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

Abstract

Purpose: 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. Methods & Materials: 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. Results: 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 <100 Colony-Forming Units (CFU) or gene equivalents (copies) per ml of sample. There was high concordance (Kappa: 0.84 – 1.0) between the qPCR methods and the culture-based Quellung method for identifying serotypes 6A, 6B and 6C. Due to the low prevalence of serogroup 18, 22, and serotype 6D in our setting in 2010-11, we were unable to assess concordance against the Quellung reaction. Conclusion: The newly designed assay-sets together with previously published serogroup 6, 18, and 22 assay-sets discriminate between current vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating these serogroups to single serotypes is important for surveillance of serotype replacement and to separately assess the impact of different PCV formulations on vaccine- and non-vaccine serotypes