METHODS FOR DETECTING NUCLEIC ACIDS IN A SAMPLE

• US 20100167294A1
• Filed: 09/23/2009
• Published: 07/01/2010
• Est. Priority Date: 09/23/2008
• Status: Abandoned Application

• Current Assignee: Nexus Dx, Inc. (Sphingotec GMBH)
• Original Assignee: Nexus Dx, Inc. (Sphingotec GMBH)
• Inventors: Mather, Elizabeth, Weisburg, William, Light, James II, Huang, Ying
• Application Number:
  US12/565,206
• Publication Number:
  US 20100167294A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Abstract

Systems and methods are provided for immobilizing nucleic acid amplicons and protein antigens on a test device. Amplicons comprising a synthetic binding unit and a detectable label are generated and immobilized at predetermined locations on a test device by specific binding interactions between the synthetic binding unit and a synthetic capture unit located at the predetermined locations. The synthetic binding unit may include a unique design such that during amplification, a region of the synthetic binding unit is not subject to the amplification reaction, and thus the amplicon remains single stranded and available for binding to the synthetic capture unit during the capture process. In certain embodiments, the synthetic binding unit and a synthetic capture unit include synthetic nucleic acid analogs that do not interact with native nucleic acids or enzymes that act thereon. In one embodiment the synthetic binding unit and synthetic capture unit comprises puranosyl RNA (pRNA).

Claims

1. A method for rapid detection of nucleic acids, the method comprising the steps of:

(a) providing a sample comprising an amplicon of one or more nucleic acid(s) generated using a first primer conjugated to a synthetic binding unit (SBU) and a second primer conjugated, directly or indirectly, to a detectable moiety;

(b) providing a surface on a substrate, the surface comprising a synthetic capture unit (SCU) immobilized at predetermined location on the surface, wherein the synthetic capture unit (SCU) selectively and reversibly binds the synthetic binding unit (SBU);

(c) contacting the surface with the amplicon under conditions wherein the amplicon is immobilized at the predetermined location by binding between the synthetic capture unit and the synthetic binding unit; and

(d) detecting the presence of the nucleic acid by detecting the presence of the detectable moiety at the predetermined location.

2. The method of claim 1, wherein the synthetic capture unit or the synthetic binding unit do not participate in the amplification reaction used to generate the amplicon.

3. The method of claim 1, wherein the synthetic binding unit does not bind native nucleic acid sequences.

4. The method of claim 1, wherein the synthetic capture unit and the synthetic binding unit comprise a nucleic acid sequence selected from the group consisting of:

a pyranosyl RNA (pRNA) sequences;

a 2′

-O-methyl oligonucleotide sequences and a 5′

-5′

inverted nucleic acid.

5. The method of claim 1, wherein the amplicon is generated by a method comprising a polymerase chain reaction (PCR).

6. The method of claim 5, further comprising:

amplifying a nucleic acid using a first primer conjugated to a first pyranosyl-RNA (p-RNA) sequence and a second primer conjugated to a detectable moiety.

7. The method of claim 1, wherein the detectable moiety is selected from the group consisting of:

a fluorophore, a chromophore, a metal, a quantum dot, an enzyme, an electrochemical moieties, a radioactive moiety, a phosphorescent group, a chemiluminescent moiety, an affinity ligand, a heavy atom, a nanoparticle light scattering label, members of a binding pair that are capable of forming complexes comprising streptavidin/biotin, or avidin/biotin, or antigen/antibody, a lanthanide, an europium bead and combinations thereof.

8. The method of claim 1, further comprising:

providing a first binding agent conjugated to a detectable moiety, wherein the first binding agent is capable of binding to a second binding agent, and further wherein the second primer is conjugated to the second binding agent.

9. The method of claim 8, wherein the first and second binding agents are streptavidin and biotin.

10. The method of claim 9, wherein the detectable moiety comprises europium beads conjugated with streptavidin and the amplicon is conjugated to biotin.

11. The method of claim 1, comprising:

performing an amplification reaction in a manner sufficient to produce reactants, the amplification reaction comprising;

providing a second primer conjugated with a second binding agent;

adding a first binding agent conjugated to a detectable moiety, wherein the first and second binding agents bind to each other; and

then contacting the test surface with the reactants.

