Enrichment Step Research Articles

Untargeted metabolomics of prostate cancer zwitterionic and positively charged compounds in urine

Prostate cancer, a leading cause of cancer-related deaths worldwide, principally occurs in over 50-year-old men. Nowadays there is urgency to discover biomarkers alternative to prostate-specific antigen, as it cannot discriminate patients with benign prostatic hyperplasia from clinically significant forms of prostatic cancer. In the present paper, 32 benign prostatic hyperplasia and 41 prostatic cancer urine samples were collected and analyzed. Polar and positively charged metabolites were therein investigated using an analytical platform comprising an up to 40-fold analyte enrichment step by graphitized carbon black solid-phase extraction, HILIC separation, and untargeted high-resolution mass spectrometry analysis. These classes of compounds are often neglected in common metabolomics experiments even though previous studies reported their significance in cancer biomarker discovery. The complex metabolomics big datasets, generated by the UHPLC-HRMS, were analyzed with the ROIMCR procedure, based on the selection of the MS regions of interest data and their analysis by the Multivariate Curve-Resolution Alternating Least Squares chemometrics method. This approach allowed the resolution and tentative identification of the metabolites differentially expressed by the two data sets. Among these, amino acids and carnitine derivatives were tentatively identified highlighting the importance of the proposed methodology for cancer biomarker research.

Development of a novel visual detection technique for Campylobacter jejuni in chicken meat and caecum using polymerase spiral reaction (PSR) with pre-added dye

Rapid detection and efficient removal of Campylobacter jejuni contaminated chicken-based food products from the food chain have become decisive for preventing outbreaks. Here, we report a novel, rapid, simple and sensitive polymerase spiral reaction (PSR) assay for screening chicken samples for contamination of C. jejuni. Our method specifically amplified the hipO gene of C. jejuni and involved cost-effective three-step centrifugation method for DNA extraction and pre-addition of HNB dye for effortless visualization. PSR, real-time PCR and conventional PCR were able to detect 50 fg, 500 fg and 5 pg C. jejuni DNA per tube, respectively. In chicken meat and caecum, the detection limits of PSR without any enrichment, after including 12 h enrichment and 24 h enrichment steps were 16 × 102 CFU/g, 160 CFU/g and 16 CFU/g, respectively which could be attained in 90 min, 13.5 h and 25.5 h, respectively. The field applicability of the PSR assay was evaluated through a field sampling study by testing 75 chicken meat and caecum samples. This assay permits timely reporting of results without the need of trained personnel and sophisticated equipments as it only relies on simple enrichment, water bath amplification and visual interpretation through colour change. This PSR assay can be a handy tool to screen C. jejuni contamination in chicken products and might subsequently help in reducing the incidence of foodborne C. jejuni infections. To our knowledge, this is the primary report to employ PSR for the detection of C. jejuni.

A guanidinium-rich polymer as a new universal bioreceptor for multiplex detection of bacteria from environmental samples

Protamine, a guanidinium rich polymer, is proposed as a universal bioreceptor for bacteria, towards rapid and handheld bacteria detection from complex environmental water samples without the need for specific antibodies or primers. Escherichia coli K12, Salmonella Typhimurium, and Staphylococcus aureus (MSSA) were assayed, representing gram-negative, gram-positive, rod- and round-shaped bacteria. Samples and the protamine conjugated fluorescent particles were sequentially loaded to the paper microfluidic chips and flowed through the channels spontaneously via capillary action. The particles were aggregated via protamine-bacteria membrane interactions and unbound particles were rinsed via capillary action. A low-cost smartphone fluorescence microscope was designed, fabricated, and imaged the paper channels. A unique image processing algorithm isolated only the aggregated particles to detect all three bacteria (p < 0.05) with a detection limit of 101−102 CFU/mL. Protamine did not induce any particle aggregation with a model protein, algae, and virus. Successful bacteria detection was also demonstrated with environmental field water samples. Total assay time was < 10 min with neither extraction nor enrichment steps. In summary, a guanidinium-rich polymer showed a promise as a universal bioreceptor for bacteria and can be used on a paper microfluidic chip and smartphone quantification towards rapid and handheld detection.

