Enrichment Step Research Articles

PM 7/20 (3) Erwinia amylovora

PM 7/20 (3) <i>Erwinia amylovora</i>

Group B Streptococcal Colonization among Pregnant Women and Neonates in a Tertiary Care Hospital in South India.

ObjectivesTo assess the prevalence of maternal and neonatal group B Streptococcal colonization, incidence of neonatal systemic illness, and antibiotic sensitivity of isolates.MethodsThis prospective cohort study was conducted in a South Indian tertiary care hospital. Rectovaginal swabs from pregnant mothers at 360/7–376/7 wk gestation and throat and rectal swabs from their neonates at 48 h of age were collected. Presence of group B Streptococcus (GBS) was identified by broth enrichment step, and traditional microbiologic methods and antibiotic sensitivity of isolates was noted. All mothers received intrapartum antibiotic prophylaxis (IAP). Culture-positive sepsis, clinical sepsis, pneumonia, meningitis, and urinary tract infection were defined as neonatal systemic illness. Neonates of colonized mothers were followed at 3 mo for late-onset sepsis.ResultsOf the 310 mothers, 40 were GBS colonized (prevalence: 12.9%; 95% CI 9.2%, 17.6%). None of the neonates were colonized. Maternal GBS colonization was significantly associated with premature rupture of membrane (RR - 2.93, 95% CI - 1.66–5.16) and neonatal systemic illness (RR - 2.78, 95% CI - 1.39–5.54). Positive correlation was noted between duration of IAP ≤ 4 h and neonatal illness and between maternal GBS colonization and Apgar at 1 min ≤ 4. Clindamycin resistance was noted in 20%. All neonates remained well at 3 mo follow-up.ConclusionHigh maternal colonization alerts the need for GBS screening in India. Clindamycin resistance among GBS isolates questions its effectiveness as alternative therapy in penicillin allergy.

A Rapid, Highly Sensitive and Open-Access SARS-CoV-2 Detection Assay for Laboratory and Home Testing.

RT-qPCR-based diagnostic tests play important roles in combating virus-caused pandemics such as Covid-19. However, their dependence on sophisticated equipment and the associated costs often limits their widespread use. Loop-mediated isothermal amplification after reverse transcription (RT-LAMP) is an alternative nucleic acid detection method that overcomes these limitations. Here, we present a rapid, robust, and sensitive RT-LAMP-based SARS-CoV-2 detection assay. Our 40-min procedure bypasses the RNA isolation step, is insensitive to carryover contamination, and uses a colorimetric readout that enables robust SARS-CoV-2 detection from various sample types. Based on this assay, we have increased sensitivity and scalability by adding a nucleic acid enrichment step (Bead-LAMP), developed a version for home testing (HomeDip-LAMP), and identified open-source RT-LAMP enzymes that can be produced in any molecular biology laboratory. On a dedicated website, rtlamp.org (DOI: 10.5281/zenodo.6033689), we provide detailed protocols and videos. Our optimized, general-purpose RT-LAMP assay is an important step toward population-scale SARS-CoV-2 testing.

Nanoparticle Biomolecular Corona-Based Enrichment of Plasma Glycoproteins for N-Glycan Profiling and Application in Biomarker Discovery.

Biomolecular corona formation has emerged as a recurring and important phenomenon in nanomedicine that has been investigated for potential applications in disease diagnosis. In this study, we have combined the “personalized protein corona” with the N-glycosylation profiling that has recently gained considerable interest in human plasma biomarker discovery as a powerful early warning diagnostic and patient stratification tool. We envisioned that the protein corona formation could be exploited as an enrichment step that is critically important in both proteomic and proteoglycomic workflows. By using silica nanoparticles, plasma fibrinogen was enriched to a level in which its proteomic and glycomic “fingerprints” could be traced with confidence. Despite being a more simplified glycan profile compared to full plasma, the corona glycan profile revealed a fibrinogen-derived glycan peak that was found to potentially distinguish lung cancer patients from controls in a pilot study.

