LAMP COMPONENT DISTRIBUTION IN A MICROFLUID CELL

Abstract

A microfluidic test device has a body, a first chamber having an outlet provided with a first valve and holding a first buffer having a first buffer volume, a primary reaction chamber, a sample inlet for receiving and feeding a sample having a sample volume, into the microfluidic test device, a first fluid path connecting the outlet of the first chamber and the sample inlet, a second fluid path connecting the sample inlet and the primary reaction chamber, a primary test part having a primary test chamber, a third primary fluid path connecting the primary reaction chamber and the primary test part, a primary valve arranged in the third primary fluid path, a flow driving device configured to move fluid from the primary reaction chamber to the primary test part, and a heating assembly configured to heat a reaction fluid in the primary reaction chamber.

Claims

1. A microfluidic test device comprising: a body; a first chamber having an outlet provided with a first valve and holding a first buffer having a first buffer volume; a primary reaction chamber; a sample inlet for receiving a sample and being configured for feeding a sample having a sample volume, into the device; a first fluid path connecting the outlet of the first chamber and the sample inlet; a second fluid path connecting the sample inlet and the primary reaction chamber; a primary test part comprising a primary test chamber; a third primary fluid path connecting the primary reaction chamber and the primary test part; a primary valve arranged in the third primary fluid path; and a flow driving device configured to move fluid from the primary reaction chamber to the primary test part, and wherein the microfluidic test device comprises a primary reaction material in a lyophilized form, and wherein the primary reaction material and the first buffer are arranged on different sides of the sample inlet.

2. The microfluidic test device according to claim 1, wherein the primary reaction material is in a pellet form (cake) and/or coated to an inner surface of the microfluidic test device.

3. The microfluidic test device according to claim 1, wherein the primary reaction material is placed in the primary reaction chamber.

4. The microfluidic test device according to claim 1, wherein the packaging of the lyophilized components is made under humidity and oxygen free conditions.

5. The microfluidic test device according to claim 1, wherein the liquids are sterile filtrated prior to packaging into the microfluidic test device.

6. The microfluidic test device according to claim 1, wherein the primary reaction material components comprise primers, dNTPs, and a polymerase.

7. The microfluidic test device according to claim 1, wherein the primary reaction material comprises components for amplifying a nucleic acid target by loop-mediated isothermal amplification (LAMP).

8. The microfluidic test device according to claim 1, wherein the polymerase is a Bsm polymerase.

9. The microfluidic test device according to claim 1, wherein the Bsm polymerase is lyophilized free of glycerol.

10. The microfluidic test device according to claim 1, wherein the primary reaction chamber is free of liquid, such as a buffer, prior to feeding a sample into the device.

11. The microfluidic test device according to claim 1, wherein the primary reaction chamber is free of (NH4).sub.2SO.sub.4 and/or zwitterions (preferably betaine) prior to feeding a sample into the device.

12. The microfluidic test device according to claim 1, wherein the first buffer comprises (NH4).sub.2SO.sub.4, a zwitterion (preferably betaine), and a biozide. wherein the biozide is ProClin950

13. The microfluidic test device according to claim 1, wherein Mg(SO.sub.4)7H.sub.2O (magnesium sulfate heptahydrate) is present in both the primary reaction material and the first buffer in a ratio 1:3.

14. The microfluidic test device according to claim 1, wherein the amplification time of the LAMP reaction in the device is less than 1 hour.

15. The microfluidic test device according to claim 1, wherein the storage time is at least 2 years from the production date at a storage temperature of 4-30 C.

Description

DESCRIPTION OF THE FIGURES

[0232] Turning now to the figures, FIG. 1 schematically illustrates a first (top) view of an exemplary microfluidic test device. The microfluidic test device 2 comprises a body 4 and a first chamber 6 having an outlet 8 provided with a first valve 10 and holding a first buffer having a first buffer volume.

[0233] The microfluidic test device 2 comprises a primary reaction chamber 12 and a sample inlet 14 for receiving a sample and being configured for feeding a sample having a sample volume, into the medical test device 2. A first fluid path 16 connects the outlet 8 of the first chamber 6 and the sample inlet 14 and a second fluid path 18 connects the sample inlet 14 and the primary reaction chamber 12. Further, the microfluidic test device 2 comprises a primary test part 20 comprising a primary test chamber 22 with a third primary fluid path 24 connecting the primary reaction chamber 12 and the primary test part 20. A primary valve 26 is arranged in the third primary fluid path 24. The microfluidic test device 2 comprises a flow driving device 28 configured to move fluid from the primary reaction chamber 12 to the primary test part 20. The flow driving device 28 comprises a second chamber 28A having an outlet optionally provided with a second valve 28B. The outlet of the second chamber is connected to the first fluid path 16 via fourth fluid path 29.

