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 (blaCTX-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 blaCTX-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.Electronic supplementary materialThe online version of this article (doi:10.1007/s10544-016-0031-9) contains supplementary material, which is available to authorized users.

Differential DNA accessibility to polymerase enables 30-minute phenotypic β-lactam antibiotic susceptibility testing of carbapenem-resistant Enterobacteriaceae

“…Additionally, the presence of cells not lysed during LAMP is evidenced by the shorter TTPs seen when an aliquot of the same sample is lysed using an extraction buffer prior to performing LAMP (explained in more detail subsequently). These differences confirm that choice of amplification chemistry is critical to the success of pol-aAST and are consistent with previous work evaluating thermal lysis [65].…”

Recombinase polymerase amplification: Basics, applications and recent advances

“…Microfluidic devices incorporating a one-step digital plasma separation platform with autonomous parallel plasma separation and sample compartmentalisation for digital nucleic acid amplification have been developed for use with RPA [20]. A valveless microfluidic chip to pre-concentrate bacteria in urine using anion exchange magnetic beads prior to heat lysis has also been reported [21], as well as an isotachophoresis chip for the extraction of DNA from Listeria monocytogenes in blood samples prior to RPA [22].…”

Microfluidic and Paper-Based Devices for Disease Detection and Diagnostic Research

“…Alternate detection methods rely on the use of electronic sensors [ 47 ], chips patterned with detection molecules [ 44 , 45 , 46 , 47 , 48 ], devices incorporating nanomaterials [ 44 , 45 ], and imaging platforms [ 46 ]. These devices differ greatly in their setup, but all share a similar construction of detection chemicals patterned onto portable metal chips.…”

“…Many of these microfluidic sensors measure changes in conductance or resistance due to changes in the concentration of biomolecule deposited onto the sensor [ 44 , 45 , 47 , 48 ]. Methods to detect these molecules include the use of carbon nanobiosensors (e.g., nanotubes and nanoparticles) [ 44 , 45 ], antibodies [ 46 , 47 ], and aptamers [ 48 ]. A device developed by Matta et al used nanotubes to detect cardiac biomarkers myoglobin, cTn I, and CK-MB by imbedding detection nanotubes within SU-8 on a metallic chip to measure conductance by biomolecule accumulation on the nanotube [ 44 ].…”