SARS CoV-2 INFECTIVITY DETERMINATION ASSAY
Methods and compositions for characterizing a biological sample (e.g., comprising an infectious agent) from a subject are provided. Methods can include detecting linkage of nucleic acids that are linked in a viable cell or organism but that become degraded and thus unlinked in inviable cells or organisms and then characterizing the subject based on the quantity of linked and unlinked sequences.
1 . A method of characterizing an infectious agent in a subject, the method comprising,
providing a first sample from the subject comprising infectious agent nucleic acids;
partitioning the first sample into a plurality of first partitions;
detecting in the first partitions the presence or absence of a first infectious agent nucleic acid and a second infectious agent nucleic acid, wherein the first infectious agent nucleic acid and the second infectious agent nucleic acid are covalently linked in a viable infectious agent nucleic acid;
determining (a) the number of first partitions that contain the first infectious agent nucleic acid linked to the second infectious agent nucleic acid and (b) the number of first partitions that contain the first infectious agent nucleic acid without the second infectious agent nucleic acid or (c) the number of first partitions that contain the second infectious agent nucleic acid without the first infectious agent nucleic acid; and
characterizing the infectious agent in the subject based on the determining of (a) and (b) or (a) and (c).
2 . The method of claim 1 , wherein the determining comprises determining (b) and (c) and the characterizing is based on the determining of (a) and (b) and (c).
3 . The method of claim 1 , wherein the characterizing comprises comparing (a), (b), (c) or a combination thereof to one or more threshold value.
4 . The method of claim 1 , further comprising,
providing a second sample from the subject comprising infectious agent nucleic acids, wherein the second sample was obtained from the subject at a later time point than the first sample;
partitioning the second sample into a plurality of second partitions;
detecting in the second partitions the presence or absence of a first infectious agent nucleic acid and a second infectious agent nucleic acid;
determining (a′) the number of second partitions that contain the first infectious agent nucleic acid linked to the second infectious agent nucleic acid, (b′) the number of second partitions that contain the first infectious agent nucleic acid without the second infectious agent nucleic acid and (c′) the number of second partitions that contain the second infectious agent nucleic acid without the first infectious agent nucleic acid;
wherein the characterizing comprises comparing (a) to (a′), (b) to (b′), (c) to (c′) or a combination thereof.
5 . The method of claim 4 , wherein the second sample was obtained from the subject at least 24 hours (e.g., 1-10, 1-5, 1-3, 1-2 days) after the first sample was obtained.
6 . The method of claim 1 , further comprising detecting in the partitions a control nucleic acid and wherein the determining comprises normalizing:
(a) the number of first partitions that contain the infectious agent nucleic acid linked to the second infectious agent nucleic acid, and
(b) the number of first partitions that contain the first infectious agent nucleic acid without the second infectious agent nucleic acid, and/or
(c) the number of first partitions that contain the second infectious agent nucleic acid without the first infectious agent nucleic acid,
to the number of partitions containing the control nucleic acid.
7 . The method of claim 1 , wherein the characterizing comprises categorizing the infectious agent as viable or degraded.
8 . The method of claim 1 , wherein the infectious agent is a virus.
9 . The method of claim 8 , wherein the infectious agent is a virus selected from the group consisting of SARS-CoV-2, influenza, and respiratory syncytial virus (RSV).
10 . The method of claim 8 , wherein the infectious agent is SARS-CoV-2.
11 . The method of claim 10 , wherein the first infectious agent nucleic acid comprises at least a detectable portion of nucleocapsid (N) gene N1 and the second infectious agent nucleic acid comprises at least a detectable portion of N gene N2.
12 . The method of claim 1 , wherein the infectious agent is a bacterium or a mycoplasma.
13 . The method of claim 1 , wherein the first infectious agent nucleic acid and the second infectious agent nucleic acid are separated by 100-10,000 nucleotides from each other in the viable infectious agent nucleic acid.
14 . The method of claim 1 , wherein the subject is a human.
15 . The method of claim 1 , wherein the partitions are droplets in an emulsion or microwells or nanowells.
16 . A method of characterizing an infectious agent in a subject, the method comprising,
providing a first sample from the subject comprising infectious agent nucleic acids;
determining (a) an amount of first infection agent nucleic acid linked to the second infection agent nucleic acid, (b) an amount of first infectious agent nucleic acid unlinked to second infectious agent nucleic acid and (c) optionally an amount of second infectious agent nucleic acid unlinked to first infectious agent nucleic acid; and
characterizing the infectious agent in the subject based on the determining of (a), (b) and optionally (c).