METHODS FOR PREPARATION OF NUCLEIC ACID-CONTAINING SAMPLES USING CLAY MINERALS AND ALKALINE SOLUTIONS

• US 20160102340A1
• Filed: 05/07/2014
• Published: 04/14/2016
• Est. Priority Date: 05/07/2013
• Status: Active Grant

• Current Assignee: Revvity Health Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Micronics Inc. (Sony Group Corp.)
• Inventors: BOUZEK, Heather K.
• Granted Patent: US 10,190,153 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/706 for hepatitis
• CPC Class Codes: C12Q 1/707 non-A, non-B Hepatitis, excluding hepatitis D

Abstract

The present invention provides a rapid and highly effective method for the preparation of biological samples for detection of both DNA and RNA target molecules. The method comprises treatment of the sample with a clay mineral followed by lysis of the sample with an alkaline solution. The method is particularly suited for preparing complex biological samples, such as blood or plasma, for the simultaneous detection of DNA and RNA targets. Samples prepared by the method of the invention may be directly used as targets in PCR amplification. The method of the invention may conveniently be coupled with further steps and devices to perform molecular analyses for diagnostic and other applications.

Claims

1. A method for treating a nucleic acid-containing sample for analysis of nucleic acids, the method comprising the steps of:

a) contacting the sample with a clay mineral;

b) mixing the sample and the clay mineral until the clay mineral is evenly dispersed in the sample;

c) substantially removing the clay mineral from the sample;

d) contacting the sample with an alkaline solution at a pH suitable for lysis of cell and viral particles to form a nucleic acid solution;

e) and, optionally, contacting the nucleic acid solution with an acidic solution suitable for neutralizing the pH of the nucleic acid solution.

2. The method of claim 1, wherein the clay mineral comprises a kaolinite, smectite, or illite clay mineral.

3. The method of claim 1, wherein the clay mineral comprises talc.

4. The method of claim 1, wherein the clay mineral comprises halloysite.

5. The method of claim 1, wherein the clay mineral comprises bentonite.

6. The method of claim 1, wherein the clay mineral comprises a synthetic clay mineral.

7. The method of claim 6, wherein the synthetic clay mineral comprises laponite.

8. The method of claim 1, wherein the alkaline solution comprises KOH, NaOH, or LiOH, or combinations thereof.

9. The method of claim 8, wherein the alkaline solution comprises KOH.

10. The method of claim 1, wherein the pH suitable for lysis is obtained from an alkaline solution of about 0.1 M base.

11. The method of claim 1, wherein the optional acidic solution comprises HCl, C₂H₄O₂,or H₂SO₄.

12. The method of claim 1, wherein the sample comprises one or more infectious agents.

13. The method of claim 12, wherein the one or more infectious agents are viral agents.

14. The method of claim 13, wherein the sample comprises at least two viral agents.

15. The method of claim 14, wherein the sample comprises a DNA virus and an RNA virus.

16. The method of claim 15, wherein the DNA virus is HBV.

17. The method of claim 16, wherein the RNA virus is HCV or HIV.

18. The method of claim 1, wherein the sample is selected from blood, plasma, serum, urine, saliva, sputum, respiratory lavage, tears, and tissue swabs.

19. The method of claim 18, wherein the sample is selected from blood, plasma, and serum.

20. The method of claim 1, further comprising a nucleic acid amplification step.

21. The method of claim 20, wherein the nucleic acid amplification step is selected from PCR, RT-PCR, qPCR, and qRT-PCR.