Method of isolating nucleic acid from specimens in liquid-based cytology preservatives containing formaldehyde

Abstract

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.

What is claimed is: 
     
       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. 
     
     
       26. The method of claim 1, wherein the incubating temperature is in the range of 80° C. to 100° C. 
     
     
       27. The method of claim 26, wherein the nucleic acid isolated is RNA. 
     
     
       28. The method of claim 1, wherein the incubating temperature is at least 80° C. 
     
     
       29. The method of claim 28, wherein the nucleic acid isolated is RNA. 
     
     
       30. The method of claim 1, wherein the protease enzyme is proteinase-K. 
     
     
       31. The method of claim 30, wherein the incubating temperature is in the range of 80° C. to 100° C. 
     
     
       32. The method of claim 31, wherein the nucleic acid isolated is RNA. 
     
     
       33. The method of claim 1, wherein step (a) is performed using a nucleic acid processing system comprising (i) a programmable controller, (ii) a pipetting device in communication with the programmable controller, (iii) a first holder for a reaction vial, and (iv) a second holder for a reagent vial, and wherein the programmable controller is configured by software instructions to cause the pipetting device to transfer an aliquot of liquid from the reagent vial to the reaction vial. 
     
     
       34. The method of claim 33, wherein the reaction vial contains the specimen before the pipetting device transfers an aliquot of liquid from the reagent vial to the reaction vial. 
     
     
       35. A method of isolating RNA from 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 hydrazine- or hydrazide-containing formaldehyde scavenger;   (b) incubating the reaction mixture at a temperature in the range of 80° C. to 100° C. for a period of time sufficient to reverse chemical modifications of RNA that may be contained in the specimen by formaldehyde in the liquid-based cytology preservative; and   (c) isolating RNA from the reaction mixture after the incubating step.   
     
     
       36. The method of claim 35, wherein the protease enzyme is proteinase-K. 
     
     
       37. The method of claim 35, wherein the formaldehyde scavenger is succinic acid dihydrazide. 
     
     
       38. A system for processing nucleic acid containing samples preserved in a liquid-based cytology preservative that comprises formaldehyde, the system comprising:
 a programmable controller;   a pipetting device in communication with the programmable controller;   a first holder for a reaction vial;   a second holder for a reagent vial; and   a heating element;   wherein the programmable controller is configured by software instructions to cause the pipetting device to transfer an aliquot of liquid from the reagent vial to the reaction vial when the reagent vial contains a solution comprising a hydrazine- or hydrazide-containing formaldehyde scavenger; proteinase K, EDTA, and a pH buffer, and   wherein the programmable controller is configured by software instructions to cause the heating element to heat the reaction vial to a temperature in the range of 80° C. to 100° C.   
     
     
       39. The system of claim 38, wherein the programmable controller is configured by software instructions to cause the heating element to heat the reaction vial for a time period of 15 minutes to 30 minutes.