Journal of Virological Methods ELSEVIER Journal of Virological Methods 51(1995) 115-124 Detection and typing of human papillomavirus by single hybridization Aldo Venuti * , Gianna Badaracco, Maria Luisa Marcante Regina Elena Institute for Cancer Research, Laboratory of Virology, Via delle Messi d’Oro 156, 00158 Rome, Italy Accepted 30 September 1994 Abstract A rapid and non-radioactive molecular hybridization test was developed which simultaneously detects and types different human papillomaviruses (HPV) DNA in fresh and paraffin-embedded clinical :specimens. The method includes reverse blot hybridization between different recombinant HF’V plasmids immobilized on nylon membrane and probe of cellular DNA amplified and biotin- or digoxigenin-labeled by the polymerase chain reaction (PCR). PCR protocol using consensus primers includes the mixing of Taq polymerase at high temperature (Hot-Start) and the addition of the hapkn-conjugated nucleotide after the first ten cycles of amplification. The sensitivity level of this method resulted in detecting about 50 copies of HPV 16 for sample, independently of hapten used. The specificity of the typing method was also validated by more laborious and conventional analyses such as Southern-blot or PCR followed by several hybridizations with specific probes. Using this test HPV-11, -16, -18, -31 and -35 were typed in a number of samples from patients attendiq hospital. The method appears suitable for the handling of clinical samples in a selected population screening for type specific infections by HPV. Keyword.c Human papillomavirus typing; Reverse-dot; PCR; Non-isotopic hybridization 1. Intmduction Human papillomaviruses are implicated in several mucosal and cutaneous abnormali- ties including specific benign and malignant lesions (for reviews see de Villiers, 1989;; zur Hausen, 1991). * Corresponding author. Fax: + 39 6 4180473. 0166-0934/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-0934(94)00152-9
Transcript
Journal of Virological Methods
ELSEVIER Journal of Virological Methods 51(1995) 115-124
Detection and typing of human papillomavirus by single hybridization
Aldo Venuti * , Gianna Badaracco, Maria Luisa Marcante Regina Elena Institute for Cancer Research, Laboratory of Virology, Via delle Messi d’Oro 156, 00158 Rome,
Italy
Accepted 30 September 1994
Abstract
A rapid and non-radioactive molecular hybridization test was developed which simultaneously detects and types different human papillomaviruses (HPV) DNA in fresh and paraffin-embedded clinical :specimens. The method includes reverse blot hybridization between different recombinant HF’V plasmids immobilized on nylon membrane and probe of cellular DNA amplified and biotin- or digoxigenin-labeled by the polymerase chain reaction (PCR). PCR protocol using consensus primers includes the mixing of Taq polymerase at high temperature (Hot-Start) and the addition of the hapkn-conjugated nucleotide after the first ten cycles of amplification. The sensitivity level of this method resulted in detecting about 50 copies of HPV 16 for sample, independently of hapten used. The specificity of the typing method was also validated by more laborious and conventional analyses such as Southern-blot or PCR followed by several hybridizations with specific probes.
Using this test HPV-11, -16, -18, -31 and -35 were typed in a number of samples from patients attendiq hospital. The method appears suitable for the handling of clinical samples in a selected population screening for type specific infections by HPV.
Keyword.c Human papillomavirus typing; Reverse-dot; PCR; Non-isotopic hybridization
1. Intmduction
Human papillomaviruses are implicated in several mucosal and cutaneous abnormali- ties including specific benign and malignant lesions (for reviews see de Villiers, 1989;;
zur Hausen, 1991).
* Corresponding author. Fax: + 39 6 4180473.
0166-0934/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved
SSDI 0166-0934(94)00152-9
116 A. Venuti et al./Journal of Virological Methods 51 (1995) 115-124
HPV infection is diagnosed expecially by DNA molecular hybridization since standard viral techniques as serological or culture assays are still inadequate (Syrjanen, 1990). To establish the epidemiological role of some HPVs in human carcinogenesis it is essential to have sensitive and accurate diagnostic tests to detect the full spectrum of cancer-associated HPV types. Techniques such as Southern blot, dot blot and in situ hybridizations require procedures which are not well suited to large-scale screening or for routine use. The development of more efficient assays is an important and critical need.
