PCR-RFLP Detection and Typisation of Human Papillomaviruses (HPVs)

Abstract

Human papillomaviruses (HPV) are the members of the family Papovaviridae. Viral particles are spherical, with icosahedral symmetry, 55 nm in diameter, containing double strand molecule of DNA (mol. weight 8xl03). HPV cause benign and malignant lesions of epithelial and mucous tissue. On the basis of epidemiological and experimental data it is

known that HPV is the main etiological causative agent of cervix cancer which can be transmitted sexual contact. This virus has very unstable genome which mutates very often. At present, there are more than 70 different HPV types divided into three following groups:

HPVs of low oncogenic risk (types 6 and 7);

HPVs of intermediate oncogenic risk (types 31, 35 and 45);

HPVs of high oncogenic risk (types 16, 18 and 33).

It is very important in therapeutical and preventive sense early detection of HPV and corresponding typization. Now it is possible to achieve this by combination of two molecular methods such as PCR (Polymerase Chain Reactiori) and RFLP (Restriction Fragment Length Polymorphisni) which are adapted for analyzing of L1-region HPV-DNA (1, 2, 3).

We analyzed total 123 specimens by HPV-DNA-PCR (Ll-ORF) method (41 cervix specimens, 41 urine-superatant and 41 urine-pellet specimens). Samples detected as positive (4, 5, 28, 33, 38, 39 and 40) in all PCR analysis are typed by RFLP.

Out of 16 HPV-DNA-PCR positive specimens analyzed by RFLP detected HPV types were: 6, 2a, 16, 33 and 10a.

The percentage of successful typization of HPVs by RFLP was 40%. For five specimens after RFLP analysis, electrophoretic patterns were unclear. These PCR products and other which showed undefined restriction fragments must be controlled and confirmed by sequencing method.