METHOD AND MEANS FOR IDENTIFYING HPV VIRUS

Abstract: A method for identifying the presence of viral DNA of HPV virus in cellular material collected from an individual, comprising the following steps: extracting DNA from said cellular material amplifying said DNA by means of polymerase, using identifying means for identifying sequences complementary to sequences of the genome of HPV virus, said identifying means comprises identifying means suitable for identifying sequences complementary to sequences of said genome in the regions E of the genome. Identifying means for identifying sequences complementary to sequences of the genome of HPV virus, characterized in that it comprises identifying means suitable for identifying sequences complementary to sequences in the regions E of said genome. A diagnostic kit for amplifying DNA of HPV virus comprising identifying means suitable for identifying sequences complementary to sequences of the genome of said HPV virus, amplifying means for amplifying DNA of said virus, marker means, mineral oil.

Description: Method and means for identifying HPV virus The invention relates to a method and means for identifying the presence of HPV virus in specimens of cells collected from the anogenital region of a patient. The HPV virus, or Human Papilloma Virus, plays an etiologic role in development of uterine cervix, cancer in women, which is the second most frequent type of tumour for women. It is further involved in tumoural lesions of low female genital tract and of penis, as well as of anal and perianal region. The HPV virus is sexually transmitted and it is a small virus without envelope, provided with a protein capsid having an icosahedral geometry with a diameter of about 45 nm, which contains inside a double chain DNA single circular molecule having a length of about 8000 bases. More than 90 varieties of HPV virus have been identified, said varieties being mutually distinguishable for differences in the genome nucleotide sequence and having various danger levels with reference to the oncogenic hazard, i.e. the hazard of forming a tumour. The various genotypes of HPV virus may be divided into three groups, from the oncogenic hazard point of view: a first group having a low oncogenic hazard, for example the genotypes 6, 11, 42, 43 and 44, for which the probability of causing a tumour is nearly null, even if it should not be completely excluded; a middle hazard group, for example the genotypes 31, 33, 35, 45, 51, 52, 56, for which there is a greater probability of causing a tumour and a high hazard group, for example the genotypes 16 and 18, for which the probability of causing a tumour is high. Since the uterine cervix cancer is a pathology which may be easily treated if early identified, it is very important being able both to identify the presence of HPV virus in the uterine cervix epithelium, and to identify the virus genotype, or at least its group, even if there are no evident epithelium injuries . The techniques actually used for identifying the onset of an HPV virus infection in women are PAP test and colposcopy observation of uterine cervix epithelium with collection of biopsy specimens in possible suspicious regions of mucous membrane, ϋrethral swabs and seminal fluid collection, or biopsy in the anal region, are used for men. The above-mentioned techniques only highlight possible cellular atypisms and therefore are not able to provide realistic information on the virus presence in the uterine cervix mucous membrane, nor on the viral genotype or the infectious content. Furthermore, the HPV virus may be present even if visible lesions and atypisms in the mucous membrane are absent, even though it belongs to the high hazard genotype. In this case, the above-mentioned techniques for identifying the virus are completely ineffective. For certainly identifying the virus presence and determining to which genotype group it belongs, i.e. a low or middle-high hazard group, the above-mentioned techniques have to be accompanied with a test for viral DNA search. In situ hybridisation and gene amplification, associated with restriction enzymes employment, are highlighted amongst the currently most used techniques. However, these techniques are arduous and not reliable enough, since they are directed on the region LI which is not always present. Actually, one of the main problems to be solved for searching viral DNA of HPV virus is that, while at infection beginning the virus is well represented in infected cells, at a rate, for example, of 2000-3000 virus copies every cell, when the infection proceeds the virus tends to integrate in the cellular DNA. Therefore, the original viral genome is substantially absent in the infected cells and only the genes integrated in DNA of cells may be detected, particularly if the infection is in an advanced stage. Determining the existence of an HPV infection is now based on identification of a sequence of nucleotides in the region Ll belonging to the late region L of the virus genome. Nevertheless, this sequence is not found if the virus is already partially or completely integrated into the genome of a cell, so that only a limited percentage of specimens effectively infected by the HPV virus, roughly 20%, is positive to a test for identifying the sequence in the above- mentioned region Ll. Furthermore, nearly 90% of said positive specimens is infected by virus strains of low hazard, since the latter have lower probability of integrating in the genome of infected cells. Therefore, the virus cannot