Systems and modules for nucleic acid amplification testing

Abstract

A system for nucleic acid amplification testing is provided. The system includes a consumable amplification module and a reader module for receiving the amplification module. The amplification module includes a reaction vessel for containing a test sample, a heater in thermal contact with the reaction vessel and including a heater element controllable to apply heat to the reaction vessel to heat the test sample, a temperature sensor for determining the temperature of at least one of the heater element and the test sample, and a heat sink or heat spreader in thermal contact with the heater. The reader module includes a heater controller for selectively controlling the heater element between an on state and an off state in response to the determined temperature of the heater element and / or the test sample, and an electrical heater interface for connecting the heater controller and the heater. The system includes a heat sink for subtracting heat from the reaction vessel to cool the test sample. The amplification module may include a heat sink. The receiver module may include a heat sink and a thermal interface, and the amplification module may include a heat spreader in thermal contact with the thermal interface.

Description

Technical Field

[0001] Background Art The present invention relates to a system for nucleic acid amplification testing and a module for such a system.

Background Art

[0002] Exemplary processes that require a reactor are DNA amplification by polymerase chain reaction (PCR), and the reactor is suitable for high-speed thermal cycling to shorten the completion time of PCR. Another example is DNA sequencing by synthesis, where base addition can be optimized by adjusting the temperature at each stage of a multi-step reaction.

[0003] PCR requires repeated temperature cycling between about 60°C and 95°C. Conventionally, heating and cooling have been performed using expensive Peltier elements to introduce heat from a heat sink to the sample when heating is required and from the sample to the heat sink when cooling is required. The heat sink is often cooled by a fan.

[0004] This method has many drawbacks, for example, as follows. The required equipment is large, expensive, and consumes a large amount of power. The heat capacity of the part of the device that changes temperature during thermal cycling is significantly larger than that of the sample, resulting in an increase in energy consumption and a slowdown in thermal cycling. The temperature gradient rate is limited, and the thermal cycling time is increased by the long heat diffusion time through the Peltier element and the parts used to accommodate the sample in thermal contact and through the sample itself. These factors result in slow and energy-inefficient PCR thermal cycling.

[0005] Conventional Peltier-based thermal cycling equipment includes several parts, including a layered bulk heat block and a complex thermal interface within the reader unit. This equipment requires a thermoelectric block and a large heat sink with fins for passive or forced convection cooling.

[0006] Due to the limited lifespan of this component due to the mechanical stress of repeated thermal cycling, it would be advantageous to discard large amounts of this material from the system, as well as to eliminate the thermoelectric (Peltier) element.

[0007] Another conventional Peltier-based thermal cycling device comprises a reaction vessel having a system for controlling the temperature of an internal sample. The reaction vessel comprises a polypropylene frame having thin heat-sealing films on both sides to seal the volume while providing a heat contact area. Thermal contact with the consumable is provided by spring clips and by the air pressure applied to the reaction vessel to expand the wall of the consumable. This arrangement has advantages over conventional thermal cycling by providing closer thermal contact with the low thermal mass part, but requires complex clamping and expansion to achieve thermal contact with the reaction mixture. The reaction volume is also considerably thicker compared to the heat contact area, which limits the gradient rate of thermal cycling as faster temperature changes are observed in the side volume compared to the central volume.

[0008] Another conventional Peltier-based thermal cycling device comprises heating and temperature sensing means at the thermal interface part of the reader. The temperature sensor occupies the central region of the sample that can be used for heat transfer, and the distance between the sensor and the heater track can result in a discrepancy between the measured temperature and the heater and sample temperatures.

[0009] The present invention aims to at least somewhat alleviate one or more of the problems of the prior art. Summary of the Invention Means for Solving the Problems

[0010] Summary of the Invention According to one aspect of the present invention, a system for nucleic acid amplification testing is provided. The system comprises a disposable amplification module and a reader module for receiving the amplification module. The amplification module includes a reaction vessel for accommodating a test sample, a heater having a heater element in thermal contact with the reaction vessel and controllable to apply heat to the reaction vessel to heat the test sample, a temperature sensor for determining the temperature of at least one of the heater element and the test sample, and a heat sink in thermal contact with the heater to draw heat from the reaction vessel to cool the test sample. The reader module includes a heater controller for selectively controlling the heater element between an on state and an off state in response to the determined temperature of the heater element and / or the test sample, and an electrical heater interface for connecting the heater controller and the heater.

