Development and evaluation of a PCR and mass spectroscopy (PCR-MS)-based method for quantitative, type-specific detection of human papillomavirus.
Knowledge of the central role of high-risk human papillomavirus (HPV) in cervical carcinogenesis, coupled with an emerging need to monitor the efficacy of newly introduced HPV vaccines, warrant development and evaluation of type-specific, quantitative HPV detection methods. In the present study, a prototype PCR and mass spectroscopy (PCR-MS)-based method to detect and quantitate 13 high-risk HPV types is compared to the Hybrid Capture 2 High-Risk HPV DNA test (HC2; Digene Corp., Gaithersburg, MD) in 199 cervical scraping samples and to DNA sequencing in 77 cervical tumor samples. High-risk HPV types were detected in 76/77 (98.7%) cervical tumor samples by PCR-MS. Degenerate and type-specific sequencing confirmed the types detected by PCR-MS. In 199 cervical scraping samples, all 13 HPV types were detected by PCR-MS. Eighteen (14.5%) of 124 cervical scraping samples that were positive for high-risk HPV by HC2 were negative by PCR-MS. In all these cases, degenerate DNA sequencing failed to detect any of the 13 high-risk HPV types. Nearly half (46.7%) of the 75 cervical scraping samples that were negative for high-risk HPV by the HC2 assay were positive by PCR-MS. Type-specific sequencing in a subset of these samples confirmed the HPV type detected by PCR-MS. Quantitative PCR-MS results demonstrated that 11/75 (14.7%) samples contained as much HPV copies/cell as HC2-positive samples. These findings suggest that this prototype PCR-MS assay performs at least as well as HC2 for HPV detection, while offering the additional, unique advantages of type-specific identification and quantitation. Further validation work is underway to define clinically meaningful HPV detection thresholds and to evaluate the potential clinical application of future generations of the PCR-MS assay.
Department of Obstetrics and Gynecology, University of Michigan Medical School, Women's Hospital, Ann Arbor, MI 48109-5276, USA. divya@umich.edu
Divya A
DA
Patel
Yang-Jen
YJ
Shih
Duane W
DW
Newton
Claire W
CW
Michael
Paul A
PA
Oeth
Michael D
MD
Kane
Anthony W
AW
Opipari
Mack T
MT
Ruffin
Linda M
LM
Kalikin
David M
DM
Kurnit
D017931:DNA Primers / Q000235:genetics; D005260:Female; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D027383:Papillomaviridae / Q000145:classification* / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D002583:Uterine Cervical Neoplasms / Q000821:virology
D017931:DNA Primers