FR2679254A1 - OLIGONUCLEOTIDE PROBES FOR THE TYPING AND DETECTION OF HUMAN PAPILLOMAVIRUSES. - Google Patents

Abstract

The invention relates to oligonucleotide probes for type determination and detection of human papillomaviruses, particularly HPV 6, HPV 11, HPV 16, HPV 18, HPV 31 and HPV 33, in a biological sample, said probes comprising all or part of at least one of the oligonucleotide sequences of formula (I) or their complementary sequences. The invention relates more particularly to specific probes of at least one type of HPV, particularly HPV 6, HPV 11, HPV 16, HPV 18, HPV 31 and HPV 33.

Description

OLIGONUCLEOTIDE PROBES FOR THE
TYPING AND DETECTION OF PAPILLOMAVIRUSES
HUMANS
The present invention relates to oligonucleotide probes constituted by nucleic acid sequences derived from Acid
Deoxyribonucleic (DNA) of at least one human papillomavirus (HPV), useful for the typing and detection of said HPV.

 Papillomaviruses are associated with a large number of more or less serious viral infections; some PVH specifically infect the epidermis, others infect the genital, oral or laryngeal mucosa. While most HPV causes benign lesions such as skin warts, others are responsible for precancerous lesions, such as skin neoplasias developing in patients with wart skin epidermodysplasia, such as intraepithelial neoplasias. Regarding genital infections, papillomavirus types 16 (HPV 16) and 18 (HPV 18) are generally associated with severe dysplasia, cervical cancer and carcinoma; Likewise, but less commonly, for papillomaviruses types 31, 33, 35 or 51. On the other hand, papillomaviruses types 6 and 11 are most often associated with lesions of the genital warts type or mild dysplasias.

 Over 60 types of HPV have been described. In all cases, they are small viruses, the capsid of which contains a double-stranded DNA molecule made up of approximately 8,000 base pairs. Their classification is based on the comparison of viral DNAs by molecular hybridization under stringent conditions; by convention, a PVH constitutes a new type if its DNA shows homology of less than 50% with the DNA of other PVH.

Recombinant DNA cloning techniques have made it possible to isolate and purify the DNA of several types of PVH, in particular those of PVH 6, 11, 16 and 18 (DURST M. et al., Proc. Natl. Acad. , USA (1983), 80: 312; BOSHART M. et al., EMBO J. (1984), 3: 1151; DE
VILLIERS E.-M. et al., J. Virol. (1981), 40: 932;
GISSMANN L. et al., J. Virol. (1982), 44: 393; LORINCZ
AT et al., J. Virol. (1986), 58: 225; BEAUDEMON S.

et al., Nature. (1986), 321: 246).

 Much of today's knowledge about HPV is the result of studying the nucleic acid sequence of these DNAs, as well as using them to prepare nucleic acid probes for typing and detecting papillomaviruses in samples by hybridization.

 Detection of HPV by immunological methods is not suitable because the capsid antigens are not specific for the type of PVH and these antigens are little or not expressed in certain stages of infection. In addition, papillomaviruses cannot be cultured in vitro.

 These are the reasons why detection by molecular hybridization is the best way to detect and type papillomaviruses.

This detection and this typing can be carried out either by hybridization on a plate or membrane in dot lot or by the Southern method from scrapings of the cervical cells or biopsies (PAO CC et al., Journal of Clinical Microbiology. (1989), 27: 168-173; INOUE M. et al., Journal of Virologicals
Methods. (1989), 26: 159-170; WEBB DH. et al., Journal of Infectious diseases. (1987), 156 n06: 912-919;
HALLAM N. et al., Journal of Medical Virology (1989), 27: 317-321; MELCHERS WJG et al., Journal of Medical
Virology. (1988), 25: 11-16), either by in situ hybridization on slides made from biopsies or from cells obtained from smears (SYRJANEN S. et al.,
Journal of Virology Methods. (1988), 19: 225-238; LEWIS
FA. et al., Journal Of Clinical Pathol. (1987), 40: 163-166; BURNS J. et al., Journal of Clinical Pathol.

(1987), 40: 858-864).

In situ hybridization screening and / or typing tests have been described in particular in European patent applications NO 294 659, 192 001 and 370 625. Most of these tests are based on the use of probes corresponding to the whole genome or to a large part of the genome of the virus, others call for the use of oligonucleotides as primers in a process of amplification of a nucleic sequence in vitro known under the name of PCR ("Polymerase
Chain Reaction "described by the company CETUS CORPORATION) (MORRIS BJ et al., Journal of Medical Virology.

(1990), 32: 22-30; RAKOCZY P. et al., Journal of
Medical Virology. (1990), 32: 10-17; WJM MELCHERS

et al., Journal of Medical Virology. (1989), 27: 329335; DALLAS P. et al., Journal of Medical Virology.

(1989), 27: 105-111).

 European patent application NO 301 968 in the name of IRE-CELLTARG S.A. describes nucleic acid probes useful for indifferently detecting the different types of human papillomavirus.

 The work carried out by the Applicant on the genomes of several types of PVH made it possible to determine oligonucleotide sequences comprising between 20 and 30 bases and having characteristics which made it possible to prepare oligonucleotide probes useful for detection and typing, according to methods hybridization, PVH, more particularly PVH 6, PVH 11, PVH 16, PVH 18, PVH 31 and PVH 33, with or without recourse to PCR type amplification.

