title: Comparison of commercial extraction systems and PCR assays for quantification of Epstein-Barr virus DNA load in whole blood.
abstract: The automation of DNA extraction and the use of commercial quantitative real-time PCR assays could help obtain more reliable results for the quantification of Epstein-Barr virus DNA loads (EBV VL). This study compared two automated extraction platforms and two commercial PCRs for measurement of EBV VL in 10 EBV specimens from Quality Control for Molecular Diagnostics (QCMD) and in 200 whole-blood (WB) specimens from transplant (n = 137) and nontransplant (n = 63) patients. The WB specimens were extracted using the QIAcube or MagNA Pure instrument; VL were quantified with the EBV R-gene quantification kit (Argene) or the artus EBV RG PCR kit (Qiagen) on the Rotor-Gene 6000 real-time analyzer; and the results were compared with those of a laboratory-developed PCR. DNA was extracted from the QCMD specimens by use of the QIAamp DNA minikit and was quantified by the three PCR assays. The extraction platforms and the PCR assays showed good correlation (R, >0.9; P, <0.0001), but as many as 10% discordant results were observed, mostly for low viral loads (<3 log(10) copies/ml), and standard deviations reached as high as 0.49 log(10) copy/ml. In WB but not in QCMD samples, Argene PCR tended to give higher VL values than artus PCR or the laboratory-developed PCR (mean difference for the 200 WB VL, -0.42 or -0.36, respectively). In conclusion, the two automated extraction platforms and the two PCRs provided reliable and comparable VL results, but differences greater than 0.5 log(10) copy/ml remained between the two commercial PCRs after common DNA extraction.
authors.affiliation: Unit of Virus Host Cell Interactions UMI 3265 UJF-EMBL-CNRS, Grenoble, France. RGermi@chu-grenoble.fr
authors.forename: Raphaële
authors.initials: R
authors.lastname: Germi
authors.forename: Julien
authors.initials: J
authors.lastname: Lupo
authors.forename: Touyana
authors.initials: T
authors.lastname: Semenova
authors.forename: Sylvie
authors.initials: S
authors.lastname: Larrat
authors.forename: Nelly
authors.initials: N
authors.lastname: Magnat
authors.forename: Laurence
authors.initials: L
authors.lastname: Grossi
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Seigneurin
authors.forename: Patrice
authors.initials: P
authors.lastname: Morand
mesh_terms: D057205:Automation, Laboratory; D004279:DNA, Viral / Q000097:blood* / Q000302:isolation & purification*; D020031:Epstein-Barr Virus Infections / Q000097:blood* / Q000175:diagnosis / Q000821:virology; D018380:Hematopoietic Stem Cell Transplantation; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D016377:Organ Transplantation; D011933:Reagent Kits, Diagnostic; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D018709:Statistics, Nonparametric; D019562:Viral Load / Q000379:methods*
chemical_list: D004279:DNA, Viral; D011933:Reagent Kits, Diagnostic