[0234] The microfluidic test device 2 comprises a heating assembly 30 configured to heat a reaction liquid in the primary reaction chamber 12. The heating assembly 30 is connected to a power source (not shown). The power source may be one or more batteries accommodated or inserted in the microfluidic test device. The power source may be an external power source connected to the microfluidic test device via a connector (not shown).

[0235] The primary test part 20 comprises a first lateral flow strip 32 having a first end 34 connected to an outlet of the primary test chamber 22. The first lateral flow strip 32 has a length of 65 mm and a width of 3.0 mm.

[0236] FIG. 2 shows an exemplary cross-sectional view of the microfluidic test device 2. The microfluidic test device 2. The body 4 has a first end 36 with a first end surface, and a second end 38 with a second end surface. The body 4 has a first surface 40 intended for facing upwards when the microfluidic test device is positioned in a test position, and a second surface 42 opposite the first surface 40 and intended for facing downwards when the microfluidic test device is positioned in a test position.

[0237] The microfluidic test device comprises a first primary foil 44 attached to the first surface 40 of the body 4. The first primary foil 44 forms a part of the third primary fluid path 24 and the primary valve 26.

[0238] The microfluidic test device comprises a first secondary foil 46 attached to the first surface 40 of the body 4. The first secondary foil 46 forms a part of the first chamber 6 and the first valve 10.

[0239] The microfluidic test device comprises a first tertiary foil 47 attached to the first surface 40 of the body 4. The first tertiary foil 47 forms a part of the flow driving device 28. The flow driving device 28 is embodied as a second chamber with an outlet and a second valve arranged at the outlet of the second chamber. The microfluidic test device comprises a second primary foil 48 attached to the second surface 42 of the body 4.

[0240] The first fluid path 16 connects the first chamber 6 and the bottom of sample inlet 14. The second fluid path 18 connects the sample inlet 14 and the first reaction chamber 12. Thus, first buffer can be moved from the first chamber 6 through the first fluid path 16 into the sample inlet 14 by pressing on the first secondary foil 46 with a force sufficient to open the first valve 10, i.e. a force to apply a first pressure larger than the first pressure threshold. The first buffer thus flushes sample in the sample inlet 14 through the second fluid path 18 and into the first reaction chamber 12, where the first buffer, the sample and a primary reaction material are mixed. After a reaction, assisted by heating with heating assembly 30, has taken place, the flow driving device 28 is activated by pressing on the first tertiary foil 47 with a force sufficient to open the second valve 28B, i.e. a force to apply a second pressure larger than the second pressure threshold. Thereby, primary reaction liquid in the primary reaction chamber 12 is moved into the primary test chamber 22 via the third primary fluid path 24 by the primary valve 26 opening upon activation of the flow driving device 28. The primary test liquid in the primary test chamber 22 flows into the first lateral flow strip 32 for obtaining a readout of the test result.

[0241] FIG. 3 schematically illustrates a first (top) view of an exemplary microfluidic test device. The microfluidic test device 2A comprises a body 4 and a first chamber 6 having an outlet 8 provided with a first valve 10 and holding a first buffer having a first buffer volume.

[0242] The microfluidic test device 2 comprises a primary reaction chamber 12 and a sample inlet 14 for receiving a sample and being configured for feeding a sample having a sample volume, into the medical test device 2. A first fluid path 16 connects the outlet 8 of the first chamber 6 and the sample inlet 14 and a second fluid path 18 connects the sample inlet 14 and the primary reaction chamber 12. Further, the microfluidic test device 2 comprises a primary test part 20 comprising a primary test chamber 22 with a third primary fluid path 24 connecting the primary reaction chamber 12 and the primary test part 20. A primary valve 26 is arranged in the third primary fluid path 24. The microfluidic test device 2 comprises a flow driving device 28 configured to move fluid from the primary reaction chamber 12 to the primary test part 20, and a heating assembly 30 configured to heat a reaction fluid in the primary reaction chamber 12. The primary test part 20 comprises a first lateral flow strip 32 having a first end 34 connected to an outlet of the primary test chamber 22.

