This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Swan, D. C. Articles by Icenogle, J. P. Search for Related Content PubMed PubMed Citation Articles by Swan, D. C. Articles by Icenogle, J. P.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, Apr 1997, 886-891, Vol 35, No. 4
Copyright © 1997 by the American Society for Microbiology. All rights reserved.

A sensitive, type-specific, fluorogenic probe assay for detection of human papillomavirus DNA

DC Swan, RA Tucker, BP Holloway and JP Icenogle
Human Papillomavirus Section, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia 30333, USA. dcs1@ciddvd1.em.cdc.gov

A simple method for the detection of a number of human papillomavirus (HPV) genotypes associated with cervical cancer has been developed. The assay exploits the 5'-->3' exonucleolytic activity of Taq DNA polymerase to increase the signal from fluorescent dyes by releasing them from genotype-specific probes during PCR. The probes are oligonucleotides with a 5' reporter dye (6-carboxyfluorescein), a quencher dye (6-carboxy-tetramethyl-rhodamine), and a phosphate-blocked 3' end. In the intact probe, the proximity of the reporter and the quencher results in suppression of reporter fluorescence by Forster- type energy transfer (V. T. Forster. Ann. Phys. 2:55-75, 1948). If the probe is bound downstream of either primer during PCR, the 5'-->3' exonucleolytic activity of Taq polymerase degrades it, allowing the reporter to diffuse away from the quencher, which results in an increase in reporter fluorescence. The increased fluorescence is directly related to the amount of target DNA and can be detected with an automated fluorometer. Probes for the L1 region of the cervical- cancer-associated HPV types 16, 18, 31, 33, and 35 were synthesized and the assays were optimized. The most sensitive assay can detect as few as two copies of HPV DNA in human cervical specimens.

This article has been cited by other articles:

