetic and epigenetic information of its origin tissues. However, cfDNA is the mixture of various cell fragments, and the efficient enrichment of cfDNA fragments with diagnostic value remains a great challenge for application in the clinical setting. Evidence from recent years shows that cfDNA fragmentomics' characteristics differ in normal and diseased individuals without the need to distinguish the source of the cfDNA fragments, which makes it a promising novel biomarker. Moreover, cfDNA fragmentomics can identify tissue origins by inferring epigenetic information. Thus, further insights into the fragmentomics of plasma cfDNA shed light on the origin and fragmentation mechanisms of cfDNA during physiological and pathological processes in diseases and enhance our ability to take the advantage of plasma cfDNA as a molecular diagnostic tool. In this review, we focus on the cfDNA fragment characteristics and its potential application, such as fragment length, end motifs, jagged ends, preferred end coordinates, as well as nucleosome footprints, open chromatin region, and gene expression inferred by the cfDNA fragmentation pattern across the genome. Furthermore, we summarize the methods for deducing the tissue of origin by cfDNA fragmentomics.

Solvent-free synthesis of zeolites: New insights into the mechanism and non-mechanochemical route

The solvent-free (SF) synthesis is a green alternative to the conventional method of synthesis for zeolites. In this study, we show for the first time the feasibility of carrying out the SF synthesis of silicalite-1 (F− route) without any grinding. We also monitor the changes during the synthesis thanks to multiple characterization techniques: XRD, TGA, SEM, EDX, and multinuclear NMR (13C, 19F, 29Si). We show how the duration of the thermal treatment impacts the zeolite crystal size and morphology, as well as the local degree of order probed by solid-state NMR. The obtained results provide new insights on the SF formation mechanism based on solid phase transformations. Our findings also show that once the organic structure directing agent (OSDA), here tetrapropylammonium, is incorporated, the crystals are formed and no other intermediate phases incorporating the OSDA exist. Further, we study the role of water in the synthesis using 17O NMR. For this purpose, we develop a simple and efficient 17O enrichment method of zeolite frameworks. The efficiency of this enrichment demonstrates the role of water in the hydrolysis and condensation reactions and its need for a successful synthesis.

An advanced protocol for profiling RNA-binding proteins in Arabidopsis using plant phase extraction

Abstract RNA-binding proteins (RBPs) are key players in regulating cell fate and essential developmental processes. Systematic profiling of the RNA-binding proteome (RBPome) is thus indispensable for researchers aiming to understand the mechanisms of post-transcriptional gene regulation. RBPome identification methods developed in humans, mice, and bacteria have successfully identified RBPomes in these organisms. However, the biochemical and genetic complexities of plant tissues have greatly hindered the effectiveness of these methods in plants. Moreover, plant RBPs have been predominantly discovered through oligo d(T) based affinity purification (RNA-interactome capture). Since polyadenylated RNA only accounts for less than 5% of the total RNA population in eukaryotic cells, there is a pressing need to develop a comprehensive, yet unbiased, method to capture the full spectrum of RBPs in plants. Here, we describe a detailed protocol of Plant Phase Extraction (PPE), a recently developed method to identify RBPs in Arabidopsis (Zhang Y, Xu Y, Skaggs TH, et al. Plant phase extraction: a method for enhanced discovery of the RNA-binding proteome and its dynamics in plants. Plant Cell 2023; 35: 2750–72.) [1]. The PPE method enables the efficient enrichment of both poly(A) and non-poly(A) RBPs from various tissues quickly and reproducibly. Most importantly, PPE allows for unveiling dynamic RBP–RNA interactions under various abiotic and biotic stress conditions and during different plant developmental stages. This provides a much broader and more accurate understanding of plant RBPs, marking a significant advancement in plant molecular biology.

