Method for detecting the presence of a target nucleic acid sequence in a sample
A method comprises loading a sample portion into a sample chamber which comprises means for minimizing diffusion of the sample portion, subjecting the sample portion to an amplification step, and determining whether the sample portion contains at least one molecule of a target nucleic acid. If the sample portion contains a single molecule of the target nucleic acid, the sample portion would attain a detectable concentration of the target nucleic acid after a single round of amplification. Also, a microfluidic device comprising a sample portion and a sample chamber comprising means for minimizing diffusion of the sample portion. Also, a microfluidic device comprising a sample chamber and an amplification targeting reagent positioned in the first sample chamber.
1. A method for detecting whether a target nucleic acid sequence is present in a biological sample, said method comprising:
loading sample portions of at least a first sample comprising nucleic acid into a plurality of respective sample chambers of a fluidic device, wherein said loaded sample portions have respective volumes ranging from 1 picoliter to 1 microliter, said sample chambers comprising means for minimizing diffusion of said sample portions such that when at least one sample portion loaded into a respective sample chamber contains a single molecule of a target nucleic acid sequence configured to produce amplified product of the target nucleic acid sequence in response to a primer-based amplification reaction, said at least one sample portion will attain a detectable concentration of amplified product of said target nucleic acid sequence within a portion of said respective sample chamber after being subjected to a primer-based amplification reaction with reagents configured and sufficient to perform primer-based amplification and fluorescence-based detection of said target nucleic acid sequence;
simultaneously subjecting said sample portions in said sample chambers to the primer-based amplification reaction with said reagents sufficient to perform primer-based amplification and fluorescence-based detection of amplified product of said target nucleic acid sequence when present; and
while in said sample chambers, detecting whether any of said sample portions contain amplified product of the target nucleic acid sequence after said subjecting.
2. A method as recited in claim 1 , wherein said means for minimizing diffusion of said sample portions comprises at least one physical restraint on diffusion.
3. A method as recited in claim 1 , wherein said means for minimizing diffusion of said sample portions comprises at least one chemical restraint on diffusion.
4. A method as recited in claim 1 , wherein said means for minimizing diffusion of said sample portions comprises at least one feature selected from among flow restriction means, glass beads, gels, absorbent particles and electrophoretic means.
5. A method as recited in claim 1 , wherein said loading of said sample portions is carried out using at least centrifugal force.
6. A method as recited in claim 1 , wherein said loading of said sample portions comprises loading said sample portions into said sample chambers via at least one ink jet.
7. A method as recited in claim 1 , wherein said loading of said sample portions is carried out using at least pressure.
8. A method as recited in claim 1 , wherein said loading of said sample portions is carried out using at least a vacuum.
9. A method as recited in claim 1 , wherein said loading of said sample portions is carried out using at least capillary action.
10. A method as recited in claim 1 , wherein said loading of said sample portions comprises loading sample portions having respective volumes ranging from 1 picoliter to 100 nanoliters.
11. A method as recited in claim 1 , wherein said loading of said sample portion comprises loading sample portions having respective volumes ranging from 1 picoliter to 10 nanoliters.
12. A method as recited in claim 1 , wherein said loading of said sample portions comprises loading sample portions having respective volumes ranging from 1 nanoliter to 10 nanoliters.
13. A method as recited in claim 1 , wherein said loading of said sample portions comprises loading sample portions having respective volumes ranging from 1 nanoliter to 60 nanoliters.
14. A method as recited in claim 1 , wherein said loading of said sample portions comprises loading sample portions having respective volumes ranging from 1 nanoliter to 100 nanoliters.
15. A method as recited in claim 1 , wherein simultaneously subjecting the sample portions to said primer-based amplification reaction comprises simultaneously subjecting the sample portions to a single round of a primer-based amplification reaction using said reagents.
16. The method as recited in claim 1 , wherein the primer-based amplification reaction comprises 30 to 40 thermal cycles.