[0011] According to another aspect of the present invention, a system for nucleic acid amplification testing is provided. The system comprises a disposable amplification module and a reader module for receiving the amplification module. The amplification module includes a reaction vessel for accommodating a test sample, a heater having a heater element in thermal contact with the reaction vessel and controllable to apply heat to the reaction vessel to heat the test sample, a temperature sensor for determining the temperature of at least one of the heater element and the test sample, and a heat spreader in thermal contact with the heater. The reader module includes a heater controller for selectively controlling the heater element between an on state and an off state in response to the determined temperature of the heater element and / or the test sample, an electrical heater interface for connecting the heater controller and the heater, a heat sink, and a thermal interface in thermal contact with the heat sink, wherein the thermal interface is adapted to be in thermal contact with the heat spreader when the amplification module is received by the reader module to draw heat from the reaction vessel to cool the test sample.

[0012] The present invention is particularly applicable to the thermal cycling PCR (polymerase chain reaction) method. As used herein, the term "disposable" has its ordinary meaning, i.e., it refers to a disposable product that typically reaches the end of its life and is discarded after single use.

[0013] Including a heater and a temperature sensor within the disposable amplification module advantageously enables rapid and accurate adjustment of the temperature of the reaction volume (reaction vessel) with a high degree of temperature uniformity. Thus, the claimed invention provides rapid and accurate thermal control in a low-cost device. By providing a heater controller in the reader module, the disposable amplification module can be conveniently made compact, avoiding the cost impact of having to discard the heater control device along with other more disposable and low-cost elements of the system provided in the amplification module.

[0014] The thermal interface and the heat sink may form an integral structure. The heat spreader may have a smaller heat capacity than the heat sink.

[0015] The reader module may include a cooling device configured to cool the heat sink. The cooling device may include a thermoelectric cooler or a fan.

[0016] The system may include a heater support configured to provide such thermal contact between the heater and the heat sink or the heat spreader. The product of the thermal resistance and the area of the heater support may be in the range of 1×10 -4 to 1×10 -2 K.m 2 / W, preferably in the range of 3×10 -4 to 3×10 -3 K.m 2 / W.

[0017] The reader module can be provided with an optical system for detecting reactions in a test sample when an amplification module is received by the reader module. The optical system includes an optical interface for connecting the optical system to the amplification module, a light source for supplying light to the test sample, and a photodetector for detecting changes in light transmission, absorption, reflection, or emission by the test sample.

[0018] The reader module can be provided with a pneumatic system for controlling the pressure and / or movement of a test sample when an amplification module is received by the reader module. The pneumatic system includes a pneumatic interface for connecting the pneumatic system to the amplification module, a pneumatic pump for providing pressure and / or movement to the test sample via the pneumatic interface, and a pneumatic controller for controlling the pneumatic pump.

[0019] The amplification module may include a detector for detecting electrochemical changes in a test sample contained in a reaction vessel, and the reader module may be adapted to receive a signal from the detector via an electrical heater interface when the amplification module is received by the reader module.

[0020] The heater element may include a temperature sensor, and the temperature of the heater element can be determined from the electrical resistance of the heater element.

[0021] The reader module may be adapted to receive a plurality of such amplification modules. The reader module may be adapted to perform synchronous and / or asynchronous tests on a plurality of test samples contained by respective amplification modules.

[0022] According to another aspect of the present invention, there is provided a consumable amplification module for insertion into a reader module of a system for nucleic acid amplification testing. The amplification module comprises a reaction vessel for containing a test sample, a heater comprising a heater element in thermal contact with the reaction vessel and adapted to receive a control signal from an external controller to apply heat to the reaction vessel to heat the test sample, a temperature sensor for determining the temperature of at least one of the heater element and the test sample, and a heat sink in thermal contact with the heater for removing heat from the reaction vessel to cool the test sample.

[0023] According to another aspect of the present invention, there is provided a consumable amplification module for insertion into a reader module of a system for nucleic acid amplification testing. The amplification module comprises a reaction vessel for containing a test sample, a heater comprising a heater element in thermal contact with the reaction vessel and adapted to receive a control signal from an external controller to apply heat to the reaction vessel to heat the test sample, a temperature sensor for determining the temperature of at least one of the heater element and the test sample, and a heat spreader in thermal contact with the heater, the heat spreader being adapted to be in thermal contact with a heat interface of a heat sink of the reader module when the amplification module is received by the reader module for removing heat from the reaction vessel to cool the test sample.

[0024] Brief Description of the Drawings Next, examples will be described with reference to the accompanying drawings.

Brief Description of the Drawings

[0025]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10A

Figure 10B

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

DETAILED DESCRIPTION OF THE INVENTION

[0026] DETAILED DESCRIPTION OF THE INVENTION Referring to FIG. 1, an exemplary system for nucleic acid amplification testing (NAAT) includes a consumable unit 100 and a reader unit 101. The consumable unit 100 includes a reaction vessel 102, a heater 103a and a temperature sensor 103b (collectively labeled 103 in FIG. 1), and a heat sink 104. The reader unit 101 includes temperature control and heater drive electronics 106. This exemplary system requires only one electrical interface 105 to perform NAAT.

