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Journal of Clinical Microbiology, November 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 Medical Microbiology and Infectious Diseases, Gelre Hospitals, Location Lukas,Apeldoorn ,2 GG&GD, Municipal Health Service, Amsterdam,3 Laboratory for Pathology and Microbiology, PAMM, Veldhoven, The Netherlands4

Received 4 March 2005/ Returned for modification 19 May 2005/ Accepted 11 August 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Early diagnosis of Neisseria gonorrhoeae infections is important with regard to patients' health and infectivity. 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 5' 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 Diagnostics 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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
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 sexual partners and lead to long-term consequences, such as pelvic inflammatory disease, chronic pelvic pain, ectopic pregnancy, 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 incidence 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 specimen collection, transport, and storage. Nucleic acid amplification-based techniques, including the ligase chain reaction, strand displacement amplification assay, nucleic acid sequence-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 application 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 Diagnostics Nederland BV, Almere, The Netherlands), for instance, produces false-positive results with certain nonpathogenic Neisseria species (Neisseria subflava and Neisseria cinerea) and lactobacilli, and a subsequent confirmation test is necessary (5, 12, 15, 30, 38). CppB- and 16S rRNA gene-based assays are used for confirmation (35); however, about 5% of N. gonorrhoeae 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 problems (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 colony 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 meningitidis 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 separate loci (opaA through -K) (4) and are subject to on/off phase variation. Changes in the repetitive sequences within the various 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 proteoglycans (3, 9, 21, 40, 42) and gonococcal interactions with polymorphonuclear leukocytes (19). Furthermore, Opa proteins enhance resistance to complement-mediated killing (6). Because 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 epidemiological 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 performed at the Public Health Laboratory in Amsterdam as described previously (8). In the context of a communal epidemiology study, we typed these N. gonorrhoeae 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 lyophilized 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 material. 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 µl 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 µl 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 µl, 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 µl 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 µl. 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 µl was added in the COBAS AMPLICOR, 5 µl was added in the 16S rRNA tests, and 10 µl was added to the opa PCR.

(iii) Other clinical samples. Dry urethra or cervical swabs (plastic minitip swab 185CS01; Copan, AMDS-Benelux, Malden, The Netherlands) were placed in 500 µl 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 x g for 15 min, and the supernatant was removed. The remaining pellet was dissolved in 300 µl of TE and incubated for 30 min at 97°C; 10 µl was used in the PCR.

If inhibition occurred in the PCR (see below), DNA was isolated from 190 µl of sample, to which 10 µl 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 µl; 10 µl 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-µl PCR was performed containing 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 3 mM MgCl2 (prepared from 10x PCR buffer delivered with Platinum Taq polymerase), 0.75 U Platinum Taq polymerase (Invitrogen BV, Breda, The Netherlands), 4% glycerol (molecular biology grade; CalBiochem, VWR International BV, Amsterdam, The Netherlands), 200 µM of each deoxynucleoside triphosphate (Amersham Bioscience, Roosendaal, The Netherlands), 0.5 µl 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 µl sample (5 µl DNA was used when analyzing the panel of 448 N. gonorrhoeae strains; 10 µl 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 µl of combined exonuclease I (10 U/ml) and shrimp alkaline phosphatase (2 U/µl) (USB Corporation; distributor, Amersham Bioscience, Roosendaal, The Netherlands) to 20 µl 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 µl purified PCR product, 4 µl 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 sequence 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 µl. DNA was quantified by photospectrometer (Eppendorf BioPhotometer; Eppendorf, Hamburg, Germany) and found to be 18.85 ng/µl (1:1 dilution [A260, 0.192; A280, 0.107; concentration, 18.6 ng/µl] and 1:4 dilution [A260, 0.099; A280, 0.056; concentration, 19.1 ng/µl]). Tenfold serial dilutions were made containing 10 µg/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 x 106 bp [11] x 665 Da/bp x 1.67 x 10–24 g/Da).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 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 Express 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.



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  		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).

 

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  		TABLE 1. Sequences of primers and probes used for real-time N. gonorrhoeae detection (13)

 
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 revealed exactly the same sequence in all 24 strains (Fig. 1). The MGB probe opa-2 was designed to cover this additional sequence, which was included in subsequent assays. We subsequently analyzed DNAs from 21 opa-1-positive N. gonorrhoeae strains from the above-mentioned panel using the N. gonorrhoeae assay with only the probe set for opa-2. One out of 21 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. gonorrhoeae 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 isolates. No signal in the opa real-time PCR was observed with any of the microorganisms tested using probes opa-1 and opa-2.


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  		TABLE 2. Reactivity of the opa-based N. gonorrhoeae assay (probes opa-1 and opa-2) with various species of Neisseria and other microorganisms

 
Sensitivity. DNA was isolated of from N. gonorrhoeae ATCC strain 49226 as described in Materials and Methods and diluted to an undetectable level. Tenfold serial dilutions ranging from 100 fg to 10 µg 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 reactions.



