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Inosine-containing primers in human papillomavirus detection by polymerase chain reaction
Eiomed &iPharmacother (1392) 46.167-169 D Elsevier, Paris
167
em~Qntainin~primers in ~~rna ~Qlyrn~r~$~ await re
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G Novelli’*, M Gennarelli’, L De Santis’, P Azgeloni3, B Dallapiccola’v2 ‘Deportment of Public Health and Cell Biology, 2nd University of Rome and University of Vrbino; ‘CSS, Hospital IRCCS San Giovanni Rotondo; ‘~orator~o Centrale CR& Rome, Italy (Received 2 April 1992; accepted 24 April 1992)
Summary - A PCR protocol has been developed using inosine-containing primers for human papilloma virus (HPV) detection. The HPV16 Ll region from CaSki-positive cells was efficiently amplified by a single reaction aud directly analyzed by agarose gel elec~ophoresis. The method was found to be sensitive and reproducible, and is easy to use for HPV typing and screening. polymerase chain reaction I papillomavirus
I inosine
R&urn& - Utilisation de la &action d’amplification ghique (PCR) pour le d&p&age et I’analyse des papillomavirus. Les auteurs ant d~velopp~ un protocole de dktection du pap~liomavirus h~ain fHPV) par une r&action d’ampli~cation g&ique fPCRj it part& d’oiigonn~l~atides synth~tiques contenant de l’inosine. La r&ion Li d’un HPVM provenant des cellules CaSki-positives a pu etre st&Zsamment amplifik in vitro par use r&action en une &ape pour &we directement analys6e en Jlectrophor&e. I1 en est conclu que cette technique sensible et reproductible peut Etre facilement utilish pour ie typage et le dt?pistage de I’HPK r&action d’amplification
ghique
i papillomavirus I inosine
Introduction The application of the polymerase chain reaction (PCR) to the study of different papillomavir~s types (HPVs) [ 1, 131 has provided new insight into the understanding of the role and the progressive development of the invasive carcinomas of the genital tract [7]. Region-s~cific oligonucleotide primers designed on HPVs sequence are currently used in the PCR assay [2, 3, 10, 1lJ However, this method requires the availability of a great number of primers and the setting up of several PCR reactions. This constitutes a major drawback in * Correspondence to ; present address : Prof G Novelli, LCICRI. Sarvizio di Genetica Via Ramazzini 15 00151 Rome, Italy
using this technique on a large scale. To partly overcome these difficulties, “universal primers” cont~ning four degeneracy positions have been developed, which allow detection of 25 HPVs in a single tube reaction [S, 12, 141. In the present study we have investigated PCR amplification of HPV 16 and 18 types using 2 primers (Pi1 and Pi2) that contain inosine at the degeneracy positions, and which provide results on genital mucosa HPV infection screening.
Materials and methods The sequence of the inosine-con~ning primers used in our protocol is shown in figure 1. The oligomers were synthesized by DNA synthesizer (model 381, Applied Biosystem, UK), PCR was carried out in a DNA thermal
168
i MY 11 GCA(C)CAGGGA(T~CATAAC(T)AATGG Y 09 CGTCCA(C)AA(G)A(G)GGAA(T)ACTGATC Pi1 GCICAGGGICATAAIAATGG Pi2 CGTTCCIAIIGGAIACTGATC Fig 1. Nucleotide sequences of universal (MY 11,MY 0% and inosine-containing (Pi I, Pi2) primer4 (Perk&Elmer Cetus) with ever 41 denaturation cycles at 94*C for 1 min, annealing at WC for 30 s, and primer extension at 72OC for 1 min. The reaction mixture in a final vol of IO0 ~1 contained a dNTi% mix of 200 yM, 1 PM of each primer, 2.5 units of Tag polymerase (Perkin-Elmer Cetus), 1.5 mM MgC12, 50 mM CL 10 mM Tris-NC1 (pH 8.0), and DMSO 2.5% (w/v). For an average assay, about 200 ng of template (genomic DNA from patient’s cervical smears and/or DNA from cancer cell lines, CaSki and HeLa, obtained from ATCC, USA) was used. Cervical punch biopsy tissue specimens were obtained from 43 patients presenting histological sigs of HPVs infection (acanthosis, papillomatosis, dyskeratosis, koilocytosis) as previousfy reported by Pasetto et al [6]. Human lymphocyte DNA and water controls cycler
were also included
in the experiments
as negative
con-
Fig 3. Restriction enzyme digestion of inosine-PCR product (HPV16 genome). Lanes 1 and 6: undigested PCR product; lane 2: PCR productdigested with EfoRI; lane 3: PCR product digested with Suu3A; lane 4: PCR product digested with PsfI; lane 5: PCR product digested with RsnI; m: pBR328 digested with BgII and HinfI.
