AMPLICON RESCUE MULTIPLEX POLYMERASE CHAIN REACTION FOR AMPLIFICATION OF MULTIPLE TARGETS
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.