Department of Medical Microbiology, Clinical Virology Section, University of Lund, University Hospital, S-205 02 Malmö, Sweden1
Departments of Dermatology2 and Surgery3, Lundby Hospital, Gothenburg, Sweden
Author for correspondence: Bengt Hansson.Fax +46 4033 7312. e-mail Bengt-Goran.Hansson{at}mikrobiol.mas.lu.se
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Abstract |
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Introduction |
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In the present study, we have designed and evaluated a single pair of degenerate PCR primers primarily aimed at the amplification of cutaneous HPV types.
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Methods |
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Generality and sensitivity analysis.
To evaluate the general applicability of the degenerate primers, cloned materials from 73 different HPV types were tested. HPV-19 was not tested since the vector was positioned between the sites of the primers. This was also true of HPV-16 and -51, and therefore the material used for analysis of these two types was obtained from cervical brush samples. HPV types 25, 46, 78 and 79 were not available for analysis. DNA (2·5 pg, corresponding to approximately 2x105 copies) of each cloned HPV type was added to each PCR tube. As negative controls, pBR322, proteinase K-treated human embryonal lung (HEL) fibroblasts and H2O without template were used.
Since 73 different HPV clones were tested in the generality analysis, there could be a theoretical risk that cross-contamination between the clones had taken place. Therefore, amplicons of two plasmids (HPV-5 and HPV-30) were selected for direct DNA sequencing. They were found to contain the expected and correct HPV type-specific sequences, ruling out the possibility of contamination.
For analysing the sensitivity of the PCR, 10-fold dilution series starting from 1x103 copies of cloned HPV-5, -20 and -30 were used. Each PCR contained a background of 100 pg human placental DNA (Sigma) in TE buffer (10 mM TrisHCl, 0·1 mM EDTA, pH 8·0).
Clinical samples.
From eight patients with different skin tumours, both tumour biopsies and biopsies from normal skin of the volar aspect of the forearm were collected and immersed in 0·9% NaCl for transport to the laboratory. The patients' characteristics are presented in Table 1. The samples were stored at -20 °C (without 0·9% NaCl) until analysed. Then DNA was extracted using a simple phenol-free method. The sample was incubated overnight at 37 °C, with 400 ml `lysis buffer' (10 mM TrisHCl, 10 mM NaCl, 10 mM EDTA, pH 7·8) with 4% SDS and proteinase K (200 µg/ml) (Boehringer Mannheim). Then, 120 µl of saturated NH4Ac was added and the tube was vortexed for 20 s and centrifuged at 17600 g for 15 min. The supernatant was transferred to a new tube and the DNA precipitated with 900 µl ethanol for 30 min at -20 °C. After centrifugation at 17600 g for 7·5 min, the resulting pellet was washed once with 500 µl 95% ethanol. The pellet was dried briefly and then dissolved in 100 µl TE buffer.
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Aliquots (2·5 µl) of each sample were added to 22·5 µl PCR mixture and 45 cycles of amplification were performed as described above. The positive controls used in the PCR were two clinical samples, one containing HPV-20, the other HPV-6. As negative control, H2O without template was used. Five µl of the amplified material was analysed by electrophoresis as described above.
Cloning and DNA sequence analysis.
For improving the quality of direct DNA sequencing, 5 µl of the amplicon was reamplified in 50 µl PCR mixture, which was subsequently separated in 1·5% agarose gel (Sea Kem) in 1x TAE buffer (Sambrook et al., 1989 ). The amplicon was then cut out from the gel and purified with a spin column (QIAquick Gel Extraction kit, Qiagen) and eluted in 30 µl H2O. Eight µl was used for the cycle sequencing reaction (ABI Prism, Dye Terminator Cycle Sequencing ready reaction kit FS, Perkin Elmer). The products were separated and analysed with an automated DNA sequencer (model 373A, Perkin Elmer). In cases where low quality DNA sequences were obtained, the amplicons were cloned using the pCR-Script cloning kit (Stratagene).
At least three recombinant clones were sequenced from each sample. Amplicons from samples collected with cotton swabs were cloned without reamplification and the cloned materials were sequenced with forward and reverse primers.
Obtained sequences were compared to available HPV sequences in the GenBank database, by using the Blast server.
Nucleotide sequence accession numbers.
The obtained DNA sequences have been submitted to GenBank under the following numbers: FA1.1, AF121419; FA1.2, AF121420; FA1.3, AF121421; FA2.1, AF121422; FA2.2, AF121423; FA2.3, AF121424; FA3, AF121425; FA4, AF121426; FA5, AF121427; FA6, AF121428; FA7, AF121429; FA8, AF121430; FA9, AF121431; FA11, AF121432; FA12, AF121433; and FA13, AF121434.
Alternative PCR.
The ability of the FAP59/64 PCR to detect HPV from the 40 skin samples was compared with that of an HPV skin type PCR described by Berkhout et al. (1995) . Briefly, 5 µl of sample was used in the first-step PCR with the CP65/CP70 primer set. In the nested PCR with the CP66/CP69 primers, 3 µl from the first step was used as input. As positive controls HPV-5 and -20 were used, and as negative controls H2O without template. Five µl of the amplified material was analysed by electrophoresis as described above.
Statistical analysis.
Statistical analysis of the HPV DNA results obtained by the FAP PCR and the alternative PCR test was done by using McNemar's test (corrected for continuity).