Validation Study for PathogenDx EnviroX-F Assay for the Detection of Listeria, L. monocytogenes, and Salmonella in Environmental Surface Samples: AOAC Performance Tested MethodSM 092001.

The PathogenDx EnviroX-F uses microarray technology to simultaneously detect Listeria species, Listeria monocytogenes, and Salmonella species from environmental samples without the need for enrichment. The validation study included a matrix study of four matrices (stainless steel, plastic, rubber, and sealed concrete) comparing the PathogenDx EnviroX-F assay to the US Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 10, "Detection of L. monocytogenes in Foods and Environmental Samples, and Enumeration of L. monocytogenes in Foods" (revised March 2017), and FDA BAM Chapter 5,"Salmonella" (revised February 2020). Other required Performance Tested MethodSM parameters evaluated included: inclusivity and exclusivity, robustness, instrument variation, and product consistency and stability. The PathogenDx EnviroX-F assay was evaluated with 30 unpaired replicate surface areas for each environmental surface. The candidate method was evaluated without an enrichment step. In the inclusivity and exclusivity study 50 out of 50 Listeria isolates were detected, 50 out of 50 L. monocytogenes strains were detected, 108 out of 108 Salmonella strains were detected, and 95 out of 95 exclusivity strains were correctly excluded. In the matrix study, the PathogenDx EnviroX-F assay showed no significant differences between confirmed results or between candidate and reference method results for 4" × 4" environmental surface areas for each matrix. The PathogenDx EnviroX-F assay is an effective method for the qualitative detection of Listeria spp., L. monocytogenes, and Salmonella spp. from environmental surface swabs. The PathogenDx EnviroX-F assay will be the first PTM-approved multiplex assay for Listeria spp., L. monocytogenes, and Salmonella without the need for an environment step.

Phage Display-Derived Monoclonal Antibodies Against Internalins A and B Allow Specific Detection of Listeria monocytogenes.

Listeria monocytogenes is the causative agent of listeriosis, a highly lethal disease initiated after the ingestion of Listeria-contaminated food. This species comprises different serovars, from which 4b, 1/2a, and 1/2b cause most of the infections. Among the different proteins involved in pathogenesis, the internalins A (InlA) and B (InlB) are the best characterized, since they play a major role in the enterocyte entry of Listeria cells during early infection. Due to their covalent attachment to the cell wall and location on the bacterial surface, along with their exclusive presence in the pathogenic L. monocytogenes, these proteins are also used as detection targets for this species. Even though huge advancements were achieved in the enrichment steps for subsequent Listeria detection, few studies have focused on the improvement of the antibodies for immunodetection. In the present study, recombinant InlA and InlB produced in Escherichia coli were used as targets to generate antibodies via phage display using the human naïve antibody libraries HAL9 and HAL10. A set of five recombinant antibodies (four against InlA, and one against InlB) were produced in scFv-Fc format and tested in indirect ELISA against a panel of 19 Listeria strains (17 species; including the three main serovars of L. monocytogenes) and 16 non-Listeria species. All five antibodies were able to recognize L. monocytogenes with 100% sensitivity (CI 29.24–100.0) and specificity (CI 88.78–100.0) in all three analyzed antibody concentrations. These findings show that phage display-derived antibodies can improve the biological tools to develop better immunodiagnostics for L. monocytogenes.