[0027] In use, the reaction vessel 102 contains the reagents and samples required to perform the reaction for NAAT. The consumable unit 100 can contain pre-loaded reagents and can add test samples during use.

[0028] Since test results are often measured by reaction rates that are highly dependent on temperature, it is important to achieve accurate and uniform thermal control in NAAT. By including the heat sink 104, heater 103a, and temperature sensor 103b in the consumable unit, it becomes possible to have uniform and permanent thermal contact between the sample volume in the reaction vessel 102 and the temperature-controlled thermal engine, i.e., the heater 103a and temperature sensor 103b as well as the heat sink 104.

[0029] Depending on the situation, for example, due to environmental sustainability or cost concerns, it may not be desirable to include a high thermal mass heat sink in the consumable unit 100 to avoid the disposal of a non-negligible amount of metal heat sink material in each test. FIG. 2 shows an alternative configuration that reduces the material in the consumable unit 200 by providing a heat sink 204 within the reader 201. In this example, the reader 201 includes a thermal interface 207 between the heat sink 204 and the consumable unit 200 in addition to the temperature-driven electronics 206 and electrical interface 205.

[0030] When using a particularly simple and low-cost connection mechanism, it may be difficult to achieve good uniformity of thermal contact between the reader surface and the consumable part. To address this problem, the consumable part comprises a thin thermal diffusion layer 206 of a material having a high thermal conductivity. In this example, the consumable part 100 includes an optional heater support layer 208 disposed between the heat spreader 206 and the heater 103a and the temperature sensor 103b. Alternatively, the heat spreader 206 is arranged to be in direct, close and uniform contact with the reaction vessel 202 and the heater temperature sensor 203b. In this example, the heater support is configured as a continuous layer of material between the heat spreader 206 and the heater 103a and the temperature sensor 103b, but it will be understood that the heater support may be configured in a variety of different ways to support the heater 103a and the temperature sensor 103b on the heat spreader 206. For example, the heater support may comprise a ribbed structure having discontinuities in the structural material between the heat spreader 206 and the heater 103a and the temperature sensor 103b.

[0031] Also, when the heater 103a is not driven, it is desirable to control the cooling rate of the heater 103a and the reaction vessel 102 by the heat flow to the heat sink 104. The cooling rate depends on the thermal resistance R T of the heater support layer 208, which can be optimized to minimize the thermal cycling time for a given temperature profile and the heat sink temperature T sink and the heater output p Heat . The time required for thermal cycling between T LOW and T HIGH is minimized when the heating time is equal to the cooling time, and this condition is satisfied when R T =R T,Opt .

[0032] R T,Opt =(T HIGH +T LOW -2 T Sink ) / p Heat . The attached Table 1 shows examples of values of heater power, optimum thermal resistance, and thermal cycle time. These are for an area of 50 mm2 and are shown for a reaction area with a heat capacity of 0.04 J / K, and circulate between 60°C and 95°C at a heat sink temperature of 30°C.

[0033] Attached Table 2 shows examples of values of heater power, optimal thermal resistance, and thermal cycle time when the thermal cycle includes a holding step at 72°C for a duration of 1 second. These are for a reaction area of area 50 mm 2 and are shown for a reaction area with a heat capacity of 0.04 J / K, and circulate between 60°C and 95°C at a heat sink temperature of 30°C.

[0034] Figure 3 shows an exemplary product for all samples to respond to a system for performing NAAT using a low-cost portable instrument for performing these tests in an environment near the patient. In this example, the reader unit 301 includes a display 302 for showing the results of the test to the user and buttons 305 for user control. The consumable unit 300 includes a space (i.e., cavity or volume) for simple sample loading 303 and a fillable reagent well 304 to enable non-laboratory staff to perform the test with little or no training.

[0035] Figure 4 is a schematic diagram of the system of the product shown in, for example, Figure 3. In this example, the consumable unit 400 includes a sample preparation function 411 for preparing a sample loaded from a swab 412 that is ready for NAAT in a reaction vessel 402. A heater, temperature sensor, and heat spreader 403 are included in the consumable unit 400. The consumable unit and reader 401 are electrically 420, thermally 421, optically 422, and fluidically 423 interfaced. The electronics within the reader 401 control the temperature of the external user interface (302 and 305 in Figure 3) and the heater 403 by supplying power from a power supply 409 mediated by a closed-loop heater control device 406. The control of the system can include active cooling 407 of the heat sink 404 via, for example, a Peltier element or a variable speed fan.