esults and discussion 2 shows the PCR product obtained with the Pi1 and Pi2 primers in CaSki-DNA target cells. The Pil and Pi2 primers promoted the amplification of a 450-bp product (fig 2) from the Ll viral genome, which was similar to the PCR products generated from MY11 and MY09 amplification [14]. Serial dilutions of CaSki cellular DNA were utilized to measure the sensitivity of the system (fig 2). The HPVl6 amplification product was detected by ethidium bromide-stained agarose gel electrophoresis starting with as little as 10 pg CaSki DNA. Similar results were obtained with HeLa cellular DNA containing HPV 18 genome (fig 4). The specificity of this product Figure
Fig 2. Inosine-containing
primer PCR products of HPV16 genome. Lane 1: 0.1 pg of CaSki cell DNA; Iane 2: 10 pg of CaSki cell DNA; lane 3: 1 ng of CaSki cell DNA; lane 4: 10 ng of CaSki ceii DNA; lane 5: 150ng of CaSki cell DNA; lane 6: I pg of patient’s positive HPV16 DNA; lane 7: 1 j.lg of patient’s positive HPV16 DNA; lane 8: negative template PCR control; lane 9: negative enzyme PCR control ; lane 10: PCR product using universal primers (IO ng CasKi ceils); m: phi x 174 DNA digested with HaeIII.
trots. Ten-@ aiiquots were analysed for the presence of HPV-DNA (fig 2). The specificity of amplified viral DNA was confirmed by restriction analysis (fig 3) and Southern blot hybridization with 32P end-1abe~Ied oligonucleotide MY I2 and WD74 [ 141 type-specific probes, for HPV16 and HPVl8 respectively (data not shown). The absence of cross-amplification as a consequence of decreasing the hybridization stringency by the inclusion of inosine was established by PCR of genomic DNA samples containing HIV and HBV genomes.
Fig 4. Inosine-containing primers PCR products of HPVl8 genome. Lane I: PCR product from HPVl8 positive HeLa cells (10 ng); lane 2: PCR product from HPVl8 positive HeLa cells (150 ng); lane 3: negative enzyme PCR control; lane 4: I pg of patient’s positive HPVl8 DNA; lane 5: I pg of patient’s positive HPV18 DNA; lane 6: negative template PCR control; m: phi x 174 DNA digested with HueIII.
169 was evaluated by restriction analysis of spe,tfiz sites in the Ll region of the HPV16 gencme (fig 3). The restriction pattern matched perfectly with that deduced from the DNA sequence of the Ll region (for either HPV16 or HPV18). The PCR product generated by Pi1 and Pi2 primers was found in a reproducible manner in all DNA from patients who had previously tested positive with the MY09 and MY11 primers (S/43 HPV16 positive and 2143 HPV18 positive). However, we found that MY 11, MY09 and or similar primers [ 123 reduced the efficiency of the p~ming reaction by the formation of complex secondary structures and base mispairing, due to the introduction of 24 different primers in a single reaction tube. In addition, the use of these “universal primers” requires the simultaneous synthesis of different oligomers which makes the protocol expensive and inefficient. The inclusion of inosine at positions of ambiguity reduces the formation of mismatches which compromise subsequent annealing. This neutral base forms stable base pairs with the four conventional bases and with similar pairing strength. The use of inosine is thus recommended rather than that of a conventional base at positions of three- or four-fold ambiguity [9]. inosine-containing primers have been successfully used to amplify cDNA fragments [S] and to detect point mutations [4]. To the best of our knowledge, the present protocol constitutes the first application of PCR technology to viral genome detection using inosine-containing primers. This technique, in conjunction with reand striction analysis, oligomer-hybridization direct sequencing also allows characterization of known and undefined types of HPV genomes. However, the utility of these primers in the detection of cutaneous HPVs needs to be demonstrated, particularly since MY09 and MY 11 primers are unable to detect many cutaneous HPV types. Studies are in progress in our laboratory in order to verify the differentiation of several HPV types using Pi1 and Pi2 primers. Finally, this protocol can be used efficiently and at low cost in HPV 16 population screening programmes.
