JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2005, p. 5653–5659 Vol. 43, No.

11
0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.11.5653–5659.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Specific and Sensitive Detection of Neisseria gonorrhoeae in Clinical

Specimens by Real-Time PCR
C. W. M. Geraats-Peters,1 M. Brouwers,1 P. M. Schneeberger,1 A. G. M. van der Zanden,2
S. M. Bruisten,3 G. Weers-Pothoff,1 C. H. E. Boel,4 A. J. C. van den Brule,4
H. G. Harmsen,2 and M. H. A. Hermans1*
Multidisciplinary Laboratory of Molecular Diagnostics and Regional Laboratory of Medical Microbiology, Jeroen Bosch
Hospital,1 and Medical Microbiology and Infectious Diseases, Gelre Hospitals, Location Lukas,2 Apeldoorn,
GG&GD, Municipal Health Service, Amsterdam,3 and Laboratory for Pathology and Microbiology,
PAMM, Veldhoven,4 The Netherlands
Received 4 March 2005/Returned for modification 19 May 2005/Accepted 11 August 2005

Early diagnosis of Neisseria gonorrhoeae infections is important with regard to patients’ health and infec-
tivity. We report the development of a specific and sensitive TaqMan assay for the detection of N. gonorrhoeae
in clinical samples. The target sequence is a 76-bp fragment of the 5ⴕ untranslated region of the opa genes that
encode opacity proteins. A panel of 448 well-defined N. gonorrhoeae isolates was used to evaluate and optimize
the assay. The method employs two minor-groove binding probes, one of them recognizing a newly identified
sequence in the opa genes. Testing a large panel of related and unrelated microorganisms revealed that other
Neisseria strains and other microorganisms tested negative in the opa test. With a lower detection limit of one
genome per reaction, the opa test appeared more sensitive than both the COBAS AMPLICOR (Roche Diag-
nostics Nederland BV, Almere, The Netherlands) and a LightCycler 16S rRNA test. Analysis of a panel of 122
COBAS AMPLICOR-positive samples revealed that 68% were negative in both the 16S rRNA test and the opa
assay (confirming that the COBAS AMPLICOR test produces false positives), while 30% were positive in both
assays. Three samples were opa positive and 16S rRNA negative, which may be due to the higher sensitivity of
the opa assay. We conclude that the opa gene-based real-time amplification assay offers a sensitive, specific,
semiquantitative, and reliable assay suitable for the detection of N. gonorrhoeae in clinical specimens and/or for
confirmation of less specific tests.

Gonorrhea is the second most prevalent sexually transmitted
disease (STD), after infection with Chlamydia trachomatis. A
significant proportion of the infections, especially in women,
are asymptomatic. Undiscovered, infections may spread to sex-
ual partners and lead to long-term consequences, such as pel-
vic inflammatory disease, chronic pelvic pain, ectopic preg-
nancy, neonatal conjunctivitis, and infertility (14). Infection
with Neisseria gonorrhoeae is known to increase the risk for
human immunodeficiency virus (HIV) infection. Odds ratio
estimates for increased risk of HIV infection due to previous
infection with an STD vary from 3.5 to 9.0 for N. gonorrhoeae
(28). Infection with N. gonorrhoeae may also be associated with
an increased risk of HIV seroconversion (28). The high inci-
dence rates of N. gonorrhoeae infections, coupled with the
prevalence with which they go undiagnosed and/or untreated,
highlights the need for accurate diagnosis of both symptomatic
and asymptomatic infections.
A number of techniques have been developed to detect
genital infections caused by N. gonorrhoeae. The current “gold
standard” for diagnosis of infection is by culture on selective
media. However, even under optimal laboratory conditions,
the sensitivity of gonococcal cultures ranges from 85 to 95%
for acute infection (31) and falls to approximately 50% for
females with chronic infections (2). This is largely due to poor
* Corresponding author. Mailing address: Multidisciplinary Labora-
tory of Molecular Diagnostics, Jeroen Bosch Hospital, M. H. A. Her-
mans, P.O. Box 90153, 5200 ME ’s-Hertogenbosch, The Netherlands.
Phone: 31-73-699.21.06. Fax: 31-73-699.21.36. E-mail: m.hermans@jbz.nl.
specimen collection, transport, and storage. Nucleic acid am-
plification-based techniques, including the ligase chain reac-
tion, strand displacement amplification assay, nucleic acid se-
quence-based amplification, and PCR, have been shown to
have both high sensitivity and specificity for the detection of N.
gonorrhoeae (1, 10, 16, 18, 20, 23, 25, 32, 37), and a number of
commercial assays are available. However, each of these tests
has limitations, including variable sensitivities to inhibitors,
cross-reactivity with other microorganisms, limited sensitivity,
high costs, and dedicated equipment. In addition, their appli-
cation is often restricted to specific specimen types due to
limited validation of the assays. The COBAS AMPLICOR test
for N. gonorrhoeae (COBAS AMPLICOR CT/NG; Roche Di-
agnostics Nederland BV, Almere, The Netherlands), for in-
stance, produces false-positive results with certain nonpatho-
genic Neisseria species (Neisseria subflava and Neisseria cinerea)
and lactobacilli, and a subsequent confirmation test is neces-
sary (5, 12, 15, 30, 38). CppB- and 16S rRNA gene-based assays
are used for confirmation (35); however, about 5% of N. gon-
orrhoeae strains do not carry the CppB plasmid (5, 7), and not
all 16S rRNA-based tests are sensitive and specific enough (12,
39).
Recently, Abbott Laboratories (Abbott Park, IL) voluntarily
recalled its LCx N. gonorrhoeae Assay because of reagent prob-
lems (8a). This prompted us to develop an N. gonorhoeae test
on the TaqMan platform present in our laboratory.
All meningococcal and gonococcal strains express opacity
(Opa) proteins, so called because of their contribution to col-

