title: Routine use of duplex real-time PCR assays including a commercial internal control for molecular diagnosis of opportunistic DNA virus infections.
abstract: The aim of this work was to improve the validity of laboratory-developed real-time PCR protocols implemented in the laboratory for molecular diagnosis of opportunistic DNA virus infections using the Simplexa™ extraction and amplification control (SEAC) which allows the monitoring of the whole extraction and amplification process. Herpes simplex virus (HSV), varicella-zoster virus (VZV), human cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK virus (BKV), and adenovirus (AdV) genomes were investigated in 152 different clinical specimens. The use of the SEAC did not influence the results of the different virus-specific PCRs. The SEAC results showed high reproducibility with a mean Cp value of 31.08±1.44, and were not influenced by the virus-specific PCR performed or the type of clinical specimen tested. The SEAC in the DNA extracts showed high stability during storage at both +4°C and -20°C. These data allowed establishing a new procedure for the validation of viral PCR results. In conclusion, the SEAC provides a reliable option for improving the diagnosis of opportunistic viral infections by laboratory-developed real-time PCR assays in quality assurance programs.
authors: UPMC Univ Paris 06, ER1 DETIV, F-75013 Paris, France.
authors: Sonia
authors: S
authors: Burrel
authors: Christelle
authors: C
authors: Fovet
authors: Christel
authors: C
authors: Brunet
authors: Lydia
authors: L
authors: Ovaguimian
authors: Nathalie
authors: N
authors: Hamm
authors: Françoise
authors: F
authors: Conan
authors: Laurence
authors: L
authors: Kalkias
authors: Henri
authors: H
authors: Agut
authors: David
authors: D
authors: Boutolleau
mesh_terms: D004266:DNA Virus Infections / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods* / Q000592:standards; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D009894:Opportunistic Infections / Q000175:diagnosis* / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D013696:Temperature; D014773:Virology / Q000379:methods* / Q000592:standards