Dynamic crack analysis in anisotropic media under wave-like thermal loading

In this paper, the eXtended finite element method (XFEM) is used to study a cracked orthotropic medium under a non-classic thermal shock considering the Green-Naghdi (GN) theory of thermoelasticity (type II). A brief critical review of the generalized thermoelasticity in anisotropic materials is presented, and outstanding features of the Green-Naghdi theory in the description of the thermal wave traveling in an anisotropic medium are discussed. Tip enrichment functions are derived for the temperature according to the governing equations of the Green-Naghdi type II model. In addition, new simple and efficient tip enrichment functions are proposed, which are identical for both displacement and temperature fields. The effect of the thermal wave speed in the material directions on the thermal wave traveling and its reflection is studied. In particular, the time variation of the Stress Intensity Factors (SIFs) and the temperature distribution based on the GN theory are numerically compared with the classic thermoelasticity (CTE) theory.

A green and designable natural deep eutectic solvent-based supramolecular solvents system: Efficient extraction and enrichment for phytochemicals

A green, novel and designable natural deep eutectic solvent-based supramolecular solvents/equilibrium solution (NADES-SUPRAS/EqS) was developed, which possesses better stability and free designability with a wider range of synthesis while compared to the typical alkyl carboxylic acid-based SUPRAS. The physicochemical properties and specific synthesis patterns of NADES-SUPRAS/EqS had been investigated. In addition, a more intelligent solvent regulated synthesis system had been developed to prepare corresponding supramolecular systems according to different polarity target phytochemicals through tailoring the components of synthetic NADES-SUPRAS/EqS. During the NADES-SUPRAS extraction of Zanthoxylum bungeanum Maxim. leaves (ZBML), 18 kinds of NADES were screened and the proportion of NADES as well as the ethanol content were varied tailor. After tailoring, all of the extracted chlorogenic acid, neochlorogenic acid, rutin, as well as 91.775 % of hyperin, 94.940 % isoquercetin and 84.405 % quercetin were distributed in the EqS phase while all of the hydroxy-α-sanshool (HAS) were distributed in the NADES-SUPRAS phase. Furthermore, NADES-SUPRAS/EqS extraction rate reached 9.741 mg/g, which were 7.604-fold, 2.352-fold, 1.141-fold, and 1.384-fold to that of water, ethanol, 70 % ethanol and methanol extraction, respectively. The excellent extraction and separation effects were due to the large number of hydrogen bonds and hydrophobic forces. Dynamic multi-stage countercurrent extraction combined with water and macroporous resin recovery enabled significant cost reductions and rapid recovery of chemical components and solvents. This novel solvent could be intelligently designed and be of great significance for the extraction of different polarity phytochemicals from plants.

Post-synthesis of a titanium-rich magnetic COF nanocomposite with flexible branched polymers for efficient enrichment of phosphopeptides from human saliva and serum.

A Ti4+-functionalized magnetic covalent organic framework material with flexible branched polymers (mCOF@ε-PL@THBA-Ti4+) built via an immobilized metal ion affinity chromatography (IMAC) enrichment strategy was proposed through post-synthesis modification. Hydrophilic ε-poly-L-lysine (ε-PL) rich in amino active groups was first introduced in the fabrication of the phosphopeptide enrichment material to increase the hydrophilicity while providing more functional modification pathways of the material. 2,3,4-Trihydroxy-benzaldehyde (THBA) provides abundant binding sites for the immobilization of numerous Ti4+, which is advantageous for the subsequent efficient phosphopeptide enrichment. The magnetic nanocomposite exhibited outstanding performance of phosphopeptide enrichment with good selectivity (1 : 5000), a low detection limit (2 fmol), and relatively high loading capacity (66.7 mg g-1). What's more, after treatment with mCOF@ε-PL@THBA-Ti4+, 16 endogenous phosphopeptides from fresh saliva of healthy people were recognized by MALDI-TOF MS, and 50 phosphopeptides belonging to 35 phosphoproteins from the serum of uremia patients were detected by nano-LC-MS/MS. Proteomics data analysis for the differential protein selection between uremia and normal controls was conducted using R software, and four down-regulated and three up-regulated proteins were obtained. The results suggested that the prepared material has potential applications in biomarker discovery.

