Capture and detection format versatility for dipstick assays

• US 20040053255A1
• Filed: 04/07/2003
• Published: 03/18/2004
• Est. Priority Date: 07/07/2000
• Status: Active Grant

• Current Assignee: Diagnostics for the Real World, Ltd.
• Original Assignee: Diagnostics for the Real World, Ltd.
• Inventors: Dineva, Magda Anastassova, Lee, Helen, Hu, Hsiang Yun
• Granted Patent: US 7,192,701 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates

Abstract

Dipsticks for testing for the presence of a target nucleic acid in a sample solution are described the dipsticks comprise a universal capture probe immobilised at a capture zone of the dipstick. The universal capture probe is capable of hybridising to a hook capture probe which is hybridised to the target nucleic acid in the sample solution. A contact end of the dipstick is contacted with the sample solution to cause hook capture probe hybridised to the target nucleic acid to move by capillary action to the capture zone where the target nucleic acid can be detected. Use of the universal and hook capture probes allows dipsticks to be prepared which can be used to capture any target nucleic acid, thereby simplifying preparation of the dipsticks. Specificity of target nucleic acid capture is then achieved by use of an appropriate hook capture probe. Methods and kits are also described.

Claims

1. A method for testing for the presence of target nucleic acid in a sample solution which comprises:

a) providing a chromatographic strip having a contact end for contacting the sample solution and a universal capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end;

b) providing a hook capture probe capable of hybridising to a first region of the target nucleic acid and to the universal capture probe;

c) incubating the sample solution with the hook capture probe under conditions for hybridisation of the hook capture probe to the first region of target nucleic acid in the sample solution;

d) contacting the sample solution with the contact end of the chromatographic strip so that a complex formed between the hook capture probe, the detection probe and target nucleic acid can travel by capillary action to the capture zone and bind to the capture zone by interaction between the hook capture probe of the complex and the universal capture probe immobilised at the capture zone; and

e) detecting for target nucleic acid at the capture zone.

2. A method for testing for the presence of target nucleic acid in a sample solution which comprises:

a) providing a chromatographic strip having;

a contact end for contacting the sample solution;

a universal capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end; and

a hook capture probe releasably immobilised to the chromatographic strip between the contact end and the capture zone, the hook capture probe being capable of hybridising to a first region of the target nucleic acid and to the universal capture probe;

b) contacting the sample solution with the contact end of the chromatographic strip to cause sample solution to move by capillary action to the capture zone, thereby releasing the hook capture probe and allowing released hook capture probe to hybridise to target nucleic acid in the sample solution as it moves to the capture zone so that a complex comprising target nucleic acid and hook capture probe can be captured at the capture zone by hybridisation of the universal capture probe to the hook capture probe of the complex; and

c) detecting for target nucleic acid at the capture zone.

3. A method for testing for the presence of target nucleic acid in a sample solution which comprises:

a) providing a chromatographic strip having a contact end for contacting the sample solution and a universal capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end;

b) providing a hook capture probe capable of hybridising to a first region of the target nucleic acid and to the universal capture probe;

c) providing a detection probe capable of attaching to the target nucleic acid thereby allowing direct or indirect detection of the target nucleic acid utilising the detection probe;

d) incubating the sample solution with the detection probe under conditions for attachment of the detection probe to target nucleic acid in the sample solution; and

incubating the sample solution with the hook capture probe under conditions for hybridisation of the hook capture probe to the first region of target nucleic acid in the sample solution;

e) contacting the sample solution with the contact end of the chromatographic strip so that a complex formed between the hook capture probe, the detection probe and target nucleic acid can travel by capillary action to the capture zone and bind to the capture zone by interaction between the hook capture probe of the complex and the universal capture probe immobilised at the capture zone; and

f) checking for the presence of detection probe at the capture zone.

6. A method according to claim 3 or 5 in which the detection probe is capable of attaching to the target nucleic acid by hybridising to a second region of the target nucleic acid.

7. A method according to claim 6 in which the detection probe comprises a hook detection probe capable of hybridising to the second region of the target nucleic acid and a universal detection probe capable of hybridising to the hook detection probe when the hook detection probe has hybridised to the target nucleic acid.

