LATERAL FLOW DETECTION OF TARGET SEQUENCES

• US 20140170649A1
• Filed: 09/27/2013
• Published: 06/19/2014
• Est. Priority Date: 09/27/2012
• Status: Abandoned Application

• Current Assignee: Bioo Scientific Corporation (Revvity, Inc.)
• Original Assignee: Bioo Scientific Corporation (Revvity, Inc.)
• Inventors: Krebs, Joseph F., Morrison, Paul, Goldrick, Marianna
• Application Number:
  US14/039,908
• Publication Number:
  US 20140170649A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates

Abstract

Provided is a sensitive method to specifically detect nucleic acid sequences using a combination of DNA replication based signal amplification (e.g., PCR) and lateral flow analyte detection. The disclosed method uses a dual-labeled DNA probe complementary to a region of the target DNA sequence. Cleavage of the probe, caused by the presence of the amplified DNA target, is detected using lateral flow methods.

Claims

1. A method for detecting the presence a target DNA sequence in a sample comprising:

amplifying the target DNA sequence by applying polymerase chain reaction (PCR), to the sample, wherein the PCR is performed using a composition comprising;

a thermostable DNA polymerase having 5′

exonuclease activity;

forward and reverse amplification primers;

deoxyribonucleotides; and

a probe oligonucleotide that is complementary to an internal region of the target DNA sequence, wherein the probe oligonucleotide comprises a 5′

modification, the 5′

modification comprising a first moiety capable of associating with a first affinity reagent, and wherein the probe oligonucleotide comprises a 3′

modification, the 3′

modification comprising a second moiety capable of associating with a second affinity reagent;

mixing a portion of the PCR composition after amplification of the target DNA sequence with a mobile reporter agent to form a mobile reporter-PCR composition mixture, wherein said mobile reporter agent comprises the second affinity reagent, wherein the second affinity reagent binds to the second moiety of the probe oligonucleotide;

introducing the mobile reporter-PCR composition mixture into a lateral flow strip under conditions that allow the probe oligonucleotide associated with the mobile reporter agent to migrate along the strip, wherein the probe oligonucleotide associated with the mobile reporter agent is captured by first affinity reagents positioned at a detection position of the lateral flow strip, and wherein the first affinity reagent binds to the first moiety on the probe oligonucleotide to inhibit further lateral flow of the probe oligonucleotide associated with the mobile reporter agent;

analyzing the detection position of the lateral flow strip to determine the intensity of the mobile reporter agent captured at the detection position, wherein the intensity of the mobile reporter agent indicates the presence of the target DNA in the sample.

2. The method of claim 1, wherein the second affinity reagent is a second antibody, and the second moiety is an antigen of the second antibody.

3. The method of claim 1, wherein the first affinity reagent at the detection position of the lateral flow strip is an antibody capable of binding to the first moiety of the probe oligonucleotide, wherein the first moiety is an antigen of the first affinity reagent.

4. The method of claim 1, wherein the second affinity reagent on the mobile reporter agent is streptavidin and the second moiety on the probe oligonucleotide is biotin.

5. The method of claim 1, where the mobile reporter agent is a fluorescent compound.

6. The method of claim 1, where the mobile reporter agent is a colloidal gold particle.

7. The method of claim 1, wherein the lateral flow strip comprises of nitrocellulose.

8. The method of claim 1, wherein the thermostable DNA polymerase is natural or recombinant Taq from Thermus aquaticus.