Cartridge for nucleic acid analysis

Abstract

A cartridge for nucleic acid analysis comprising: a cartridge body; a plurality of reagent ports each in fluid connection with an external reagent storage tube; a reaction chamber configured for nucleic acid extraction and in fluid communication with a first number of the reagent ports, the reaction chamber projecting from the cartridge body; a PCR chamber configured for mixing a PCR mix from one of the reagent ports with eluted nucleic acid to form a PCR mixture and in fluid communication with the reaction chamber and in fluid communication with a second number of the reagent ports; a PCR chamber configured for performing PCR on the PCR mixture and in fluid communication with the pre-PCR chamber; and a flow control system configured to control fluid flow in the cartridge body and comprising a plurality of capillary valves actuated by at least one of: pressurized air and vacuum pressure.

Description

CARTRIDGE FOR NUCLEIC ACID ANALYSISTechnical Field

[0001] The present disclosure relates to a cartridge for nucleic acid analysis.Background

[0002] Nucleic acid-based analysis offers precise and early detection capabilities for infectious disease diagnosis. The process involves steps such as cell lysis, DNA / RNA extraction, nucleic acid amplification and detection, and so forth. Conventional approaches for nucleic acid analysis requires use of expensive instruments such as biosafety cabinets, refrigerators and / or freezers, centrifuges, qPCR analysers and so on, and is thus normally only performed in central labs by well-trained personnel. This makes nucleic acid-based analysis currently unavailable in areas with limited resources and impractical for point-of-care testing.Summary

[0003] Disclosed is an integrated cartridge for automated nucleic acid analysis, offering a comprehensive solution for various processes involved in nucleic acid extraction and analysis. The fully enclosed cartridge enhances efficiency, flexibility, and contamination prevention, thereby addressing key challenges associated with existing microfluidics-based nucleic acid analysis cartridges.

[0004] According to a first aspect, there is provided a cartridge for nucleic acid analysis comprising: a cartridge body; a plurality of reagent ports provided in the cartridge body, each reagent port configured to establish a fluid connection with an external reagent storage tube for accommodating a range of reagent volumes; a reaction chamber configured for nucleic acid extraction therein from a sample placed in the reaction chamber during use of the cartridge, the reaction chamber provided in fluid communication with a first number of the reagent ports via a first number of reagent fluid channels provided within the cartridge body, the reaction chamber projecting from a top surface of the cartridge body for accommodating a range of reagent and sample volumes therein; a pre-polymerase chain reaction (PCR) chamber configured for mixing therein a PCR mix from one of the reagent ports with eluted nucleic acid from the reaction chamber to form a PCR mixture, the pre-PCR chamber provided within the cartridge body in fluid communication with the reaction chamber via a sample fluid channel provided within the cartridge body, the pre-PCR chamber being in fluid communication with a second number of the reagent ports via a second number of reagent fluid channels providedwithin the cartridge body; a PCR chamber configured for performing PCR, RT-PCR or RT- qPCR therein on the PCR mixture from the pre-PCR chamber, the PCR chamber provided within the cartridge body in fluid communication with the pre-PCR chamber via a PCR fluid channel provided within the cartridge body; and a flow control system configured to control fluid flow in the cartridge body; the flow control system comprising a plurality of capillary valves actuated by at least one of: pressurized air and vacuum pressure.

[0005] The cartridge may further comprise a waste chamber in fluid communication with the reaction chamber via a waste fluid channel provided within the cartridge body.

[0006] The cartridge may further comprise a waste vacuum port provided in the cartridge body in fluid communication with the waste chamber via a waste air channel provided within the cartridge body for controllable application of the vacuum pressure to the waste chamber for drawing used reagents from the reaction chamber into the waste chamber.

[0007] The capillary valves may be provided along at least some of: the reagent fluid channels, the sample fluid channel and the waste fluid channel, wherein each capillary valve may comprise a narrowed fluid channel having a fluid inlet surrounded by a conical protrusion provided within an enlarged space where the capillary valve is provided, wherein the conical protrusion is configured to increase a burst pressure that must be overcome in order for liquid to enter the fluid inlet, and wherein the burst pressure is overcome by controlled application of one of: the pressurised air and the vacuum pressure.

[0008] The wedge angle of the conical protrusion may range from 25° to 45° and diameter of the fluid channel and fluid inlet may range from 100 pm to 300 pm.

