Description:
ObjectiveTo date, CDC's HPV vaccine effectiveness studies have relied on one highly quality‐controlled commercial assay that uses consensus PCR of the HPV L1 gene followed by type‐specific hybridization. However, the reporting process is complex and additional testing is needed to resolve ambiguous results. Alternative assays with increased automation, faster turnaround and digital readout are needed.MethodsWe evaluated NanoString technology as a method for digital readout of the L1 gene for 48 HPV types in a single reaction. Unique probe pairs targeting the L1 and globin genes were designed in collaboration with NanoString. Forty residual DNA extracts from epidemiologic specimens and eight controls were tested directly (no amplification step) as well as after L1 consensus PCR. After overnight hybridization, type‐specific hybridization signals from scans were normalized and background assessed per the recommendations of NanoString. A positive result was defined as specimen‐normalized counts greater than the calculated sample specific background (average+2SD).ResultsThe NanoString assay produces a direct unambiguous readout of type‐specific results directly populating a database without manipulation. HPV detection in plasmid controls was successful only at the highest copy number tested (10,000 copies) and comparison of results of prior method and NanoString in residual extracts showed good type‐specific agreement (k=0.621) but lower sensitivity (65%) of NanoString. With PGMY primer L1 amplification, 45 cycles, hybridizing the highly diluted product gave almost‐perfect type‐specific agreement (k=0.862) between NanoString and prior method. Relative to prior results, NanoString had 82% sensitivity and 99% specificity. Initial evaluation of reduced amplification cycles suggest that sensitivity is maintained with only 15 cycles.ConclusionsThis proof‐of‐principle study demonstrates the potential of using the NanoString platform for highly multiplexed, high throughput and digital genotyping of HPV with a low number of PCR cycles. The platform improves the reliability of data management. NanoString technology is a candidate to improve the quality and reduce the cost of surveillance in studies of HPV and other infectious diseases.Support or Funding InformationSource of research support: Centers for Disease Control and PreventionThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.