In recent years, PCR-based systems (Saiki et al., 1985; Mullis and Faloona, 1987;
Saiki et al., 19881, that amplify the target DNA viral sequences, seem to offer more sensitive methods only if conducted accurately and carefully controlled (Gravitt and Manos, 1992). Furthermore, nonisotopic detection systems are replacing successfully
radioisotopes and numerous methods for labeling and detecting nucleic acids have been described (for review see Guesdon, 1992; Kessler, 1992).
Many PCR protocols available now use either primers recognizing specific HPV
ORFs (Shibata et al., 1988; Comelissen et al., 1989; Marcante and Venuti, 1991) or common highly conserved sequences among different HPV types (Manes et al., 1989; Snijders et al., 1990; Contomi and Leoncini, 1993).
At present, consensus primers developed by Manos et al. (1989) are mainly employed because they can detect a major number of HPV types including those considered at high risk in carcinogenesis. All the above PCR protocols suffer the limitation of performing several hybridization steps to type HPV DNA after the amplification.
To overcome these difficulties, a rapid hybridization technique was developed and named ‘strip-test’, for the detection and the typing of HPV in a single reaction with an amplified HPV DNA. The PCR product directly labeled with biotin or digoxigenin during the amplification reaction was hybridized against several HPV recombinant plasmids, immobilized on solid support and then visualized by colourimetric or chemilu- minescent procedures.
2. Materials and methods
2.1. Plasmids and cell lines
The cloned HPV types were kindly provided by Drs E.M. de Villiers, Heidelberg, Germany (HPV 1, 2, 3, 4, 5, 6b, 7, 8, 10, 11, 13, 16, 18, 30, 32, 37, 38, 41, 571, A. Lorincz, Silver Spring, USA (HPV 31), G. Orth, Paris, France (HPV 33). HPV 35, 43, 44 and 57 were purchased from American Type Culture Collection (Rockville, USA). The Herpes Simplex type 2 recombinant plasmid was a gift of Dr. A.C. Minson, Cambridge, UK.
The SiHa cell line containing one integrated copy of HPV 16 per cell (Baker et al., 1987) and the human rhabdomyosarcoma cell line TE 671, were kindly provided by Dr. M.S. Campo, Glasgow, U.K. and by Dr. A. Delpino, Rome, Italy, respectively.
A. Venuti et al./Journal of Virological Methods 51 (1995) 115-124 117
2.2. Cellular DNA preparation
Cellular specimens from cervical scrapes of women attending for routine Pap-test were collected in PBS and centrifuged. The pellet was lysed and protease digested by incubation at 56°C for l-3 h in 200-300 ~1 of 50 mM KCl, 10 mM Tris (pH 8.3), 2.5 mM MgCl,, 100 pg/ml gelatin, 0.45% Nonidet P-40, 0.45% Tween-20 and 60 pg/ml Proteinase K, followed by heat inactivation at 95°C for 10 min. Paraffin-embedded sections (5 pm) were processed essentially as described by Manos et al. (1989). A single section was placed in a microcentrifuge tube, dewaxed twice in xylene, dehy- drated in absolute ethanol and protease digested as above mentioned. DNA from frozen biopsies was purified by the ‘DNA Single Tube’ kit (Biotecx Lab., Houston USA) accordmg the manufacturer’s instructions. Five or ten microliters of each clinical sample DNA were employed for PCR amplification. In experiments determining the sensitivity of the method, log-dilutions starting from 107/ml SiHa cells were carried out into HPV negative clinical samples. Five microliters of the above dilutions corresponding to
5 X 103, 5 X lo’, 5 X 10, 5, and 5 X 10-l. SiHa cells per reaction were utilized.
2.3. Primers and probe
Synthesis of biotin or digoxigenin labeled DNA cellular probes was carried out by PCR using consensus single primer pair MY09 and MY11 (Perkin Elmer Cetus Norwalk, USA), able to detect more than 25 different HPV types within the Ll open reading frame (ORF) (Schiffman et al., 1991).
