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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.


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