Complete genotyping of mucosal human papillomavirus using a restriction fragment length polymorphism analysis and an original typing algorithm.
BACKGROUND
Due to the differences in the oncogenic activity of human papillomaviruses (HPV), it is clinically important to accurately identify HPV types in a simple and time effective manner.


OBJECTIVE
We aimed at developing a straightforward and cost-effective assay to individually identify all mucosal HPVs, based on the amplification of L1 gene using MY09/11 primers, and subsequent restriction fragment length polymorphism (RFLP) analysis.


METHODS
We made use of bioinformatic tools to analyze all published DNA sequences of 49 mucosal HPV types for PstI, HaeIII, DdeI and RsaI restriction sites. Based on the RFLP patterns, we have designed an original genotyping algorithm.


RESULTS
Each HPV type presented a distinct RFLP pattern, which was visually distinguishable on polyacrylamide gels. A set of 27 pre-selected patient samples of known HPV types was confirmed positive for the same HPV type using this RFLP assay. Furthermore, in a random and blind HPV typing experiment performed in 30 untyped clinical samples, RFLP data consistently matched DNA sequencing results.


CONCLUSIONS
Our polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, using 4 restriction enzymes (PstI, HaeIII, DdeI, RsaI) and an original genotyping algorithm, allows discrimination of all individual mucosal HPV types in single infections, and even detection of multiple infections. This assay gives complementary information to commercially available methods, and may also be financially advantageous, particularly when financial resources are scarce.
Nobre
Rui Jorge
RJ
Molecular Pathology Laboratory, Portuguese Institute for Oncology at Coimbra, EPE, Avenida Bissaya Barreto 98, 3000-075 Coimbra, Portugal. rui.jorge.nobre@gmail.com
de Almeida
Luís Pereira
LP
Martins
Teresa C
TC
D019295:Computational Biology; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D005260:Female; D006801:Humans; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics*; D030361:Papillomavirus Infections / Q000821:virology*; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004279:DNA, Viral