A Highly Sensitive, Multiplex Broad-Spectrum PCR-DNA-Enzyme Immunoassay and Reverse Hybridization Assay for Rapid Detection and Identification of Chlamydia.

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A Highly Sensitive, Multiplex Broad-Spectrum PCR-DNA-Enzyme Immunoassay and Reverse Hybridization Assay for Rapid Detection and Identification of Chlamydia trachomatis Serovars Koen D. Quint, Leen-Jan van Doorn, Bernhard Kleter, Maurits N.C. de Koning, Henk A.M. van den Munckhof, Servaas A. Morre, Bram ter Harmsel, Elisabete Weiderpass, Gonneke Harbers, Willem J.G. Melchers, Wim G.V. Quint The Journal of Molecular Diagnostics Volume 9, Issue 5, Pages 631-638 (November 2007) DOI: 10.2353/jmoldx.2007.070011 Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Alignment of the 157- to 160-bp target sequences from the VD2 omp1 gene, which are amplified by the Ct-amplification step, generating region D. Regions A and E are the targets for forward and reverse primers, respectively. The interprimer region (B) is used as target for serovar-specific probes. Serogroup-specific probes are aimed at region C. The reference sequences that were used for this alignment are as follows: Serovar A, M58938; Serovar B, DQ064281; Serovar C, AF352789; Serovar D, AF063196; Serovar E, AY535104; Serovar F, AY535123; Serovar G, AY535136; Serovar H, AY535137; Serovar Ia, AY535150; Serovar J, AY535161; Serovar K, AY535167; Serovar L1, M36533; Serovar L2, DQ064295; and Serovar L3, X55700. The Journal of Molecular Diagnostics 2007 9, 631-638DOI: (10.2353/jmoldx.2007.070011) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Outline and representative example of the RHA C. trachomatis genotyping step. The positions of the control line and the 19 specific probes for the cryptic plasmid, three different serogroups, and 15 serovars are shown at the left. The letter above each strip indicates the Ct serovar for which DNA was amplified by the multiplex broad spectrum PCR. The exact interpretation of hybrid patterns is described in Materials and Methods. J* was found as a clinical isolate. The Journal of Molecular Diagnostics 2007 9, 631-638DOI: (10.2353/jmoldx.2007.070011) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Limiting dilution series of C. trachomatis DNA, analyzed for the presence of the plasmid and the omp1 gene. A: Agarose gel (3%) showing amplicons from the dilution series of Ct serovar L1. B: DEIA and Cobas TaqMan results. The Cobas TaqMan internal control shows no inhibition. C: RHA results of the corresponding amplicons, which agree with the results of the agarose gel. The Journal of Molecular Diagnostics 2007 9, 631-638DOI: (10.2353/jmoldx.2007.070011) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Examples of results that were found with the RHA. Table 3 shows the results after interpretation of the RHA strip (X, unknown). The Journal of Molecular Diagnostics 2007 9, 631-638DOI: (10.2353/jmoldx.2007.070011) Copyright © 2007 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions