METHOD TO DETECT HCV SPECIFIC NUCLEIC ACIDS

Abstract: A process to detect hepatitis C virus (HCV) specific nucleic acids comprising the steps of: (a) reverse transcribing the viral RNA by means of a primer having specified sequences; (b) amplifying by a single polymerase chain reaction (PCR) with a specific primer; wherein the Mg<++>/Taq polymerase ratio in the reaction mix is of approx. 100 nmoles/enzyme unit; (c) revealing the amplified product by means of the DEIA method using a specific probe.

Description: METHOD TO DETECT HCV SPECIFIC NUCLEIC ACIDS The invention concerns a method to detect hepatitis C virus specific (HCV) nucleic acids. In particular the invention refers to an improved method to detect HCV amplified DNA, by means of a single step polymerase chain reaction (PCR) , under controlled and optimized reaction parameters, and of a revealing system of amplified products. One of the most used methods to detect HCV specific nucleic acids is based upon the reverse transcription of viral RNA to cDNA, followed by a double amplification step (nested PCR) of the most conserved genome region (5'UTR) . The amplified product of the second amplification step may be identified by means of revealing techniques as electrophoresis or enzyme mediated signals. The double amplification allows to reach a very high sensitivity able to identify even few viral RNA molecules. On the other hand the double PCR step has many disadvantages mainly due to DNA contamination from previous amplifications, length of time, high costs. In order to overcome said disadvantages there is the need to set up a single step amplification protocol, which reaches similar levels of sensitivity than the nested PCR. The authors of the instant invention have optimized the nested PCR reaction conditions in order to eliminate the second step. Moreover the system used to reveal amplified products is the DNA Enzyme Immunoassay (DEIA) , _ hich mekes the use of a specific capturing probe and of a monoclonal antibody able to recognize double strand DNA (Mantero G. et al . Clin Chem. 37, 422-429, here incorporated by references) . The combination of all of optimized parameters, both of the amplification and of the revealing step, allowed to set up a method able to detect HCV specific DNA with a sensitivity equal, if not higher than the nested PCR, but with no disadvantages. In order to optimize the PCR reaction parameters a comparison of first (not revealed by DEIA assay) and second (revealed by DEIA assay) amplification step, and an analysis to individuate the capturing probe with best features for a revealing step by DEIA assay as well, were performed. C. Payan et al. J. Virol. Meth. (1995) 53, 167-175 describe a process to reveal HCV nucleic acids characterised by a single step for both the reverse transcription and the amplification. The amplification is a single step, no nested-PCR. Primer sequences are deduced from the HCV genome nucleotide sequence, and in particular from the high conserved 5'end region. The magnesium chloride final concentration in the PCR solution is of 1 mM, the ratio [Mg " ]/U Taq being 35 nmol/U Taq. C. Payan et al. Res. Virol. (1995) 1_46, 363-70 optimize the above referred process by modifying the MgCl 2 concentration to 2 mM, the units of Mu-MLV RNAse and of Taq polymerase, to 10 U and 1 U respectively, with a ratio of 42 nmol [Mg ++ _/U Taq, and thus diminishing the RNA copy number to be revealed from 15 to 10. It is therefore evident that even minimal variations of general reaction conditions may interfere and increase significantly the yield and sensitivity. It is an object of the instant invention a method to detect hepatitis C virus (HCV) specific nucleic acids comprising the steps of: a) reverse transcribing the viral RNA with a primer having a sequence substantially homologous to one of the sequences SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3; b) amplifying by means of a single