Methods and apparatus for point-of-care nucleic acid amplification and detection
Methods and apparatus are provided for point-of-care nucleic acid amplification and detection. One embodiment of the invention comprises a fully integrated, sample-to-answer molecular diagnostic instrument that optionally may be used in a multiplexed fashion to detect multiple target nucleic acid sequences of interest and that optionally may be configured for disposal after one-time use. The instrument preferable utilizes an isothermal nucleic acid amplification technique, such as loop-mediated isothermal amplification (LAMP), to reduce the instrumentation requirements associated with nucleic acid amplification. Detection of target amplification may be achieved, for example, via detection of a color shift or fluorescence in a dye added to the amplification reaction. Such detection may be performed visually by an operator or may be achieved utilizing an imaging technique, e.g., spectrophotometric imaging.
1. A method for amplification and detection of a target nucleic acid sequence in a sample, the method comprising: transferring the sample through an inlet into a diagnostic apparatus; distributing the sample among a plurality of microfluidic channels of the diagnostic apparatus, each microfluidic channel extending to one of a plurality of reaction chambers in the diagnostic apparatus, at least one of the reaction chambers having a nucleic acid amplification reagent therein, so that a portion of the sample is received in each reaction chamber; heating the reaction chambers in order to amplify any target nucleic acid sequence within the sample portion; manually sliding a locking valve within a void of the diagnostic apparatus from an open position to a closed position after the transferring step to block all of the microfluidic channels and prevent backflow of the sample portion from each reaction chamber through the microfluidic channel extending to it during heating; and detecting in the at least one reaction chamber amplification of the target nucleic acid sequence within the sample.
2. The method of claim 1 , further comprising venting overflow from the reaction chambers.
3. The method of claim 2 , wherein venting overflow further comprises venting to at least one sequestered overflow chamber.
4. The method of claim 2 , wherein venting overflow further comprises venting gases through flow control media while precluding venting of fluids.
5. The method of claim 2 , wherein venting comprising moving air from the reaction chambers through a medium that permits the passage of air but not fluid.
6. The method of claim 1 , further comprising disposing of the diagnostic apparatus and sample after one-time use.
7. The method of claim 1 , wherein heating further comprises: heating the reaction chambers with a heating element; and disposing of the heating element after one-time use.
8. The method of claim 1 , wherein detecting amplification further comprises detecting a color shift in a colorimetric dye, detecting an increase in turbidity, or detecting fluorescence.
9. The method of claim 1 , wherein distributing further comprises transferring the sample through at least one branching microfluidic channel that distributes the sample matrix across the reaction chambers.
10. The method of claim 1 , wherein preventing backflow further comprises preventing cross-contamination between the plurality of reaction chambers.
11. The method of claim 1 , further comprising amplifying and detecting different target nucleic acid sequences in different reaction chambers.
12. The method of claim 1 , further comprising collecting the sample with a sample collector.
13. The method of claim 12 , further comprising eluting the sample from the sample collector.
14. The method of claim 13 , wherein eluting comprises depressing a plunger of a syringe.
15. The method of claim 14 , further comprising connecting the syringe and sample collector to the diagnostic apparatus prior to the step of depressing the plunger.
16. The method of claim 1 , wherein a first reaction chamber of the plurality of reaction chambers contains a first nucleic acid amplification reagent therein and a second reaction chamber of the plurality of reaction chambers contains a second nucleic acid amplification reagent different from the first nucleic acid amplification reagent.
17. The method of claim 1 , wherein at least one reaction chamber of the plurality of reaction chambers contains a quantity of the target nucleic acid sequence prior to the step of transferring the sample through the inlet into the diagnostic apparatus.
18. The method of claim 1 , wherein the diagnostic apparatus further comprises at least one additional reaction chamber not connected to any of the plurality of microfluidic channels, the additional reaction chamber containing a nucleic acid amplification reagent.
19. The method of claim 1 , wherein the locking valve comprises a plurality of lumens, each microfluidic channel aligning with one of the lumens when the valve is in an open position, the sliding step comprising sliding the locking valve to a closed position in which the microfluidic channels are not aligned with the lumens.
20. The method of claim 1 , wherein each of the reaction chambers has a volume of less than 100 microliters.
21. The method of claim 1 , wherein the detecting step comprises viewing the reaction chambers through a transparent cover.
22. The method of claim 1 , wherein the sample is not lysed with lysing chemicals prior to the transferring step.