[0009] Each reagent port may be provided with an upstanding piercing pin configured to pierce a foil covering of an external reagent storage tube for allowing outflow of reagent from the external reagent storage tube into the cartridge body.

[0010] At least some of the reagent ports may be configured to function as pressurized air ports for controllable introduction of pressurized air into at least some of the reagent fluid channels for moving reagent in the at least some of the reagent fluid channels into the reaction chamber.

[0011] The cartridge may further comprise a pressurized air port provided in the cartridge body in fluid communication with the pre-PCR chamber via a pre-PCR air channel for controllableintroduction of pressurized air into the pre-PCR chamber to prevent reagents from the reaction chamber from moving into the pre-PCR chamber.

[0012] The pressurized air port may further be provided for controllable introduction of pressurized air into the pre-PCR chamber to push all used reagents to the waste chamber.

[0013] The cartridge may further comprise a pre-PCR vacuum port provided in the cartridge body in fluid communication with the pre-PCR chamber via the pre-PCR air channel for controllable application of the vacuum pressure to the pre-PCR chamber for drawing eluted nucleic acid from the reaction chamber into the pre-PCR chamber and for drawing the PCR mix from the one of the reagent ports into the pre-PCR chamber.

[0014] The cartridge may further comprise a PCR vacuum port provided in the cartridge body in fluid communication with the PCR chamber via a PCR air channel for controllable application of the vacuum pressure to the PCR chamber for drawing the PCR mixture from the pre-PCR chamber into the PCR chamber.

[0015] The cartridge may further comprise a debubbler membrane provided along the PCR fluid channel for removal of air from the PCR mixture moving in the PCR fluid channel from the pre-PCR chamber to the PCR chamber by application of the vacuum pressure across the debubbler membrane from the PCR vacuum port.

[0016] The cartridge may further comprise a sealing mechanism provided to form a permanent seal over a PCR valve of the PCR chamber, the sealing mechanism may comprise a non- porous sheet provided in a cavity within the cartridge body over the PCR valve and an elastic insert configured to be permanently inserted into the cavity above the non-porous sheet when it is desired to seal the PCR valve, wherein when the elastic insert has been permanently inserted into the cavity, the elastic insert is compressed within the cavity and exerts pressure on the non-porous sheet against the PCR valve to thereby cut off liquid flow through the PCR valve.

[0017] PCR valve may be at least one of: an inlet valve of the PCR chamber and an outlet valve of the PCR chamber.

[0018] The reaction chamber may be provided with a venting port configured for release of extra air from the reaction chamber during loading of reagents into the reaction chamber while preventing contaminants from entering the reaction chamber.Brief Description of the Drawings

[0019] In order that the invention may be fully understood and readily put into practical effect there shall now be described by way of non-limitative example only exemplary embodiments of the present invention, the description being with reference to the accompanying illustrative drawings.Fig. 1 is an isometric view of an exemplary cartridge for nucleic analysis with reagent storage tubes connected thereto.Fig. 2 is a top view of the cartridge and reagent storage tubes of Fig. 1 .Fig. 3 is a close-up isometric view of a reagent port in the cartridge of Figs. 1 and 2.Fig. 4A is a top view of a debubbler membrane.Fig. 4B is an isometric view of the debubbler membrane of Fig. 4A.Fig. 5A is a cross-sectional view of a capillary valve in the cartridge with close-up inset of the valve structureFig. 5B is an isometric view of the capillary valve of Fig. 5A.Fig. 6A is a close-up plan view of PCR valves at the inlet and the outlet of the PCR chamber.Fig. 6B is an isometric view of the PCR valves and PCR chamber of Fig. 6A.Fig. 6C is a schematic cross-sectional view of a valve of Fig. 6B before sealing.Fig. 6D is a schematic cross-sectional view of the valve of Fig. 6C after sealing.Fig. 7 is a graph of amplification curves of different COVID-19 targets using the cartridge ofFig. 1.Fig. 8 is a graph of amplification curves of different respiratory viruses using the cartridge of Fig. 1.Detailed Description

[0020] Exemplary embodiments of the cartridge for nucleic acid analysis will be described with reference to Figs. 1 to 8 in which the same reference numerals are used across the figures to refer to the same or similar parts.