5653
5654 GERAATS-PETERS ET AL.
ony opacity during growth of the bacteria on agar plates (34).
They are a family of basic integral outer membrane proteins of
approximately 27 kDa. Eleven to 13 individual opa genes have
been identified in N. gonorrhoeae, whereas Neisseria meningi-
tidis has fewer (three of the four) opa genes. Opa-like proteins
are expressed in a number of commensal Neisseriaceae as well
(36). The opa genes in N. gonorrhoeae are contained in sepa-
rate loci (opaA through -K) (4) and are subject to on/off phase
variation. Changes in the repetitive sequences within the var-
ious opa loci result in this variable expression of different Opa
proteins in a single bacterium (33). Opa expression has been
found to promote gonococcal adherence to epithelial cells and
entry into epithelial cells via binding to cell surface proteogly-
cans (3, 9, 21, 40, 42) and gonococcal interactions with poly-
morphonuclear leukocytes (19). Furthermore, Opa proteins
enhance resistance to complement-mediated killing (6). Be-
cause the opa genes are multicopy genes that harbor conserved
regions and encode proteins with physiological functions, we
thought them suitable as target sequences for a real-time PCR
amplification assay.
(European Patent Application 04077241.0 covers the assay
described in this report.)
MATERIALS AND METHODS
Panel of 448 N. gonorrhoeae strains. From September 2002 to April 2003,
patients with complaints indicative of gonorrhea visited the Sexually Transmitted
Infections clinic in Amsterdam, The Netherlands, where clinical and epidemio-
logical data were registered and samples were taken (7). Urethral, cervical,
proctal, or tonsil specimens were used to inoculate GC-Lect agar plates (Becton-
Dickinson). Culturing and determination of N. gonorrhoeae identity were per-
formed at the Public Health Laboratory in Amsterdam as described previously
(8). In the context of a communal epidemiology study, we typed these N. gon-
orrhoeae strains by PCR-restriction fragment length polymorphism of the opa
and por genes, further confirming that true N. gonorrhoeae strains were used for
DNA isolation (24, 29).
QCMD (Glasgow, United Kingdom) N. gonorrhoeae 2003 and 2004 panels. In
order to assess the performance of nucleic acid amplification technologies for the
detection of N. gonorrhoeae, proficiency panels are distributed by the Quality
Control for Molecular Diagnostics (QCMD) Working Party on STD (Chair,
Jurjen Schirm, Groningen, The Netherlands). Both panels consisted of lyophi-
lized urine samples. As indicated by QCMD, 1.2 ml and 1.6 ml water (for the
2003 and 2004 panels, respectively) were used to dissolve the lyophilized mate-
rial. Specimens were processed as for urine samples (described below).
Nucleic acid extraction. (i) Bacterial strains. For the isolation of nucleic acids
from the panel of 448 N. gonorrhoeae strains, DNAs were isolated from one to
three colonies using isopropanol precipitation, followed by dissolving the pellet
in 50 ␮l 10 mM Tris-HCl, pH 8.0 (26). Such pellets were diluted 10,000 times in
10 mM Tris-HCl, pH 8.0, and 5 ␮l was added to PCR mixtures. For the isolation
of nucleic acids from other bacterial strains, bacteria were suspended in TE (1
mM EDTA in 10 mM Tris-HCl buffer, pH 8.0) to a suspension of approximately
0.5 McFarland units and incubated for 15 min at 100°C. Because of the low
threshold cycle (Ct ⫽ 12 to 14, indicating a high DNA load) when analyzed
directly in the real-time PCR, 1-in-1,000 dilutions in TE were prepared and
analyzed. Five microliters was added to each PCR mixture.
(ii) COBAS AMPLICOR N. gonorrhoeae-positive clinical samples. Between 1
and 1.5 ml of urine was centrifuged for 15 min at 13,000 rpm. The supernatant
was discarded, except for approximately 100 ␮l, which was left on the pellet.
Samples in STM or 2-SP medium (Roche Diagnostics Nederland BV, Almere,
The Netherlands) were processed directly. DNA was isolated from 100 ␮l of
material using the DNA Isolation Kit III (Bacterial Fungi; Roche Diagnostics
Nederland BV, Almere, The Netherlands) and the MagnaPure LC Isolation
station (Roche Diagnostics Nederland BV, Almere, The Netherlands) exactly as
described by the manufacturer. The nucleic acids were eluted in a final volume
of 100 ␮l. Isolates were split for COBAS AMPLICOR, 16S rRNA confirmation
tests, and opa-based N. gonorrhoeae assay. DNA isolation and all tests were