The probes of the invention are characterized in that they comprise all or part of at least one of the sequences of the following formulas
(5 ') CGGTGGACCGGTCGATGTATGT (3') (I)
(5 ') CTGAAGTGGAAACTCAGCAGATG (3') (Il)
(5 ') ATCAGATGACGAGGACGAAAATGC (3') (III)
(5 ') CGAGCAATTAAGCGACTCAGAG (3') (1V)
(5 ') GACCCTGTAGGGTTACATTGCT (3') (V)
(5 ') CTGAAGTGGAAGCTGGAACGGGA (3') (VI)
(5 ') TAGATCAGTTTCCTTTAGGACGCAA (3') (VII)
(5 ') TATGAGCAATTAAATGACAGCTCAGA (3') (VIII)
In these formulas, the letters represent the following nucleotides: A for the adenine base, C for the cytosine base, G for the guanine base and T for the thymine base; (5 ') and (3') indicate the 5 'and 3' ends of the oligonucleotide.

Each of the sequences of the oligonucleotides of formulas (I), (II), (III), (IV), (V), (VI), (VII) and (VIII) corresponds to a DNA fragment of at least a
PVH, in particular of PVH 6, PVH 11, PVH 16, PVH 18, PVH 31 and PVH 33.

Consequently, the subject of the invention more particularly is oligonucleotide probes allowing, under suitable hybridization conditions, ie screening in general for
PVH implicated in genital lesions, i.e. the specific detection of PVH 6, PVH 11, PVH 16 and PVH 18 which are the most frequently encountered and PVH 31 and
PVH 33 which are less frequent.

I1 will be referred in the following to the appended figures in which
FIG. 1 represents the nucleotide sequence of the PVH6 DNA,
FIG. 2 represents the nucleotide sequence of the DNA of PVH11,
FIG. 3 represents the nucleotide sequence of the PVH16 DNA,
FIG. 4 represents the nucleotide sequence of the DNA of PVH18,
FIG. 5 represents the nucleotide sequence of the DNA of PVH31,
- Figure 6 shows the nucleotide sequence of PVH33 DNA.

A probe according to the invention, useful for the detection and typing of PVH 16 comprises all or part of the sequence of following formula
5 'CGGTGGACCGGTCGATGTATGT 3 t (I)
The oligonucleotide of formula (I) is derived from the DNA sequence of PVH 16 and has 100% homology with the fragment thereof delimited by the nucleotides located at positions 494 to 515. Among the probes of l The invention comprising the sequence of formula (I), mention may be made of those consisting of the sequence of formula (I) extended on either side by 1 to 10 corresponding nucleotides in the genome of PVH 16.

A preferred probe according to the invention, useful for the detection and typing of PVH 16 in a biological sample, consists of the sequence of following formula
5 'CGGTGGACCGGTCGATGTATGT 3' (I)
A probe according to the invention, useful for the detection and typing of PVH 16, PVH 31 and PVH 33 comprises all or part of the sequence of following formula
5 'CTGAAGTGGAAACTCAGCAGATG 3' (Il)
The oligonucleotide of formula (II) is derived from the DNA sequence of PVH 16 and from the DNA sequence of PVH 33. It has 100% homology with the fragment of DNA from PVH 16 delimited by the nucleotides located at positions 1273 to 1295, and 100% homology with the DNA fragment of PVH 33 delimited by the nucleotides located at positions 1285 and 1307. In addition, the oligonucleotide of formula (II) differs only of a base with the fragment of the DNA of PVH 31 delimited by the nucleotides located at positions 1268 to 1290. Among the probes of the invention comprising the sequence of formula (II), mention may be made of those consisting of the sequence of formula (II) extended on both sides, by 1 to 10 corresponding nucleotides in the genome of PVH 16, PVH 31 and PVH 33.

A preferred probe according to the invention, useful for the detection and typing of PVH 16, PVH 31 and PVH 33 in a biological sample consists of the sequence of following formula
5 'CTGAAGTGGAAACTCAGCAGATG 3' (Il)
A probe according to the invention, useful for the detection and typing of PVH 16 and PVH 18 comprises all or part of the sequence of following formula
5 'ATCAGATGACGAGGACGAAAATGC 3' (III)
The oligonucleotide of formula (III) is derived from the DNA sequence of PVH 18 and has 100% homology with the DNA fragment thereof delimited by the nucleotides located at positions 1009 to 1032. En in addition to the oligonucleotide of formula (III) differs only by 2 bases with the DNA fragment of PVH 16 delimited by the nucleotides located at positions 963 to 986. Among the probes of the invention comprising the sequence of formula (III ), mention may be made of those consisting of the sequence of formula (III) extended on either side by 1 to 10 corresponding nucleotides in the genome of PVH 16 and PVH 18.

A preferred probe according to the invention, useful for the detection and typing of PVH 16 and PVH 18 in a biological sample consists of the sequence of following formula
5 'ATCAGATGACGAGGACGAAAATGC 3' (III)
A probe according to the invention, useful for the detection and typing of PVH 18 comprises all or part of the sequence of following formula
(5 ') CGAGCAATTAAGCGACTCAGAG (3') (IV)
The oligonucleotide of formula (IV) is derived from the DNA sequence of PVH 18 and has 100% homology with the DNA fragment thereof delimited by the nucleotides located at positions 673 to 694. Among the probes of the invention comprising the sequence of formula (IV), mention may be made of those consisting of the sequence of formula (IV) extended on either side by 1 to 10 corresponding nucleotides in the genome of PVH 18.

A preferred probe according to the invention, useful for the detection and typing of PVH 18 in a biological sample consists of the sequence of following formula
(5 ') CGAGCAATTAAGCGACTCAGAG (3') (IV)
A probe according to the invention, useful for the detection and typing of PVH 6 and PVH 11 comprises all or part of the sequence of following formula
(5 ') GACCCTGTAGGGTTACATTGCT (3') (V)
The oligonucleotide of formula (V) is derived from the DNA sequence of PVH 6 and from the DNA sequence of PVH 11. It has 100% homology with the fragments of DNA from PVH 6 and PVH 11 delimited by the nucleotides located at positions 584 to 605. Among the probes of the invention comprising the sequence of formula (V), mention may be made of those consisting of the sequence of formula (V) extended on either side by 1 10 corresponding nucleotides in the PVH 6 genome and
PVH 11.

