title: A miniaturized and integrated gel post platform for multiparameter PCR detection of herpes simplex viruses from raw genital swabs.
abstract: Herpes simplex virus (HSV) is one of the most prevalent viruses, with acute and recurrent infections in humans. The current gold standard for the diagnosis of HSV is viral culture which takes 2-14 days and has low sensitivity. In contrast, DNA amplification by polymerase chain reaction (PCR) can be performed within 1-2 h. We here describe a multiparameter PCR assay to simultaneously detect HSV-1 and HSV-2 DNA templates, together with integrated positive and negative controls, with product detection by melting curve analysis (MCA), in an array of semi-solid polyacrylamide gel posts. Each gel post is 0.67 μL in volume, and polymerized with all the components required for PCR. Both PCR and MCA can currently be performed in one hour and 20 min. Unprocessed genital swabs collected in universal transport medium were directly added to the reagents before or after polymerization, diffusing from atop the gel posts. The gel post platform detects HSV templates in as little as 2.5 nL of raw sample. In this study, 45 genital swab specimens were tested blindly as a preliminary validation of this platform. The concordance of PCR on gel posts with conventional PCR was 91%. The primer sequestration method introduced here (wherein different primers are placed in different sets of posts) enables the simultaneous detection of multiple pathogens for the same sample, together with positive and negative controls, on a single chip. This platform accepts unprocessed samples and is readily adaptable to detection of multiple different pathogens or biomarkers for point-of-care diagnostics.
authors.affiliation: Department of Oncology, University of Alberta and Cross Cancer Institute, Edmonton, AB, Canada.
authors.forename: Dammika P
authors.initials: DP
authors.lastname: Manage
authors.forename: Jana
authors.initials: J
authors.lastname: Lauzon
authors.forename: Alexey
authors.initials: A
authors.lastname: Atrazhev
authors.forename: Yuen C
authors.initials: YC
authors.lastname: Morrissey
authors.forename: Ann L
authors.initials: AL
authors.lastname: Edwards
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Stickel
authors.forename: H John
authors.initials: HJ
authors.lastname: Crabtree
authors.forename: Kanti
authors.initials: K
authors.lastname: Pabbaraju
authors.forename: George
authors.initials: G
authors.lastname: Zahariadis
authors.forename: Stephanie K
authors.initials: SK
authors.lastname: Yanow
authors.forename: Linda M
authors.initials: LM
authors.lastname: Pilarski
mesh_terms: D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D004867:Equipment Design; D005835:Genitalia / Q000821:virology*; D006558:Herpes Genitalis / Q000175:diagnosis / Q000821:virology; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000302:isolation & purification*; D018258:Herpesvirus 2, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D057230:Limit of Detection; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012107:Research Design; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D017931:DNA Primers; D004279:DNA, Viral