Reproducibility of HPV 16 and HPV 18 viral load quantitation using TaqMan real-time PCR assays

Patti E. Gravitt, Cheri Peyton, Cosette Wheeler, Raymond Apple, Russell Higuchi, Keerti V. Shah

Research output: Contribution to journalArticle

Abstract

A reproducibility study was designed to assess within-assay, between-day, and interlaboratory variability of three real-time PCR assays targeting HPV 16, HPV 18, and the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pseudogenes. Fifteen HPV 16 and fifteen HPV 18 cervical swab samples were amplified in triplicate by GAPDH and HPV 16 and by GAPDH and HPV 18 assays, respectively. All samples were amplified undiluted and at a 1:10 dilution on 2 separate days in the same laboratory, and the same samples were amplified in a separate laboratory. HPV 16 and HPV 18 normalized viral load is reported as the number of HPV genomes per 20 000 GAPDH copies. The analytic specificity of the HPV 16 and 18 assays was 100 and 97%, respectively. The intraclass correlation coefficients (ICC) were 0.99, 0.97, and 0.98 for HPV 16, HPV 18, and GAPDH, respectively, indicating that the variability due to experimental error was very low. Ten-fold differences in viral load could be readily discriminated across a six order of magnitude dynamic range (ca. 5-5 × 106 copies). Power of discrimination was increased at higher target concentrations (> 5000 copies). The correlation of normalized HPV 16 and 18 viral load was high between the two laboratories (Spearman rho (ρ) = 0.96 and 0.87, respectively). These HPV 16 and HPV 18 quantitative PCR assays with GAPDH normalization are reproducibly quantitative over a broad linear dynamic range allowing for application in epidemiologic studies for measurement of viral load.

Original languageEnglish (US)
Pages (from-to)23-33
Number of pages11
JournalJournal of Virological Methods
Volume112
Issue number1-2
DOIs
StatePublished - Sep 2003

Keywords

  • Human papillomavirus
  • Quantitation
  • Real-time PCR
  • Reproducibility
  • Viral load

ASJC Scopus subject areas

  • Virology

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