In some preliminary experiments a set of primers, whose sequences span the El conserved region of some mucosotropic HPV, were also tested (Contorni and Leoncini,
1993). Primers described by Saiki et al. (1985) recognizing human B globin gene sequence were mixed in some PCR reaction to determine the adequacy of the sample.
Probes for Southern blot hybridization consisted of HPV recombinant plasmids DNA labeled with [32P]a-dCTP (222 TBq/mmol) by a random primed DNA labeling kit (Boehringer Mannheim, Germany).
Primers for the El region and P-globin gene were provided by Dr. E.D’Ambrosio, Rome, Italy.
2.4. PCR amplification and hapten labeling
Five or ten microliters of the samples were added to a final 50 ~1 of solution containi:ng 50 mM KCl, 10 mM Tris (pH 9.0), 1% Triton X-100, 1.5 mM MgCl,, 200
mM of each dNTPs, and 25 pmol of each primer. After the first sample denaturation 1.25 units of Taq DNA Polymerase (Promega, Madison, USA) were mixed at 65°C to avoid mispriming. The reaction was done in a thermal cycler (Biostar, Violet, Italy) with cycles consisting of 94°C for 5 min, 45°C for 1 min, and 72°C for 1 min. After ten cycles, biotin-1ldUTP (BRL, Gaithersburg, USA) or digoxigenin-14dATP (Boehringer Mannheim) and 1.25 units of Taq enzyme were added for the remaining twenty cycles of amplification at the above-mentioned temperatures, with an additional 10 min for the last primer extension segment. The final ratio between the concentrations of cold and
118 A. Venuti et al./Journul of Virological Methods 51 (1995) 115-124
hapten-labeled deoxynucleotide was 3:l. One third of the amplified mixture was analysed by 4% agarose gel and ethidium bromide staining. The efficiency of labeling was ascertained by alkaline capillary transfer (Sambrook et al., 1989) and calorimetric
or chemiluminescent detection.
2.5. Reverse dot blot hybridization
HPV recombinant plasmids and negative DNA controls were heat denaturated in 0.4 M NaOH, 10 mM EDTA at 100°C for 10 min. 500 ng of denaturated DNA for each dot were fixed to the nylon membrane (Zeta Probe, Biorad, USA) by filtration throughout a Dot apparatus (Biorad, USA). The wells were rinsed further in 0.4 M NaOH.
The membranes were baked at 80°C for 30 min after a brief washing in 2 X SSC and
stored dry at room temperature before to be undertaken to the hybridizations. Two sets of filters were prepared; one with all HPV types available (cutaneous and mucosal) and the other one with only the mucosotropic viruses.
The membranes were prehybridized at 43°C for 30 min in a mixture of 45% formamide, 0.25 M NaHPO, (pH 7.21, 0.25 M NaCl, 1 mM EDTA, 7% SDS, 100 ng/pl DNA herring sperm in sealed plastic bags. The prehybridization solution was replaced with the hybridization solution which contained the same components with the addition of the PCR amplified probe denatured at 95°C for 10 min. Filters were hybridized at 43°C (low stringency) or 55°C (high stringency) for 16-20 h, then washed
twice at room temperature for 15 min in 2 X SSC with 0.1% SDS, twice at 50°C or 60°C for 15 min in 0.1 X SSC with 0.1% SDS.
All steps were carried out with agitation in separate plastic bags or tubes to avoid any overlapping among filters and to reduce the background.
2.6. Probe detection
DNA-DNA hybrids were visualized with calorimetric or chemiluminescent proce- dures for biotin or digoxigenin-bound probes, respectively. Both methods depend on a common final visualization obtained by the reaction of alkaline phosphatase on 5- bromo-4-chloro-3-indolyl phopsphate and Nitroblue-tetrazolium (BCIP/NTB) or Lu- miphos (Boehringer Mannhein) substrates.