A preferred probe according to the invention, useful for the detection and typing of PVH 6 and PVH 11 in a biological sample consists of the sequence of following formula
(5 t) GACCCTGTAGGGTTACATTGCT (3 ') (V)
A probe according to the invention, useful for the detection and typing of PVH 6 comprises all or part of the sequence of following formula
(5 ') CTGAAGTGGAAGCTGGAACGGGA (3') (VI)
The oligonucleotide of formula (VI) is derived from the DNA sequence of PVH 6 and has 100% homology with the fragment thereof delimited by the nucleotides located at positions 1250 to 1272.

Among the probes of the invention comprising the sequence of formula (VI), mention may be made of those consisting of the sequence of formula (VI) extended on either side by 1 to 10 corresponding nucleotides in the genome of
PVH 6.

A preferred probe according to the invention, useful for the detection of PVH 6 in a biological sample consists of the sequence of following formula
(5 ') CTGAAGTGGAAGCTGGAACGGGA (3') (VI)
A probe according to the invention, useful for the detection and typing of PVH 6, PVH 11, PVH 16, PVH 18,
PVH 31 and PVH 33, includes all or part of the sequence of the following formula
(5 ') TAGATCAGTTTCCTTTAGGACGCAA (3') (VII)
The oligonucleotide of formula (VII) is derived from the DNA sequence of PVH 16 and has a homology of 100 * with the DNA fragment thereof delimited by the nucleotides located at positions 7012 to 7036. In in addition to the oligonucleotide of formula (VII) has more than 80% homology with a DNA fragment from PVH 6, PVH 11, PVH 31 and PVH 33, and 76% homology with a DNA fragment PVH 18. Among the probes of the invention comprising the sequence of formula (VII), mention may be made of those consisting of the sequence of formula (VII) extended on either side by 1 to 10 corresponding nucleotides in the genome of
PVH 16.

A preferred probe according to the invention, useful for the detection of PVH 6, PVH 11, PVH 16, PVH 18, PVH 31 and PVH 33 in a biological sample, consists of the sequence of following formula
(5 ') TAGATCAGTTTCCTTTAGGACGCAA (3') (VII)
A probe according to the invention, useful for the detection of PVH 6, PVH 11, PVH 16, PVH 18, PVH 31 and
PVH 33, includes all or part of the sequence of the following formula
(5 ') TATGAGCAATTAAATGACAGCTCAGA (3') (VIII)
The oligonucleotide of formula (VIII) is derived from the DNA sequence of PVH 16 and has 100% homology with the fragment thereof delimited by the nucleotides located at positions 634 to 659. In addition, the oligonucleotide of formula (VIII) has more than 88% homology with a fragment of the DNA of PVH 6,
PVH 11, PVH 31 and PVH 33, and 60% homology with a fragment of the DNA of PVH 18. Among the probes of the invention comprising the sequence of formula (VIII), mention may be made of those consisting of the sequence of formula (VIII) extended on both sides, by 1 to 10 corresponding nucleotides in the genome of PVH 16.

The probes constituted by the oligonucleotide sequences of formulas I, II, III, IV, V and VI allow respectively, under appropriate hybridization conditions, the specific detection of
PVH 16, PVH 16/31/33, PVH 18/16, PVH 18, PVH 6/11 and PVH 6. The probes constituted by the oligonucleotide sequences of formulas VII and VIII allow, under the same hybridization conditions, screening for l one of these people
The invention naturally also relates to the oligonucleotide probes complementary to the preceding ones and therefore capable of pairing with the same portion of PVH DNA due to its double-stranded nature.

 Also part of the invention are the above oligonucleotide probes in the sequence of which the thymines are replaced by uracils or in which one of the four bases is replaced by an inosine.

 The invention also relates to any probe which is distinguished from the preceding ones at the level of the oligonucleotide sequence only by additions and / or deletions and / or substitutions of one or more nucleotides, provided that these do not involve any modification of the properties. hybridization of said probes.

 The subject of the invention is also any association of the above oligonucleotide probes with each other or with already known probes, for screening or specific typing of HPV; such mixtures make it possible to improve the sensitivity and the specificity of detection in order to obtain better discrimination of papillomavirus infections.

 The invention further relates to any recombinant nucleic acid containing all or part of at least one of the oligonucleotide sequences of the probes of the invention, as well as the probes prepared from these recombinant nucleic acids.

 The probes of 1 t invention are used with hybridization media and according to hybridization conditions suitable for screening or typing of HPV.

Preferred hybridization media and conditions will be described in the examples below and may be modified without affecting the pairing properties of the probes and the DNAs of the HPVs; in particular the temperature and washing conditions can be modified between 180 ° C., 25 ° C. and 37 ° C., as well as the percentage of formamide, whether or not in conjunction with the hybridization temperature.

 The probes of the invention are used in hybridization methods for the typing and detection of HPV contained in a biological sample.

 Such methods include contacting a probe of the invention or a mixture of these probes with the biological sample treated so that the cells which it contains are lysed and possibly so that the nucleic acids contained in said cells are fragmented using restriction enzymes, then hybridized under conditions allowing the production of a hybridization complex between the probe (s) and the target DNA of PVH contained in I e the sample. Detection is carried out using any suitable means to demonstrate said hybrid. These methods can be implemented in tests on a membrane or on a plate or on any other suitable support, according to dot blot or Southern methods blot or filtration.

