WO1996000795B1 - Method of amplification for increasing the sensitivity of detecting nucleic acid-probe target hybrids - Google Patents
Method of amplification for increasing the sensitivity of detecting nucleic acid-probe target hybridsInfo
- Publication number
- WO1996000795B1 WO1996000795B1 PCT/CA1995/000369 CA9500369W WO9600795B1 WO 1996000795 B1 WO1996000795 B1 WO 1996000795B1 CA 9500369 W CA9500369 W CA 9500369W WO 9600795 B1 WO9600795 B1 WO 9600795B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- probe
- sequence
- nucleic acid
- label
- target
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Ceased
Links
Abstract
The present invention provides a method for simplifying and significantly enhancing the sensitivity of nucleic acid hybridization assays. A method is described whereby a single-stranded primary nucleic acid sequence that includes a region of sequences complementary to a single-stranded target nucleic acid sequence is hybridized to the target molecule. Stability of the double-stranded complex thereby formed can be enhanced by using RNA as the probe if DNA is the target or DNA as the probe if RNA is the target. The probe-target complex is subsequently immunocaptured for detection. After washing away extraneous material, a secondary nucleic acid sequence containing many repeating sequence units is hybridized to the probe component of the immobilized probe-target complex. Detection occurs following hybridization of many labelled nucleic acid sequence probes to each of the repeating sequence units of a nucleic acid amplification probe. Thus, attachment of multiple labelling probes to an amplification probe that is hybridized to an immobilized probe-target complex, provides a simplified method for amplifying the detection signal and therefore the sensitivity of nucleic acid hybridization assays.
Claims
1. An amplification probe comprising two regions of nucleic acid sequences: a first region including a homopolymeric nucleotide tail and a second region including a plurality of discretely labelable sequence units, said units ranging from two to fifty in number.
2. An amplification probe as claimed in Claim 1 , wherein the number of nucleotide molecules ranges from ten to sixty in number.
3 . An amplif ication probe as claimed in Claim 1 , wherein each said discretely labelable sequence unit comprises a sequence of nucleotide bases hybridizable to complementary sequence on a labelling probe , said labelling probe covalently attached to a detectable chemicle label .
4 . An ampl if ication probe as claimed in Claim 3 , wherein the length of each sequence unit ranges from 16 to 100 nucleot ides .
5. A method for enhancing detectable labeling of probe-target complexes in nucleic acid hybridization assays comprising hybridizing a complex of a selected nucleic acid target and a selected primary probe with [incorporating] an amplification probe adapted to permit enhanced detectable labelling of [a] said selected nucleic acid target, [such] said probe comprising at least two regions of nucleic acid sequences: a first region including a sequence complementary to a sequence on [a] said selected primary probe which also contains a sequence complimentary to a sequence of said selected nucleic acid target, and a second region including a plurality of discretely labelable sequence units.
6. A method as claimed in Claim 5 wherein each said discretely labelable sequence unit comprises a sequence of nucleotide bases hybridizable to a complementary sequence on a labelling probe, said labelling probe covalently attached to a detectable chemical label.
7. A method as claimed in claim 6 wherein the detectable chemical label is selected from an enzymatically active group, a fluorescer, a chromophore, a luminescer, a specifically bindable ligand, or a radioisotope.
8. A method as claimed in Claim 5 wherein a label attached to each said discretely labelable sequence units interacts with a reagent member of a label detection system to provide the detectable response.
9. The method as claimed in Claim 8 wherein the detectable chemical label is a substrate, cofactor, or inhibitor of an enzyme which is the member of the label detection system with which the label interacts to provide the detectable response.
10. The method as claimed in Claim 9 wherein the label is a substrate which is acted on by the enzyme to produce a colori etric, fluorescent or luminescent signal.
11. The method as claimed in Claim 9 wherein the label is a prosthetic group of an enzyme and wherein the apoenzy e of such enzyme is the member of the label detection system with which the label interacts to produce the catalytically active holoenzyme.
12. The method as claimed in Claim 11 wherein the prosthetic group is [PAD] flavin adenine dinucleotide and the apoenzyme is apo(glucose oxidase) .
13. A method for detecting specific nucleic acid sequences comprising:
(a) hybridizing a first sequence of a primary polynucleotide probe to a selectable target nucleic acid sequence wherein the primary probe has a means for binding to an amplification probe comprising a nucleic acid sequence adapted to permit enhanced detectable labelling, the amplification probe being capable of hybridizing to at least one labelling probe comprising a nucleic acid sequence conjugated to a chemical label;
(b) immobilizing the targe -probe complex;
(c) exposing the immobilized target-probe complex to said amplification probe, such probe comprising at least two regions of nucleic acid sequences: a first region including a sequence complementary to a sequence on a selected primary probe which also contains a sequence complementary to a sequence of said selected nucleic acid target, and a second region including a plurality of discretely labelable sequence units, under conditions that allows the amplification probe to hybridize to the target-probe complex;
(d) exposing the hybridized amplification probe to a labelling probe covalently attached to a detectable chemical label, such probe comprising sequences complementary to sequences on the amplification probe, under conditions that allow many labelling probes to hybridize to the amplification probe;
(e) observing the presence or absence of the detectable chemical label, covalently attached to said labelling probe, in association with the sample as indicating the presence or absence of the target sequence.
14. A method as claimed in Claim 13 wherein the means for immobilizing the probe-target complex involves an antibody reagent capable of binding to DNA/DNA, DNA/RNA or RNA/RNA duplexes formed between the selectable target sequence and the complementary primary probe sequence .
15. A method as claimed in Claim 14 wherein the antibody reagent is:
(i) selective for binding DNA/RNA hybrids wherein one of the probe and the sequence to be detected is DNA and the other is RNA; (ii) selective for binding RNA/RNA hybrids wherein both the probe and the sequence to be detected are RNA; or (iii) selective for binding intercalation complexes wherein the duplexes formed in the assay comprise a nucleic acid intercalator bound thereto in the form of intercalation complexes.
16. A method as claimed in Claim 13 wherein the detectable chemical label is selected from a enzymatically active group, a [flourescer] fluorescer, a chromophere, a luminescer, a specifically bindable ligand, or a radioscope.
17. A method as claimed in Claim 13 wherein a label attached to each said discretely labelable sequence units interacts with a reagent member of a label detection system to provide the detectable response.
18. The method as claimed in Claim 17 wherein the label is a substrate, cofactor, or inhibitor of an enzyme which is the [ember] member of the label detection system with which the label interacts to provide the detectable response.
19. The method as claimed in Claim 18 wherein the label is a substrate which is acted on by the enzyme to produce a colorimetric, fluorescent or luminescent signal.
20. The method as claimed in Claim 18 wherein the label is a prosthetic group of an enzyme and wherein the apoenzy e of such an enzyme is the [ember] member of the label detection system with which the label interacts to produce the catalytically active holoenzyme.
21. The method as claimed in Claim 20 wherein the prosthetic group is [FAD] flavin adenine dinucleotide and the apoenzyme is apo(glucose oxidase) .
22. The method as claimed in Claim 13 applied to the detection of a particular nucleic acid sequence in a test medium wherein the test medium comprises a biological sample which has been subjected to conditions to release and denature nucleic acids present therein.
23. A method as claimed in Claim 22 wherein the biological sample includes food substances and the target nucleic acid sequence is of a bacterial microorganism.
24. A method as claimed in Claim 22 wherein the biological sample includes food substances and the target nucleic acid sequence is of a virus. 53
25. A reagent for detecting a particular polynucleotide sequence in a test sample, comprising:
(i) a primary nucleic acid probe comprising at least one single stranded base sequence that is substantially complementary to the sequence to be detected; (ii) an antibody reagent capable of binding to hybrids formed between any of the particular polynucleotide sequences to be detected in the sample and the primary probe, but incapable of binding substantially to single stranded nucleic acids;
(iii) an amplification probe adapted to permit enhanced detectable labelling of a selected nucleic acid target, such probe comprising at least two regions of nucleic acid sequences: a first region including a sequence complementary to a sequence of said selected nucleic acid target, and a second region including a plurality of discreetly labelable sequence units;
(iv) a labelling probe covalently attached to a detectable chemical label, such probe comprising sequences complementary to sequences on the amplification probe.
26. The reagent system as claimed in Claim 2Scapable to converting double stranded nucleic acids in a test sample into single stranded form.
27. A diagnostic kit for detecting a particular polynucleotide sequence within a sample comprising:
(i) a primary nucleic acid probe comprising at least one single stranded base sequence that is substantially complementary to the sequence to be detected; (ii) an antibody reagent capable of binding to hybrids formed between any of the particular polynucleotide sequences to be detected in the sample and the primary probe, but incapable of binding substantially to single stranded nucleic acids;
(iii) an amplification probe adapted to permit enhanced detectable labelling of a selected nucleic acid target, such probe comprising at least two regions of nucleic acid sequences: a first region including a sequence complementary to a sequence on a selected primary probe which also contains a sequence complementary to a sequence of said selected nucleic acid target, and a second region including a plurality of discretely labelable sequence units;
(iv) a labelling probe covalently attached to a detectable chemical label, such probe comprising sequences complementary to sequences on the amplification probe.
28. The diagnostic kit of Claim 27 which additionally comprises a denaturation agent capable to converting double stranded nucleic acids in a test sample into single stranded form.
29. A diagnostic kit for the detection of Escheri c ia coli in a test sample comprising the diagnostic kit as claimed in Claim 27.
30. A diagnostic kit for the detection of Salmonella typhi in a test sample comprising the diagnostic kit as claimed in Claim 27.
31. A diagnostic kit for the detection of Lis teria monocytogenes in a test sample comprising the diagnostic kit as claimed in Claim 27.
Priority Applications (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT95922381T ATE188744T1 (en) | 1994-06-28 | 1995-06-27 | METHOD FOR INCREASING THE DETECTION SENSITIVITY OF NUCLEIC ACID HYBRIDS (TARGET/SAMPLE) |
| JP8502671A JPH10508741A (en) | 1994-06-28 | 1995-06-27 | Amplification method for increasing sensitivity of nucleic acid-probe target hybrid detection |
| EP95922381A EP0770142B1 (en) | 1994-06-28 | 1995-06-27 | Method for increasing the sensitivity of detecting nucleic acid-probe target hybrids |
| DE69514521T DE69514521D1 (en) | 1994-06-28 | 1995-06-27 | METHOD FOR INCREASING THE DETECTION SENSITIVITY OF NUCLEIC ACID HYBRIDS (TARGET / SAMPLE) |
| AU27099/95A AU2709995A (en) | 1994-06-28 | 1995-06-27 | Method of amplification for increasing the sensitivity of detecting nucleic acid-probe target hybrids |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA2126952A CA2126952C (en) | 1994-06-28 | 1994-06-28 | Probe, kit, and method of amplification for increasing the sensitivity of nucleic acid hybridization assays |
| CA2,126,952 | 1994-06-28 |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| WO1996000795A2 WO1996000795A2 (en) | 1996-01-11 |
| WO1996000795A3 WO1996000795A3 (en) | 1996-02-22 |
| WO1996000795B1 true WO1996000795B1 (en) | 1996-05-02 |
Family
ID=4153909
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CA1995/000369 Ceased WO1996000795A2 (en) | 1994-06-28 | 1995-06-27 | Method of amplification for increasing the sensitivity of detecting nucleic acid-probe target hybrids |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US5627030A (en) |
| EP (1) | EP0770142B1 (en) |
| JP (1) | JPH10508741A (en) |
| AT (1) | ATE188744T1 (en) |
| AU (1) | AU2709995A (en) |
| CA (1) | CA2126952C (en) |
| DE (1) | DE69514521D1 (en) |
| WO (1) | WO1996000795A2 (en) |
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| US6077668A (en) * | 1993-04-15 | 2000-06-20 | University Of Rochester | Highly sensitive multimeric nucleic acid probes |
| US7135312B2 (en) | 1993-04-15 | 2006-11-14 | University Of Rochester | Circular DNA vectors for synthesis of RNA and DNA |
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| US5853993A (en) * | 1996-10-21 | 1998-12-29 | Hewlett-Packard Company | Signal enhancement method and kit |
| GB2324370B (en) * | 1997-04-14 | 1999-03-03 | Stuart Harbron | Detection of hybrid double-stranded DNA with antibody after enzyme degradation of excess single-standed DNA |
| CA2309384A1 (en) * | 1997-11-06 | 1999-05-20 | Mosaic Technologies | Multiple sequential polynucleotide displacement reactions for signal amplification and processing |
| US6355424B1 (en) * | 1997-12-12 | 2002-03-12 | Digene Corporation | Assessment of human papillomavirus-related disease |
| US6287772B1 (en) * | 1998-04-29 | 2001-09-11 | Boston Probes, Inc. | Methods, kits and compositions for detecting and quantitating target sequences |
| US6503747B2 (en) | 1998-07-14 | 2003-01-07 | University Of Hawaii | Serotype-specific probes for Listeria monocytogenes |
| JP2002525078A (en) * | 1998-09-15 | 2002-08-13 | イェール ユニバーシティ | Artificial long terminal repeat |
| AU6412799A (en) | 1998-10-05 | 2000-04-26 | Mosaic Technologies | Reverse displacement assay for detection of nucleic acid sequences |
| KR20010102992A (en) * | 1999-01-29 | 2001-11-17 | 추후보정 | Non-invasive method for detecting target rna |
| WO2004061126A2 (en) * | 2002-03-25 | 2004-07-22 | Datascope Investment Corp. | Nucleic acid hybridization assay using bridging sequence |
| US7601497B2 (en) | 2000-06-15 | 2009-10-13 | Qiagen Gaithersburg, Inc. | Detection of nucleic acids by target-specific hybrid capture method |
| US7439016B1 (en) * | 2000-06-15 | 2008-10-21 | Digene Corporation | Detection of nucleic acids by type-specific hybrid capture method |
| US20040029142A1 (en) * | 2002-02-11 | 2004-02-12 | Schon Eric A. | Concatenation-based nucleic acid detection compositions and methods |
| US20030198966A1 (en) * | 2002-04-19 | 2003-10-23 | Stojanovic Milan N. | Displacement assay for detection of small molecules |
| US7015317B2 (en) * | 2002-05-02 | 2006-03-21 | Abbott Laboratories | Polynucleotides for the detection and quantification of hepatitis B virus nucleic acids |
| US7807802B2 (en) | 2002-11-12 | 2010-10-05 | Abbott Lab | Polynucleotides for the amplification and detection of Chlamydia trachomatis and Neisseria gonorrhoeae |
| US9487823B2 (en) | 2002-12-20 | 2016-11-08 | Qiagen Gmbh | Nucleic acid amplification |
| US7470516B2 (en) * | 2003-04-14 | 2008-12-30 | The Trustees Of Columbia University In The City Of New York | Cross reactive arrays of three-way junction sensors for steroid determination |
| US20040203007A1 (en) * | 2003-04-14 | 2004-10-14 | Stojanovic Milan N. | Cross reactive arrays of three-way junction sensors for steroid determination |
| US20050019916A1 (en) * | 2003-04-14 | 2005-01-27 | Stojanovic Milan N. | Cross reactive arrays of three-way junction sensors for steriod determination |
| EP1762627A1 (en) | 2005-09-09 | 2007-03-14 | Qiagen GmbH | Method for the activation of a nucleic acid for performing a polymerase reaction |
| US9102986B2 (en) * | 2007-04-13 | 2015-08-11 | Abbott Molecular Inc. | Primer and probe sequences for detecting Chlamydia trachomatis |
| TWI394839B (en) * | 2008-10-27 | 2013-05-01 | Qiagen Gaithersburg Inc | Fast results hybrid capture assay and system |
| AU2009334505B2 (en) * | 2008-12-31 | 2013-05-09 | Abbott Point Of Care Inc. | Method and device for immunoassay using nucleotide conjugates |
| CA2750338C (en) * | 2009-01-28 | 2019-06-25 | Qiagen Gaithersburg, Inc. | Sequence-specific large volume sample preparation method and assay |
| AU2010242867B2 (en) | 2009-05-01 | 2016-05-12 | Qiagen Gaithersburg, Inc. | A non-target amplification method for detection of RNA splice-forms in a sample |
| JP5826752B2 (en) | 2009-09-14 | 2015-12-02 | キアジェン ゲイサーズバーグ インコーポレイテッド | Compositions and methods for recovering nucleic acids or proteins from tissue samples fixed in cytological media |
| EP2529031B1 (en) | 2010-01-29 | 2014-07-09 | QIAGEN Gaithersburg, Inc. | Method of determining and confirming the presence of hpv in a sample |
| AU2011210734B2 (en) * | 2010-01-29 | 2017-02-09 | Qiagen Gaithersburg, Inc. | Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids |
| US20120301886A1 (en) * | 2010-02-26 | 2012-11-29 | Michael Farrell | Polytag probes |
| JP2013528049A (en) | 2010-05-19 | 2013-07-08 | キアゲン ガイサーズバーグ アイエヌシー． | Methods and compositions for sequence-specific purification and multiplex analysis of nucleic acids |
| EP2663658B1 (en) | 2011-01-12 | 2017-10-25 | Abbott Molecular Inc. | Materials and method for detecting cytomegalovirus (cmv) |
| US9885092B2 (en) | 2011-02-24 | 2018-02-06 | Qiagen Gaithersburg Inc. | Materials and methods for detection of HPV nucleic acids |
| US10077474B2 (en) | 2012-05-29 | 2018-09-18 | Abbott Molecular, Inc. | Method of designing primers, method of detecting single nucleotide polymorphisms (SNPs), method of distinguishing SNPs, and related primers, detectable oligonucleotides, and kits |
| EP2971104A2 (en) | 2013-03-15 | 2016-01-20 | Abbott Molecular Inc. | Method for amplification and assay of rna fusion gene variants, method of distinguishing same and related primers, probes, and kits |
| CN106715718B (en) | 2014-07-24 | 2021-07-06 | 雅培分子公司 | Compositions and methods for detection and analysis of Mycobacterium tuberculosis |
| HK1255315A1 (en) | 2015-07-14 | 2019-08-16 | Abbott Molecular Inc. | Compositions and methods for identifying drug resistant tuberculosis |
| CN114480585B (en) * | 2020-10-28 | 2024-02-23 | 清华大学 | Nucleic acid probe composition, pretreatment liquid, nucleic acid detection kit and detection method |
| CN113025690B (en) * | 2021-05-27 | 2021-08-10 | 广东品博易视生物科技有限公司 | Nucleic acid probe group and application thereof |
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-
1994
- 1994-06-28 CA CA2126952A patent/CA2126952C/en not_active Expired - Fee Related
- 1994-07-15 US US08/275,849 patent/US5627030A/en not_active Expired - Lifetime
-
1995
- 1995-06-27 EP EP95922381A patent/EP0770142B1/en not_active Expired - Lifetime
- 1995-06-27 JP JP8502671A patent/JPH10508741A/en not_active Ceased
- 1995-06-27 DE DE69514521T patent/DE69514521D1/en not_active Expired - Lifetime
- 1995-06-27 AT AT95922381T patent/ATE188744T1/en not_active IP Right Cessation
- 1995-06-27 WO PCT/CA1995/000369 patent/WO1996000795A2/en not_active Ceased
- 1995-06-27 AU AU27099/95A patent/AU2709995A/en not_active Abandoned
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