8. A method according to claim 6 or 7 in which the detection probe or the universal detection probe comprises a label thereby allowing direct detection of target nucleic acid utilising the detection probe or the universal and hook detection probes.

9. A method according to claim 6 or 7 which further comprises contacting the detection probe or the universal detection probe with a detection moiety capable of binding by non base-pairing interaction to the detection probe when the detection probe has hybridised to the target nucleic acid, thereby allowing indirect detection of target nucleic acid utilising the detection probe or the universal and hook detection probes.

10. A method according to claim 9 in which the detection probe or the universal detection probe comprises a detection ligand which can be bound by the detection moiety.

11. A method according to claim 9 or 10 in which the detection moiety comprises an antibody or antibody fragment.

13. A method according to any of claims 9 to 12 in which the detection moiety is releasably immobilised at a conjugate zone located between the contact end and the capture zone of the chromatographic strip so that the detection moiety is released and can bind to the detection probe as the complex travels by capillary action to the capture zone.

14. A method according to any of claims 9 to 13 in which the detection moiety comprises a label allowing detection of the detection moiety.

15. A method according to claim 8 or 14 in which the label is a non radioactive label.

16. A method according to claim 15 in which the label is a colour label.

17. A method according to claim 16 in which the colour label comprises a textile dye, a metal sol such as colloidal gold, or coloured particles such as coloured latex is particles.

4. A method for testing for the presence of target nucleic acid in a sample solution which comprises:

a) providing a chromatographic strip having a contact end for contacting the sample solution, a universal capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end, and a hook capture probe hybridised to the universal capture probe, the hook probe being capable of hybridising to a first region of the target nucleic acid;

b) contacting the sample solution with the contact end of the chromatographic strip so that target nucleic acid in the sample solution can travel by capillary action to the capture zone and bind to the capture zone by interaction between the hook capture probe immobilised at the capture zone and the first region of the target nucleic acid; and

c) detecting for target nucleic acid at the capture zone.

5. A method for testing for the presence of target nucleic acid in a sample solution which comprises:

a) providing a chromatographic strip having a contact end for contacting the sample solution, a universal capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end, and a hook capture probe hybridised to the universal capture probe, the hook probe being capable of hybridising to a first region of the target nucleic acid;

b) providing a detection probe capable of attaching to the target nucleic acid thereby allowing direct or indirect detection of the target nucleic acid utilising the detection probe;

c) incubating the sample solution with the detection probe under conditions for attachment of the detection probe to target nucleic acid in the sample solution;

d) contacting the sample solution with the contact end of the chromatographic strip so that target nucleic acid attached to the detection probe can travel by capillary action to the capture zone and bind to the capture zone by interaction between the hook capture probe immobilised at the capture zone and the first region of the target nucleic acid; and

e) checking for the presence of detection probe at the capture zone.

12. A method according to claim 12 in which the detection ligand comprises biotin and the detection moiety comprises an anti-biotin antibody, or in which the detection ligand comprises fluorescein and the detection moiety comprises an anti-fluorescein antibody, or in which the detection ligand comprises 2,4-dinitrophenol (DNP) and the detection moiety comprises an anti-DNP antibody.

18. A method for testing for the presence of target nucleic acid in a sample solution which comprises:

a) providing a chromatographic strip having a contact end for contacting the sample solution and a capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end, the capture probe being capable of hybridising to a first region of the target nucleic acid;

b) providing a hook detection probe capable of hybridising to a second region of the target nucleic acid;

c) providing a universal detection probe capable of hybridising to the hook detection probe when the hook detection probe has hybridised to the second region of the target nucleic acid, thereby allowing direct or indirect detection of target nucleic acid utilising the universal and hook detection probes;

d) incubating the sample solution with the hook detection probe and the universal detection probe under conditions for hybridisation of the hook detection probe to the second region of the target nucleic acid and for hybridisation of the universal detection probe to the hook detection probe;

e) contacting the sample solution with the contact end of the chromatographic strip so that a complex formed between the universal detection probe, the hook detection probe and target nucleic acid can travel by capillary action to the capture zone and bind to the capture zone by interaction between target nucleic acid of the complex and the capture probe immobilised at the capture zone; and

f) checking for the presence of universal detection probe at the capture zone.

