Point of care and improved detection and quantification of biomolecules
The present disclosure provides materials and methods for detecting the presence of a target, such as a nucleic acid, biomolecule or biological particle, or virus genome in a sample. The present disclosure further provides point of care detection methods that cane done quickly and simply, optionally without sophisticated instrumentation. The present disclosure further provides improved methods for detecting and quantifying detection signals from various emulsions.
1 . A method of detecting a target nucleic acid in a sample from a patient collected at a point of care comprising:
(a) combining in a vessel (i) a sample comprising a target nucleic acid; (ii) a solution comprising reagents for amplifying and detecting the target nucleic acid; and (iii) an immiscible carrier, thereby forming a combined solution;
(b) agitating the combined solution of (a) under conditions that allow encapsulation of the target nucleic acid with the reagents for amplifying and detecting the target nucleic acid in multiple individual droplets, thereby forming a polydispersed emulsion;
(c) incubating the polydispersed emulsion of step (b) under conditions that allow amplification of the target nucleic acid in the multiple individual droplets; and
(d) detecting the target nucleic acid in the polydispersed emulsion;
wherein steps (a)-(d) occur at the point of care, and
wherein the detecting comprises simultaneously quantifying detection signals from (I) multiple layers of the multiple individual droplets and (II) a monolayer of the multiple individual droplets, and
wherein the detecting occurs in a dual-height 2-d chamber.
2 . The method according to claim 1 , wherein the method does steps de not use a microwell, microdroplet array or microfluidic device.
3 . The method according to claim 1 , wherein the sample comprising the target nucleic acid has not undergone purification steps prior to combining in the vessel of step (a).
4 . The method according to claim 1 , wherein the detecting comprises quantifying a detection signal comprising a quantitation assay selected from the group consisting of sequencing, fluorescent signal reading, and turbidity reading.
5 . The method according to claim 1 , wherein the target nucleic acid is present in the sample at 1 to 50 copies.
6 . The method according to claim 1 , wherein the agitating comprises mixing the reagents by pipetting, shaking by hand, stirring, beating, bubbling, vortexing and sonicating.
7 . The method according to claim 1 , wherein the sample comprising the target nucleic acid is a sample obtained from a human subject.
8 . The method according to claim 7 , wherein the sample is a saliva sample, blood sample, urine sample, or tissue sample.
9 . The method according to claim 1 , wherein the target nucleic acid is from a virus, a bacteria, or a parasite.
10 . The method according to claim 9 , wherein the target nucleic acid is coronavirus, SARS-COV-2, human immunodeficiency virus (HIV), herpes simplex virus (HSV), human papilloma virus (HPV), influenza virus, or respiratory syncytial virus (RSV).
11 . The method according to claim 1 , wherein the target nucleic acid is amplified by PCR, RT-PCR, qPCR, digital droplet PCR (ddPCR), LAMP, or NASBA.
12 . A method of detecting a target nucleic acid in a sample from a patient comprising:
(a) combining in a vessel (i) a sample comprising a target nucleic acid; (ii) a solution comprising reagents for amplifying and detecting the target nucleic acid; and (iii) an immiscible carrier, thereby forming a combined solution;
(b) agitating the combined solution of (a) under conditions that allow encapsulation of the target nucleic acid with the reagents for amplifying and detecting the target nucleic acid in multiple individual droplets, thereby forming a polydispersed emulsion;
(c) incubating the polydispersed emulsion of step (b) under conditions that allow amplification of the target nucleic acid in the multiple individual droplets; and
(d) detecting the target nucleic acid in the polydispersed emulsion;
wherein the detecting comprises simultaneously quantifying detection signals from (I) multiple layers of the multiple individual droplets and (II) a monolayer of the multiple individual droplets, and wherein the detecting occurs in a dual-height 2-d chamber.