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Journal of Clinical Microbiology, July 2004, p. 3176-3184, Vol. 42, No. 7
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.7.3176-3184.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Detection and Typing of Human Papillomavirus by E6 Nested Multiplex PCR

K. Sotlar,1* D. Diemer,1 A. Dethleffs,1 Y. Hack,1 A. Stubner,1 N. Vollmer,2 S. Menton,2 M. Menton,2 K. Dietz,3 D. Wallwiener,2 R. Kandolf,1 and B. Bültmann1

Institute of Pathology,1 Department of Obstetrics and Gynecology,2 Department of Medical Biometry, University of Tübingen, Tübingen, Germany3

Received 21 August 2003/ Returned for modification 9 October 2003/ Accepted 25 March 2004

A nested multiplex PCR (NMPCR) assay that combines degenerate E6/E7 consensus primers and type-specific primers was evaluated for the detection and typing of human papillomavirus (HPV) genotypes 6/11, 16, 18, 31, 33, 35, 39, 42, 43, 44, 45, 51, 52, 56, 58, 59, 66, and 68 using HPV DNA-containing plasmids and cervical scrapes (n = 1,525). The performance of the NMPCR assay relative to that of conventional PCR with MY09-MY11 and GP5+-GP6+ primers, and nested PCR with these two primer sets (MY/GP) was evaluated in 495 cervical scrapes with corresponding histologic and cytologic findings. HPV prevalence rates determined with the NMPCR assay were 34.7% (102 of 294) in the absence of cervical intraepithelial neoplasia (CIN 0), 94.2% (113 of 120) in the presence of mild or moderate dysplasia (CIN I/II), and 97.8% (44 of 45) in the presence of severe dysplasia (CIN III). The combination of all four HPV detection methods applied in the study was taken as "gold standard": in all three morphological subgroups the NMPCR assay had significantly (P < 0.0001) higher sensitivities than the MY09-MY11 and GP5+-GP6+ assays and sensitivities comparable or equal to those of the MY/GP assay. All 18 HPV genotypes investigated were detected among the clinical samples. The ratio of high- to low-risk HPV genotypes increased from 4:1 (80 of 103) in CIN 0 to 19:1 (149 of 157) in CIN I to III. Multiple infections were detected in 47.9% (124 of 259) of the patients. In conclusion, the novel NMPCR method is a sensitive and useful tool for HPV DNA detection, especially when exact HPV genotyping and the identification of multiple HPV infections are required.

* Corresponding author. Mailing address: Institute of Pathology, University of Tübingen, Liebermeisterstraße 8, 72076 Tübingen, Germany. Phone: 49 7071 2982265. Fax: 49 7071 292258. E-mail: klsotlar{at}med.uni-tuebingen.de.

Journal of Clinical Microbiology, July 2004, p. 3176-3184, Vol. 42, No. 7
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.7.3176-3184.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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