[0036] The sample preparation process 411 can be controlled by a fluid system 410 that includes, for example, an air pump and a pressure sensor to provide metered pressure and volume to the fluid of the consumable unit 400 via an air pressure interface. This exemplary system uses an optical detection method to detect the results of the test. The optical interface 422 between the reader 401 and the consumable reaction vessel (or fluid cell) 402 is port-connected to a reader optical system that includes, in this example, a light source and lens 413, excitation and emission filters 414, and a photodetector 415 in this example. This configuration can be used to detect the presence of amplified DNA via a fluorescent probe by exciting at one wavelength of light and detecting at the wavelength of fluorescence.

[0037] Figure 5 shows a cross-section of an exemplary small reader 501 with the consumable unit 500 inserted (received). The electrical connection 520 and the pneumatic connection 523 are arranged on the same side of the reader 501 to enable a single-sided consumable unit 500 that can be conveniently inserted into the instrument 530. In one example, a secondary mechanical interface is provided between the consumable heat spreader or heat sink 504 and the reader heat sink 521 to increase the thermal mass for tests that require rapid cooling thermal cycling. The thermal interface between the heat sink 504 of the consumable unit 500 and the heat sink 521 of the reader 501 can include a sliding contact for simple mechanical assembly and insertion of the consumable unit 500.

[0038] The analyte can be detected directly using the optical interface 522 or via the same electrical interface 520 as the heater control. When the optical interface is used, it can be configured as shown in Figure 5 using illumination and detection perpendicular to the plane of the fluid section including the reaction volume 502 to reduce the number of optical surfaces on the consumable unit 500. A series of detection methods can be used to detect and / or quantify nucleic acids in the sample, and an overview is included in the attached Table 3.

[0039] FIG. 6 shows an exemplary product for a multi-up system in which a desktop reader 601 can accept several consumables 630 and, at the same time, execute multiple tests synchronously or asynchronously with each other. The reader 601 includes a display 602 for outputting test results. The advantage of this system in a patient-proximate setting is that part of the system control can be shared among separate consumable interfaces, thus increasing the throughput of the tests and reducing the per-test cost of the reader.

[0040] The reaction vessel should maintain good temperature uniformity and control. In particular, the structure of the reaction volume of the vessel can be made thin compared to its width, and the reaction volume dominates the heat capacity of the vessel by means of uniform and good thermal contact between the heater, the temperature sensor, and the reaction volume.

[0041] FIG. 7 is a cross-sectional view of an exemplary system consumable. In this example, the reaction volume cavity 710 is formed from a fluid substrate material 702 via a process such as embossing, injection molding, etc., and is closed via a thin sealing layer 711. The thickness of the sealing layer 711 is determined by the requirements for temperature accuracy and thermal cycling speed within the reaction volume 710. This film may be attached to the fluid substrate material via an adhesive or a heat-sealing process, and the other side of this film may be attached to the heater and the temperature sensor via the same or a similar process.

[0042] In this example, heating and temperature sensing are performed via resistance traces 703 on an insulating substrate 705 that is inexpensively manufactured using standard laminated and etched printed circuit board (PCB) or flexible circuit processes. These traces may also be formed by processes such as sputtering, evaporation, or electroplating. The back surface of the insulating substrate material 705 is joined to a heat sink 704 having a high thermal mass with a high uniform thermal conductivity in order to enable passive cooling of the reaction volume 710 over the course of the test. The cooler heat sink 704 and the hotter reaction volume 710 thermally equilibrate, and thus, in order to maintain a stable and consistent cooling rate, the temperature rise of the heat sink 704 must be minimal, e.g., less than 10 °C, over the course of the test.

[0043] FIG. 8 shows a cross-sectional view of an example system as shown in FIG. 2, where the heat sink portion 804 is provided in the reader portion and the system includes a thermal interface 820 between the consumable portion and the reader. Conventional systems have difficulty achieving a uniform and low-resistance thermal contact. In contrast, in this example, the consumable portion includes a thermal diffusion layer 806 for providing a laterally uniform thermal surface to the reaction vessel 810, reducing the accuracy required of the thermal interface 820. This thermal diffusion layer 806 can be constructed by the same process as the electrical circuit traces 803 on the back side of the insulating substrate 805, e.g., another copper layer on a multilayer printed circuit board. The reaction volume 810, the fluid substrate material 802, and the thin fluid seal layer 811 are as shown in FIG. 7.