Assembly of hyperhalophilic complex consortia of isolates from anchovy ripening attaining histamine degradation and their microbiome configuration

High histamine content in salted-ripened fish results in food safety detriment. The present study focused on the assembly of mixed cultures from halophilic prokaryotes isolated during salting-ripening anchovy process in order to determine their histamine-degrading capacity. For this purpose, 57 halophilic prokaryotes were inoculated together and submitted to several enrichment steps with strong shifts in culturing conditions and three microbial consortia were subsequently assembled: original consortium named C1 (without histamine exposure), consortium C2 (enriched in nutritive culture medium with high histamine content ~900 mg/kg) and consortium C3 (against histamine ~200 mg/kg) and reduced amounts of nutrients. These consortia were submitted to histamine-degrading capacity studies and 16S rRNA gene amplicons analysis. In the absence of histamine (C1), Salinivibro costicola (16.83%), Haloarcula spp. (11.08%) and Halococcus spp. (8.73%) were the most abundant. C2 presented ability to degrade histamine and major OTUs belonged to the family Halobacteriaceae and the genus Halomonas. C3 degraded histamine and predominant OTUs were from the family Halomonadaceae and the genus Chomohalobacter. Therefore, members of the archaeal family Halobacteriaceae and bacterial genera Halomonas and Chomohalobacter would be associated to histamine degradation and their potential as single or mixed starter cultures for improving salted-ripened foodstuff safety is promising.

Machine learning-enabled non-destructive paper chromogenic array detection of multiplexed viable pathogens on food.

Fast and simultaneous identification of multiple viable pathogens on food is critical to public health. Here we report a pathogen identification system using a paper chromogenic array (PCA) enabled by machine learning. The PCA consists of a paper substrate impregnated with 23 chromogenic dyes and dye combinations, which undergo colour changes on exposure to volatile organic compounds emitted by pathogens of interest. These colour changes are digitized and used to train a multi-layer neural network (NN), endowing it with high-accuracy (91-95%) strain-specific pathogen identification and quantification capabilities. The trained PCA-NN system can distinguish between viable Escherichia coli, E. coli O157:H7 and other viable pathogens, and can simultaneously identify both E. coli O157:H7 and Listeria monocytogenes on fresh-cut romaine lettuce, which represents a realistic and complex environment. This approach has the potential to advance non-destructive pathogen detection and identification on food, without enrichment, culturing, incubation or other sample preparation steps.

Determination of antibiotics in meat samples using analytical methodologies: A review.

Antibiotics are widely used to prevent or treat some diseases in human and veterinary medicine and also as animal growth promoters. The presence of these compounds in foods derived from food-producing animals can be a risk for human health. Consequently, regulatory agencies have set maximum residue limits for antibiotics in food samples. Therefore, the development of novel methodologies for its determination in food samples is required. Specifically, the analysis and quantification of these substances in meat tissues is a challenge for the analytical chemistry research community. This is due to the complexity of the matrix and the low detection limits required by the regulatory agencies. In this sense, a comprehensive review on the development of new sample preparation treatments involving extraction, cleanup, and enrichment steps of antibiotics in meat samples in combination with sensitive and sophisticated determination techniques that have been carry out in the last years is necessary. Therefore, the aim of this work is to summarize the published methodologies for the determination of antibiotics from 2016 until the beginning of the second semester of 2020. The first part of this review includes an introduction about antibiotic families, followed by sample preparation and determination techniques applied to the different families. Finally, a detailed discussion of the current trends and the future possible perspectives in this field are also included.