Impact of wastewater treatment plants on microbiological contamination for evaluating the risks of wastewater reuse

BackgroundWastewater reuse represents a promising alternative source of water supply considering the water scarcity related to climate change. However, if not adequately treated, wastewater represents a source of microbiological health risk. The purpose of this work was to investigate the role of wastewater treatment on microbiological contamination by evaluating the possible risks associated with wastewater effluent reuse, taking into account new EU legislation (2020/741) on minimum requirements for water reuse. E. coli that produce Shiga toxins (STEC) and thermotolerant Campylobacter were monitored using an enrichment step associated with specific PCR, while Salmonella spp. and Legionella were detected with both cultural and molecular methods (PCR and q-PCR, respectively). Culture method was also used for the enumeration of different microbial indicators. The bacteria detection was compared in different wastewater plants with membrane bioreactor (MBR) system or with disinfection step with chlorine dioxide (ClO2). Moreover a comparison between molecular and culture methods was discussed.ResultsThe results obtained showed good abatement performance for WWTPs equipped with MBR. The high concentrations of E. coli (range between 0.88 and 5.21 Log MPN/100 mL) and contamination by Salmonella spp. in effluent disinfected with ClO2 (17% of samples) showed the need to control the quality of this effluent. In addition, despite the absence of Legionella spp. with the culture method required by EU regulation, high concentrations of Legionella spp. (range between 2 and 7 log GU/L) and the presence of Leg. pneumophila with qPCR (15% of samples) highlight the need to carry out further investigations for reuse associated with aerosol formation (e.g. spray irrigation in agriculture).ConclusionsThe results obtained underline that the MBR technology can be suitable for wastewater reuse applications allowing to achieve the requirement proposed by the new European legislation. More attention should be given to wastewater reuse of effluents treated with ClO2. The use of the molecular methods for pathogens detection in wastewater could allow a more precautionary risks estimation associated with reuse. The overall results highlight that an evaluation of the effectiveness of the wastewater treatments is required for the prevention of a possible risk to public health.

A Tyrosine Phosphoproteome Analysis Approach Enabled by Selective Dephosphorylation with Protein Tyrosine Phosphatase.

Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, low sensitivity, poor reproducibility, and high cost are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphotyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of the PTPs including PTP1B, TCPTP, and SHP1 can efficiently and selectively dephosphorylate phosphotyrosine peptides. We then designed a workflow by combining two Ti4+-IMAC-based phosphopeptide enrichment steps with PTP-catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was first validated by selective detection of phosphotyrosine peptides from semicomplex samples and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 μg of cell lysate. The tyrosine phosphosites on the majority of these peptides could be unambiguously determined for over 70% of them possessing only one tyrosine residue. It was also found that the tyrosine sites identified by this method were highly complementary to those identified by the SH2 superbinder-based method. Therefore, the combination of Ti4+-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.

Independent Study for the Detectx Combined Assay for the Detection of Aspergillus, Salmonella, and STEC (stx1 and/or 2) in Dried Cannabis Flower and Dried Hemp Flower: Level 3 Modification Study 012201.

The PathogenDx family of assays uses microarray technology to simultaneously detect the presence of bacterial and fungal pathogens in food products, environmental surfaces, and cannabis products. The Detectx Combined assay was validated for the detection of Aspergillus, (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus), Salmonella, and a broad range of STEC (stx1 and/or 2) species. The validation consisted of two matrix studies in dried hemp flower and dried cannabis flower (>0.3% delta-9 tetrahydrocannabinol) flower, product consistency, stability, robustness, and inclusivity and exclusivity for two targets: Aspergillus and STEC. The PathogenDx Detectx Combined assay was evaluated with 30 replicates in each matrix and confirmed according to the instructions outlined in this study. Results of the validation study met the requirements of AOAC Standard Method Performance Requirement (SMPR®) 2020.002 and 2020.012. In the inclusivity and exclusivity study, all target isolates (Aspergillus and STEC) were correctly detected. For the exclusivity study, 26 out of 30 Aspergillus and 30 out of 30 STEC non-target strains were correctly excluded. In the matrix study, the PathogenDx Detectx Combined assay showed no significant statistical differences between confirmed results for dried hemp and cannabis flower. Robustness testing indicated that small changes to the method parameters did not impact the performance of the assay. Stability and consistency studies verified that the assay's shelf-life claims were appropriate, and manufacturing of the assay was consistent. The validation study indicated that the PathogenDx DetectX Combined assay was successful in detection of the new target analytes (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus and STEC containing stx1 and/or 2) and could successfully recover these organisms and Salmonella from dried hemp flower and dried cannabis flower (>0.3% delta-9 tetrahydrocannabinol). The PathogenDx DetectX Combined Assay will be the first PTM approved multiplex assay for Aspergillus, E. coli and Salmonella that does not require an enrichment step.