Biotinylated probe was essentially detected according to the ‘DNA Detection System’ protocol (BRL, Gaithersburg, USA) while digoxigenin-probe following the ‘Dig Lumi- nescent Detection Kit’protocol (Boehringer Mamrheim).
3. Results
3.1. Sensitivity of method
One of the major problem with PCR-based test for HPV detection is to check the sensitivity of the methods with standardized controls. In this way SiHa and HeLa cells represent well recognized systems used by almost all groups working on HPV field. In
A. Venuti et al. /Journal of Virological Methods 51 (1995) 115-124
abcdefaM
B
abcdefg
119
Fig. 1. Sensitivity of amplification and labelling. (Panel A) Ethidium bromide staining of 4% agarose gel of
amplified products from dilutions of SiHa cells as in methods. The length of amplified products by consensus
and &globin primers are 450 bp and 110 bp, respectively. (Panel B) Chemiluminescent detection of
DIG-labelled product after alkaline transfer. a-e, log-dilutions of SiHa cells from 5 X lo3 to 5 X 10-l. f,
negative sample in which SiHa dilutions were made. g, PCR without input DNA. M, Molecular mass marker
type V (Boehringer Mannheim).
b C d
Fig. 2. Specifity of strip-test. The strip test has been carried out on SiHa dilutions as in Section 2. Letters over
dot-strips (correspond to samples as in Fig. 1; numbers are related to HPV types; R is DNA extracted from
human rhabdomyosarcoma cell line TE 671; H is a recombinant plasmid for Herpes virus type 2 sequences.
120 A. Venuti et al./Journal of VirologicalMethods 51 (1995) 115-124
A B
S P R
Fig. 3. Strip-test on laryngeal papillomatosis biopsy. (Panel A) Strip-test has been carried out at low
stringency, without P_globin primers and by calorimetric detection. Numbers correspond to HPV types; R is
DNA extracted from human rhabdomyosarcoma cell line TE 671; H is a recombinant plasmid for Herpes virus
type 2 sequences. (Panel B) Southern-blot of PstI-digested DNA hybridized with radiolabelled genomic probe
of HPV 11. S, 10 pg of SiHa DNA; P, 10 pg of DNA extracted from papillomatosis biopsy; R, 10 pg of TE
671 cell DNA. The arrowheads indicate the position of HindIII-digested lambda markers of 4.3, 2.3, 2.02 and
0.56 kb. Higher bands were cut out of the photograph.
our experiments SiHa cells have been chosen to determine the maximum level of achievable detection because of the presence of one copy of HPV 16 genome per cell. Hot-Start technique has been employed to direct the synthesis of only target DNA (Chou et al., 1992) whereas the highest level of labeling was achieved when the modified base was added later in the PCR cycles as in methods.
Fig. 1 (panel A) represents the determination of the sensitivity using dilutions of the SiHa DNA reconstituted into HPV-negative DNA specimens and then subjected to biotin incorporation and amplification with the consensus primers. A clear band corresponding to the amplified product of HPV 16 is present up to the DNA dilution representing about 50 copies of viral DNA. Transfer of these bands to nylon membrane and NTB-BCIP detection confirmed the labeling of these amplified products (Fig. 1,
A. Venuti et al. /Journal of Virological Methods 51 (1995) 115-124 121
a PCR was done with specific primers for HPV 11-16 and 18.
panel B). In digoxigenin labeling experiments a significant increase in sensitivity was
not found but the results were similar to biotin labeling (data not shown).
3.2. Typmg
The major goal of this study was to type the HPVs in a single reaction. For this
reason the labeled amplified product has been used in reverse dot hybridization with mucosotropic HPV types in condition of high stringency. As shown in Fig. 2, stained dots appear only on HPV 16 DNA with a decreasing intensity corresponding to SiHa
dilutions. The lowest quantity of HPV DNA detected was again 50 viral genomes in a total of 50 SiHa cells per sample.
