Use of Applied Biosystems 7900HT Sequence Detection System and Taqman Assay for Detection of Quinolone-Resistant Neisseria gonorrhoeae

Mutations in quinolone resistance-determining regions (QRDRs)have been associated with quinolone-resistant Neisseria gonorrhoeae(QRNG). Since diagnostic nucleic acid amplification tests forgonococci are now in frequent use, molecular detection of QRNGcould facilitate surveillance in the absence of culture. Herewe describe a real-time molecular assay for detecting QRDR mutationsin gonococci.

INTRODUCTION

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Introduction

Quinolone-resistant Neisseria gonorrhoeae (QRNG) strains arerapidly emerging. Well-characterized quinolone resistance-determiningregion (QRDR) mutations correlate with decreased gonococcalantimicrobial susceptibility to fluoroquinolones (MIC, $≥$ 1 µg/ml)(2-6, 8-10, 13, 19, 21, 24, 26-28).

Using well-characterized isolates, we developed a method fordetecting QRDR mutations utilizing Taqman chemistry and theABI 7900HT Prism sequence detector (Applied Biosystems, FosterCity, Calif.).

N. gonorrhoeae strains.

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N. gonorrhoeae strains.

We evaluated 80 isolates collected in 2000 to 2001 in Israelthat were characterized previously (9, 29).

DNA isolation, QRDR amplification, and direct sequencing.

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Dna isolation, qrdr...

Genomic DNA was purified (Promega Wizard, Promega Corp., Madison,Wis.), and QRDRs were amplified (11, 12). The forward primersused were GyrA Forward (NG-GYRA-Z; 5'-CAAATTCGCCCTCGAAACCCT-3';nucleotides [nt] 30 to 50 of the gyrA gene, 368-bp product)and ParC Forward (NG-PARC-Z; 5'-GCCCGTGCAGCGGCGCAT-3'; nt 138to 155 of the parC gene, 219-bp product). Reaction mixtureshad a 50-µl total volume: 25 µl of PCR Master mix,22 µl of sterile water, 1 µl each of forward andreverse primers, and 1 µl of DNA template. PCR productswere sequenced with forward primers, and data were aligned withQRDR sequences for amino acids (aa) 91 to 95 of gyrA (GenBankaccession no. U08817) and aa 86 to 92 of parC (GenBank accessionno. U08907). The three mutation patterns identified are shownin Table 1.

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TABLE 1. QRDR mutation patterns for gonococcal samples as determined by direct sequencing and real-time PCR detection with the ABI 7900HT sequence detection system

ABI primer and probe design.

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Abi primer and probe...

Primers and probes (Table 2) were developed by using PrimerExpress 2.0 software (Applied Biosystems, Foster City, Calif.).Probes encompassed aa 91 and 95 of gyrA and aa 86, 87, and 88of parC, the loci most often associated with resistance (2-6,8-10, 13, 19, 21, 24, 26-28). Using National Center for BiotechnologyInformation BLAST analysis, primers and probes were designedto match only gonococcal target sequences.

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TABLE 2. Primers and probes for the ABI QRNG QRDR mutation detection system

ABI real-time PCR.

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Abi real-time pcr.

Findings.

Conclusions.

References

Diplex PCRs were performed in 96-well plates with the followingper well: 25 µl of Promega PCR Master mix, 21 µlof sterile water, 0.2 µl of each set of forward and reverseprimers (0.2 µM final concentration of each), 0.5 µlof each probe (0.2 µM final concentration of each), and1 µl of DNA template, for a total reaction volume of 50µl. Cycles included one 2-min hold (50°C); a 10-mindenaturation (95°C); and 40 cycles of denaturation (95°Cfor 30s), annealing (60°C for 30s), and extension (72°Cfor 30s). Control wells included blanks, wild-type (WT) strains,strains with one mutation, and strains with multiple mutations,as determined by sequencing. The lower-level detection limitwas established at five genome copies by using published methods(15).

Findings.

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Abi real-time pcr.