2
3
4
5
6
7 8
9
10
11
12
13
14
reverences 1 Eurmer GC, Parker JD, Sates J, East K, Kulander BG (1990) Comparative analysis of human papil-
toma virus detection by polymerase chain reaction and virapaplviratype kits. Am .I Clin Park01 94, 554 Cole ST, Danos 0 (1987) Nucleotide sequence and comparative analysis of the human papillomavims type 18 genome. J Mel Biol 193, 599 Dartmann K, Schwarz E, Gissmann L, Zur Hausem 1’ (1985) The nucleotide sequence and genome organization of human papilloma virus type 11. i,i’rology 151, 124 Ehlen T, Dubeau L (1989) Detection of Ras point ~~utations by polymerase chain reaction using muration-s~~ifi~, inoslne-confining oligonucleot~de primers. Biockem Biopkys Res Commun 160, 441 Knoth K, Roberds S, Potee C, Bamkun M (1988) Highly degenerate, inosine-containing primers specifically amplify rare cDNA using the polymerase chain reaction. Nucleic Acids Res 16, 10932 Pasetto N, Sesti F, De Santis L, Piccione E, Novelli G, Dallapiccola B (1992) The prevalence of HPV16 DNA in normal and pathological cervical scrapes using the polymerase chain reaction. Gynewl Cncol (in press) Pfister H (1987) Human papillomavirus and genital cancer. Adv Cancer Res 48, I1 3 Rodu B, Christian C, Snyder RC, Ray R, Miller DM (1991) Simplified PCR-based detection and typing strategy for human papilloma viruses utilizing a single oligonucleotide primer set. BioT’eckniques IO, 632 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Schwarz E, Durst M, Demankowski C (1983) DNA sequence and genome organization of genital human papillomavirus type 6B. EMBO J 2, 2341 Seedorf K, Krammer G, Durst M, Suhai S, Roweekamp WG (1985) Human papillomavirus type 16 DNA sequence. ecology 145, 181 Snijders PJF, van den Brule AJC, Schrijnemakers HFJ, Snow G, Meijer CJLN, Walboomers JMM (1990) The use of general primers in the polymerase chain reaction permits the detection of a broad sptrum of human papillomavirus genotypes. J Gen Viral 71, 173 Tidy JA, Vousden KH, Farrell PJ (1989) Evaluation between infection with a subtype of HPV 16 and cervical neoplasia. Lancer i, 1225 Ting Y, Manos M (1990) Detection and typing Of genital human papillomavi~ses. In.- PCR Protocots: A Guide to Methods and Applicufio~ (MA Innis, DH Gelfand, JJ Sninsky, TJ White, eds) Academic Press, San Diego, CA, 356
167
em~Qntainin~primers in ~~rna ~Qlyrn~r~$~ await re
avi
G Novelli’*, M Gennarelli’, L De Santis’, P Azgeloni3, B Dallapiccola’v2 ‘Deportment of Public Health and Cell Biology, 2nd University of Rome and University of Vrbino; ‘CSS, Hospital IRCCS San Giovanni Rotondo; ‘~orator~o Centrale CR& Rome, Italy (Received 2 April 1992; accepted 24 April 1992)
Summary - A PCR protocol has been developed using inosine-containing primers for human papilloma virus (HPV) detection. The HPV16 Ll region from CaSki-positive cells was efficiently amplified by a single reaction aud directly analyzed by agarose gel elec~ophoresis. The method was found to be sensitive and reproducible, and is easy to use for HPV typing and screening. polymerase chain reaction I papillomavirus
I inosine
R&urn& - Utilisation de la &action d’amplification ghique (PCR) pour le d&p&age et I’analyse des papillomavirus. Les auteurs ant d~velopp~ un protocole de dktection du pap~liomavirus h~ain fHPV) par une r&action d’ampli~cation g&ique fPCRj it part& d’oiigonn~l~atides synth~tiques contenant de l’inosine. La r&ion Li d’un HPVM provenant des cellules CaSki-positives a pu etre st&Zsamment amplifik in vitro par use r&action en une &ape pour &we directement analys6e en Jlectrophor&e. I1 en est conclu que cette technique sensible et reproductible peut Etre facilement utilish pour ie typage et le dt?pistage de I’HPK r&action d’amplification
ghique
i papillomavirus I inosine
Introduction The application of the polymerase chain reaction (PCR) to the study of different papillomavir~s types (HPVs) [ 1, 131 has provided new insight into the understanding of the role and the progressive development of the invasive carcinomas of the genital tract [7]. Region-s~cific oligonucleotide primers designed on HPVs sequence are currently used in the PCR assay [2, 3, 10, 1lJ However, this method requires the availability of a great number of primers and the setting up of several PCR reactions. This constitutes a major drawback in * Correspondence to ; present address : Prof G Novelli, LCICRI. Sarvizio di Genetica Via Ramazzini 15 00151 Rome, Italy
using this technique on a large scale. To partly overcome these difficulties, “universal primers” cont~ning four degeneracy positions have been developed, which allow detection of 25 HPVs in a single tube reaction [S, 12, 141. In the present study we have investigated PCR amplification of HPV 16 and 18 types using 2 primers (Pi1 and Pi2) that contain inosine at the degeneracy positions, and which provide results on genital mucosa HPV infection screening.