[0243] Further, the microfluidic test device 2A comprises a secondary reaction chamber 50, wherein the second fluid path 18 connects the sample inlet 14 and the secondary reaction chamber 50. Further, the microfluidic test device 2A comprises a secondary test part 52 comprising a secondary test chamber 54 with a third secondary fluid path 56 connecting the secondary reaction chamber 50 and the secondary test part 52. A secondary valve 58 is arranged in the third secondary fluid path 56. The flow driving device 28 is configured to move fluid from the secondary reaction chamber 50 to the secondary test part 52, and the heating assembly 30 is optionally configured to heat a reaction fluid in the secondary reaction chamber 50. The secondary test part 52 comprises a second lateral flow strip 60 having a first end 62 connected to an outlet of the secondary test chamber 54. The second lateral flow strip 60 has a length of 65 mm and a width of 3.0 mm.

[0244] FIGS. 4-10 show different views of exemplary microfluidic test system(s) and parts thereof. The microfluidic test system comprises a microfluidic test device (housing not shown) and a sample plug.

[0245] FIGS. 4 and 5 show perspective views of parts of microfluidic test device 2B with a sample plug 63 inserted in the sample inlet of the microfluidic test device. The microfluidic test device 2B comprises a body 4A and one or more foils attached to surfaces of the body. The microfluidic test device 2B comprises a first primary foil 44 attached to a first primary surface 40A of the body 4A and forming a part of the third fluid paths (primary and secondary), the primary valve, and the secondary valve of the microfluidic test device 2B. The first primary foil 44 optionally is a flexible plastic foil made of polypropylene that has been laser-welded to the body 4A made of polypropylene. The microfluidic test device 2B comprises a first secondary foil 46 attached to a first secondary surface 40B (See FIG. 6) of the body 4A and forming a part of the first chamber 6, first valve 10, second chamber 28A, and second valve 28B. The first secondary foil 46 optionally is a metal foil, such as an aluminium foil, and/or comprises one or more metal layers.

[0246] The first surfaces 40A, 40B are intended for facing upwards when the microfluidic test device 2B is positioned in a test position, and the second surface 42 is intended for facing downwards when the microfluidic test device 2B is positioned in a test position. The microfluidic test device 2B comprises a second primary foil 48 attached to the second surface 42 (See FIG. 7) of the body 4A. Further, the microfluidic test device 2B comprises a heating assembly 30 comprising a primary heating element. The heating element is arranged on the second primary foil 48 adjacent the primary reaction chamber and the secondary reaction chamber and configured to heat the primary reaction chamber (and primary reaction liquid) and the secondary reaction chamber (and secondary reaction liquid). The second primary foil 48 forms a part of the first, second, third (primary and secondary), and fourth fluid paths, primary reaction chamber, secondary reaction chamber, primary test chamber, secondary test chamber, and sample inlet (sample chamber).

[0247] The microfluidic test device 2B comprises a primary reaction chamber 12 and a sample inlet 14 for receiving a sample and being configured for feeding a sample having a sample volume, into the medical test device 2B. The microfluidic test device 2B comprises a primary reaction chamber plug 12A forming a part of the primary reaction chamber 12. The sample inlet 14 is configured for receiving the sample plug 63, the sample plug 63 carrying the sample. A first fluid path connects the outlet of the first chamber 6 and the sample inlet 14 and a second fluid path connects the sample inlet 14 and the primary reaction chamber 12. Further, the microfluidic test device 2B comprises a primary test part comprising a primary test chamber and a first lateral flow strip with a third primary fluid path connecting the primary reaction chamber 12 and the primary test part (primary test chamber). A primary valve is arranged in the third primary fluid path. The microfluidic test device 2B comprises a flow driving device 28 configured to move fluid from the primary reaction chamber 12 to the primary test part. The flow driving device 28 comprises a second chamber 28A having an outlet optionally provided with a second valve. The outlet of the second chamber is connected to the first fluid path via fourth fluid path.

[0248] Further, the microfluidic test device 2B comprises a secondary reaction chamber 50, wherein the second fluid path connects the sample inlet 14 and the secondary reaction chamber 50. The microfluidic test device 2B comprises a secondary reaction chamber plug 50A forming a part of the secondary reaction chamber 50. Further, the microfluidic test device 2B comprises a secondary test part comprising a secondary test chamber and a second lateral flow strip, with a third secondary fluid path connecting the secondary reaction chamber 50 and the secondary test part (secondary test chamber). A secondary valve is arranged in the third secondary fluid path. The flow driving device 28 is configured to move fluid from the secondary reaction chamber 50 to the secondary test part.

[0249] FIG. 6 shows a perspective view of a body of the microfluidic test device. A first valve body part 10A of the body 4A forms a part of the first valve 10. The first valve body part 10A is formed as a recess in the first secondary surface 40B of the body 4A. A first through-going bore 16A from the first secondary surface 40B to the second surface 42 forms a part of the first fluid path 18.