12. The method of claim 1, wherein the substrate comprises a lateral flow membrane.

13. The method of claim 12, wherein the surface comprises a lateral flow membrane comprising a test pad adjacent a sample pad, and the test surface is contacted by the amplicons at the sample pad, wherein the test pad and the sample pad are fluidably coupled.

14. The method of claim 13, further comprising providing an absorbent pad adjacent to the test pad, wherein the absorbent pad is distal to the sample pad.

15. The method of claim 13, wherein the test pad comprises nitrocellulose and the synthetic capture unit is a p-RNA sequence conjugated to a protein.

16. The method of claim 15, wherein the protein is selected from the group consisting of an immunoglobulin and bovine serum albumin (BSA).

17. The method of claim 1, wherein the synthetic capture unit is immobilized along predetermined test lines on the surface.

18. The method of claim 1, wherein the synthetic capture unit is immobilized along predetermined test spots on the surface.

19. The method of claim 1, further comprising:

providing a sample pad adjacent to the surface, wherein a sample contacted with the sample pad is capable of flowing to the surface;

applying the sample comprising the amplicon to the sample pad under conditions allowing lateral flow to the surface;

immobilizing the nucleic acid at the predetermined location on the surface by hybridization between the first SBU sequence and the second SCU sequence immobilized on the surface; and

detecting the presence of the nucleic acid by detecting the presence of the detectable moiety at the predetermined location.

20. The method of claim 19, further comprising providing an absorbent pad adjacent to the surface, wherein the absorbent pad is distal to the sample pad.

21. The method of claims 20, wherein the sample pad, surface and optional absorbent pad comprise a lateral flow test strip comprising a lateral flow membrane, the method further comprising:

scanning the lateral flow test strip for the presence of the detectable moiety using a device capable of detecting the detectable moiety, wherein the presence of the nucleic acid is determined by detecting the presence of the detectable moiety at the predetermined location on the surface.

22. A method for simultaneously detecting a nucleic acid and a protein antigen in a sample, the method comprising:

(a) providing a sample comprising an amplicon of one or more nucleic acid(s) generated using a first primer conjugated to a first synthetic binding unit (SBU) and a second primer conjugated, directly or indirectly, to a first detectable moiety;

(b) providing a surface on a substrate, the surface comprising a first synthetic capture unit (SCU) immobilized at predetermined location on the surface, wherein the first synthetic capture unit (SCU) selectively and reversibly binds the first synthetic binding unit (SBU), but the first synthetic capture unit or the first synthetic binding unit do not participate in the amplification reaction used to generate the amplicon;

(c) contacting the surface with the amplicon under conditions wherein the amplicon is immobilized at the predetermined location by binding between the first synthetic capture unit and the first synthetic binding unit; and

(d) detecting the presence of the nucleic acid by detecting the presence of the first detectable moiety at the predetermined location.

(e) forming a mixture by mixing a sample being tested for the presence of at least one target antigen with a solution comprising a plurality of reagents comprising;

(i) an antibody-synthetic binding unit conjugate comprising a first antibody which specifically binds a target antigen and a second synthetic binding unit (SBU) that specifically binds a second synthetic capture unit (SCU), and

(ii) a labeled second antibody, wherein the second antibody specifically binds the same target antigen including when the target antigen is bound to the first antibody, wherein the second antibody is labeled with a second detectable moiety;

(f) applying the mixture and the amplicon solution to the lateral flow membrane comprising a plurality of detection regions, each of said detection region having immobilized thereto a first or second synthetic capture unit;

(g) flowing the mixture and the amplicon solution across the membrane, whereby the first or second synthetic capture unit captures a complex having a first or second synthetic binding unit to which it is directed; and

(h) scanning the membrane for presence of the first and second detectable moieties in at least one of said detection region, whereby detection of the first or second detectable moieties indicates the presence of a target nucleic acid or a target protein in said sample.

23. The method of claim 22, wherein the synthetic capture unit and the synthetic binding unit are complementary pRNA sequences.

24. The method of claim 22, wherein the detectable moiety is selected from the group consisting of:

a fluorophore, a chromophore, a metal, a quantum dot, an enzyme, an electrochemical moieties, a radioactive moiety, a phosphorescent group, a chemiluminescent moiety, an affinity ligand, a heavy atom, a nanoparticle light scattering label, members of a binding pair that are capable of forming complexes comprising streptavidin/biotin, or avidin/biotin, or antigen/antibody, a lanthanide, an europium bead and combinations thereof.