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Results |
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Of the four healthy volunteers, three were found to harbour HPV on their skin. Of the samples from one of the male volunteers (no. 4), those from four of the five sampling sites were HPV-positive, HPV-12, -49 and an HPV isolate provisionally designated FA9 being found in the left thigh sample, and five different HPV isolates of previously undefined types in samples from the other sites (Table 2). At repeat testing of this HPV-positive healthy volunteer 16 days later, samples from three of five sites were again found to be HPV-positive, manifesting three putatively novel HPV types, two of which had not been found in the earlier samples. Thus, in all, four different HPV types, including two which were previously unknown, were isolated from the left thigh of this healthy volunteer. Three of the four volunteers yielded HPV-positive forehead samples, one putatively novel type in one case, and two putatively novel HPV types in another, no typing having been performed in the third case (Table 2
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Discussion |
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We also found AmpliTaq Gold to be the only DNA polymerase from Thermus aquaticus that gave the desired amplicon without non-specific background on the gels.
The generality of the designed primers was satisfactory for most of the EV and other cutaneous types, since only HPV-1, -2, -41 and -63 could not be detected. The reason for the negative result with these HPV types appears to have been inefficient base pairing in the 3' region between one or both the primers and the templates. The negative result for HPV-35 was also probably due to inefficient base pairing in the 3' region of both primers. The detection failures for HPV-44 and -55 could be traced to three mismatches in each of the primers, and the negative result of HPV-66 was probably due to five mismatches of the primer FAP64. The reason for the failure to detect HPV-71 could not be determined, since no sequence data were available. The negative result of HPV-74 was probably due to four mismatches of the primer FAP59.
The reduced amplification yield of HPV-15 might have been due to the presence of two mismatches, one and four nucleotides from the 3' terminal nucleotide of the primer FAP59. The poor amplification of HPV-33 was unexpected, since the mismatches were manifested close to the 5' terminus of both primers. Surprisingly, the low HPV-38 amplification efficiency could be explained by only one A mismatch (A·C, primer/template) two nucleotides from the 3' terminal nucleotide of the primer FAP64. The low efficiency of HPV-56 amplification was probably attributable to four mismatches of the primer FAP59. The weak amplification of HPV-72 might have been due to a mismatched T (T·G, primer/template) one nucleotide from the 3' terminal nucleotide of the primer FAP59, in combination with three mismatches in the 5' region of the primer FAP64.
The shorter amplicon of HPV-58 was calculated to be 264 bp, since 10 matching nucleotides (positions 60036012) at the 3' terminus of the FAP59 were found downstream of the predicted primer site. The longer amplicon of HPV-40 was calculated to be 740 bp, if seven nucleotides (positions 56585664) upstream of the ordinary primer site matching the 3' terminus of the FAP59 were considered. The ordinary FAP59 primer site was probably affected by a mismatched T (T·G, primer/template) one base from the 3' terminal nucleotide.
Although no correlation was found to exist between certain HPV types and skin tumours in our small series, all HPV findings were associated with the skin and EV types. By means of different PCR approaches, the EV types have also been found to predominate in skin tumours from renal transplant patients (Shamanin et al., 1994b ; Berkhout et al., 1995
; de Jong-Tieben et al., 1995
;de Villiers et al., 1997
; Hopfl et al., 1997
; Bens et al., 1998
). The EV HPV types have been found to occur at high frequency in hairs plucked from normal skin, suggesting HPV to be subclinically present in normal skin (Boxman et al., 1997
). Possibly, the stem cells of the hair follicles represent a reservoir for HPV of the skin. However, in our material the HPV DNA was frequently found on the skin by sampling with cotton swabs.
In recent years, a substantial number of putatively novel HPV types has been presented in studies of renal transplant patients (Shamanin et al., 1994b ; Berkhout et al., 1995
; de Jong-Tieben et al., 1995
;de Villiers et al., 1997
; Hopfl et al., 1997
; Bens et al., 1998
). In our series, 12 novel HPV candidate types were found. However, it cannot be ruled out that some of these have been reported earlier by others, since the region for the amplicon was positioned upstream of the amplicons presented in other studies.
Direct sequencing of the amplicons from clinical material was unsuccessful, yielding overlapping peak patterns, indicating the presence of more than one type of HPV template in each sample. This was confirmed in 37% of the samples through sequence analysis of a number of clones from each sample. Findings of multiple HPV types from skin lesions have also been reported by others (Shamanin et al., 1994b ; Berkhout et al., 1995
;de Jong-Tieben et al., 1995
; Boxman et al., 1997
).
Finally, by using the described PCR method we suggest that it will be possible to perform reliable studies of HPV prevalence in larger series of skin carcinomas, benign lesions and normal skin of the healthy population. Such studies might not only contribute to our understanding of the involvement of HPV in the pathogenesis of skin cancer, but also prove to be useful in investigating the general epidemiology of cutaneous HPV types.
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Acknowledgments |
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Thanks are also due to Hkan Jansson (Department of Medical Microbiology, Lund University, University Hospital, Malmö, Sweden) for the suggestion of using AmpliTaq Gold.
This work was supported by the Cancer Foundation of University Hospital, Malmö, the Alfred Österlund Foundation, and the local programme for the control of human immunodeficiency virus and AIDS.
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Footnotes |
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References |
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Received 4 March 1999;
accepted 1 June 1999.