[0250] A primary reaction chamber body part 12B forms a part of the primary reaction chamber 12. The primary reaction chamber body part 12B is formed as a through-going bore in the body with the primary reaction chamber plug 12A and the second primary foil 48 forming top and bottom of the primary reaction chamber 12.

[0251] A primary test chamber body part 22A of the body 4A forms a part of the primary test chamber 22. The primary test chamber body part 22A is formed as a through-going bore from the first primary surface 40A to the second surface 42. The first primary foil 44 and the second primary foil 48 form top and bottom of the primary test chamber 22.

[0252] A primary valve body part 26A of the body 4A forms a part of the primary valve 26. The primary valve body part 26A is formed as a recess in the first primary surface 40A of the body 4A. A primary through-going bore 24A from the first primary surface 40A to the second surface 42 forms a part of the third primary fluid path 24.

[0253] A second valve body part 28C of the body 4A forms a part of the second valve 28B. The second valve body part 28C is formed as a recess in the first secondary surface 40B of the body 4A. A fourth through-going bore 29A from the first secondary surface 40B to the second surface 42 forms a part of the fourth fluid path 29.

[0254] A secondary reaction chamber body part 50B forms a part of the secondary reaction chamber 50. The secondary reaction chamber body part 50B is formed as a through-going bore in the body with the secondary reaction chamber plug 50A and the second primary foil 48 forming top and bottom of the secondary reaction chamber 50.

[0255] A secondary test chamber body part 54A of the body 4A forms a part of the secondary test chamber 54. The secondary test chamber body part 54A is formed as a through-going bore from the first primary surface 40A to the second surface 42. The first primary foil 44 and the second primary foil 48 form top and bottom of the secondary test chamber 54.

[0256] A secondary valve body part 58A of the body 4A forms a part of the secondary valve 58. The secondary valve body part 58A is formed as a recess in the first primary surface 40A of the body 4A. A secondary through-going bore 56A from the first primary surface 40A to the second surface 42 forms a part of the third secondary fluid path 56.

[0257] The microfluidic test device comprises a first flow strip chamber and a second flow strip chamber partly formed in the body 4A. The first flow strip chamber is accessible through a first opening 68 in the first primary surface 40A, and the second flow strip chamber is accessible through a second opening 70 in the first primary surface 40A. The first and second openings enables optical readout of lateral flow strips.

[0258] FIG. 7 shows another perspective view of the body 4A, where a first recess 16B forms a part of the first fluid path 16 between the first chamber 6 and the sample inlet 14 partly formed by sample inlet body part 14A. The second fluid path comprises a second primary fluid path 18A and a second secondary fluid path 18B. The second primary fluid path 18A connects the sample inlet 14 and the primary reaction chamber 12, and the second secondary fluid path 18B connects the sample inlet 14 and the secondary reaction chamber 50. A second primary recess 18C in the second surface 42 forms, together with the second primary foil 48, a part of the second primary fluid path 18A. A second secondary recess 18D in the second surface 42 forms, together with the second primary foil 48, a part of the second secondary fluid path 18B.

[0259] A fourth recess 29B in the second surface 42 forms, together with the second primary foil 48, a part of the fourth fluid path 29. The fourth fluid path 29 connects the outlet of the second chamber 28A at second valve 28B and the first fluid path 16. A third primary recess 24B in the second surface 42 forms, together with the second primary foil 48, a part of the third primary fluid path 24.

[0260] A third secondary recess 56B in the second surface 42 forms, together with the second primary foil 48, a part of the third secondary fluid path 56.

[0261] A first strip chamber recess 64A forms a part of first flow strip chamber for accommodating a first lateral flow strip. A second strip chamber recess 66A forms a part of second flow strip chamber 66 for accommodating a second lateral flow strip. The second primary foil 48 forms the bottom of the flow strip chambers 64, 66. A first end of the first lateral flow strip is arranged to overlap with the primary test chamber body part 22A such that test liquid in the primary test chamber 22 is fed to the first lateral flow strip. A first end of the second lateral flow strip is arranged to overlap with the secondary test chamber body part 54A such that test liquid in the secondary test chamber 54 is fed to the second lateral flow strip.

[0262] First holding elements 72, 74 are provided in the first flow strip chamber 64 of the body 4A. The first primary holding element 72 and the first secondary holding element 74 are configured to hold or fixate the first lateral flow strip in position between the body 4A and the second primary foil 48. Second holding elements 76, 78 are provided in the second flow strip chamber 66 of the body 4A. The second primary holding element 76 and the second secondary holding element 78 are configured to hold or fixate the second lateral flow strip in position between the body 4A and the second primary foil 48.

