Compositions and methods for the simultaneous detection and quantification of multiple specific nucleic acid sequences

• US 5,658,737 A
• Filed: 08/06/1996
• Issued: 08/19/1997
• Est. Priority Date: 10/28/1994
• Status: Expired due to Term

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Weeks, Ian, Woodhead, James Stuart, Cheikh, Azzouz Ben, Nelson, Norman Charles
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US08/692,610
• Time in Patent Office: 378 Days
• Field of Search: 435/5, 435/6, 435/91.1, 435/91.2, 435/810, 252/700, 436/164, 436/171, 436/174, 436/94, 436/826, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 935/1, 935/16, 935/19, 935/76, 935/77
• US Class Current: 435/6.11
• CPC Class Codes: C07D 219/04 with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/108 Hybridisation protection assay [HPA]
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10S 436/826 Additives, e.g. buffers, diluents, preservatives
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

The invention relates to methods for simultaneously or sequentially detecting multiple nucleic acid analytes in a single medium utilizing oligonucleotide hybridization probes coupled to different chemiluminescent labeling reagents. The methods may be used in a heterogeneous, homogeneous or non-homogeneous assay system. The invention also relates to specific combinations of chemiluminescent labeling reagents suitable, when coupled to an oligonucleotide probe, for use together in methods for the detection of multiple nucleic acid analytes. The invention also concerns kits useful in these methods.

Claims

1. A kit for the detection of two or more nucleic acid analytes suspected of being present in a sample, comprising, in one or more container:

a) at least two different oligonucleotide hybridization assay probes, each able to specifically hybridize to a different nucleic acid target analyte,

b) at least one different chemiluminescent label coupled to each said probe, wherein each chemiluminescent label in said kit is chosen so that the chemiluminescence emitted by said label upon induction of light emission is separately distinguishable on the basis of wavelength of light emission, kinetics of the chemiluminescent reaction, or pH of reaction from the chemiluminescence emitted by at least one other of said labels; and

said coupled labels are able to react with a destabilizing agent which will selectively destroy or inhibit the chemiluminescent potential of said labels when said probe has not formed a stable nucleic acid hybrid with an analyte without extinguishing the chemiluminescent potential of said labels coupled to hybridized probe; and

the rate of destruction or inhibition of at least one hybrid-unassociated coupled label by said destabilizing agent is no more than 50 fold the rate of destruction or inhibition of at least one other of said hybrid-unassociated coupled labels by said agent.

2. The kit of claim 1 in which at least one of said chemiluminescent labels is an acridinium ester derivative.

3. The kit of claim 1 in which at least two of said chemiluminescent labels are acridinium ester derivatives.

4. The kit of claim 3 in which one of said acridinium esters is selected from the group:

a) standard AE,

b) naphthyl AE,

c) o-diBr AE,

d) 1- and 3- Me AE,

e) 4,5-diMe AE,

f) 2,7-diMe AE,

g) o-Me AE,

h) o-MeO(cinnamyl) AE,

i) o-MeO AE,

j) ortho AE,

k) o-F-AE,

l) 1- and 3-Me-o-F AE,

m) 2,7-diMe-o-F AE, and

n) 1- and 3-Me-m-diF AE.

5. The kit of claim 3 wherein the acridinium ester labels are chosen so that the wavelength of light emitted from at least one of said labels after initiation of a chemiluminescent reaction is sufficiently different from the wavelength of light emitted from at least one other said label to permit each label'"'"'s separate detection in a single sample.

6. The kit of claim 5 wherein said acridinium ester derivatives are 2,7-diMe AE and standard AE.

7. The kit of claim 3 wherein the acridinium ester labels are chosen so that the chemiluminescent reaction kinetics of at least one said label is sufficiently different from the reaction kinetics of at least one other said label to permit each label'"'"'s separate detection in a single sample.

8. The kit of claim 7 in which a first acridinium ester is standard AE and a second acridinium ester is selected from the group:

naphthyl AE, o-diBr AE, o-diMe AE, o-Me AE, o-MeO (cinnamyl) AE, o-MeO AE, o-AE, o-F AE, 1-Me-o-F AE, and 1-Me-m-diF AE.

