Nanofluidic Real-Time PCR to Discriminate Vaccine-Associated Pneumococcal Groups 6, 18 and 22 to Individual Serotypes

Current quantitative real-time Polymerase Chain Reaction (qPCR) methods are unable to distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) to distinguish between these individual serotypes by including Dual Priming Oligonucleotide (DPO) primers, a Locked Nucleic Acid (LNA)-modified probe and standard TaqMan assay-sets within a high-throughput serotyping reaction-set. In serogroup 6, the capsular gene wciP for 6A/C (wciPα) that differs from 6B/D (wciPβ) by two single nucleotide polymorphisms (SNPs) was targeted using a DPO based assay-set. In serogroup 18, capsular genes wciX and wxcM were targeted using a DPO- and LNA-based assay-set as well as standard TaqMan assay-sets. A standard TaqMan assay-set was designed to discriminate serotype 22F from serotype 22A within serogroup 22, based on the wcwA region of 22F. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and the newly designed sets for 6A/C; 18B/C/F; 18C/F, 18F (16F/18F/28AF) and 22F was applied and validated through a blind analysis of 490 archived clinical samples collected from South African children ≤5 years old (2010-11), previously serotyped with standard culture and Quellung methods. All assay-sets including those containing DPO-primers and an LNA-modified probe were efficient (92-101%), had a low variation between replicates (R2>0.98), and were able to correctly detect their respective targets at a limit of detection (LOD) at