step polymerase chain reaction (PCR) wherein the primer has a sequence substantially homologous to sequences of SEQ ID No. 4 or SEQ ID No. 5, wherein the ratio between the Mg" ion concentration and of Taq polymerase in the reaction mix is of approximately of 100 nmoles/enzyme unit; c) revealing the amplified product by means of DEIA method using a probe having the sequence of SEQ ID No. 6, or a probe having a sequence substantially homologous or complementary thereof. It is a further object of the invention the use of the of an oligonucleotide of SEQ ID No. 6 or of an oligonucleotide having a sequence substantially homologous or complementary thereof as probe for revealing HCV specific nucleic acids by means of the DEIA method. It is a further object of the invention a composition to reveal HCV specific nucleic acids by means of the DEIA method comprising an oligonucleotide of SEQ ID No. 6 or of sequence substantially homologous or complementary thereof. By comparing the two steps of the nested- PCR, as in Table 2, it is clear that main differences are: 1) primer sequences; 2) MgCl 2 concentration (which is a Taq polymerase activator) ; 3) buffer concentration (TRIS-HCI pH 8.3); 4) solution ionic strength (mainly related to the KCI concentration) ; 5) deoxinucleotidetriphosphate concentration (dNTP) . Primer sequences are shown in Table la. Table la Primer sequences name se qυence SEQ ID No. 1 CH 5' GGT GCA CGG TCT ACG AGA CCT 3* SEQ ID No. 1 2 CH 5' AAC TAC TGT CTT CAC GCA GAA 3' SEQ ID No. 4 1 TS 5' GCG ACC CAA CAC TAC TCG GCT 3' SEQ ID No. 2 4 CH 5' ATG GCG TTA GTA TGA GTG 3' SEQ ID No. 5 PT2 5' CGG TGT ACT CAC CGG TTC 3' SEQ ID No. 3 PKY78 5' CTC GCA AGC ACC CTA TCA GGC AGT 3' SEQ ID No. 7 PKYΘO 5' GCA GAA AGC GTC TAG CCA TGG CGT 3' SEQ ID No. 8 Probe sequences for the revealing step are shown in Table lb. Table lb 3 CH 5' CGG TGA GTA CAC CGG AAT TGC CAG GAC CGG SEQ ID No. 9 GTC CTT TCT 3' WT 5' GCC ATA GTG GTC TGC GG 3' SEQ ID No. 10 PT 21 5' GGG AGA GCC ATA GTG GTC TGC 3' SEQ ID No. 6 KY 150 5' CAT AGT GGT CTG CGG AAC CGG TGA GT 3' SEQ ID No. 11 HCV40 5' CCA TAG TGG TCT GCG GAA CCG TGA GTA CA 3' SEQ ID No. 12 CH5 5' TAG TGG TCT GCG GAA CCG GT 3' SEQ ID No. 13 Table 2 Two step nested PCR parameters param. I step II step primer lCH(50pmol) /2CH(50pmol) lTS(50pmol)/4CH(50pmol) [Tris-HCI] 22.5 mM 10 mM [KCI] 62.5 mM 50 mM [ gCl 2 ] 4 mM 1.5 mM [dNTP] 260 μM ** 200 μM Taq pol. 2.5 U 2.5 U ★ * The concentration accc junts also for added dl during the reverse transcription step. 1) SAMPLE PREPARATION Sera to be tested by nested or single step PCR were treated to isolate RNA. Commercially available products, as RNAzol B e ULTRASPEC (Biotecx) , were used following instructions of producer. 2) NESTED PROTOCOL The nested PCR to detect HCV RNA at the 5'UTR region was used as control. 5 μl of extracted RNA were reverse transcribed in 25 μl volume containing 22.5 mM TRIS-HCI pH 8.3, 62.5 mM KCI, 4 mM MgCl , 250 μM dNTPs, 4U AMV-RT, 2 μM 1CH antisense primer, 25 U RNAse inhibitors (HRPI) . The reverse transcription was performed at 42°C for 1 hr and the enzyme was further denatured at 100°C for 10 min. The nested PCR first step was performed in a 100 μl volume containing cDNA (25 μl from the reverse transcription), 22.5 mM TRIS-HCI pH 8.3, 62.5 mM KCI, 4 mM MgCl 2 , 200 μM dNTPs (only for this step), 0.5 μM 2CH sense primer, 2.5 U Taq polymerase; thermal cycle: 94°C 1 min., 50°C 1 min., 72°C 2 min., 35 cycles. For the second step 3 μl from the first step were amplified in a 100 μl volume containing 10 mM TRIS-HCI pH 8.3, 50 mM KCI, 1.5 mM MgCl 2 , 200 μM dNTPs, 0.5 μM ITS internal antisense primer, 0.5 μM 4CH internal sense primer; 2.5 U Taq polymerase; thermal cycle: 9 °C 1 min., 50°C 1 min., 72°C 2 min.; 25 cycles. 20 μl of each amplified product were tested by the DEIA assay using as probe the 3CH oligonucleotide. Experimental tests are hereinbelow reported, according to different parameters of the first and second PCR steps. In all of reactions the amount of AMV-RT enzyme was of 15 U/sample. 3) CHANGE of PRIMER SEQUENCES Different combinations of primers were assayed in the single step RT-PCR. Nested external primers: ICH (antisense) / 2CH (sense) Nested internal primers: ITS (antisense) / 4CH (sense) Nested external/internal primers: ICH (antisense) / 4CH (sense) Nested internal/external primers: ITS (antisense) / 2CH (sense) The antisense primer PT2 and those described in the EP 529493 application, identified as PKY78 antisense and PKY80 sense, were also assayed. Reaction conditions were those for all of primer combinations and are as follows: - reverse transcription was performed as for the nested protocol, but of AMV-RT units; PCR was performed in 100 μl final volume containing cDNA, 22.5 mM TRIS-HCI pH 8.3, 62.5 mM KCI, 4 mM MgCl 2 , 200 μM dNTP (only those added in this step) , 0.5 μM sense primer (according to different combinations), 2.5 U Taq polymerase; thermal cycle: 94°C 1 min., 50°C 1 min., 72°C 2 min.; 45 cycles; - 20 μl of each amplified product were assayed by means of the DEIA Enzyme Immunoassay with the 3CH probe. By only varying primer combinations ICH, 2CH, 4CH, ITS, PT2 no detectable amplified product was made in a single step PCR amplification. Even when "alternative" sequences were use (primers as in EP 529493) no clear positive results were obtained (positive result only in one sample) , see Table 3 (positive values in bold character) . Table 3 nested single step PCR PCR primer combination 1CH/2CH 1TS/4CH 1CH/4CH 1TS/2CH PKY78/PKY80 3ample O.D. 450/630 nm 0 RNA 0,061 0,068 0,076 0,176 0, 110 0,084 N. 1 Neg 0,084 0,062 0,067 0,103 0, 112 0,089 N. 2 Pos 2,185 0,123 0,078 0,108 0,103 1,159 N. 3 Pos 1,979 0,066 0,081 0,109 0,109 0,092 N. 4 Pos 2,566 0,054 0,084 0,105 0,113 0,069 cut-off 0,192 0, 192 0,192 0,231 0,231 0,220 4) MODIFICATIONS OF MgCl CONCENTRATION It has been found that the MgCl : concentration (a Taq polymerase activator) has to be finely modulated to obtain the best yield of the amplification reaction. During the amplification, MgCl ; concentrations of 1.5 mM, 2.5 mM, 4 mM, corresponding, respectively, to 60nmol, lOOnmol, lδOnmol of Mg" + per Taq unit were used. The reverse transcription reaction was performed as in the nested PCR protocol, but of enzyme units. The PCR reaction was performed in a 100 μl volume containing cDNA (25 μl from the reverse transcription mix), 22.5 mM TRIS-HCI pH 8.3, 62.5 mM KCI, MgCl. at different concentrations, 200 μM dNTPs (only dNTPs added in this step), 0,5 μM 2CH primer and 2,5 U Taq polymerase. Data obtained by means of the DEIA immunoenzyme assay (with the 3CH probe), as in Table 4, show that: a) very low MgCl 2 concentrations (1.5 mM, 60 nmol/U) reduce the yield of the single step PCR reaction; in fact, only 50% of nested positive samples are still positive; b) very high MgCl 2 concentrations (4 mM, 160 nmol/U) are not workable since no nested positive sample was confirmed as such in the single step reaction; c) a MgCl concentration of 2.5 mM (100 nmol/U) is the best, confirming all of data obtained by the nested PCR. Table 4 5) MODIFICATIONS OF REACTION BUFFER CONCENTRATION As opposite to MgCl 2 , differences in the concentration of TRIS-HCI (12.5 mM instead of 22.5 mM) and KCI (50 mM instead of 62.5 mM) do not interfere in a significant way with the efficacy of the single step amplification, as DEIA obtained data are comparable to controls, as in Table 5. Table 5 Reverse transcription and amplification reactions were performed as described in 4) . 6) MODIFICATION OF dNTP CONCENTRATION Experimental tests were performed in order to evaluate the nucleotide concentration during the single step amplification. Results obtained by DEIA assays (as in Table 6) show that a dNTP concentration lower than the single step optimized concentration (83 μM instead of 200 μM, as referred to dNTP added to the amplification step) lowers the reaction yield. Table 6 Reverse transcription and amplification reactions were performed as described in 4) . 