Magnetic enrichment of immuno-specific extracellular vesicles for mass spectrometry using biofilm-derived iron oxide nanowires.

Immuno-specific enrichment of extracellular vesicles (EVs) can provide important information into cellular pathways underpinning various pathologies and for non-invasive diagnostics, including mass spectrometry-based analyses. Herein, we report an optimised protocol for immuno-magnetic enrichment of specific EV subtypes and their subsequent processing with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specifically, we conjugated placental alkaline phosphatase (PLAP) antibodies to magnetic iron oxide nanowires (NWs) derived from bacterial biofilms and demonstrated the utility of this approach by enriching placenta-specific EVs (containing PLAP) from cell culture media. We demonstrate efficient PLAP+ve EV enrichment for both NW-PLAP and Dynabeads™-PLAP, with high PLAP protein recovery (83.7 ± 8.9% and 83.2 ± 5.9%, respectively), high particle-to-protein ratio (7.5 ± 0.7 × 109 and 7.1 ± 1.2 × 109, respectively), and low non-specific binding of non-target EVs (7 ± 3.2% and 5.4 ± 2.2%, respectively). Furthermore, our optimized EV enrichment and processing approach identified 2518 and 2545 protein groups with LC-MS/MS for NW-PLAP and Dynabead™-PLAP, respectively, with excellent reproducibility (Pearson correlation 0.986 and 0.988). These findings demonstrate that naturally occurring iron oxide NWs have comparable performance to current gold standard immune-magnetic beads. The optimized immuno-specific EV enrichment for LC-MS/MS method provides a low-cost and highly-scalable yet efficient, high-throughput approach for quality EV proteomic studies.

Rapid Real-time Squiggle Classification for Read until using RawMap.

ReadUntil enables Oxford Nanopore Technology's (ONT) sequencers to selectively sequence reads of target species in real-time. This enables efficient microbial enrichment for applications such as microbial abundance estimation and is particularly beneficial for metagenomic samples with a very high fraction of non-target reads (> 99% can be human reads). However, read-until requires a fast and accurate software filter that analyzes a short prefix of a read and determines if it belongs to a microbe of interest (target) or not. The baseline Read Until pipeline uses a deep neural network-based basecaller called Guppy and is slow and inaccurate for this task (~60% of bases sequenced are unclassified). We present RawMap, an efficient CPU-only microbial species-agnostic Read Until classifier for filtering non-target human reads in the squiggle space. RawMap uses a Support Vector Machine (SVM), which is trained to distinguish human from microbe using non-linear and non-stationary characteristics of ONT's squiggle output (continuous electrical signals). Compared to the baseline Read Until pipeline, RawMap is a 1327X faster classifier and significantly improves the sequencing time and cost, and compute time savings. We show that RawMap augmented pipelines reduce sequencing time and cost by ~24% and computing cost by 22%. Additionally, since RawMap is agnostic to microbial species, it can also classify microbial species it is not trained on. We also discuss how RawMap may be used as an alternative to the RT-PCR test for viral load quantification of SARS-CoV-2.

Enrichment of rosmarinic acid from Salvia przewalskii Maxim. leaves using macroporous resin: Adsorption/desorption behavior, process optimization followed by scale-up

To valorize Salvia przewalskii Maxim. leaves as a renewable source of rosmarinic acid, an efficient macroporous resin enrichment technology was established in this work. X-5 resin was considered as the most appropriate adsorbent in view of its highest adsorption/desorption capacity for rosmarinic acid, and the adsorption data were best expounded by Langmuir isotherm model and pseudo-second-order kinetics model. The influences of sample volume, loading and elution flow rates, eluent type and volume were evaluated to obtain the optimum enrichment procedure, which was as follows: for adsorption, the rosmarinic acid concentration, loading flow rate and sample volume were respectively 0.24 mg/mL, 3 BV/h, and 12 BV; for desorption, the eluent type, elution flow rate and volume were respectively 50% ethanol, 3 BV/h and 9 BV. Furthermore, an enrichment process scale-up of rosmarinic acid was performed under optimal procedure. After one-step large-scale enrichment, the content of rosmarinic acid improved 3.65-fold from 12.41% to 45.30% with a satisfactory recovery of 80.17%. Results suggested that X-5 resin chromatographic enrichment technique developed in the present study offered a feasible scheme for the industrial enrichment of rosmarinic acid from S. przewalskii leaves. • X-5 reisn was the optimum resin for the enrichment of rosmarinic acid. • Adsorption/desorption behavior of rosmarinic acid on X-5 reisn was studied. • An effective resin chromatography to enrich rosmarinic acid was developed. • The enrichment method showed a promising prospect for industrial application.

