Lateral flow system for nucleic acid detection

• US 20040110167A1
• Filed: 04/14/2003
• Published: 06/10/2004
• Est. Priority Date: 07/13/1995
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Mondesire, Roy R., Hansen, Lara A., Gerdes, John C.
• Application Number:
  US10/413,433
• Publication Number:
  US 20040110167A1
• 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 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates

Abstract

The invention provides a complete, one-step, fully functional, ready to use lateral flow assay device for the rapid, accurate detection of a target nucleic acid in a fluid sample, wherein the device contains all reagents necessary for the assay in an anhydrous format. The device comprises a sample receiving zone, a labeling zone, and a capture zone. The sample receiving zone may contain one or more oligonucleotides coupled to binding partners and reversibly bound to the capture zone membrane, the labeling zone comprises a visible moiety coupled to a ligand specific for one of the binding partners and reversibly bound to the labeling zone membrane, and the capture zone comprises an capture moiety specific for the second binding partner and immobilized on the capture zone membrane.

Claims

1. A lateral flow assay device for detecting the presence or absence of at least one single-stranded target nucleic acid in a fluid sample, said device having a first and second end and comprising:

a sample receiving zone at or near said first end for receiving an aliquot of said sample and comprising a porous material having first and second oligonucleotide probes coupled to first and second binding partners, respectively, wherein said probes specifically hybridize to said target nucleic acid to form a complex having said first and second binding partners, said sample receiving zone being in lateral flow contact with

a labeling zone comprising a porous material having at least a first visible moiety reversibly bound thereto and coupled to a first ligand which specifically binds to said first binding partner to form a visible complex, said labeling zone being in lateral flow contact with

a capture zone comprising a microporous membrane which contains in a portion thereof a first capture moiety immobilized thereto which specifically binds said second binding partner, said capture zone being in lateral flow contact with

an absorbent zone positioned at or near the second end of said device,

wherein said visible complex is captured by said capture moiety in said portion of the capture zone.

2. The device of claim 1, wherein said sample receiving zone porous material retains said probes prior to contact with said fluid sample and releases said probes after contact with said fluid sample.

3. The device of claim 2, wherein said sample receiving zone porous material is selected from the group consisting of glass, cotton, cellulose, polyester, rayon, nylon, polyethersulfone, and polyethylene.

4. The device of claim 1, wherein said first and second binding partners are selected from the group consisting of antibodies or fragments thereof, proteins, haptens, antigens or fragments thereof, avidin, streptavidin, biotin, fluorescein isothiocyanate, folic acid, folate binding protein, protein A, protein G, immunoglobulins, digoxigenin, anti-digoxigenin F(ab′

)₂, complementary nucleic acid segments, protein A, protein G, immunoglobulins, lectin, carbohydrate, enzymes, viruses, maleimides, haloacetyl derivatives, isotriocyanates, succinimidyl esters, sulfonyl halides, steroids, halogens and 2,4-dinitrophenyl.

5. The device of claim 1, wherein said labeling zone porous material is selected from the group consisting of glass, cotton, cellulose, polyester, polyethylene, rayon or nylon.

6. The device of claim 1, wherein said first visible moiety comprises a ligand coupled to a colored microparticle.

7. The device of claim 6, wherein said microparticle is selected from the group consisting of polymers or copolymers of olefinically unsaturated monomers, glass, acrylamide, methacrylate, nylon, acrylonitrile, polybutadiene, metals, metal oxides and their derivatives, dextran, cellulose, liposomes, red blood cells, pollens, and bacteria.

8. The device of claim 1, wherein said capture zone membrane comprises a microporous material selected from the group consisting of nitrocellulose, polyethersulfone, polyvinylidine fluoride, nylon, charge-modified nylon, and polytetrafluoroethylene.

9. The device of claim 1, wherein said first capture moiety is selected from the group consisting of antibodies or fragments thereof, proteins, haptens, antigens or fragments thereof, avidin, streptavidin, biotin, fluorescein isothiocyanate, folic acid, folate binding protein, protein A, protein G, immunoglobulins, digoxigenin, anti-digoxigenin F(ab′

)₂, complementary nucleic acid segments, protein A, protein G, immunoglobulins, lectin, carbohydrate, enzymes, viruses, maleimides, haloacetyl derivatives, isotriocyanates, succinimidyl esters, sulfonyl halides, steroids, halogens and 2,4-dinitrophenyl.

10. The device of claim 1, wherein said capture zone is prepared by applying a solution containing said capture moiety to said membrane under conditions wherein the capture moiety becomes immobilized on said membrane, followed by drying said membrane.

11. The device of claim 10, wherein said solution is applied to said membrane in the form of a line.

12. The device of claim 1, wherein said labeling zone further comprises a second visible moiety reversibly affixed to said matrix and coupled to a second ligand, and said capture zone further comprises in a portion thereof a second capture moiety immobilized thereon which specifically binds said second ligand.

13. The device of claim 12, wherein said portion of said capture zone containing said first capture moiety is separate from said portion containing second capture moiety.