[0044] In systems that use optical detection methods, it may be necessary to include an optical layer in the stack of materials to prevent spurious signals and improve the noise floor. FIG. 9 shows the addition of this optical layer 921 in a cross-section of the reaction vessel 910, and the optical substrate 902 is sufficiently transparent to observe changes within the reaction volume 910. For example, PCB materials such as FR4 have fluorescent properties that can interfere with the fluorescence detection of samples, and thus it is beneficial to include an opaque layer between or within the fluid encapsulation layers 911 and 903 heater circuit traces. The opaque layer may be a thin metal layer that functions as a barrier to light transmission and as a reflector to increase the signal received by the photodetector.

[0045] Using a metal or other high thermal conductivity layer at the location indicated by 921 in FIG. 9 has the additional advantage of being a thermal diffusion layer. The thermal diffusion layer can increase the temperature uniformity within the sample volume 910 while having a thermal capacity significantly smaller than that of the sample or the surrounding fluid cell, so including a heat spreader layer does not significantly increase the heating and cooling power required to change the sample temperature.

[0046] FIG. 10A shows an exemplary layout of the printed circuit traces 1003 for two reaction volumes 1000 of the heater-equipped consumable, and the temperature measurement traces 1001. A single layer is designed to form an electrical interface 1004 with the reader to be compatible with a standard off-the-shelf PCIE edge connector. The traces 1003 connect another set of electrodes for heaters, temperature sensing, and fluid measurement and proceed through the consumable. In this example, to detect that the fluid within the chip has reached this stage of the assay, capacitance sensing electronics within the reader unit are used to measure the change in the dielectric constant of the fluid layer on the circuit board as the liquid flows past the electrodes.

[0047] The advantage of detecting the presence or flow of liquid on the consumable part is that it enables fluid control without the need to measure or control the displacement generated by the reader to calculate the position of the liquid on the consumable part. There are several techniques that can be used with this consumable part structure, and both capacitance and resistance sensing can detect the presence of liquid near a set of electrodes. The resistance sensing technique is more stable but requires an electrode that makes electrical contact with the reaction volume, while capacitance measurement requires more sensitive electronics but can be measured through a thin fluid encapsulation layer and can use electrical tracks manufactured on the same printed circuit layer as the heater track.

[0048] A further technique for detecting the presence or flow of liquid within the consumable part is to perform thermal measurements using heaters and sensing traces. Flow is measured by observing the time of flight of a thermal pulse using a central heater trace located between upstream and downstream temperature sensing traces. The presence of liquid can be measured by observing an increase in heat capacity and a corresponding decrease in temperature change near or at a heater track located near the fluid channel.

[0049] Figure 10B shows an enlarged view of the heater region on the consumable part circuit board and shows various electrical connections to the heater. A four-wire Kelvin connection to the heater track is provided for accurate measurement of the heater resistance and thus the reaction vessel temperature. By dividing the heater electrical connection into drive currents 1032, 1034 and voltage sense 1036, 1038, the voltage across the heater region is measured at the points of entry and exit of the current to the heater track. Little current flows across the voltage sense interface, and the effect of the electrical resistance within the electrical interface between the reader and the consumable part on the measurement of the heater trace resistance is minimized. This is advantageous for separate / detachable readers and consumable parts as it uses resistance temperature measurement to improve the accuracy and reliability of reaction vessel temperature control.

[0050] The two outer connection parts 1042 and 1044 are provided to drive the guard heater track to a temperature higher than that of the main heater, and by compensating for the edge effect, improve the temperature uniformity within the heater zone, thereby maintaining a uniform temperature across the entire reaction volume and improving the efficiency of the reaction.

[0051] In one example, the consumable portion can be designed to perform a polymerase chain reaction (PCR) assay to detect the presence of a specific sequence. FIG. 11 shows a process flowchart of how such an assay can be performed by the disclosed system. The sample is collected using a sampling device 1101, i.e., a swab, and loaded into the consumable portion. The consumable portion or the sampling device may contain an eluent that contains enzymes and reagents for eluting cells or DNA from the sampling device 1102, and the fluid system pushes the liquid into a filtration system 1104 to remove contaminants and reaction inhibitors while retaining DNA and enzymes. If the assay is designed to examine cellular DNA, the cells need to undergo lysis 1105 prior to the amplification step 1106 to break down the DNA strands without the cell wall. If a thermal lysis method is used, lysis and amplification are performed within a temperature-controlled reaction vessel.

[0052] An advantage over conventional systems and methods is that the temperature of the entire reaction volume can be accurately controlled and changed (heated or cooled) at a fast gradient rate. When thermal cycling amplification technology is used, this system can typically perform the required number of thermal cycles between 20 and 60 to quantitatively detect DNA amplification and determine the presence and concentration of the target DNA sequence in a much shorter time than conventional DNA detection devices. After thermal cycling, the amplified DNA detected via one of the methods described in Table 1 above and the results are displayed to the user or uploaded to an online database.

