High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides.

Abstract

Current 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) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009-11), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92–101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/ml of sample. There was high concordance (Kappa = 0.73–0.92); sensitivity (85–100%) and specificity (96–100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

Optimization and validation of a high-throughput nanofluidic real-time PCR assay to evaluate nasopharyngeal carriage of 15 bacterial species and 92 Streptococcus pneumoniae serotypes

“…Control strains were grown according to standard microtiter culture methods to quantify their relative density as colony-forming units (CFU/ml) for use as quanti cation calibrators and to assess analytical e cacy. Total DNA was extracted and stored at -30 C until assayed, as described previously [35] . DNA from 89 isolates that were representative of the included pneumococcal serotypes and 15 other bacteria were used to optimize the PCR assay-sets and assess the analytical speci city of the assay-sets to their respective targets (pneumococcal serotypes: 1; 2…”

“…These were applied to assess the analytical sensitivity (Limit of Detection) and repeatability (intra-and inter-assay variation and Levy-Jennings plots) of the reaction-set. The design, properties and sequences of each gBlock have been detailed previously [35] . Brie y, for each of the three pools (A, B and C, Supplementary Table 4) three gBlocks were designed (nine in total) that each included nine to thirteen target regions.…”

“…2.6 Fluidigm BioMark™ HD Nano uidic qPCR loading, sample, and assay preparation Speci c target ampli cation (STA) was done as per manufactures recommendations as the initial step (pre-ampli cation of DNA) for the Biomark HD system (Fluidigm) as discussed previously [35] . In short, assay-sets were separated into three STA multiplex pools (Supplementary Table 3) to prevent any primers' cross-reactivity.…”

“…A ow diagram of the preparation and loading of the Fluidigm 96.96 IFC is detailed in Supplementary Fig. 2 and has been described in detail previously [35] . Brie y, assay premix for each target was aspirated into the IFC assay inlets for a nal concentration of 9µM primers and 2.5µM probe per reaction and samples (2.25 µL Pooled STA product, 2.50µL Applied Biosystems™ TaqMan™ Gene Expression Master Mix, catalog number 4370074 and 0.25µL Fluidigm sample loading reagent) were aspirated into sample inlets.…”

“…Previously we developed a nano uidic qPCR reaction-set that was able to simultaneously detect and quantify 55 pneumococcal serotypes [32] , as well as a detection and typing method to distinguish serogroups 6A/B/C/D, 18A/B/C, and 22A/F into single serotypes [35] using the same platform. Here we expand on our method to detect and quantify an additional 38 serotypes -allowing for comprehensive pneumococcal serotyping (92 serotypes) and detection of 15 other bacterial species to assess colonization and bacterial load in respiratory samples.…”