be identified just in the most dangerous cases, i.e. in cases of infection with virus strains of middle and high hazard. An object of the invention is to improve the effectiveness of diagnostic tests for identifying the HPV virus. A further object of the invention is to make available a test for identifying the presence of HPV virus in cells infected by said virus, in particular when the virus genome is integrated in DNA of cells. The present invention is based on identifying sequences of nucleotides of the HPV virus genome, said sequences allowing the virus to be identified in all significant phases of infection and further allowing low hazard virus typologies to be distinguished from middle and high hazard virus typologies. In particular, two sequences have been identified in the regions E6 and E7 belonging to the early region E of the virus genome, which keeps even when the virus is partially or totally integrated in a cell genome. By detecting the above-mentioned sequences and the sequence of the region Ll it is possible to identify the presence of HPV virus in every stage of infection and to distinguish between virus strains having low hazard and virus strains having middle/high hazard. In a first aspect of the present invention, a method is provided for identifying the presence of viral DNA of HPV virus in cellular material collected from an individual, comprising the following steps: - extracting DNA from said cellular material amplifying said DNA by means of polymerase, using identifying means for identifying sequences complementary to sequences of the genome of HPV virus, characterized in that said identifying means comprises identifying means suitable for identifying sequences complementary to sequences of said genome in the regions E of genome . In a second aspect of the present invention, identifying means are provided for identifying sequences complementary to sequences of the genome of HPV virus, characterized in that said identifying means comprises identifying means suitable for identifying sequences complementary to sequences of said genome in the regions E of genome . In a third aspect of the present invention a diagnostic kit is provided for amplifying DNA of HPV virus comprising identifying means suitable for identifying sequences complementary to sequences of the genome of said virus in the regions Ll, E6 and E7 of genome, amplifying means for amplifying DNA of said virus, marker means, mineral oil. Some embodiments of the invention will be described below, only for exemplifying and non restrictive purposes. In order to carry out an identifying test for identifying the presence of HPV virus, first of all DNA has to be extracted from specimens of organic tissue to be examined. The above mentioned organic tissue . may be in form of biopsy fresh material, or fixed and embedded in paraffin, or specimens of desquamation cells obtained through vaginal or urethral swab and material collected from vagina using a spatula. DNA preparation from tissue fixed and embedded in paraffin: 1) 1-6 small slices of waxed tissue are introduced in a 1,5 ml test tube of the Eppendorf type; 2) one ml of Xylene (Reactant A) is added and the whole is shaken in a vortex for 10 sec; 3) the whole is centrifuged for 5 minutes at 13000 rpm; 4) the floating material is removed and the operations of points 2) and 3) above are repeated; 5) the floating material is discarded and 1 ml of absolute alcohol (Reactant B) is added to the pellet; 6) the whole is shaken in a vortex for 10 sec, then it is centrifuged as in point 3) above and the floating material is removed; 7) the pellet is left to dry for about 20 minutes; 8) 340 μl of solution , 30 ml of reactant 1; 15 μl of Proteinase K (18 mg/ml) are added; 9) the whole is incubated at a temperature of 55 °C for a period of some 30 minutes to 60 minutes, or for an entire night at a temperature of 37 °C; 10) 200 μl of Reactant 2 are added and the whole is shaken in the vortex for 20 sec; 11) the whole is centrifuged for 5 minutes at 13000 rpm; 12) the floating material containing the nucleic acids is collected and transferred into another test tube; 13) 500 μl of isopropyl alcohol are added, the whole is mixed and DNA is caused to precipitate; 14) the whole is centrifuged for 1 minute at 13000 rpm and the floating material is removed; 15) 500 μl of 70% ethanol are added and the whole is centrifuged for 1 minute at 13000 rpm; 16) the ethanol is removed without moving the sediment and the whole is left to dry for about 20 minutes; 17) the whole is suspended again in 25-50 μl of ddH20 and kept at a temperature between -20 °C and -70 °C. The solution W, the reactant 1 and the reactant 2 have the following compositions: Solution W: Tris pH 7,6 10 mM EDTA pH 8 10 mM NaCl 50 mM Reactant 1 : Sodium Dodecyl Sulfate - sodium salt - 10% Chloride in milliQ or bi-distilled sterile H20 Reactant 2: Sodium acetate 3M DNA preparation from fresh tissue The preparing process previously described for specimens of tissue fixed and embedded in paraffin is used, starting from point 8) of the process. DNA preparation from desquamation cells obtained through vaginal swab or material collected from vagina using a spatula. 