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A PCR-based RFLP
method for the determination of HSV serotypes was more sensitive
than a PCR using type-specific primers and more cost-effective than
DNA sequencing.
Phenotypic and
genotypic methods for the detection of herpes simplex virus
serotypes.
Madhavan HN, Priya K, Bagyalakshmi R.
J Virol Meth. 2003;108:97-102.
Summary:
Question
How does a PCR-based restriction fragment length polymorphism (RFLP)
method compare to an antibody neutralization test, PCR using type-specific
primers, and DNA sequencing of PCR amplicons for the identification of
HSV-1 and HSV-2 clinical isolates, and how well does it compare to a
type-common PCR assay for HSV detection in clinical specimens?
Design
This study describes the development and evaluation of a PCR-based RFLP
assay compared to the antibody neutralization test, a type-specific PCR
assay, and DNA sequencing for typing of HSV-1 and HSV-2 clinical isolates.
Participants
One standard strain each of HSV-1 and HSV-2, 23 clinical HSV isolates
(from 16 genital ulcer swabs, 2 throat swabs, four corneal scrapings, and
1 skin scraping), and 20 clinical specimens (8 CSF, 2 genital swabs, and
10 ocular samples) were tested. The clinical specimens were negative by
culture but positive by an HSV type-common PCR assay that amplifies a 179
bp region of the HSV DNA polymerase gene. The clinical isolates from the
16 genital and 2 throat swabs were obtained from the Christian Medical
College and Hospital in Vellore, India. All the other isolates and
specimens were collected at hospitals in Chennai, India.
Description of Tests and Diagnostic
Standard
Serotyping by a conventional antibody neutralization method was done using
type-specific HSV polyclonal antisera (DAKO, A/S Denmark). For PCR
amplification, DNA was extracted by a standard Proteinase
K/phenol-chloroform method, and tested with a previously described PCR
using HSV-1 and 2 type-specific primers that amplify a 469 and 391 bp
fragment, respectively, of the DNA polymerase gene. The PCR products were
detected by gel electrophoresis in 2% agarose containing ethidium bromide.
The specificity of this assay, originally described as a separate PCR
reaction for each HSV type, and that of a duplex assay combining both
primer sets in one reaction, were evaluated on 7 viral, 8 bacterial, 1
yeast, and human DNA. The sensitivity was determined using serial 10-fold
dilutions of HSV DNA. DNA sequencing by a fluorescent dye labeled
terminator reaction mix analyzed in a PRISM 310 Genetic Analyzer (Applied
Biosystems, USA) was performed on a 179 bp fragment of the HSV DNA
polymerase gene generated by PCR amplification using previously described
type-common primers. For the PCR-based RFLP method, the 179 bp region of
the DNA polymerase gene, obtained by amplification using type-common
primers, was gel purified and digested with the restriction enzymes Hae
III and Taq I. The cleavage fragment patterns were visualized using
a 25% polyacrylamide gel stained by 0.1% silver nitrate.
The two standard strains of HSV types 1
and 2 and the 23 HSV clinical isolates were analyzed for specific HSV type
using the antibody neutralization test, PCR using type-specific primers,
DNA sequencing, and PCR-based RFLP. The HSV 20 culture negative,
type-common PCR positive clinical specimens were tested by the PCR-based
RFLP.
Main Outcome Measures
The HSV detection and typing results of the PCR-based RFLP method were
compared to those obtained using DNA sequencing of the PCR-amplified 179
bp fragment and the PCR using type-specific primers as the gold standard
methods.
Main Results
The type-specific PCR assays (uniplex and duplex) were equally sensitive
(2 pfu/ml and 3 pfu/ml with HSV-1 and HSV-2, respectively) and specific
for HSV DNA. The analytical sensitivity of the type-specific primers was
10-fold less than the type-common primers. The results of the
neutralization test, type-specific PCR, DNA sequencing, and RFLP method
were concordant for 23 HSV clinical isolates, 12 were typed HSV-1 and 11
were typed HSV-2. The RFLP for the identification of HSV serotypes
performed on 20 culture negative, PCR positive clinical specimens showed
that 15 were HSV-1 and 5 were HSV-2 positive.
Authors' Conclusions
The PCR-based RFLP could be used as a rapid
and specific method to differentiate HSV-1 and HSV-2. It is a reliable,
less laborious, and cost-effective molecular biological tool for the
determination of HSV serotypes both for clinical isolates and culture
negative clinical specimens.
Source of funding:
None given
For correspondence: H. N. Madhavan,
L&T Microbiology Research Centre, Vision Research Foundation, Sankara
Nethralaya, 18 College Road, Chennai 600 006, India. E-mail address: drhnm@sankaranethralaya.org.
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