Amplification with molecular beacon primers and reverse line blotting for the detection and typing of human papillomaviruses
Introduction
Human papillomaviruses (HPVs) are classified into approximately 100 genotypes, defined on the basis of DNA sequence of the L1 (capsid), E6, and upstream regulatory regions (van Ranst et al., 1993). Sexually transmitted HPV infections in women divide into symptomatic warts on the external genitalia, most commonly caused by types 6 or 11, and asymptomatic cervical infections. A small proportion of women with cervical HPV infection progress to cervical intraepithelial neoplasia (CIN) which, if not detected and treated, may develop into cervical cancer. The risk of cervical cancer developing is the highest for HPV types 16, 18, 31 and 45 and lowest for types 6 and 11. There is growing evidence that HPV infection is an essential prerequisite for the development of cervical cancer (Walboomers et al., 1999).
Cervical cancer is the commonest cancer in women aged under 35 and is a major cause of death in this age group in developing countries such as India and Brazil which cannot resource a comprehensive cervical screening programme (Nandakumar et al., 1995). Even in Western countries an unacceptable proportion of cervical smears are falsely reported as normal (Fricker, 1997). The publicity surrounding the development of cancer in women missed by the cervical screening programme has led to the suggestion that testing for potentially oncogenic HPV viruses should be added (Cuzick et al., 1995). One suitable commercially available test (Digene Corp. Beltsville, MD) uses the technique of direct hybridisation of HPV DNA with a cocktail of probes designed to detect the most common high and intermediate risk HPV types. The limitations of such a test for studies on the natural history of cervical HPV infection in different populations include; (1) an inability to identify the infecting HPV type or even discriminate between high and intermediate risk types; (2) inability to identify mixed HPV infections; and (3) all the infections with the many HPV types not included in the probe cocktail will be recorded as negative. A significant advance was made by Gravitt et al. (1998) who used biotinylated primers for the L1 gene and probed the PCR amplification products with a panel of 27 HPV genotypes. This approach identifies the HPV types involved, including mixed infections, but records as negative HPV infections not included in the probe panel. Further, 75% of samples tested by probing were negative generating a considerable unnecessary workload.
Using molecular beacon labelled primers (Nazarenko et al., 1997) we have developed a PCR method which permits the real-time detection of any HPV infection. Further, the fluorescent amplicons detected in positive samples are suitably labelled for direct testing against a panel of HPV genotype-specific probes. To confirm the validity of the method for rapid screening and the detection of HPV types not covered by the probe panel, we compared it with conventional PCR in a study of cervical samples from women attending a sexually transmitted diseases (STD) clinic in Colombo, Sri Lanka.
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Cervical samples
Women attending the central STD clinic from September through December 1997 were invited to participate in the study. All the patients were interviewed in Sinhalese or Tamil and completed a questionnaire designed to identify risk factors for the acquisition of genital tract HPV infections. The 352 women who agreed to participate in the study were managed in accordance with standard clinical procedures but with the addition of a cervical cytobrush (Cellpath, UK) sample taken for HPV detection.
Detection of HPV infection by conventional PCR
Cervical samples from 352 Sri Lankan women were tested for HPV by PCR using conventional GP5+/GP6+ primers. Positive products were detected in 129 of the samples (36%) using agarose gels stained with ethidium bromide. These results show that for women under 26 years of age (n=52) the carriage rate of 47.7% was significantly greater (P=0.0003, χ2 analysis) than the 28.6% carriage rate detected in women over 26 (n=238). There was no significant difference in the HPV carriage rate in commercial
Discussion
The present study developed a novel PCR for the detection of HPV infection based on molecular beacon primers which only fluoresce when incorporated into an amplicon. This enabled the PCR reaction to be monitored in real-time using an ABI Prism™ 7700 Sequence Detector. The method was shown to detect <10 copies of the HPV genome. To the best of our knowledge, this is the first application of molecular beacon primers for the detection of microorganisms. Cervical samples from women attending an STD
Acknowledgements
We are grateful to the staff and clients at the STD clinic, Colombo, Sri Lanka for providing samples. We thank J. Robertson and S. Green, Southampton Public Health Laboratory (PHL), and J. Gray and U. Desselberger, Public Health Laboratory Service HPV Reference Laboratory, Cambridge PHL, for useful discussions. This study benefitted from the use of the Seqnet facility at Daresbury Laboratory. The Storm analyser was purchased with a Wellcome Trust equipment grant.
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