Characteristics of the m2000 automated sample preparation and multiplex real-time PCR system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

We evaluated a new real-time PCR-based prototype assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae developed by Abbott Molecular Inc. This assay is designed to be performed on an Abbott m2000 real-time instrument system, which consists of an m2000sp instrument for sample preparation and an m2000rt instrument for real-time PCR amplification and detection. The limit of detection of this prototype assay was determined to be 20 copies of target DNA for both C. trachomatis and N. gonorrhoeae, using serially diluted linearized plasmids. No cross-reactivity could be detected when 55 nongonococcal Neisseria isolates and 3 non-C. trachomatis Chlamydia isolates were tested at 1 million genome equivalents per reaction. Concordance with the Roche Amplicor, BDProbeTec ET, and Gen-Probe APTIMA Combo 2 tests was assessed using unlinked/deidentified surplus clinical specimens previously analyzed with these tests. For C. trachomatis, concordance for positive results ranged from 93.7% to 100%, while concordance for negative results ranged from 98.2% to 100%. For N. gonorrhoeae, concordance for positive and negative results ranged from 91.4% to 100% and 99.3% to 100%, respectively. A workflow analysis of the prototype assay was conducted to obtain information on throughput under laboratory conditions. At 48 samples/run, the time to first result for both C. trachomatis and N. gonorrhoeae was 4.5 h. A total of 135 patient specimens could be analyzed in 8.9 h, with 75 min of hands-on time. This study demonstrated the technical and clinical feasibility of the new Abbott real-time PCR C. trachomatis/N. gonorrhoeae assay.

Insertion mutations in pilE differentially alter gonococcal pilin antigenic variation.

Pilus antigenic variation in Neisseria gonorrhoeae occurs by the high-frequency, unidirectional transfer of DNA sequences from one of several silent pilin loci (pilS) into the expressed pilin gene (pilE), resulting in a change in the primary pilin protein sequence. Previously, we investigated the effects of large or small heterologous insertions in conserved and variable portions of a pilS copy on antigenic variation. We observed differential effects on pilin recombination by the various insertions, and the severity of the defect correlated with the disruption or displacement of a conserved pilin DNA sequence called cys2. In this study, we show that disruption or displacement of the pilE cys2 sequence by the same insertions or a deletion also affects pilin recombination. However, in contrast to the insertions in pilS, the analogous insertions in pilE impaired, but did not block, recombination of the flanking pilin sequences. These results, the change in the spectrum of donor silent copies used during variation, and our previous results with pilS mutations show that the donor pilS and recipient pilE play different roles in antigenic variation. We conclude that when high-frequency recombination mechanisms are blocked, alternative mechanisms are operative.

Molecular models accounting for the gene conversion reactions mediating gonococcal pilin antigenic variation.

The pilus antigenic variation (Av) system of Neisseria gonorrhoeae is one of several high-frequency variation systems that utilize gene conversion to switch between numerous forms of an antigen on the cell surface. We have tested three predictions of the first models that explain the movement of DNA during pilin Av: (i) Av requires two recombinations at short regions of identity, (ii) circular intermediates exist that carry pilE/pilS hybrid loci and (iii) these pilE/pilS hybrid loci target the pilS sequences to a recipient pilE gene. We confirm that normal pilin Av utilizes recombination at very short regions of DNA sequence identity and that these recombination events can occur independent of homologous recombination functions. We have isolated covalently closed circular DNA molecules carrying hybrid pilin loci, but propose that an alternative hybrid molecule is the intermediate of pilin Av. Our most striking finding is that transformation of isolated pilE/pilS hybrid loci targets the pilS sequences of the hybrid to a recipient pilE at frequencies much higher than normal recombination frequencies. These results show that the different steps of a model that explains pilin Av can be separately tested to support the validity of these novel models that account for the high-frequency gene conversions that mediate pilin Av.

The size and position of heterologous insertions in a silent locus differentially affect pilin recombination in Neisseria gonorrhoeae.

Gonococcal pilus antigenic and phase variation result from unidirectional, RecA-dependent recombination of DNA sequences from a silent pilin copy (pilS) into the expressed pilin gene (pilE). To develop a quantitative assay for pilin gene recombination that is independent of phase variation, a promoterless cat gene was inserted into pilS, and recombination of "cat into pilE was detected by selection of chloramphenicol-resistant (CmR) variants expressing "cat from the pilin promoter. Although RecA-dependent CmR variants occurred, none were generated by the simple transfer of "cat into pilE. Instead, each CmR variant contained a new pilin locus that was a hybrid of sequences from the pilE and the pilS1::cat loci in addition to the two starting loci. Therefore, this system could not be used to quantify antigenic variation. However, combined studies of these hybrid loci and of recombination products generated during additional pilS mutational analyses demonstrated that both the size and position of an insertion in pilS differentially affect pilin recombination. Also, the hybrid loci appear to be intermediates of antigenic variation. This enabled the creation of molecular models for the recombination reactions that result in pilin antigenic variation.