carried out on the same day; 25 ␮l was added in the COBAS AMPLICOR, 5 ␮l
was added in the 16S rRNA tests, and 10 ␮l was added to the opa PCR.
J. CLIN. MICROBIOL.
(iii) Other clinical samples. Dry urethra or cervical swabs (plastic minitip swab
185CS01; Copan, AMDS-Benelux, Malden, The Netherlands) were placed in 500
␮l of TE, incubated for 30 min at 97°C, and centrifuged for 1 min at 8,000 rpm.
Ten microliters was used in the PCR. For urine samples, 1 ml was centrifuged at
10,000 ⫻ g for 15 min, and the supernatant was removed. The remaining pellet
was dissolved in 300 ␮l of TE and incubated for 30 min at 97°C; 10 ␮l was used
in the PCR.
If inhibition occurred in the PCR (see below), DNA was isolated from 190 ␮l
of sample, to which 10 ␮l of a seal herpesvirus (PhHV-1) was added using the
QIAGEN Blood Kit, following the manufacturer’s guidelines but omitting the
protease treatment and eluting in 50 ␮l; 10 ␮l was used in the PCRs.
PCR inhibition control. To monitor the real-time N. gonorrhoeae detection, a
separate PCR was run on all samples to which PhHV-1 was added at a final
concentration of approximately 5,000 to 10,000 DNA copies per ml, equivalent
to a Ct value of approximately 30 (38). If the Ct was within range of the mean ⫾2
standard deviations, the PCR was considered not to be inhibited.
Opa-based N. gonorrhoeae assay. A 25-␮l PCR was performed containing 20
mM Tris-HCl, pH 8.4, 50 mM KCl, 3 mM MgCl2 (prepared from 10⫻ PCR
buffer delivered with Platinum Taq polymerase), 0.75 U Platinum Taq polymer-
ase (Invitrogen BV, Breda, The Netherlands), 4% glycerol (molecular biology
grade; CalBiochem, VWR International BV, Amsterdam, The Netherlands), 200
␮M of each deoxynucleoside triphosphate (Amersham Bioscience, Roosendaal,
The Netherlands), 0.5 ␮l Rox Reference Dye (Invitrogen BV), 150 nM probe
opa-1 (when indicated, 150 nM probe opa-2) (Applied Biosystems, Nieuwerkerk
a/d IJssel, The Netherlands), 300 nM opa-Fw primer and 300 nM opa-Rv primer
(Sigma-Genosys Ltd., Haverhill, United Kingdom), and 5 or 10 ␮l sample (5 ␮l
DNA was used when analyzing the panel of 448 N. gonorrhoeae strains; 10 ␮l was
used for all other assays).
ABI Prism sequence detection system 7000 (Applied Biosystems, Nieuwerkerk
a/d IJssel, The Netherlands) was used for amplification and detection (2 min at
50°C, 10 min at 95°C, 45 cycles of 15 s at 95°C and 60 s at 60°C).
COBAS AMPLICOR test for N. gonorrhoeae. The COBAS AMPLICOR test
was performed according to the manufacturer’s instructions.
16S rRNA confirmation test. The 16S rRNA confirmation test was carried out
as previously described (5).
PhHV detection. PhHV was detected as previously described (38).
Sequence analysis. M13-opa-Fw (TGT AAA ACG ACG GCC AGT GTT
GAA ACA CCG CCC GG) and M13-opa-Rv (CAG GAA ACA GCT ATG
ACC CGG TTT GAC CGG TTA AAA AAA GAT) primers (300 nM each)
were used for amplification. The PCR was carried out as described above. The
PCR product was purified by adding 4 ␮l of combined exonuclease I (10 U/ml)
and shrimp alkaline phosphatase (2 U/␮l) (USB Corporation; distributor, Am-
ersham Bioscience, Roosendaal, The Netherlands) to 20 ␮l of PCR product and
incubating the mixture for 15 min at 37°C, followed by inactivation for 15 min at
80°C (PTC-200 thermocycler; MJ Research, Biozym TC BV, Landgraaf, The
Netherlands). Fragments were sequenced using M13 primers. Twenty-microliter
reaction mixtures contained 5 ␮l purified PCR product, 4 ␮l BigDye Terminator
Cycle Sequencing Ready Reaction Mix (Applied Biosystems), and 7.5 pmol
forward or reverse primer. Twenty-five cycles of 10 s at 96°C, 5 s at 50°C, and 2.5
min at 60°C were run, and the products were purified over Sephadex (G-50
Superfine) before being analyzed on an ABI Prism 3700 DNA Analyzer (Applied
Biosystems). For each N. gonorrhoeae strain, one forward and one reverse se-
quence were determined.
Sensitivity. N. gonorrhoeae bacteria (ATCC 49226) were resuspended in TE
buffer to a density of 0.96 McFarland units. DNA was extracted as described
above by means of the QIAGEN Blood kit and eluted in 50 ␮l. DNA was
quantified by photospectrometer (Eppendorf BioPhotometer; Eppendorf, Ham-
burg, Germany) and found to be 18.85 ng/␮l (1:1 dilution [A260, 0.192; A280,
0.107; concentration, 18.6 ng/␮l] and 1:4 dilution [A260, 0.099; A280, 0.056; con-
