Amplicon Rescue Multiplex Polymerase Chain Reaction for Amplification of Multiple Targets

Abstract

Disclosed is a method for amplifying and detecting polynucleotides which can provide sensitive, specific detection of multiple targets from a clinical specimen within a relatively short time.

1 . A method comprising
 amplifying one or more target nucleic acids using high-concentration target-specific primers in a first amplification reaction, thereby producing at least one nucleic acid amplicon containing at least one common primer binding site;   rescuing the at least one nucleic acid amplicon; and   amplifying the at least one nucleic acid amplicon in a second amplification reaction using at least one common primer which binds to the at least one common primer binding site.   
     
     
         2 . A method as in  claim 1  wherein the high-concentration target-specific primers are nested primers. 
     
     
         3 . A method as in  claim 1  wherein the one or more target nucleic acids are chosen from among the group consisting of viral, bacterial, and fungal nucleic acids. 
     
     
         4 . A method as in  claim 1  wherein the one or more target nucleic acids are obtained from a human clinical sample. 
     
     
         5 . A method as in  claim 1  wherein the one or more target nucleic acids are obtained from a clinical sample from an animal. 
     
     
         6 . A method as in  claim 1  wherein the at least one common primer is chosen from among the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and combinations thereof. 
     
     
         7 . A method as in  claim 1  wherein the step of rescuing the at least one nucleic acid amplicon further comprises taking a small sampling from a completed amplification in a first reaction system to provide amplicons for a second amplification in a second reaction system.