21. A method according to claim 18, 19, or 20 in which the universal detection probe comprises a label.

19. A method for testing for the presence of target nucleic acid in a sample solution which comprises:

a) providing a chromatographic strip having;

a contact end for contacting the sample solution;

a capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end, the capture probe being capable of hybridising to a first region of the target nucleic acid; and

a universal detection probe releasably immobilised to the chromatographic strip between the contact end and the capture zone;

b) incubating the sample solution with a hook detection probe capable of hybridising to a second region of the target nucleic acid, under conditions for hybridisation of the hook detection probe to the second region;

c) contacting the sample solution with the contact end of the chromatographic strip to cause sample solution to travel by capillary action to the capture zone, thereby releasing universal detection probe so that released universal detection probe can hybridise to hook detection probe in the sample solution as it travels to the capture zone and a complex comprising the universal detection probe, the hook detection probe and target nucleic acid can bind to the capture zone by interaction between target nucleic acid of the complex and the capture probe immobilised at the capture zone; and

f) checking for the presence of universal detection probe at the capture zone.

20. A method for testing for the presence of target nucleic acid in a sample solution which comprises:

a) providing a chromatographic strip having a contact end is for contacting the sample solution and a capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end, the capture probe being capable of hybridising to a first region of the target nucleic acid;

b) providing a hook detection probe capable of hybridising to a second region of the target nucleic acid;

c) providing a universal detection probe capable of hybridising to the hook detection probe when the hook detection probe has hybridised to the second region of the target nucleic acid, thereby allowing direct or indirect detection of target nucleic acid utilising the universal and hook detection probes;

d) contacting the sample solution with the contact end of the chromatographic strip to cause sample solution to travel by capillary action to the capture zone and be captured at the capture zone by the capture probe;

e) contacting the capture zone with a hook detection probe and a universal detection probe; and

f) checking for the presence of universal detection probe at the capture zone.

22. A method according to claim 20 which further comprises contacting the universal detection probe with a detection moiety capable of binding by non base pairing interaction to the universal detection probe when the universal detection probe has hybridised to the hook detection probe.

23. A method according to claim 22 in which the universal detection probe comprises a detection ligand which can be bound by the detection moiety.

24. A method according to claim 22 or 23 in which the detection moiety comprises an antibody or antibody fragment.

25. A method according to claim 24 in which the detection ligand comprises biotin and the detection moiety comprises an anti-biotin antibody, or in which the detection ligand comprises fluorescein and the detection moiety comprises an anti-fluorescein antibody, or in which the detection ligand comprises 2,4-dinitrophenol (DNP) and the detection moiety comprises an anti-DNP antibody.

26. A method according to any of claims 22 to 25 in which the detection moiety is releasably immobilised at a conjugate zone located between the contact end and the capture zone of the chromatographic strip so that the detection moiety is released and can bind to the universal detection probe as the complex travels by capillary action to the capture zone.

27. A method for testing for the presence of a target nucleic acid in a sample solution which comprises:

a) providing a chromatographic strip having a contact end for contacting the sample solution and a capture moiety immobilised at a capture zone of the chromatographic strip remote from the contact end, the capture moiety being capable of binding by non base pairing interaction to a universal capture probe bound to the target nucleic acid by a hook capture probe;

b) providing a hook capture probe capable of hybridising to the target nucleic acid;

c) providing a universal capture probe capable of hybridising to the hook capture probe;

d) contacting the sample solution with;

the hook capture probe under conditions for hybridisation of the hook capture probe to the target nucleic acid; and

the universal capture probe under conditions for hybridisation of the universal capture probe to the hook capture probe;

e) contacting the sample solution with the contact end of the chromatographic strip so that a complex formed between the universal capture probe, the hook capture probe, and target nucleic acid can travel by capillary action to the capture zone and bind to the capture zone by binding (by non base pairing interaction) of the capture moiety to the capture ligand of the complex; and

f) detecting for target nucleic acid at the capture zone.

