LATERAL FLOW NUCLEIC ACID DETECTOR

• US 20090305290A1
• Filed: 07/14/2009
• Published: 12/10/2009
• Est. Priority Date: 06/10/2008
• Status: Active Grant

• Current Assignee: Rapid Pathogen Screening, Inc
• Original Assignee: Rapid Pathogen Screening, Inc
• Inventors: Sambursky, Robert P., VanDine, Robert W., Babu, Uma Mahesh
• Granted Patent: US 8,669,052 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 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
• CPC Class Codes: G01N 33/526 the element being adapted for a specific analyte
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Point-of-care binding assays include at least one target nucleic acid binding in a multiplex structure with at least one sequence in a partner nucleic acid associated with a label, due to complementary base pairings between at least one sequence in the target nucleic acid and at least one sequence in the partner nucleic acid. The assays overcome the inherent deficiencies of antibody-protein antigen assays. In a preferred embodiment, color tagged nucleic acid sequences are used to bind a complementary target nucleic acid. The tagged nucleic acid sequences are preferably made from deoxyribonucleotides, ribonucleotides, or peptide nucleotides.

Claims

1. A test strip for detection of at least one target nucleic acid in a sample comprising:

at least one detection zone comprising at least one test zone;

a sample application zone for applying the sample to the test strip;

at least one first complex comprising at least one first nucleic acid sequence associated with at least one label, wherein the first complex is loaded in a first complex application zone, and wherein the first nucleic acid sequence comprises a sequence complementary to a first portion of a sequence of the target nucleic acid; and

at least one second complex comprising at least one second nucleic acid sequence associated with at least one immobilization agent, wherein the immobilization agent is immobilizable in a test zone of the assay, and wherein the second nucleic acid sequence comprises a sequence complementary to a second portion of the sequence of the target nucleic acid; and

wherein the sample, the first complex, and the second complex are all loaded on the test strip at locations such that the sample encounters both the first complex and the second complex while running an assay to detect the target nucleic acid in the sample.

2. The test strip of claim 1, wherein the target nucleic acid is a single-stranded nucleic acid.

3. The test strip of claim 1, wherein the target nucleic acid is a double-stranded nucleic acid.

4. The test strip of claim 1, wherein the detection zone further comprises a control zone located beyond the test zone for immobilizing any portion of the first complex that does not bind to the target nucleic acid.

5. The test strip of claim 1, further comprising a receptor that recognizes and immobilizes the immobilizing agent, wherein the second complex is loaded at a location upstream of the test zone and the immobilization site is located at the test zone.

6. The test strip of claim 1, further comprising a receptor that recognizes and immobilizes the immobilizing agent, wherein the second complex is loaded at the test zone and the receptor is loaded at a location upstream of the test zone.

7. The test strip of claim 1, wherein the label is selected from the group consisting of:

a) at least one colloidal gold particle mixed with at least one fluorescent microbead; and

b) at least one dyed latex microbead.

8. The test strip of claim 1, wherein the first nucleic acid sequence and the second nucleic acid sequence are each a polymerase chain reaction (PCR) type primer of the target nucleic acid selected from the group consisting of:

a) an inner primer;

b) an outer primer;

c) a sense primer; and

d) an anti-sense primer.

9. The test strip of claim 1, further comprising a lysis agent or a denaturant.

10. A polymerase chain reaction primer associated with at least one label.

11. A nucleic acid immobilized in a detection zone of a test strip for a point-of-care assay.

12. The nucleic acid of claim 11, wherein the nucleic acid is a polymerase chain reaction primer.

13. A method of detecting at least one target nucleic acid in a sample using a point-of-care assay, the method comprising the steps of:

a) exposing the sample to at least one first complex comprising at least one first nucleic acid sequence associated with at least one label; and

b) exposing the sample to at least one second complex comprising at least one second nucleic acid sequence;

wherein, when the target nucleic acid is present in the sample, the first nucleic acid sequence and the second nucleic acid sequence associate with the target nucleic acid such that the first complex, the target nucleic acid, and the second complex are immobilized in the test zone at a completion of the assay.

14. The method of claim 13, wherein the second complex is associated with at least one immobilization agent, wherein the immobilization agent is immobilizable in a test zone of the assay

15. The method of claim 13, wherein the second complex is located in a test zone of the assay.

16. The method of claim 13, wherein the first nucleic acid sequence and the second nucleic acid sequence are each a polymerase chain reaction (PCR) type primer of the target nucleic acid selected from the group consisting of:

a) an inner primer;

b) an outer primer;

c) a sense primer; and

d) an anti-sense primer.

17. The method of claim 13 further comprising the step of taking the sample from the patient.

18. The method of claim 13, wherein the sample is from a body fluid selected from the group consisting of:

a) saliva;

b) mucus;

c) tissue;

d) tears;

e) spinal fluid;

f) vaginal fluid;

g) penile fluid;

h) nasopharygeal secretions;

i) skin ulcerations;

j) blood; and

k) urine.

19. The method of claim 13 further comprising the step of exposing the sample to a lysis agent or a denaturant, wherein the target nucleic acid is a double-stranded nucleic acid.

20. The method of claim 13 further comprising the step of detecting for a presence or an absence of the label in the test zone.

21. The method of claim 20, wherein the step of detecting the label comprises using a method selected from the group consisting of:

fluorescence, chemoluminescence, radioactivity, colorimetry, gravimetry, X-ray diffraction, X-ray absorption, magnetism, and enzymatic activity.

22. The method of claim 13, wherein the label is selected from the group consisting of:

a) at least one colloidal gold particle mixed with at least one fluorescent microbead; and

b) at least one dyed latex microbead.

23. The method of claim 13, wherein the sample is run on the assay without an amplification step to amplify the target nucleic acid.

24. The method of claim 13, where in the assay is run on a chromatographic test strip.

25. A test strip for detection of at least one target nucleic acid in a sample comprising:

at least one detection zone comprising at least one test zone;

a sample application zone for applying the sample to the test strip;

at least one first complex comprising at least one first nucleic acid sequence associated with at least one label, wherein the first complex is loaded in a first complex application zone; and

at least one second complex immobilized in a test zone of the assay, comprising at least one second nucleic acid sequence;

wherein the sample and the first complex are loaded on the test strip at locations such that the sample encounters the first complex; and

wherein the target nucleic acid comprises at least one first target nucleic acid sequence complementary to the first nucleic acid sequence and at least one second target nucleic acid target complementary to the second nucleic acid sequence.

26. The test strip of claim 25, wherein the target nucleic acid is a single-stranded nucleic acid.

27. The test strip of claim 25, wherein the target nucleic acid is a double-stranded nucleic acid.

28. The test strip of claim 25, wherein the detection zone further comprises a control zone located beyond the test zone for immobilizing any portion of the first complex that does not bind to the target nucleic acid.

29. The test strip of claim 25, wherein the first nucleic acid sequence and the second nucleic acid sequence are each a polymerase chain reaction (PCR) type primer of the target nucleic acid selected from the group consisting of:

a) an inner primer;

b) an outer primer;

c) a sense primer; and

d) an anti-sense primer.