[0263] FIG. 8 shows a second or bottom view of the body 4A. The first fluid path 16 comprises and is split into a first branch formed by first branch recess 17A and second primary foil 48 and a second branch formed by second branch recess 17B and second primary foil 48. The first branch of the first fluid path feeds first buffer into a first inlet of the sample chamber formed by sample inlet and sample plug. The second branch of the second fluid path feeds first buffer into a second inlet of the sample chamber formed by sample inlet and sample plug. A plurality of sample chamber inlets in the microfluidic test device provides improved flushing of the sample. In one or more exemplary, the sample chamber only has a single inlet.

[0264] The second fluid path 18 comprises a first branch formed by first branch recess 19A and second primary foil 48 and a second branch formed by second branch recess 19B and second primary foil 48. Liquid from the sample chamber enters the first branch and the second branch of the second fluid path via respective first outlet and second outlet of the sample chamber. The first branch and the second branch of the second fluid path are joined in second fluid path joint 80 before splitting into second primary fluid path 18A and second secondary fluid path 18B.

[0265] FIG. 9 shows a cut out side view of the microfluidic test device 4A illustrating the heating assembly in further detail. The heating assembly 30 comprises a primary heating element 30A sandwiched between a first electrode layer 90 and a second electrode layer 92 for applying a primary voltage to the primary heating element. The heating assembly 30 (first electrode layer 90) is attached to the second primary foil 48 adjacent to and overlapping the primary and secondary reaction chambers 12, 50. A primary pellet of primary reaction material (not shown) is arranged in the primary reaction chamber, and a secondary pellet 94 of secondary reaction material is arranged in the secondary reaction chamber 50. The thin second primary foil 48 provides a good heat transport to reaction liquid in the reaction chambers 12, 50. Thus, low heat loss from the heating assembly to the reaction chambers and precise control of the reaction liquid temperature is provided.

[0266] FIG. 10 shows an exemplary body 4B of a microfluidic test device. The sample chamber/sample inlet 14A has a single inlet from the first fluid path. Further, the sample chamber/sample inlet 14A has a single outlet to the second fluid path. In one or more exemplary microfluidic test devices, a single inlet from the first fluid path may be combined with two or more outlets to the second fluid path. Further, two or more inlets from the first fluid path may be combined with a single outlet to the second fluid path.

[0267] The use of the terms first, second, third, fourth, primary, secondary, etc. does not imply any particular order, but are included to identify individual elements. Moreover, the use of the terms first, second, third, fourth, primary, secondary, etc. does not denote any order or importance, but rather these terms are used to distinguish one element from another. Note that the words first, second, third, fourth, primary, and secondary, are used here and elsewhere for labelling purposes only and are not intended to denote any specific spatial or temporal ordering. Furthermore, the labelling of a first element does not imply the presence of a second element and vice versa.

[0268] Although features have been shown and described, it will be understood that they are not intended to limit the claimed invention, and it will be made obvious to those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the claimed invention. The specification and drawings are, accordingly to be regarded in an illustrative rather than restrictive sense. The claimed invention is intended to cover all alternatives, modifications, and equivalents.

[0269] FIG. 11

[0270] Comparing the amplification time of pellet 1 and pellet 2 with the liquid LAMP-Assay as a reference, only pellet 2 performed similar to the reference. qLAMP Amplification time of reactions including different excipients compared to H2O control without excipient.

[0271] FIG. 12

[0272] LAMP results of reactions including different excipients compared to H2O control without excipient.

[0273] FIG. 13

[0274] qLAMP results testing different reaction buffer components lyophilized in the pellet. [0275] Pellet 1 (RD_4138) (RED) included the original 10 reaction buffer ([(NH4)2SO4] and was resuspended in Resuspension buffer (RD_4224) including betaine, MgSO4 and water. [0276] Pellet 2 (RD_4159) (GREEN) included 10 reaction buffer without (NH4)2SO4 and was resuspended in Resuspension buffer (RD_4225) including betaine, MgSO4, (NH4)2SO4 and water

[0277] Pellet 3 (RD_4158) (PINK) included 10 reactionBuffer with NH4(CH3COO) instead of (NH4)2SO4] and was resuspended in Resuspension buffer (RD_4224) including betaine, MgSO4 and water.

