JCM Figure table search 04
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Josefsson, A.
Right arrow Articles by Gyllensten, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Josefsson, A.
Right arrow Articles by Gyllensten, U.

Journal of Clinical Microbiology, March 1999, p. 490-496, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Detection and Quantitation of Human Papillomavirus by Using the Fluorescent 5' Exonuclease Assay

Agnetha Josefsson,1 Ken Livak,2 and Ulf Gyllensten1,*

Department of Genetics and Pathology, Unit of Medical Genetics, University of Uppsala, S-751 23 Uppsala, Sweden,1 and Applied Biosystems Division of Perkin-Elmer, Inc., Foster City, California2

Received 13 May 1998/Returned for modification 20 July 1998/Accepted 13 October 1998

A method for the detection and quantitation of oncogenic human papillomavirus (HPV) was developed by using the fluorescent 5' exonuclease assay. The method is based on the amplification of a 180-bp fragment from the 3' part of the E1 open reading frame in a single PCR with type-specific probes for HPV types 16, 18, 31, 33, and 35. The probes can be used separately or in combinations of up to three probes per assay. Quantitation over a range of 101 to 106 initial HPV copies was possible by using real-time detection of the accumulation of fluorescence with cycle number. Reconstitution experiments, performed to mimic mixed infections, showed that individual HPV types can be detected down to a ratio of about 1% in a mixture. The performance of the assay depends on DNA quality, the presence of PCR inhibitors, and the number of different probes used simultaneously. This homogeneous assay provides a fast and sensitive way of screening for oncogenic HPV types in biopsy specimens as well as cervical smear samples. The closed-tube nature of the assay and the inclusion of uracil N'-glycosylase reduces cross contamination of PCR products to a minimum. A similar assay for beta -actin was used in parallel for quantitation of genomic DNA. After normalizing the samples for genomic DNA content, the mean number of HPV copies per cell could be calculated.


* Corresponding author. Mailing address: Department of Genetics and Pathology, Unit of Medical Genetics, Box 589, University of Uppsala, S-751 23 Uppsala, Sweden. Phone: 46-18-4714909. Fax: 46-18-510792. E-mail: ulf.gyllensten{at}medgen.uu.se.


Journal of Clinical Microbiology, March 1999, p. 490-496, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.



This article has been cited by other articles:




Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Antimicrob. Agents Chemother. Clin. Microbiol. Rev.
Clin. Vaccine Immunol. ALL ASM JOURNALS

Copyright © 1999 by the American Society for Microbiology. All rights reserved.