|
A real-time nested
multiplex PCR for the detection of HSV types 1 and 2 and VZV
provides a specific result without the need for further typing.
Real-time nested multiplex PCR for the
detection of Herpes simplex virus types 1 and 2 and Varicella zoster
virus.
O'Neill HJ, Wyatt DE, Coyle PV, McCaughey C,
Mitchell F.
Journal of Medical Virology 2003;71:557-560.
Summary:
Question
How well does a real-time nested multiplex PCR assay compare to a
conventional multiplex nested PCR using agarose gel electrophoresis for
the detection and typing of HSV types 1 and 2 and VZV in cutaneous
lesions?
Design
A nested, multiplex PCR using agarose gel electrophoresis for the
detection and typing of HSV types 1 and 2 and VZV, which had proven
sensitivity and specificity, was adapted to a real-time nested multiplex
PCR using the LightCycler system to identify amplicons by melting
temperatures.
Participants
One hundred forty-nine specimens were tested by both PCR methods. One
hundred two were tested as they arrived in the laboratory; 47 archived
specimens that had been stored at -20oC for between 1 and 12
months were also tested. These 47 were selected based on previously
determined results. Seventeen, 28, and 2 were HSV-2 positive, VZV
positive, and negative, respectively. Specimens were obtained from 70
males, 68 females, and 11 from patients for which gender was not
specified. Penile, vulval, vaginal, or anal specimens were collected from
81 patients attending genitourinary medicine clinics and non-genital
vesicular lesion specimens were collected from 68 other patients.
Description of Tests and Diagnostic
Standard
Swabs were placed in 2 ml of viral transport medium and the specimens were
vortexed. Anogenital specimens were tested without extraction. Non-genital
specimens were extracted using the Qiagen DNA blood kit. The standard
routine nested, multiplex PCR used primers targeting 221 and 138 bp
fragments of the HSV-1 gpD gene, 221 and 101 bp fragments of the
HSV-2 gpG gene, and 646 and 552 bp fragments of the VZV ORF38. The
assay had previously been optimized with respect to primer and MgCl2
concentration and annealing temperature. For first round PCR, 2μl
of DNA was added to 8μl of PCR reaction mix. For second round PCR, 0.2μl
of first round product was added to 9.8μl of reaction mix. Both the first
and second round products were run on 2% agarose gels that were stained
with ethidium bromide. The first round of the real-time nested multiplex
PCR was carried out using the same procedure as for the standard PCR. The
second round was performed in a LightCycler (Roche, Lewes, UK), with
modified VZV primers that amplied a smaller amplicon (165 bp) and SYBR
green. Melting curve analysis was performed after amplification to
identify the PCR amplicon. Positive and negative controls were included
for each target in each test run.
Main Outcome Measures
The results of the standard nested multiplex PCR assay were compared to
those of the real-time nested multiplex PCR assay for 149 swab specimens.
Main Results
The results of the standard and real-time PCR assays are shown in the
table. The results of both assays were the same except for one specimen,
which was repeatedly HSV-2 positive by the real-time assay and repeatedly
negative by the standard assay. Each target gave a distinct Tm
on melting curve analysis.
Authors' Conclusions
The real-time assay differentiated between
HSV-1, HSV-2, and VZV without further testing of the amplified products
and gave a similar level of sensitivity and specificity as the standard
assay. The results of the real-time assay were available approximately 2
hr earlier than the standard assay, and allowed product detection and
identification in a single, closed tube system, reducing the possibility
of specimen contamination by amplified products.
Source of funding:
Not given
For correspondence:
Hugh J. O'Neill, Regional Virus Laboratory, Royal Victoria Hospital,
Belfast, BT12 6BA. E-mail address: hugh.oneill@bll.n-i.nhs.uk
|
.gif){kind=small}