The DNA extracted can also, prior to hybridization with the probes of the invention, be amplified according to an enzymatic amplification process using DNA polymerase and primers specific for
HPV to be typed, known as PCR, consisting in repeating cycles of denaturation of the target DNA, hybridization of the primers and extension from the primers, a number of times sufficient to increase the quantity of the starting sequence in a exponential proportion compared to the number of cycles implemented. An aliquot of the amplification product is removed and treated under the conditions described below.

According to a preferred protocol, the amplification products are placed in the wells of the microtitration plates overnight at room temperature, before being brought into contact with a probe of the invention, specific for the fragment to be amplified.

 According to another embodiment of a hybridization method according to the invention, histological sections are made from the biological sample and the probe or the mixture of probes of the invention are brought into direct contact with said histological sections for detection of HPV DNA by in situ hybridization.

 Advantageously, the oligonucleotide probes of the invention are labeled with any label conventionally used.

 The probes can be marked using a radioactive tracer like 32p, 3sus, 125I, 3H, 14C. The radioactive labeling can be carried out according to any method known to those skilled in the art.

The probes can be labeled in (3 ') by addition of one or more deoxyribonucleotides or ribonucleotides or of a nucleotide analog such as a dideoxynucleotide, labeled in the alpha position by 32p, in the presence of the Deoxynucleotidyl Terminal Transferor; the probes can also be labeled in (5 ') using a kinase, for example the T4 Polynucleotide
Kinase; in this case, it is the transfer to the probe of the radioactive phosphate of a nucleotide labeled in the gamma position. The probes can also be labeled at each end by the addition of any radiolabelled sequence in the presence of a ligase.

 The probes can also be marked with "Random Priming" or also during their chemical synthesis by incorporating one or more radioactive ribonucleotides or deoxyribonucleotides therein.

 The method of detection of hybridization will depend on the radioactive marker used and may be based on autoradiography, liquid scintillation, counting of gamma radiation or any other technique making it possible to detect the radiation emitted by the radioactive marker.

 Non-radioactive labeling can also be used, by associating with the probes of the invention groups having immunological properties, such as an antigen or a hapten, a specific affinity for certain reagents such as a ligand, properties allowing the completion of enzymatic reactions such as an enzyme or an enzyme substrate. the probes can be labeled with "Random Priming" or during chemical synthesis by incorporating one or more ribonucleotides or deoxyribonucleotides labeled non-radioactively. Non-radioactive labeling can also be carried out by incorporation at the 3 ′ end of one or more deoxyribonucleotides or ribonucleotides or of a nucleotide analog such as a dideoxynucleotide comprising one of these groups. Labeling can also be done directly by chemical modification of the oligonucleotide, such as photobiotinylation or sulfonation. It can also be carried out by adding 3 'or 5' of tracer molecules by chemical reaction after synthesis; the probes can also be labeled at each end by adding any sequence carrying tracer molecules, in the presence of a ligase. The method of detection and revealing of hybridization will depend on the non-radioactive marker used.

 The subject of the invention is also kits for implementing the methods of detection and typing of HPV in vitro defined above.

By way of example, such kits include in particular
- a medium for processing the sample to be tested,
- a determined quantity of a probe or a mixture of probes in accordance with the invention,
a medium suitable for carrying out a hybridization reaction between the DNA of PVH and said probes,
- reagents for the detection of the hybrids formed between the DNA of PVH and said probe during the hybridization reaction.

 In addition to the foregoing characteristics, the invention includes other characteristics which will appear during the description which follows and which refer to examples of embodiment and implementation of the present invention, it being understood that these examples should not constitute any limitation on the scope of the claims.

I - Preparation of cells
The various studies carried out to control the sensitivity and specificity of detection of the selected oligonucleotide probes were carried out on the CaSki and HeLa cell lines or
SiHa containing respectively 1000-500 copies of PVH 16, 50-10 copies of PVH 18 and 10-1 copies of PVH 16 per cell.

 The cells are cultured on a monolayer in DMEM medium (Dulbecco - modified Eagle minimum essential medium) containing 10% fetal calf serum. The cells are counted, centrifuged and resuspended in 100 Wl of 10 mM Tris-HCl buffer pH 7.5, 5 mM EDTA, 100 mM NaCl. They are treated for 1 hour at 60 OC in the presence of proteinase K, 1% SDS.

 After stopping the reaction, the DNA is precipitated with alcohol and then resuspended in 20 μl of Tris-HCl buffer pH 8.0, 1 mM Na4EDTA.

II - Preparation and marking of probes
The oligonucleotides of formulas I, II, III,
IV, V, VI, VII and VIII are prepared by chemical synthesis. They are marked in 5 'with a tracer molecule, adding a cycle during chemical synthesis in order to introduce an aminolinker arm in 5'.

In the preferred protocol, the molecule incorporated is a biotin. It is added by treatment of the aminolinker oligonucleotide with
N-hydroxysuccinimide biotin overnight at room temperature. The purification of the labeled oligonucleotide is carried out by ethanol precipitation.

 When the probe is labeled at 3 ′, the labeling is carried out by incubation in the presence of deoxynucleotidyl terminal transferase, according to the protocol described by RILEY et al. (DNA. (1986), 5: 333337) and KUMAR et al. (Anal. Biochem. (1988), 169: 376382).

III - Preparation of samples for dowry hybridization. in Southern by filtration on membrane or on microtiter plate
The cells taken from the patients are diluted in a transport buffer containing 10 mM
Tris-HCl pH 7.5, 5 mM EDTA, 100 mM NaCl and then centrifuged. The pellet is taken up in 100 l of the buffer
Tris described above and treated for 1 hour in the presence of 500 fg / ml of proteinase K, 1% SDS, at 60 OC.

