|
Differentiation of
HSV types 1 and 2 by melting curve analysis following real-time PCR
was problematic for the three assays evaluated.
Preliminary comparison of three
LightCycler PCR assays for the detection of herpes simplex virus in swab
specimens.
Whiley DM, Syrmis MW, Mackay IM, Sloots TP.
European Journal of Clinical Microbiology & Infectious Diseases
2003;22:764-767.
Summary:
Question
How do three HSV real-time PCR assays compare for the detection and typing
of HSV in swab samples?
Design
Three real-time PCR assays, including an in-house assay and two
commercially available kits, which enable HSV types 1 and 2 to be
differentiated in a single reaction mix by melting curve analysis on the
LightCycler instrument, were compared for the detection and typing of HSV
types 1 and 2 in swab specimens. Discrepant results were resolved by
conventional PCR.
Participants
Forty-eight patients (33 females, 15 males) presenting for a sexual health
screen at a hospital in Queensland, Australia were tested for HSV. The age
range was 17 to 40 years. Swab specimens were collected from 21 genital, 5
oral, 5 anal, and 17 miscellaneous sites.
Description of Tests and Diagnostic
Standard
Specimens were collected using Virocult swabs and tested within 24 h of
collection. DNA was extracted using the High Pure Viral Nucleic Acid kit
(Roche Diagnostics, Australia), according to the manufacturer's
instructions. Equal amounts of DNA were analyzed in three LightCycler
real-time PCR assays, which all used hybridization probes to detect HSV
DNA, melting curve analysis to distinguish HSV types 1 and 2, and included
internal control reactions. The in-house assay employed primers that
target the HSV glycoprotein D gene and the human endogenous retrovirus.
Two HSV LightCycler kits, HSV LC RealArt PCR kit (Artus, Germany) and the
LightCycler HSV 1/2 Detection kit (Roche Diagnostics), were performed
according to the manufacturers' instructions. The conventional PCR assay,
which was used for analysis of discrepant samples, targets the HSV
glycoprotein D gene. Two separate reactions, which were specific for
either HSV type 1 or type 2 were performed. Amplicons were detected using
a biotinylated probe in a colorimetric assay.
Main Outcome Measures
The agreement between the results of the three real-time PCR assays for
the detection and typing of HSV was determined.
Main Results
The results of each of the three real-time PCR assays for the detection of
HSV in 48 swab specimens from various sites are shown in table 1. Among
the 32 samples found HSV positive by all tests, 13 were typed as HSV-1 and
16 were typed as HSV-2 by all tests, and 3 samples had discrepant results.
The two specimens positive only by the in-house and Artus PCRs were both
typed as HSV-2 by both assays. The PCR typing results by the three
real-time and one conventional PCR assay for the three specimens that had
discrepant typing results are shown in table 2. No inhibitory substances
were detected in any of the specimens by any assay. All three assays
exhibited equivalent analytical sensitivity when dilutions of an HSV type
2 isolate of known concentration was tested.
Authors' Conclusions
Differentiation of HSV types 1 and 2 by
melting curve analysis was problematic in all assays. None of the assays
were able to successfully identify the HSV type present in all positive
samples.
Source of funding: Royal Children's
Hospital Foundation, which is sponsored by the Woolworth's "Care for
Kids" campaign
For correspondence:
T. P. Sloots, Clinical Virology Research Unit, Sir Albert Sakzewski Virus
Research Centre, Royal Children's Hospital and Health Service District,
Herston Road, 4029 Herston, Queensland, Australia. E-mail address:
t.sloots@mailbox.uq.edu.au
|
.gif){kind=small}