25. The method of claim 22, further comprising:

providing a first binding agent conjugated to a detectable moiety, wherein the first binding agent is capable of binding to a second binding agent, and further wherein the second primer is conjugated to the second binding agent.

26. The method of claim 25, wherein the first and second binding agents are streptavidin and biotin.

27. The method of claim 26, wherein the detectable moiety comprises europium beads conjugated with streptavidin and the amplicon or the antigen-antibody is conjugated to biotin.

28. The method of claim 22, wherein the synthetic capture unit and the synthetic binding unit comprise a nucleic acid sequence selected from the group consisting of:

a pyranosyl RNA (pRNA) sequences;

a 2′

-O-methyl oligonucleotide sequences and a 5′

-5′

inverted nucleic acid.

29. The method of claim 22, wherein the nucleic acid and the protein antigen are from an infectious agent.

30. The method of claim 29, wherein the infectious agent is selected from a virus, a bacteria, a fungus and a parasite.

31. The method of claim 30, wherein the infectious agent is selected from the group consisting of:

a strain of influenza virus, parainfluenza virus, a type of HIV, a type of hepatitis virus, a herpes simplex virus, adenovirus, enterovirus, Streptococcus pneumoniae, Staphylococcus aureus, Bordetella pertussis, Mycoplasma pneumoniae, and Coccidioides immitis.

32. A test device comprising a lateral flow strip, the test device comprising:

(a) a test device body;

(b) a lateral flow membrane in the body and exposed through a single or a plurality of windows in the body;

(c) an optional absorbent pad in communication with the lateral flow membrane, the absorbent pad comprising a wicking material and being positioned upstream of said plurality of windows;

(d) a sample pad in communication with the lateral flow membrane, the sample pad comprising an absorbent material and being positioned downstream of said plurality of windows; and

(e) a plurality of addressable regions, each addressable region having immobilized thereto a synthetic capture unit, each synthetic capture unit capable of specifically binding with a synthetic binding unit conjugated to a target analyte.

33. The test device of claim 29, wherein the lateral flow membrane is capable of being scanned by a detection apparatus.

34. The test device of claim 32, wherein at least one of the synthetic capture unit and the synthetic binding unit comprise complementary sequences selected from the group consisting of:

a pRNA sequence, 2′

-O-methyl oligonucleotide sequence, and a 5′

-5′

inverted nucleic acid sequence.

35. The test device of claim 32, wherein said target analyte is an infectious agent.

36. The test device of claim 35, wherein the infectious agent is selected from a virus, a bacteria, a fungus and a parasite.

37. The test device of claim 32 further comprising an identifying marker.

38. The test device of claim 32 further comprising control spots or control lines for binding a control reagent for standardization.

39. The test device of claim 32, wherein the plurality of addressable regions include a plurality of different capture agents.

40. The test device of claim 39, wherein each of the plurality of different types of capture agents recognize and bind to a different synthetic binding unit.

41. The test device of claim 40, wherein each different synthetic binding agent comprises a different detection moiety.

42. The test device of claim 41, wherein each different detection moiety comprises a different fluorescent label.

43. The test device of claim 42, wherein the detection apparatus is configured for detecting and distinguishing emitted light from each different fluorescent label.

44. A system comprising:

(a) a test device of claim 24;

(b) a reader that reads a signal generated at any addressable region of the test device; and

(c) machine readable encoded instructions capable of directing said reader to detect the signal or signals.

45. The system of claim 44, wherein said signal is generated by a detectable moiety is selected from the group consisting of:

a fluorophore, a chromophore, a metal, a quantum dot, an enzyme, an electrochemical moieties, a radioactive moiety, a phosphorescent group, a chemiluminescent moiety, an affinity ligand, a heavy atom, a nanoparticle light scattering label, members of a binding pair that are capable of forming complexes comprising streptavidin/biotin, or avidin/biotin, or antigen/antibody, a lanthanide, an europium bead and combinations thereof.

46. The system of claim 44, wherein said signal is generated by a fluorophore and said reader is a fluorescence reader capable of distinguishing a fluorescent signal from each of the one or more addressable regions on said test strip, said reader comprising a light emitting diode which emits in the UV region of the spectrum and a photodiode capable of detecting the emitted fluorescent signal.

47. The system of claim 46 wherein said fluorescent signal is generated by europium.

48. A kit comprising a lateral flow test device of claim 24, and a streptavidin conjugated detectable label.