Lateral flow nucleic acid detector

• US 8,669,052 B2
• Filed: 07/14/2009
• Issued: 03/11/2014
• Est. Priority Date: 06/10/2008
• Status: Expired due to Fees

• Current Assignee: Rapid Pathogen Screening, Inc
• Original Assignee: Rapid Pathogen Screening, Inc
• Inventors: Sambursky, Robert P., Babu, Uma Mahesh, VanDine, Robert W.
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US12/502,626
• Publication Number:
  US 20090305290A1
• Time in Patent Office: 1,701 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 double stranded 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;

a lysis zone comprising at least one lysis or denaturing agent that partially opens the target double stranded nucleic acid into a first single strand and a second single strand;

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 portion of a sequence of the first single strand 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 test strip and wherein the second nucleic acid sequence comprises a sequence complementary to a portion of a sequence of the second single strand of the target nucleic acid;

wherein the sample, the first complex, and the second complex are all loaded on the test strip at locations such that the sample encounters the lysis zone, and 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 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.

3. 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.

4. 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.

5. 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.

11. The test strip of claim 1, wherein the first nucleic acid sequence is a primer or oligonucleotide anti-sense sequence to the first single strand of the target nucleic acid sequence.

12. The test strip of claim 1, wherein the second nucleic acid sequence is a primer or oligonucleotide anti-sense sequence to the second single strand of the target nucleic acid sequence.

13. The test strip of claim 1, wherein the first nucleic acid sequence and the second nucleic acid sequence are non-overlapping sequences.

19. The test strip of claim 1, wherein the sample is run on the assay without an amplification step to enhance assay sensitivity for the target nucleic acid.

6. A test strip for detection of at least one target double stranded 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;

a lysis zone comprising at least one lysis or denaturing agent that partially opens the target double stranded nucleic acid into a first single strand and a second single strand;

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 portion of a sequence of the first single strand 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 second complex is not immobilized in a test zone at the start of an assay and wherein the immobilization agent is immobilizable in the test zone of the assay, and wherein the second nucleic acid sequence comprises a sequence complementary to a portion of a sequence of the second single strand 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 the lysis zone, and both the first complex and the second complex while running the assay to detect the target nucleic acid in the sample.

7. The test strip of claim 6, wherein the sample is run on the assay without an amplification step.

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

9. The test strip of claim 6, 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.

10. The test strip of claim 6, wherein the lysis zone is located in the sample application zone.

14. The test strip of claim 6, wherein the first nucleic acid sequence is a primer or oligonucleotide anti-sense sequence to the first single strand of the target nucleic acid sequence.

15. The test strip of claim 6, wherein the second nucleic acid sequence is a primer or oligonucleotide anti-sense sequence to the second single strand of the target nucleic acid sequence.

16. The test strip of claim 6, wherein the first nucleic acid sequence and the second nucleic acid sequence are non-overlapping sequences.

17. A test strip for detection of at least one target double stranded 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 assay condition that partially opens the target double stranded nucleic acid into a first single strand and a second single strand;

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 first single strand 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 second complex is not immobilized in a test zone at the start of an assay and wherein the immobilization agent is immobilizable in the test zone of the assay, and wherein the second nucleic acid sequence comprises a sequence complementary to a second portion of a the sequence of the second single strand 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 the an assay to detect the target nucleic acid in the sample.

18. The test strip of claim 17, wherein the assay condition is selected from the group consisting of:

a) a tonicity that partially opens the target double stranded nucleic acid;

b) a temperature that partially opens the target double stranded nucleic acid;

c) a pH that partially opens the target double stranded nucleic acid;

d) a lysis agent or denaturant that partially opens the target double stranded nucleic acid, wherein the lysis agent is added to the sample before the sample is applied to the test strip; and

e) any combination of a) through d).