The composition of the buffer and the proteolytic digestion temperature are given by way of example.

 Depending on the type of support used for hybridization, the lysed cells are used in the screening or typing test after the reaction is stopped or the DNA is precipitated with ethanol, then resuspended in 20 l of Tris-HCl pH 8.0, 1 mM Na4EDTA.

IV - Rybridation in dowry or in Southern
1) Preparation of the membrane for the dowry
The DNA is deposited on a nitrocellulose membrane or on a charged or uncharged nylon membrane. In the preferred protocol, a nylon membrane is used. After drying, the DNA is treated in order to be denatured with a solution such as 0.4 M NaOH, 0.6 M
NaCl for 10 minutes, then neutralized using a solution such as 0.5 M Tris-HCl pH 7.5, 1.5 M NaCl for 10 minutes. After drying, the membrane is treated with W in order to fix the DNA.

2) Preparation of the samples and the membrane for the Southern
The DNA is deposited on an agarose gel after enzymatic digestion using restriction enzymes.

After electrophoresis and staining of the ethidium bromide gel in order to visualize the restriction fragments obtained, the gel is treated for 30 minutes in a solution of 0.4 M NaOH, 0.6 M NaCl, then for 30 minutes in a solution 0.5 M Tris HC1 pH 7.5, 1.5 M NaCl. The DNA is transferred from the gel to a nitrocellulose membrane or to a membrane of
Nylon charged or not, by active transfer type
Southern or by passive transfer. In the preferred protocol, the nylon membrane used is fixed to UV for 10 minutes after drying.

3) Hybridization
The membrane is prehybridized for 15 minutes at room temperature in the presence of the hybridization medium containing for example 45% formamide, 6 x SSC, 0.1% SDS, 1 x Denhart and 100 Ag / ml of denatured salmon sperm DNA. . The SSC buffer is defined as follows: 1 x SSC = 0.15 M NaCl, 0.015 M Na3 citrate, pH 7. The Denhart solution is defined as follows: 50 x Denhart = 1% Ficoll, 1% polyvinylpyrrolidone, 1%
BSA. Denhart can also be combined with or replaced by PEG or Dextran Sulfate. The labeled oligonucleotide probe (s) are added at a concentration of between 10 and 1000 ng / ml depending on the tracer molecule used. In the example cited above, the concentration is 100 ng / ml.

Hybridization is carried out at 18 OC for one hour.

After rapid rinsing in a buffer containing SSC and
SDS, for example 1 x SSC, 0.1% SDS, three washes of 10 minutes are carried out with this same solution at room temperature with stirring.

The membrane, rinsed in TEN buffer (50 mM
Tris-HCl pH 8.0, 1 mM EDTA, 50 mM NaCl) is ready for the detection step.

4) Detection
The membrane is blocked with a solution containing skim milk, for example 0.3 g / ml and heparin. The concentration of this can be 100 Rg / ml in the TEN buffer. The incubation is preferably carried out for 15 minutes. A conjugate recognizing the tracer molecule attached to the probe, or a substrate if the molecule in question is an enzyme, is added. In the preferred protocol, the tracer molecule is biotin. The conjugate used is therefore composed of a molecule of streptavidin, of avidin or of an anti-biotin antibody, coupled to an enzyme, for example peroxidase or with fluorescent molecules, such as FITC or colloidal gold .

In the preferred protocol, streptavidin is used in conjunction with alkaline phosphatase. The alkaline streptavidin phosphatase conjugate is incubated for 7 minutes 30 at room temperature at a concentration of 40 fg / ml. After rapid rinsing in a solution containing 0.1 M Na sulphate and 0.3% of a 30% solution of BRIJ 35, three washes of 15 minutes are carried out with this same solution at room temperature.

5) Revelation
When the conjugate used is a streptavidin alkaline phosphatase conjugate, different substrates of alkaline phosphatase can be used such as 4-bromo-5-chloro-indolylphosphate (BCIP) associated or not with tetrazolium blue nitro (NBT) or naphthol phosphate and fast red or other molecules with a bond which can be hydrolyzed by alkaline phosphatase.

 If the substrate is NBT / BCIP, the membrane is covered with the development buffer, 100 mM Tris-HCl pH 9.5, 100 mM NaCl, 10 mM MgCl2 in the presence of 0.33 mg / ml of NBT and 0.17 mg / ml BCIP.

 When the conjugate used is a streptavidin peroxidase conjugate, different combinations of substrates can be used, such as hydrogen peroxide and tetramethylbenzidine or alternatively hydrogen peroxide and aminoethylcarbazole.

V - Hybridization in membrane filtration
1) Preparation of the membrane
The DNA is treated to be denatured with a concentrated NaOH solution to obtain a final concentration of 0.4 M. After 10 minutes of treatment at room temperature, the DNA is deposited on a nitrocellulose or nylon membrane. , charged or not. This membrane is itself adapted to an absorbent of the POREX or other type, the whole being placed in a plastic support of the type of those used in immunofiltration.

2) Hybridization in filtration
All steps are performed at room temperature. Each reagent is pipetted, deposited on the membrane until it is absorbed, and incubated as follows. The membrane is prehybridized for 30 seconds in the presence of 100 Al of the hybridization medium containing for example 45% formamide, 6 x SSC, 0.1% SDS and 1 x
Denhart. Denhart can also be combined with or replaced by PEG or Dextran Sulfate.

