Method and device for collection and amplification of circulating nucleic acids

1. A method for amplification of circulating nucleic acids that are present in a non-cellular fraction of a biological sample, the method comprising:

filtering the biological sample to separate the non-cellular fraction from intact cells;

collecting the separated, non-cellular fraction onto a dry solid matrix;

extracting the circulating nucleic acids from the collected, non-cellular fraction;

circularizing the extracted, circulating nucleic acids to form single-stranded nucleic acid circles; and

amplifying the single-stranded nucleic acid circles via random-primed rolling circle amplification using completely random primers comprising a nucleotide analogue to form an amplified, circulating nucleic acid product,

wherein the circularizing, and amplifying steps take place in a single reaction vessel without any intervening isolation or purification steps.

2. The method of claim 1 , further comprising drying the collected, non-cellular fraction to a substantially dry state prior to extraction.

3. The method of claim 1 , further comprising denaturing the extracted, circulating nucleic acids prior to circularization.

4. The method of claim 2 , wherein the circularization is performed using a TS2126 RNA ligase.

5. The method of claim 1 , further comprising detecting a presence, absence or quantity of a specific circulating nucleic acid sequence in the amplified, circulating nucleic acid product.

6. The method of claim 1 , wherein the biological sample is whole blood and the non-cellular fraction is plasma or serum.

7. The method of claim 6 , wherein the plasma or serum is collected from less than 150 μL of the whole blood.

8. The method of claim 6 , wherein the plasma or serum is separated from the whole blood in an absence of an anticoagulant.

9. The method of claim 1 , wherein the circulating nucleic acids are circulating DNAs or circulating RNAs.

10. The method of claim 9 , wherein the circulating nucleic acids are circulating DNAs, and the circulating DNAs comprise a tumor-derived DNA, a fetus-derived DNA, a donated organ-derived DNA, a transplanted cell-derived DNA, a transplanted tissue-derived DNA, or a combination thereof.

11. The method of claim 9 , wherein the circulating nucleic acids are circulating DNAs, and the circulating DNAs comprise a tumor-derived DNA.

12. The method of claim 1 , wherein the filtration of the biological sample is performed by using a membrane having a pore size between 0.01 micron and 5 micron.

13. The method of claim 12 , wherein the filtration of the biological sample is performed by using a membrane having a pore size between 1 micron and 2 micron.

14. The method of claim 1 , wherein the dry solid matrix is a cellulose matrix that is devoid of any detergent.

15. The method of claim 1 , wherein the dry solid matrix is impregnated with a chaotropic salt.

16. The method of claim 1 , wherein the biological sample is whole blood, the circulating nucleic acids are circulating DNAs present in the whole blood and the dry solid matrix is a cellulose matrix that is devoid of any detergent, and wherein the non-cellular fraction of the whole blood is separated via filtration through a filtration membrane having a pore size between 1 micron and 2 micron.

17. A method for processing whole blood at a point-of-collection for circulating nucleic acid analysis, the method comprising:

providing the whole blood at a sample application zone of a filtration membrane;

filtering the whole blood to separate plasma or serum at the point-of-collection, wherein the plasma or serum is separated from the whole blood via filtration through the filtration membrane in an absence of an anticoagulant;

collecting the separated plasma or serum on to a dry solid matrix, wherein the dry solid matrix is configured to establish a direct contact with the filtration membrane, and wherein the dry solid matrix is devoid of any detergent and wherein the dry solid matrix is a cellulose matrix;

drying the collected plasma or serum in the dry solid matrix;

extracting circulating nucleic acids from the dried sample of plasma or serum;

performing a whole genome amplification of the extracted circulating DNA to generate an amplified, circulating nucleic acid product; and

detecting a presence, absence, or quantity of a specific circulating nucleic acid sequence in the amplified, circulating nucleic acid product.

18. The method of claim 17 , wherein the plasma or serum is collected from less than 150 μL of whole blood.

19. The method of claim 17 , wherein the filtering, collecting, and drying, take place in a single device.

20. The method of claim 1 , wherein the nucleotide analogue comprises a locked nucleic acid (LNA), 2-amino dA, 2-thio dT, or a combination thereof.

21. The method of claim 1 , wherein the nucleotide analogue comprises a phosphorothioate group.