28. A dipstick for testing for the presence of target nucleic acid in a sample solution which comprises a chromatographic strip having:

a contact end for contacting the sample solution;

a universal capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end; and

a hook capture probe hybridised to the universal capture probe and which is capable of hybridising to a first region of the target nucleic acid.

30. A dipstick according to claim 28 or 29 which further comprises a detection moiety releasably immobilised at a conjugate zone of the chromatographic strip between the contact end and the capture zone, the detection moiety being capable of binding by non base pairing interaction to a detection probe hybridised to a second region of the target nucleic acid.

32. A dipstick according to claim 30 or 31 in which the detection moiety comprises a label.

33. A dipstick according to claim 32 in which the label is non radioactive.

34. A dipstick according to claim 33 in which the label is a colour label.

35. A dipstick according to claim 34 in which the colour label comprises a textile dye, a metal sol such as colloidal gold, or a coloured particle such as a coloured latex particle.

36. A dipstick according to any of claims 30 to 35 in which the detection moiety comprises an antibody or an antibody fragment.

37. A dipstick according to claim 36 in which the detection moiety comprises an anti-biotin antibody, an anti-fluorescein antibody, or an anti-DNP antibody.

38. A dipstick according to any of claims 30 to 35 in which the detection moiety comprises avidin, streptavidin, or a derivative thereof which retains biotin binding activity.

42. A dipstick according to any of claims 28 to 41 in which the target nucleic acid is nucleic acid of a disease causing micro-organism, preferably a sexually transmitted disease causing micro-organism.

43. A dipstick according to claim 42 in which the target nucleic acid is Chlamydia trachomatis nucleic acid.

44. Use of a dipstick according to any of claims 28 to 43 to test for the presence of a target nucleic acid in a sample solution.

29. A dipstick for testing for the presence of target nucleic acid in a sample solution which comprises a chromatographic strip having:

a contact end for contacting the sample solution;

a universal capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end; and

a hook capture probe releasably immobilised to the chromatographic strip between the contact end and the capture zone, the hook capture probe being capable of hybridising to a first region of the target nucleic acid.

31. A dipstick for testing for the presence of target nucleic acid in a sample solution which comprises a chromatographic strip having:

a contact end for contacting the sample solution;

a universal capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end, the universal capture probe being capable of hybridising to a hook capture probe bound to a first region of the target nucleic acid; and

a detection moiety releasably immobilised at a conjugate zone of the chromatographic strip between the contact end and the capture zone, the detection moiety being capable of binding by non base pairing interaction to a detection probe hybridised to a second region of the target nucleic acid.

39. A dipstick for testing for the presence of target nucleic acid in a sample solution which comprises a chromatographic strip having:

a contact end for contacting the sample solution;

a capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end, the capture probe being capable of binding directly or indirectly to the target nucleic acid;

a universal detection probe releasably immobilised to the chromatographic strip between the contact end and the capture zone, the universal detection probe being capable of hybridising to a hook detection probe; and

optionally

a hook detection probe releasably immobilised to the chromatographic strip between the contact end and the capture zone, the hook detection probe being capable of hybridisng to the target nucleic acid.

40. A dipstick according to claim 39 which comprises a detection moiety releasably immobilised at a conjugate zone of the chromatographic strip between the contact end and the capture zone, the detection moiety being capable of binding by non base pairing interaction to the universal detection probe.

41. A dipstick for testing for the presence of a target nucleic acid in a sample solution which comprises:

a dipstick having a chromatographic strip with a contact end for contacting the sample solution and a capture moiety immobilised at a capture zone of the chromatographic strip remote from the contact end, the capture moiety being capable of binding by non base pairing interaction to a universal capture probe bound to the target nucleic acid by a hook capture probe; and

a hook capture probe and/or a universal capture probe releasably immobilised to the chromatographic strip between the contact end and the capture zone, the hook capture probe being capable of hybridising to the target nucleic acid, and the universal capture probe being capable of hybridising to the hook capture probe.