centration, 19.1 ng/␮l]). Tenfold serial dilutions were made containing 10 ␮g/ml
to 100 fg/ml. Ten microliters of each dilution was used for duplicate PCRs. The
results were analyzed using ABI Prism 7000 SDS Software version 1.1 with
manual baseline setting from cycle 3 to 10 and manual Ct at 0.200. To calculate
the standard curve, the 1-pg/ml and 100-fg/ml dilutions were omitted. One N.
gonorrhoeae genome is 2.45 fg (2.2 ⫻ 106 bp [11] ⫻ 665 Da/bp ⫻ 1.67 ⫻ 10⫺24
g/Da).
RESULTS
Primers and probe design. Sequences covering a conserved
region within the 5⬘ untranslated regions of the opa genes were
obtained from the NCBI database. Based on homology, the
VOL. 43, 2005 DETECTION OF N. GONORRHOEAE BY REAL-TIME TaqMan PCR
FIG. 1. Design of primers and probe. Sequences of opa genes 92 to 16 bases 5⬘ of the start codon retrieved from NCBI database. The light-gray
shading indicates the positions of the primers; the dark-gray shading indicates the position of the probe (sequences as in upper line).
corresponding sequences of N. gonorrhoeae, N. meningitidis,
and N. flava were retrieved and aligned (Fig. 1). Primers and
the minor-groove binding (MGB) probe opa-1 (Table 1) were
designed and adapted to TaqMan standards using Primer Ex-
press software (Applied Biosystems). Minor-groove binding
probes form stable duplexes with single-stranded DNA targets,
thus allowing short probes to be used for hybridization-based
assays.
Optimization of the opa-based N. gonorrhoeae assay. Of the
panel of 448 clinical N. gonorrhoeae strains (see Materials and
Methods), 424 generated a positive fluorescent signal in the
TaqMan PCR employing probe opa-1. The fluorescent signals
from the remaining 24 strains were undetectable. The PCR
products of those 24 “aberrant” N. gonorrhoeae strains were
analyzed on agarose gels. All 24 showed ample PCR products
of the expected size. Sequencing of these PCR products re-
vealed exactly the same sequence in all 24 strains (Fig. 1). The
MGB probe opa-2 was designed to cover this additional se-
quence, which was included in subsequent assays. We subse-
quently analyzed DNAs from 21 opa-1-positive N. gonorrhoeae
strains from the above-mentioned panel using the N. gonor-
rhoeae assay with only the probe set for opa-2. One out of 21
TABLE 1. Sequences of primers and probes used for real-time
N. gonorrhoeae detection (13)
Primer or probe Sequence
opa-Fw........................GTT GAA ACA CCG CCC GG
opa-Rv ........................CGG TTT GAC CGG TTA AAA AAA GAT
Probe opa-1 ...............CCC TTC AAC ATC AGT GAA A-MGB
Probe opa-2 ...............CTT TGA ACC ATC AGT GAA A-MGB
5655
appeared positive with probe opa-2, as well as opa-1, indicating
the presence of both sequences in that particular strain (data
not shown). The Ct values showed a 10-fold-higher signal with
probe opa-1 than with opa-2.
Specificity. In addition to the performance with 448 N. gon-
orrhoeae strains, the specificity of the assay was assessed by
testing a panel of non-N. gonorrhoeae microorganisms (Table
2), including DNAs from 10 other different Neisseriaceae iso-
lates. No signal in the opa real-time PCR was observed with
any of the microorganisms tested using probes opa-1 and
opa-2.
Sensitivity. DNA was isolated of from N. gonorrhoeae ATCC
strain 49226 as described in Materials and Methods and di-
luted to an undetectable level. Tenfold serial dilutions ranging
from 100 fg to 10 ␮g DNA per ml were made, and these
samples were amplified in the opa assay. N. gonorrhoeae DNA
could be measured in a linear fashion over a range of 8 log
scales (Fig. 2). The PCR efficiency was calculated to be 93%
when probes opa-1 and opa-2 were present in the PCR (the
efficiency was 98% when only probe opa-1 was employed). One
femtogram of N. gonorrhoeae DNA (equivalent to 0.41 N.
gonorrhoeae genome) was detectable in four out of six reac-
tions.
Panel of 122 clinical COBAS AMPLICOR-positive samples.
From January 2003 to March 2004, a total of 3,957 clinical
samples from patients of the health care region of the Gelre
Hospital were analyzed in the COBAS AMPLICOR test for
the presence of N. gonorrhoeae. One hundred twenty-two sam-
ples (3.1%) tested positive for N. gonorrhoeae. These samples,
consisting of 36 urine, 8 urethra, 47 cervix, 29 throat, and 2 anal
samples (Table 3), were analyzed in a real-time 16S rRNA test
5656 GERAATS-PETERS ET AL. J. CLIN. MICROBIOL.