 The hybridization is carried out for 15 minutes in the presence of 100 F1 of the hybridization medium containing the labeled oligonucleotide probe (s) at a concentration of between 10 and 100 ng / ml depending on the tracer molecule used. In the example cited the concentration is 50 ng / ml.

 Then the membrane is washed with 300 μl of a solution containing for example 1 x SSC and 0.1% SDS until complete absorption.

3) Filtration detection
The membrane is blocked for 30 seconds with 100 Al of a solution containing skim milk, for example 1 g per 100 ml of a 100 mM, 150 mM Tris buffer.
NaCl, and heparin at a concentration of 100 Ag / ml.

 A conjugate recognizing the tracer molecule attached to the probe, or a substrate if said molecule is an enzyme, is added. According to the preferred protocol, the tracer molecule is biotin. The conjugate used is therefore composed of a molecule of streptavidin, avidin or even anti-biotin antibody, coupled to an enzyme, for example peroxidase, or to fluorescent molecules such as FITC or colloidal gold. According to the preferred protocol, streptavidin is used in conjunction with alkaline phosphatase. The streptavidin alkaline phosphatase conjugate can be diluted in the blocking solution and incubated for 30 seconds at a concentration of 6 fg / ml.

 The membrane is then washed with 300 μl of a solution containing 0.1 M of Na sulphate and 0.3% of a 30% BRIJ 35 solution until complete absorption.

4) Revelation in filtration
When the conjugate used is a streptavidin alkaline phosphatase conjugate, different substrates of alkaline phosphatase can be used such as 4-bromo-5-chloro-indolylphosphate (BCIP) associated or not with tetrazolium blue nitro (NBT) or naphthol phosphate and fast red or other molecules with a bond which can be hydrolyzed by alkaline phosphatase.

If the substrate is NBT / BCIP, 200 Al of the 100 mM Tris-HCl development buffer pH 9.5, 100 mM
NaCl, 10 mM MgCl2 containing 0.33 mg / ml of NBT and 0.17 mg / ml of BCIP is added until complete absorption. When the conjugate used is a streptavidin peroxidase conjugate, different combinations of substrates can be used, such as hydrogen peroxide and tetramethylbenzidine or alternatively hydrogen peroxide and aminoethylcarbazole.

VI - In situ hybridization
This protocol can be used on frozen sections, paraffin sections containing epithelial cells as well as on cytological smears.

1) Preparation of samples
The tissues are placed in 10% formalin and prepared within the next 4 to 18 hours. They can also be fixed using the other fixers conventionally used in immunohistochemistry, such as PFA 4% or Carnoy.

 The cells in the case of a smear are preferably fixed on the slides with acetone or Carnoy.

 When the sections are paraffin, one to three sections (4 to 6 Fm thick) of specimen are deposited on slides.

 Whether cells or sections, the slides are pretreated to promote their adhesion to the slide.

 The waxing of the sections is carried out according to a conventional protocol by treatment with xylene. These are two 5-minute baths in xylene followed by two 5-minute baths in 100% ethanol and then drying at room temperature.

 When the sections are frozen, apply one to three sections (6 to 8 Fm thick) of fixed and frozen tissue on the pretreated slides; then the slides are cooked for one hour at 60 ° C., they are fixed in an acetone bath and dried at room temperature.

2) Proteinase K treatment
Slides prepared from paraffin biopsies or frozen are incubated in the presence of proteinase K or pronase for 15 minutes at 37 ° C., at a concentration depending on the type and time of fixation. A concentration of 1 Ag / ml was used in the examples cited below for the paraffin sections. The reaction is stopped by three successive rinses of 5 minutes in distilled water.

3) Dehydration of the slides
The slides are dehydrated by incubation in two successive baths of 95 and 100% ethanol. The slides are dried at room temperature.

4) Hybridization
Each section is covered with 25 to 30 W1 of hybridization medium containing for example 45% formamide, 1 x Denhart, 6 x SSC, 100 Rg / ml of salmon sperm DNA and 1 Rg / ml of labeled oligonucleotide probe. Denhart can be combined with or replaced by PEG or Dextran Sulfate. The sections are covered with coverslips and are denatured by incubation for 10 minutes at 95 ° C. They are then hybridized for 1 to 2 hours at room temperature. The slides are removed and the slides are washed in the presence of SSC buffer at a concentration which can vary between 4 and 0.1 × SSC. The slides are rinsed in 0.1 M Tris-HCl pH 7.4, 0.1 M NaCl, 10 mM
MgCl2, three times 5 minutes at room temperature.

5) Detection
A conjugate recognizing the tracer molecule attached to the probe, or a substrate if the molecule in question is an enzyme, is added. In the preferred protocol, the tracer molecule is biotin.

The conjugate used is therefore composed of a molecule of streptavidin, of avidin or of an antibiotin antibody; coupled to an enzyme, for example peroxidase or with fluorescent molecules, such as FITC or colloidal tor. In the preferred protocol, streptavidin is used in conjunction with alkaline phosphatase.

 The slides are incubated in the presence of streptavidin alkaline phosphatase at 1 Fg / ml for 30 minutes at room temperature and then washed in the development buffer, 0.1 M Tris-HCl pH 9.5, 0.1 M NaCl, 50 mM MgCl2 .

6) Revelation
When the conjugate used is a streptavidin alkaline phosphatase conjugate, different substrates of alkaline phosphatase can be used such as 4-Bromo-5-chloro-indolylphosphate (BCIP) combined or not with tetrazolium blue nitro (NBT) or
Naphtol phosphate and Fast Red or other molecules with a bond which can be hydrolyzed by alkaline phosphatase.

 If the substrate is NBT / BCIP, the development is carried out in the development buffer in the presence of 0.33 mg / ml of NBT and 0.17 mg / ml of BCIP for 1 to 2 hours 30 minutes at room temperature and at shelter from light.

