Detection and typing of low-risk human papillomavirus genotypes HPV 6, HPV 11, HPV 42, HPV 43 and HPV 44 by polymerase chain reaction and restriction fragment length polymorphism.
A novel PCR-restriction fragment length polymorphism assay (PCR-RFLP) was developed for sensitive detection and reliable differentiation of five low-risk human papillomavirus (lr-HPV) genotypes: HPV 6, HPV 11, HPV 42, HPV 43 and HPV 44, as well as differentiation of prototypic and non-prototypic HPV 6 genomic variants. The assay is based on the amplification of a 320-bp fragment of the HPV E1 gene and subsequent analysis of PCR-products with BsaJI and HinFI. Testing on plasmid standards showed that PCR-RFLP enabled simple and reliable identification and differentiation of five targeted lr-HPV genotypes and could detect reproducibly down to 10 copies of viral genome equivalents per PCR. The PCR-RFLP showed almost complete agreement with previously obtained genotyping results on 42 HPV-DNA negative samples and 223 HPV-DNA positive samples (45 HPV 6, 34 HPV 11, 35 HPV 42, 10 HPV 43, 24 HPV 44 positive samples and 75 samples containing 28 non-targeted HPV genotypes). The novel assay is simple and robust, does not require any sophisticated equipment and can be of great value for epidemiological studies, particularly in settings in which financial resources are limited.
Maver
Polona J
PJ
Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1105 Ljubljana, Slovenia.
Poljak
Mario
M
Seme
Katja
K
Kocjan
Bostjan J
BJ
D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005838:Genotype; D006801:Humans; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*
D004279:DNA, Viral; D014764:Viral Proteins; C047012:endodeoxyribonuclease SecI; D015252:Deoxyribonucleases, Type II Site-Specific; C032229:GANTC-specific type II deoxyribonucleases