TABLE 2. Reactivity of the opa-based N. gonorrhoeae assay (probes and the opa assay. The 16S rRNA confirmation test was carried
opa-1 and opa-2) with various species of Neisseria and out in two independent laboratories, and both laboratories
other microorganisms obtained exactly the same results. Thirty-six samples were
Species n Strain no.a/origin opa assay found to be positive and 83 samples were negative in all three
Neisseria cinerea 1 A841390b Negative
tests (Table 4). The remaining three samples that were nega-
Neisseria denitrificans 1 A841389b Negative tive in the 16S rRNA test were positive in the opa assay. They
Neisseria elongata 1 NRBM 901301b Negative encompassed a urine sample (COBAS AMPLICOR 1.018,
Neisseria flavescens 1 NRBM 930649b Negative
Neisseria lactamica 2 NRBM 900295b,c Negative 0.429, and 0.366; 16S rRNA negative; opa assay, Ct ⫽ 34.9 and
Neisseria meningitidis 11 ATCC 13102c,d Negative 35.2) and two STM cervix swabs (COBAS AMPLICOR 1.553
Neisseria mucosa 2 40489b,c Negative and 1.855; 16S rRNA negative; opa assay, Ct ⫽ 34.6 and 34.9,
Neisseria perflava 1 A841399b Negative
Neisseria polysaccharea 1 BD02-00484e Negative and COBAS AMPLICOR 3.876 and ⬎3.999; 16S rRNA neg-
Neisseria sicca 1 A841401b Negative ative; opa assay, Ct ⫽ 36.2 and 38.0). The fact that the three
Neisseria subflava 1 –c Negative
Neisseria subflava var. flava 1 NRBM 921185b Negative
samples showed the highest opa assay Ct values of all the
Bacteroides fragilis 2 ATCC 25285c Negative positive samples in the panel suggested that the discrepancy
Bacteroides vulgatus 1 ATCC 10583 Negative could be due to a slight difference in the detection levels of the
Campylobacter jejuni 1 ATCC 11392 Negative
Chlamydia trachomatis 2 –e Negative 16S rRNA PCR and the opa assay. We therefore analyzed a
Candida albicans 2 ATCC 90028c Negative dilution series of N. gonorrhoeae DNA in both assays on the
Candida glabrata 1 ATCC 90030 Negative same day. The results of this test revealed a 5- to 10-fold
Candida krusei 1 ATCC 6258 Negative
Candida parapsilosis 1 ATCC 90018 Negative difference in sensitivity between the 16S rRNA PCR and the
Corynebacterium aquaticum 1 –f Negative opa PCR (Table 5), which might be partly due to the difference
Corynebacterium diphteriae subsp. mitis 1 SKMM panel 1996 Negative
Corynebacterium diphteriae subsp. belfanti 1 SKMM panel 1996 Negative
in sample input volume (5 ␮l in the 16S rRNA PCR versus 10
Corynebacterium diphteriae 2 SKMM panel 2000 Negative ␮l in the opa PCR).
subsp. mitis/belfanti QCMD panels. We analyzed QCMD panels that were dis-
Corynebacterium diphteriae 1 SKMM panel 2001 Negative
Corynebacterium ulcerans 2 SKMM panel 2000 Negative tributed in 2003 and 2004 in the COBAS AMPLICOR (two
Cryptococcus neoformans 1 ATCC 90112 Negative laboratories), in the 16S rRNA test (two laboratories), and in
Enterococcus faecalis 1 ATCC 29212 Negative the opa-based assay. The results are shown in Table 6. Sample
Enterococcus casseliflavus 1 ATCC 700327 Negative
Escherichia coli 3 ATCC 25922, 35218c Negative NG03-04 was found to be negative in one laboratory in the
Gardnerella vaginalis 1 ATCC 14018 Negative COBAS AMPLICOR test and in both laboratories in the 16S
Haemophilus influenzae 3 ATCC 49247, 49766, Negative
9006c
rRNA test. Samples NG03-05, NG03-07, NG04-03, and
Haemophilus parainfluenzae 1 –c Negative NG04-09 were missed in one of the two laboratories in the 16S
