Rapid nucleic acid assay

Abstract

This invention relates to an improved process for detecting and quantifying a desired nucleic acid sequence. The process involves synthesizing single stranded RNA, single stranded DNA, double-stranded DNA followed by detection using an electrochemiluminescent labeled binding species.

1 - 20 . (canceled)  
     
     
         21 . A process for detecting a specific nucleic-acid sequence comprising: 
 (a) forming a first composition comprising 
 (i) a sample,  
 (ii) a first oligonucleotide primer which comprises a promoter sequence,  
 (iii) a second oligonucleotide primer,  
 (iv) a DNA-directed RNA polymerase,  
 (v) an RNA-directed DNA polymerase,  
 (vi) a DNA-directed DNA polymerase, and  
 (vii) a ribonuclease that hydrolyzes RNA of an RNA-DNA hybrid without hydrolyzing single or double-stranded RNA or DNA;  
   (b) incubating the first composition for a sufficient time to amplify said specific nucleic acid sequence to form a mixture comprising an amplified nucleic acid sequence;    (c) forming a second composition by adding to a sample of said mixture the following reagents 
 (i) at least one detection probe sequence which specifically hybridizes to said amplified nucleic-acid sequence, said detection probe sequence being labeled with an electrochemiluminescent (ECL) species,  
 (ii) at least one capture probe sequence which specifically hybridizes to said amplified nucleic-acid sequence, said capture probe sequence being covalently attached to a solid phase;  
   (d) incubating said second composition for a time sufficient to allow hybridization of said probes to said amplified nucleic-acid sequence to form a solid phase-bound hybridization complex; and    (e) detecting said specific nucleic acid sequence by measuring electrochemiluminescence from said solid phase-bound hybridization complex.    
     
     
         22 . The process of  claim 21 , wherein said amplified nucleic-acid sequence is the anti-sense copy of the specific nucleic-acid sequence and wherein said amplification of said specific nucleic-acid sequence is carried out under conditions which permit 
 (i) said second oligonucleotide primer to hybridize to an RNA template which comprises the specific nucleic-acid sequence or an anti-sense copy of the specific nucleic-acid sequence,    (ii) said RNA-directed DNA polymerase to utilize said RNA template to synthesize a DNA template by extension of said second oligonucleotide primer and thereby form an RNA-DNA hybrid intermediate,    (iii) said ribonuclease to hydrolyze RNA contained in said RNA-DNA hybrid intermediate,    (iv) said first oligonucleotide primer to hybridize to said DNA template,    (v) said DNA-directed DNA polymerase to utilize said DNA template to synthesize a double-stranded DNA product by extension of said first oligonucleotide primer, said double-stranded DNA product comprising said promoter, and    (vi) said DNA-directed RNA polymerase to recognize said promoter and transcribe said double-stranded DNA product so as to form more copies of said RNA template.    
     
     
         23 . The process of  claim 21 , wherein said ECL species comprises ruthenium-tris-bipyridine.  
     
     
         24 . The process of  claim 21 , wherein said solid phase is a bead.  
     
     
         25 . The process of  claim 24 , wherein said bead is a magnetic bead.  
     
     
         26 . The process of  claim 21 , wherein ECL-labeled nucleotides are added to step (b) in sufficient quantity to produce ECL-labeled amplified target molecules.  
     
     
         27 . The process of  claim 21 , wherein the capture probe or the detection probe is either the first oligonucleotide primer or the second oligonucleotide primer.  
     
     
         28 . The process of  claim 21 , wherein said amplified nucleic-acid sequence is the anti-sense copy of the specific nucleic acid sequence and wherein said amplification of said specific nucleic acid sequence is carried out under conditions which permit 
 (i) said second oligonucleotide primer to hybridize to an RNA template which comprises the specific nucleic acid sequence or an anti-sense copy of the specific nucleic acid sequence,    (ii) said RNA-directed DNA polymerase to utilize said RNA template to synthesize a DNA template by extension of said second oligonucleotide primer and thereby form an RNA-DNA hybrid intermediate,    (iii) said ribonuclease to hydrolyze RNA contained in said RNA-DNA hybrid intermediate,    (iv) said first oligonucleotide primer to hybridize to said DNA template,    (v) said DNA-directed DNA polymerase to utilize said DNA template to synthesize a double-stranded DNA product by extension of said first oligonucleotide primer, said double-stranded DNA product comprising said promoter, and    (vi) said DNA-directed RNA polymerase to recognize said promoter and transcribe said double-stranded DNA product so as to form more copies of said RNA template.    
     