7) SINGLE STEP PCR According to the above described tests the best reaction conditions to amplify HCV RNA are as follows: A) REVERSE TRANSCRIPTION 5 μl of RNA were reverse transcribed in a 25 μl volume containing 50 mM TRIS-HCI pH 8.3, 50 mM KCI, 10 mM MgCl 2 , 250 μl dNTP, 2 μM ICH antisense primer, 25 U HRPI and 15 U AMV-RT. The reverse transcription reaction was performed at 42°C for 1 hr, followed by an enzyme denaturation step at 100°C for 10 min. B) AMPLIFICATION The amplification reaction was performed in a 100 -μl volume containing the cDNA from the reverse transcription step (25 μl) , 22.5 mM TRIS-HCI pH 8.3, 62.5 mM KCI, 2.5 mM MgCl 2 , 0.5 μM 2CH sense primer, 200 μM dNTP (referring only to dNTP added during said step, and 2.5 U Taq polymerase. C) THERMAL CYCLE 94°C 1 min - 50°C 1 min - 72°C 2 min 45 cycles 8) REVEALING PROBE In all of above experiments the 3CH revealing probe was used (Sorin Biomedica Diagnostics SpA PS000C Hepatitis C) . Different probes were tested, having sequences deduced from a different region of the 5' UTR terminus. Various parameters were evaluated, as the oligonucleotide length that may allow an efficient hybridisation to the complementary sample, and a sequence excluding the formation of thermodyna ically stable loops or dimers, due to the presence of internal homologous stretches, as well. In order to select the optimal sequence an analysis of thermodynamic features of some oligonucleotides, as derived from the internal region of the amplified sequence (HCV genome 5' UTR region) was performed. Table 7 probe ΔG ΣΔG ∑ΔG ΣΔG hybrid loops dimers (loops + dimers) PROBE Kcal/mol WT - 29,6 1,3 0,0 1,3 PT 21 - 35,5 1,1 0,0 1,1 KY150 - 45,7 - 0,6 - 7,4 - 8,0 HCV40 - 52,9 - 0,3 - 7,4 - 7,7 CH 5 - 35,9 - 0,3 - 7,4 - 7,7 3 CH - 83,1 - 8,1 no dimers - 8,1 Some of the selected sequences were deduced from prior art literature, others designed to satisfy the above requisites. The thermodynamic analysis was performed with the OLIGO.EXE structure © (ver 3.3) program distributed by MedProbe A.S. (Norvegy) , by maintaining as constant two parameters: the probe (30 nM) and the salt (188 mM) concentration. These parameters are those experimentally used during the DEIA assay. The thermodynamic analysis of sequences, as in Table 7, shows that hybridisation reactions of all of oligos with complementary sequences are thermodynamically favoured. As expected the lowest ΔG values are those of the longest probes (3CH, HCV40, KY150) , which on the other hand favour the formation of either loops or dimers. On the other side, among the shortest probes, PT21 and WT are those that, according to their sequence typology, show the best features, whereas CH5, though having analogous dimensions to the other two probes, may give rise to undesired thermodynamically stable dimers. The PT21 probe is able to form a more stable specific hybrid than the WT probe. The two PT21 and 3CH probes were then compared. The reaction conditions were standard conditions of the DEIA kit (Sorin Biomedica Diagnostics SpA, GEN-ETI-K-DEIA cod. PS0001) to reveal amplified HCV and foresee a specific hybridisation at 50°C. Experimental results, reported in Table 8, confirm that the PT21 probe allows a more efficient hybridisation with the amplified complementary strand. Approximately 96% of analyzed positive samples show absorbance values higher than 1,0 O.D., whereas with the 3CH probe only 40% of samples overcomes this threshold. Table 8 probe sample 3CH PT21 O.D. 450/630 nm N 1 0,633 2,259 N 2 0,215 1,430 N 3 0,579 2,105 N 4 0,610 2,361 N 5 0,230 0,560 N 6 0,833 2,051 N 7 1,734 2,926 N 8 1,872 2,852 N 9 0,574 2,546 N 10 1,147 2,829 N 11 1,562 2,788 N 12 1,206 2,897 N 13 2,002 2,876 N 14 0,499 2,253 N 16 1, 600 2,715 N 17 1,522 2, 656 N 18 2,281 2, 682 N 19 0,690 2,238 N 20 0,275 