Unlabeled probes for the detection and typing of herpes simplex virus.
BACKGROUND
Unlabeled probe detection with a double-stranded DNA (dsDNA) binding dye is one method to detect and confirm target amplification after PCR. Unlabeled probes and amplicon melting have been used to detect small deletions and single-nucleotide polymorphisms in assays where template is in abundance. Unlabeled probes have not been applied to low-level target detection, however.


METHODS
Herpes simplex virus (HSV) was chosen as a model to compare the unlabeled probe method to an in-house reference assay using dual-labeled, minor groove binding probes. A saturating dsDNA dye (LCGreen Plus) was used for real-time PCR. HSV-1, HSV-2, and an internal control were differentiated by PCR amplicon and unlabeled probe melting analysis after PCR.


RESULTS
The unlabeled probe technique displayed 98% concordance with the reference assay for the detection of HSV from a variety of archived clinical samples (n = 182). HSV typing using unlabeled probes was 99% concordant (n = 104) to sequenced clinical samples and allowed for the detection of sequence polymorphisms in the amplicon and under the probe.


CONCLUSIONS
Unlabeled probes and amplicon melting can be used to detect and genotype as few as 10 copies of target per reaction, restricted only by stochastic limitations. The use of unlabeled probes provides an attractive alternative to conventional fluorescence-labeled, probe-based assays for genotyping and detection of HSV and might be useful for other low-copy targets where typing is informative.
Dames
Shale
S
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA. shale.dames@aruplab.com
Pattison
David C
DC
Bromley
L Kathryn
LK
Wittwer
Carl T
CT
Voelkerding
Karl V
KV
D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D005838:Genotype; D018259:Herpesvirus 1, Human / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D018258:Herpesvirus 2, Human / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012015:Reference Standards; D016415:Sequence Alignment; D044366:Transition Temperature; D014759:Viral Envelope Proteins / Q000235:genetics; D014773:Virology / Q000379:methods
D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1