     
         29 . A process for detecting a specific nucleic-acid sequence comprising: 
 (a) forming a first composition comprising 
 (i) a sample,  
 (ii) a first oligonucleotide primer which comprises a promoter sequence,  
 (iii) a second oligonucleotide primer,  
 (iv) a DNA-directed RNA polymerase,  
 (v) an RNA-directed DNA polymerase,  
 (vi) a DNA-directed DNA polymerase, and  
 (vii) a ribonuclease that hydrolyzes RNA of an RNA-DNA hybrid without hydrolyzing single or double-stranded RNA or DNA;  
   (b) incubating the first composition for a sufficient time to amplify said specific nucleic-acid sequence to form a mixture comprising an amplified nucleic-acid sequence;    (c) forming a second composition by adding to a sample of said first composition at least one capture probe sequence which specifically hybridizes to said amplified nucleic-acid sequence, said capture probe sequence being covalently attached to a solid phase to form a solid phase-bound complex and incubating said second composition for a time sufficient to allow hybridization of said capture probe to said amplified nucleic-acid sequence;    (d) forming a third composition by adding to a sample of said second composition at least one detection probe sequence which specifically hybridizes to said amplified nucleic-acid sequence, said detection probe sequence being labeled with an ECL species and incubating said third composition for a time sufficient to allow hybridization of said detection probe to said amplified nucleic-acid sequence; and    (e) detecting said specific nucleic-acid sequence by measuring electrochemiluminescence from said solid phase-bound hybridization complex.    
     
     
         30 . A process for detecting a specific nucleic-acid sequence comprising: 
 (a) forming a first composition comprising 
 (i) a sample,  
 (ii) a first oligonucleotide primer which comprises a promoter sequence,  
 (iii) a second oligonucleotide primer,  
 (iv) a DNA-directed RNA polymerase,  
 (v) an RNA-directed DNA polymerase,  
 (vi) a DNA-directed DNA polymerase, and  
 (vii) a ribonuclease that hydrolyzes RNA of an RNA-DNA hybrid without hydrolyzing single or double-stranded RNA or DNA;  
   (b) incubating the first composition for a sufficient time to amplify said specific nucleic-acid sequence to form a mixture comprising an amplified nucleic-acid sequence;    (c) forming a second composition by adding to a sample of said first composition at least one detection probe sequence which specifically hybridizes to said amplified nucleic-acid sequence, said detection probe sequence being labeled with an ECL species and incubating said second composition for a time sufficient to allow hybridization of said detection probe to said amplified nucleic-acid sequence,    (d) forming a third composition by adding to a sample of said second composition at least one capture probe sequence which specifically hybridizes to said amplified nucleic-acid sequence, said capture probe sequence being covalently attached to a solid phase to form a solid phase-bound hybridization complex and incubating said second composition for a time sufficient to allow hybridization of said capture probe to said amplified nucleic-acid sequence;    (e) incubating said second composition for a time sufficient to allow hybridization of said probes to said amplified nucleic-acid sequence; and    (f) detecting said specific nucleic acid sequence by measuring electrochemiluminescence from said solid phase-bound hybridization complex.    
     
     
         31 . In a cycling DNA/RNA amplification assay involving an initial nucleic acid template and having at least one amplification cycle which results in an amplification reaction mixture wherein the improvement comprises: 
 (a) obtaining at least one sample from the amplification reaction mixture;    (b) adding to said sample the following reagent mixture comprising, 
 (i) at least one probe sequence labeled with an ECL species wherein said probe sequence specifically hybridizes to said initial nucleic acid template;  
 (ii) at least one capture probe sequence which specifically hybridizes to said initial nucleic acid template wherein said capture probe sequence is bound to a solid phase support;  
   (c) providing conditions of temperature and buffer to allow hybridization of the probe sequences to said first nucleic acid template to form a solid phase-bound complex; and then    (d) detecting said solid phase-bound complex using said ECL species.    
     
     
         32 . The cycling DNA/RNA amplification assay of  claim 31  wherein the solid phase is a bead.  
     
     
         33 . The cycling DNA/RNA amplification assay of  claim 32  wherein the bead is a magnetic bead.