Multiplex sorting of foodborne pathogens by on-chip free-flow magnetophoresis

This study reports multiplex sorting of Salmonella typhimurium and Escherichia coli 0157, from broth cultures and from pathogen-spiked skinned chicken breast enrichment broths by employing microfluidic free-flow magnetophoresis. Magnetic beads of different sizes and magnetite content, namely Dynabeads anti-salmonella and Hyglos-Streptavidin beads together with the corresponding pathogen-specific biotinylated recombinant phages, were utilised as affinity solid phases for the capture and concentration of viable S. typhimurium and E. coli 0157. Following optimisation, the protocol was used to demonstrate continuous magnetophoretic sorting of the two pathogen-bound magnetic bead populations from mixed cultures and from pathogen-spiked chicken pre-enrichment broths under the influence of a Halbach magnet array. For example, in the la tter case, a pure population of S. typhimurium-bound Dynabeads (72% recovery) was sorted from a 100 μL mixture containing E. coli 0157-bound Hyglos beads (67% recovery) within 1.2 min in the presence of 0.1% Tween 20. This proof-of-principle study demonstrates how more than one pathogen type can be simultaneously isolated/enriched from a single food pre-enrichment broth (e.g. Universal food enrichment broth)

Paper-based analytical devices for colorimetric detection of: S. aureus and E. coli and their antibiotic resistant strains in milk

Animal derived milk which is an important part of human diet due to its high nutritional value not only supports humans but also presents a growth environment for pathogenic bacteria. Milk may become contaminated with bacteria through udder infections or through contact within the dairy farm environment. Infections are treated with antibiotics, with β-lactams most commonly used in veterinary medicine. However, their frequent use leads to the emergence of β-lactam resistant bacterial strains, which causes difficulties in the treatment of infections in both humans and animals. Detection of pathogens as well as their antibiotic sensitivity is a pre-requisite for successful treatment and this is generally achieved with laboratory-based techniques such as growth inhibition assays, enzyme-linked immunosorbent assays (ELISA) or polymerase chain reactions (PCRs), which are unavailable in resource-limited settings. Here, we investigated paper-based analytical devices (μPADs) for the presumptive detection of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) and their antibiotic resistant bacterial strains in milk samples. The μPADs were fabricated on filter paper using wax printing, and then impregnated with chromogenic substrates, which reacted with bacterial enzymes to form coloured products. Limits of detection of S. aureus and E. coli and their antibiotic resistant strains in milk samples were found to be 106 cfu mL-1. Enrichment of milk samples in a selective medium for 12 h enabled detection as low as 10 cfu mL-1. The paper devices tested on a set of 640 milk samples collected from dairy animals in Pakistan demonstrated more than 90% sensitivity and 100% selectivity compared to PCR, showing promise to provide inexpensive and portable diagnostic solutions for the detection of pathogenic bacteria in resource-limited settings

On-chip acoustophoretic isolation of microflora including S. typhimurium from raw chicken, beef and blood samples

Pathogen analysis in food samples routinely involves lengthy growth-based pre-enrichment and selective enrichment of food matrices to increase the ratio of pathogen to background flora. Similarly, for blood culture analysis, pathogens must be isolated and enriched from large excess of blood cells to allow further analysis. Conventional techniques of centrifugation and filtration are cumbersome, suffer from low sample throughput, are not readily amenable to automation and carry a risk of damaging biological samples. We report on-chip acoustophoresis as a pre-analytical technique for the resolution of total microbial flora from food and blood samples. The resulting ‘clarified’ sample is expected to increase the performance of downstream systems for the specific detection of the pathogens. A microfluidic chip with three inlets, a central separation channel and three outlets was utilized. Samples were introduced through the side inlets, buffer through the central inlet. Upon ultrasound actuation, large debris particles (10–100 μm) from meat samples were continuously partitioned into the central buffer channel, leaving the “clarified” outer sample streams containing both, the pathogenic cells and the background flora (ca. 1 μm) to be collected over a 30 min operation cycle before further analysis. The system was successfully tested with Salmonella typhimurium-spiked (ca. 103 CFU mL⁻¹) samples of chicken and minced beef, demonstrating a high level of the pathogen recovery (60–90%). When applied to S. typhimurium contaminated blood samples (107 CFU mL⁻¹), acoustophoresis resulted in a high depletion of the red blood cells (99.8%) which partitioned in the buffer stream, whilst sufficient numbers of the viable S. typhimurium remained in the outer channels for further analysis. These results indicate that the technology may provide a generic approach for pre-analytical sample preparation prior to integrated and automated downstream detection of bacterial pathogens

A lab-on-a-chip platform for integrated extraction and detection of SARS-CoV-2 RNA in resource-limited settings

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the unprecedented global pandemic of coronavirus disease-2019 (COVID-19). Efforts are needed to develop rapid and accurate diagnostic tools for extensive testing, allowing for effective containment of the infection via timely identification and isolation of SARS-CoV-2 carriers. Current gold standard nucleic acid tests require many separate steps that need trained personnel to operate specialist instrumentation in laboratory environments, hampering turnaround time and test accessibility, especially in low-resource settings. We devised an integrated on-chip platform coupling RNA extraction based on immiscible filtration assisted by surface tension (IFAST), with RNA amplification and detection via colorimetric reverse-transcription loop mediated isothermal amplification (RT-LAMP), using two sets of primers targeting open reading frame 1a (ORF1a) and nucleoprotein (N) genes of SARS-CoV-2. Results were identified visually, with a colour change from pink to yellow indicating positive amplification, and further confirmed by DNA gel electrophoresis. The specificity of the assay was tested against HCoV-OC43 and H1N1 RNAs. The assay based on use of gene N primers was 100% specific to SARS-CoV-2 with no cross-reactivity to HCoV-OC43 nor H1N1. Proof-of-concept studies on water and artificial sputum containing genomic SARS-CoV-2 RNA showed our IFAST RT-LAMP device to be capable of extracting and detecting 470 SARS-CoV-2 copies mL−1 within 1 h (from sample-in to answer-out). IFAST RT-LAMP is a simple-to-use, integrated, rapid and accurate COVID-19 diagnostic platform, which could provide an attractive means for extensive screening of SARS-CoV-2 infections at point-of-care, especially in resource-constrained settings

Rapid Detection of Group B Streptococcus (GBS) from artificial urine samples based on IFAST and ATP Bioluminescence Assay: from development to practical challenges during protocol testing in Kenya

© The Royal Society of Chemistry 2019. We report the rapid detection (20 min) of Streptococcus agalactiae, Group B Streptococcus (GBS) employing on-chip magnetic isolation of GBS based on immiscible filtration assisted by surface tension (IFAST), followed by detection of the isolated GBS using an adenosine triphosphate (ATP) bioluminescence assay. Up to 80% GBS cells were isolated from spiked artificial urine samples with linear responses of bioluminescence signals from isolated cells at 2.3 × 102-9.1 × 105 CFU mL-1, demonstrating great promise for point-of-care detection of pathogenic bacteria in screening urine samples from pregnant women. Practical challenges during initial testing of the developed protocol with urine samples in Kenya are also described