CReVIS-Seq: A highly accurate and multiplexable method for genome-wide mapping of lentiviral integration sites

Lentiviruses have been widely used as a means of transferring exogenous DNAs into human cells to treat various genetic diseases. Lentiviral vectors are fundamentally integrated into the host genome, but their integration sites are generally unpredictable, which may increase the uncertainty for their use in therapeutics. To determine the viral integration sites in the host genome, several PCR-based methods have been developed. However, the sensitivities of the PCR-based methods are highly dependent on the primer sequences, and optimized primer design is required for individual target sites. In order to address this issue, we developed an alternative method for genome-wide mapping of viral insertion sites, named CReVIS-seq (CRISPR-enhanced Viral Integration Site Sequencing). The method is based on the sequential steps: fragmentation of genomic DNAs, in vitro circularization, cleavage of target sequence in a CRISPR guide RNA-specific manner, high-throughput sequencing of the linearized DNA fragments in an unbiased manner, and identification of viral insertion sites via sequence analysis. By design, CReVIS-seq is not affected by biases that could be introduced during the target enrichment step via PCR amplification using site specific primers. Furthermore, we found that multiplexed CReVIS-seq, using collections of different single-guide RNAs (sgRNAs), enables simultaneous identification of multiple target sites and structural variations (i.e., circularized viral genome), in both single cell clones and heterogeneous cell populations.

Determination of Pharmaceutical Residues by UPLC-MS/MS Method: Validation and Application on Surface Water and Hospital Wastewater.

In this study, an analytical method for the simultaneous determination of 7 major pharmaceutical residues in Vietnam, namely, carbamazepine, ciprofloxacin, ofloxacin, ketoprofen, paracetamol, sulfamethoxazole, and trimethoprim, in surface water and hospital wastewater has been developed. The method includes enrichment and clean-up steps by solid phase extraction using mix-mode cation exchange, followed by identification and quantification using an ultrahigh-performance liquid chromatography and tandem mass spectrometry and employing electrospray ionization (UPLC-ESI-MS/MS). Seven target compounds were separated on the reversed phase column and detected in multiple reaction monitoring (MRM) mode within 6 minutes. The present study also optimized the operating parameters of the mass spectrometer to achieve the highest analytical signals for all target compounds. All characteristic parameters of the analytical method were investigated, including linearity range, limit of detection, limit of quantification, precision, and accuracy. The important parameter in UPLC-ESI-MS/MS, matrix effect, was assessed and implemented via preextraction and postextraction spiking experiments. The overall recoveries of all target compounds were in the ranges from 55% to 109% and 56 % to 115% for surface water and hospital wastewater, respectively. Detection limits for surface water and hospital wastewater were 0.005–0.015 µg L−1 and 0.014–0.123 µg L−1, respectively. The sensitivity of the developed method was allowed for determination of target compounds at trace level in environmental water samples. The in-house validation of the developed method was performed by spiking experiment in both the surface water and hospital wastewater matrix. The method was then applied to analyze several surface water and hospital wastewater samples taken from West Lake and some hospitals in Vietnam, where the level of these pharmaceutical product residues was still missed. Sulfamethoxazole was present at a high detection frequency in both surface water (33% of analyzed samples) and hospital wastewater (81% of analyzed samples) samples.

Comparison of single and multi-host enrichment approach for harnessing lytic phages against antimicrobial-resistant E. coli: Repurposing the enrichment step

Escherichia coli has been enlisted as a pathogen of antimicrobial resistance (AMR) importance posing a threat to human health. In the wake of the widespread AMR, lytic bacteriophages remain a promising alternative to antibiotics for controlling the multidrug-resistant (MDR) pathogens. However, the major hindrance for phage therapy is the lack of a rapid procedure to harness broad host range phages. Hitherto in vogue, lytic phage isolation methods are based on conventional single host enrichment (M1). Limitation to the available methods includes a higher number of steps, additional instrumentation, cost and time involved in the screening of samples and more recovery of narrow host range phages.To improve the conventional method of isolation, a multiple host enrichment approach (modified method; M2) is developed and validated with a classical approach using 24 MDR extended-spectrum β-lactamase producing (ESBL) E. coli and universal coliphage host E. coli NCIM 2089. A total of 58 phages were isolated using both the methods (27 phages from M1 and 31 phages from M2) and were categorized, based on the DNA restriction digestion pattern, to 25 groups with a maximum host range of 17 in host range analysis. Even though there is no significant difference (P > 0.01) in recovering number of phages between M1 and M2 methods, excepting in one occasion where MFB13 host yielded 3 more morphologically distinct phages in M2 (p ≤ 0.05), the M2 approach was better in terms of reduction in time (4.72%) and cost (23.5%) and same instrumentation requirement. This emphasizes that the proposed method has the potential to yield broad host range lytic phages against localized strain repository of AMR E. coli which can be leading to its application in food, food contact surfaces as well as phage therapy.

