A new approach for the evaluation of the human papillomavirus type 16 variability with high resolution melting analysis.
Studies on the variability of human papillomavirus (HPV) type 16 are based mostly on DNA sequencing of the viral oncogenes E6 and E7. In order to simplify variant identification, high resolution melting (HRM) analysis, which has been shown to distinguish amplicons differing in a single nucleotide, was employed. Optimised HRM analysis was applied to 255 anogenital samples positive for HPV 16. The E6/E7 region of the HPV 16 genome was amplified using nested PCR with subsequent melting of the amplicons. Samples giving ambiguous melting profiles were melted again in the presence of reference HPV 16 DNA to define and confirm the novel melting profiles. Out of 219 samples of Croatian origin, 65 reference variants, 119 E6-360G variants and 35 novel melting profiles were found. Samples containing unusual profiles were sequenced for identification. In addition, a subset of samples with two common variants, 23 reference and 34 E6-350G variants, was also sequenced to confirm the findings of high resolution melting. Concordance between the melting analysis and sequencing was 93.9%, while HRM sensitivity and specificity were 92.9% and 94.7%, respectively. This study showed that HRM analysis can be useful for the identification of HPV 16 variants. The HRM method will be useful in low resource settings as it saves considerable time and resources compared to sequencing.
Sabol
Ivan
I
Laboratory of Molecular Virology and Bacteriology, Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka cesta 54, 10002 Zagreb, Croatia.
Cretnik
Maja
M
Hadzisejdić
Ita
I
Si-Mohamed
Ali
A
Matovina
Mihaela
M
Grahovac
Blazenka
B
Levanat
Sonja
S
Grce
Magdalena
M
D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D005260:Female; D014644:Genetic Variation; D052162:Human papillomavirus 16 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D030361:Papillomavirus Infections / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D044366:Transition Temperature; D002583:Uterine Cervical Neoplasms / Q000821:virology; D014626:Vaginal Smears; D002578:Uterine Cervical Dysplasia / Q000821:virology
D004279:DNA, Viral