title: Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens.
abstract: A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA together with an internal control was developed on the BD Max platform combining automated DNA extraction and an open amplification procedure. Its performance was compared to those of PCR assays routinely used in the laboratory, namely, a laboratory-developed test for HSV DNA on the LightCycler instrument and a test using a commercial master mix for VZV DNA on the ABI7500fast system. Using a pool of negative cerebrospinal fluid (CSF) samples spiked with either calibrated controls for HSV-1 and VZV or dilutions of a clinical strain that was previously quantified for HSV-2, the empirical limit of detection of the BD Max assay was 195.65, 91.80, and 414.07 copies/ml for HSV-1, HSV-2, and VZV, respectively. All the samples from HSV and VZV DNA quality control panels (Quality Control for Molecular Diagnostics [QCMD], 2013, Glasgow, United Kingdom) were correctly identified by the BD Max assay. From 180 clinical specimens of various origins, 2 CSF samples were found invalid by the BD Max assay due to the absence of detection of the internal control; a concordance of 100% was observed between the BD Max assay and the corresponding routine tests. The BD Max assay detected the PCR signal 3 to 4 cycles earlier than did the routine methods. With results available within 2 h on a wide range of specimens, this sensitive and fully automated PCR assay exhibited the qualities required for detecting simultaneously HSV and VZV DNA on a routine basis.
authors: Laboratory of Infectious Agents and Hygiene and GIMAP-EA3064, University Hospital of Saint-Etienne, Saint-Etienne, France.
authors: Sylvie
authors: S
authors: Pillet
authors: Laboratory of Infectious Agents and Hygiene and GIMAP-EA3064, University Hospital of Saint-Etienne, Saint-Etienne, France.
authors: Paul O
authors: PO
authors: Verhoeven
authors: Laboratory of Infectious Agents and Hygiene and GIMAP-EA3064, University Hospital of Saint-Etienne, Saint-Etienne, France.
authors: Amélie
authors: A
authors: Epercieux
authors: Laboratory of Infectious Agents and Hygiene and GIMAP-EA3064, University Hospital of Saint-Etienne, Saint-Etienne, France.
authors: Thomas
authors: T
authors: Bourlet
authors: Laboratory of Infectious Agents and Hygiene and GIMAP-EA3064, University Hospital of Saint-Etienne, Saint-Etienne, France bruno.pozzetto@univ-st-etienne.fr.
authors: Bruno
authors: B
authors: Pozzetto
mesh_terms: D004279:DNA, Viral / Q000134:cerebrospinal fluid; D006566:Herpesviridae Infections / Q000175:diagnosis* / Q000821:virology; D014645:Herpesvirus 3, Human / Q000235:genetics*; D006801:Humans; D057230:Limit of Detection; D058889:Molecular Typing / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012189:Retrospective Studies; D018139:Simplexvirus / Q000235:genetics*
chemical_list: D004279:DNA, Viral