Isothermal transcription based amplification assay for the detection and quantitation of macrophage derived chemokine RNA

• US 6,093,542 A
• Filed: 01/09/1998
• Issued: 07/25/2000
• Est. Priority Date: 01/09/1998
• Status: Expired due to Term

• Current Assignee: Biomerieux Incorporated (Compagnie Merieux Alliance SAS)
• Original Assignee: Akzo Nobel N.V. (Nouryon BV)
• Inventors: Romano, Joseph, Pal, Ranajit, Shurtliff, Roxanne
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US09/005,165
• Time in Patent Office: 928 Days
• Field of Search: 435/6, 435/91.3, 536/23.5, 536/24.3, 536/24.33
• US Class Current: 435/6.14
• CPC Class Codes: C07K 14/523 Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Abstract

An isothermal transcription based amplification assay for MDC RNA uses primer combinations for sequences within the MDC gene. A quantitative control uses a mutant RNA for comparison.

Claims

1. A method for the detection of quantitation of MDC RNA in a sample, comprising:

a) obtaining a sample which may contain MDC RNA;

b) performing an isothermal transcription based amplification on the sample with two primers, a first primer being about 15-26 nucleotides in length and comprising at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO;

2 and SEQ ID NO;

3, and a second primer being about 15-26 nucleotides in length and comprising at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO;

6 and SEQ ID NO;

7; and

c) detecting the amplification product using at least one probe, whereby detection of the amplification product indicates the presence and/or quantity of MDC RNA in the sample.

2. The method of claim 1, wherein the sample comprises cells and RNA is extracted from the cells in the sample prior to step b).

3. The method of claim 1, wherein the detection step uses a labelled probe, wherein the labelled probe comprises a sequence selected from the group consisting of SEQ ID NO:

9, and SEQ ID NO;

10, whereby hybridization of the probe to the amplification product indicates the presence and/or quantity of MDC RNA in the sample.

4. The method of claim 1, wherein the at least one probe comprises SEQ ID NO:

8, SEQ ID NO;

9 and SEQ ID NO;

10.

5. The method of claim 1, wherein the at least one probe comprises a wild type probe selected from the group consisting of SEQ ID NO:

8 and SEQ ID NO;

9.

6. The method of claim 5, further comprising adding a known amount of control RNA Q at step b), and detecting amplification product of Q by using a labeled probe comprising the sequence of SEQ ID NO:

10, whereby the quantity of MDC RNA in the sample is calculated by comparing the signals of the probes for Q and the wild-type probe.

7. The method of claim 1, wherein said first primer further comprises an RNA polymerase promoter sequence covalently bonded to the 5'"'"' end thereof.

8. The method of claim 7, wherein said RNA polymerase promoter sequence is a T7 RNA polymerase promoter sequence as set forth in SEQ ID NO:

1.

9. The method of claim 8, wherein said first primer is SEQ ID NO:

4 and said second primer is SEQ ID NO;

6.

10. The method of claim 9, wherein the at least one probe comprises SEQ ID NO:

8, SEQ ID NO;

9 and SEQ ID NO;

10.

11. A kit for the detection or quantitation of MDC RNA in a sample, comprising, two primers selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, and SEQ ID NO;

7.

12. The kit of claim 11, wherein the primers are SEQ ID NO:

4 and SEQ ID NO;

6.

13. The kit of claim 11, wherein the primers are SEQ ID NO:

4 and SEQ ID NO;

7.

14. The kit of claim 11, wherein the primers are SEQ ID NO:

5 and SEQ ID NO;

6.

15. The kit of claim 11, wherein the primers are SEQ ID NO:

5 and SEQ ID NO;

7.

16. The kit of claim 11, further comprising at least one probe comprising a sequence selected from the group consisting of SEQ ID NO:

8, SEQ ID NO;

9, and SEQ ID NO;

10.

17. An oligonucleotide selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9 and SEQ ID NO;

10.

18. An oligonucleotide of about 15-26 nucleotides, comprising at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

6, and SEQ ID NO;

7.

19. An oligonucleotide of about 15-26 nucleotides, comprising at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO:

2 and SEQ ID NO;

3, further comprising an RNA polymerase promoter sequence covalently bonded to the 5'"'"' end thereof.

20. The oligonucleotide of claim 19, wherein the RNA polymerase promoter sequence is a T7 RNA polymerase promoter sequence as set forth in SEQ ID NO:

1.

21. An oligonucleotide selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, and SEQ ID NO;

7.

22. A pair of oligonucleotides for the detection or quantitation of MDC RNA, a first oligonucleotide of said pair being about 15-26 nucleotides in length and comprising at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO:

2 and SEQ ID NO;

3, and a second oligonucleotide of said pair being about 15-26 nucleotides in length and comprising at least 10 consecutive nucleotides of a sequence selected from the group consisting of SEQ ID NO;

6 and SEQ ID NO;

7.

23. The pair of oligonucleotides of claim 22, wherein said first oligonucleotide is selected from the group consisting of SEQ ID NO:

2 and SEQ ID NO;

3, and said second oligonucleotide is selected from the group consisting of SEQ ID NO;

6 and SEQ ID NO;

7.

24. The pair of oligonucleotides of claim 22, wherein said first oligonucleotide further comprises an RNA polymerase promoter sequence covalently bonded to the 5'"'"' end thereof.

25. The pair of oligonucleotides of claim 24, wherein the RNA polymerase promoter sequence is a T7 RNA polymerase promoter sequence as set forth in SEQ ID NO:

1.

26. A kit for the detection or quantitation of MDC RNA in a sample, comprising the pair of oligonucleotides of claim 22.

27. The kit of claim 26, further comprising at least one probe comprising a sequence selected from the group consisting of SEQ ID NO:

8, SEQ ID NO;

9, and SEQ ID NO;

10.