Detection and differentiation of human papillomavirus genotypes HPV-6 and HPV-11 by FRET-based real-time PCR.
A real-time PCR (RT-PCR) assay was developed based on fluorescence resonance energy transfer (FRET) hybridization probe technology, allowing very sensitive and specific detection of HPV-6 and HPV-11, reliable differentiation of HPV-6 and HPV-11, as well as prototypic and non-prototypic HPV-6 genomic variants, in a single PCR reaction. The primers and probe were designed on the basis of multiple alignments of 74 HPV-6 E2 gene sequences and 20 HPV-11 E2 gene sequences. Testing on defined plasmid standards showed that the RT-PCR allowed simple and reliable identification of HPV-6 and HPV-11 using type specific amplification followed by probe-specific post-amplification dissociation analysis. Sensitivity, assessed by probit analysis at a 95% detection level, was 42.9, 43.4, and 25.3 DNA copies per assay for prototypic and non-prototypic HPV-6 variants and HPV-11, respectively. The results obtained by the developed assay on 51 HPV DNA-negative samples and 149 HPV DNA-positive samples, including 81 HPV-6 positive samples (19 prototypic and 62 non-prototypic HPV-6 variants), 28 HPV-11 positive samples, 10 samples of HPV-44 and HPV-74 (the closest relatives of HPV-6 and HPV-11) and 30 samples of 15 other important alpha HPV, showed complete agreement with those obtained with the INNO-LiPA human papillomavirus (HPV) Genotyping Assay and HPV-6 E2 and E6 gene sequencing.
Kocjan
Bostjan J
BJ
Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana, Slovenia.
Seme
Katja
K
Poljak
Mario
M
D017931:DNA Primers; D005260:Female; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005838:Genotype; D052140:Human papillomavirus 11 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D052163:Human papillomavirus 6 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D030361:Papillomavirus Infections / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; C117355:E2 protein, Human papillomavirus type 11; D014764:Viral Proteins