LIST OF REFERENCES

[0278] 2, 2A, 2B microfluidic test device [0279] 4, 4A, 4B body [0280] 6 first chamber [0281] 8 outlet [0282] 10 first valve [0283] 10A first valve body part [0284] 12 primary reaction chamber [0285] 12A primary reaction chamber plug [0286] 12B primary reaction chamber body part [0287] 14 sample inlet [0288] 16 first fluid path [0289] 16A first through-going bore [0290] 16B first recess [0291] 17A first branch recess of first fluid path [0292] 17B second branch recess of first fluid path [0293] 18 second fluid path [0294] 18A second primary fluid path [0295] 18B second secondary fluid path [0296] 18C second primary recess [0297] 18D second secondary recess [0298] 19A first branch recess of second fluid path [0299] 19B second branch recess of second fluid path [0300] 20 primary test part [0301] 22 primary test chamber [0302] 22A primary test chamber body part [0303] 24 third primary fluid path [0304] 24A primary through-going bore [0305] 24B third primary recess [0306] 26 primary valve [0307] 26A primary valve body part [0308] 28 flow driving device [0309] 28A second chamber [0310] 28B second valve [0311] 28C second valve body part [0312] 29 fourth fluid path [0313] 29A fourth through-going bore [0314] 29B fourth recess [0315] 30 heating assembly [0316] 30A primary heating element [0317] 32 first lateral flow strip [0318] 34 first end of first lateral flow strip [0319] 36 first end of body [0320] 38 second end of body [0321] 40 first surface of body [0322] 40A first primary surface of body [0323] 40B first secondary surface of body [0324] 42 second surface of body [0325] 44 first primary foil [0326] 46 first secondary foil [0327] 47 first tertiary foil [0328] 48 second primary foil [0329] 50 secondary reaction chamber [0330] 50A secondary reaction chamber plug [0331] 50B secondary reaction chamber body part [0332] 52 secondary test part [0333] 54 secondary test chamber [0334] 54A secondary test chamber body part [0335] 56 third secondary fluid path [0336] 56A secondary through-going bore [0337] 56B third secondary recess [0338] 58 secondary valve [0339] 58A secondary valve body part [0340] 60 second lateral flow strip [0341] 62 first end of second lateral flow strip [0342] 63 sample plug [0343] 64 first flow strip chamber [0344] 64A first strip chamber recess [0345] 66 second flow strip chamber [0346] 66A second strip chamber recess [0347] 68 first opening [0348] 70 second opening [0349] 72 first primary holding element [0350] 74 first secondary holding element [0351] 72 first primary holding element [0352] 74 first secondary holding element [0353] 76 second primary holding element [0354] 78 second secondary holding element [0355] 80 second fluid path joint [0356] 90 first electrode [0357] 92 second electrode [0358] 94 secondary pellet of secondary reaction material.

EXAMPLES

Example 1Stage of Components

[0359] Experiments were performed to compare the LAMP reaction with the components in two states, lyophilized as pellets and in liquid form. Liquid components were mixed together fresh from frozen individual components and used the same day (see Table 1).

[0360] Pellets were created through freeze drying while several components are left in a buffer that is used later to dissolve the pellet (see Tables 4 and 5).

TABLE-US-00003 TABLE 6 Results of example experiment. pellet 3xLOD 4-urin 30 min pos Negative pos controls: 2/2 pellet 3xLOD 4-urin 30 min pos neg. pos Positive pellet 3xLOD 4-urin 30 min pos controls: 2/2 pos pos.

[0361] The controls were made with fresh master mix as outlined above. DNA was spiked into urine to be used as the sample material. The reaction was left for 30 minutes in the chip with pellets and 25 minutes for the controls, since the heater in the chip needs longer to heat up.

[0362] The workflow is slightly different for both cases however. With the fresh samples (control), the urine is mixed into the master mix and then incubated. All components are liquid. Whereas, in the chip, the buffer does not contain all components and some are in the pellet. Firstly, the sample is mixed with the buffer and then the rest of the components are introduced (as the pellet is dissolved).

[0363] In conclusion, even at the limit of detection both versions of the assay perform equally well. Splitting the components of the LAMP assay into two groups while lyophilizing one and keeping the other in the liquid buffer is a good option that does not impact the performance of the assay. At the same time it allows to make the components stable since they can be stored in dry form. The components that remain in the liquid buffer would impede the lyophilisation process, but remain stable in liquid form.

Example 2Determination of Ammonium Ions Placement

[0364] The 10 reaction buffer included in LAMP assay includes 100 mM (NH.sub.4).sub.2SO.sub.4. However, (NH.sub.4).sub.2SO.sub.4 could be disadvantageous for the stability of the pellet. Therefore, we tested different pellet compositions (with and without (NH.sub.4).sub.2SO.sub.4) compared to our liquid LAMP Assay in a quantitative LAMP assay (qLAMP).

