Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
  • C12Q1/708—Specific hybridization probes for papilloma

Definitions

• 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.
• 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.
• 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
• 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, ⁇ rethral swabs and seminal fluid collection, or biopsy in the anal region, are used for men.
• 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.
• 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.
• 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.
• 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.
• 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, 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 .
• 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 .
• a diagnostic kit 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.
• 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;
• DNA preparation from desquamation cells obtained through vaginal swab or material collected from vagina using a spatula obtained through vaginal swab or material collected from vagina using a spatula.
• 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:
• said first mixture comprising:
• said second mixture comprising:
• said third mixture comprising:
• 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.
• 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.
• 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.
• 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.
• 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 ⁇ l, 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.

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• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

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• 2000
  • 2000-05-05 IT IT2000MO000091A patent/IT1316070B1/it active
• 2001
  • 2001-05-02 AU AU2001252465A patent/AU2001252465A1/en not_active Abandoned
  • 2001-05-02 EP EP01925788A patent/EP1409747A2/en not_active Withdrawn
  • 2001-05-02 WO PCT/IB2001/000771 patent/WO2001085994A2/en not_active Application Discontinuation

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