1) The cellular material is suspended in 10 ml of physiologic solution in a 15 ml centrifuge test tube; 2) the whole is centrifuged at 2000 rpm for 10 minutes at room temperature; 3) the floating material is removed and the pellet is suspended again in 10 ml of physiologic solution; 4) the whole is centrifuged as in point 2) and the floating material is discarded; the procedure is then carried out as described for the process for preparing DNA from specimens of tissue fixed and embedded in paraffin, starting from point 8) of said process. After DNA has been prepared, DNA is amplified through polymerase reaction, using amplifying means for amplifying DNA, said amplifying means comprising identifying means for identifying sequences of nucleotides of region Ll and of regions E6 and E7 of the HPV virus genome. The identifying means for identifying said sequences of said regions E6 and E7 comprises a first primer representative of said region E7, with sequence 5' -GAG-CTG-TCG-CTT-AAT-TGC-TC- 3' , a second primer with sequence 5' -TGT-CAA-AAA-CCG-TTG-TGT- CC-3' representative of said region E6 of strains of HPV virus having high-middle hazard, a third primer representative of said region E6 of strains of HPV virus having low hazard with sequence 5' -TGC-TAA-TTC-GGT-GCT-ACC-TG-3' . The identifying means for identifying said sequences in said region Ll comprises a fourth primer with sequence 5'-CGT-CCM- ARR-GGA-WAC-TGA-TC-3' and a fifth primer with sequence 5'-GCM- CAG-GGW-CAT-AAY-AAT-GG-3' . DNA amplification is carried out separately for the late region L of the virus and for the early region E, as described below: the following components are introduced into a thin-walled test tube, for each amplification reaction: for the late region L: - a first reaction mixture containing said fourth primer and said fifth primer for sequences in the region Ll: 45 μl - DNA polymerase 0,5 μl - sample of DNA 5 μl said first mixture comprising: - buffer of PCR without MgC12 IX - dNTPs 200 μM - MgC12 6 mM - Fourth primer 0,5 μM - Fifth primer 0,5 μM - milliQ or bi-distilled sterile H20 for the early region E, for HPV virus belonging to the high- middle hazard group: - a second reaction mixture containing said first primer and said second primer for sequences in the regions E6 and E7 : 45 μl - DNA polymerase 5 μl - sample of DNA 5 μl said second mixture comprising: - buffer of PCR without MgCl2 IX - dNTPs 200 μM - MgC12 6 mM - First primer 0, 5 μM - Second primer o ( 5 μM - milliQ or bi-distilled sterile H20 for the early region E, for HPV virus belonging to the low hazard group: - a third reaction mixture containing said first primer and said third primer for sequences in the regions E6 and E7: 45 μl - DNA polymerase o, 5 μl - sample of DNA 5 μl said third mixture comprising: - buffer of PCR without MgCl2 IX - dNTPs 200 μM - MgC12 6 mM - First primer o, 5 μM - Third primer o, 5 μM - milliQ or bi-distilled sterile H20 The amplification reaction of DNA may be subsequently carried out according to the following table: Samples of DNA obtained from the amplifying reaction are then sown on an agarose gel in a concentration of 2-4%, together with marker means suitable for highlighting the viral sequences identified by primers. The samples are eventually examined with ultraviolet light in order to highlight possible positive results of the amplification of sequences of regions Ll, E6/E7 of the genome of HPV virus . The positive results are represented by a band of 450 pb for the region Ll, contained therefore between the first and the second restriction fragment of the molecular weight marker, and by a second band of 233-268 pb for the regions E6, E7, contained between the third and the fifth restriction fragment of the molecular weight marker. If the amount of DNA is high, the primers for the sequences of the region E of the strains of low hazard virus, may cause unspecific amplification, i.e. an amplification which does not correspond to an effective presence of virus, but which is represented by a band at about 400 pb, clearly distinguished from the band of 233-268 pb of the specific amplification in presence of virus. In this case, it is advisable to denature the sample for 3 minutes at 94 °C and to repeat the test at a temperature of 0 °C. An interpretative table regarding the result of the above described test on specimens of tissue infected by HPV virus is here attached: After electrophoresis, both the band corresponding to strains of virus with high-middle hazard and the band corresponding to strains of virus with low hazard may be present. This means that a co-infection is present, said co-infection being caused by two different types of HPV virus. Owing to the present invention, by searching for the regions E6 and E7 of virus genome, the presence of infection from HPV virus may be detected even in specimens in which the region Ll of the genome is not present, thus reducing significantly the possibility of errors in identifying the virus presence; furthermore, by searching for the above-mentioned regions E6 and E7 an indication may be obtained about the possible integration of the virus into DNA of the infected cells and, therefore, on the progression level of infection. Eventually, the present invention allows even the group of the viral strain responsible for the infection to be identified, i.e. the invention allows to establish whether a strain of low oncogenic hazard or middle-high oncogenic hazard is involved. The components for the amplifying reaction of DNA may be provided in a reaction kit containing: The final volumes of first, second and third mixture may also be halved, i.e. reduced to 742,5 μl, without modifying the final concentrations of single components. The mineral oil has the function of preventing the buffer of PCR from evaporating if a thermo-cycler without thermo-heated cover is used.

