US 2014/0349299 A1Published application

Methods And Systems For Quantitative Fluorescence-Based Detection Of Molecules And Proteins

AssigneeSRC, Inc.

InventorsJeffrey H. Mills, Huda S. Suliman, Stacey A. Massulik, Frances L. Stites, Timothy F. Moshier, Kenton A. Doctor, Lisa H. Chamberlin, Justin P. Andrews, Stephen R. Houghton

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Abstract

A kit and method for detection of a target in a sample. An assay mixture provided in the kit and used in the method includes a first probe with a first antibody recognizing a first epitope of the target and conjugated to an RNA oligonucleotide; a second probe with a second antibody recognizing a second epitope of the target and conjugated to a DNA oligonucleotide; a reverse primer with a first region complimentary to the RNA oligonucleotide and a second region complimentary to the DNA oligonucleotide; and a reverse transcriptase that creates a DNA transcription product from the RNA oligonucleotide using the reverse primer only if the RNA oligonucleotide and the DNA oligonucleotide are in close proximity. If the target is present in the sample, the reverse primer binds the RNA oligonucleotide and the DNA oligonucleotide to bring the RNA oligonucleotide and the DNA oligonucleotide in close proximity.

Claims20

Dependent claims

A method for detection of a target in a sample, the method comprising the steps of;

providing an assay mixture comprising:
(i) a first probe comprising a first antibody recognizing a first epitope of the target, the first antibody conjugated to an RNA oligonucleotide;
(ii) a second probe comprising a second antibody recognizing a second epitope of the target, the second antibody conjugated to a DNA oligonucleotide;
(iii) a reverse primer, wherein the reverse primer comprises a first region complimentary to the RNA oligonucleotide, and a second region complimentary to said DNA oligonucleotide;
and (iv) a reverse transcriptase, wherein said reverse transcriptase creates a DNA transcription product from the RNA oligonucleotide using said reverse primer only if said RNA oligonucleotide and said DNA oligonucleotide are in close proximity;
adding the sample to the assay mixture to create a reaction mixture;
incubating the reaction mixture for a predetermined period of time under conditions suitable for reverse transcription by said reverse transcriptase;
and
analyzing said reaction mixture for the presence of said DNA transcription product of the RNA oligonucleotide;
wherein when the target is present in the sample, and the first antibody is interacting with the first epitope, and the second antibody is interacting with the second epitope, the first region of the reverse primer binds the RNA oligonucleotide and the second region of the reverse primer binds the DNA oligonucleotide to bring the RNA oligonucleotide and the DNA oligonucleotide in close proximity;
wherein the presence of said DNA transcription product indicates the presence of the target in said sample.

8 more elements

A method for detection of a target in a sample, the method comprising the steps of;

providing an assay mixture comprising:
(i) a first probe comprising a first antibody recognizing a first epitope of the target, the first antibody conjugated to the 5′ end of an RNA oligonucleotide;
(ii) a second probe comprising a second antibody recognizing a second epitope of the target, the second antibody conjugated to the 3′ end of a DNA oligonucleotide;
(iii) a reverse primer, wherein the reverse primer comprises a first region complimentary to 3′ end of the RNA oligonucleotide, and a second region complimentary to the 5′ end of said DNA oligonucleotide;
(iv) a reverse transcriptase, wherein said reverse transcriptase creates a DNA transcription product from the RNA oligonucleotide using said reverse primer only if said RNA oligonucleotide and said DNA oligonucleotide are in close proximity;
(v) a DNA polymerase;
(vi) a forward primer complimentary to at least a portion of a DNA transcription product;
and (vii) a detection probe comprising an oligonucleotide complimentary to at least a portion of said DNA transcription product, and further comprising a fluorophore at one end of the oligonucleotide and a quencher at the opposite end of the oligonucleotide;
adding the sample to the assay mixture to create a reaction mixture;
incubating the reaction mixture for a predetermined period of time under conditions suitable for reverse transcription by said reverse transcriptase;
inactivating said reverse transcriptase;
and
incubating the reaction mixture for a predetermined period of time under conditions suitable for qPCR;
wherein when the target is present in the sample, and the first antibody is interacting with the first epitope, and the second antibody is interacting with the second epitope, the first region of the reverse primer binds the RNA oligonucleotide and the second region of the reverse primer binds the DNA oligonucleotide to bring the RNA oligonucleotide and the DNA oligonucleotide in close proximity.

11 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/283,294Filed 21 May 2014
Priority: 21 May 2013
Published: 27 Nov 2014
Term ends: Not estimatedThe source records don’t support an estimate.

Classification4 codes

C12Q 1/6804
Involving nucleic acids: nucleic acid analysis using immunogens
G01N 2458/10
Labels used in chemical analysis of biological material: oligonucleotides as tagging agents for labelling antibodies
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms
G01N
Investigating or analysing materials by determining their chemical or physical properties