The dark proteome: translation from noncanonical open reading frames.

Omics-based technologies have revolutionized our understanding of the coding potential of the genome. In particular, these studies revealed widespread unannotated open reading frames (ORFs) throughout genomes and that these regions have the potential to encode novel functional (micro-)proteins and/or hold regulatory roles. However, despite their genomic prevalence, relatively few of these noncanonical ORFs have been functionally characterized, likely in part due to their under-recognition by the broader scientific community. The few that have been investigated in detail have demonstrated their essentiality in critical and divergent biological processes. As such, here we aim to discuss recent advances in understanding the diversity of noncanonical ORFs and their roles, as well as detail biologically important examples within the context of the mammalian genome.

MRNA-mediated delivery of gene editing tools to human primary muscle stem cells

Muscular dystrophies are approximately 50 devastating, untreatable monogenic diseases leading to progressive muscle degeneration and atrophy. Gene correction of transplantable cells using CRISPR/Cas9-based tools is a realistic scenario for autologous cell replacement therapies to restore organ function in many genetic disorders. However, muscle stem cells have so far lagged behind due to the absence of methods to isolate and propagate them and their susceptibility to extensive ex vivo manipulations. Here, we show that mRNA-based delivery of SpCas9 and an adenine base editor results in up to >90% efficient genome editing in human muscle stem cells from many donors regardless of age and gender and without any enrichment step. Using NCAM1 as an endogenous reporter locus expressed by all muscle stem cells and whose knockout does not affect cell fitness, we show that cells edited with mRNA fully retain their myogenic marker signature, proliferation capacity, and functional attributes. Moreover, mRNA-based delivery of a base editor led to the highly efficient repair of a muscular dystrophy-causing SGCA mutation in a single selection-free step. In summary, our work establishes mRNA-mediated delivery of CRISPR/Cas9-based tools as a promising and universal approach for taking gene edited muscle stem cells into clinical application to treat muscle disease.

Cold Enrichment Methods for the Detection of Foodborne Yersiniosis: Friend or Foe?

Yersinia enterocolitica and Y. pseudotuberculosis are important causes of enteric illness worldwide. Rapid response to suspected foodborne outbreaks is hampered by the widespread use of cold enrichment methods that require incubation periods of 10–21 days. Although these species grow faster at elevated temperatures, part of the rationale for cold enrichment is that a key pathogenicity marker (pYV virulence plasmid) is said to be lost at elevated temperatures. Experimental data on this claim seems scarce. We previously described an approach involving an enrichment step at 37 °C for Yersinia detection, applied this approach to additional strains, and examined the presence of plasmids in reisolates, as well as those recovered in our original study. Plasmids were recovered from every reisolate examined; the presence of marker genes yadA and virF denoted the virulence plasmid in 10 of the 11 strains examined. Use of an enrichment step at 37 °C does not appear to promote loss of the pYV or other plasmids harboured by foodborne pathogenic Y. enterocolitica and Y. pseudotuberculosis; wider adoption of this approach may assist the development of more rapid detection methods.

Comparable Results of Helicobacter pylori Antibiotic Resistance Testing of Stools vs Gastric Biopsies Using Next-Generation Sequencing

Comparable Results of Helicobacter pylori Antibiotic Resistance Testing of Stools vs Gastric Biopsies Using Next-Generation Sequencing

Infectious Endometritis in Mares: Microbiological Findings in Field Samples.

