Devices and methods for nucleic acid identification in samples

1 . A device for nucleic acid identification of material in a sample, the device comprising:

a microfluidics system; and

a heat source;

the microfluidics system comprising a movable cartridge assembly;

the movable cartridge assembly comprising:

a surface configured to receive the sample;

at least one target-specific set of reagent components deposited on the surface at a target location;

at least a second target-specific set of reagent components deposited on the surface at a second target location;

at least one set of RCA-LAMP reaction components deposited on the surface at an RCA-LAMP location;

the at least one target-specific set of reagent components including:

at least one target-specific padlock probe reagent component;

at least one target probe-associated ligase enzyme component; and

at least one target probe-associated set of ligase buffer components;

the second target-specific set of reagent components including:

a second target-specific padlock probe reagent component;

the at least one target probe-associated ligase enzyme component; and

the at least one target probe-associated set of ligase buffer components; and

the at least one set of RCA-LAMP reaction components including:

at least one polymerase buffer component;

at least one polymerase enzyme with strand displacement activity;

a betaine additive;

a poloxamine surfactant additive;

a sequence-specific probe;

dNTPs; and

a primer mix;

the primer mix including:

a forward inside primer specific to a backbone of the at least one target-specific padlock probe reagent component; and

a backward inside primer specific to the backbone of the at least one target-specific padlock probe reagent component;

wherein the second target-specific padlock probe reagent component includes the backbone of the at least one target-specific padlock probe reagent component.

2 . The device of claim 1 ,

wherein the ligase enzyme component is a Taq DNA ligase enzyme component; and

wherein the set of ligase buffer components are a set of Taq DNA ligase buffer components.

3 . The device of claim 1 ,

wherein the ligase enzyme component is a RNA ligase enzyme component; and

wherein the set of ligase buffer components are a set of RNA ligase buffer components.

4 . The device of claim 1 ,

where the microfluidics system is a digital microfluidics system.

5 . The device of claim 1 ,

where the sequence-specific probe is an oligonucleotide strand displacement probe;

where the at least one polymerase buffer component is a Bst 3.0 polymerase buffer component; and

where the at least at least one polymerase enzyme with strand displacement activity is a Bst 3.0 polymerase enzyme.

6 . The device of claim 5 , further comprising:

a camera system for monitoring fluorescence emanating from the target location and the second target location in response to excitation radiation from a radiation source.

7 . The device of claim 1 , further comprising:

a system for cooling a heated fluid at the target location and for cooling a heated fluid at the second target location;

wherein the heat source is configured to heat fluid located on at least the target location and fluid located on at least the second target location to approximately 95 degrees Celsius; and

wherein the heat source is further configured to heat fluid located on at least the target location and fluid located on at least the second target location to approximately 65 degrees Celsius.

8 . The device of claim 1 ,

wherein the movable cartridge assembly is a consumable cartridge.

9 . The device of claim 1 ,

wherein the at least one target-specific set of reagent components deposited on the surface at the target location are printed at the target location; and

wherein the second target-specific set of reagent components deposited on the surface at the second target location are printed at the second target location.

10 . The device of claim 1 ,

wherein the microfluidics system is configured to transport an aliquot of the sample received on the surface to the target location and is further configured to transport a second aliquot of the sample received on the surface to the second target location; and

wherein the microfluidics system is further configured to transport an aliquot of RCA-LAMP reaction components on the surface to the target location and is further configured to transport a second aliquot of RCA-LAMP reaction components on the surface to the second target location.

11 . The device of claim 10 ,

wherein the at least one set of RCA-LAMP reaction components deposited on the surface at the RCA-LAMP location are deposited in a dried form; and

wherein the microfluidics system is further configured to transport an aliquot of hydrated RCA-LAMP reaction components on the surface to the target location and is further configured to transport a second aliquot of hydrated RCA-LAMP reaction components on the surface to the second target location.

