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

Abstract

Mutations in quinolone resistance-determining regions (QRDRs) have been associated with quinoloneresistant Neisseria gonorrhoeae (QRNG). Since diagnostic nucleic acid amplification tests for gonococci are now in frequent use, molecular detection of QRNG could facilitate surveillance in the absence of culture. Here we describe a real-time molecular assay for detecting QRDR mutations in gonococci.Quinolone-resistant Neisseria gonorrhoeae (QRNG) strains are rapidly emerging. Well-characterized quinolone resistancedetermining region (QRDR) mutations correlate with decreased gonococcal antimicrobial susceptibility to fluoroquinolones (MIC, Ն1 g/ml) (2-6, 8-10, 13, 19, 21, 24, 26-28).Using well-characterized isolates, we developed a method for detecting QRDR mutations utilizing Taqman chemistry and the ABI 7900HT Prism sequence detector (Applied Biosystems, Foster City, Calif.).N. gonorrhoeae strains. We evaluated 80 isolates collected in 2000 to 2001 in Israel that were characterized previously (9, 29).DNA isolation, QRDR amplification, and direct sequencing. Genomic DNA was purified (Promega Wizard, Promega Corp., Madison, Wis.), and QRDRs were amplified (11, 12). The forward primers used were GyrA Forward (NG-GYRA-Z; 5Ј-CAAATTCGCCCTCGAAACCCT-3Ј; nucleotides [nt] 30 to 50 of the gyrA gene, 368-bp product) and ParC Forward (NG-PARC-Z; 5Ј-GCCCGTGCAGCGGCGCAT-3Ј; nt 138 to 155 of the parC gene, 219-bp product). Reaction mixtures had a 50-l total volume: 25 l of PCR Master mix, 22 l of sterile water, 1 l each of forward and reverse primers, and 1 l of DNA template. PCR products were sequenced with forward primers, and data were aligned with QRDR sequences for amino acids (aa) 91 to 95 of gyrA (GenBank accession no. U08817) and aa 86 to 92 of parC (GenBank accession no. U08907). The three mutation patterns identified are shown in Table 1.ABI primer and probe design. Primers and probes (Table 2) were developed by using Primer Express 2.0 software (Applied Biosystems, Foster City, Calif.). Probes encompassed aa 91 and 95 of gyrA and aa 86, 87, and 88 of parC, the loci most often associated with resistance (2-6, 8-10, 13, 19, 21, 24, 26-28). Using National Center for Biotechnology Information BLAST analysis, primers and probes were designed to match only gonococcal target sequences.ABI real-time PCR. Diplex PCRs were performed in 96-well plates with the following per well: 25 l of Promega PCR Master mix, 21 l of sterile water, 0.2 l of each set of forward and reverse primers (0.2 M final concentration of each), 0.5 l of each probe (0.2 M final concentration of each), and 1 l of DNA template, for a total reaction volume of 50 l. Cycles included one 2-min hold (50°C); a 10-min denaturation (95°C); and 40 cycles of denaturation (95°C for 30s), annealing (60°C for 30s), and extension (72°C for 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 limit was established at five genome copies by using publi...

A 5′ Nuclease Genotyping Assay for Identification of Macrolide-Resistant Mycoplasma genitalium in Clinical Specimens

“…Highresolution melting (HRM) analysis is another powerful tool to detect mutations in a target sequence, and PCR assays using HRM analysis for detecting mutations associated with macrolide resistance have recently been reported (10,11). An alternative method for identifying known mutations is genotyping assays using differentially labeled hydrolysis probes targeting wild-type and mutated alleles (12,13). In the present study, we report such a 5= nuclease genotyping assay for the detection of the macrolide resistance-associated mutations in M. genitalium.…”

Real-Time PCR Assay for Detection of Quinolone-Resistant Neisseria gonorrhoeae in Urine Samples

“…In response, we restricted our assay to the detection of one codon (Ser91) and shortened our amplification length to 133 bp in an attempt to increase sensitivity in specimens with lower DNA concentrations. We accomplished this by using a primer already documented as effective in generating amplicons within gyrA (9). Pairwise comparison of the modified assay described here with that described by Li et al revealed an average sensitivity increase of 3.44 crossing point values when generating the smaller amplicon (Table 2).…”

“…The sequencing and biochip methods might be untenable in locales with limited resources. Real-time PCR techniques using TaqMan-style probes (Applied Biosystems, Foster City, CA) exclusively detect wild-type strains, with negative results interpreted as presumed resistant strains (9). The lack of internal control might therefore indicate resistance when, in actuality, laboratory error or other causes of failed wild-type amplification (e.g., low concentrations of DNA) has led to this result.…”

“…In response, methods for detecting QRNG have been developed and reported in the scientific literature that include sequencing (12), microarray biochip (5,13,19), and other molecular techniques (9,11). However, each of these techniques has disadvantages with regard to detecting resistance in certain settings.…”

Rapid Screening of Fluoroquinolone Resistance Determinants in Streptococcus pneumoniae by PCR-Restriction Fragment Length Polymorphism and Single-Strand Conformational Polymorphism

“…Methods for detection of known mutations in the QRDRs have included PCR-restriction fragment length polymorphism (PCR-RF) (1), oligonucleotide probe assay (6), TaqMan assay (8), and single-strand conformational polymorphism (SSCP) (27). Often, these methods are not designed to have, or do not have, a sufficiently high sensitivity or resolution to identify new mutations.…”