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  		FIG. 2. (A)Fluorescence profiles of 10-fold serial dilutions in duplicate from 100 fg to 10 µg/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 ({Delta}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.

 
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 samples (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 and the opa assay. The 16S rRNA confirmation test was carried out in two independent laboratories, and both laboratories obtained exactly the same results. Thirty-six samples were found to be positive and 83 samples were negative in all three tests (Table 4). The remaining three samples that were negative in the 16S rRNA test were positive in the opa assay. They encompassed a urine sample (COBAS AMPLICOR 1.018, 0.429, and 0.366; 16S rRNA negative; opa assay, Ct = 34.9 and 35.2) and two STM cervix swabs (COBAS AMPLICOR 1.553 and 1.855; 16S rRNA negative; opa assay, Ct = 34.6 and 34.9, and COBAS AMPLICOR 3.876 and >3.999; 16S rRNA negative; opa assay, Ct = 36.2 and 38.0). The fact that the three samples showed the highest opa assay Ct values of all the positive samples in the panel suggested that the discrepancy could be due to a slight difference in the detection levels of the 16S rRNA PCR and the opa assay. We therefore analyzed a dilution series of N. gonorrhoeae DNA in both assays on the same day. The results of this test revealed a 5- to 10-fold difference in sensitivity between the 16S rRNA PCR and the opa PCR (Table 5), which might be partly due to the difference in sample input volume (5 µl in the 16S rRNA PCR versus 10 µl in the opa PCR).


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  		TABLE 3. Composition of panel for evaluation of 16S rRNA N. gonorrhoeae test and opa assay on 122 clinical materials tested positive in the COBAS AMPLICOR test for N. gonorrhoeaea

 

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  		TABLE 4. Results of analysis for evaluation of 16S rRNA N. gonorrhoeae test and opa assay on 122 clinical materials tested positive in the COBAS AMPLICOR test for N. gonorrhoeae

 

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  		TABLE 5. Detection limits of the 16S rRNA test (single assay) and the opa assay (duplicate assays)

 
QCMD panels. We analyzed QCMD panels that were distributed in 2003 and 2004 in the COBAS AMPLICOR (two laboratories), in the 16S rRNA test (two laboratories), and in the opa-based assay. The results are shown in Table 6. Sample NG03-04 was found to be negative in one laboratory in the COBAS AMPLICOR test and in both laboratories in the 16S rRNA test. Samples NG03-05, NG03-07, NG04-03, and NG04-09 were missed in one of the two laboratories in the 16S rRNA test. Sample NG04-06 was false positive in the COBAS AMPLICOR test. The opa assay detected all samples that contained N. gonorrhoeae correctly. The Cts of samples NG03-04 and NG03-07 (indicated as Pos [+/–] by QCMD) were 33.2 and 33.5, respectively.


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

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We developed a real-time PCR assay for detection of N. gonorrhoeae. The assay targets the opa genes and is highly specific and very sensitive. The design of the assay is based on opa gene sequences from N. gonorrhoeae and N. gonorrhoeae-related strains obtained from the NCBI database. Primers and a TaqMan MGB probe were designed covering a 5' untranslated region of the opa genes. Of the 448 N. gonorrhoeae strains that were analyzed in the assay, 24 strains tested negative. These 24 strains generated PCR products that were not detected by the opa-1 probe. Sequence analysis of these PCR products revealed the same newly identified sequence in all 24 strains. A second probe (opa-2) was designed and included in the assay to cover this sequence. Because 11 opa genes have been reported to be present in the genome of N. gonorrhoeae, it somewhat surprised us to detect only one sequence in the PCR products of the 24 opa-1-negative N. gonorrhoeae strains. Analysis of 21 opa-1-positive N. gonorrhoeae strains using the probes opa-1 and opa-2 separately showed hydrolysis of both probes in only 1 of the 21 strains. Although theoretically a double infection cannot be excluded, it might be possible that both probe sequences are found in one N. gonorrhoeae strain. Whether the other strains harbor just one of the two sequences 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 known N. gonorrhoeae opa genes and to three known N. meningitidis opa genes (Fig. 1). Transfer of opa alleles between neisserial species is rare in nature (22, 41). However, the possibility of genetic recombination between gonococci and meningococci 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 partners (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 patterns, 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 correlation was found.

We evaluated the specificity of the opa assay by testing non-gonorrhoeae Neisseriaceae and other bacteria and by assaying 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 discrepancy 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 infections, a low detection limit is of crucial importance. By measuring the DNA content in a spectrophotometer and theoretically 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 sensitivity of the opa assay. Two samples from the 2003 panel with low N. gonorrhoeae copy numbers, which were reported correctly 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 amplification assay that we have developed is a sensitive and reliable assay for the detection of N. gonorrhoeae in clinical specimens.


   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 sequencing the "aberrant" opa PCR fragments. We cordially thank Colin Ingham for his valuable comments on the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: Multidisciplinary Laboratory of Molecular Diagnostics, Jeroen Bosch Hospital, M. H. A. Hermans, 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{at}jbz.nl. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Clinical Microbiology, November 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.



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