3.3. Diagnostic use
Once optimized, the strip-test was used for various clinical samples. A frozen sample from laryngeal papillomatosis has been tested by our method and by a classical Southern blot hybridization. In this experiment a nylon membrane with a broad spectrum of immobilized HPV and low stringency conditions were employed to ensure a testing for all HPV available.
A clear dot was present m the area of immobilized HPV 11 DNA but weak signals
were evident in other spots due the low stringency conditions (Fig. 3, panel A). Concordance of results has been obtained with Southern blot of PstI digested DNA hybridized with radiolabeled HPV 11 probe (Fig. 3, panel B).
Freshly scraped cervical cells, frozen biopsies and archival paraffin- embedded samples from the histopathological collection were also tested. The typing experiments with SiHa cells have shown that high stringency conditions are able to avoid cross-hy- bridization among dotted HPV DNA. Thus, the same hybridization conditions have been chosen for the analysis of clinical samples. Results on screening of these specimens are
122 A. Venuti et al. /Journal of Virological Methods 51 (1995) 115-124
shown in Table 1 together with the data from PCR by type specific primers (Marcante and Venuti, 1991). A perfect agreement among data from specific primers-PCRs and those from strip-test were found, and moreover multiple HPV types were detected in three samples.
4. Discussion
In PCR-based methods the major problem is that the primers may bind to non-target DNA or to themselves and therefore they can induce Tuq polymerase to synthesize undesiderate DNA. The Hot-Start technique used in the present protocol has largely extent solved this problem, improving the specificity and the amplification of low copy-number sequences (Chou et al., 1992). The addition of modified nucleotide after ten cycles of amplification has increased the labeling of amplified product (data not shown). In fact, in clinical samples the enzyme works in conditions which are not ideal (Goldsborough et al., 1992) and in the presence of modified nucleotides the efficiency could be so decreased that the enzyme cannot amplify low copy number sequences. In the present protocol the first ten cycles ensure higher copy number of sequence available for the subsequent labeling step.
Under this optimized condition both sets of primers, consensus and El primers, are
effective in amplifying as little quantity of specific HPV DNA as that present in 50 SiHa cells mixed with thousands of negative cells. It is noteworthy that negative cells were clinical samples where PCR functions less efficiently than in purified DNA (Goldsbor- ough et al., 1992). This fact could also explain the slight reduced level of SiHa DNA detection reported by Manos et al. (1989).
The amplified and labeled product is suitable for reverse hybridization with cloned HPV DNA immobilized onto nylon membrane. In this way a single amplification with a single hybridization can define a positive sample and simultaneously the HPV type. We obtain precise and rapid results with different specimens, demonstrating the ductility of our method. The analysis of the same sample by conventional methods such as Southern blot (Fig. 3) and HPV-specific PCR (Table 1) confirmed the high sensitivity and specificity of the strip-test.
The detection of multiple HPVs in human DNA samples (Table 1) demonstrates that this assay may overcome the problem with using consensus primers that have been reported to amplify human DNA sequences of equivalent size (Gravitt et al., 1991) and to misidentify common HPV if the reaction contained a large amount of human DNA product (Tucker et al., 1993). In fact the final reverse blot discriminates true positive samples from other labeled products. In this way our protocol works better in conditions of high stringency, as shown by the weak non-specific crossreactions obtained at lower temperatures (Fig. 3).
The use of hapten-labeled deoxynucleotide could increase the cost of HPV diagnostic procedures, and therefore the method will find maximum use in screening of selected population groups with other associate risk factors such as age and sexual habits (Muiioz et al., 1992; Venuti et al., 1994).
A. Venuti et al./Joumal of Virological Methods 51 (199.5) 11.5-124 123
Acknowliedgements
This :study was partially supported by grants of AIRC, CNR/ACRO, and the Ministry of Health.
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