[0365] Besides the (NH.sub.4).sub.2SO.sub.4 composition of pellet 1 and 2 was the same.

[0366] Liquid qLAMP-Assay:

TABLE-US-00004 Final MM1 concentration qLAMP primer mix 1x MgSO.sub.4 (100 mM) 6 mM dNTP mix (4 25 mM) 4 1.4 mM 10x reaction Buffer 1x Betaine (5M) 0.8M EvaGreen (EG) (20x) 0.5x Bsm polymerase (8 U/l) 3.2 U/reaction Qia-water TOTAL DNA (ATCC VR886D)

[0367] 10 reaction Buffer: 200 mM TrisHCl (pH 8.8 at 25 C.), 100 mM KCL, 100 mM (NH.sub.4).sub.2SO.sub.4, 20 mM MgSO.sub.4, 1% (v/v) Tween 20

[0368] Pellet 1 (RD_4138) included the original 10 reaction buffer and was resuspended in 2 Resuspension buffer (RD_4224) and water.

TABLE-US-00005 Final MM2 for RD_4138 concentration qLAMP primer mix 1x RD_4224 (2x 1x Resusp Buff) EvaGreen (EG) (20x) 0.5x.sup. Qia-water TOTAL DNA (ATCC VR886D)/H.sub.2O RD_4224: 1.6M Betaine, 12 mM MgSO.sub.4

[0369] Pellet 2 (RD_4159) included 10 reaction buffer without (NH.sub.4).sub.2SO.sub.4 and was resuspended in 2 Resuspension buffer with (NH.sub.4).sub.2SO.sub.4 (RD_4225) and water.

TABLE-US-00006 Final MM3 for RD_4159 concentration qLAMP primer mix 1x RD_4225 (2x 1x Resusp Buff) EvaGreen (EG) (20x) 0.5x.sup. Qia-water TOTAL DNA (ATCC VR886D)/H.sub.2O RD_4225: 1.6M Betaine, 12 mM MgSO.sub.4, 20 mM (NH.sub.4).sub.2SO.sub.4

[0370] The MasterMixes (MM) were prepared. MM1 was distributed in a 96-well plate. MM2 and MM3 were used to re-suspend pellet 1 or pellet 2 respectively and afterwards this mixture was transferred into a 96-well plate. Genomic DNA was added in an appropiate concentration and qLAMP was performed in a CFX96 Touch and analysed with the BioRad CFX Manager Software.

[0371] Comparing the amplification time of pellet 1 and pellet 2 with the liquid LAMP-Assay as a reference where all the components are the same and in the same concentration but only in liquid form, only pellet 2 performed similar to the reference. With pellet 1 amplification was delayed 6 to 7 min compared to the reference assay. Pellet 2 showed a delay of only 2 min compared to a standard liquid LAMP Assay, which is in the range of our acceptance criteria (3 min)see FIG. 11.

[0372] Therefore, we concluded that the removal of (NH.sub.4).sub.2SO.sub.4 from the pellet supports the lyophilisation process and the stability of the pellets. Consequently, (NH.sub.4).sub.2SO.sub.4 is included in the First Buffer.

Example 3Freeze Drying Process of Components

[0373] All components that need to be included in the pellet for freeze drying are prepared by taking them out of storage (and thawing them). Most can now be mixed together directly, however Glycerol is interfering with the freeze drying process and is removed via filter columns from the polymerase prior to mixing. Then the mixed components are frozen solid by placing them in a 80 freezer for 30 minutes. Now the frozen components are placed in a lyophilisation machine and the water is removed under low pressure and low temperature. After 24 hours the samples are removed from the lyophilisation machine and used or placed in a dry environment for storage (for example an evacuated aluminium pouch).

Example 4Lysis of the Bacterial Cells

[0374] Urine samples 20 ml5 ml were collected and treated for 5 min with 10.1 ml 21 lysis buffer to achieve a final concentration of lysis buffer in urine of 10 mM EDTA, 0.4% Triton X-100 and 0.1 M AMP.

[0375] 10 l of the urine lysis buffer mixture including the released DNA was inserted in the device by a specially designed sample plug, said sample plug extending along an axis and having a first end with a first end surface and comprising a sample part and a handle part, the sample part comprising a first sample recess and a second sample recess formed in the first end surface for feeding a sample into a sample inlet of the microfluidic test device.

Example 5(NH4)2SO4 placement.