Haemophilus ducreyi 1 –c Negative rRNA test. Sample NG04-06 was false positive in the COBAS
Klebsiella oxytoca 1 ATCC 700324 Negative
Lactobacillus sp. 1 ATCC 314 Negative AMPLICOR test. The opa assay detected all samples that
Legionalle pneumophila serogroup 1 1 RMM 220186 Negative contained N. gonorrhoeae correctly. The Cts of samples
Moraxella catarrhalis 1 –c Negative NG03-04 and NG03-07 (indicated as Pos [⫹/⫺] by QCMD)
M. atlantii (??) 1 –c Negative
Moraxella catarrhalis 1 –c Negative were 33.2 and 33.5, respectively.
Peptostreptococcus magnus 1 ATCC 29328 Negative
Pseudomonas aeruginosa 2 ATCC 27853c Negative
Proteus mirabilis 1 –c Negative DISCUSSION
Salmonella 2 Group B: S36198403c Negative
Serratia odorifera 1 ATCC 33077 Negative We developed a real-time PCR assay for detection of N.
Shigella 1 –c Negative
Staphylococcus aureus 4 ATCC 25923, 29213, Negative gonorrhoeae. The assay targets the opa genes and is highly
43300c specific and very sensitive. The design of the assay is based on
Staphylococcus coagulase negative 1 –c Negative opa gene sequences from N. gonorrhoeae and N. gonorrhoeae-
Staphylococcus epidermidis 1 ATCC 12228 Negative
Staphylococcus marcescens 1 –c Negative related strains obtained from the NCBI database. Primers and
Streptococcus pneumoniae 3 ATCC 49619, 6306c Negative a TaqMan MGB probe were designed covering a 5⬘ untrans-
Streptococcus haemolyticus group B 2 –g Negative
MRSA 2 –c,h Negative
lated region of the opa genes. Of the 448 N. gonorrhoeae strains
Treponema pallidum 1 –c Negative that were analyzed in the assay, 24 strains tested negative.
a
These 24 strains generated PCR products that were not de-
ATCC, American Type Culture Collection; NRBM, Netherlands Reference
Laboratory for Bacterial Meningitis; SKMM, Dutch Organization for Quality tected by the opa-1 probe. Sequence analysis of these PCR
Control in Medical Microbiology. products revealed the same newly identified sequence in all 24
b
Amsterdam Medical Center, Academic Medical Center, Department of
Medical Microbiology, Amsterdam, The Netherlands.
strains. A second probe (opa-2) was designed and included in
c
S. M. Bruisten et al. (7), GG&GD, Municipal Health Service, Amsterdam, the assay to cover this sequence. Because 11 opa genes have
The Netherlands, 2004. been reported to be present in the genome of N. gonorrhoeae,
d
Determined by Vitek NHI (Vitek Systems, Inc., Hazelwood, Mo.); confirmed
by NRBM. it somewhat surprised us to detect only one sequence in the
e
Special Reference Department for Identification of Bacteria (LIS-BBD), PCR products of the 24 opa-1-negative N. gonorrhoeae strains.
National Institute of Public Health and the Environment (RIVM), Bilthoven, Analysis of 21 opa-1-positive N. gonorrhoeae strains using the
The Netherlands.
f
Determined by API-Coryne (bioMérieux, Boxtel, The Netherlands). probes opa-1 and opa-2 separately showed hydrolysis of both
g
Determined by PathoDx latex Strep Grouping Kit (Diagnostic Products probes in only 1 of the 21 strains. Although theoretically a
Corporation, Los Angeles, Calif.).
h
Determined by Staphaurex Plus (Remel Inc., Lenexa, KS); confirmed by
double infection cannot be excluded, it might be possible that
National Institute of Public Health and the Environment (RIVM), Bilthoven, both probe sequences are found in one N. gonorrhoeae strain.
The Netherlands. Whether the other strains harbor just one of the two sequences
VOL. 43, 2005 DETECTION OF N. GONORRHOEAE BY REAL-TIME TaqMan PCR 5657