[0021] In general, as shown in Figs. 1 and 2, the cartridge 100 for nucleic analysis comprises a cartridge body 10, a plurality of reagent ports 20 provided in the cartridge body 10, a reaction chamber 30, a pre-polymerase chain reaction (pre-PCR) chamber 40, a PCR chamber 50, and a flow control system comprising a plurality of capillary valves 70 that are actuated by pressurized air such as compressed dry air (CDA), vacuum pressure, or a combination of pressurized air and vacuum pressure for precise reagent delivery and control of fluid flow inthe cartridge 100. As there are many capillary valves 70 in the cartridge 100, it is impractical to label all of them in the figures. Accordingly, a legend showing the symbol representing a capillary valve 70 is provided in Fig. 2, allowing readers to see where the capillary valves 70 in the exemplary cartridge 100 shown in Fig. 2 are located.

[0022] In the cartridge body 10, each reagent port 20 is configured to establish a fluid connection with an external reagent storage tube 22 for accommodating a range of reagent volumes therein when the external reagent storage tube 22 is placed in the reagent port 20. As shown in Fig. 3, each reagent port 20 may be provided with an upstanding piercing pin 21 configured to pierce a foil covering of the external reagent storage tube 22 to allow outflow of reagent from the external reagent storage tube 22 into the cartridge body 10 for precise reagent delivery. The external reagent tubes 22 may be used for storage of reagents therein before use of the cartridge 100, with a volume range of 1 uL to 1 mL for volume adjustment flexibility, and at temperatures ranging from room temperature to a frozen state (e.g. -20 °C). The reagents may include magnetic beads, lysis buffer, binding buffer, washing buffer, elution buffer, and so on. For different testing protocols and applications, it is only needed to change the testing programme and the pre-stored reagent tubes 22 for the correct reagents to be used.

[0023] The reaction chamber 30 is configured for nucleic acid extraction (including cell lysis, binding, washing, elution and so on) from a sample placed in the reaction chamber 30 during use of the cartridge 10. As can be seen in Fig. 2, the reaction chamber 30 is provided in fluid communication with a first number of the reagent ports 20 (C1-C6 in Fig. 2) via a first number of reagent fluid channels 23 provided within the cartridge body 10 for the supply of sample preparation reagents that are normally stored at ambient temperature. Preferably, at least some of the reagent ports 20 are configured to function as pressurized air ports for controllable introduction of pressurized air into at least some of the reagent fluid channels 23 for moving reagent in the at least some of the reagent fluid channels 23 into the reaction chamber 30 after the sample has been loaded into the reaction chamber 30 during use. In an exemplary embodiment, the pressurized air may comprise CDA with a pressure ranging from 2 kPa to 20 kPa.

[0024] Notably, the reaction chamber 30 is configured to project from a top surface 11 (which may be planar) of the cartridge body 10 for accommodating a range of reagent and sample volumes in the reaction chamber 30, as shown in Fig. 1 , allowing volume adjustment flexibility without requiring different cartridge sizes to be provided or modifying the cartridge structure for different applications. In an exemplary embodiment, the reaction chamber 30 may accommodate a reaction volume of 50 uL to 2mL.

[0025] A cap 31 at the top of the reaction chamber 30 may be provided with a venting port 32 that is covered by a porous layer (such as a filter or membrane) with an appropriate pore size to allow extra air to be released from the reaction chamber 30 during loading of reagents into the reaction chamber 30 while preventing contaminants (such as pathogens or aerosols) from passing through the venting port 32 into the reaction chamber 30 to prevent contamination.

[0026] The reaction chamber 30 may include a magnetic stirrer (such as a magnetic stirring bar, not shown) provided therein that can be activated to mix the reagents with the sample in the reaction chamber 30 after reagent loading.

[0027] In use, the reaction chamber 30 may be heated to facilitate lysis or nucleic acid elution. Drying of magnetic beads before next reagent loading may be speeded up by applying pressurized air, vacuum, and heating together to the reaction chamber 30.

[0028] The cartridge 10 preferably also comprises a waste chamber 90 in fluid communication with the reaction chamber 30 via a waste fluid channel 39 provided within the cartridge body 100. A waste vacuum port 81 may be provided in the cartridge body 10 in fluid communication with the waste chamber 90 via a waste air channel 41 provided within the cartridge body. The waste vacuum port 81 is provided for controllable application of the vacuum pressure to the waste chamber 90 for drawing used reagents from the reaction chamber 30 into the waste chamber 90. In use, a vacuum ranging from -3 to -30 kPa may be applied to the first vacuum port 81 to vacuum aspirate used reagents into the waste chamber 90.

