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

• US 6,280,930 B1
• Filed: 04/25/1997
• Issued: 08/28/2001
• Est. Priority Date: 03/11/1996
• Status: Expired due to Term

• Current Assignee: Johnson & Johnson Clinical Diagnostics Inc. (Johnson & Johnson)
• Original Assignee: Johnson & Johnson Clinical Diagnostics Inc. (Johnson & Johnson)
• Inventors: Backus, John W., Kramer, Marcia L., Falvo, Joseph
• Primary Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/845,739
• Time in Patent Office: 1,586 Days
• Field of Search: 435/6, 435/91.2, 435/91.21
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2527/101 Temperature

Abstract

The present invention relates to a method for amplifying and detecting a target nucleic acid. The method comprising contacting a sample suspected of containing the target nucleic acid with a thermostable DNA polymerase and two primers that are substantially complementary to the target nucleic acid, under conditions such that the target nucleic acid is amplified. The amplified target nucleic acids are then denatured to form single stranded nucleic acids. Following amplification, the sample is subject to a pre-detection incubation step. The sample is incubated for between 1 second and 30 minutes at between 95° C. and 120° C. to inactivate said polymerization agent. Finally, the presence or absence of the amplified target nucleic acids is determined. Preferably, amplification, incubation and detection are carried out in a closed reaction vessel.

Claims

1. 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 at least two oligonucleotides and a thermostable amplification enzyme, wherein said oligonucleotides are substantially complementary to a portion of 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.

2. The method according to claim 1, wherein four oligonucleotides and a thermostable DNA ligase are used.

3. 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.

4. The method according to claim 3, wherein said post amplification incubation step is carried out for between 2 minutes to 10 minutes at between 100°

C. to 110°

C.

5. The method according to claim 3, wherein said post amplification incubation step is carried out for between 2 minutes to 5 minutes at about 103°

C.

6. The method of claim 3, wherein said target nucleic acid is DNA or RNA.

7. The method of claim 6, wherein said target nucleic acid is DNA.

8. The method of claim 6, wherein said target nucleic acid is RNA.

9. The method of claim 3, wherein said nucleoside triphosphates are deoxyribonucleoside triphosphates.

10. The method of claim 9, wherein said deoxyribonucleoside triphosphates are dATP, dCTP, dGTP, and dTTP.

11. The method of claim 3, wherein said thermostable DNA polymerase is selected from the group consisting of thermus aquaticus polymerase, therimus thermophilus polymerase, and Thermococcus litoralis polymerase.

12. The method of claim 3, wherein at least one of said primers is labeled.

13. The method of claim 3, wherein said primers are labeled.

14. The method of claim 12, wherein at least one of said primers is labeled with a specific binding ligand.

15. The method of claim 14, wherein said specific binding ligand is biotin.

16. The method of claim 3, wherein said amplified target nucleic acids are detected using a labeled probe that can hybridize with one of the one or more target nucleic acids.

17. The method of claim 16, wherein said labeled probe is attached to a solid support.

18. The method of claim 3, wherein at least one of said primers are labeled with a specific binding moiety and said amplified target nucleic acids are detected using a probe that can hybridize with one of the one or more target nucleic acids.

19. The method of claim 18, wherein said probe is attached to a solid support.

20. 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).

21. The method of claim 20, wherein said biotinylated amplified target nucleic acids are detected by contacting them with an avidin-peroxidase conjugate, followed by reaction of peroxidase, in the presence of an oxidant, with either:

luminol to produce a detectable chemiluminescent signal, or a leuco dye to produce a detectable calorimetric signal.