Method of isolating nucleic acid from specimens in liquid-based cytology preservatives containing formaldehyde
Method, composition, kit and system for isolating amplifiable nucleic acid from specimens preserved in a liquid-based cytology preservative that contains formaldehyde. The technique relies on the use of 2-imidazolidone and a protease enzyme, such as proteinase K, at elevated temperatures. Advantageously, RNA can be isolated and used as a template in nucleic acid amplification reactions.
1. A method of isolating a nucleic acid from processing a specimen that includes a clinical sample disposed in a liquid-based cytology preservative that comprises formaldehyde, the method comprising the steps of:
(a) combining the specimen with a protease enzyme and a formaldehyde scavenger that is 2-imidazolidone to create a reaction mixture;
(b) incubating the reaction mixture at an elevated temperature for a period of time sufficient to reverse chemical modifications of nucleic acid that may be contained in the specimen by formaldehyde in the liquid-based cytology preservative; and
(c) isolating a nucleic acid from the reaction mixture after the incubating step.
2. The method of claim 1 , wherein the reversing releases nucleic acids in the specimen from formaldehyde-induced crosslinking to polypeptides in the specimen.
3. The method of claim 2 , wherein the protease enzyme frees the nucleic acids from the formaldehyde-induced crosslinking.
4. The method of claim 1 , wherein the clinical sample has been disposed in the liquid-based cytology preservative for from about 7 to about 120 days before performing step (a).
5. The method of claim 1 , wherein the incubating step is for a period of time no greater than 30 minutes.
6. The method of claim 1 , wherein at least 90% of the chemical modifications of the nucleic acid molecules in the specimen are reversed after the incubating step.
7. The method of claim 1 , wherein final concentration of the 2-imidazolidone before the incubation step is from about 1 to about 5 fold by moles greater than the final maximum concentration of the formaldehyde.
8. The method of claim 7 , wherein the protease enzyme is proteinase-K present at a concentration of from about 4.3 to about 43 U/ml.
9. The method of claim 1 , wherein the temperature of the incubating step is about 90° C.
10. The method of claim 1 , wherein the protease enzyme and formaldehyde scavenger are combined simultaneously with the specimen.
11. The method of claim 1 , wherein the protease enzyme is combined with the specimen before formaldehyde scavenger.
12. The method of claim 1 , wherein the formaldehyde scavenger is combined with the specimen before the protease enzyme.
13. The method of claim 1 , wherein the clinical sample comprises RNA.
14. The method of claim 13 , wherein the isolated nucleic acid is RNA.
15. The method of claim 1 , wherein the nucleic acid is isolated in step (c) by a target capture assay with a target capture probe hybridizing to the nucleic acid to be isolated and to an immobilized probe that is immobilized to a solid support.
16. The method of claim 13 , wherein the isolated nucleic acid is human papillomavirus (HPV) RNA target nucleic acid.
17. The method of claim 1 , wherein the specimen is a cervical cell clinical sample disposed in a liquid-based cytology preservative.
18. The method of claim 1 , wherein the protease enzyme is proteinase-K present at a concentration of from about 4.3 to about 43 U/ml.
19. The method of claim 15 , wherein the solid support comprises a magnetic bead.
20. The method of claim 1 , wherein the incubating step is for a period of time between about 5 minutes and about 30 minutes.
21. The method of claim 1 , wherein the incubating step is for a period of time between about 5 minutes and about 15 minutes.
22. The method of claim 1 , wherein the incubating step is for a period of time no greater than 15 minutes.
23. The method of claim 1 , wherein the temperature of the incubating step is from about 91° C. to about 95° C.
24. The method of claim 5 , wherein the temperature of the incubating step is from about 60° C. to about 100° C.
25. The method of claim 1 , wherein the temperature of the incubating step is from about 85° C. to about 95° C.