Method of detecting the presence of a target nucleic acid via a multiplex amplification reaction using unequal primer concentration

Abstract

A method for evaluating a sample for the presence or absence of multiple virus infections is disclosed. In one embodiment, this method comprises the step of evaluating a biological sample for nucleic acid sequences complementary to nucleotide primers and probes derived from the sequences of human parainfluenza virus 1, 2 and 3, respiratory syncytial virus A and B and influenza virus A and B. In another embodiment, the invention is an improved PCR method.

1 - 51 . (cancelled)  
     
     
         52 . A method of detecting the presence of at least two target nucleic acids in a biological sample comprising the steps of 
 exposing a nucleic acid sample to at least two primer pairs specific for at least two target nucleic acids under conditions suitable for nucleic acid amplification, wherein the nucleic acid sample is a nucleic acid obtained from a biological sample or is a cDNA obtained from the nucleic acid of the biological sample, wherein the primer pairs comprise a 5′ primer and a 3′ primer, wherein the 5′ primer and the 3′ primer are present in unequal concentrations, wherein double stranded amplification product is formed if the nucleic acid sample contains the target nucleic acids and wherein the double-stranded amplification product is present in a greater amount as measured by optical density compared to product formed in a separate control reaction with equal primer concentrations.    
     
     
         53 . The method of  claim 52  wherein the ratio of 5′ to 3′ primer is selected from the group consisting of approximately 50:25, 25:50, 12.5:50 and 12.5:25.  
     
     
         54 . The method of  claim 52  wherein the product is present at least 3.59 times the amount of product produced with equal primer concentration.  
     
     
         55 . A method of detecting the presence of at least two target nucleic acids in a biological sample comprising the steps of exposing a nucleic acid sample to at least two primer pairs specific for at least two target nucleic acids under conditions suitable for nucleic acid amplification, wherein the nucleic acid sample is a nucleic acid obtained from a biological sample or is a cDNA obtained from the nucleic acid of the biological sample, wherein the primer pairs comprise a 5′ primer and a 3′ primer, wherein the 5′ primer and the 3′ primer are present in unequal concentrations, wherein double stranded amplification product is formed if the nucleic acid sample contains the target nucleic acids and wherein the double-stranded amplification product is present in a greater amount as measured by optical density compared to product formed in a separate control reaction with equal primer concentrations, wherein the nucleic acid or cDNA created from the nucleic acid is exposed to at least two primers pairs specific for sequences selected from the group consisting of parainfluenza virus-1, 2 and 3, respiratory syncytial virus A and B and influenza virus A and B sequences.  
     
     
         56 . The method of  claim 55  wherein the product is present at least 3.59 times the amount of product produced with equal primer concentration.  
     
     
         57 . The method of  claim 55  wherein the ratio of 5′ to 3′ primer is selected from the group consisting of approximately 50:25, 25:50, 12.5:50 and 12.5:25.  
     
     
         58 . A method of detecting the presence of at least two target nucleic acids in a biological sample comprising the steps of 
 exposing a nucleic acid sample to at least two primer pairs specific for at least two target nucleic acids under conditions suitable for nucleic acid amplification, wherein the nucleic acid sample is a nucleic acid obtained from a biological sample or is a cDNA obtained from the nucleic acid of the biological sample, wherein the primer pairs comprise a 5′ primer and a 3′ primer, wherein the 5′ primer and the 3′ primer are present in unequal concentrations, wherein double stranded amplification product is formed if the nucleic acid sample contains the target nucleic acids and wherein the double-stranded amplification product is present in a greater amount as measured by optical density compared to product formed in a separate control reaction with equal primer concentrations; and    denaturing the double-stranded product at least once.    
     
     
         59 . The method of  claim 58  wherein the product is present at least 3.59 times the amount of product produced with equal primer concentration.  
     
     
         60 . The method of  claim 58  wherein the ratio of 5′ to 3′ primer is selected from the group consisting of approximately 50:25, 25:50, 12.5:50 and 12.5:25.  
     
     
         61 . A method of detecting the presence of at least two target nucleic acids in a biological sample comprising the steps of 
 exposing a nucleic acid sample to at least two primer pairs specific for at least two target nucleic acids under conditions suitable for nucleic acid amplification, wherein the nucleic acid sample is a nucleic acid obtained from a biological sample or is a cDNA obtained from the nucleic acid of the biological sample, wherein the primer pairs comprise a 5′ primer and a 3′ primer, wherein the 5′ primer and the 3′ primer are present in unequal concentrations, wherein double stranded amplification product is formed if the nucleic acid sample contains the target nucleic acids and wherein the double-stranded amplification product is present in a greater amount as measured by optical density compared to product formed in a separate control reaction with equal primer concentrations, wherein the nucleic acid or cDNA created from the nucleic acid is exposed to at least two primers pairs specific for sequences selected from the group consisting of parainfluenza virus-1, 2 and 3, respiratory syncytial virus A and B and influenza virus A and B sequences and denaturing the double-stranded product.    
     
     
         62 . The method of  claim 61  wherein the product is present at least 3.59 times the amount of product produced with equal primer concentration.  
     
     
         63 . The method of  claim 61  wherein the ratio of 5′ to 3′ primer is selected from the group consisting of approximately 50:25, 25:50, 12.5:50 and 12.5:25.