[0053] In one example, when the nucleic acid of interest is ribonucleic acid (RNA), the consumable can be designed to perform a "reverse transcription" PCR test (RT-PCR). Referring to FIG. 12, after a sample is taken and loaded into the device 1201, the sample is eluted from the swab with an enzyme-free eluent 1203, filtered, lysed, and the RNA is released into the reaction mixture. Next, the mixture is introduced into the RT enzyme. This is done at this stage to prevent damage to the RT enzyme during the lysis step and to allow the enzyme to be lyophilized to extend the shelf life of the consumable. A known problem associated with the resuspension 1206 of the lyophilized enzyme is that the enzyme can be foamy and has a tendency to generate air bubbles in the mixture, which can make amplification detection difficult. To mitigate this risk, a degassing or bubble trap step 1207 is used to ensure that a non-gaseous mixture ends up in the amplification chamber, where the mixture is heated to reverse transcribe the RNA into DNA 1208 and thermocycled to detect the presence of a specific DNA sequence 1209. The amplified DNA is detected via one of the methods described in Table 1 above.

[0054] The thermal design of the reaction vessel within the consumable of the system can be optimized to perform the NAAT temperature-dependent process described in the attached Table 4.

[0055] Figure 13 shows a plan view of an example of a consumable part of a NAAT system having areas designated for various assay processes. The snap closure lid mechanism 1301 seals the sample within the consumable part. This feature can also be used to pressurize the fluid system, drive liquid through channels, or provide the operating force to break a foil seal. The electrical connection area 1302 is designed to be plugged into a standard PCB edge connector, and on the same surface, ports for push-fit pneumatic connections 1303 are arranged. A reservoir for storing the eluate 1304 may be sealed from the dry part of the consumable part using a foil seal or the like, and the seal may be broken after sample loading into area 1305. The eluate carries the sample from the loading area 1305 through the filter 1306 and then to a reaction area 1307 designed to dissolve any sample cells or virus particles to release nucleic acids. The lysis may be performed by heating the sample, in which case the reaction area 1307 is located over the first heater 1312.

[0056] After the lysis step, the mixture of the sample and the eluate can be mixed with dried or lyophilized reagents and enzymes at 1308 and then flowed into the bubble trap / degassing area 1309. Next, the bubble-free mixture is moved into a second reaction vessel 1310 located over the second heater 1313. The sample may be divided into separate detection chambers within the second reaction vessel 1310, each containing a different primer set for amplifying a specific nucleic acid sequence. A separate detection chamber may be used for each test or control sequence to be detected. The second heater 1313 may be used to provide thermal cycling for PCR amplification. The results of the test can be optically detected within the optical detection area 1311. After amplification, optionally, the temperature of the reaction vessel can be raised and a melting curve can be measured by detecting the thermal denaturation of the amplified DNA.

[0057] Figure 14 shows a plan view of an exemplary consumable reaction vessel for detecting four specific nucleic acid sequences, or three test sequences and a positive control sequence. In this example, the detection chamber 1401 is formed from a serpentine channel, ensuring uniform filling of all four channels without air pockets. As the chamber fills, the input to the reaction vessel 1402 is much wider than the outlet 1403 to increase the viscous resistance, and it can be seen that the capillary pressure surely bursts at each detection chamber input.

[0058] Figure 15 shows a preferred range 1501 of thermal resistance when the holding step is included in the thermal cycle. The PCR cycle consists of melting, annealing, and extension steps, and the extension is often the most time-consuming part of the reaction and may require a holding step. In this example, a holding step of 1 second at 72 °C is included to allow time for the extension in the PCR reaction. The time required for extension can vary depending on the polymerase speed and the length of the DNA sequence to be amplified. The 1-second holding step may be appropriate for the rapid amplification of DNA sequences having a length in the range of 100 to 150 base pairs, typically the length used in nucleic acid-based diagnostic tests, and longer sequences generally require longer extension times. The duration of the holding step should optimally be long enough to allow extension but not significantly long, otherwise it will dominate the overall cycle time and undesirably extend it. Those skilled in the art will readily understand that the adjustment of the holding step duration exemplified as 1 second in the above example can be made without significantly affecting the overall operation of the present invention.

[0059] The graph in Figure 15 shows a preferred range of values of thermal resistance to enable both low thermal cycle time and low energy consumption per cycle, and the minimum thermal cycle time t cycle 1504 becomes undesirably large (>5 seconds) when the thermal resistance is greater than the maximum preferred value 1503, while the energy consumption E cycle 1505 becomes undesirably large (>10 J) when the thermal resistance is less than the minimum preferred value 1502. In summary, the range 3×10-3 from 3×10 -2 K.m 2 The product R of the thermal resistance × cell area from 2 T、Opt ×A cell is such that the energy consumption per cycle is low (A cell =5×10 -5 m 2 in which case E cycle <10 J) for high-speed thermal cycling (t cycle <5 seconds) is preferable.

