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A PCR-microtiter
plate hybridization assay was effective in detecting human
mycoplasmas and ureaplasmas in urine from patients with
nongonococcal urethritis.
Rapid detection of Mycoplasma
genitalium, Mycoplasma hominis, Ureaplasma parvum, and Ureaplasma
urealyticum organisms in genitourinary samples by PCR-microtiter
plate hybridization assay.
Yoshida T, Maeda S-I, Deguchi T,
Miyazawa T, Ishiko H.
Journal of Clinical
Microbiology 2003;41:1850-1855.
Summary:
Question
What are the analytical sensitivity and specificity of a PCR-microtiter
plate hybridization assay for the detection of mycoplasma and ureaplasma?
Design
This study describes the evaluation of a PCR-microtiter plate
hybridization assay consisting of amplification of a part of the 16S rRNA
gene followed by a hybridization assay using four species-specific capture
probes for the detection of M. genitalium, M. hominis, U.
parvum, and U. urealyticum organisms in clinical urine samples
from men.
Participants
Seventy-nine M. genitalium, M. hominis, U. parvum, or U.
urealyticum positive urine samples from men identified using a
previously described PCR and phylogenetic analysis method were tested in
this study. Prototype strains of 13 species of human mycoplasma and 2 of
ureaplasma were also used to evaluate the method.
Description of Tests and Diagnostic
Standard
A diagnosis of urethritis was based on the observation of 5 or more
polymorphonuclear leukocytes per oil immersion field in a Gram-stained
urethral swab. DNA was extracted from 1 ml of urine by centrifugation, and
proteinase K digestion and phenol-chloroform extraction of the pellet. A
forward primer (My-ins) and two biotinylated reverse primers (MGSO-2-Bi
and UGSO-Bi) were used to amplify an approximately 520 base region of the
16S rRNA gene of mycoplasmas and ureaplasmas. Ten copies of an internal
control plasmid consisting of the positive M. genitalium control
DNA with the probe binding region deleted, was added to each PCR reaction
to monitor the presence of inhibitors. The PCR products were separated in
3% agarose gels and stained with ethidium bromide and identified by
separate hybridizations with 5 oligonucleotide probes specific to M.
genitalium, M. hominis, U. parvum, U. urealyticum and the internal
control. The 5' aminated probes were immobilized in microtiter plate wells
(DNA-BIND, Corning Inc., Corning, NY). The denatured amplicons were added
to the wells, incubated, and detected using streptavidin-peroxidase and a
colorimetric substrate. The cutoff OD value for a positive test (>0.300)
was calculated from the mean value of 20 negative control readings plus 4
standard deviations. The sensitivity of the assay was determined by
analyzing 10-fold dilutions of a plasmid DNA that contained the target
fragments of all 4 organisms. The specificity of the assay was determined
by testing 13 mycoplasma strains, 2 ureaplasma strains, and other genital
tract organisms including C. trachomatis, N. gonorrhoeae, C. albicans,
HSV types 1 and 2, and adenovirus types 40 and 41.
Main Outcome Measures
The analytical sensitivity and specificity of the PCR-microtiter plate
method and its performance for the detection of mycoplasmas and
ureaplasmas in urine samples that were positive by a PCR-phylogenetic
method were determined.
Main Results
The sensitivity of the assay was 10 copies of DNA per reaction. There were
no cross-reactions with the other human genital tract organisms tested,
and each probe was specific for its respective target. In samples prepared
with mixtures of 2 organisms, the assay was able to detect the minor
population of DNA when its content was as low as 1%. The results of 79
urine samples from men with and without urethritis, which were positive by
the PCR-phylogenetic assay, are shown in the table. The same results were
obtained by both methods. In some of the samples, two species were
detected by the PCR-microtiter plate method.
Authors' Conclusions
The PCR-microtiter plate hybridization assay
is an effective tool for the diagnosis of genitourinary infections with
mycoplasmas or ureaplasmas.
Source of funding: None given.
For correspondence: Hiroaki Ishiko,
Research and Development Department, Mitsubishi Kagaku Bio-Clinical
Laboratories, Inc., 3-30-1 Shimura, Itabashi, Tokyo 174-8555, Japan.
E-mail address: ishiko@m1.mbcl.co.jp.
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