Claims: CLAIMS 1. Method for identifying the presence of viral DNA of HPV virus in cellular material collected from an individual, comprising the following steps: - extracting DNA from said cellular material amplifying said DNA by means of polymerase, using identifying means for identifying sequences complementary to sequences of the genome of HPV virus, characterised in that said identifying means comprises identifying means suitable for identifying sequences complementary to sequences of said genome in the regions E of the genome. 2. Method according to claim 1, wherein said identifying means is suitable for identifying sequences complementary to sequences in the region E6 of said genome. 3. Method according to claim 1, or 2, wherein said identifying means is suitable for identifying sequences complementary to sequences in the region E7 of said genome. 4. Method according to claim 2, or 3, wherein said identifying means is suitable for identifying sequences complementary to sequences in the region E6 of the genome of strains of HPV virus having middle/high oncogenic hazard. 5. Method according to any of claims 2 to 4, wherein said identifying means is suitable for identifying sequences complementary to sequences in the regions E6 of strains of the genome of HPV virus having low oncogenic hazard. 6. Method according to any of claims 3 to 5, wherein said identifying means comprises a first primer directed on said region E7. 7. Method according to claim 6, wherein said first primer comprises the following sequence: 5' -GAG-CTG-TCG-CTT-AAT-TGC- TC-3' . 8. Method according to any of claims 4 to 7, wherein said identifying means comprises a second primer directed on said region E6 of strains of HPV virus having middle/high oncogenic hazard. 9. Method according to claim 8, wherein said second primer comprises the following sequence: 5' -TGT-CAA-AAA-CCG-TTG-TGT- CC-3' . 10. Method according to any of claims 5 to 9, wherein said identifying means comprises a third primer directed on said region E6 of strains of HPV virus having low oncogenic hazard. 11. Method according to claim 11, wherein said third primer comprises the following sequence: 5' -TGC-TAA-TTC-GGT-GCT-ACC- TG-3' . 12. Method according to any of preceding claims wherein said identifying means comprises identifying means suitable for identifying sequences complementary to sequences of the region Ll of the genome of said HPV virus . 13. Method according to claim 12, wherein said identifying means comprises a fourth and/or a fifth primer directed on said Ll region. 14. Method according to claim 13, wherein said fourth primer comprises the following sequence: 5' -CGT-CCM-ARR-GGA-WAC-TGA- TC-3' . 15. Method according to claim 15, wherein said fifth primer comprises the following sequence: 5' -GCM-CAG-GGW-CAT-AAY-AAT- GG-3' . 16. Identifying means for identifying sequences complementary to sequences of the genome of HPV virus, characterized in that it comprises identifying means suitable for identifying sequences complementary to sequences in the regions E of said genome . 17. Identifying means according to the claim 16, characterized in that it is suitable for identifying sequences complementary to sequences in the region E7 of said genome. 18. Identifying means according to claim 16, or 17, characterized in that it is suitable for identifying sequences complementary to sequences in the region E6 of the genome of strains of HPV virus having middle/high oncogenic hazard. 19. Identifying means according to any of claims 16 to 18, characterized in that it is suitable for identifying sequences complementary to sequences in the regions E6 of the genome of strains of HPV virus having low oncogenic hazard. 20. Identifying means according to any of claims 17 to 19, characterized in that it comprises a first primer directed on said region E7. 21. Identifying means according to claim 20, wherein said first primer comprises the following sequence: 5' -GAG-CTG-TCG- CTT-AAT-TGC-TC-3' . 