Rt-late-pcr

Abstract

An assay comprising more than one primer pair and more than one detection probe, a low copy number synthetic amplicon corresponding to each of the primer pairs. The assay can detect and distinguish between various sub-types and strains of an influenza virus using any suitable nucleic acid amplification technique. Related kits and methods are also described.

1 . (canceled) 
     
     
         2 . A reverse transcription-polymerase chain reaction (PCR) method comprising:
 a) adding to a reaction vessel a reaction mixture comprising at least one RNA comprising a target sequence, reverse transcriptase, DNA amplification reagents that include an excess primer and a limiting primer for the target sequence, and DNA polymerase, wherein the concentration of the excess primer is 500-2000 nM and at least five times higher than the concentration of the limiting primer, and wherein the concentration-adjusted melting temperature of the limiting primer to the target sequence is equal to or greater than the concentration-adjusted melting temperature of the excess primer to the target sequence;   b) incubating the reaction mixture at a first temperature to reverse transcribe the target sequence from the RNA utilizing the excess primer and the reverse transcriptase to create a cDNA template for LATE-PCR amplification, said first temperature being sufficiently high to disrupt secondary structure of the RNA but sufficiently low to provide a reverse-transcriptase half-life of at least 2.2 minutes, wherein the effective melting temperature of the excess primer to the RNA is not lower than 2° C. below said first temperature; and   c) thermally cycling the reaction mixture to amplify the cDNA template using the excess primer, the limiting primer and the DNA polymerase to produce both double-stranded amplification product and single-stranded amplification product.   
     
     
         3 . The method of  claim 2  wherein the concentration of the excess primer is at least ten times higher than the concentration of the limiting primer. 
     
     
         4 . The method of  claim 2  wherein the concentration of the excess primer is at least twenty times higher than the concentration of the limiting primer. 
     
     
         5 . The method of  claim 1  wherein the RNA is added to the reaction mixture as at least one lysed cell or at least one lysed virus. 
     
     
         6 . The method of  claim 1  wherein the reverse transcriptase and the DNA polymerase are a single enzyme that has both reverse transcription activity and DNA polymerase activity. 
     
     
         7 . The method of  claim 1  wherein the reaction mixture includes a fluorescently-labeled hybridization probe for detection of the single-stranded amplification product. 
     
     
         8 . The method of  claim 1  wherein said at least one RNA comprises at least two target sequences, and the DNA amplification reagents include an excess primer and a Limiting primer for each target sequence. 
     
     
         9 . The method of  claim 1  wherein said first temperature is in the range of 50° C. to 60° C. 
     
     
         10 . A reaction mixture comprising:
 (a) at least one RNA, wherein said RNA comprises a target sequence;   (b) reverse transcriptase;   (c) DNA amplification reagents including an excess primer and a limiting primer for the target sequence, wherein the concentration of the excess primer is 500-2000 nM and at least five times higher than the concentration of the limiting primer, and wherein the concentration-adjusted melting temperature of the limiting primer to the target sequence is equal to or greater than the concentration-adjusted melting temperature of the excess primer to the target sequence; and   (d) DNA polymerase.   
     
     
         11 . The reaction mixture of  claim 10  wherein the concentration of the excess primer is at least ten times higher than the concentration of the limiting primer. 
     
     
         12 . The reaction mixture of  claim 11  wherein the concentration of the excess primer is at least twenty times higher than the concentration of the limiting primer. 
     
     
         13 . The reaction mixture of  claim 10  wherein the RNA is added to the reaction mixture as at least one lysed cell or at least one lysed virus. 
     
     
         14 . The reaction mixture of  claim 10  wherein the reverse transcriptase and the DNA polymerase are a single enzyme that has both reverse transcription activity and DNA polymerase activity. 
     
     
         15 . The reaction mixture of  claim 10  further comprising a fluorescently-labeled hybridization probe for detection of the single-stranded amplification product. 
     
     
         16 . The reaction mixture of  claim 10  wherein said at least one RNA comprises at least two target sequences, and the DNA amplification reagents include an excess primer and a Limiting primer for each target sequence.