45. A kit for testing for the presence of target nucleic acid in a sample solution which comprises:

i) a dipstick comprising a chromatographic strip with a contact end for contacting the sample solution, and a universal capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end;

ii) a hook capture probe capable of hybridising to a first region of the target nucleic acid and to the universal capture probe; and

optionally

iii) a detection probe capable of attaching to the target nucleic acid thereby allowing direct or indirect detection of the target nucleic acid utilising the detection probe.

47. A kit according to claim 45 or 46 in which the detection probe is capable of attaching to the target nucleic acid by hybridising to a second region of the target nucleic acid.

48. A kit according to claim 47 in which the detection probe comprises a hook detection probe capable of hybridising to the second region of the target nucleic acid and a universal detection probe capable of hybridising to the hook detection probe when the hook detection probe has hybridised to the target nucleic acid.

50. A kit according to any of claims 45 to 49 in which the detection probe or the universal detection probe comprises a label thereby allowing direct detection of target nucleic acid utilising the detection probe or the universal and hook detection probes.

51. A kit according to any of claims 45 to 49 in which the detection probe or the universal detection probe comprises a detection ligand which can be bound by a detection moiety thereby allowing indirect detection of target nucleic acid utilising the detection probe or the universal and hook detection probes.

52. A kit according to claim 51 which further comprises a detection moiety capable of binding to the detection ligand.

53. A kit according to claim 52 in which the detection moiety comprises an antibody or antibody fragment.

54. A kit according to claim 53 in which the detection ligand comprises biotin and the detection moiety comprises an anti-biotin antibody, or in which the detection ligand comprises fluorescein and the detection moiety comprises an anti-fluorescein antibody, or in which the detection ligand comprises 2,4-dinitrophenol (DNP) and the detection moiety comprises an anti-DNP antibody.

55. A kit according to any of claims 52 to 54 in which the detection moiety is releasably immobilised at a conjugate zone located between the contact end and the capture zone of the chromatographic strip.

56. A kit according to any of claims 52 to 55 in which the detection moiety comprises a label allowing detection of the detection moiety.

57. A kit according to claim 50 or 55 in which the label is non radioactive.

58. A kit according to claim 57 in which the label is a colour label.

59. A kit according to claim 58 in which the colour label comprises a textile dye, a metal sol such as colloidal gold, or a coloured particle such as a coloured latex particle.

63. Use of a kit according to any of claims 45 to 60 to test for the presence of a target nucleic acid in a sample solution.

46. A kit for testing for the presence of target nucleic acid in a sample solution which comprises:

i) a dipstick comprising a chromatographic strip with a contact end for contacting the sample solution, a universal capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end, and a hook capture probe hybridised to the universal capture probe, the hook probe being capable of hybridising to a first region of the target nucleic acid; and

optionally

ii) a detection probe capable of attaching to the target nucleic acid thereby allowing direct or indirect detection of the target nucleic acid utilising the detection probe.

49. A kit for testing for the presence of target nucleic acid in a sample solution which comprises:

i) a dipstick comprising a chromatographic strip with a contact end for contacting the sample solution, and a capture probe immobilised at a capture zone of the chromatographic strip remote from the contact end, the capture probe being capable of hybridising to a first region of the target nucleic acid, or being capable of binding indirectly to the target nucleic acid;

ii) a hook detection probe capable of hybridising to a second region of the target nucleic acid; and

iii) a universal detection probe capable of hybridising to the hook detection probe when the hook detection probe has hybridised to the second region of the target nucleic acid, thereby allowing direct or indirect detection of the target nucleic acid utilising the detection probe.

60. A kit for testing for the presence of a target nucleic acid in a sample solution which comprises:

a dipstick having a chromatographic strip with a contact end for contacting the sample solution and a capture moiety immobilised at a capture zone of the chromatographic strip remote from the contact end, the capture moiety being capable of binding by non base pairing interaction to a universal capture probe bound to the target nucleic acid by a hook capture probe;

a hook capture probe capable of hybridising to the target nucleic acid; and

a universal capture probe capable of hybridising to the hook capture probe.

61. Use of a universal and a hook capture probe in a dipstick assay for testing for the presence of a target nucleic acid in a sample solution.

62. Use of a universal and a hook detection probe in a dipstick assay for testing for the presence of a target nucleic acid in a sample solution.