Endometritis is a major cause of infertility and subfertility in the mare. Early diagnosis and identification of the pathogens involved in infectious endometritis are crucial to initiate correct treatments in time, in order to optimize fertility and reduce the risk of bacterial resistance development. In this retrospective study (from 2014 to 2018), 394 samples (uterine swabs and lavages) obtained from mares before breeding, regardless of clinical history of endometritis were analyzed. Our bacteriological procedure included the subculturing from the enrichment in Brain Heart Infusion Broth of the samples resulted negative after direct smearing. A total of 386 microorganisms were isolated from 230 positive samples (58%). At least one microorganism was isolated from 33% of the samples after direct smearing and from another 25% after enrichment. The results, obtained from both direct smearing and enrichment, also show a significative difference between positive uterine lavages (80%) and swabs (53%). The most frequently isolated bacteria were α-haemolytic Streptococcus (27%), Escherichia coli (27%), β-haemolytic Streptococcus (26.1%) and Staphylococcus spp. (19.1%). In monoculture, the most common isolated microorganisms were α-haemolytic Streptococcus (13%), Staphylococcus spp. (12.2%), β-haemolytic Streptococcus (11.4%) and Escherichia coli (9.8%). Focusing on the samples with a pure culture, Gram-negative bacteria grew preferably after direct smearing, while Gram-positive after enrichment. In conclusion, the present study shows that uterine lavage with high volume of fluid statistically significantly increased the sensitivity of the bacteriological examination and highlights the key role of the enrichment step in the routine bacteriological laboratory procedure by increasing the isolation rate.

Prognosis and correlation with Ca19.9 of circulating tumor cells (CTCs) in locally advanced and metastatic pancreatic cancer.

586 Background: In recent years CTCs have been extensively studied in different neoplasms. However, in PC CTCs are still emerging as a potential prognostic tool and the significance in different stages of the disease and the correlation with tumor markers are poorly understood. Methods: We conducted an observational, prospective, cohort study in two Spanish centers. Isolation was carried out with Isoflux technology (Fluxion Biosciences, Inc. San Francisco, CA, USA), that is combining cell separation by immunomagnetic particles (positive selection) with a microfluidic system. Before isolation, it was performed a first cell enrichment step using the density gradient cell separation technique (Ficoll). Four different markers were used, including two mesenchymal markers, EpCAM and EGFR (EMT Enrichment Kit:EpCAM/EGFR/Mesenchymal). The count of CTCs was done in a confocal microscope using the iMSRC (intelligent Matrix Screen Remote Control). A high throughput system was performed over the entire area, using a 20x objective, increasing to a 60x objective. Primary endpoint was the prognostic significance of CTCs, correlating the number of CTCs at diagnosis with overall survival. Secondary endpoints: Correlation between number of CTCs with Ca19.9/CEA values, and the difference in the median value of CTCs in metastatic compared to locally advanced disease. CTCs were analyzed at day 0 (CTC-0), just before first chemotherapy cycle was administered. Results: Thirty-four patients were analyzed. Clinical characteristics: Table 1. Median follow up 12.0 months. Median value of CTC-0 was 347 (range 104 - 3273) for metastatic and 436 (81 - 1082) for locally advanced patients (p = 0.942). Correlation coefficient for Ca 19.9 0.006 (p = 0.97); CEA correlation coefficient 0.191 (p = 0.303). For a cutoff of 500 CTCs, we found an AUC of 0.8 for mortality. Kaplan-Meier analysis showed an estimated median overall survival for patients with ≥500 CTCs of 8.6 months (95% confident interval [95% CI] 5.3 – 12) vs not reached for patients with &lt; 500 CTCs (p = 0.007). HR for mortality 3.3 (95% CI 1.3 – 8.4; p = 0.011) for patients with ≥ 500 CTCs. Conclusions: Our data suggest 500 CTCs might be a potential cutoff for prognostic assessment. The absence of differences of CTC-0 between metastatic and locally advanced patients supports the idea of a systemic disease irrespective of the presence of metastases at diagnosis. CTC-0 seems not to correlate with tumor markers at diagnosis.[Table: see text]

Simultaneous Detection of Four Main Foodborne Pathogens in Ready-to-Eat Food by Using a Simple and Rapid Multiplex PCR (mPCR) Assay.