Self-assembly of hydrazide-linked porous organic polymers rich in titanium for efficient enrichment of glycopeptides and phosphopeptides from human serum.

In this work, titanium-rich hydrazide-linked porous organic polymers (hydrazide-POPs-Ti4+) were synthesized using hydrazine, 2,3-dihydroxyterephthalaldehyde (DHTA) and trimethyl 1,3,5-benzenetricarboxylate (TP) as the ligands. Hydrazide-POPs-Ti4+ combined with HILIC and IMAC can be used for simultaneous enrichment of glycopeptides and phosphopeptides. The detection limit of this protocol is 0.1 fmol μL-1 for glycopeptides and 0.005 fmol μL-1 for phosphopeptides, and the selectivities are 1 : 1000 and 1 : 2000 for glycopeptides and phosphopeptides, respectively. For practical bio-sample analysis, 201 glycopeptides associated with 129 glycoproteins and 26 phosphopeptides associated with 21 phosphoproteins were selectively captured from healthy human serum, and 186 glycopeptides associated with 117 glycoproteins and 60 phosphopeptides associated with 50 phosphoproteins were enriched in the serum of breast cancer patients. Gene Ontology analysis indicated that the identified glycoproteins and phosphoproteins were linked to breast cancer, including the binding of complement component C1q and low-density lipoprotein particles, protein oxidation and complement activation, suggesting that these connected pathways are probably engaged in the disease pathology of breast cancer.

Magnetic resin composites for the enrichment of proteins, peptides and phosphopeptides.

There is growing interest in the development of materials for enriching proteins and phosphoproteins from complex sample matrices for mass spectrometric analysis. Herein, we designed and synthesized two types of magnetic resin composites, i.e., MTS9200@Fe3O4 and FPA90CL@Fe3O4, and assessed their applications as adsorbents for enriching proteins, peptides and phosphopeptides. With the combination of Fe3+-IMAC interaction (MTS9200) or electrostatic attraction (FPA90CL) of resins and the adsorption of Fe3O4, the prepared composites exhibited higher capacities for adsorbing a protein (bovine serum albumin, at 195.71 and 135.03 mg g-1 for MTS9200@Fe3O4 and FPA90CL@Fe3O4, respectively) than MTS9200, FPA90CL and Fe3O4. In addition, due to the contributions of the hydrophobic skeleton of resins and Fe3O4, the magnetic resin composites allowed for efficient enrichment of peptides. Moreover, through Fe3+-IMAC interaction or electrostatic attraction of resins and Fe-O MOAC interaction of Fe3O4 with phosphate groups, phosphopeptides could also be captured. Furthermore, we employed the prepared composites for enriching proteins and phosphopeptides from human serum, where 466 and 506 proteins, and 434 and 356 phosphorylation sites, were detected from human serum after being processed with FPA90CL@Fe3O4 and MTS9200@Fe3O4, respectively. Together, our work revealed the great potential of magnetic resin composites as enrichment materials for proteomics and phosphoproteomics analysis.

Solvent-assisted dispersive micro-solid phase extraction of bisphenols using iron(III) thenoyltrifluoroacetonate complex (Fe(TTA)3) as a new nanostructured sorbent: a proof of concept.

