A rapid sequencing based prototype assay for detection of high risk HPV strains in cervical samples

Abstract number: 1134_03_389

Marlowe N., Bruce R., Owens M., White T., Zoccoli M.

Background:  

Human papillomavirus (HPV) is one of the most common causes of sexually transmitted diseases resulting in an estimated 288,000 deaths yearly from cervical cancer worldwide. Of more than 30 types found in the anogenital tract, at least 13 are considered high risk (HR), as they are significantly associated with progression to invasive cervical cancer. In an effort to accommodate high throughput screening for HR HPV types, we recently developed a prototype HPV assay using a Single Base Dye Primer Profiling (SBDPP) approach. In the present study, we evaluate prototype assay performance on 74 patient samples.

Objective:  

To evaluate SBDPP assay for the detection of HR HPV strains in cervical samples.

Methods:  

Genomic DNA purified from 74 retrospective cervical samples was used for this feasibility study. Fifty-four out of 74 were previously typed successfully with the Digene Hybrid Capture® 2 (HC2) assay. The remaining 20 samples yielded inconclusive results with the HC2 assay. Using consensus PCR primer sequences conserved among HPV types, a fragment of the L1 region was amplified in the presence of an intercalating dye. HPV-positive samples were identified by amplification Ct values and dissociation curve melting profiles, sequenced with a rapid SBDPP protocol and analysed on the ABI PRISM® 3100 Genetic Analyzer. HR HPV types were identified by their unique single base distribution profiles.

Results:  

The presence of HR HPV types was determined in 28/74 clinical samples using the SBDPP assay. HPV types were confirmed by NCBI BLAST (v2.2.9) analysis using four-color sequencing data. Twenty-four of the 54 HC2-typed samples were typed as HR HPV, while 30 were HPV-negative/low risk HPV. Four of the twenty previously untyped samples were genotyped as HR HPV. Type determinations for 24 HR HPV samples were concordant between the SBDPP and HC2. Of the 28 cervical samples identified as HR types, 5 samples were determined to be HPV mixed infections.

Conclusion:  

This feasibility study demonstrates the potential of the SBDPP assay to type patients harbouring HR HPV strains. Genotyping results obtained with the prototype assay are highly comparable to the four-color DNA sequencing data. These findings confirm that a rapid SBDPP method can be used for high-throughput screening of cervical samples for the presence of HR HPV types.