 When the conjugate used is a streptavidin peroxidase conjugate, different combinations of substrates can be used, such as hydrogen peroxide and tetramethylbenzidine or alternatively hydrogen peroxide and aminoethylcarbazole.

VII - Hybridization on microtiter plate
1) Freezing samples
The DNA extracted from the cells according to the protocol described above is denatured for 10 minutes in the presence of sodium hydroxide at a final concentration of 0.4 M, then diluted in the presence of ammonium acetate in order to obtain a final concentration 1 M DNA is then deposited directly into the microtiter plates at the rate of 50 W1 per well.

The DNA extracted can also, prior to hybridization with the probes of the invention, be amplified according to an enzymatic amplification process using DNA polymerase and primers specific for
HPV to be typed, known as PCR, consisting in repeating the cycle of denaturation of the target DNA, hybridization of the primers and extension from the primers, a number of times sufficient to increase the quantity of the starting sequence in a exponential proportion compared to the number of cycles implemented.

 An aliquot of the amplification product is removed and treated under the conditions described above.

According to a preferred protocol, the plates are incubated overnight at room temperature.

Such an amplification method can in particular be implemented according to standard PCR conditions, with the following specific primers of PVH 16
- ACCGAAACCGGTTAGTATAAAAGC
- TCATTTAATTGCTCATAACAGTAG
2) Hybridization
The wells are emptied and 100 μl of hybridization medium are added. The preferred hybridization medium contains 30% formamide, 6 x SSC, 0.1% SDS, 1 x
Denhart and 100 fg / ml denatured salmon sperm DNA.

Denhart can also be combined with or replaced by PEG or Dextran Sulfate.

 The labeled oligonucleotide probe is added at a concentration of between 10 and 1000 ng / ml depending on the tracer molecule used. In the example cited, the probe is labeled with biotin and used at a concentration of 100 ng / ml. Hybridization is carried out at 18 OC for one hour. The wells are emptied, rinsed twice then washed once 15 minutes in the presence of a buffer containing SSC and SDS, for example 1 x SSC and 0.1% SDS.

3) Detection
The wells are blocked with a solution containing skim milk, for example 100 mg / ml and heparin. The concentration of this can be 100 fg / ml in the TEN buffer (50 mM Tris HCl pH 8.0, 1 mM EDTA, 50 mM NaCl). The incubation is preferably carried out for 15 minutes. The wells are emptied and 100 μl of TEN buffer containing a conjugate recognizing the tracer molecule attached to the probe, or a substrate if the molecule in question is an enzyme, are added. In the preferred protocol, the tracer molecule is biotin. The conjugate used is therefore composed of a molecule of streptavidin, of avidin or of anti-biotin antibodies, coupled to an enzyme, for example alkaline phosphatase or peroxidase.

In the preferred protocol, streptavidin is used coupled with alkaline phosphatase. The alkaline streptavidin phophatase conjugate is incubated for 10 minutes at room temperature at a concentration of 20 Rg / ml. The wells are emptied, rinsed twice, then washed for 15 minutes at room temperature in a solution containing 0.1 M Na sulfate and 0.3% of a 30% BRIJ 35 solution.

4) Revelation
When the conjugate used is a streptavidin alkaline phosphatase conjugate, different substrates of alkaline phosphatase can be used, such as p-Nitrophenyl Phosphate (PNPP) or other molecules having a bond which can be hydrolyzed by alkaline phosphatase.

 If the substrate is PNPP, a 2 mg / ml solution of PNPP in diethanolamine buffer (1 M diethanolamine, pH 9.8, 0.5 mM MgCl 2) is added at the rate of 100 A1 per well. This solution is prepared immediately. The revelation is carried out in 3 hours at 37 OC, protected from light.

 When the conjugate used is a streptavidin peroxidase conjugate, different combinations of substrates can be used such as hydrogen peroxide and tetramethylbenzidine or alternatively hydrogen peroxide and O-phenylene diamine.

Claims (17)

