Direct nucleic acid analysis
Methods and apparatus are described for nucleic acid analysis of swab samples without the need for purification.
1. A method comprising steps of:
obtaining a swab sample comprising nucleic acid from a source;
contacting the swab sample with a nucleic acid amplification reagent in a reaction vessel, wherein the swab sample is directly contacted with the nucleic acid amplification reagent without any intervening steps, wherein the nucleic acid amplification reagent comprises:
a) a DNA polymerase at a concentration of at least 1.0 U/reaction;
b) a primer at a concentration of at least 0.2 μM;
c) a probe at a concentration of at least 0.2 μM; and
performing a nucleic acid amplification reaction on the swab sample.
2. The method of claim 1 , wherein the DNA polymerase is at a concentration of at least 2.0 U/reaction.
3. The method of claim 1 , further comprising the step of, determining whether an amplification product was produced as a result of the nucleic acid amplification reaction.
4. The method of claim 1 , wherein the nucleic acid amplification reaction is performed within 120 minutes of contacting the swab sample with the nucleic acid amplification reagents.
5. The method of claim 1 , wherein the nucleic acid amplification reaction is performed within 60 minutes of contacting the swab sample with the nucleic acid amplification reagents.
6. The method of claim 1 , wherein the nucleic acid amplification reaction is performed within 30 minutes of contacting the swab sample with the nucleic acid amplification reagents.
7. The method of claim 1 , wherein the nucleic acid amplification reaction is performed within 15 minutes of contacting the swab sample with the nucleic acid amplification reagents.
8. The method of claim 1 , wherein the nucleic acid amplification reaction is performed within 10 minutes of contacting the swab sample with the nucleic acid amplification reagents.
9. The method of claim 1 , wherein the nucleic acid amplification reaction is performed within 5 minutes of contacting the swab sample with the nucleic acid amplification reagents.
10. The method of claim 1 , wherein the nucleic acid amplification reaction is performed within 1 minute of contacting the swab sample with the nucleic acid amplification reagents.
11. The method of claim 1 , wherein the step of performing a nucleic acid amplification reaction on the swab sample comprises an initial heat denaturation step of 15 minutes or less.
12. The method of claim 1 , wherein the step of performing a nucleic acid amplification reaction on the swab sample comprises an initial heat denaturation step of 5 minutes or less.
13. The method of claim 1 , wherein the step of performing a nucleic acid amplification reaction on the swab sample comprises an initial heat denaturation step of 3 minutes or less.
14. The method of claim 1 , wherein the step of performing a nucleic acid amplification reaction on the swab sample comprises an initial heat denaturation step of 1 minute or less.
15. The method of claim 1 , wherein the step of obtaining comprises collecting the swab sample.
16. The method of claim 15 , wherein the swab sample is collected from a mammal.
17. The method of claim 16 , wherein the mammal is a human.
18. The method of claim 16 , wherein the swab sample is collected from an open body cavity.
19. The method of claim 16 , wherein the swab sample is collected from a body surface.
20. The method of claim 16 , wherein the swab sample is a buccal sample.
21. The method of claim 16 , wherein the swab sample is collected from the group consisting of the palm of a hand, inside the folds of the pinna of an ear, an armpit, inside a nasal cavity and combinations thereof.
22. The method of claim 15 , wherein the swab sample is collected from a foodstuff.
23. The method of claim 22 , wherein the foodstuff is raw.
24. The method of claim 22 , wherein the foodstuff is a fruit, a vegetable, a meat, a fish, or a shellfish.
25. The method of claim 24 , wherein the meat is pork, beef, chicken or lamb.
26. The method of claim 15 , wherein the reaction vessel comprises a removable cap and the cap is used to collect the swab sample.
27. The method of claim 26 , wherein a means for holding the cap is used when collecting the swab sample.
28. The method of claim 26 , wherein the cap is contacted with the inside of a cheek.
29. The method of claim 26 , wherein the cap is contacted with a tongue.
30. The method of claim 26 , wherein the cap is contacted with a body region selected from the group consisting of the palm of a hand, the inside of the folds of the pinna of an ear, an armpit, the inside of a nasal cavity and combinations thereof.
31. The method of claim 26 , wherein the cap is contacted with a foodstuff.
32. The method of claim 31 , wherein the foodstuff is raw.
33. The method of claim 31 , wherein the foodstuff is selected from the group consisting of a fruit, a vegetable, a meat, a fish, a shellfish and combinations thereof.
34. The method of claim 33 , wherein the meat is pork, beef, chicken or lamb.
35. The method of claim 15 , wherein the swab sample is collected from a source and the method is repeated with at least one additional swab sample from the same source.
36. The method of claim 1 , wherein the nucleic acid amplification reaction is performed in a reaction vessel having a removable cap and the cap is used to collect the swab sample.
37. The method of claim 36 , wherein the reaction vessel comprises the nucleic acid amplification reagent and the cap is used to seal the reaction vessel after the swab sample has been collected.
38. The method of claim 36 , wherein a means for holding the cap is used when collecting the swab sample.
39. The method according to claim 1 , wherein contacting the nucleic acid amplification reagent with the swab sample comprises agitating the container.
40. The method according to claim 1 , wherein the nucleic acid amplification reagent is not modified to resist inhibitors.
41. A method comprising the steps of:
(a) obtaining a swab sample comprising nucleic acid from a source; and
(b) contacting the swab sample with a nucleic acid amplification reagent in a reaction vessel without any intervening steps; and
(c) performing a nucleic acid amplification reaction on the swab sample, wherein the nucleic acid amplification reaction is performed within 60 minutes of contacting the swab sample with the nucleic acid amplification reagents.
42. The method of claim 2 , wherein the primer is at a concentration of at least 0.3 μM; and the probe is at a concentration of at least 0.4 μM.
43. The method of claim 1 , wherein the primer is at a concentration of at least 0.5 μM; and the probe is at a concentration of at least 0.7 μM.
44. The method of claim 2 , wherein the primer is at a concentration of at least 0.3 μM; and the probe is at a concentration of at least 0.4 μM.
45. The method of claim 2 , wherein the primer is at a concentration of at least 0.5 μM; and the probe is at a concentration of at least 0.7 μM.
46. The method of claim 38 , wherein the cap is a solid plug cap which comprises:
a) a tip that is capable of protruding into and mating with the reaction vessel; and
b) an end opposing the tip that is capable of mating with the means for holding the cap, wherein the solid cap is arranged and constructed to sealingly nest within an upper cylindrical portion of the reaction vessel and partially within a lower frustoconical portion of the reaction vessel.