Materials and methods The sequence of the inosine-con~ning primers used in our protocol is shown in figure 1. The oligomers were synthesized by DNA synthesizer (model 381, Applied Biosystem, UK), PCR was carried out in a DNA thermal
168
i MY 11 GCA(C)CAGGGA(T~CATAAC(T)AATGG Y 09 CGTCCA(C)AA(G)A(G)GGAA(T)ACTGATC Pi1 GCICAGGGICATAAIAATGG Pi2 CGTTCCIAIIGGAIACTGATC Fig 1. Nucleotide sequences of universal (MY 11,MY 0% and inosine-containing (Pi I, Pi2) primer4 (Perk&Elmer Cetus) with ever 41 denaturation cycles at 94*C for 1 min, annealing at WC for 30 s, and primer extension at 72OC for 1 min. The reaction mixture in a final vol of IO0 ~1 contained a dNTi% mix of 200 yM, 1 PM of each primer, 2.5 units of Tag polymerase (Perkin-Elmer Cetus), 1.5 mM MgC12, 50 mM CL 10 mM Tris-NC1 (pH 8.0), and DMSO 2.5% (w/v). For an average assay, about 200 ng of template (genomic DNA from patient’s cervical smears and/or DNA from cancer cell lines, CaSki and HeLa, obtained from ATCC, USA) was used. Cervical punch biopsy tissue specimens were obtained from 43 patients presenting histological sigs of HPVs infection (acanthosis, papillomatosis, dyskeratosis, koilocytosis) as previousfy reported by Pasetto et al [6]. Human lymphocyte DNA and water controls cycler
were also included
in the experiments
as negative
con-
Fig 3. Restriction enzyme digestion of inosine-PCR product (HPV16 genome). Lanes 1 and 6: undigested PCR product; lane 2: PCR productdigested with EfoRI; lane 3: PCR product digested with Suu3A; lane 4: PCR product digested with PsfI; lane 5: PCR product digested with RsnI; m: pBR328 digested with BgII and HinfI.
esults and discussion 2 shows the PCR product obtained with the Pi1 and Pi2 primers in CaSki-DNA target cells. The Pil and Pi2 primers promoted the amplification of a 450-bp product (fig 2) from the Ll viral genome, which was similar to the PCR products generated from MY11 and MY09 amplification [14]. Serial dilutions of CaSki cellular DNA were utilized to measure the sensitivity of the system (fig 2). The HPVl6 amplification product was detected by ethidium bromide-stained agarose gel electrophoresis starting with as little as 10 pg CaSki DNA. Similar results were obtained with HeLa cellular DNA containing HPV 18 genome (fig 4). The specificity of this product Figure
Fig 2. Inosine-containing
primer PCR products of HPV16 genome. Lane 1: 0.1 pg of CaSki cell DNA; Iane 2: 10 pg of CaSki cell DNA; lane 3: 1 ng of CaSki cell DNA; lane 4: 10 ng of CaSki ceii DNA; lane 5: 150ng of CaSki cell DNA; lane 6: I pg of patient’s positive HPV16 DNA; lane 7: 1 j.lg of patient’s positive HPV16 DNA; lane 8: negative template PCR control; lane 9: negative enzyme PCR control ; lane 10: PCR product using universal primers (IO ng CasKi ceils); m: phi x 174 DNA digested with HaeIII.
trots. Ten-@ aiiquots were analysed for the presence of HPV-DNA (fig 2). The specificity of amplified viral DNA was confirmed by restriction analysis (fig 3) and Southern blot hybridization with 32P end-1abe~Ied oligonucleotide MY I2 and WD74 [ 141 type-specific probes, for HPV16 and HPVl8 respectively (data not shown). The absence of cross-amplification as a consequence of decreasing the hybridization stringency by the inclusion of inosine was established by PCR of genomic DNA samples containing HIV and HBV genomes.
Fig 4. Inosine-containing primers PCR products of HPVl8 genome. Lane I: PCR product from HPVl8 positive HeLa cells (10 ng); lane 2: PCR product from HPVl8 positive HeLa cells (150 ng); lane 3: negative enzyme PCR control; lane 4: I pg of patient’s positive HPVl8 DNA; lane 5: I pg of patient’s positive HPV18 DNA; lane 6: negative template PCR control; m: phi x 174 DNA digested with HueIII.
