|
A real-time PCR assay exhibited enhanced detection of HSV by incorporating an internal control reaction and by accurately typing HSV positive specimens.
Detection and differentiation of herpes simplex virus types 1 and 2 by a duplex LightCycler PCR that incorporates an internal control PCR reaction.
Whiley DM, Mackay IM, Syrmis MW, Witt MJ, Sloots TP.
Journal of Clinical Virology 2004;30:32-38.
Summary:
Question
What are the performances of a LightCycler-based real-time, duplex PCR assay targeting the HSV-1 and HSV-2 glycoprotein D genes and an internal control human endogenous retrovirus and a real-time PCR targeting the HSV DNA polymerase gene for the detection and typing of HSV-1 and HSV-2 in clinical specimens?
Design
Two LightCycler-based real-time PCR assays, one targeting the HSV-1 and HSV-2 glycoprotein D genes and an internal control human endogenous retrovirus and the other targeting the HSV DNA polymerase gene, were compared for the detection and typing by melting curve analysis of HSV-1 and HSV-2 in clinical specimens.Discrepant results were resolved by a conventional PCR assay.
Participants
Swab specimens from 300 patients (181 females, 119 males) that were submitted to the hospital laboratory for detection of HSV were analyzed.The specimens included 180 ano-genital, 33 oral, and 87 skin, throat, or unspecified swabs.The median patient age was 29 years (range = 1 to 87 years).
Description of Tests and Diagnostic Standard
Swabs were placed in 2 ml of viral transport medium.After vortexing, 2 µl of specimen was added directly to each real-time PCR reaction.The HSVgG-dPCR assay utilized primers and fluorescence resonance energy transfer (FRET) probes that targeted highly conserved sequences of the glycoprotein D gene of HSV types 1 and 2.Each primer contained a single degenerate base and amplified a 260 bp product.Both probes were 100% similar to the HSV type 1 sequence.The downstream probe was labeled on the
5'
end with LC-Red640.The detection of the human endogenous retrovirus internal control (ERV) was performed using primers that amplified a 135 bp product.The ERV FRET downstream probe was labeled on the
5'
end with LC-Red705.The two fluorescent dyes were detected by separate channels of the LightCycler instrument.The HSV primers were added at a greater concentration than the ERV primers to limit the competition between the internal control PCR reaction with the HSV PCR reaction. The Dpol-HSV-LCPCR assay primers targeted the HSV DNA polymerase gene and did not contain primers and probes for the internal control.The FRET probes were 100% similar to the HSV type 2 sequence.The PCRs were carried out for 55 cycles; HSV typing was performed by melting curve analysis following PCR amplification.
Discrepant analysis was performed using two separate type specific conventional HSV PCR reactions that targeted a conserved region of the HSV glycoprotein D gene.The assays contained either an HSV type-1 or type-2 specific forward primer and a consensus HSV reverse primer.Five
µl of specimen was added to each reaction.Digoxigenin labeled products were detected in an enzyme-linked amplicon hybrdization assay using a biotinylated probe.Amplification products from the HSV positive specimens that could not be typed by melting curve analysis were
sequenced.
Main Outcome Measures
The HSV typing results of the two real-time PCR assays were compared for 300 specimens.
Main Results
The HSV detection and typing results of the HSVgG-dPCR and the Dpol-HSV-LCPCR real time PCR assays for 300 clinical specimen are shown in the table.All discrepant specimens were confirmed for HSV type by the conventional PCR method.Sequencing of amplicons from the 3 specimens that could not be typed by the Dpol-HSV-LCPCR showed that one amplicon contained 2 mismatches and 2 amplicons contained 3 mismatches with the probe sequence in the probe binding region.The 4 specimens that were positive by the Dpol-HSV-LCPCR assay and negative by the HSVgG-dPCR assay were HSV positive for the correct type when the HSVgG-dPCR assay was run without the internal control primers and probe, indicating that the sensitivity of the HSV glycoprotein D PCR was reduced when combined with the internal control PCR.Negative HSV and internal control results were obtained for 15 (5%) of the 300 specimens.The results of conventional PCR on these 15 specimens were HSV negative.
Results of two real-time PCR assays for detection and typing of HSV in 300 clinical specimens
|
HSV DNA polymerase gene PCR result
|
HSV glycoprotein D gene PCR result
|
|
HSV 1
|
HSV 2
|
Negative
|
Total
|
|
HSV 1
|
46
|
0
|
3
|
49
|
|
HSV 2
|
0
|
42
|
1
|
43
|
|
Not typed
|
0
|
3
|
0
|
3
|
|
Negative
|
1
|
0
|
204
|
205
|
|
Total
|
47
|
45
|
208
|
300
|
Authors '
Conclusions
The newly developed HSVgD-dPCR enhanced the diagnostic utility of real-time detection of HSV by enabling the simultaneous detection and differentiation of HSV types 1 and 2 and an internal control within the same reaction mix.The HSV glycoprotein D PCR assay gave good agreement with the DNA polymerase PCR assay and correctly typed all HSV positive specimens identified.The low incidence of inhibition in the specimens suggested that it may not be worthwhile to incorporate the internal control assay, given that it reduced detection of HSV.This decision should be an individual choice based on local requirements in any given laboratory.
Source of funding:Royal Children 's Foundation grant sponsored by the
Woolworth's
"Care for Kids" campaign.
For correspondence:Theo P. Sloots, Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal
Children 's Hospital and Health Service District, Herston Road, Herston, Qld 4029,
Australia. E-mail address: t.sloots@mailbox.uq.edu.au.
|
.gif){kind=small}