[0029] The pre-PCR chamber 40 is provided within the cartridge body 10 in fluid communication with the reaction chamber 30 via a sample fluid channel 34 provided within the cartridge body 10. The pre-PCR chamber 40 is also in fluid communication with a second number of the reagent ports 20 (C7 in Fig. 2) via a second number of reagent fluid channels 24 provided within the cartridge body 10 for the supply of PCR reagents and other reagents that are normally stored at a low temperature. The pre-PCR chamber 40 is configured for mixing therein a polymerase chain reaction (PCR) mix from one of the reagent ports 20 (C7 in Fig. 2) with eluted nucleic acid from the reaction chamber 30 to form a PCR mixture.

[0030] Preferably, the cartridge 100 further comprises a pressurized air port 60 provided in the cartridge body 10 in fluid communication with the pre-PCR chamber 40 via a pre-PCR air channel 41 . The pressurized air port 60 is provided for controllable introduction of pressurized air into the pre-PCR chamber 40. This is to prevent reagents from the reaction chamber 30 from moving into the pre-PCR chamber 40. For example, to prevent any extraction reagentsin the reaction chamber 30 from leaking into the pre-PCR chamber 40, CDA from the pressurized air port 60 may be applied after each bead-washing step in the reaction chamber 30. CDA may also be applied from the pressurized air port 60 to push all used reagents to the waste chamber 90.

[0031] The cartridge 100 may further comprise a pre-PCR vacuum port 82 provided in the cartridge body 10 in fluid communication with the pre-PCR chamber 40 via the pre-PCR air channel 42. The pre-PCR vacuum port 82 is provided for controllable application of the vacuum pressure to the pre-PCR chamber 40. Applying vacuum pressure to the pre-PCR chamber 40 is for drawing eluted nucleic acid from the reaction chamber 30 into the pre-PCR chamber 40 and for drawing the PCR mix from the one of the reagent ports 20 (C7 in Fig. 2) into the pre- PCR chamber 40. In use, the vacuum pressure introduced through the pre-PCR vacuum port 82 may be applied intermittently for thorough mixing of the eluted nucleic acid with the PCR mix in the pre-PCR chamber 40 while opening the reagent port 20 (C7 in Fig. 2) from which the PCR mix is drawn.

[0032] The PCR chamber 50 is configured for performing PCR, reverse transcription PCR (RT-PCR) or reverse transcription qualitative PCR (RT-qPCR) therein on the PCR mixture from the pre-PCR chamber 40. The PCR chamber 50 is provided within the cartridge body 10 in fluid communication with the pre-PCR chamber 40 via a PCR fluid channel 45 provided within the cartridge body 10.

[0033] The cartridge 100 may further comprise a PCR vacuum port 83 provided in the cartridge body 10 in fluid communication with the PCR chamber 50 via a PCR air channel 43. The PCR vacuum port 83 is provided for controllable application of the vacuum pressure to the PCR chamber 50. Applying vacuum pressure to the PCR chamber 50 is for drawing the PCR mixture from the pre-PCR chamber 40 into the PCR chamber 50.

[0034] As can be seen in Fig. 2, the PCR chamber 50 has two PCR valves: an inlet valve 51 and an outlet valve 52 that allow inflow and outflow of fluid to and from the PCR chamber 50 respectively. A debubbler membrane 53 is preferably provided between the pre-PCR chamber 40 and the inlet valve 51 of the PCR chamber 50 to prevent entry of air bubbles into the PCR chamber 50. The debubbler membrane 53 comprises a piece of semi-permeable hydrophobic membrane embedded between two layers of the cartridge body 10, as shown in Fig. 4. Vacuum pressure is applied from the PCR vacuum port 83 to both sides of the debubbler membrane 53 to draw the PCR mixture into the PCR chamber 50 from the pre-PCR chamber 40. Simultaneously, air or bubbles are removed from the PCR mixture when the vacuum pressureor suction (ranging from -10 kPa to -3 kPa) is applied to one side of the debubbler membrane 53 as only air passes through the minuscule pores of the debubber membrane 53 while the PCR mixture is kept contained by the debubbler membrane 53.

