Multi-target plasmid controls for conventional and real-time PCR-based serotyping of Streptococcus pneumoniae

Background: Serotyping of Streptococcus pneumoniae is an integral part of disease surveillance, with over 92 serotypes characterized to date using traditional serotyping. To identify the most predominant disease causing serotypes, molecular serotyping methods are now increasingly being used, like conventional and real-time multiplex PCR (cmPCR and rmPCR, respectively). Given that cmPCR consists of eight reactions spanning 41 targets, and rnWCR consists of seven triplex reactions, standardizing positive controls for these assays is challenging. As such, a 43-target plasmid for cmPCR (pSpn-CM1) and a 23 target plasmid for rmPCR (pSpn-RM1) were designed and validated. Methods: Plasmid pSpn-RM1 was designed and synthesized as chimeric DNA sequences to include all PCR target primer binding sites sequences for cmPCR. Plasmid pSpn-RM1 consisted of all primer and probe sequences required for rmPCR. Additional targets (lytA and cpsA) were included in both plasmids for quantification, following their propagation and purification from Escherichia coli. Results: When tested using the cmPCR reactions, all targets could be reproducibly be detected using pSpn-CM1 as template, with good amplicon visibility at a concentration of 1.4 (+/- 0.3) x 10(5) copies/ml was used. For the rmPCR reactions, all targets were reproducibly amplified with a concentration of 1.1 (+/- 0.2) x 10(4) copies/ml of pSpn-RM1, and the PCR efficiency for each target was equivalent to DNA extracted from representative S. pneumoniae serotypes. Conclusions: These quantifiable multi-target plasmids simplify the preparation of controls for PCR-based serotyping of S. pneumoniae, and methods herein could be extended to other highly multiplexed PCR assays.