Viral detection using template emulsification
The disclosure provides methods and systems for multiplex viral detection using monodisperse emulsion droplets and template particles.
1. A virus detection method, the method comprising:
obtaining a sample comprising virus particles;
capturing virus particles from the sample with template particles in a first fluid within a vessel, wherein the template particles comprise a first plurality of first capture probes and a second plurality of second capture probes, and wherein the template particles comprise antibodies that capture the virus particles;
adding a second fluid immiscible to the first fluid to the vessel;
vortexing the vessel to simultaneously generate a plurality of monodisperse emulsion droplets that encapsulate a single template particle and a single virus particle from the sample;
lysing the virus particles contained within the monodisperse emulsion droplets to release a plurality of distinct viral genomic RNA;
capturing the plurality of distinct viral genomic RNA using a first capture probe of the first plurality of first capture probes, wherein the first capture probe comprises a first primer binding site and a first capture sequence;
forming cDNA using the captured plurality of distinct viral genomic RNA;
capturing the cDNA using a second capture probe of the second plurality of second capture probes, wherein the second capture probe comprises a second primer binding site different from the first primer binding site, wherein the second capture probe comprises a second capture sequence different from the first capture sequence, wherein the second capture sequence binds cDNA, wherein the second capture sequences of at least two individual second capture probes are different, and wherein the first capture probe, the second capture probe, or both, comprises a barcode sequence that is unique to a template particle;
providing each cDNA within a droplet the barcode unique to the template particles; and
sequencing the plurality of distinct viral RNA based on the cDNA to detect the presence of one or more viruses in the sample.
2. The method of claim 1 , wherein the first fluid is an aqueous solution and the second fluid comprises an oil.
3. The method of claim 2 , wherein the template particles comprise a hydrogel that includes agarose, alginate, a polyethylene glycol (PEG), a polyacrylamide (PAA), acrylate, acrylamide/bisacylamide copolymer matrix, azide-modified PEG, poly-lysine, polyethyleneimine, or any combination thereof.
4. The method of claim 3 , wherein the barcode unique to the droplet is provided to the droplet by the template particle encapsulated by the droplet.
5. The method of claim 1 , wherein the first capture sequence is a random N-mer between 6 and 12 nucleotides in length.
6. The method of claim 1 , wherein the second capture sequence is a random hexamer.
7. The method of claim 1 , further comprising fragmenting the RNA prior to reverse transcribing the plurality of distinct viral genomic RNAs.
8. The method of claim 1 , wherein the antibodies are specific to a particular virus and the plurality of monodisperse emulsion droplets encapsulate a single template particle with a single virus particle of the particular virus from the sample.
9. The method of claim 8 , wherein the antibodies are specific to SARS-COV-2 and the plurality of monodisperse emulsion droplets encapsulate a single template particle with a single SARS-COV-2 virus particle from the sample.
10. The method of claim 1 , wherein the template particles are a plurality of different template particles that each capture a virus particle of a different virus.
11. The method of claim 10 , wherein the different template particles comprise different antibodies.
12. The method of claim 11 , wherein the different antibodies are each specific to a different virus and the plurality of monodisperse emulsion droplets encapsulate a single template particle with a single virus particle of the virus specific to antibody of the encapsulated template particle.
13. The method of claim 12 , wherein the different antibodies are specific to SARS-COV-2 or seasonal influenza and the plurality of monodisperse emulsion droplets encapsulate a single template particle with a single SARS-COV-2 virus particle or a single seasonal influenza virus particle from the sample.
14. The method of claim 11 , wherein the different antibodies are specific to SARS-COV-2, seasonal influenza, rhinovirus, orthopneumovirus, parainfluenza viruses, or human metapneumovirus and the plurality of monodisperse emulsion droplets encapsulate a single template particle with a single SARS-COV-2, seasonal influenza, rhinovirus, orthopneumovirus, parainfluenza viruses, or human metapneumovirus virus particle from the sample.
15. A virus detection method, the method comprising:
obtaining a sample comprising virus particles;
lysing the virus particles to release a plurality of distinct viral genomic RNA;
combining the plurality of distinct viral genomic RNA with template particles in a first fluid in a vessel, wherein the template particles comprise a first plurality of first capture probes and a second plurality of second capture probes, and wherein the template particles comprise antibodies that capture the virus particles;
adding a second fluid immiscible to the first fluid to the vessel;
vortexing the vessel to simultaneously generate a plurality of monodisperse emulsion droplets that encapsulate a single template particle and viral genomic nucleic acid from the sample;
capturing the plurality of distinct viral genomic RNA using a first capture probe of the first plurality of first capture probes, wherein the first capture probe comprises a first primer binding site and a first capture sequence;
forming cDNA using the captured plurality of distinct viral genomic RNA;
capturing the cDNA using a second capture probe of the second plurality of second capture probes, wherein the second capture probe comprises a second primer binding site different from the first primer binding site, wherein the second capture probe comprises a second capture sequence different from the first capture sequence, wherein the second capture sequence binds cDNA, wherein the second capture sequences of at least two individual second capture probes are different, and wherein the first capture probe, the second capture probe, or both, comprises a barcode sequence that is unique to a template particle;
providing each viral cDNA within the droplet a barcode unique to the template particles; and
sequencing the viral RNA based on the cDNA encapsulated in the monodisperse emulsion droplets to detect the presence of one or more viruses in the sample.
16. The method of claim 15 , wherein the first fluid is an aqueous solution and the second fluid comprises an oil.
17. The method of claim 16 , wherein the template particles comprise a hydrogel that includes agarose, alginate, a polyethylene glycol (PEG), a polyacrylamide (PAA), acrylate, acrylamide/bisacylamide copolymer matrix, azide-modified PEG, poly-lysine, polyethyleneimine, or any combination thereof.
18. The method of claim 17 , wherein the barcode unique to the droplet is provided to the droplet by the template particle encapsulated by the droplet.
19. The method of claim 15 , wherein the first capture sequence is a random N-mer between 6 and 12 nucleotides in length.
20. The method of claim 15 , wherein the second capture sequence is a random hexamer.
21. The method of claim 15 , wherein the capture probes further comprise a unique molecular identifier (UMI).
22. The method of claim 15 , comprising detecting RNA viruses, and the viral particles release a plurality of distinct viral genomic RNAs.
23. The method of claim 15 , further comprising reverse transcribing the plurality of distinct viral genomic RNAs.
24. The method of claim 23 , wherein during vortexing the capture probes capture viral genomic RNAs and reverse transcribe the viral genomic RNAs into cDNAs.