[0035] In the cartridge 100, movement of reagents, buffers, waste and other fluids are controlled accurately by a combination of pressurized air, vacuum pressure and strategic placement of the capillary valves 70 of the flow control system. This prevents any undesirable reagent migration and ensures precise control of fluid flow within the cartridge 100. The capillary valves 70 of the flow control system are passive valves provided along at least some of the reagent fluid channels 23, 24, the sample fluid channel 34 and the waste fluid channel 39. Preferably, as shown in Figs. 5A and 5B, each capillary valve 70 comprises a narrowed valve channel 74 having a fluid inlet 71 surrounded by a conical protrusion 72 provided within an enlarged space 73 in the fluid channel 23, 24, 34, 39 where the capillary valve 70 is provided. The conical protrusion 72 is configured to increase a burst pressure that must be overcome in order for liquid to enter the fluid inlet 71 . The conical protrusion 72 may have the form of a conical frustum having a central through-hole along its axis that defines the valve channel 74 and fluid inlet 71 , as shown in Fig. 5B. The burst pressure is overcome by controlled application of one of: the pressurised air and the vacuum pressure of the flow control system.

[0036] Burst pressure p of the capillary valve 70 can be estimated by the following equation:4 p = y — cos(a — 0) a where y is surface tension, 9 is contact angle of reagent on the cartridge body 10, d is diameter of the valve channel 74 and the fluid inlet 71 , and a is the wedge angle of the conical protrusion 72. The smaller the channel diameter d, the better the performance of the capillary valve 70 (in the sense that the required burst pressure is greater for liquid to enter the fluid inlet 71 ); the wedge angle a should also be close to the contact angle 9. In an exemplary embodiment, the wedge angle a of the conical protrusion 72 ranges from 25° to 45° and diameter d of the valve channel 74 and fluid inlet 71 ranges from 100 pm to 300 pm.

[0037] The capillary valves 70 serve to prevent reagents and other liquids in the cartridge body 10 from migrating to undesired locations of the cartridge 100 until the controlled pressurized air or vacuum pressure / suction force is applied to overcome the burst pressure at the capillary valves 70. Multiple capillary valves 70 may be provided to prevent reverse flow of reagents that have added solvent or surfactants.

[0038] The cartridge 100 preferably further comprises a sealing mechanism provided to form a permanent seal over each PCR valve 51 , 52 of the PCR chamber 50. Each sealing mechanism may comprise a non-porous sheet 54 provided in a cavity 56 within the cartridge body 10 over the PCR valve 51 , 52 and an elastic insert 55 configured to be permanently inserted into the cavity 56 above the non-porous sheet 54 when it is desired to seal the PCR valve 51 , 52. As shown in Fig. 6C, before the elastic insert 55 is permanently inserted into the cavity 56, the non-porous sheet 54 allows liquid to flow through the PCR valve 51 , 52 and the elastic insert 55 is provided in a through hole 13 extending from the top surface 11 of the cartridge body 10 into the cavity 46. The elastic insert 55 preferably extends slightly out of the through hole 13 above the top surface 11 of the cartridge body 10 as shown in Fig. 6C.

[0039] To seal the PCR valve, 51 , 52, the elastic insert 55 is simply pushed down fully and permanently into the cavity 56. This compresses the elastic insert 55 within the cavity 56 and the compressed elastic insert 55 functions as a compressed spring that exerts or applies a continuous force or pressure on the non-porous sheet 54 against the PCR valve 51 , 52, thereby cutting off liquid flow through the PCR valve 51 , 52. This allows the PCR mixture to be permanently sealed within the PCR chamber 50 throughout and after the PCR process in the PCR chamber 50, thus achieving a fully enclosed and automated PCR preparation within the cartridge 100.

