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Journal of Clinical Microbiology, August 1999, p. 2508-2517, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Development and Clinical Evaluation of a Highly Sensitive PCR-Reverse Hybridization Line Probe Assay for Detection and Identification of Anogenital Human Papillomavirus

Bernhard Kleter,1 Leen-Jan van Doorn,1 Lianne Schrauwen,1 Anco Molijn,1 Suprapto Sastrowijoto,2 Jan ter Schegget,3 Jan Lindeman,4 Bram ter Harmsel,5 Matthé Burger,6 and Wim Quint1,3,*

Delft Diagnostic Laboratory1 and Department of Pathology2 and Department of Gynaecology and Obstetrics,5 R. de Graaf Hospital, Delft, and Department of Virology3 and Department of Gynaecology and Obstetrics,6 Academic Medical Center, University of Amsterdam, and Department of Pathology, Slotervaart Hospital,4 Amsterdam, The Netherlands

Received 23 November 1998/Returned for modification 16 February 1999/Accepted 27 April 1999

Human papillomavirus (HPV) can be detected by amplification of viral DNA. A novel PCR primer set generating a short PCR fragment (SPF PCR) was used for amplification of a fragment of only 65 bp from the L1 region and permitted ultrasensitive detection of a broad spectrum of HPV genotypes. The intra- and intertypic sequence variations of the 22-bp interprimer region of this amplimer were studied. Among 238 HPV sequences from GenBank and clinical specimens, HPV genotypes were correctly identified based on the 22-bp sequence in 232 cases (97.2%). Genotype-specific probes for HPV genotypes 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, 51 to 54, 56, 58, 59, 66, 68, 70, and 74 were selected, and a reverse hybridization line probe assay (LiPA) (the INNO-LiPA HPV prototype research assay) was developed. This LiPA permits the use of amplimers generated by the SPF as well as the MY 09/11 primers. The assay was evaluated with a total of 1,354 clinical specimens, comprising cervical scrapes (classifications ranging from normal cytology to severe dyskaryosis) and formalin-fixed, paraffin-embedded cervical carcinoma samples. LiPA results were highly concordant with sequence analysis of the SPF amplimer, genotype-specific PCR, and sequence analysis of amplimers generated by MY 09/11 primers. The sensitivity of the SPF primers was higher than that of the GP5+/6+ primers over a broad range of HPV types, especially when multiple HPV genotypes were present. In conclusion, the SPF LiPA method allows extremely sensitive detection of HPV DNA as well as reliable identification of HPV genotypes in both cervical smears and paraffin-embedded materials.

* Corresponding author. Mailing address: Delft Diagnostic Laboratory, R. de Graafweg 7, P.O. Box 5100, 2600 GA Delft, The Netherlands. Phone: 31-15-2604581. Fax: 31-15-2604550. E-mail: wquint{at}ddl.nl.

Journal of Clinical Microbiology, August 1999, p. 2508-2517, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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