Findings.

Conclusions.

References

All 42 ciprofloxacin-resistant (Cip^r) isolates had mutationsin both gyrA and parC QRDRs, identical in 93%. One intermediatelyresistant (Cipⁱ) isolate had one mutation in gyrA. Susceptiblestrains (Cip^s) were WT (Table 1).

Fluorescence data were analyzed with ABI 7900HT Prism sequencedetector software (Applied Biosystems, Foster City, Calif.)and are shown in Fig. 1. Ct is defined as the first PCR cyclein which parametric increases of fluorescence are detected andis an indicator of successful PCR, as well as specific annealingof probe and successful exonucleic cleavage of reporter molecule.

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FIG. 1. QRDR amplification curves and multicomponent data plots from the ABI 7900HT sequence detection system. A, B, and C refer to sample types for which sequence data are given in Table 1. Each plot represents diplex results for one sample. Upper set of curves show relative fluorescence versus PCR cycle number. The lower set of curves depict the relative fluorescence of each fluorophore as compared to that of the background over time.

Real-time PCR, sequencing, and MICs correlated 100%. The amplificationplot for a WT strain (Table 1, type A; ciprofloxacin MICs, <0.125µg/ml) shows exponential signal increase. This indicatesWT strains were positively amplified, with mean Cts of 30.6± 3.21 cycles (n = 38) and 25.2 ± 2.11 cycles(n = 38) for the gyrA and parC loci, respectively.

For mutant strains (Table 1, types B and C), no exponentialfluorescence increases were observed (n = 42). The Cipⁱ strain(Table 1, type B, ciprofloxacin MIC = 0.25 µg/ml) showedsignal amplification for the parC locus only (Ct for parC =23.2, Ct for gyrA = 40.0). Cip^r strains (Table 1, type C; ciprofloxacinMICs, $≥$ 1 µg/ml) showed signal amplification at neitherlocus (Ct = 40.0). Curves were analogous when either one ortwo mutations were present.

Multicomponent data analysis was used to set baseline backgroundlevels (Fig. 1) and shows acceptable background and significantincreases in fluorescence over time for the WT. Combined withrelative fluorescence increase, this indicates a successfulassay.

Conclusions.

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Abi real-time pcr.

Findings.

Conclusions.

References

With widespread use of nucleic acid amplification tests (NAATs),antimicrobial resistance detection will require molecular methods,as has been described for other organisms (1, 7, 14, 16-18,25). QRNG detection is a good model for this approach sincethe resistance mechanisms are based on stepwise accumulationof point mutations correlating with increased MICs (20, 22,23).

This approach has disadvantages. Because mutations prevent reportercleavage, negative results would give results similar to thosefor mutant strains. Thus, this screening tool can only be appliedto samples which test gonococcus positive by other methods,such as commercially available NAATs. Our lower detection limitwas 5 WT genome copies, similar to that of widely used NAATs.However, further investigations using different NAATs with variousgonococcus-DNA concentrations need to be performed. Anotherlimitation is the possibility of detecting synonymous mutations,which translate as "false mutants," although the frequency ofsynonymous mutations at these loci appears to be very low. Outliermutations have been observed at loci not targeted by our probes(6, 19, 27, 28), but these occur infrequently and have onlybeen observed in the presence of at least one mutation detectablewith our probes. We intended to develop an assay to screen forclinically important mutations (i.e., those associated witha ciprofloxacin MIC of $≥$ 4 µg/ml, requiring a change intherapy) and not for definitive genetic analysis. Real-timefluorometric PCR systems can, however, be adapted to screenfor resistance-associated gonococcal QRDR mutations and canpotentially be applied to NAAT samples.

FOOTNOTES

* Corresponding author. Mailing address: Division of Allergies and Infectious Disease, Johns Hopkins University School of Medicine, Baltimore, MD 21205. Phone: (410) 614-4480. Fax: (410) 614-9775. E-mail: jgiles{at}jhmi.edu.

REFERENCES

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