22. Identifying means according to any of claims 18 to 21, characterized in that it comprises a second primer directed on said region E6 of the genome of strains of HPV virus having high/middle hazard. 23. Identifying means according to claim 22 wherein said second primer comprises the following sequence: 5'-TGT-CAA- AAA-CCG-TTG-TGT-CC-3' . 24. Identifying means according to any of claims 19 to 23, characterized in that it comprises a third primer directed on said region E6 of strains of HPV virus having low oncogenic hazard*. 25. Identifying means according to claim 24, wherein said third primer comprises the following sequence: 5' -TGC-TAA-TTC- GGT-GCT-ACC-TG-3' . 26. Identifying means according to any of claims 16 to 25, characterized in that it is suitable for identifying sequences complementary to sequences of the region Ll of the genome of HPV virus. 27. Identifying means according to claim 26, characterized in that it comprises a fourth and/or a fifth primer directed on said region Ll . 28. Identifying means according to claim 27, wherein said fourth primer comprises the following sequence: 5'-CGT-CCM- ARR-GGA-WAC-TGA-TC-3' . 29. Identifying means according to claim 27, or 28, wherein said fifth primer comprises the following sequence: 5'-GCM- CAG-GGW-CAT-AAY-AAT-GG-3' . 30. Diagnostic kit for amplifying DNA of HPV virus comprising identifying means suitable for identifying sequences complementary to sequences of the genome of said HPV virus, amplifying means for amplifying DNA of said virus, marker means, mineral oil. 31. Diagnostic kit according to claim 30, wherein said identifying means comprises identifying means according to any of claims 16 to 25. 32. Diagnostic kit according to claim 30, or 31, wherein said identifying means comprises identifying means according to any of claims 26 to 29. 33. Diagnostic kit according to any of claims 30 to 32, characterized in that it comprises: - a first reaction mixture comprising identifying means according to any of claims 27 to 29 1485 μl - a second reaction mixture comprising identifying means according to any of claims 20 to 23 1485 μl - a third reaction mixture comprising identifying means according to any of claims 20, 21, 24, 25 1485 μl - DNA Polymerase 5ϋ/μl 45 μl - marker means 10 μl - mineral oil 2 ml x 3 34. Diagnostic kit according to any of claims 30 to 32, characterized in that it comprises: - a first reaction mixture comprising identifying means according to any of claims 27 to 29 742,5 μl - a second reaction mixture comprising identifying means according to any of claims 20 to 23 742,5 μl - a third reaction mixture comprising identifying means according to any of claims 20, 21, 24, 25 742,5 μl - DNA Polymerase 5ϋ/μl 45 μl - marker means 10 μl - mineral oil 2 ml x 3 35. Diagnostic kit according to claim 33, or 34, wherein said first mixture comprises: - buffer of PCR without MgC12 IX - dNTPs 200 μM - MgC12 6 mM - Fourth primer o, .5 μM - Fifth primer o, ,5 μM - milliQ or bi-distilled sterile H20. 36. Diagnostic kit according to any o )ff c (cllaaiimmss 33 to 35, wherein said second mixture comprises: - buffer of PCR without MgC12 IX - dNTPs 200 μM - MgC12 6 mM - First primer o, ,5 μM - Second primer 0, .5 μM - milliQ or bi-distilled sterile H20. 37. Diagnostic kit according to any of claims 33 to 36, wherein said third mixture comprises: - buffer of PCR without MgC12 IX - dNTPs 200 μM - MgCl2 6 mM - First primer 0,5 μM - Third primer 0,5 μM - milliQ or bi-distilled sterile H20.

Inventors: DE MONTIS ANTONELLA, FLORIS MONICA MANUELA, PERSEU SINIBALDO, DE MONTIS, ANTONELLA, FLORIS, MONICA, MANUELA, PERSEU, SINIBALDO
Applicants: BIOANALISI CT SUD S N C DI PER, FLORIS MONICA MANUELA, MONTIS ANTONELLA DE, PERSEU SINIBALDO, BIOANALISI CENTRO SUD S.N.C. DI PERSEU SINIBLADO EC., DE MONTIS, ANTONELLA, FLORIS, MONICA, MANUELA, PERSEU, SINIBALDO
Application year: 2001
Main classification: C12Q1
IPC codes: C12Q1