Amplifying and detecting target nucleic acids using a post amplification incubation step

A method of inactivating a thermostable amplification enzyme in a method for amplifying and detecting a target nucleic acid in a closed reaction vessel comprising:

• a) contacting a sample suspected of containing said target nucleic acid with four different nucleoside triphosphates, a thermostable DNA polymerase, and two primers, wherein said primers are substantially complementary to said target nucleic acid, under conditions such that said target nucleic acid is amplifiable:
• b) amplifying said target nucleic acid;
• c) denaturing amplified target nucleic acids to form single stranded nucleic acids;
• d) after the denaturation step and prior to detection, inactivating the thermostable amplification enzyme by incubating said sample for between 2 minutes and 30 minutes at between 97° C. and 120° C.;
• and
• e) detecting the presence or absence of the amplified target nucleic acids, wherein the reaction vessel remains closed during steps (a) through (e), and wherein the inactivating step results in an increase in detection sensitivity over background as compared to the detection sensitivity without the inactivating step.

A method of inactivating a thermostable amplification enzyme in a method for amplifying and detecting a target nucleic acid in a closed reaction vessel comprising:

• a) contacting a sample suspected of containing said target nucleic acid with four different nucleoside triphosphates, a thermostable DNA polymerase, and two primers, wherein at least one of said primers is labeled with biotin and said primers are substantially complementary to said target nucleic acid, under conditions such that said target nucleic acid is amplifiable.
• b) amplifying said target nucleic acid,
• c) denaturing, amplified target nucleic acids to form single stranded nucleic acids;
• d) after the denaturation step and prior to detection, inactivating the thermostable amplification enzyme by incubating said sample for between 2 minutes and 5 minutes at about 103° C.;
• and
• e) detecting(the presence or absence of the biotinylated amplified target nucleic acids by reacting the biotinylated amplified target nucleic acids with an avidin-enzyme conjugate, followed by reaction of the enzyme with a substrate reagent to produce a detectable colorimetric or chemiluminescent signal,
• wherein the reaction vessel remains closed during steps (a) through (e).