[0376] (NH4)2SO4 was removed from the reaction buffer and put it into the blister buffer or displace it with NH4(CH3COO) in the reaction buffer, since it can have some disadvantages on lyophilisation process and the stability of the pellets.

[0377] Different pellets produced were tested in our qLAMP reaction (FIG. 3). For better understanding the following reaction was developed (Tab. 1):

TABLE-US-00007 TABLE 1 LAMP reagents included in liquid reaction Stock conc. End concentration LAMP-reagents (to be prepared) in LAMP (50 ul) Water Primer-Mix 100 M 5.2 uM MgSO4 1M 6 mM dNTPs 4 25 mM 4 1.4 mM reaction 10x 1x Buffer* Betaine 5M 0.8M Polymerase 1600 U (8 U/l) 16 U (Bsm) *10x reaction buffer: 200 mM TrisHCl (pH 8.8 at 25 C.), 100 mM KCL, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% (v/v) Tween 20

[0378] We decided to exclude betaine from the reaction buffer and to clean up Bsm (glycerol-free; +5%) and additionally to exclude the 6 mM MgSO4 from the pellet (see 2a+b). Therefore, at this point the reaction was split into pellet and blister buffer according to table 2+3.

TABLE-US-00008 TABLE 2 LAMP reagents included in pellet Pellet reagents End concentration (50 ul) Primer-Mix 5.2 uM dNTPs 4 1.4 mM 10x reactionBuffer* 1x Polymerase (Bsm) 16.8 U *10x reaction buffer: 200 mM TrisHCl (pH 8.8 at 25 C.), 100 mM KCL, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% (v/v) Tween 20

TABLE-US-00009 TABLE 3 LAMP reagents included in the first buffer End concentration 1.1x BlisterBuffer-reagents stock concentration (50 ul) Water MgSO4 6.67 mM 6 mM Betaine 0.89M 0.8M

[0379] For testing the (NH4)2SO4 issue we now used the quantitative LAMP assay (qLAMP), which additionally includes EvaGreen and tested different pellet compositions (with and without (NH4)2SO4 or with NH4(CH3COO)) compared to our liquid LAMP Assay in.

[0380] Besides the (NH4)2SO4 composition pellet 1, 2 and 3 included the same substancessee FIG. 13. Comparing the amplification time of pellet 1, 2 and 3 with the liquid LAMP-Assay as a reference, only pellet 2 performed similar to the reference. With pellet 1+3 amplification was delayed 5 to 7 min compared to the reference assay. Pellet 2 showed a delay of only 2 min compared to SelfD reference, which is in the range of our acceptance criteria (3 min). [0381] Pellet 1 (RD_4138) (RED) included the original 10 reaction buffer ([(NH4)2SO4] and was resuspended in Resuspension buffer (RD_4224) including betaine, MgSO4 and water. [0382] Pellet 2 (RD_4159) (GREEN) included 10 reaction buffer without (NH4)2SO4 and was resuspended in Resuspension buffer (RD_4225) including betaine, MgSO4, (NH4)2SO4 and water [0383] Pellet 3 (RD_4158) (PINK) included 10 reactionBuffer with NH4(CH3COO) instead of (NH4)2SO4] and was resuspended in Resuspension buffer (RD_4224) including betaine, MgSO4 and water.

[0384] Therefore, we concluded that the removal of (NH4)2SO4 from the pellet supports the lyophilisation process and the stability of the pellets in the device. NH4(CH3COO) did not compensate the delay. Consequently (NH4)2SO4 is included in the BlisterBuffer.

[0385] Additionally, we added ProClin950 a biozide to the BlisterBuffer in order to inhibit microbial growth. ProClin950 did not inhibit the LAMP reaction and may be beneficial for long term storage of BlisterBuffer at RT.

[0386] The optimal distribution of reagents in pellet and BlisterBuffer is:

TABLE-US-00010 TABLE 4 LAMP reagents included in pellet End concentration Pellet reagents (50 ul) Primer-Mix 5.2 uM dNTPs 4 1.4 mM 10x reactionBuffer* 1x Polymerase (Bsm) 16.8 U *10x reaction buffer: 200 mM TrisHCl (pH 8.8 at 25 C.), 100 mM KCL, 20 mM MgSO4, 1% (v/v) Tween 20

TABLE-US-00011 TABLE 6 LAMP reagents included in BlisterBuffer End concentration 1.1x BlisterBuffer-reagents stock concentration (50 ul) Water MgSO4 6.67 mM 6 mM Betaine 0.89M 0.8M (NH4)2SO4 11.1 mM 10 mM ProClin 0.1% 0.09%