The increasing consumption of organic or ready-to-eat food may cause serious foodborne disease outbreaks. Developing microbiological culture for detection of food-borne pathogens is time-consuming, expensive, and laborious. Thus, alternative methods such as polymerase chain reaction (PCR) are usually employed for outbreaks investigation. In this work, we aimed to develop a rapid and simple protocol for the simultaneous detection of Escherichia coli (E coli), Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus (S. aureus) and Salmonella enterica (S. enterica), by the combination of an enrichment step in a single culture broth and a multiplex PCR (mPCR) assay. The effectiveness of several enrichment media was assessed by culture and PCR. Buffered peptone water (BPW) was selected as the optimum one. Then, mPCR conditions were optimized and applied both to pure co-cultures and artificially inoculated food samples (organic lettuce and minced meat). In the culture medium inoculated at 100 CFU/mL, mPCR was able to detect the four microorganisms. When performed on artificially food samples, the mPCR assy was able to detect E. coli, S. enterica, and L. monocytogenes. In conclusion, BPW broth can effectively support the simultaneous growth of E. coli, S. aureus, L. monocytogenes, and S. enterica and could be, thus, used prior to a mPCR detection assay in ready-to-eat food, thereby considerably reducing the time, efforts and costs of analyzes.

Real-time and visual detection of viable Salmonella in milk by a competitive annealing mediated isothermal amplification (CAMP) combined with propidium monoazide (PMA).

Salmonella is a common pathogen in raw milk. The conventional isothermal amplification assay cannot distinguish viable bacteria from dead bacteria, which may cause false positive results or overestimate the number of viable bacteria. This study proposed a competitive annealing mediated isothermal amplification (CAMP) combined with propidium monoazide (PMA) for real-time and visual detection of viable Salmonella in milk. Based on the invA gene, specific CAMP primers were constructed. Moreover, the primers for accelerating the CAMP reaction were also designed and added to the reaction system. The real-time PMA-CAMP showed a LOD of 102 CFU mL-1 for quantitative detection of viable Salmonella in spiked milk samples, and the recovery rate was 80-106%. The visual PMA-CAMP can be performed under isothermal conditions using a portable dry bath, and the positive results can be directly observed by the colorimetric change from violet to sky blue. Without enrichment step, viable Salmonella could be detected with a LOD of 102 CFU mL-1. With enrichment step, even if the initial inoculation level is 1 CFU mL-1, the visual PMA-CAMP could still detect the presence of viable Salmonella in milk samples. Therefore, the developed PMA-CAMP assays are suitable for the monitoring of viable Salmonella contamination in milk.

Improving Arabic Cognitive Distortion Classification in Twitter using BERTopic

Social media platforms allow users to share thoughts, experiences, and beliefs. These platforms represent a rich resource for natural language processing techniques to make inferences in the context of cognitive psychology. Some inaccurate and biased thinking patterns are defined as cognitive distortions. Detecting these distortions helps users restructure how to perceive thoughts in a healthier way. This paper proposed a machine learning-based approach to improve cognitive distortions’ classi-fication of the Arabic content over Twitter. One of the challenges that face this task is the text shortness, which results in a sparsity of co-occurrence patterns and a lack of context information (semantic features). The proposed approach enriches text rep-resentation by defining the latent topics within tweets. Although classification is a supervised learning concept, the enrichment step uses unsupervised learning. The proposed algorithm utilizes a transformer-based topic modeling (BERTopic). It employs two types of document representations and performs averaging and concatenation to produce contextual topic embeddings. A comparative analysis of F1-score, precision, recall, and accuracy is presented. The experimental results demonstrate that our enriched representation outperformed the baseline models by different rates. These encouraging results suggest that using latent topic distribution, obtained from the BERTopic technique, can improve the classifier’s ability to distinguish between different CD categories.

