Methods and compositions for rapid direct detection and differentiation of infectious from noninfectious virus
Methods of detecting presence of a virus in a sample are provided, the method including contacting the sample with a solid state nanopore comprising a plurality of virus-specific aptamers and measuring a current-voltage curve in the solid state nanopore, wherein a decrease in the current indicates presence of the virus in the sample. Solid state nanopores comprising a plurality of virus-specific aptamers covalently linked to the interior of the solid state nanopore are also provided. Membranes including a plurality of solid state nanopores including a plurality of covalently attached virus-specific aptamers and kits and systems with a membrane including a plurality of solid state nanopores including a plurality of covalently attached virus-specific aptamers are also provided.
1 . A method of detecting presence of a virus particle in a sample, comprising:
contacting the sample with a solid state nanopore comprising a plurality of virus-specific nucleic acid aptamers covalently linked to an inner wall of the solid state nanopore, wherein the solid state nanopore passes from a first surface to a second surface of a membrane and wherein the solid state nanopore comprises a diameter of about 50-60 nm at the first surface of the membrane and a diameter of about 800-1000 nm at the second surface of the membrane; and
measuring a current-voltage curve in the solid state nanopore,
wherein a decrease in current indicates presence of the virus particle in the sample.
2 . The method of claim 1 , wherein the membrane is present in a reservoir, wherein the reservoir is separated into two compartments by the membrane and the current-voltage curve is measured using two or more electrodes.
3 . The method of claim 1 , wherein the aptamer comprises a 3′ or 5′ amino modification.
4 . The method of claim 3 , wherein the aptamer comprises a spacer between the aptamer and the amino-terminal modification.
5 . The method of claim 1 , wherein the aptamer selectively binds an infectious, intact virus particle.
6 . The method of claim 5 , wherein the aptamer does not bind to a non-infectious, intact virus particle.
7 . The method of claim 1 , wherein the virus is a human adenovirus or a coronavirus.
8 . The method of claim 7 , wherein:
the human adenovirus is a human adenovirus type 2, a human adenovirus type 5, or a human adenovirus type 40; or
the coronavirus is a SARS-COV-2 coronavirus.
9 . The method of claim 8 , wherein the aptamer comprises the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 11.
10 . The method of claim 1 , wherein the method detects 10 pfu/ml or less of virus, 1 pfu/ml or less of virus, or wherein the method detects a single virus particle.
11 . A solid state nanopore comprising a plurality of virus-specific aptamers covalently linked to an inner wall of the solid state nanopore, wherein the solid state nanopore passes from a first surface to a second surface of a membrane and wherein the solid state nanopore comprises a diameter of about 50-60 nm at the first surface of the membrane and a diameter of about 800-1000 nm at the second surface of the membrane.
12 . The solid state nanopore of claim 11 , wherein the aptamer comprises a 3′ or 5′ amino modification.
13 . The solid state nanopore of claim 12 , wherein the aptamer comprises a spacer between the aptamer and the amino-terminal modification.
14 . The solid state nanopore of claim 11 , wherein the aptamer selectively binds an infectious, intact virus particle.
15 . The solid state nanopore of claim 14 , wherein the aptamer does not bind to a non-infectious, intact virus particle.
16 . The solid state nanopore of claim 11 , wherein the virus is a human adenovirus or a coronavirus.
17 . The solid state nanopore of claim 16 , wherein:
the human adenovirus is a human adenovirus type 2, a human adenovirus type 5, or a human adenovirus type 40; or
the coronavirus is a SARS-COV-2 coronavirus.
18 . The solid state nanopore of claim 17 , wherein the aptamer comprises the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 11.
19 . A membrane comprising a plurality of the solid state nanopores of claim 11 .
20 . A kit comprising the membrane of claim 19 and instructions for use.
21 . A system comprising:
the membrane of claim 19 ; and
one or more electrodes that are electrically coupled to the membrane.