FIG. 2. (A) Fluorescence profiles of 10-fold serial dilutions in duplicate from 100 fg to 10 ␮g/ml of N. gonorrhoeae DNA obtained from ATCC
strain 49226 and analyzed in the opa-based real-time PCR using probes opa-1 and opa-2. The intensity of fluorescence is given on the y axis (⌬Rn
⫽ reporter signal [FAM]/passive reference signal [ROX]). (B) Standard curve calculated from the Ct values: y ⫽ ⫺3.40 * log{concentration of
DNA in ng/ml} ⫹ 24.35; R2 ⫽ 0.9993.

or harbor both, with one of them being preferentially amplified

during the PCR, remains to be established.
The opa-2 sequence shows 97% homology to one of the
TABLE 4. Results of analysis for evaluation of 16S rRNA
known N. gonorrhoeae opa genes and to three known N. men- N. gonorrhoeae test and opa assay on 122 clinical materials tested
ingitidis opa genes (Fig. 1). Transfer of opa alleles between positive in the COBAS AMPLICOR test for N. gonorrhoeae
neisserial species is rare in nature (22, 41). However, the pos-
opa assayb
Material Assaya
Result No. neg No. pos

TABLE 3. Composition of panel for evaluation of 16S rRNA Urine 16S rRNA Neg 13 1
N. gonorrhoeae test and opa assay on 122 clinical materials tested Pos 0 22
positive in the COBAS AMPLICOR test for N. gonorrhoeaea Cervical swab 16S rRNA Neg 37 2
Pos 0 8
No. of specimens Urethra swab 16S rRNA Neg 2 0
Material
No medium STM medium 2-sp medium Pos 0 6
Throat swab 16S rRNA Neg 29 0
Urine 36 Pos 0 0
Cervical swab 40 7 Anal swab 16S rRNA Neg 2 0
Urethra swab 8 0 Pos 0 0
Throat swab 13 16 Total 16S rRNA Neg 83 3
Anal swab 2 0 Pos 0 36
a a
The 16S rRNA confirmation test was performed in two independent labo- Exactly the same results were obtained in two independent laboratories.
b
ratories. Neg, negative; pos, positive.
5658 GERAATS-PETERS ET AL. J. CLIN. MICROBIOL.

