Lateral flow nucleic acid detector
View Patent ↗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.
1. A method of detecting at least one target double stranded nucleic acid in a sample using a point-of-care assay, the method comprising the steps of:
a) partially opening the target double stranded nucleic acid into a first sink strand and a second single strand;
b) exposing the sample to at least one first complex located on a chromatographic test strip, wherein the first complex comprises at least one first nucleic acid sequence associated with at least one label, 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
c) exposing the sample to at least one second complex located on the test strip, wherein the second complex comprises at least one second nucleic acid sequence, 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, 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.
2. The method of claim 1 , wherein the second complex is associated with at least one immobilization agent, wherein the immobilization agent is immobilizable in a test zone of the test strip.
3. The method of claim 1 , wherein the second complex is located in a test zone of the test strip.
4. The method of claim 1 further comprising the step of taking the sample from the patient.
5. The method of claim 1 , wherein the sample is from a body fluid selected from the group consisting of:
a) saliva;
b) mucus;
c) tears; and
d) nasopharyngeal secretions.
6. The method of claim 1 , wherein step a) comprises exposing the sample to a lysis agent or a denaturant.
7. The method of claim 1 further comprising the step of detecting for a presence or an absence of the label in the test zone of the test strip.
8. The method of claim 7 , wherein the step of detecting the label comprises using colorimetry.
9. The method 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.
10. The method of claim 6 , further comprising the step of applying the sample to a sample application zone on the test strip, wherein the sample is unlysed when it is applied to the test strip and wherein the lysis agent is located on the test strip.
11. The method of claim 1 , wherein the step of partially opening the target nucleic acid includes an assay condition 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; and
d) any combination of a) through c).
12. The method of claim 1 , wherein the chromatographic test strip comprises a lysis zone comprising at least one lysis or denaturing agent that partially opens the target double stranded nucleic acid into the first single strand and the second single strand.
13. A method of detecting at least one target double stranded 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 assay condition that partially opens the target double stranded nucleic acid into a first single strand and a second single strand;
b) exposing the sample to at least one first complex located on the chromatographic test strip, the 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
c) exposing the sample to at least one second complex located on the chromatographic test strip, the 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 the target nucleic acid;
wherein, when the target double stranded 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 a test zone of the chromatographic test strip at a completion of the assay.
14. The method of claim 13 , wherein the assay condition in step a) comprises a lysis agent or denaturant that partially opens the target double stranded nucleic acid into the first single strand and the second single strand.
15. The method of claim 14 , wherein the lysis agent is added to the sample before the sample is applied to the test strip.
16. The method of claim 14 , wherein the chromatographic test strip comprises a lysis zone comprising the lysis or denaturing agent that partially opens the target double stranded nucleic acid to have the first single strand portion and the second single strand portion in step a).
17. The method of claim 13 , wherein the step of partially opening the target nucleic acid includes an assay condition 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; and
d) any combination of a) through c).
18. The method of claim 1 , wherein the step of partially opening the target nucleic acid includes 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.
19. A method of detecting at least one target double stranded 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 assay condition that partially opens the target double stranded nucleic acid into a first single strand and a second single strand;
b) exposing the sample to at least one first complex located on the chromatographic test strip, the 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
c) exposing the sample to at least one second complex located on the chromatographic test strip, the 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;
wherein, when the target double stranded 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 a test zone of the chromatographic test strip at a completion of the assay.
20. The method of claim 19 , wherein the assay condition in step a) comprises a lysis agent or denaturant that partially opens the target double stranded nucleic acid into the first single strand and the second single strand.
21. The method of claim 20 , wherein the lysis agent is added to the sample before the sample is applied to the test strip.
22. The method of claim 20 , wherein the chromatographic test strip comprises a lysis zone comprising the lysis or denaturing agent that partially opens the target double stranded nucleic acid into the first single strand and the second single strand in step a).
23. The method of claim 19 , wherein the step of partially opening the target nucleic acid includes an assay condition 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; and
d) any combination of a) through c).
24. The method of claim 1 , wherein the sample is run on the assay without an amplification step to enhance assay sensitivity for the target nucleic acid.
25. The method of claim 13 , wherein the sample is run on the assay without an amplification step to enhance assay sensitivity for the target nucleic acid.
26. The method of claim 19 , wherein the sample is run on the assay without an amplification step to enhance assay sensitivity for the target nucleic acid.
27. The method of claim 1 , wherein the sample is from a body fluid selected from the group consisting of:
a) vaginal fluid; and
b) penile fluid.
28. The method of claim 1 , wherein the sample is a sample selected from the group consisting of:
a) a sample of tissue;
b) a sample of cerebrospinal fluid; and
c) a sample of skin ulcerations.
29. The method of claim 1 , wherein the sample is from a body fluid selected from the group consisting of:
a) exudates; and
b) secretions.
30. The method of claim 1 , wherein the sample is from a body fluid selected from the group consisting of:
a) blood; and
b) urine.
31. The method of claim 7 , wherein the step of detecting the label comprises using a method selected from the group consisting of fluorescence and chemiluminescence.
32. The method of claim 7 , wherein the step of detecting the label comprises using a method selected from the group consisting of radioactivity, gravimetry, and magnetism.
33. The method of claim 7 , wherein the step of detecting the label comprises using an enzymatic activity method.
34. The method of claim 7 , wherein the step of detecting the label comprises using a method selected from the group consisting of X-ray diffraction and X-ray absorption.