Real-time PCR diagnostics failure caused by nucleotide variability within exon 4 of the human cytomegalovirus major immediate-early gene.
Here we report how variability in the human cytomegalovirus genome sequence may seriously affect the outcome of its molecular diagnosis. A real-time quantitative PCR assay targeting the major immediate-early gene failed to detect the viral load in some, but not all, clinical samples from hematooncological patients. By amplification and sequencing the DNA across the regions targeted by this assay we found a number of nucleotide substitutions which accounted for decreased primer/probe binding. This decreased the sensitivity of the assay up to 1,000-fold.
Lengerova
Martina
M
Center of Molecular Biology and Gene Therapy, Department of Internal Medicine-Hematooncology, University Hospital Brno, Brno, Czech Republic. mlengerova@fnbrno.cz
Racil
Zdenek
Z
Volfova
Pavlina
P
Lochmanova
Jana
J
Berkovcova
Jitka
J
Dvorakova
Dana
D
Vorlicek
Jiri
J
Mayer
Jiri
J
D001483:Base Sequence; D003587:Cytomegalovirus / Q000235:genetics*; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D005091:Exons / Q000235:genetics*; D005188:False Negative Reactions; D017781:Genes, Immediate-Early; D014644:Genetic Variation; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004279:DNA, Viral