TABLE 5. Detection limits of the 16S rRNA test (single assay) and
the opa assay (duplicate assays)
DNA (fg/␮l) Ct 16S rRNA testa Ct opa assayb
10,000 27.1 24.3 ⫾ 0.0
1,000 32.2 28.4 ⫾ 0.0
100 36.2 32.5 ⫾ 0.1
10 ⬎41.0 35.9 ⫾ 0.3
1 Undetectable 42.4; undetectable
a
5 ␮l of sample volume was added to the reaction mixture.
b
10 ␮l of sample volume was added to the reaction mixture.
sibility of genetic recombination between gonococci and me-
ningococci at the opa gene level might be considered.
The 24 opa-1-negative strains were not related to a certain
time interval; new strains with opa sequences identical to the
second probe are currently being identified. Some sexual part-
ners (two pairs within two separate clusters; clusters are based
on analysis of por and opa genes [reference 7 and M. Kolader,
personal communication]) were both infected with an opa-1-
negative opa-2-positive strain. Based on the observed opa pat-
terns, the 24 strains are not identical but do resemble each
other; they are divided into three subclusters (of nine, nine,
and six strains). With regard to antibiotic resistance, no corre-
lation was found.
We evaluated the specificity of the opa assay by testing
non-gonorrhoeae Neisseriaceae and other bacteria and by as-
saying clinical samples by means of various N. gonorrhoeae
tests. Analysis of a large panel of N. gonorrhoeae-related and
other microorganisms displayed no cross-reactivity with other
Neisseriaceae or with any other microorganisms tested so far.
Of 122 clinical samples that tested positive in the COBAS
AMPLICOR test, 36 tested positive in the 16S rRNA test and
in the opa assay. This confirms that the COBAS AMPLICOR
test produces false-positive results and needs a subsequent
confirmation assay(s) (12, 15, 30, 38). In addition to the 36 16S
rRNA-positive (and opa assay-positive) samples, the opa assay
showed three more samples as positive. The fact that the three
samples showed the highest opa assay Ct values of all the
positive samples present in the panel suggests that the discrep-
ancy is due to the difference in detection levels of the 16S
rRNA PCR and the opa assay. The third sample of the three,
an STM cervical swab, showed a very weak signal in one of two
cppB PCRs carried out on the specimen.
For the detection of asymptomatic N. gonorrhoeae infec-
tions, a low detection limit is of crucial importance. By mea-
suring the DNA content in a spectrophotometer and theoret-
ically calculating the number of bacteria based on genome
weight, we determined that approximately 0.4 bacterial DNA
copies were detected in four out of six reactions in the opa
assay. Comparison of the opa assay with the 16S rRNA PCR
showed a fivefold-higher sensitivity of the opa assay. Besides
the larger specimen volume added to the opa PCR, this might
be due to the higher copy number of the opa gene versus the
16S rRNA gene (11 [4] versus 4 [17], respectively) in the N.
gonorrhoeae genome.
Analyses of the QCMD panels exemplified the high sensi-
tivity of the opa assay. Two samples from the 2003 panel with
low N. gonorrhoeae copy numbers, which were reported cor-
rectly by only half the laboratories that participated in the
evaluation of the panel and by only 33% of the laboratories
that used the COBAS AMPLICOR test, were easily detected
in the opa-based assay (Cts, 33.2 and 33.5).
We conclude that the opa gene-based real-time amplifica-
tion assay that we have developed is a sensitive and reliable
assay for the detection of N. gonorrhoeae in clinical specimens.

TABLE 6. Evaluation of QCMD N. gonorrhoeae panels 2003 and 2004

Resultsa % Correct results

COBAS
QCMD code 16S rRNA opa assay COBAS
AMPLICOR QCMD All laboratories
AMPLICOR
Lab 1 Lab 2 Lab 1 Lab 2 Result Ct

NG03-01 0.005 0.004 Neg Neg Neg Undet Neg 100 100
NG03-02 0.004 0.004 Neg Neg Neg Undet Neg 100 100
NG03-03 3.873 ⬎4.000 Pos Pos Pos 28.4 Pos 100 97
NG03-04 0.006 2.771 Neg Neg Pos 33.2 Pos 33 48
NG03-05 2.541 ⬎4.000 Neg Pos Pos 31.2 Pos 85 92
NG03-06 0.004 0.004 Neg Neg Neg Undet Neg 100 98
NG03-07 2.157 2.598 Neg Pos Pos 33.5 Pos 33 53
NG03-08 3.873 3.402 Pos Pos Pos 30.8 Pos 82 90
NG03-09 3.873 ⬎4.000 Pos Pos Pos 26.5 Pos 91 81
NG03-10 ⬎4.000 3.946 Pos Pos Pos 28.1 Pos 91 95

NG04-01 2.353 3.208 Pos Pos Pos 29.8 Pos 100 96

NG04-02 0.004 0.001 Neg Neg Neg Undet Neg 96 93
NG04-03 2.359 ⬎4.000 Neg Pos Pos 31.3 Pos 81 84
NG04-04 ⬎4.000 ⬎4.000 Pos Pos Pos 25.7 Pos 100 87
NG04-05 1.223 1.213 Pos Pos Pos 31.6 Pos 54 71
NG04-06 ⬎4.000 ⬎4.000 Neg Neg Neg Undet Neg 19 62
NG04-07 1.998 3.647 Pos Pos Pos 28.9 Pos 96 97
NG04-08 0.003 0.002 Neg Neg Neg Undet Neg 92 77
NG04-09 2.064 3.404 Neg Pos Pos 30.7 Pos 69 81
NG04-10 3.092 3.948 Pos Pos Pos 29.8 Pos 89 91
a
Neg, negative; Pos, positive; Undet, undetermined.
VOL. 43, 2005 DETECTION OF N. GONORRHOEAE BY REAL-TIME TaqMan PCR
ACKNOWLEDGMENTS
We thank Birgitta Duim and Lodewijk Spanjaard of the Amsterdam
Medical Center, Amsterdam, The Netherlands, for providing us with
non-gonorrhoeae strains of Neisseria. We thank Roelof Pruntel of the
Dutch Cancer Institute (NKI), Amsterdam, The Netherlands, for se-
quencing the “aberrant” opa PCR fragments. We cordially thank Colin
Ingham for his valuable comments on the manuscript.
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