Synthesis and Characterization of Activated Carbon from Biowaste-Peanut Shell and Application to Preconcentration/Removal of Uranium

This study aims to synthesize and characterize an economical and ecological adsorbent with high adsorption capacity. For this purpose, the peanut shells (Pistacia vera L.) were modified chemically. After the synthesis of activated carbon (AC), the optimum conditions for enrichment steps were performed using parameters: pH and contact time for uranium in the model solutions. The measurements were carried out by inductively coupled plasma-mass spectrometry (ICP-MS). From the shapes of the BET isotherms, the AC obtained exhibits type I. The study indicated that the surface area and total pore volume of the AC were found to be 679.9m2 g-1 and 0.31cc g-1, respectively. The adsorption capacity was found to be 260mg g-1. The optimum pH was found to be 6.0 for enrichment using the AC obtained by sulfuric acid as a chemical-modifier. The optimized method was applied to enrichment of U at ppb levels in the model solutions.

CRISPR/Cas9-Mediated Introduction of Specific Heterozygous Mutations in Human Induced Pluripotent Stem Cells.

Advances in genome editing and our ability to derive and differentiate human induced pluripotent stem cells (hiPSCs) into a wide variety of cell types present in the body is revolutionizing how we model human diseases in vitro. Central to this has been the development of the CRISPR/Cas9 system as an inexpensive and highly efficient tool for introducing or correcting disease-associated mutations. However, the ease with which CRISPR/Cas9 enables genetic modification is a double-edged sword, with the challenge now being to introduce changes precisely to just one allele without disrupting the other.In this chapter, we describe strategies to introduce specific mutations into hiPSCs without enrichment steps. Monoallelic modification is contingent on the target activity of the guide RNA, delivery method of the CRISPR/Cas9 components and design of the oligonucleotide(s) transfected. As well as addressing these aspects, we detail high throughput culturing, freezing and screening methods to identify clonal hiPSCs with the desired nucleotide change. This set of protocols offers an efficient and ultimately time- and labor-saving approach for generating isogenic pairs of hiPSCs to detect subtle phenotypic differences caused by the disease variant.

Integration of FISH and Microfluidics.

Suitable molecular methods for a faster microbial identification in food and clinical samples have been explored and optimized during the last decades. However, most molecular methods still rely on time-consuming enrichment steps prior to detection, so that the microbial load can be increased and reach the detection limit of the techniques.In this chapter, we describe an integrated methodology that combines a microfluidic (lab-on-a-chip) platform, designed to concentrate cell suspensions and speed up the identification process in Saccharomyces cerevisiae , and a peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) protocol optimized and adapted to microfluidics. Microfluidic devices with different geometries were designed, based on computational fluid dynamics simulations, and subsequently fabricated in polydimethylsiloxane by soft lithography. The microfluidic designs and PNA-FISH procedure described here are easily adaptable for the detection of other microorganisms of similar size.

Identification of the gamma irradiation dose applied to ground beef that reduces Shiga toxin producing Escherichia coli but has no impact on consumer acceptance

The aims of the present study were: a) to estimate the minimal dose of gamma irradiation required to reduce 5 log CFU/g of native O157 and non-O157 Shiga toxin-producing Escherichia coli population in ground beef samples inoculated with high inoculum; b) to assess its effectiveness in samples with low inoculum and 3) to evaluate consumer acceptance. Based on the results, 1 kGy was estimated as the minimal dose of gamma irradiation required to reduce 5 log CFU/g of STEC in ground beef. However, when samples with low inoculum level were subjected to 1 kGy, 3.9% of the samples were positive for stx and eae genes after an enrichment step. Consumer acceptance analysis was carried out with samples subjected to 2.5 kGy and no significant differences were found between irradiated and control samples. Therefore, 2.5 kGy was identified as the gama irradiation dose that reduces STEC but has no impact on consumer acceptance of ground beef.

Flow Cytometry Analysis of Circulating Extracellular Vesicle Subtypes from Fresh Peripheral Blood Samples.

Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance.

MAPt: A Rapid Antibiotic Susceptibility Testing for Bacteria in Environmental Samples as a Means for Bioterror Preparedness

Antibiotic resistance of bio-threat agents holds major concerns especially in light of advances in methods for engineering pathogens with antibiotic resistance. Preparedness means for rapid identification and prompt proper medical treatment are of need to contain the event and prevent morbidity and spreading of the disease by properly treating exposed individuals before symptoms appearance. Herein, we describe a novel, rapid, simple, specific, and sensitive method named Micro-Agar-PCR-test (MAPt), which determines antibiotic susceptibility of bio-terror pathogens, directly from environmental samples, with no need for any prior isolation, quantification, or enrichment steps. As proof of concept, we have used this approach to obtain correct therapeutic antibiotic minimal inhibitory concentration (MIC) values for the Tier-1 select agents, Bacillus anthracis, Yersinia pestis, and Francisella tularensis, spiked in various environmental samples recapitulating potential bioterror scenarios. The method demonstrated efficiency for a broad dynamic range of bacterial concentrations, both for fast-growing as well as slow-growing bacteria and most importantly significantly shortening the time for accurate results from days to a few hours. The MAPt allows us to address bioterror agents-contaminated environmental samples, offering rational targeted prophylactic treatment, before the onset of morbidity in exposed individuals. Hence, MAPt is expected to provide data for decision-making personal for treatment regimens before the onset of symptoms in infected individuals.

Rapid detection for low numbers of Escherichia coli O157:H7 by real-time PCR in cabbage using a combination of filtration, short microbial enrichment, and DNA concentration within 4 h

Methodsfocused on conventional foodborne pathogen detection by selective media are cumbersome and require long time for culture enrichment. Several rapid tests have been developed for the detection of Escherichia coli O157:H7 in the food industry. However, the E. coli O157:H7 method takes very long and requires more than one day to achieve the detection threshold using PCR based enrichment in food. We reported a combination of filtration-based accelerated sample concentration methods to detect <9 CFU/25g of E. coli O157:H7 in cabbage. Filtration, short concentration, as well as DNA concentration contribute to making the detection level the target for real-time PCR analysis. The short enrichment was performed after the filtration, which was shorter than the conventional enrichment method. It was concluded that 0–7 CFU/25 g of E. coli O157:H7 was detected within 4 h after 2 h of incubation following filtration. This combination of filter-based enrichment methods is a faster and more efficient way for reaching the desired bacterial concentration compared to the conventional culture enrichment steps, and can significantly reduce the time required to obtain assay results.

Variability in lag duration of Listeria monocytogenes strains in half Fraser enrichment broth after stress affects the detection efficacy using the ISO 11290-1 method

A collection of 23 Listeria monocytogenes strains of clinical and food origin was tested for their ability to recover and grow out in half Fraser enrichment broth following the ISO 11290-1:2017 protocol. Recovery of sub-lethally heat-injured cells in half Fraser broth was compared to reference cells with no stress pre-treatment. The enrichments were followed over time by plate counts and the growth parameters were estimated with the 3-phase model which described the data best. The reference cells without stress pre-treatment showed a short lag duration, which ranged from 1.4 to 2.7 h. However, significant variation in the ability to recover after 60 °C heat stress was observed among the tested strains and resulted in a lag duration from 4.7 to 15.8 h. A subset of strains was also exposed to low-temperature acid stress, and the lag duration showed to be also stress dependent. Scenario analyses and Monte Carlo simulations were carried out using the growth parameters obtained in the enrichments. This demonstrated that when starting with one cell, the detection threshold for efficient transfer of at least one cell to the secondary enrichment step, i.e. 2 log10 CFU/ml, was not reached by 11 of 23 strains tested (48%) after exposure to 60 °C heat stress. Increasing the incubation time from 24 to 26 h and the transfer volume from 0.1 to 1.0 ml can increase the average probability to transfer at least one cell to the secondary enrichment step from 79.9% to 99.0%. When optimizing enrichment procedures, it is crucial to take strain variability into account as this can have a significant impact on the detection efficacy.