[0060] In this detection chamber configuration, single-wavelength fluorescence detection can be used. By dividing the reaction mixture into several chambers within the reaction vessel, it becomes possible to evaluate multiple probes with only one type of light source.

[0061] The rapid diagnosis of patients presenting symptoms of viral infections can enable a faster treatment path for patients and a reduction in the burden on healthcare services, as there are fewer patients who need to be isolated while waiting for test results. Conventionally, molecular tests (e.g., NAAT) are performed by central laboratories and take at least several hours, if not days, to return test results to healthcare providers. The described system brings this test to the patient and has the potential to reduce the sample and bring the response time down to minutes.

[0062] One specific use of this system is to detect influenza virus infection in an environment close to the patient. Samples from the patient may be collected from throat, nasal, or cheek swabs, loaded into the consumable part, whereby the virus is eluted by the eluent, and the reaction mixture is filtered to remove large contaminants before moving to the lysis reaction vessel. After lysis, the mixture is presented to reverse transcriptase and degassed if necessary. Next, the mixture is divided into the detection chamber in reaction vessel 1402, and each of the four chambers 1401 may contain probes and a positive control for the major strains of influenza A, influenza B, and respiratory syncytial virus (RSV). This reaction vessel is controlled to a high temperature for reverse transcription, and then the mixture is thermally cycled to perform PCR.

[0063] The system can analyze the results of the test using the thermal characteristics of the reaction mixture measured using a temperature sensor within the consumable section.

[0064] All mechanical interfaces to the consumable section, such as pneumatic and electrical contacts, may be provided on a single side to enable a simple and robust consumable insert.

[0065] The consumable section can include a macroscopic fluid substrate layer for accommodating the sample preparation and reaction vessel volume, a thin fluid sealing layer, an electrical circuit trace forming a heater and temperature sensing region on an insulating substrate material, and a thermal diffusion layer to avoid the need for exact thermal contact between the consumable section and the reader.

[0066] In the consumable section, a thin thermal diffusion layer may be sandwiched between the electrical circuit trace layer and the fluid reaction chamber.

[0067] The consumable section can be provided with a fluorescence blocking layer between the electrical circuit trace and the reaction volume to eliminate optical background noise from intrinsic fluorescence within the substrate layer, such as an optically opaque solder mask on the circuit or metallization within or on the thin fluid sealing layer.

[0068] The consumable section may be composed of a fluid layer having a thermally fusible or adhesive coating polymer film laminated to form a bond between the heater and the reaction vessel.

[0069] The consumable section can be provided with a liquid sensing electrode configured to detect changes in capacitance or resistance interfaces on the same electrical substrate as the heater to detect the fluid filling state at important process stages.

[0070] The consumable section can be provided with an electrode interfacing with the same electrical substrate as the heater to detect the presence or flow of fluid via a thermal detection method.

[0071] The consumable part can include an area for performing an analysis process for DNA amplification, and the area includes a sample loading area, a storage area for pumping an eluate containing enzymes and reagents, an elution and filtration area, a container for thermal or chemical cell lysis, a reaction container equipped with an accompanying heater and control device for DNA amplification, an area for pneumatic or mechanical connection for driving reagents through the consumable part assay area, and an electrical connection.

[0072] The method of using the consumable part can include manual user loading during processing, i.e., pre-filtration and concentration of the sample by operating a syringe of elution buffer through a swab during sample loading.

[0073] The consumable part can include a microfluidic mechanism on the chip after the thermal lysis and resuspension steps to capture or remove air bubbles from the fluid cell.

[0074] The method of using the consumable part can include lysing the sample cell in the range of 60 - 90 °C in a temperature-controlled reaction container, with 75 - 80 °C being preferred.

[0075] The method of using the consumable part can include reverse transcription of target RNA to DNA in a temperature-controlled reaction container in the range of 50 - 70 °C, preferably 60 - 65 °C.

[0076] The consumable part can include a process area within the fluid substrate, such as a serpentine channel for creating a lysis reaction container or an amplification reaction container.

[0077] The presence of amplicons can be detected using spatially multiplexed fluorescence of a single wavelength, the consumable part includes a plurality of spatially separated amplification regions with different primer sequences, and the reader includes a plurality of detectors that coincide with the consumable part amplification regions.

