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Journal of Clinical Microbiology, March 2002, p. 779-787, Vol. 40, No. 3
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.3.779-787.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

GP5+/6+ PCR followed by Reverse Line Blot Analysis Enables Rapid and High-Throughput Identification of Human Papillomavirus Genotypes

Adriaan J. C. van den Brule,1* René Pol,1 Nathalie Fransen-Daalmeijer,1 Leo M. Schouls,2 Chris J. L. M. Meijer,1 and Peter J. F. Snijders1

Department of Pathology, Vrije Universiteit Medical Center, Amsterdam,1 Department of Bacteriology, National Institute of Public Health and the Environment, Bilthoven, The Netherlands2

Received 30 July 2001/ Returned for modification 22 September 2001/ Accepted 21 December 2001

In this study, we developed a simple and fast typing procedure for 37 mucosotropic human papillomavirus (HPV) types using a nonradioactive reverse line blotting (RLB) procedure for general primer (GP5+/6+) PCR products. This system has the advantages not only that in a simple format, up to 42 PCR products can be simultaneously typed per membrane per day, but also that after stripping, the membranes can be easily rehybridized at least 15 times without a loss of signal. RLB appeared highly specific, and its sensitivity was identical to that of conventional typing performed with type-specific oligonucleotide probes in an enzyme immunoassay (EIA). The performance of RLB typing was evaluated with samples of HPV-positive cervical scrapings (n = 196) and biopsies of cervical premalignant lesions (n = 100). The distribution of HPV genotypes detected in these samples was in line with the distribution expected on the basis of literature data. In addition, RLB and EIA typing procedures were compared for the typing of high-risk HPV types in GP5+/6+ PCR products of 210 cervical scrapings from high-risk HPV-positive women who participated in a population-based screening program. The typing procedures had an excellent overall agreement rate of 96.5% (kappa value, 0.77). RLB was successful in detecting multiple HPV infections as well as single infections. In conclusion, the GP5+/6+ PCR-RLB procedure appeared to be a reliable and simple approach that may be of great value for large epidemiological studies, population-based cervical cancer screening programs, and vaccination trials that require high-throughput HPV typing.

* Corresponding author. Mailing address: Department of Pathology, Vrije Universiteit Medical Center, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Phone: 31-20-444.4023. Fax: 31-20-444.2964. E-mail: vandenbrule{at}vumc.nl.

Journal of Clinical Microbiology, March 2002, p. 779-787, Vol. 40, No. 3
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.3.779-787.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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