Methods for multiplexing recombinase polymerase amplification

Abstract

This disclosure provides for methods and reagents for rapid multiplex RPA reactions and improved methods for detection of multiplex RPA reaction products. In addition, the disclosure provides new methods for eliminating carryover contamination between RPA processes.

1 - 78 . (canceled) 
     
     
         79 . A method of amplifying and detecting a target nucleic acid, said method comprising:
 (a) amplifying a target nucleic acid by recombinase polymerase amplification to produce an amplified target nucleic acid, wherein the amplified target nucleic acid is a DNA duplex extension product of a labelled amplification primer and a differently labelled opposing primer, wherein said amplification primer and opposing primer are different oligonucleotide primers; and   (b) detecting the amplified target nucleic acid by determining the association of the labelled amplification primer and the labelled opposing primer in a common DNA duplex using a lateral flow strip, wherein antibodies or streptavidin that bind the oligonucleotide labels are used to immobilize and detect the amplified target nucleic acid.   
     
     
         80 . The method of  claim 79 , wherein a first antibody is coupled to visible particles and a second antibody is immobilized on a flow membrane in a line or spot. 
     
     
         81 . The method of  claim 80 , wherein the visible particles are colloidal gold or latex. 
     
     
         82 . The method of  claim 80 , further comprising applying a sample of an amplification reaction comprising the amplified target nucleic acid to a sample pad comprising the first antibody, such that the visible particles become stably associated with the labelled amplification primer, wherein the sample then moves by capillary action up the flow membrane causing the labelled opposing primer to become bound to the immobilized second antibody and the visible particles to accumulate at the line or spot, thus producing a visible signal. 
     
     
         83 . The method of  claim 79 , wherein each of the labelled amplification primer and the labelled opposing primer comprises a label that is an antigen, biotin, a fluorophore, an enzyme, a quencher, an enzyme inhibitor, a radioactive label, a member of a binding pair, a digoexygenin residue, a peptide, or a combination thereof. 
     
     
         84 . The method of  claim 79 , wherein the recombinase polymerase amplification comprises:
 (a) contacting a recombinase agent with a first nucleic acid primer, a second nucleic acid primer, and a third extension blocked primer comprising one or more noncomplementary or modified internal residues to form a first nucleoprotein primer, a second nucleoprotein primer, and a third nucleoprotein primer;   (b) contacting the first nucleoprotein primer and the second nucleoprotein primer to said target nucleic acid to provide a first double stranded structure comprising a first strand of the target nucleic acid and said first nucleoprotein primer at a first portion of said first strand and forming a second double stranded structure comprising a second strand of the target nucleic acid and said second nucleoprotein primer at a second portion of said second strand such that a 3′ end of said first nucleoprotein primer and a 3′ end of said second nucleoprotein primer are oriented toward each other on the same target nucleic acid molecule and a third portion of the target nucleic acid molecule is between the 3′ end of said first nucleoprotein primer and the 3′ end of said second nucleoprotein primer;   (c) extending the 3′ end of said first nucleoprotein primer and the 3′ end of said second nucleoprotein primer with one or more polymerases and dNTPs to generate an amplified target nucleic acid having an internal region;   (d) contacting said amplified target nucleic acid to said third nucleoprotein primer to provide a third double stranded structure at the internal region of said amplified target nucleic acid in the presences of a nuclease; wherein said nuclease specifically cleaves said noncomplementary or modified internal residue only after the formation of said third double stranded structure to form a 3′ extension blocked primer and a 5′ primer;   (e) extending the 3′ end of said 5′ primer with one or more polymerases and dNTPs to generate a second amplified target nucleic acid comprising said first nucleic acid primer and said 5′ primer;   (f) continuing the reaction through repetition of (b) and (e) until a desired amount of the second amplified target nucleic acid is produced;   wherein the first nucleic acid primer and the 5′ primer are both labelled, further wherein the first nucleic acid primer is the labelled amplification primer and the 5′ primer is the labelled opposing primer.   
     
     
         85 . The method of  claim 84 , wherein the nuclease recognizes a base mismatch between the noncomplementary or modified internal residue of the third nucleoprotein primer and said target nucleic acid and cleaves said third nucleoprotein primer at said noncomplementary or modified internal residue. 
     
     
         86 . The method of  claim 84 , wherein said 3′ extension blocked primer comprises a blocked 3′ residue which is resistant to extension by DNA polymerase. 
     
     
         87 . The method of  claim 86 , wherein the blocked 3′ residue comprises a blocking moiety attached to the 3′ or 2′ site of the 3′ residue sugar. 
     
     
         88 . The method of  claim 86 , wherein the blocking moiety is a detectable label. 
     
     
         89 . The method of  claim 88 , wherein the detectable label is a fluorophore, an enzyme, a quencher, an enzyme inhibitor, a radioactive label, a member of a binding pair, or a combination thereof. 
     
     
         90 . The method of  claim 86 , wherein the blocked 3′ residue is a dideoxy nucleotide. 
     
     
         91 . The method of  claim 84 , wherein said method is performed in the presence of 1% to 12% PEG 
     
     
         92 . The method of  claim 84 , wherein the recombinase polymerase amplification is performed at a temperature of between 14° C. and 43° C. 
     
     
         93 . The method of  claim 84 , wherein the recombinase polymerase amplification is performed at a temperature of between 14° C. and 48° C. 
     
     
         94 . The method of  claim 84 , wherein each of the first nucleic acid primer, the second nucleic acid primer, and the third extension blocked primer is 12 to 60 residues in length. 
     
     
         95 . The method of  claim 79 , wherein the method of amplifying and detecting a target nucleic acid is performed a plurality of times for a plurality of different target nucleic acids and each target nucleic acid is detected.