3149 – COMPARISON OF PRE-ENRICHMENT STRATEGIES FOR SORTING OF MOUSE LIN-CKIT+SCA1+ CELLS

The short life span of blood cells requires hematopoiesis to be a continuous ongoing process. In adult mammals, blood production takes place in the bone marrow. This tissue is home to hematopoietic stem cells (HSCs) that possess the ability to both self-renew and differentiate into mature blood cells. HSCs are widely studied in cancer research, as they are the source of various hematological malignancies. Such cancers develop in response to a progressive accumulation of mutations that confer a selective growth advantage to HSCs. In the case of leukemia, the mutations will lead to a differentiation blockade, causing dysfunctional blood cells to accumulate. Mouse models are the system of choice to study blood cancers given the complexity of the hematopoietic system. Thus, in order to study HSCs, we need to be able to isolate them from mice. Several HSC subpopulations have been defined, however, the most widely studied are the Lin-cKit+Sca1+ (LKS) cells. The best way to isolate LKS cells is by FACS sorting. Yet, the frequency of LKS cells in the bone marrow being very low (< 0.1%), a pre-enrichment step is necessary to decrease the sorting time. With the aim of identifying the most suitable pre-enrichment strategy, we tested several magnetic kits and compared them based on enrichment efficiency and final LKS yield. Four approaches were tested: lineage depletion (Lin-), cKit-enrichment (cKit+), Sca1-enrichment (Sca1+) and a combination of lineage depletion followed by Sca1-enrichment (Lin- Sca1+). Our results show that the best strategy are lineage depletion and Sca1-enrichment. Combining these two methods highly increased the LKS enrichment, however, the final LKS yield was low due to increased cell loss from performing a second enrichment step.

Biofunctionalized Magnetic Nanoparticles with Multiplex Touchdown PCR for Simultaneous and Rapid Detection/Identification of Campylobacter jejuni and Campylobacter coli

The simple, accurate, and rapid detection of foodborne pathogens is essential for public health. Development of an immunomagnetic separation (IMS) multiplex touchdown PCR (IMS–multiplex TD–PCR) assay for simultaneous detection and distinguishing of C. jejuni and C. coli is reported herein. Polyclonal antibody (pAb) against multiepitope antigen (MEA) was conjugated to ferromagnetic nanoparticles (FMNs) to produce anti‐MEA FMNs. Optimal anti‐MEA FMNs loading yielded 26.7 μg of immunoglobulin G (IgG) molecules per mg of FMNs with an average size of 72 ± 9 nm, corresponding to an 83% rate of pAb conjugation. Anti‐MEA FMNs (20 μg) for IMS captured culturable C. jejuni cells at 3.54 × 102 colony‐forming unit (CFU)/mL in pure culture, while higher amounts (40 and 60 μg) reduced the recovery. The scanning electron microscope (SEM) analysis revealed the attachment of anti‐MEA FMNs to target bacteria, forming aggregated cells and magnetic nanoparticles in ellipse‐like shapes. The subsequent multiplex TD–PCR assay simultaneously detected and distinguished C. jejuni and C. coli at 104 CFU/mL in mixed culture and at 103 CFU/mL for each individual species. Furthermore, the limit of detection (LOD) of the IMS–multiplex TD–PCR assay was 104 CFU/g in spiked chicken breast samples. Specificity was 100% for both C. jejuni and C. coli as none of the amplicons were detected in control samples where Campylobacter was absent. This assay is able to detect and distinguish C. jejuni and C. coli simultaneously and is simple, accurate, and rapid with a time to result of 4 h without an enrichment step, making it a promising approach for rapid and culture‐free detection of Campylobacter in chicken products.