title: Rapid and fully automated bacterial pathogen detection on a centrifugal-microfluidic LabDisk using highly sensitive nested PCR with integrated sample preparation.
abstract: Diagnosis of infectious diseases suffers from long turnaround times for gold standard culture-based identification of bacterial pathogens, therefore impeding timely specific antimicrobial treatment based on laboratory evidence. Rapid molecular diagnostics-based technologies enable detection of microorganisms within hours however cumbersome workflows and complex equipment still prevent their widespread use in the routine clinical microbiology setting. We developed a centrifugal-microfluidic "LabDisk" system for rapid and highly-sensitive pathogen detection on a point-of-care analyser. The unit-use LabDisk with pre-stored reagents features fully automated and integrated DNA extraction, consensus multiplex PCR pre-amplification and geometrically-multiplexed species-specific real-time PCR. Processing merely requires loading of the sample and DNA extraction reagents with minimal hands-on time of approximately 5 min. We demonstrate detection of as few as 3 colony-forming-units (cfu) of Staphylococcus warneri, 200 cfu of Streptococcus agalactiae, 5 cfu of Escherichia coli and 2 cfu of Haemophilus influenzae in a 200 μL serum sample. The turnaround time of the complete analysis from "sample-to-result" was 3 h and 45 min. The LabDisk consequently provides an easy-to-use molecular diagnostic platform for rapid and highly-sensitive detection of bacterial pathogens without requiring major hands-on time and complex laboratory instrumentation.
authors.affiliation: Hahn Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany. Gregor.Czilwik@Hahn-Schickard.de.
authors.forename: G
authors.initials: G
authors.lastname: Czilwik
authors.forename: T
authors.initials: T
authors.lastname: Messinger
authors.forename: O
authors.initials: O
authors.lastname: Strohmeier
authors.forename: S
authors.initials: S
authors.lastname: Wadle
authors.forename: F
authors.initials: F
authors.lastname: von Stetten
authors.forename: N
authors.initials: N
authors.lastname: Paust
authors.forename: G
authors.initials: G
authors.lastname: Roth
authors.forename: R
authors.initials: R
authors.lastname: Zengerle
authors.forename: P
authors.initials: P
authors.lastname: Saarinen
authors.forename: J
authors.initials: J
authors.lastname: Niittymäki
authors.forename: K
authors.initials: K
authors.lastname: McAllister
authors.forename: O
authors.initials: O
authors.lastname: Sheils
authors.forename: J
authors.initials: J
authors.lastname: O'Leary
authors.forename: D
authors.initials: D
authors.lastname: Mark
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics; D015373:Bacterial Typing Techniques / Q000295:instrumentation / Q000379:methods; D002498:Centrifugation / Q000295:instrumentation / Q000379:methods; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D056656:Lab-On-A-Chip Devices; D060885:Multiplex Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods
chemical_list: D004269:DNA, Bacterial