In this work, we describe for the first time the use of iron(III) thenoyltrifluoroacetonate complex (Fe(TTA)3) as a novel sorbent for solvent-assisted dispersive micro-solid phase extraction (SA-dμSPE) of bisphenols from water samples. The extraction procedure is based on the formation of nanoparticles in situ following the rapid injection of a methanolic solution of Fe(TTA)3 into the stirred aqueous sample. Herein, the synthesis of Fe(TTA)3 and study of the essential parameters of the preparative procedure are described. The optimized procedure allowed for efficient enrichment of bisphenols from various water samples, chosen as model contaminants and matrix, within 2.5 min. The sorbent was collected by centrifugation, dissolved in methanol, and injected to perform HPLC with spectrophotometric detection. The limits of detection and quantification obtained ranged from 1.0-3.1 and 3.1-7.5 μg L-1, respectively. Intraday and interday precisions of <7% relative standard deviation (RSD) and <8% RSD with analyte recoveries ranging between 70-117% (103.8% on average) were obtained for the analysis of river water, wastewater treatment plant effluent, and bottled water.

Room temperature synthesis of flower-like hollow covalent organic framework for efficient enrichment of microcystins.

The morphology of nanomaterials is one of essential factors for their unique properties. Herein, a hollow covalent organic framework with a flower-like structure (HFH-COF) was synthesized at room temperature. The synthesized HFH-COF has a very large specific surface area, mesoporous structure, excellent chemical stability, and good crystallinity. The special morphology endowed HFH-COF with high specific surface area utilization and rapid mass transfer rate, resulting in faster equilibration time and better extraction performance than spherical COF (S-COF). Subsequently, combined with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), an efficient and sensitive method was established for microcystins (MCs) detection. The developed method has low detection limits (0.6-0.8 pg mL-1), wide linear ranges (1.5-1000.0 pg mL-1, R ≥ 0.9993), and acceptable reproducibility (RSD ≤ 7.6%, n = 6). Real biological samples were analyzed by the developed method, and trace levels of MC-YR, MC-RR and MC-LR were detected. The results indicate that the synthesized HFH-COF is an ideal sorbent for efficient extraction of MCs from complex biological samples.

Three-Dimensional MAX-Ti3 AlC2 Nanomaterials for Dual-Selective and Highly Efficient Enrichment of Phosphorylated and Glycosylated Peptides.

Dual-selective enrichment of phosphopeptides and glycopeptides of post-translational modifications (PTMs) in the complex biological samples are challenging. In this work, considering the versatile properties including surface abundant metal sites and electrostatic attraction between Ti3 C2 -layers and Al-layers, layered ternary carbides Ti3 AlC2 nanomaterials was successfully applied for the first time as an affinity adsorbent for the dual-selective capture of phosphopeptides and glycopeptides. Especially, the Ti3 AlC2 nanomaterials had an excellent detection sensitivity for phosphopeptides (1×10-11 M) and a good selectivity for glycopeptides with a low molar ratio of 1 : 500 of HRP (horseradish peroxidase) to BSA (bovine serum albumin). Furthermore, Ti3 AlC2 nanomaterials was also applied for dual-selective enrichment of phosphopeptides and glycopeptides from mouse brain neocortex lysate and human serum lysate respectively before mass spectrometry (MS) analysis, yielding twenty-two unique phosphopeptides from thirteen phosphoproteins and fifty-three unique glycopeptides from thirty-seven glycoproteins, respectively. This work will open a new avenue and will greatly promote sample preparation for mass spectrometric analysis in phosphoproteomics and glycoproteomics research.

