LATERAL FLOW ASSAYS

• US 20110086359A1
• Filed: 12/01/2010
• Published: 04/14/2011
• Est. Priority Date: 06/10/2008
• Status: Abandoned Application

• Current Assignee: Rapid Pathogen Screening, Inc
• Original Assignee: Rapid Pathogen Screening, Inc
• Inventors: Sambursky, Robert P., VanDine, Robert W., Babu, Uma Mahesh
• Application Number:
  US12/957,683
• Publication Number:
  US 20110086359A1
• 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 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Assays and methods including mobile tagged single stranded nucleic acid reagents pre-loaded on an analysis device, which are preferably tagged, but not labeled and are complementary to a strand (preferably the anti-sense strand in double stranded DNA targets) of the target nucleic acid. The assay also includes a running buffer that includes a dye or other detectable label that nonspecifically binds only to double stranded nucleic acids. In addition, the analysis device includes a detection zone including one or more test zones that have an immobilized tag that binds to the tag on the mobile nucleic acid reagent.

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 mobile tagged nucleic acid reagent comprising a plurality of nucleic acid sequences complementary to a first portion of a sequence of the target nucleic acid and at least one tag portion comprising a first tag, wherein the mobile tagged nucleic acid reagent does not include a detectable label and wherein the mobile tagged nucleic acid reagent is loaded in a reagent zone; and

at least one second immobilized tag that binds to the first tag, wherein the immobilized tag is immobilized in a test zone on the test strip; and

wherein the sample, the mobile tagged nucleic acid reagent, and the immobilized tag are all loaded on the test strip at locations such that the sample encounters the mobile tagged nucleic acid reagent and the immobilized tag while running an assay to detect the target nucleic acid in the sample.

2. The test strip of claim 1, further comprising at least one lysis agent, wherein the lysis agent is loaded in a lysis zone located such that the lysis agent lyses the sample and a plurality of single strands of the target nucleic acid are accessible to the mobile tagged nucleic acid reagent at some point during the assay when the sample encounters the mobile tagged nucleic acid reagent.

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

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

5. The test strip of claim 1, wherein the detection zone further comprises a control zone located beyond the test zone.

6. The test strip of claim 1, further comprising at least one label that nonspecifically binds double stranded nucleic acids.

7. The test strip of claim 6, wherein the label is selected from the group consisting of SYBR®

green dye, PicoGreen®

reagents and ethidium bromide.

8. The test strip of claim 6, further comprising a running buffer, wherein the label is in the running buffer.

9. The test strip of claim 1, wherein the mobile tagged nucleic acid reagents comprises peptide nucleic acid (PNA) sequences.

10. The test strip of claim 1, wherein the tag and the immobilized tag comprise a pair selected from the group consisting of:

a) a first biotin tag and a second immobilized tag selected from the group consisting of avidin, neutravidin and streptavidin;

b) a first tag selected from the group consisting of avidin, neutravidin and streptavidin and a second biotin immobilized tag;

c) a first lectin tag and a second glycosyl moiety immobilized tag; and

d) a first glyosyl moiety tag and a second lectin immobilized tag.

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

12. The test strip of claim 1, wherein the target nucleic acid is not amplified.

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 mobile tagged nucleic acid reagent comprising a plurality of nucleic acids complementary to a portion of a sequence of the target nucleic acid and a first tag but not a detectable label; and

b) exposing the sample to a detectable label, wherein the label nonspecifically binds to double stranded nucleic acids;

wherein, when the target nucleic acid is present in the sample, the mobile tagged nucleic acid reagent binds to the target nucleic acid sequence and the first tag of the mobile tagged nucleic acid reagent binds to a second immobilized tag in a test zone of the assay at a completion of the assay.

14. The method of claim 13, wherein the detectable label is in a running buffer.

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

16. The method of claim 13, wherein the mobile tagged nucleic acid reagents comprises peptide nucleic acid (PNA) sequences.

17. The method of claim 13, wherein the label is selected from the group consisting of SYBR®

green dye, PicoGreen®

reagents and ethidium bromide.

18. The method of claim 13, wherein the first tag and the second immobilized tag comprise a pair selected from the group consisting of:

a) a first biotin tag and a second immobilized tag selected from the group consisting of avidin, neutravidin and streptavidin;

b) a first tag selected from the group consisting of avidin, neutravidin and streptavidin and a second biotin immobilized tag;

c) a first lectin tag and a second glycosyl moiety immobilized tag; and

d) a first glyosyl moiety tag and a second lectin immobilized tag.

19. 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) cerebrospinal fluid;

f) vaginal fluid;

g) penile fluid;

h) nasopharygeal secretions;

i) skin ulcerations;

j) exudates;

k) secretions;

l) blood;

m) inanimate hard surfaces;

n) food sources;

o) environmental water;

p) soil; and

q) urine.

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

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

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

visual detection, phosphorescence, fluorescence, chemiluminescence, radioactivity, colorimetry, gravimetry, X-ray diffraction, X-ray absorption, magnetism, fluorescent resonant emissions, and enzymatic activity.

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

24. The method of claim 13, further comprising the step of exposing the mobile tagged nucleic acid reagent to a control zone comprising a plurality of immobilized nucleic acids complementary to a portion of a sequence of the mobile tagged nucleic acid reagent;

wherein the mobile tagged nucleic acid reagent binds to the immobilized nucleic acids to form a plurality of double stranded nucleic acids such that the detectable label binds to the double stranded nucleic acids.

25. The method of claim 13, wherein the target nucleic acid is not amplified.

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

a test strip 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 mobile tagged nucleic acid reagent comprising a plurality of nucleic acids complementary to a first portion of a sequence of the target nucleic acid and at least one tag portion but not including a label, wherein the mobile tagged nucleic acid reagent is loaded in a reagent zone;

at least one lysis agent loaded in a lysis zone wherein the lysis agent processes the sample such that the target nucleic acid can bind to the mobile tagged nucleic acid reagent when the target nucleic acid encounters the mobile tagged nucleic acid reagent; and

at least one immobilized tag that binds to the tag portion of the mobile tagged nucleic acid reagent, wherein the immobilized tag is immobilized in a test zone of an assay; and

a running buffer comprising at least one detectable label that nonspecifically binds to double stranded nucleic acids.

27. The test kit of claim 26, wherein the sample, the mobile tagged nucleic acid reagent, the lysis agent and the immobilized tag are all loaded on the test strip at locations such that:

if the target nucleic acid is present in the sample, the target nucleic acid binds to the mobile tagged nucleic acid reagent to form a double stranded nucleic acid complex;

the double stranded nucleic acid complex binds to the label in the running buffer to form a labeled duplex complex; and

the immobilized tag binds to the tag portion on the mobile tagged nucleic acid reagent of the labeled duplex complex while running an assay to detect the target nucleic acid in the sample.