14. The device of claim 1, wherein said absorbent zone comprises a material selected from the group consisting of nitrocellulose, cellulose esters, glass, polyethersulfone, and cotton.

15. The device of claim 1, wherein said entire test strip except for a portion of said sample receiving zone is completely sheathed in a transparent film.

16. The device of claim 15, wherein said film is a polyester, polycarbonate, polystyrene, polypropylene, glycol modified polyethylene terphthalate, a heat resistant acrylic, or a butyrate.

17. The device of claim 1, wherein said sample receiving zone microporous material is affixed to a first end of the top side of said capture zone membrane, said labeling zone is affixed to the top side of said capture zone membrane and position between said sample receiving zone and said capture zone, and said absorbent pad is affixed to the top side of said capture zone membrane near the second end of said membrane.

18. The device of claim 17, wherein said capture zone membrane is affixed to the top side of a rigid or semi-rigid support.

19. The device of claim 18, wherein said rigid or semi-rigid support comprises polypropylene, poly(vinyl chloride), propylene, or polystyrene).

20. The device of claim 18, further comprising a heating sheet affixed to the bottom side of said rigid or semi-rigid support.

21. The device of claim 1, wherein said sample receiving zone, said labeling zone, said capture zone, and said absorbent zone are affixed to the top side of a rigid or semi-rigid support.

22. The device of claim 21, wherein said support comprises polypropylene, poly(vinyl chloride), propylene, or polystyrene).

23. The device of claim 21, further comprising a heating sheet affixed to the bottom side of said support.

24. The device of claim 1, further comprising a piercing means at said first end.

25. The device of claim 1 for detecting the presence of two or more target nucleic acids, wherein the sample receiving zone comprises a first and second oligonucleotide probe specific for each of said target nucleic acid, the labeling zone comprises a first visible moiety specific for each of said target nucleic acids and distinguishable from the other visible moieties, and the capture zone comprises a capture moiety specific for each of said target nucleic acids.

26. A lateral flow assay device for detection of the presence or absence of at least one target nucleic acid in a fluid sample, wherein said target nucleic acid is coupled to a first binding partner, said device comprising a test strip having a first and second end and comprising:

a sample receiving zone at or near said first end for receiving an aliquot of said sample and comprising a porous material having an oligonucleotide probe coupled to a second binding partner, wherein said probe is reversibly bound to said microporous material and specifically hybridizes to said target nucleic acid to form a complex comprising said first and second binding partners, said sample receiving zone being in lateral flow contact with

a labeling zone comprising a porous material having at least a first visible moiety reversibly bound thereto and coupled to a first ligand which specifically binds to said first binding partner to form a Visible complex, said labeling zone being in lateral flow contact with

a capture zone comprising a microporous membrane which contains in a portion thereof at least a first capture moiety immobilized thereto which specifically binds said second binding partner, said capture zone being in lateral flow contact with

an absorbent zone positioned at or near the second end of said test strip, wherein said visible complex is captured by said capture moiety in said portion of the capture zone.

27. The lateral flow assay device of claim 26 for detecting the presence of two or more target nucleic acids, wherein the sample receiving zone comprises an oligonucleotide probe specific for each of said target nucleic acids, the labeling zone comprises a first visible moiety specific for each of said target nucleic acids and distinguishable from the other visible moieties, and the capture zone comprises a capture moiety specific for each of said target nucleic acids.

33. The method of claim 27, wherein said assay device is affixed to a heating sheet, said method further comprising heating said device while said sample is wicking along said test strip.

34. The method of claim 27, wherein said device is heated to a temperature between about 20 and 95°

C.

28. A lateral flow assay device for detection of the presence or absence of at least one target nucleic acid in a fluid sample, wherein said target nucleic acid is coupled to a first and second binding partner, said device comprising a test strip having a first and second end and comprising:

a sample receiving zone at or near said first end of said test strip for receiving an aliquot of said sample and comprising a porous material, said sample receiving zone being in lateral flow contact with

a labeling zone comprising a porous material having at least a first visible moiety coupled to a first ligand which specifically binds to said first binding partner to form a visible complex, said labeling zone being in lateral flow contact with

a capture zone comprising a membrane which contains in at least a portion thereof at least a first capture moiety immobilized thereon which specifically binds said second binding partner, said capture zone being in lateral flow contact with

an absorbent zone at or near said second end of said test strip,

wherein said visible complex is captured by said capture moiety in said portion of the capture zone.

29. The lateral flow assay device of claim 28 for detecting the presence of two or more target nucleic acids, wherein the labeling zone comprises a first visible moiety specific for each of said target nucleic acids and distinguishable from the other visible moieties, and the capture zone comprises a capture moiety specific for each of said target nucleic acids.

