Failure to genotype herpes simplex virus by real-time PCR assay and melting curve analysis due to sequence variation within probe binding sites.
Real-time PCR with melting curve analysis of PCR products is a rapid procedure for detecting and genotyping herpes simplex virus (HSV). When testing mucocutaneous samples for HSV by a real-time PCR assay targeting the DNA polymerase gene, we found that some PCR products had atypical melting curves that did not conform to the expected melting temperatures for HSV type 1 or 2. Sequence analysis showed that these strains had base-pair mismatches over the probe binding sites. An alternative assay is required to type such atypical isolates.
Microbiology Unit, Canterbury Health Laboratories, Christchurch, New Zealand. Trevor.Anderson@cdhb.govt.nz
Trevor P
TP
Anderson
Anja M
AM
Werno
Kirsten A
KA
Beynon
David R
DR
Murdoch
D020137:Base Pair Mismatch; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D005814:Genes, Viral; D014644:Genetic Variation; D005838:Genotype; D018259:Herpesvirus 1, Human / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D018258:Herpesvirus 2, Human / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods; D012689:Sequence Homology, Nucleic Acid; D018139:Simplexvirus / Q000145:classification / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification
D015342:DNA Probes; D004259:DNA-Directed DNA Polymerase