• Tabora, N., Ferrera, A., Bakkers, J. M. J. E., Massuger, L. F. A. G., Melchers, W. J. G. (2008). High HPV 16 Viral Load is Associated with Increased Cervical Dysplasia in Honduran Women. Am J Trop Med Hyg 78: 843-846 [Abstract] [Full Text]  
• Fontaine, J., Gravitt, P., Duh, L.-M., Lefevre, J., Pourreaux, K., Hankins, C., Coutlee, F., The Canadian Women's HIV Study Group, (2005). High Level of Correlation of Human Papillomavirus-16 DNA Viral Load Estimates Generated by Three Real-time PCR Assays Applied on Genital Specimens. Cancer Epidemiol. Biomarkers Prev. 14: 2200-2207 [Abstract] [Full Text]  
• Flores-Munguia, R., Siegel, E., Klimecki, W. T., Giuliano, A. R. (2004). Performance Assessment of Eight High-Throughput PCR Assays for Viral Load Quantitation of Oncogenic HPV Types. J. Mol. Diagn. 6: 115-124 [Abstract] [Full Text]  
• Moberg, M., Gustavsson, I., Gyllensten, U. (2003). Real-Time PCR-Based System for Simultaneous Quantification of Human Papillomavirus Types Associated with High Risk of Cervical Cancer. J. Clin. Microbiol. 41: 3221-3228 [Abstract] [Full Text]  
• Iftner, T., Villa, L. L. (2003). Chapter 12: Human Papillomavirus Technologies. J Natl Cancer Inst Monogr 2003: 80-88 [Abstract] [Full Text]  
• Murphy, N, Ring, M, Killalea, A G, Uhlmann, V, O'Donovan, M, Mulcahy, F, Turner, M, McGuinness, E, Griffin, M, Martin, C, Sheils, O, O'Leary, J J (2003). p16INK4A as a marker for cervical dyskaryosis: CIN and cGIN in cervical biopsies and ThinPrepTM smears. J. Clin. Pathol. 56: 56-63 [Abstract] [Full Text]  
• Garcia-Quintanilla, A., Gonzalez-Martin, J., Tudo, G., Espasa, M., Jimenez de Anta, M. T. (2002). Simultaneous Identification of Mycobacterium Genus and Mycobacterium tuberculosis Complex in Clinical Samples by 5'-Exonuclease Fluorogenic PCR. J. Clin. Microbiol. 40: 4646-4651 [Abstract] [Full Text]  
• Mackay, I. M., Arden, K. E., Nitsche, A. (2002). Real-time PCR in virology. Nucleic Acids Res 30: 1292-1305 [Abstract] [Full Text]  
• Szuhai, K., Sandhaus, E., Kolkman-Uljee, S. M., Lemaitre, M., Truffert, J.-C., Dirks, R. W., Tanke, H. J., Fleuren, G. J., Schuuring, E., Raap, A. K. (2001). A Novel Strategy for Human Papillomavirus Detection and Genotyping with SybrGreen and Molecular Beacon Polymerase Chain Reaction. Am. J. Pathol. 159: 1651-1660 [Abstract] [Full Text]  
• Takai, K., Horikoshi, K. (2000). Rapid Detection and Quantification of Members of the Archaeal Community by Quantitative PCR Using Fluorogenic Probes. Appl. Environ. Microbiol. 66: 5066-5072 [Abstract] [Full Text]  
• Lanciotti, R. S., Kerst, A. J., Nasci, R. S., Godsey, M. S., Mitchell, C. J., Savage, H. M., Komar, N., Panella, N. A., Allen, B. C., Volpe, K. E., Davis, B. S., Roehrig, J. T. (2000). Rapid Detection of West Nile Virus from Human Clinical Specimens, Field-Collected Mosquitoes, and Avian Samples by a TaqMan Reverse Transcriptase-PCR Assay. J. Clin. Microbiol. 38: 4066-4071 [Abstract] [Full Text]  
• Allen, M. I., Gauthier, J., DesLauriers, M., Bourne, E. J., Carrick, K. M., Baldanti, F., Ross, L. L., Lutz, M. W., Condreay, L. D. (1999). Two Sensitive PCR-Based Methods for Detection of Hepatitis B Virus Variants Associated with Reduced Susceptibility to Lamivudine. J. Clin. Microbiol. 37: 3338-3347 [Abstract] [Full Text]  
• Norton, D.-M., Batt, C. A. (1999). Detection of Viable Listeria monocytogenes with a 5' Nuclease PCR Assay. Appl. Environ. Microbiol. 65: 2122-2127 [Abstract] [Full Text]  
• Swan, D. C., Tucker, R. A., Tortolero-Luna, G., Mitchell, M. F., Wideroff, L., Unger, E. R., Nisenbaum, R. A., Reeves, W. C., Icenogle, J. P. (1999). Human Papillomavirus (HPV) DNA Copy Number Is Dependent on Grade of Cervical Disease and HPV Type. J. Clin. Microbiol. 37: 1030-1034 [Abstract] [Full Text]  
• Josefsson, A., Livak, K., Gyllensten, U. (1999). Detection and Quantitation of Human Papillomavirus by Using the Fluorescent 5' Exonuclease Assay. J. Clin. Microbiol. 37: 490-496 [Abstract] [Full Text]  
• Higgins, J. A., Ezzell, J., Hinnebusch, B. J., Shipley, M., Henchal, E. A., Ibrahim, M. S. (1998). 5' Nuclease PCR Assay To Detect Yersinia pestis. J. Clin. Microbiol. 36: 2284-2288 [Abstract] [Full Text]  
• Brandt, M. E., Padhye, A. A., Mayer, L. W., Holloway, B. P. (1998). Utility of Random Amplified Polymorphic DNA PCR and TaqMan Automated Detection in Molecular Identification of Aspergillus fumigatus. J. Clin. Microbiol. 36: 2057-2062 [Abstract] [Full Text]