NanoString Technology for Human Papillomavirus Typing

Abstract

High-throughput HPV typing assays with increased automation, faster turnaround and type-specific digital readout would facilitate studies monitoring the impact of HPV vaccination. We evaluated the NanoString nCounter^® platform for detection and digital readout of 48 HPV types in a single reaction. NanoString (NS) used proprietary software to design CodeSets: type-specific probe pairs targeting 48 HPV types and the globin gene. We tested residual DNA extracts from epidemiologic specimens and defined samples (HPV plasmids at 10 to 10⁴ copies/reaction) directly (No-PCR) as well as after L1 consensus PCR of 45 (PCR-45) or 15 cycles (PCR-15). Assay and interpretation followed NS recommendations. We evaluated analytic performance by comparing NanoString results for types included in prior assays: Roche Linear Array (LA) or HPV TypeSeq assay. No-PCR results on 40 samples showed good type-specific agreement with LA (k = 0.621) but sensitivity was 65% with lower limit of detection (LOD) at 10⁴ plasmid copies. PCR-45 results showed almost perfect type-specific agreement with LA (k = 0.862), 82% sensitivity and LOD at 10 copies. PCR-15 results on 75 samples showed substantial type-specific agreement with LA (k = 0.796, 92% sensitivity) and TypeSeq (k = 0.777, 87% sensitivity), and LOD at 10 copies of plasmids. This proof-of-principle study demonstrates the efficacy of the NS platform with HPV CodeSet for type-specific detection using a low number of PCR cycles (PCR-15). Studies are in progress to evaluate assay reproducibility and analytic validation with a larger number of samples.

Keywords: HPV detection; Linear Array; NanoString; PCR cycles; TypeSeq.

Figure 1

A schematic illustration of the conceptual framework using a sample with multiple infection of HPV types 16, 18 and 52 to evaluate NanoString technology for HPV typing. A CodeSet for an HPV type consists of sequence-specific capture and reporter probes, each 35–50 bases long. The CodeSets are hybridized to DNA extract, either directly or to amplified product. Following hybridization, the tripartide complex is purified, aligned and immobilized in a sample cartridge using the automated nCounter Prep Station. Cartridges are then transferred to the Digital Analyzer for imaging and digital counting of molecular barcodes carried in the reporter probes. HPV type determination is based on normalized count of barcodes that pass a cut-off.

Figure 2

Individual HPV type-specific agreement as indicated by kappa values between PCR-15 and LA for 37 types (A), and between PCR-15 and TypeSeq for 47 types (B). HPV types are shown on the X-axis (yellow bars: HR types and others: LR types) and corresponding kappa values on the Y-axis. HPV types with kappa values crossing the black line represent >substantial agreement (k ≥ 0.61) between tests, whereas HPV types that fall between the black line and dashed black line represent moderate (k = 0.41–0.6) agreement between tests.