WO1997046716A1 - Method to detect hcv specific nucleic acids - Google Patents
Method to detect hcv specific nucleic acids Download PDFInfo
- Publication number
- WO1997046716A1 WO1997046716A1 PCT/IT1997/000128 IT9700128W WO9746716A1 WO 1997046716 A1 WO1997046716 A1 WO 1997046716A1 IT 9700128 W IT9700128 W IT 9700128W WO 9746716 A1 WO9746716 A1 WO 9746716A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- deia
- primer
- probe
- pcr
- Prior art date
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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
- C12Q1/706—Specific hybridization probes for hepatitis
- C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D
Definitions
- the invention concerns a method to detect hepatitis C virus specific (HCV) nucleic acids.
- HCV hepatitis C virus specific
- 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.
- PCR polymerase chain reaction
- 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.
- the double PCR step has many disadvantages mainly due to DNA contamination from previous amplifications, length of time, high costs.
- the authors of the instant invention have optimized the nested PCR reaction conditions in order to eliminate the second step.
- 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) .
- DEIA DNA Enzyme Immunoassay
- HCV hepatitis C virus
- 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.
- PCR polymerase chain reaction
- RNAzol B e ULTRASPEC Biotecx
- the nested PCR to detect HCV RNA at the 5'UTR region was used as control.
- 5 ⁇ l of extracted RNA were reverse transcribed in 25 ⁇ l volume containing 22.5 mM TRIS-HCI pH 8.3, 62.5 mM KCI, 4 mM MgCl , 250 ⁇ M dNTPs, 4U AMV-RT, 2 ⁇ M 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 ⁇ l volume containing cDNA (25 ⁇ l from the reverse transcription), 22.5 mM TRIS-HCI pH 8.3, 62.5 mM KCI, 4 mM MgCl 2 , 200 ⁇ M dNTPs (only for this step), 0.5 ⁇ M 2CH sense primer, 2.5 U Taq polymerase; thermal cycle: 94°C 1 min., 50°C 1 min., 72°C 2 min., 35 cycles.
- ⁇ l from the first step were amplified in a 100 ⁇ l volume containing 10 mM TRIS-HCI pH 8.3, 50 mM KCI, 1.5 mM MgCl 2 , 200 ⁇ M dNTPs, 0.5 ⁇ M ITS internal antisense primer, 0.5 ⁇ M 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.
- 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)
- PCR was performed in 100 ⁇ l final volume containing cDNA, 22.5 mM TRIS-HCI pH 8.3, 62.5 mM KCI, 4 mM MgCl 2 , 200 ⁇ M dNTP (only those added in this step) , 0.5 ⁇ M 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 ⁇ l of each amplified product were assayed by means of the DEIA Enzyme Immunoassay with the 3CH probe.
- N. 2 Pos 2,185 0,123 0,078 0,108 0,103 1,159
- Taq polymerase activator has to be finely modulated to obtain the best yield of the amplification reaction.
- MgCl concentrations of 1.5 mM, 2.5 mM, 4 mM, corresponding, respectively, to 60nmol, lOOnmol, l ⁇ Onmol 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 ⁇ l volume containing cDNA (25 ⁇ l from the reverse transcription mix), 22.5 mM TRIS-HCI pH 8.3, 62.5 mM KCI, MgCl. at different concentrations, 200 ⁇ M dNTPs (only dNTPs added in this step), 0,5 ⁇ M 2CH primer and 2,5 U Taq polymerase.
- RNA 5 ⁇ l of RNA were reverse transcribed in a 25 ⁇ l volume containing 50 mM TRIS-HCI pH 8.3, 50 mM KCI, 10 mM MgCl 2 , 250 ⁇ l dNTP, 2 ⁇ M 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.
- the amplification reaction was performed in a 100 m
- 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.
- thermodynamic analysis of sequences shows that hybridisation reactions of all of oligos with complementary sequences are thermodynamically favoured.
- the lowest ⁇ G values are those of the longest probes (3CH, HCV40, KY150) , which on the other hand favour the formation of either loops or dimers.
- 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.
- 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.
- 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.
