engleski

Evaluation of RFLP, DNA Sequencing, PCR/SSCP and Line Probe Assay HPV Genotyping

Cervical cancer is one of the most common malignancy both in incidence and in mortality in women worldwide. In Croatia, in 1993, cervical cancer took the third place following breast and stomach cancer. Therefore, early detection of HPV infection, as a leading cause of cervical cancer, is of major importance. In Croatian population, approximately 40% of samples positive by polymerase chain reaction (PCR) with universal HPV primers (MY09/MY11 and L1C1/L1C2) remained undetermined with type-specific primers for HPV 6/11, 16, 18, 31 and 33. The aim of this study was to identify HPV genotypes (HPV X) that remained undetermined, in order to determine the prevalence of other supposed high risk HPV types other than 16, 18, 31 and 33. For that purpose, we compared restriction fragment length polymorphism (RFLP) analysis, DNA sequencing (Alf-express system, Amersham Pharmacia Biotech), PCR-single-strand conformational polymorphism (PCR-SSCP) analysis and line probe assay (LiPA, Innogenetics). MY09/MY11 amplicons were analysed by RFLP using DdeI, DraI, PstI, Sau3AI, BamHI, HaeIII and/or RsaI restriction enzymes, DNA sequencing and PCR-SSCP. Amplicons obtained with general primer set, SPF10, which allows identification of 25 HPV genotypes, were hybridized according to the manufacturer’ s instuctions (LiPA). Out of 35 HPV X samples, single HPV infection was determined in 20 (57%) and 22 (63%) cases, multiple infections in 4 (11%) and 9 (26%) cases, by RFLP and LiPA, respectively. The most frequently observed types were HPV 53 and 58, both in 5 cases (14%). RFLP and LiPA did not allow the identification of HPV types in 11 (32%) and 4 (11%) cases, respectively. The remaining HPV-positive unresolved specimens were identificated by DNA sequencing. PCR-SSCP analysis was used to confirm multiple infections detrmined by RFLP and LiPA. The advantage of RFLP and DNA sequencing of PCR products over LiPA is the ability of genotype larger number of HPV types. However, multiple HPV infections can not be discriminated clearly enough. PCR-SSCP analysis proved to be a good method of choice for confirmation of multiple HPV infections. Yet, our preliminary results should be confirmed on a larger number of samples.

PCR; RFLP; SSCP; Sequencing; HPV

nije evidentirano