Method and device for collection and amplification of circulating nucleic acids
Provided herein are methods for the collection and amplification of circulating nucleic acids from a non-celluar fraction of a biological sample. Circulating nucleic acids are extracted from the non-cellular fraction and are circularized to generate single-stranded nucleic acid circles, which are then subsequently amplified by rolling circular amplification using random primers to produce an amplified library. Devices for the collection of a non-cellular fraction from a biological sample are also provided. The device includes a filtration membrane and a dry solid matrix, which is in direct contact with the filtration membrane.
1. A method for processing a whole blood sample at a point of sample collection to collect circulating nucleic acid in plasma or serum, the method comprising:
a) applying the whole blood sample directly on to a filtration membrane at the point of sample collection;
b) separating, at the point of sample collection, the non-cellular fraction by filtering the whole blood sample through the filtration membrane in the absence of an anti-coagulant, wherein the intact cells in the whole blood are retained on the filtration membrane, and wherein the non-cellular fraction comprises plasma or serum;
c) collecting the separated non-cellular fraction onto a dry solid matrix, wherein the dry solid matrix is configured to establish a direct contact with the filtration membrane, wherein the dry solid matrix comprises cellulose and wherein the dry solid matrix is devoid of any detergent lysis buffer;
d) drying the collected non-cellular fraction on the solid matrix; and
e) storing the dry solid matrix containing the collected circulating nucleic acids in the non-cellular fraction.
2. The method of claim 1 , wherein the separating step is performed using vertical flow.
3. The method of claim 1 , wherein the separating step is performed using lateral flow.
4. The method of claim 1 , wherein the filtration membrane comprises pores with a diameter of less than or equal to about 5 microns.
5. The method of claim 1 , wherein the drying occurs under ambient conditions or by vacuum-assisted evaporations.
6. The method of claim 1 , wherein the whole blood sample comprises a circulating nucleic acid.
7. The method of claim 6 , wherein the circulating nucleic acid is derived from a tumor, a fetus, a donated organ after implantation, a transplanted cell, a transplanted tissue, or a diseased state.
8. The method of claim 6 , wherein the circulating nucleic acid is a circulating DNA or a circulating RNA, wherein the circulating DNA comprises 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.
9. The method of claim 1 , wherein the dried, collected non-cellular fraction is stored on the dry solid matrix for at least 30 minutes.
10. The method of claim 1 , wherein the dried, collected non-cellular fraction is stored on the dry solid matrix under dry or desiccated conditions or under inert atmospheres.
11. The method of claim 6 , wherein the circulating nucleic acid is stabilized for at least 24 hours at ambient conditions.
12. The method of claim 1 , wherein an intervening layer is positioned between the filtration membrane and the dry solid matrix.
13. The method of claim 1 , wherein the filtration membrane and the dry solid matrix are fully overlapping.
14. The method of claim 1 , wherein the filtration membrane and the dry solid matrix are partially overlapping.
15. The method of claim 1 , wherein the dry solid matrix comprises a cellulose paper.
16. The method of claim 1 , wherein the dry solid matrix is impregnated with a chaotropic salt.
17. The method of claim 16 , wherein the chaotropic salt is selected from a group consisting of: guanidine thiocyanate, guanidine chloride, guanidine hydrochloride, guanidine isothiocyanate, sodium thiocyanate, and sodium iodide.
18. The method of claim 1 , wherein a volume of whole blood sample applied to the filtration membrane is about 10 μL to about 500 μL.
19. The method of claim 1 , wherein the filtering step is performed without a prior pre-treatment of the whole blood.
20. The method of claim 1 , further comprising:
f) amplifying the collected circulating nucleic acids using random primers comprising a nucleotide analogue in a single reaction vessel; and
g) detecting a presence, absence, or quantity of a specific circulating nucleic acid sequence in the amplified circulating nucleic acid product.