title: Detection of Panton-Valentine Leukocidin DNA from methicillin-resistant Staphylococcus aureus by resistive pulse sensing and loop-mediated isothermal amplification with gold nanoparticles.
abstract: This report describes a novel diagnostic assay for rapid detection of the Panton-Valentine Leukocidin (PVL) toxin of methicillin-resistant Staphylococcus aureus (MRSA) utilizing resistive pulse sensing (RPS), loop-mediated isothermal DNA amplification (LAMP) in combination with gold nanoparticles (AuNPs). The PVL DNA from MRSA was specifically amplified by LAMP using four primers at one temperature (65 °C). The DNA products with biotin were then conjugated to a first AuNP1 (55±2 nm) through biotin-avidin binding. A second AuNP2 (30±1.5 nm) coated with a specific DNA probe hybridized with the LAMP DNA products at the loop region to enhance assay sensitivity and specificity, to generate supra-AuNP1-DNA-AuNP2 assemblies. Scanning electron microscopy confirmed the presence of these supra-assemblies. Using RPS, detection and quantitation of the agglomerated AuNPs were performed by a tunable fluidic nanopore sensor. The results demonstrate that the LAMP-based RPS sensor is sensitive and rapid for detecting the PVL DNA. This technique could achieve a limit of detection (LOD) up to about 500 copies of genomic DNA from the bacteria MRSA MW2 and the detection can be completed within two hours with a straightforward signal-to-readout setup. It is anticipated that this LAMP-based AuNP RPS may become an effective tool for MRSA detection and a potential platform in clinical laboratory to report the presence or absence of other types of infectious agents.
authors.affiliation: Program of Biochemistry, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong. s0907465@cuhk.mail.serv.edu.hk
authors.forename: Alice Kar Lai
authors.initials: AK
authors.lastname: Yang
authors.forename: Haifei
authors.initials: H
authors.lastname: Lu
authors.forename: Shu Yuen
authors.initials: SY
authors.lastname: Wu
authors.forename: Ho Chin
authors.initials: HC
authors.lastname: Kwok
authors.forename: Ho Pui
authors.initials: HP
authors.lastname: Ho
authors.forename: Samuel
authors.initials: S
authors.lastname: Yu
authors.forename: Anthony Ka Lun
authors.initials: AK
authors.lastname: Cheung
authors.forename: Siu Kai
authors.initials: SK
authors.lastname: Kong
mesh_terms: D001427:Bacterial Toxins / Q000032:analysis* / Q000235:genetics*; D001483:Base Sequence; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis*; D005098:Exotoxins / Q000032:analysis* / Q000235:genetics*; D006046:Gold; D007956:Leukocidins / Q000032:analysis* / Q000235:genetics*; D057230:Limit of Detection; D055624:Methicillin-Resistant Staphylococcus aureus / Q000235:genetics*; D008969:Molecular Sequence Data; D053758:Nanoparticles; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: D001427:Bacterial Toxins; D015342:DNA Probes; D004269:DNA, Bacterial; D005098:Exotoxins; D007956:Leukocidins; C078284:Panton-Valentine leukocidin; D006046:Gold