A new analytical platform for potential point-of-care testing of circulating tumor cells

It is of significance to detect circulating tumor cells (CTCs) in whole blood using transportable instruments at the point of care to assist evaluating chemotherapeutic efficacy and recurrence risk of cancer patients. However, the current widely used detection methods either require expensive and complex equipments, need complicated enrichment steps, or produce high rates of false positive and/or negative results. Aiming for solving the two critical challenges involved in instrumentation miniaturization and simplification of sample preparation for POCT of CTCs without sacrificing the detection sensitivity and accuracy, this work reports a custom-built, automatic, large field-of-view microscopic CTC cytometer and a novel enrichment strategy based on a synthesized peptide ligand discovered from One-Bead One-Compound library screening. The custom-built microscope has compact size, low weight and efficient cost while still maintaining a detection limit of as low as 5 target objects. The simplified sample preparation utilized a novel peptide LXW7 functionalized to magnetic beads and allows for rapid, highly selective and sensitive detection of CTCs. This analytical platform may fulfill the unmet need for possible point-of-care CTC counting, and provide a new option for early diagnosis of cancers and convenient evaluation of chemotherapeutic efficacy and cancer recurrence.

Point-of-Need Diagnostics for Foodborne Pathogen Screening.

Foodborne illness is a major public health issue that results in millions of global infections annually. The burden of such illness sits mostly with developing countries, as access to advanced laboratory equipment and skilled lab technicians, as well as consistent power sources, is limited and expensive. Current gold standards in foodborne pathogen screening involve labor-intensive sample enrichment steps, pathogen isolation and purification, and costly readout machinery. Overall, time to detection can take multiple days, excluding the time it takes to ship samples to off-site laboratories. Efforts have been made to simplify the workflow of such tests by integrating multiple steps of foodborne pathogen screening procedures into a singular device, as well as implementing more point-of-need readout methods. In this review, we explore recent advancements in developing point-of-need devices for foodborne pathogen screening. We discuss the detection of surface markers, nucleic acids, and metabolic products using both paper-based and microfluidic devices, focusing primarily on developments that have been made between 2015 and mid-2020.

Isotope fractionation of micropollutants during large-volume extraction: heads-up from a critical method evaluation for atrazine, desethylatrazine and 2,6-dichlorobenzamide at low ng/L concentrations in groundwater

ABSTRACT Micropollutants are frequently detected in groundwater. Thus, the question arises whether they are eliminated by natural attenuation so that pesticide degradation would be observed with increasing residence time in groundwater. Conventional analytical approaches rely on parent compound/metabolite ratios. These are difficult to interpret if metabolites are sorbed or further transformed. Compound-specific stable isotope analysis (CSIA) presents an alternative for identifying degradation based on the analysis of natural isotope abundances in pesticides and their changes during degradation. However, CSIA by gas chromatography–isotope ratio mass spectrometry is challenged by the low concentrations (ng/L) of micropollutants in groundwater. Consequently, large amounts of water need to be sampled requiring enrichment and clean-up steps from interfering matrix effects that must not introduce artefacts in measured isotope values. The aim of this study was to evaluate the accuracy of isotope ratio measurements of the frequently detected micropollutants atrazine, desethylatrazine and 2,6-dichlorobenzamide after enrichment from large water volumes (up to 100 L) by solid-phase extraction with consecutive clean-up by HPLC. Associated artefacts of isotope discrimination were found to depend on numerous factors including organic matter content and extraction volume. This emphasizes the necessity to perform a careful method evaluation of sample preparation and sample pre-treatment prior reliable CSIA.