Specific andSensitive Detection of Neisseria gonorrhoeae in ClinicalSpecimens by Real-Time PCR

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 (C_t = 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 100μ 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 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 10μ l 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 × g for 15 min, and the supernatant was removed. The remaining pellet was dissolved in 300μ l of TE and incubated for 30 min at 97°C; 10μ l 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 C_t value of approximately 30 (38). If the C_t 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 10× 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), 0.5μ 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 μl sample (5μ l 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 C_t 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).

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).

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 C_t 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.

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 (ΔRn = reporter signal [FAM]/passive reference signal [ROX]). (B) Standard curve calculated from the C_t 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, C_t = 34.9 and 35.2) and two STM cervix swabs (COBAS AMPLICOR 1.553 and 1.855; 16S rRNA negative; opa assay, C_t = 34.6 and 34.9, and COBAS AMPLICOR 3.876 and >3.999; 16S rRNA negative; opa assay, C_t = 36.2 and 38.0). The fact that the three samples showed the highest opa assay C_t 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 (5μ l in the 16S rRNA PCR versus 10 μl in the opa PCR).

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 C_ts of samples NG03-04 and NG03-07 (indicated as Pos [+/−] by QCMD) were 33.2 and 33.5, respectively.