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A real-time PCR assay
for the detection of Neisseria gonorrhoeae in urine samples is a
rapid but less sensitive alternative to an end-point PCR assay.
A real-time PCR assay for the detection of Neisseria gonorrhoeae
by LightCycler.
Whiley DM, LeCornec GM, Mackay IM,
Siebert DJ, Sloots TP.
Diag Microbiol Infect Dis. 2002;42:85-89.
Summary:
Question
How does the performance of a real-time PCR assay using the
LightCycler and fluorophore-labeled hybridization probes (with
fluorescence resulting from fluorescence resonance energy transfer or
FRET) compare to an in-house end-point PCR assay using a solid-phase
colorimetric hybridization detection of amplicons for the detection of N.
gonorrhoeae in urine samples?
Design
This study describes a direct comparison of two in-house PCR methods to
detect N. gonorrhoeae in urine samples.
Participants
Patient specimens were urines (n=152) submitted to the Queensland Health
Pathology Service in Australia for routine investigation of N.
gonorrhoeae infection. Participants were not otherwise described.
Description of Tests and Diagnostic
Standard
The real-time PCR assay was performed using a LightCycler (Roche
Diagnostics, Australia) and two target-specific hybridization probes that
bind to adjacent positions on the target DNA. The primers and probes used
in the PCR targeted a sequence of the cppB gene of N. gonorrhoeae.
The specificity of the real-time PCR assay was determined by melting curve
analysis. The end-point PCR assay was a single tube nested PCR using
primers that also amplified the N. gonorrhoeae cppB gene, and
digoxigenin-labeled dUTP. For amplicon detection, a biotin-labeled probe
was hybridized to the PCR amplicons, captured on streptavidin-coated
microwells, and detected with a colorimetric enzyme immunoassay.
Main Outcome Measures
The sensitivity and specificity of the real-time PCR assay compared to the
end-point PCR assay for the detection of N. gonorrhoeae DNA in
urine samples were determined.
Main Results
The specificity of the real-time PCR assay was tested with 29 bacterial
and fungal isolates, including 3 other Neisseria species, and all failed
to amplify. Testing of 10-fold dilutions of a laboratory isolate of N.
gonorrhoeae showed that the real-time PCR assay could a detect 10-5
dilution, whereas the end-point PCR detected a 10-6 dilution. The
comparison of results with the two assays for 152 urine samples is shown
in the table. The clinical sensitivity of the real-time PCR assay was 94%
compared to the end-point assay.
| Comparison
of results for N. gonorrhoeae real-time and end-point PCR assays on
152 urine samples |
| Real-time
PCR |
End-point
PCR |
Total |
| Positive |
Negative |
| Positive |
29 |
0 |
29 |
| Negative |
2 |
121 |
123 |
| Total |
31 |
121 |
152 |
Authors' Conclusions
Although the real-time PCR assay was less
sensitive than end-point PCR on one experiment with a laboratory isolate,
and was negative in two urine specimens positive by the end-point PCR,
these differences were not statistically significant. The real-time PCR is
a faster method for the detection of N. gonorrhoeae DNA in urine
samples, potentially giving results within 1.5 h of receipt of the
specimen compared with the 5 to 6 h required for end-point PCR.
Source of funding: Royal Children's
Hospital Foundation Gran, sponsored by the Woolworths "Care for
Kids" campaignt.
For correspondence: Theo Sloots,
Clinical Virology Research Unit, Royal Children's Hospital and Health
Service District, Brisbane, Queensland, Australia. E-mail address: lsloots@mailbox.ug.edu.au.
expert
review
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