30. A method for detecting the presence or absence of at least one target nucleic acid in a fluid sample, said method comprising:

(a) applying said sample to a sample receiving zone of a lateral flow test strip of a lateral flow assay device, wherein prior to said application said nucleic acid present in a double-stranded form are rendered single-stranded, wherein said sample wicks sequentially from said sample receiving zone to a labeling zone and to a capture zone of said test strip, said sample receiving zone comprising first and second oligonucleotide probes coupled to first and second binding partners, respectively, and reversibly bound to said test strip, wherein said probes are released from said test strip and specifically hybridize to said target nucleic acid upon contact with said sample to form a complex comprising said first and second binding partners,

said labeling zone comprising at least a first visible moiety coupled to a first ligand and reversibly bound to said test strip, wherein said first ligand specifically binds said first binding partner, and

said capture zone comprising a capture moiety immobilized on a portion of said test strip, wherein said capture moiety specifically binds said second binding partner; and

(b) detecting the presence of said first visible moiety in said portion of said capture zone.

31. The method of claim 30, wherein prior to step (a) said target nucleic acid is amplified.

32. The method of claim 31, wherein said amplification methodology is polymerase chain reaction (PCR), ligase chain reaction, Qβ

replicase, strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), loop amplification (LAMP), ramification amplification (RAM), or cascade rolling circle amplification (CRCA).

35. The method of claim 30, wherein said assay is performed under high stringency conditions.

36. The method of claim 30, wherein said assay is performed under low stringency conditions.

37. The method of claim 30, wherein said labeling zone further comprises a second visible moiety reversibly bound to said test strip and coupled to a second ligand, and said capture zone further comprises a second capture moiety immobilized on said test strip, wherein said second capture moiety specifically binds said second ligand.

38. The method of claim 30, wherein said entire test strip except for a portion of said sample receiving zone is sheathed in a transparent film.

39. The method of claim 30 for detecting the presence of two or more target nucleic acids, wherein the sample receiving zone comprises a first and second oligonucleotide probe specific for each of said target nucleic acid, the labeling zone comprises a first visible moiety specific for each of said target nucleic acids and distinguishable from the other visible moieties, and the capture zone comprises a capture moiety specific for each of said target nucleic acids.

40. A method for detecting the presence or absence of at least one target nucleic acid in a fluid sample, said method comprising:

(a) coupling said target nucleic acid to a first binding partner to provide a labeled target nucleic acid;

(b) applying said labeled target nucleic acid to a sample receiving zone of a lateral flow test strip of a lateral flow assay device, wherein prior to said application said nucleic acid present in a double-stranded form are rendered single-stranded, wherein said labeled target nucleic acid wicks sequentially from said sample receiving zone to a labeling zone and to a capture zone of said test strip, said sample receiving zone comprising an oligonucleotide probe coupled to a second binding partner and reversibly bound to said test strip, wherein said probes are released from said test strip and specifically hybridize to said labeled target nucleic acid upon contact with said labeled target nucleic acid to form a complex comprising said first and second binding partners,

said labeling zone comprising at least a first visible moiety coupled to a first ligand and reversibly bound to said test strip, wherein said first ligand specifically binds said first binding partner, and

said capture zone comprising a capture moiety immobilized on a portion of said test strip, wherein said capture moiety specifically binds said second binding partner; and

(c) detecting the presence of said first visible moiety in said portion of said capture zone.

41. The method of claim 40, wherein said target nucleic acid is coupled to said first binding partner by amplifying said target nucleic acid with a primer comprising said first binding partner.

42. The method of claim 40 for detecting the presence of two or more target nucleic acids, wherein the sample receiving zone comprises an oligonucleotide probe specific for each of said target nucleic acids, the labeling zone comprises a first visible moiety specific for each of said target nucleic acids and distinguishable from the other visible moieties, and the capture zone comprises a capture moiety specific for each of said target nucleic acids.

43. A method for detecting the presence or absence of at least one target nucleic acid in a fluid sample, said method comprising the steps of:

(a) coupling said target nucleic acid to a first and second binding partner to provide a labeled target nucleic acid;

(b) applying said labeled target nucleic acid to a sample receiving zone of a lateral flow test strip of a lateral flow assay device, wherein said labeled target nucleic acid laterally wicks sequentially from said sample receiving zone through a labeling zone to a capture zone of said test strip,

said labeling zone comprising at least a first visible moiety reversibly bound to said test strip and coupled to a first ligand, wherein said first ligand specifically binds said first binding partner, and

said capture zone comprising a capture moiety immobilized on a portion of said test strip, wherein said capture moiety specifically binds said second binding partner; and

(c) detecting the presence of said first visible moiety in said portion of said capture zone.

44. The method of claim 43, wherein said target nucleic acid is coupled to said first and second binding partners by amplifying said target nucleic acid with first and second primers comprising said first and second binding partners, respectively, wherein said amplified nucleic acid is rendered single-stranded prior to step (b).

45. The method of claim 43 for detecting the presence of two or more target nucleic acids, wherein the labeling zone comprises a first visible moiety specific for each of said target nucleic acids and distinguishable from the other visible moieties, and the capture zone comprises a capture moiety specific for each of said target nucleic acids.