title: Simple and rapid sample preparation system for the molecular detection of antibiotic resistant pathogens in human urine.
abstract: Antibiotic resistance in urinary tract infections (UTIs) can cause significant complications without quick detection and appropriate treatment. We describe a new approach to capture, concentrate and prepare amplification-ready DNA from antibiotic resistant bacteria in human urine samples. Klebsiella pneumoniae NCTC13443 (bla CTX-M-15 positive) spiked into filtered human urine was used as a model system. Bacteria were captured using anion exchange diaethylaminoethyl (DEAE) magnetic microparticles and concentrated 200-fold within ~3.5 min using a custom, valve-less microfluidic chip. Eight samples were processed in parallel, and DNA was released using heat lysis from an integrated resistive heater. The crude cell lysate was used for real time Recombinase Polymerase Amplification (RPA) of the bla CTX-M-15 gene. The end to end processing time was approximately 15 min with a limit of detection of 1000 bacteria in 1 mL urine.
authors: Electronics and Computer Science, University of Southampton, Southampton, SO17 1BJ, UK.|Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, UK.
authors: Martha
authors: M
authors: Valiadi
authors: Electronics and Computer Science, University of Southampton, Southampton, SO17 1BJ, UK.|Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, UK.
authors: Sumit
authors: S
authors: Kalsi
authors: Electronics and Computer Science, University of Southampton, Southampton, SO17 1BJ, UK.|Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, UK.
authors: Isaac G F
authors: IG
authors: Jones
authors: National Infections Service, Public Health England, Porton Down, Salisbury, SP4 0JG, UK.
authors: Carrie
authors: C
authors: Turner
authors: National Infections Service, Public Health England, Porton Down, Salisbury, SP4 0JG, UK.
authors: J Mark
authors: JM
authors: Sutton
authors: Electronics and Computer Science, University of Southampton, Southampton, SO17 1BJ, UK. hm@ecs.soton.ac.uk.|Institute for Life Sciences, University of Southampton, Southampton, SO17 1BJ, UK. hm@ecs.soton.ac.uk.
authors: Hywel
authors: H
authors: Morgan
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005260:Female; D006801:Humans; D007710:Klebsiella Infections / Q000235:genetics / Q000652:urine; D007711:Klebsiella pneumoniae / Q000235:genetics*; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D014552:Urinary Tract Infections / Q000235:genetics / Q000652:urine; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D001618:beta-Lactamases
keywords: Antibiotic resistant bacteria; Magnetic beads; Microfluidics; Recombinase polymerase amplification; Sample preparation; Urine