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

Abstract

BackgroundIdentification 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.MethodsSingle-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.ResultsSpecificity 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 (R2>0.982); pNUversa (R2>0.991)] and efficiency of qPCR reactions using synthetic DNA were similar to those utilizing chromosomal DNA (R2>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).ConclusionWe 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.