169 was evaluated by restriction analysis of spe,tfiz sites in the Ll region of the HPV16 gencme (fig 3). The restriction pattern matched perfectly with that deduced from the DNA sequence of the Ll region (for either HPV16 or HPV18). The PCR product generated by Pi1 and Pi2 primers was found in a reproducible manner in all DNA from patients who had previously tested positive with the MY09 and MY11 primers (S/43 HPV16 positive and 2143 HPV18 positive). However, we found that MY 11, MY09 and or similar primers [ 123 reduced the efficiency of the p~ming reaction by the formation of complex secondary structures and base mispairing, due to the introduction of 24 different primers in a single reaction tube. In addition, the use of these “universal primers” requires the simultaneous synthesis of different oligomers which makes the protocol expensive and inefficient. The inclusion of inosine at positions of ambiguity reduces the formation of mismatches which compromise subsequent annealing. This neutral base forms stable base pairs with the four conventional bases and with similar pairing strength. The use of inosine is thus recommended rather than that of a conventional base at positions of three- or four-fold ambiguity [9]. inosine-containing primers have been successfully used to amplify cDNA fragments [S] and to detect point mutations [4]. To the best of our knowledge, the present protocol constitutes the first application of PCR technology to viral genome detection using inosine-containing primers. This technique, in conjunction with reand striction analysis, oligomer-hybridization direct sequencing also allows characterization of known and undefined types of HPV genomes. However, the utility of these primers in the detection of cutaneous HPVs needs to be demonstrated, particularly since MY09 and MY 11 primers are unable to detect many cutaneous HPV types. Studies are in progress in our laboratory in order to verify the differentiation of several HPV types using Pi1 and Pi2 primers. Finally, this protocol can be used efficiently and at low cost in HPV 16 population screening programmes.
2
3
4
5
6
7 8
9
10
11
12
13
14
reverences 1 Eurmer GC, Parker JD, Sates J, East K, Kulander BG (1990) Comparative analysis of human papil-
toma virus detection by polymerase chain reaction and virapaplviratype kits. Am .I Clin Park01 94, 554 Cole ST, Danos 0 (1987) Nucleotide sequence and comparative analysis of the human papillomavims type 18 genome. J Mel Biol 193, 599 Dartmann K, Schwarz E, Gissmann L, Zur Hausem 1’ (1985) The nucleotide sequence and genome organization of human papilloma virus type 11. i,i’rology 151, 124 Ehlen T, Dubeau L (1989) Detection of Ras point ~~utations by polymerase chain reaction using muration-s~~ifi~, inoslne-confining oligonucleot~de primers. Biockem Biopkys Res Commun 160, 441 Knoth K, Roberds S, Potee C, Bamkun M (1988) Highly degenerate, inosine-containing primers specifically amplify rare cDNA using the polymerase chain reaction. Nucleic Acids Res 16, 10932 Pasetto N, Sesti F, De Santis L, Piccione E, Novelli G, Dallapiccola B (1992) The prevalence of HPV16 DNA in normal and pathological cervical scrapes using the polymerase chain reaction. Gynewl Cncol (in press) Pfister H (1987) Human papillomavirus and genital cancer. Adv Cancer Res 48, I1 3 Rodu B, Christian C, Snyder RC, Ray R, Miller DM (1991) Simplified PCR-based detection and typing strategy for human papilloma viruses utilizing a single oligonucleotide primer set. BioT’eckniques IO, 632 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Schwarz E, Durst M, Demankowski C (1983) DNA sequence and genome organization of genital human papillomavirus type 6B. EMBO J 2, 2341 Seedorf K, Krammer G, Durst M, Suhai S, Roweekamp WG (1985) Human papillomavirus type 16 DNA sequence. ecology 145, 181 Snijders PJF, van den Brule AJC, Schrijnemakers HFJ, Snow G, Meijer CJLN, Walboomers JMM (1990) The use of general primers in the polymerase chain reaction permits the detection of a broad sptrum of human papillomavirus genotypes. J Gen Viral 71, 173 Tidy JA, Vousden KH, Farrell PJ (1989) Evaluation between infection with a subtype of HPV 16 and cervical neoplasia. Lancer i, 1225 Ting Y, Manos M (1990) Detection and typing Of genital human papillomavi~ses. In.- PCR Protocots: A Guide to Methods and Applicufio~ (MA Innis, DH Gelfand, JJ Sninsky, TJ White, eds) Academic Press, San Diego, CA, 356