[0040] In an exemplary embodiment of use of the cartridge 100, first, a sample is loaded into the reaction chamber 30. Reagents from reagent storage tubes 22 are then introduced into the reaction chamber 30 from the first number of the reagent ports 20 (C1-C6 in Fig. 2) by using CDA with a pressure ranging from 2 kPa to 20 kPA from said reagent ports 20. When the reagents are loaded into the reaction chamber 30, any extra air is released through the venting port 32 at the cap 31 of the reaction chamber 30. Following reagent loading, the magnetic stirrer in the reaction chamber 30 is activated to mix the reagents with the sample in the reaction chamber 30 where cell lysis, binding of DNA / RNA to the magnetic beads, beads washing and drying, and elution of the DNA / RNA take place. After each step, a magnetic force may be applied from a bottom surface 12 of the cartridge 100 to attract the magnetic beads and the magnetic stirring bar to a bottom of the reaction chamber 30. CDA is applied after each beads washing step to prevent any extraction reagents from leaking from the reaction chamber 30 into the pre-PCR chamber 40. Vacuum pressure is applied from the waste vacuum port 81 to aspirate used reagents from the reaction chamber 30 to the waste chamber 90. The eluted nucleic acid in the reaction chamber 30 is then drawn into the pre-PCR chamber 40 by applying vacuum pressure to the pre-PCR vacuum port 82. A PCR mix is also drawn into the pre-PCR chamber 40 from the second number of reagent ports 20 (C7 in Fig. 2) to form a PCR mixturewith the eluted nucleic acid in the pre-PCR chamber 40. When the PCR mixture is ready, it is drawn from the pre-PCR chamber 40 into the PCR chamber 50 by applying vacuum pressure to the PCR vacuum port 83. As the PCR mixture moves from the pre-PCR chamber towards the PCR chamber 50, the PCR mixture passes by the debubbler membrane 52 to remove air from the PCR mixture. When the debubbled PCR mixture has been fully drawn into the PCR chamber 50, the elastic inserts 55 at the PCR valves 51 , 52 are pushed down into the cavities 56 to permanently seal the PCR chamber 50. When the PCR chamber 50 has been sealed, qPCR or RT-qPCR analysis takes place.

[0041] Use of the above-disclosed cartridge 100 was successfully demonstrated for automatic nucleic acid and RT-qPCR analysis for COVID-19 detection from swab samples with a limit of detection LOD of 200 copies / mL in 2 hours, as illustrated by the results presented in Table 1 below for different target fluorophores (FAM, HEX, ROX) and different sample concentrations. Fig. 7 shows the amplification curves that were obtained for the different targets (FAM, Hex, ROX). For reference, results obtained using a conventional method (where samples were manually prepared and RT-qPCR analysis was performed using commercial PCR instruments) are also shown in Table 1. As can be seen, results obtained using the cartridge 100 are comparable to those using the conventional method.PC: positive controlNTC: no template controlTable 1

[0042] Comparing the assay time between the conventional method and using the cartridge 100, sample preparation time was found to be reduced by 54.5% when the cartridge 100 was used with an express protocol, and reduced by 72.7% when the cartridge was used with a fast protocol, as can be calculated from the durations shown in Table 2 below.Table 2

[0043] Fig. 8 shows the amplifications curves obtained when the cartridge 100 was expanded for use in the detection of various different respiratory diseases, namely, COVID-19 (Cov19- N), Influenza A (FluA), Influenza B (FluB), and Respiratory Syncytial Virus (RSV) with LOD of 1000 copies / mL in 1 hr.

[0044] The presently disclosed cartridge 100 thus provides an all-in-one integrated enclosed device having dedicated chambers 30, 40, 50, 90 for cell lysis, nucleic acid extraction, pre- PCR mixing, PCR or RT-PCR performance, and waste storage. Precise reagent delivery and fluid flow control is achieved using pressurized air through the air ports and vacuum pressure through the vacuum ports together with the strategically placed capillary valves. Volume adjustment flexibility is achieved by providing the reaction chamber 30 that projects above the top surface 11 of the cartridge body 10 and by using pre-stored reagent tubes 22. Little manual operation or training is required for use of the cartridge 100, which is thus applicable for point- of-care testing, especially in areas with limited resource, while its enclosed system lessens the risks of contamination.

[0045] While there has been described in the foregoing description exemplary embodiments of the present invention, it will be understood by those skilled in the technology concerned that many variations in details of design, construction and / or operation may be made without departing from the present invention. It will be appreciated that many further alterations, modifications and permutations of various aspects of the described embodiments are possible that fall within the spirit and scope of the claims.

