Detection and typing of herpes simplex DNA in genital swabs by real-time polymerase chain reaction.
The LightCycler polymerase chain reaction (PCR) is a sensitive assay for the detection of Herpes simplex virus (HSV) DNA in muco-cutaneous swabs. Software-based analysis of the probe melting temperature (Tm) can be used to discriminate between HSV types (HSV-1 and HSV-2). Among 76 HSV DNA positive genital swabs, atypical Tms were observed in 14 (18%). The 14 samples were all typed as HSV-2 by sequence alignment. In 4/14 samples, the atypical Tm was associated with sequence variation at the probe-binding site. Among 10 samples with conserved sequences, Tms were influenced by the specimen preparation method prior to PCR. These findings indicate that multiple factors including, but not limited to sequence variation complicate melting curve analysis following real-time PCR. Alternative typing methods are recommended for specimens with atypical melting curves.
Ramaswamy
Meghna
M
Department of Virology, Royal Free and University College Medical School, Rowland Hill Street, London NW3 2PF, UK.
Smith
Melvyn
M
Geretti
Anna Maria
AM
D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D005835:Genitalia / Q000821:virology; D005838:Genotype; D006558:Herpes Genitalis / Q000175:diagnosis* / Q000821:virology; D018258:Herpesvirus 2, Human / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D013048:Specimen Handling / Q000379:methods; D044366:Transition Temperature
D004279:DNA, Viral