Development and Characterization of a Synthetic DNA, NUversa, to Be Used as a Standard in All Quantitative PCR Reactions for Molecular Pneumococcal Serotyping

BACKGROUND: Identification of Streptococcus pneumoniae (Spn) and its more than 90 serotypes is routinely conducted by culture and Quellung reactions. Quantitative (q)PCR reactions have been developed for molecular detection, including a pan-Spn lytA assay, and assays targeting 78 serotypes. Reactions require genomic DNA from every target to prepare standards, which can be time consuming. In this study we have developed a synthetic DNA molecule as a surrogate for genomic DNA and present new single-plex qPCR reactions to increase molecular detection to 94 pneumococcal serotypes. METHODS: Single-plex qPCR reactions (N = 11) that detect 16 pneumococcal serotypes/serogroups were developed and concentration of primer and probe optimized to obtain a recommended efficiency between 90 and 110%. Specificity for the target serotype/serogroup of these new reactions was investigated using a collection of strains belonging to our laboratory and strains kindly donated by the “StrepLab” at CDC. A synthetic DNA (NUversa, ~8.2 kb) was then engineered to contain all available qPCR targets for serotyping and lytA. NUversa was cloned into pUC57-Amp-modified to generate pNUversa (~10.2 kb). Standards prepared from pNUversa and NUversa were compared against standards made out of genomic DNA. RESULTS: Specificity of these new reactions was confirmed, and after optimization, the obtained limit of detection (LOD) was between 2 and 20 genome equivalents/reaction. Molecular studies demonstrated that linearity [NUversa (R(2)>0.982); pNUversa (R(2)>0.991)] and efficiency of qPCR reactions using synthetic DNA were similar to those utilizing chromosomal DNA (R(2)>0.981). Quantification, however, with plasmid pNUversa (Y-Int=43.0 ± 1.12) was affected whereas that using synthetic NUversa (Y-Int=40.3 ± 1.08) was comparable to genomic DNA (Y-Int=39.9 ± 0.62). CONCLUSION: We validated new single-plex reactions that, together with published qPCR reactions, now make possible to detect and quantify 94 pneumococcal serotypes/serogroups. NUversa can be utilized as a control in most, if not all, published single-plex qPCR reactions, for the identification (i.e., detection), and quantification (i.e., genome equivalents) of pneumococcal serotypes. DISCLOSURES: All authors: No reported disclosures.