- NAME SORIN BIOMEDICA DIAGNOSTICS S.p.A.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002257191A CA2257191A1 (en) | 1996-06-07 | 1997-06-03 | Method to detect hcv specific nucleic acids |
AU30477/97A AU3047797A (en) | 1996-06-07 | 1997-06-03 | Method to detect hcv specific nucleic acids |
EP97925277A EP0937161A1 (en) | 1996-06-07 | 1997-06-03 | Method to detect hcv specific nucleic acids |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IT96RM000404A IT1284847B1 (en) | 1996-06-07 | 1996-06-07 | PROCEDURE FOR THE DETECTION OF HCV SPECIFIC NUCLEIC ACIDS |
ITRM96A000404 | 1996-06-07 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1997046716A1 true WO1997046716A1 (en) | 1997-12-11 |
Family
ID=11404271
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/IT1997/000128 WO1997046716A1 (en) | 1996-06-07 | 1997-06-03 | Method to detect hcv specific nucleic acids |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0937161A1 (en) |
AU (1) | AU3047797A (en) |
CA (1) | CA2257191A1 (en) |
IT (1) | IT1284847B1 (en) |
WO (1) | WO1997046716A1 (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19832050A1 (en) * | 1998-07-16 | 2000-01-27 | Biotest Pharma Gmbh | Detection of Hepatitis C and B viral genomes in serum or plasma using specific oligonucleotide primers and probes |
EP1026262A2 (en) * | 1999-02-03 | 2000-08-09 | Ortho-Clinical Diagnostics, Inc. | Oligonucleotide primers for efficient detection of hepatitis C virus (HCV) and methods of use thereof |
JP2000279198A (en) * | 1999-02-03 | 2000-10-10 | Ortho Clinical Diagnostics Inc | Oligonucleotide primer for efficient multiplex detection of hepatitis c virus(hcv) and human immunodeficiency virus(hiv) and use thereof |
KR100451049B1 (en) * | 2001-04-12 | 2004-10-02 | 바이오코아 주식회사 | Oligonucleotide chip composition for analyzing Hepatitis C virus (HCV) genotype and detecting method thereof |
KR100658606B1 (en) | 2005-04-22 | 2006-12-15 | (주)팬바이오넷 | Method for determination of hepatitis c virus genotype |
AU2006203092B2 (en) * | 1999-02-03 | 2009-11-12 | Ortho-Clinical Diagnostics, Inc. | Oligonucleotide primers for efficient detection of hepatitis C virus (HCV) and methods of use thereof |
US20140134611A1 (en) * | 2012-10-18 | 2014-05-15 | Roche Molecular System, Inc. | Dual probe assay for the detection of heterogeneous amplicon populations |
US11274998B2 (en) | 2012-12-26 | 2022-03-15 | Ventana Medical Systems, Inc. | Specimen processing systems and methods for holding slides |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992019743A2 (en) * | 1991-05-08 | 1992-11-12 | Chiron Corporation | Hcv genomic sequences for diagnostics and therapeutics |
EP0518313A2 (en) * | 1991-06-11 | 1992-12-16 | Mitsubishi Chemical Corporation | Gene of hepatitis C virus or fragment thereof, polypeptide encoded by the same |
EP0529493A2 (en) * | 1991-08-27 | 1993-03-03 | F. Hoffmann-La Roche Ag | Methods and reagents for hepatitis C detection |
WO1995006753A1 (en) * | 1993-09-03 | 1995-03-09 | The United States of America, represented by The Secretary, Department of Health & Human Services | Method and compositions for primer specific solid-phase detection of pcr products |
JPH07250700A (en) * | 1994-03-15 | 1995-10-03 | Tonen Corp | Simple quantification of nucleic acid by competitive pcr process |
EP0699751A1 (en) * | 1992-08-25 | 1996-03-06 | Mitsubishi Chemical Corporation | Antisense complementary to HCV genome |
-
1996
- 1996-06-07 IT IT96RM000404A patent/IT1284847B1/en active IP Right Grant
-
1997
- 1997-06-03 CA CA002257191A patent/CA2257191A1/en not_active Abandoned
- 1997-06-03 WO PCT/IT1997/000128 patent/WO1997046716A1/en not_active Application Discontinuation
- 1997-06-03 AU AU30477/97A patent/AU3047797A/en not_active Abandoned
- 1997-06-03 EP EP97925277A patent/EP0937161A1/en not_active Withdrawn
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992019743A2 (en) * | 1991-05-08 | 1992-11-12 | Chiron Corporation | Hcv genomic sequences for diagnostics and therapeutics |
EP0518313A2 (en) * | 1991-06-11 | 1992-12-16 | Mitsubishi Chemical Corporation | Gene of hepatitis C virus or fragment thereof, polypeptide encoded by the same |
