Method of isolating nucleic acid from specimens in liquid-based cytology preservatives containing formaldehyde
View Patent ↗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 composition of matter, comprising:
(a) 2-imidazolidone
(b) proteinase K
(c) EDTA
(d) a pH buffer.
2. A kit for processing a specimen preserved in a liquid-based cytology preservative comprising formaldehyde, the kit comprising:
(a) a first vial containing a lyophilized proteinase K enzyme;
(b) a second vial containing a reconstitution buffer for reconstituting the lyophilized proteinase K enzyme,
wherein the reconstitution buffer comprises an amount of a pH buffer, an amount of EDTA, and an amount of 2-imidazolidone.
3. A system for processing nucleic acid-containing samples preserved in a liquid-based cytology preservative that comprises formaldehyde, the system components 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 2-imidazolidone, 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 from 65° C. to 95° C.
4. The system of claim 3 , wherein the programmable controller is configured by software instructions to cause the heating element to heat the reaction vial to a temperature from 85° C. to 95° C., or wherein the programmable controller is configured by software instructions to cause the heating element to heat the reaction vial to a temperature from 90° C. to 95° C., or wherein the programmable controller is configured by software instructions to cause the heating element to heat the reaction vial to a temperature from 90° C. to 95° C. for a time period of 15 minutes to 30 minutes.
5. The system of claim 4 , wherein the reagent in the reaction vial comprises 2-imidazolidone.
6. The system of claim 5 , wherein the reagent in the reaction vial comprises a concentration of 42 to 45 U of proteinase K.
7. A reaction mixture for treating a specimen, wherein the specimen is a clinical sample comprising nucleic acids and is disposed in a liquid-based cytology preservative containing formaldehyde, and wherein the reaction mixture comprises proteinase K and 2-imidazolidone.
8. The reaction mixture of claim 7 , wherein the reaction mixture further comprises EDTA.
9. The reaction mixture of claim 7 , wherein the reaction mixture comprises proteinase K, 2-imidazolidone, EDTA, nucleic acids from a clinical sample that is a cervical swab sample, and a pH buffer.
10. The reaction mixture of claim 9 , wherein the nucleic acids from the cervical swab comprise an HPV RNA nucleic acid.