12 . A method for monitoring fluorescence associated with nucleic acid material in a sample, the method comprising:

providing a microfluidics system;

the microfluidics system including a movable cartridge assembly;

the movable cartridge assembly comprising:

a surface configured to receive the sample;

at least one target-specific set of reagent components deposited on the surface at a target location;

at least a second target-specific set of reagent components deposited on the surface at a second target location;

at least one set of RCA-LAMP reaction components deposited on the surface at an RCA-LAMP location;

the at least one target-specific set of reagent components including:

at least one target-specific padlock probe reagent component;

at least one target probe-associated ligase enzyme component; and

at least one target probe-associated set of ligase buffer components;

the second target-specific set of reagent components including:

a second target-specific padlock probe reagent component;

the at least one target probe-associated ligase enzyme component; and

the at least one target probe-associated set of ligase buffer components; and

the at least one set of RCA-LAMP reaction components including:

at least one polymerase buffer component;

at least one polymerase enzyme with strand displacement activity;

a betaine additive;

a poloxamine surfactant additive;

a sequence-specific probe;

dNTPs; and

a primer mix;

the primer mix including:

a forward inside primer specific to a backbone of the at least one target-specific padlock probe reagent component; and

a backward inside primer specific to the backbone of the at least one target-specific padlock probe reagent component;

wherein the second target-specific padlock probe reagent component includes the backbone of the at least one target-specific padlock probe reagent component;

transporting an aliquot of the sample received on the surface to the target location;

transporting a second aliquot of the sample received on the surface to the second target location;

applying heat to the target location and the second target location;

transporting an aliquot of RCA-LAMP reaction components on the surface to the target location;

transporting a second aliquot of RCA-LAMP reaction components on the surface to the second target location;

applying heat to the target location and the second target location;

providing excitation radiation to the target location and the second target location; and

monitoring fluorescence emanating from the target location and the second target location in response to the excitation radiation using a camera system.

13 . The method of claim 12 ,

wherein the ligase enzyme component is a Taq DNA ligase enzyme component; and

wherein the set of ligase buffer components are a set of Taq DNA ligase buffer components.

14 . The method of claim 12 ,

wherein the ligase enzyme component is a RNA ligase enzyme component; and

wherein the set of ligase buffer components are a set of RNA ligase buffer components.

15 . The method of claim 12 ,

where the microfluidics system is a digital microfluidics system.

16 . The method of claim 12 ,

where the sequence-specific probe is an oligonucleotide strand displacement probe;

where the at least one polymerase buffer component is a Bst 3.0 polymerase buffer component; and

where the at least at least one polymerase enzyme with strand displacement activity is a Bst 3.0 polymerase enzyme.

17 . The method of claim 12 , further comprising:

cooling heated fluid at the target location prior to the step of transporting an aliquot of RCA-LAMP reaction components on the surface to the target location; and

cooling heated fluid at the second target location prior to the step of transporting a second aliquot of RCA-LAMP reaction components on the surface to the second target location;

wherein the step of applying heat to the target location prior to the step of transporting an aliquot of RCA-LAMP reaction components on the surface to the target location includes heating fluid located on at least the target location to approximately 95 degrees Celsius;

wherein the step of applying heat to the second target location prior to the step of transporting a second aliquot of RCA-LAMP reaction components on the surface to the second target location includes heating fluid located on at least the second target location to approximately 95 degrees Celsius;

wherein the step of applying heat to the target location after the step of transporting an aliquot of RCA-LAMP reaction components on the surface to the target location includes heating fluid located on at least the target location to approximately 65 degrees Celsius; and

wherein the step of applying heat to the second target location after the step of transporting a second aliquot of RCA-LAMP reaction components on the surface to the second target location includes heating fluid located on at least the second target location to approximately 65 degrees Celsius.

18 . The method of claim 12 ,

wherein the movable cartridge is a consumable cartridge.

19 . The method of claim 12 ,

wherein the at least one target-specific set of reagent components deposited on the surface at the target location are printed at the target location; and

wherein the second target-specific set of reagent components deposited on the surface at the second target location are printed at the second target location.

20 . The method of claim 12 , wherein the at least one set of RCA-LAMP reaction components deposited on the surface at the RCA-LAMP location are deposited in a dried form, the method further comprising:

hydrating the RCA-LAMP reaction components;

wherein the step of transporting an aliquot of RCA-LAMP reaction components on the surface to the target location comprises transporting an aliquot of hydrated RCA-LAMP reaction components on the surface to the target location; and

wherein the step of transporting a second aliquot of RCA-LAMP reaction components on the surface to the second target location comprises transporting a second aliquot of hydrated RCA-LAMP reaction components on the surface to the second target location.