EP0529493A2 (en) * | 1991-08-27 | 1993-03-03 | F. Hoffmann-La Roche Ag | Methods and reagents for hepatitis C detection |
EP0699751A1 (en) * | 1992-08-25 | 1996-03-06 | Mitsubishi Chemical Corporation | Antisense complementary to HCV genome |
WO1995006753A1 (en) * | 1993-09-03 | 1995-03-09 | The United States of America, represented by The Secretary, Department of Health & Human Services | Method and compositions for primer specific solid-phase detection of pcr products |
JPH07250700A (en) * | 1994-03-15 | 1995-10-03 | Tonen Corp | Simple quantification of nucleic acid by competitive pcr process |
Non-Patent Citations (5)
Title |
---|
GUNJI T ET AL: "SPECIFIC DETECTION OF POSITIVE AND NEGATIVE STRANDED HEPATITIS C VIRAL RNA USING CHEMICAL RNA MODIFICATION", ARCHIVES OF VIROLOGY, vol. 134, no. 3/04, pages 293 - 302, XP000615872 * |
IMBERTI L. ET AL.,: "An immunoassay for specific amplified HCV sequences", J. VIROLOGICAL METHODS, vol. 34, - 1991, pages 233 - 243, XP002042919 * |
IMBERTI L. ET AL.,: "Non-radioisotopic methods for DNA probes", ANN. BIOL. CLIN., vol. 50, - 1992, pages 163 - 167, XP002042920 * |
MANTERO G ET AL: "DNA ENZYME IMMUNOASSAY: GENERAL METHOD FOR DETECTING PRODUCTS OF POLYMERASE CHAIN REACTION", CLINICAL CHEMISTRY, vol. 37, no. 3, pages 422 - 429, XP000371646 * |
PATENT ABSTRACTS OF JAPAN vol. 096, no. 002 29 February 1996 (1996-02-29) * |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19832050C2 (en) * | 1998-07-16 | 2002-10-10 | Biotest Pharma Gmbh | Procedure for the detection of hepatitis B or hepatitis C virus genomes in plasma samples and specific primers |
DE19832050A1 (en) * | 1998-07-16 | 2000-01-27 | Biotest Pharma Gmbh | Detection of Hepatitis C and B viral genomes in serum or plasma using specific oligonucleotide primers and probes |
US6638714B1 (en) | 1999-02-03 | 2003-10-28 | Ortho-Clinical Diagnostics, Inc. | Oligonucleotide primers for efficient detection of hepatitis C virus (HCV) and methods of use thereof |
JP2000279200A (en) * | 1999-02-03 | 2000-10-10 | Ortho Clinical Diagnostics Inc | Oligonucleotide primer for efficient detection of hepatitis c virus(hcv) and use thereof |
EP1026262A3 (en) * | 1999-02-03 | 2001-11-21 | Ortho-Clinical Diagnostics, Inc. | Oligonucleotide primers for efficient detection of hepatitis C virus (HCV) and methods of use thereof |
JP2000279198A (en) * | 1999-02-03 | 2000-10-10 | Ortho Clinical Diagnostics Inc | Oligonucleotide primer for efficient multiplex detection of hepatitis c virus(hcv) and human immunodeficiency virus(hiv) and use thereof |
EP1026262A2 (en) * | 1999-02-03 | 2000-08-09 | Ortho-Clinical Diagnostics, Inc. | Oligonucleotide primers for efficient detection of hepatitis C virus (HCV) and methods of use thereof |
CN100441700C (en) * | 1999-02-03 | 2008-12-10 | 奥索临床诊断有限公司 | Oligonucleotide primers for efficient detection of hepatitis c virus (HCV) and methods of use thereof |
AU2006203092B2 (en) * | 1999-02-03 | 2009-11-12 | Ortho-Clinical Diagnostics, Inc. | Oligonucleotide primers for efficient detection of hepatitis C virus (HCV) and methods of use thereof |
KR100451049B1 (en) * | 2001-04-12 | 2004-10-02 | 바이오코아 주식회사 | Oligonucleotide chip composition for analyzing Hepatitis C virus (HCV) genotype and detecting method thereof |
KR100658606B1 (en) | 2005-04-22 | 2006-12-15 | (주)팬바이오넷 | Method for determination of hepatitis c virus genotype |
US20140134611A1 (en) * | 2012-10-18 | 2014-05-15 | Roche Molecular System, Inc. | Dual probe assay for the detection of heterogeneous amplicon populations |
US9963737B2 (en) * | 2012-10-18 | 2018-05-08 | Roche Molecular Systems, Inc. | Dual probe assay for the detection of heterogeneous amplicon populations |
US11274998B2 (en) | 2012-12-26 | 2022-03-15 | Ventana Medical Systems, Inc. | Specimen processing systems and methods for holding slides |
Also Published As
Publication number | Publication date |
---|---|
CA2257191A1 (en) | 1997-12-11 |
EP0937161A1 (en) | 1999-08-25 |
AU3047797A (en) | 1998-01-05 |
ITRM960404A0 (en) | 1996-06-07 |
IT1284847B1 (en) | 1998-05-22 |
ITRM960404A1 (en) | 1997-12-07 |
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