Detection of nucleic acid amplification in a porous substrate
The present disclosure relates to characterization of biological samples by amplification detection in a porous substrate. By way of example, a porous substrate may include amplification reagents configured to provide a signal when released during amplification. When a sample is applied, amplification occurs as a wavefront from the application point, and the time that the wavefront reaches a distance on the porous substrate is related to an initial concentration of the sample applied. By detecting the distance traveled by the amplification products at one or more time points, an initial concentration of the sample may be estimated.
1. A method of assessing amplification of nucleic acids comprising:
providing a lateral flow strip comprising a porous substrate and comprising a plurality of amplification reagents distributed throughout the body of the porous substrate, wherein the amplification reagents comprise nucleotides, one or more nucleic acid primers for a nucleic acid sequence of interest, and amplification enzymes;
applying a sample having the nucleic acid sequence of interest to a predetermined initial location on the lateral flow strip at an initial time;
allowing the sample to react with the amplification reagents in the lateral flow strip at the initial location for a predetermined duration of time to generate amplification products of the nucleic acid sequence of interest, wherein amplification products comprise one or more detectable moieties configured to provide a visible signal on the lateral flow strip when the amplification products are generated during amplification and wherein the amplification products and the visible signal move outwards on the lateral flow strip from the initial location during amplification;
determining a distance between a farthest location relative to the predetermined initial location of the visible signal on the lateral flow strip at an end of the predetermined duration of time, wherein the farthest location is spaced apart from the initial location by the distance; and
determining a starting copy number of the nucleic acid sequence of interest in the sample using a plot or table showing a relationship between a plurality of farthest distances and a corresponding plurality of control samples having known copy numbers of the nucleic acid sequence of interest.
2. The method of claim 1 , wherein the porous substrate comprises a printed marker corresponding to the initial location and a plurality of printed markers corresponding to different distances from the initial location.
3. The method of claim 1 , wherein the amplification reagents comprise isothermal nucleic acid amplification reagents.
4. The method of claim 1 , wherein the amplification reagents comprise DNA polymerase and one or more nucleic acid primers.
5. The method of claim 1 , wherein the amplification reagents comprise RNA polymerase.
6. The method of claim 1 , comprising determining that the sample does not contain a threshold amount of the sequence of interest if the detected signal does not reach a minimum distance from the initial location.
7. The method of claim 1 , comprising determining a clinical characteristic of a patient from whom the sample was taken based on the initial concentration of the nucleic acid sequence of interest in the sample, wherein the clinical characteristic is associated with a presence or absence of the nucleic acid sequence of interest and wherein the amplification reagents comprise one or more primers complementary to the nucleic acid sequence of interest.
8. The method of claim 1 , wherein the porous substrate is saturated with liquid.
9. The method of claim 1 , wherein liquid moves through the substrate during amplification.
10. The method of claim 1 , wherein providing the porous substrate comprises applying the amplification reagents to the porous substrate in a single solution.
11. The method of claim 1 , comprising heating the porous substrate to activate one or more of the amplification reagents.
12. The method of claim 11 , wherein the heating is to an isothermal temperature.
13. The method of claim 1 , wherein the signal comprises a visible signal and/or a fluorescent signal.
14. The method of claim 1 , wherein the signal comprises an electrochemical signal or a pH change.
15. The method of claim 1 , wherein the porous substrate has a non-uniform porosity.
16. The method of claim 15 , wherein the porous substrate comprises an open channel with a porosity of one.
17. The method of claim 1 , wherein the signal is generated by release of a dye.
18. The method of claim 1 , wherein the signal is generated by one or more of amplicons, H + , or pyrophosphate.
19. The method of claim 1 , wherein the amplification reagents are distributed generally evenly throughout the body of the porous substrate.
20. The method of claim 1 , wherein the initial location is on or near an edge of the lateral flow strip.
21. The method of claim 1 , wherein the visible signal is generated by production of a labeled triphosphate from dye-labeled nucleotides of the amplification reagents and liberation of the dye by reaction with a phosphatase.
22. The method of claim 1 , wherein the starting copy number is determined based on the control sample whose furthest distance is closest to the furthest distance of the sample.
23. The method of claim 1 , wherein the relationship is driven by reaction diffusion and not wicking.