 1) Oligonucleotide probe for the typing and detection of human papillomaviruses, in particular of PVH 6, PVH 11, PVH 16, PVH 18, PVH 31, PVH 33, in a biological sample, characterized in that it comprises all or part of at least one of the following oligonucleotide sequences  (5 ') CGGTGGACCGGTCGATGTATGT (3') (I)  (5 ') CTGAAGTGGAAACTCAGCAGATG (3') (II)  (5 ') ATCAGATGACGAGGACGAAAATGC (3') (III)  (5 ') CGAGCAATTAAGCGACTCAGAG (3') (IV)  (5 ') GACCCTGTAGGGTTACATTGCT (3') (V)  (5 ') CTGAAGTGGAAGCTGGAACGGGA (3') (VI)  (5 ') TAGATCAGTTTCCTTTAGGACGCAA (3') (VII)  (5 ') TATGAGCAATTAAATGACAGCTCAGA (3') (VIII)  or their complementary sequences.  2) oligonucleotide probe for typing and detecting PVH 16, in a biological sample, according to claim 1, characterized in that it comprises all or part of the following oligonucleotide sequence  (5 ') CGGTGGACCGGTCGATGTATGT (3') (I)  or its complementary sequence.  3) oligonucleotide probe for typing and detecting PVH 16, PVH 31 and PVH 33, in a biological sample, according to claim 1, characterized in that it comprises all or part of the following oligonucleotide sequence  (5 ') CTGAAGTGGAAACTCAGCAGATG (3') (II)  or its complementary sequence.  4) Oligonucleotide probe for the typing and detection of PVH 16 and PVH 18, in a biological sample, according to claim 1, characterized in that it comprises all or part of the oligonucleotide sequence of the following formula  (5 ') ATCAGATGACGAGGACGAAAATGC (3') (III)  or its complementary sequence.  5) oligonucleotide probe for typing and detecting PVH 18, in a biological sample, according to claim 1, characterized in that it comprises all or part of the oligonucleotide sequence of the following formula  (5 ') CGAGCAATTAAGCGACTCAGAG (3') (IV)  or its complementary sequence.  6) Nucleotide probe for typing and detecting PVH 6 and PVH 11, in a biological sample, according to claim 1, characterized in that it comprises all or part of the oligonucleotide sequence of the following formula  (5 ') GACCCTGTAGGGTTACATTGCT (3') (V)  or its complementary sequence.  7) oligonucleotide probe for typing and detecting PVH 6, in a biological sample, according to claim 1, characterized in that it comprises all or part of the oligonucleotide sequence of the following formula  (5 ') CTGAAGTGGAAGCTGGAACGGGA (3') (VI)  or its complementary sequence.  8) Oligonucleotide probe for the detection of PVH, in particular PVH 6 PVH 11, PVH 16, PVH 18, PVH 31, PVH 33, in a biological sample, according to claim 1, characterized in that it comprises all or part of the oligonucleotide sequence of the following formula  (5 ') TAGATCAGTTTCCTTTAGGACGCAA (3') (VII)  or its complementary sequence.  9) Oligonucleotide probe for the detection of PVH, in particular PVH 6, PVH 11, PVH 16, PVH 18, PVH 31, PVH 33, in a biological sample, according to claim 1, characterized in that it comprises all or part of the oligonucleotide sequence of the following formula  (5 ') TATGAGCAATTAAATGACAGCTCAGA (3') (VIII)  or its complementary sequence.  10) Oligonucleotide probe according to one of claims 1 to 9, characterized in that it is radioactively labeled or with a marker of the enzymatic, antigenic, ligand, luminescent or fluorescent type.  11) Composition for the detection and / or typing of PVH, in particular of PVH 6, PVH 11, PVH 16, PVH 18, PVH 31, PVH 33, in a biological sample, characterized in that it comprises several distinct probes according to the claims 1 to 10.  12) Method for typing and detecting PVH, in particular PVH 6, PVH 11, PVH 16, PVH 18, PVH 31, PVH 33, in a biological sample, characterized in that it comprises  - bringing a probe according to any one of claims 1 to 10 or a mixture of probes according to claim 11 into contact with the biological sample previously treated so that the cells which it contains are lysed and optionally for the nucleic acids contained in said cells to be fragmented using restriction enzymes,  - the achievement of a dowry hybridization or Southern on membrane between the probe or the mixture of probes and the PVH DNA contained in the sample,  - Detection using any appropriate means, of the hybridization complex possibly formed.  13) Method for typing and detecting PVH, in particular PVH 6, PVH 11, PVH 16, PVH 18, PVH 31, PVH 33, in a biological sample, characterized in that it comprises  - bringing a probe according to any one of claims 1 to 10 or a mixture of probes according to claim 11 into contact with the biological sample previously treated so that the cells which it contains are lysed and optionally for the nucleic acids contained in said cells to be fragmented using restriction enzyme,  - hybridization on a membrane by filtration between the probe or the mixture of probes and the PVH DNA contained in the sample,  - detection by filtration using any appropriate means, of the hybridization complex possibly formed.  14) Method for typing and detecting PVH, in particular PVH 6, PVH 11, PVH 16, PVH 18, PVH 31, PVH 33, in a biological sample, characterized in that it comprises  bringing a probe according to any one of claims 1 to 10 or a mixture of probes according to claim 11 into contact with histological sections or with cytological samples made from the biological sample,  - carrying out in situ hybridization between the probe or the mixture of probes and the PVH DNA contained in the sample,  - Detection using any appropriate means, of the hybridization complex possibly formed.  15) Method for typing and detecting PVH, in particular PVH 6, PVH 11, PVH 16, PVH 18, PVH 31, PVH 33, in a biological sample, characterized in that it comprises  - bringing a probe according to any one of claims 1 to 10 or a mixture of probes according to claim 11 into contact with the biological sample previously treated so that the cells which it contains are lysed and optionally for the nucleic acids contained in said cells to be fragmented using restriction enzyme,  - carrying out a hybridization on a microtiter plate between the probe or the mixture of probes and the PVH DNA contained in the sample,  - Detection using any appropriate means, of the hybridization complex possibly formed.  16) Method for typing and detecting PVH, in particular PVH 6, PVH 11, PVH 16, PVH 18, PVH 31, PVH 33, in a biological sample, characterized in that it comprises  - extraction of the PVH DNA contained in the cells of the biological sample,  - the genetic amplification of said DNA by means of suitable primers,  bringing a probe according to any one of claims 1 to 10 or a mixture of probes according to claim 11 into contact with the amplified PVH DNA,  - carrying out a hybridization on a microtiter plate between the probe or the mixture of probes and the amplified PVH DNA,  - Detection using any appropriate means, of the hybridization complex possibly formed.  17) Kit for implementing an in vitro typing and detection process for HPV, in particular PVH 6, PVH 11, PVH 16, PVH 18, PVH 31 and PVH 33, according to one of claims 12 to 15, characterized in that it comprises  - a medium for processing the sample to be tested;  a determined quantity of a probe according to any one of claims 1 to 10 or of a mixture of probes according to claim 11,  a medium suitable for carrying out a hybridization reaction between the PVH DNA and the probe or the mixture of probes,  - reagents for the detection of the hybridization complexes formed during the hybridization reaction.