Development of a broad spectrum PCR assay for papillomaviruses and its application in screening lung cancer biopsies.
A PCR assay was developed to detect known and as yet unidentified papillomaviruses (PVs). For this purpose we analysed the conserved amino acid sequences in the L1 and E1 open reading frames of 45 human and nine animal PVs. Candidate regions for the design of a primer were identified as those having the least number of amino acid and nucleotide sequence variants among the different PVs. These regions in the L1 ORF have been described previously. We modified the sequences of the backward and the forward primers, as well as the sequence of the oligonucleotide used as the degenerate probe, in order to cover a broader spectrum of PVs. The sensitivity of the assay for the human and animal PVs tested after hybridization with a 32P-labelled degenerate oligonucleotide probe was one genome copy per cell for integrated PV DNA and 10 genome copies per cell for plasmid PV DNA. The only exceptions were human papillomavirus (HPV) type 4, HPV60 and HPV65, for which a lower sensitivity was obtained. This group could be detected only by using additional primers. The assay was used to analyse 85 lung cancer biopsies representing different histological types. Using this system no PV DNA sequences were detected in the biopsies when compared with human placental DNA (a negative control) and PV DNA-positive standards.
Shamanin
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Division Tumourvirus-Characterisation, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Delius
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de Villiers
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EM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001706:Biopsy; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D015342:DNA Probes; D006801:Humans; D008175:Lung Neoplasms / Q000382:microbiology*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D027383:Papillomaviridae / Q000235:genetics* / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014412:Tumor Virus Infections / Q000175:diagnosis*
D017931:DNA Primers; D015342:DNA Probes