Mechanistic insight of hydrophobic agglomeration of rhodochrosite fines Co-enhanced by Oleic-Kerosene emulsion and static magnetic field

Efficient enrichment of fine rhodochrosite is key to purifying manganese carbonate ore and reducing the discharge of electrolytic manganese slag at the source. Enhancing the agglomeration of rhodochrosite fines is important for efficient flotation separation. In this study, we investigated the agglomeration mechanism of rhodochrosite fines co-enhanced by oleic-kerosene emulsion and static magnetic field using agglomerate and surface properties analysis, electrical conductivity and atomic force microscopy measurements, and extended Derjaguin, Landau, Verwey, and Overbeek theoretical calculations. The results showed that the agglomerate size and regularity degree increased with increasing shearing time, achieving the maximum values of the average size at 20 min with magnetic field and at 40 min without magnetic field. Larger-sized and tighter agglomerates can be formed in a shorter time by the Lorentz force in magnetic field. As magnetic field was applied, the surface electronegativity of rhodochrosite in emulsion solution was stronger and positively correlated with magnetic field intensity. In addition, due to the higher solubility of oleic acid, the adsorption capacity and net heat of emulsion increased, resulting in a stronger hydrophobicity of rhodochrosite with a shorter hydrophobization time. Interestingly, almost no attraction existed between particles with magnetic field in aqueous solution, while a larger attraction existed without magnetic field in emulsion solution, and the attraction and action distance both increased markedly with increasing magnetic field intensity. However, the magnetic attraction was much smaller than hydrophobic attraction. As a result, agglomeration of rhodochrosite fines could be enhanced by stronger hydrophobic attraction under the synergy effect of magnetic field and oleic-kerosene emulsion. This research will be conducive to in-depth insight into the enhancement mechanisms of rhodochrosite agglomeration and will provide a basis for flotation regulation.

Combination of Fe(OH)3 modified diatomaceous earth and qPCR for the enrichment and detection of African swine fever virus in water.

Water is one of the primary vectors for African swine fever virus (ASFV) transmission among swine herds. However, the low concentrations of ASFV in water represent a challenge for the detection of the virus by conventional PCR methods, and enrichment of the virus would increase the test sensitivity. In this study, aiming to enrich ASFV in water quickly and efficiently, a rapid and efficient water-borne virus enrichment system (MDEF, modified diatomaceous earth by ferric hydroxide colloid) was used to enrich ASFV in water. After enrichment by MDEF, conventional real-time PCR (qPCR) was used for ASFV detection. ASFV were inactivated and diluted in 10 L of water, of which 4 mL were collected after 60 min treatment using the MDEF system. Two thousand five hundred times reduction of the sample volume was achieved after enrichment. A high adsorption rate of about 99.99 (±0.01)% and a high recovery rate of 64.01 (±10.20)% to 179.65 (±25.53)% was achieved by using 1g modified diatomaceous earth for 10 L ASFV contaminated water. The limit of qPCR detection of ASFV decreased to 1 × 10-1.11 GU ml-1 (genomic units per milliliter) from 1 × 102.71 GU ml-1 after concentrating the spiked water from 10 L to 4 ml. Preliminary application of MDEF allowed successful detection of African swine fever virus (ASFV), porcine circovirus type 2 (PCV2), and pseudorabies virus (PRV) in sewage. Thus, the combination of modified diatomaceous earth and real-time PCR is a promising strategy for the detection of viruses in water.

Magnetic Nanoseparation Technology for Efficient Control of Microorganisms and Toxins in Foods: A Review.

Outbreaks of foodborne diseases mediated by food microorganisms and toxins remain one of the leading causes of disease and death worldwide. It not only poses a serious threat to human health and safety but also imposes a huge burden on health care and socioeconomics. Traditional methods for the removal and detection of pathogenic bacteria and toxins in various samples such as food and drinking water have certain limitations, requiring a rapid and sensitive strategy for the enrichment and separation of target analytes. Magnetic nanoparticles (MNPs) exhibit excellent performance in this field due to their fascinating properties. The strategy of combining biorecognition elements with MNPs can be used for fast and efficient enrichment and isolation of pathogens. In this review, we describe new trends and practical applications of magnetic nanoseparation technology in the detection of foodborne microorganisms and toxins. We mainly summarize the biochemical modification and functionalization methods of commonly used magnetic nanomaterial carriers and discuss the application of magnetic separation combined with other instrumental analysis techniques. Combined with various detection techniques, it will increase the efficiency of detection and identification of microorganisms and toxins in rapid assays.