title: A novel set of real-time PCRs for rapid differentiation between human cytomegalovirus wild-type and highly passaged laboratory strains.
abstract: Restricting amplification of human cytomegalovirus (HCMV) DNA to wild-type (WT) HCMV is useful to exclude PCR contaminations by laboratory strains from viral cultures. A set of UL141-specific TaqMan PCRs was developed to amplify (1) WT HCMV and laboratory strain Towne(long), but not strain AD169, (2) only WT HCMV. The performance was compared to a PCR targeting the conserved sequence of HCMV glycoprotein B using 46 serum and urine samples from blood donors with primary CMV infection. Amplification was restricted to the targeted strains with the exception of Towne(long) being amplified also by PCR (2), but at a distinctly lower efficiency than WT HCMV. The coefficient of regression for linear dilutions of two clinical samples with a high concentration of HCMV DNA was 0.999 and 0.997, respectively. The correlation between both WT PCRs and the generic HCMV PCR was good, with coefficients of regression of 0.891 and 0.871 for PCR (1) and (2), respectively. The limit of detection was calculated to be 1.5 genome equivalents per PCR. The set of HCMV TaqMan PCRs enables rapid differentiation between WT and laboratory strains, which can be especially useful as even virus lysate can contaminate sensitive PCRs without prior DNA isolation. A standardized WT HCMV control would be useful to evaluate WT-specific PCR methods.
authors.affiliation: Institute of Transfusion Medicine, University Hospital of Schleswig-Holstein, Ratzeburger Allee 160, Lübeck, Germany. Malte.Ziemann@uk-sh.de
authors.forename: Malte
authors.initials: M
authors.lastname: Ziemann
authors.forename: Alexander
authors.initials: A
authors.lastname: Unmack
authors.forename: Holger
authors.initials: H
authors.lastname: Hennig
mesh_terms: D001483:Base Sequence; D017124:Conserved Sequence; D003587:Cytomegalovirus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000821:virology*; D059018:DNA Contamination; D017931:DNA Primers; D004279:DNA, Viral / Q000097:blood / Q000235:genetics; D005189:False Positive Reactions; D005784:Gene Amplification; D005838:Genotype; D006801:Humans; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D014759:Viral Envelope Proteins / Q000097:blood / Q000235:genetics
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus