Replicative RNA-based amplification/detection systems

• US 5,620,851 A
• Filed: 10/17/1994
• Issued: 04/15/1997
• Est. Priority Date: 04/16/1986
• Status: Expired due to Fees

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Kramer, Fred R., Axelrod, Vladimir D., Lizardi, Paul M., Mills, Donald R.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): TRAN, PAUL
• Application Number:
  US08/323,631
• Time in Patent Office: 911 Days
• Field of Search: 435/4, 435/6, 435/7.1, 435/15, 435/91.21, 435/14, 435/172.3, 435/320.1, 536/22.1, 536/24.1, 935/77, 935/78
• US Class Current: 435/5
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6867 Replicase-based amplification, e.g. using Q-beta replicase
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2521/119 RNA polymerase
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals

Abstract

This invention relates to the use of functional reporter molecules in the detection and measurement of targets in a sample. The invention is predicated on the utilization of a transcription step between the production of an appropriate reporter molecule and replication based amplification in order to increase the number of detectable species as an indirect reference to target.

Claims

1. An adduct for detecting a target, which adduct comprises:

(a) a probe moiety capable of specifically forming a complex with said target;

linked to

(b) a single-stranded DNA moiety comprising a promoter sequence which recognizes and binds to a DNA-dependent RNA polymerase when hybridized to a nucleic acid sequence complementary thereto operably linked to a DNA template sequence encoding an RNA transcript replicatable by an RNA-dependent RNA polymerase.

2. An adduct according to claim 1, wherein the probe moiety is capable of specifically forming a complex with a polysaccharide.

3. An adduct according to claim 1, wherein the probe moiety is capable of specifically forming a complex with a protein.

4. An adduct according to claim 1, wherein the probe moiety is an antibody.

5. An adduct according to claim 3, wherein the probe moiety is an antibody.

6. An adduct according to claim 1, wherein the promoter sequence in the DNA moiety is positioned 3'"'"' to the DNA template sequence in the DNA moiety.

7. An adduct according to claim 1, wherein the DNA-dependent RNA polymerase is T7 RNA polymerase.

8. An adduct according to claim 1, wherein the DNA-dependent RNA polymerase is SP6 RNA polymerase.

9. An adduct according to claim 1, further comprising a single-stranded nucleic acid molecule containing a nucleic acid sequence complementary to the promoter sequence hybridized to the promoter sequence.

10. A method for detecting the presence of a target in a sample, which method comprises:

(a) contacting the sample with the adduct of claim 1 under conditions which would permit the probe moiety of the adduct to specifically form a complex with the target if present in the sample;

(b) removing any uncomplexed adduct from the sample;

(c) contacting the sample with a single-stranded nucleic acid molecule comprising a nucleic acid sequence which is complementary to the promoter sequence of the DNA moiety of the adduct under conditions permitting the nucleic acid sequence to hybridize with the promoter sequence;

(d) contacting the sample with a DNA-dependent RNA polymerase under suitable conditions so as to produce RNA transcripts, thereby producing RNA transcripts from the DNA template sequence of the DNA moiety of any of the adduct remaining in the sample;

(e) incubating the sample with an appropriate RNA-dependent RNA polymerase under suitable conditions so as to produce complementary single-stranded RNA molecules from any RNA transcripts so produced in step (d);

(f) thereafter simultaneously and repeatedly contacting the sample with the appropriate RNA-dependent RNA polymerase under conditions permitting simultaneous and repeated synthesis of RNA copies having the same sequence as the RNA transcripts in step (d) and RNA copies having the same sequence as complementary single-stranded RNA molecules in step (e); and

(g) detecting any RNA copies so produced, so as to thereby detect the presence of the target in the sample.

11. The method of claim 10, further comprising measuring the RNA copies detected in step (g) in a standardized manner for detecting an RNA molecule so as to measure the amount of target contained in the sample.

12. The method of claim 10, wherein the detecting in step (g) comprises:

(i) labeling the RNA copies with a detectable marker; and

(ii) detecting the marker on the RNA copies so labeled.

13. The method of claim 12, wherein the detectable marker is selected from the group consisting of a radioactive substance, a chromophore, and a fluorophore.

14. A method for detecting the presence of a target in a sample, which method comprises:

(a) contacting the sample with the adduct of claim 9 under conditions which would permit the probe moiety of the adduct to specifically form a complex with the target if present in the sample;

(b) removing any uncomplexed adduct from the sample;

(c) contacting the sample with a DNA-dependent RNA polymerase under suitable conditions so as to produce RNA transcripts, thereby producing RNA transcripts from the DNA template sequence of the DNA moiety of any of the adduct remaining in the sample;

(d) incubating the sample with an appropriate RNA-dependent RNA polymerase under suitable conditions so as to produce complementary single-stranded RNA molecules from any RNA transcripts so produced in step (c);

(e) thereafter simultaneously and repeatedly contacting the sample with the appropriate RNA-dependent RNA polymerase under conditions permitting simultaneous and repeated synthesis of RNA copies having the same sequence as the RNA transcripts in step (c) and RNA copies having the same sequence as complementary single-stranded RNA molecules in step (d); and

(f) detecting any RNA copies so produced, so as to thereby detect the presence of the target in the sample.

15. The method of claim 14, further comprising measuring the RNA copies detected in step (f) in a standardized manner for detecting an RNA molecule so as to measure the amount of target contained in the sample.

16. The method of claim 14, wherein the detecting in step (f) comprises:

(i) labeling the RNA copies with a detectable marker; and

(ii) detecting the marker on the RNA copies so labeled.

17. The method of claim 16, wherein the detectable marker is selected from the group consisting of a radioactive substance, a chromophore, and a fluorophore.

18. A kit for detecting a target in a sample, which kit comprises the adduct of claim 1.

19. A kit according to claim 18, further comprising a single-stranded nucleic acid molecule comprising a nucleic acid sequence complementary to the promoter sequence of the DNA moiety of the adduct, A DNA-dependent RNA polymerase capable of transcribing the DNA template sequence of the DNA moiety of the adduct, and an RNA-dependent RNA polymerase capable of replicating RNA transcripts produced by transcribing the DNA template sequence of the DNA moiety of the adduct.

20. A kit for detecting a target in a sample, which kit comprises the adduct of claim 9.

21. A kit according to claim 20, further comprising a DNA-dependent RNA polymerase capable of transcribing the DNA template sequence of the DNA moiety of the adduct and an RNA-dependent RNA polymerase capable of replicating RNA transcripts produced by transcribing the DNA template sequence of the DNA moiety of the adduct.

22. A kit for preparing an adduct for detection of a target, which kit comprises a probe moiety capable of specifically forming a complex with said target, a DNA moiety comprising a promoter sequence which recognizes and binds to a DNA-dependent RNA polymerase when hybridized to a nucleic acid sequence complementary thereto operably linked to a DNA template sequence encoding an RNA transcript replicatable by an RNA-dependent RNA polymerase, and a means for linking said probe moiety to said DNA moiety.

23. A kit according to claim 22, wherein the probe moiety is an antibody.