2,274 cut off 0,190 0, 198 Finally, experimental analysis of PT21 and 3CH probes were performed by evaluating their specificity in the DEIA conditions as above. The analytical specificity was determined with probe unrelated DNA samples, which are representative of molecular dimensions and of sequence heterogeneity: different concentrations of salmon sperm DNA; different concentrations of calf thymus DNA; unrelated amplified DNA. Results, as reported in Table 9, did not show significant aspecific reactions. being absorbance values lower than cut-off (cut-off = average absorbance of negative samples + 0.150 O.D., as suggested in the DEIA assay) . Table 9 SPECIFICITY PT21 - 3CH SS 0,05 μg/μl 0,061 0,049 0,045 0,059 average neg. Ctrl. 0,049 0,047 average pos. Ctrl. 2,103 2,331 cut off 0,199 0,197 TV: new-born calf time DNA; SS: salmon sperm DNA. The setting of the single step DNA HCV amplification and revealing protocol was performed by optimizing of amplification reaction conditions (in particular by changing the MgCl 2 concentration) , and by using a new revealing probe (PT21) . Said approach was able to give results comparable to the NESTED plus DEIA method. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: SORIN BIOMEDICA DIAGNOSTICS S.p.A. (B) STREET: Via Borgonuovo 14 (C) CITY: Milan (E) COUNTRY: Italy (F) POSTAL CODE (ZIP) : 20121 (ii) TITLE OF INVENTION: Method to detect HCV specific nucleic acids (ϋi) NUMBER OF SEQUENCES: 13 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: " GGTGCACGGT CTACGAGACC T 21 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GCGACCCAAC ACTACTCGGC T 21 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CGGTGTACTC ACCGGTTC 18 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: " AACTACTGTC TTCACGCAGA A 21 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ATGGCGTTAG TATGAGTG 18 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GGGAGAGCCA TAGTGGTCTG C 21 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CTCGCAAGCA CCCTATCAGG CAGT 24 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GCAGAAAGCG TCTAGCCATG GCGT 24 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CGGTGAGTAC ACCGGAATTG CCAGGACGAC CGGGTCCTTT CT 42 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GCCATAGTGG TCTGCGG 17 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CATAGTGGTC TGCGGAACCG GTGAGT 26 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CCATAGTGGT CTGCGGAACC GTGAGTACA 29 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: - TAGTGGTCTG CGGAACCGGT 20

Claims: CLAIMS 1. Process to detect hepatitis C virus (HCV) specific nucleic acids comprising the steps of: a) reverse transcribing the viral RNA by means of a primer having a nucleotide sequence substantially homologous to SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3; b) amplifying by a single polymerase chain reaction (PCR) wherein the primer has a nucleotide sequence substantially homologous to SEQ ID No. 4 SEQ ID No. 5; wherein the Mg ++ / Taq polymerase ratio in the reaction mix is of approx. 100 nmoles/enzyme unit; c) revealing the amplified product by means of the DEIA method using as probe an oligonucleotide of SEQ ID No. 6, or having a sequence substantially homologous or complementary thereof. 2. Use of the oligonucleotide of SEQ ID No. 6 or having a sequence substantially homologous or complementary thereof as probe to reveal HCV specific nucleotides by means of the DEIA method. 3. Composition to reveal HCV specific nucleotides with the DEIA assay comprising the oligonucleotide of SEQ ID No. 6 or an oligonucleotide of sequence substantially homologous or complementary thereof.

Inventors: BOSIO PAOLO, CLEMENZA FILIPPO, STRUMIA CLAUDIA, BOSIO, PAOLO, CLEMENZA, FILIPPO, STRUMIA, CLAUDIA
Applicants: BOSIO PAOLO, CLEMENZA FILIPPO, STRUMIA CLAUDIA, WABCO B V, BOSIO, PAOLO, CLEMENZA, FILIPPO, STRUMIA, CLAUDIA, WABCO B.V.
Application year: 1997
Main classification: C12Q1
IPC codes: C12Q1