9. The kit of claim 7 in which a first acridinium ester is naphthyl AE and a second acridinium ester is selected from the group:

1-Me AE, 4,5-diMe AE, 2,7-di Me AE, o-Me AE, o-MeO (cinnamyl) AE, and o-AE.

10. The kit of claim 7 in which a first acridinium ester is o-diBr AE and a second acridinium ester is selected from the group:

1-Me AE, 4,5-diMe AE, 2,7-diMe AE, o-diMe AE, o-Me AE, o-MeO (cinnamyl) AE, and o-AE.

11. The kit of claim 7 in which a first acridinium ester is 1-Me AE and a second acridinium ester is selected from the group:

o-diMe AE, o-Me AE, o-MeO (cinnamyl) AE, o-MeO AE, o-AE, o-F AE, 1-Me-o-F AE, and 1-Me-m-diF AE.

12. The kit of claim 7 in which a first acridinium ester is 4,5-diMe AE and a second acridinium ester is selected from the group:

o-diMe AE, o-Me AE, o-MeO (cinnamyl) AE, o-MeO AE, o-AE, o-F AE, 1-Me-o-F AE, and 1-Me-m-diF AE.

13. The kit of claim 7 in which a first acridinium ester is 2,7-diMe AE and a second acridinium ester is selected from the group:

o-diMe AE, o-Me AE, o-MeO (cinnamyl) AE, o-MeO AE, o-AE, o-F AE, 1-Me-o-F AE, and 1-Me-m-diF AE.

14. The kit of claim 7 in which a first acridinium ester is o-diMe AE and a second acridinium ester is selected from the group:

o-Me AE, o-MeO (cinnamyl) AE, o-MeO AE, o-AE, o-F AE, 1-Me-o-F AE, and 1-Me-m-diF AE.

15. The kit of claim 7 in which a first acridinium ester is o-Me AE and a second acridinium ester is selected from the group:

o-MeO (cinnamyl) AE, o-MeO AE, o-AE, o-F AE, 1-Me-o-F AE, and 1-Me-m-diF AE.

16. The kit of claim 7 in which a first acridinium ester is o-MeO (cinnamyl) AE and a second acridinium ester is selected from the group:

o-MeO AE, o-F AE, 1Me-o-F AE, and 1-Me-m-diF AE.

17. The kit of claim 7 in which a first acridinium ester is o-MeO AE and a second acridinium ester is o-AE.

18. The kit of claim 7 in which a first acridinium ester is o-AE and a second acridinium ester is selected from the group consisting of o-F AE, 1-Me-o-F AE, and 1-Me-m-diF AE.

19. The kit of claim 3 in which said acridinium ester labels are chosen so that the optimal pH for the chemiluminescent reaction of at least one said coupled label is sufficiently different from that of at least one other said coupled label that the light emitted by each of said labels present in a single solution, following initiation of a chemiluminescent reaction, can be separately detected by adjustment of the solution from a first pH to one or more different pH.

20. The kit of claim 19 in which a first acridinium ester label is selected from the group consisting of:

a) o-diBr AE, and

b) o-Fl AE,

and a second acridinium ester label is selected from the group consisting of

c) standard AE, and

d) o-MeO AE.

21. The kit of claim 1 in which all said chemiluminescent labels are acridinium ester derivatives.

22. The kit of claim 1 further comprising at least five said chemiluminescent labels, in which said at least five chemiluminescent labels are acridinium ester derivatives, and said two or more said acridinium ester derivatives are selected from the group consisting of:

a) o-diBr AE

b) 2,7-diMe AE

c) o-MeO(cinnamyl) AE,

d) o-Me AE, and

e) o-diMe AE.

23. The kit of claim 1 further comprising at least five said chemiluminescent labels, in which said at least five chemiluminescent labels are acridinium ester derivatives, and said two or more said acridinium ester derivatives are selected from the group consisting of:

a) o-diBr AE,

b) 1-Me AE,

c) o-AE,

d) o-Me AE, and

e) o-diMe AE.

24. The kit of claim 1 further comprising at least seven said chemiluminescent labels, in which said at least seven chemiluminescent labels are acridinium ester derivatives, and said two or more said acridinium ester derivatives are selected from the group consisting of:

a) o-diBr AE

b) 2,7-diMe AE

c) 1-Me AE,

d) o-AE,

c) o-MeO(cinnamyl) AE,

d) o-Me AE, and

e) o-diMe AE.