Quantitative multiplex PCR assay for the detection of the seven clinically most relevant high-risk HPV types

Schmitz, Martina; Scheungraber, Cornelia; Herrmann, Jörg; Teller, Karin; Gajda, Mieczyslaw; Runnebaum, Ingo B.; Dürst, Matthias

doi:10.1016/j.jcv.2009.01.006

« Previous Next »Journal of Clinical Virology
Volume 44, Issue 4 , Pages 302-307, April 2009

Quantitative multiplex PCR assay for the detection of the seven clinically most relevant high-risk HPV types

Martina Schmitz
- Klinik für Geburtshilfe und Frauenheilkunde, Universitätsklinikum Jena, Germany
Cornelia Scheungraber
- Klinik für Geburtshilfe und Frauenheilkunde, Universitätsklinikum Jena, Germany
Jörg Herrmann
- Klinik für Geburtshilfe und Frauenheilkunde, Universitätsklinikum Jena, Germany
Karin Teller
- Klinik für Geburtshilfe und Frauenheilkunde, Universitätsklinikum Jena, Germany
Mieczyslaw Gajda
- Institut für Pathologie, Universitätsklinikum Jena, Germany
Ingo B. Runnebaum
- Klinik für Geburtshilfe und Frauenheilkunde, Universitätsklinikum Jena, Germany
Matthias Dürst
- Klinik für Geburtshilfe und Frauenheilkunde, Universitätsklinikum Jena, Germany
- Corresponding author at: Abteilung Frauenheilkunde, Klinik für Geburtshilfe und Frauenheilkunde, Universitätsklinikum Jena, Bachstr. 18, 07743 Jena, Germany. Tel.: +49 3641 933720; fax: +49 3641 934272.

Received 10 October 2008; received in revised form 29 December 2008; accepted 14 January 2009. published online 17 February 2009.

Abstract

Background

High-risk HPV DNA detection has become a valuable tool for the triage of borderline, questionable and abnormal cytologic findings in cervical carcinoma screening programs. This knowledge is largely based on studies which could only discriminate between low-risk (LR-) and high-risk (HR-) HPV groups. However, it is becoming increasingly clear that HPV genotyping may allow further risk stratification and may offer different treatment options in the future.

Objectives

To establish a fast and cost-effective system not only for genotyping but also for quantification of viral DNA.

Study design

Development and validation of a 5′ exonuclease fluorescent probe multiplex real-time PCR assay (TaqMan format) for the detection and quantification of the 7 most frequent HR-HPV types (16, 18, 31, 33, 45, 52 and 58) which account for over 87% of cervical carcinomas world-wide. Two PCR reactions are required to detect the designated HPV types.

Results

Experiments with plasmid constructs of all 18 HR-HPV DNA showed that the multiplex real-time PCR assay was highly sensitive and specific. Evaluation of DNA extracted from archived cell pellets of cervical scrapes by the multiplex assay and the GP5+/6+-EIA showed identical genotyping for 234 of 261 (89.6%) samples and an almost perfect agreement when considering all typing results (kappa 0.901). Viral load did not correlate with disease progression within the CIN spectrum but significant differences were evident when comparing all CIN with the group lacking CIN (p=0.0028) or with the cancer group (p=0.0001).

Conclusion

Our multiplex assay will be useful to address questions related to viral persistence at the genotype level, the kinetics of viral load and disease recurrence.

Abbreviations: HPV, human papillomavirus, HR, high risk, LR, low risk, PCR, polymerase chain reaction, CIN, cervical intraepithelial neoplasia