[0078] The system can be used to detect the presence of viruses. A virus sample is collected, eluted from the sampling device, filtered to remove as many cells and other large contaminants as possible, and then the virus medium is lysed to release RNA. Reverse transcriptase is mixed with the eluted RNA, then air bubbles are extracted, and the medium is transferred to a detection reaction vessel where reverse transcription occurs and PCR primers and / or probes are mixed. Here, thermal cycling occurs and the presence of the target virus is detected.

[0079] Using this system, the presence of one or more strains of influenza virus, such as influenza A and influenza B, as well as the presence of human orthopneumovirus, previous human respiratory syncytial virus, and a positive control for evaluating the correct operation of the system can be detected.

[0080] It will be understood that the present invention has been described in connection with its preferred embodiments and can be modified in many different ways without departing from the scope of the invention as defined by the appended claims.

[0081]

Table 1

[0082]

Table 2

[0083]

Table 3

[0084]

Table 4

Claims

1. A system for nucleic acid amplification testing, the system comprising a consumable amplification module and a reader module for receiving the amplification module, wherein the consumable amplification module a reaction vessel for containing a test sample, a heater in thermal contact with the reaction vessel and comprising a heater element controllable to apply heat to the reaction vessel to heat the test sample, a temperature sensor for determining the temperature of at least one of the heater element and the test sample, a heat spreader layer in thermal contact with the heater and adapted to transfer heat from the reaction vessel in the amplification module to the reader module, and wherein the reader module a heater controller for selectively controlling the heater element in the amplification module between an on state and an off state in response to the determined temperature of the heater element and / or the test sample, an electrical heater interface for connecting the heater controller in the reader module to the heater in the amplification module, a thermal interface adapted to be in thermal contact with the heat spreader layer when the amplification module is inserted into the reader module for transferring heat from the reaction vessel to cool the test sample, and a heat sink in thermal contact with the heat spreader layer via the thermal interface for removing heat from the reaction vessel. A system.

2. The system according to claim 1, wherein the thermal interface and the heat sink form an integral structure.

3. The system according to claim 1 or 2, wherein the heat spreader layer has a smaller heat capacity than the heat sink.

4. The system according to any one of claims 1 to 3, wherein the reader module further comprises a cooling device configured to cool the heat sink, preferably the cooling device comprises a thermoelectric cooler or a fan.

5. The system according to any one of claims 1 to 4, comprising a heater support configured to provide the thermal contact between the heater and the heat sink or the heat spreader layer.

6. The product of the thermal resistance and the area of the heater support is from 1×10 －４ to 1×10 －２ K·m ２ / W, preferably from 3×10 －４ to 3×10 －３ K·m ２ / W, and the system according to claim 5.

7. The reader module comprises an optical system for detecting a reaction in the test sample when the amplification module receives it from the reader module, and the optical system comprises an optical interface for connecting the optical system to the amplification module, a light source for supplying light to the test sample, a photodetector for detecting changes in light transmission, absorption, reflection, or luminescence by the test sample The system according to any one of claims 1 to 6. **Claim 8** The reader module comprises a pneumatic system for controlling the pressure and / or movement of the test sample when the amplification module receives it from the reader module, and the pneumatic system comprises a pneumatic interface for connecting the pneumatic system to the amplification module, a pneumatic pump for providing pressure and / or movement to the test sample via the pneumatic interface, a pneumatic controller for controlling the pneumatic pump The system according to any one of claims 1 to 7. **Claim 9** The amplification module comprises a detector for detecting electrochemical changes in the test sample contained in the reaction vessel, The reader module is adapted to receive a signal from the detector via the electrical heater interface when the amplification module receives it from the reader module, The system according to any one of claims 1 to 8. **Claim 10** The heater element comprises the temperature sensor, and the temperature of the heater element is determinable from the electrical resistance of the heater element. The system according to any one of claims 1 to 9. **Claim 11** The reader module is adapted to receive a plurality of the amplification modules. The system according to any one of claims 1 to 10. **Claim 12** The reader module is adapted to perform synchronous and / or asynchronous tests on a plurality of test samples contained by respective ones of the amplification modules. The system according to claim 11. **Claim 13** A consumable amplification module for insertion into a reader module of a system for nucleic acid amplification testing, the amplification module comprising a reaction vessel for containing a test sample, A heater comprising a heater element adapted to receive a control signal from an external controller to heat the reaction vessel by applying heat to the reaction vessel in thermal contact therewith to heat the test sample; A temperature sensor for determining the temperature of at least one of the heater element and the test sample; A heat spreader layer in thermal contact with the heater, the heat spreader layer being adapted to be in thermal contact with a heat interface of the heat sink of the reader module when the amplification module is inserted into the reader module to transfer heat from the reaction vessel to the heat sink to cool the test sample; A consumable amplification module comprising.

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