Claims

Claims1. A cartridge for nucleic acid analysis comprising: a cartridge body; a plurality of reagent ports provided in the cartridge body, each reagent port configured to establish a fluid connection with an external reagent storage tube for accommodating a range of reagent volumes; a reaction chamber configured for nucleic acid extraction therein from a sample placed in the reaction chamber during use of the cartridge, the reaction chamber provided in fluid communication with a first number of the reagent ports via a first number of reagent fluid channels provided within the cartridge body, the reaction chamber projecting from a top surface of the cartridge body for accommodating a range of reagent and sample volumes therein; a pre-polymerase chain reaction (PCR) chamber configured for mixing therein a PCR mix from one of the reagent ports with eluted nucleic acid from the reaction chamber to form a PCR mixture, the pre-PCR chamber provided within the cartridge body in fluid communication with the reaction chamber via a sample fluid channel provided within the cartridge body, the pre-PCR chamber being in fluid communication with a second number of the reagent ports via a second number of reagent fluid channels provided within the cartridge body; a PCR chamber configured for performing PCR, RT-PCR or RT-qPCR therein on the PCR mixture from the pre-PCR chamber, the PCR chamber provided within the cartridge body in fluid communication with the pre-PCR chamber via a PCR fluid channel provided within the cartridge body; and a flow control system configured to control fluid flow in the cartridge body; the flow control system comprising a plurality of capillary valves actuated by at least one of: pressurized air and vacuum pressure.

2. The cartridge of claim 1 , further comprising a waste chamber in fluid communication with the reaction chamber via a waste fluid channel provided within the cartridge body.

3. The cartridge of claim 2, further comprising a waste vacuum port provided in the cartridge body in fluid communication with the waste chamber via a waste air channel provided within the cartridge body for controllable application of the vacuum pressure to the waste chamber for drawing used reagents from the reaction chamber into the waste chamber.

4. The cartridge of any one of the preceding claims, wherein the capillary valves are provided along at least some of: the reagent fluid channels, the sample fluid channel and the waste fluid channel, wherein each capillary valve comprises a narrowed fluid channel having a fluid inlet surrounded by a conical protrusion provided within an enlarged space where the capillary valve is provided, wherein the conical protrusion is configured to increase a burst pressure that must be overcome in order for liquid to enter the fluid inlet, and wherein the burst pressure is overcome by controlled application of one of: the pressurised air and the vacuum pressure.

5. The cartridge of claim 4, wherein a wedge angle of the conical protrusion ranges from 25° to 45° and diameter of the fluid channel and fluid inlet ranges from 100 pm to 300 pm.

6. The cartridge of any one of the preceding claims, wherein each reagent port is provided with an upstanding piercing pin configured to pierce a foil covering of an external reagent storage tube for allowing outflow of reagent from the external reagent storage tube into the cartridge body.

7. The cartridge of any one of the preceding claims, wherein at least some of the reagent ports are configured to function as pressurized air ports for controllable introduction of pressurized air into at least some of the reagent fluid channels for moving reagent in the at least some of the reagent fluid channels into the reaction chamber.

8. The cartridge of any one of the preceding claims, further comprising a pressurized air port provided in the cartridge body in fluid communication with the pre-PCR chamber via a pre-PCR air channel for controllable introduction of pressurized air into the pre-PCR chamber to prevent reagents from the reaction chamber from moving into the pre-PCR chamber.

9. The cartridge of claim 8, wherein the pressurized air port is further provided for controllable introduction of pressurized air into the pre-PCR chamber to push all used reagents to the waste chamber.

10. The cartridge of any one of the preceding claims, further comprising a pre-PCR vacuum port provided in the cartridge body in fluid communication with the pre-PCR chamber via the pre-PCR air channel for controllable application of the vacuum pressure to the pre- PCR chamber for drawing eluted nucleic acid from the reaction chamber into the pre-PCR chamber and for drawing the PCR mix from the one of the reagent ports into the pre-PCR chamber.11 . The cartridge of any one of the preceding claims, further comprising a PCR vacuum port provided in the cartridge body in fluid communication with the PCR chamber via a PCR air channel for controllable application of the vacuum pressure to the PCR chamber for drawing the PCR mixture from the pre-PCR chamber into the PCR chamber.

12. The cartridge of claim 11 , further comprising a debubbler membrane provided along the PCR fluid channel for removal of air from the PCR mixture moving in the PCR fluid channel from the pre-PCR chamber to the PCR chamber by application of the vacuum pressure across the debubbler membrane from the PCR vacuum port.

13. The cartridge of any one of the preceding claims, further comprising a sealing mechanism provided to form a permanent seal over a PCR valve of the PCR chamber, the sealing mechanism comprising a non-porous sheet provided in a cavity within the cartridge body over the PCR valve and an elastic insert configured to be permanently inserted into the cavity above the non-porous sheet when it is desired to seal the PCR valve, wherein when the elastic insert has been permanently inserted into the cavity, the elastic insert is compressed within the cavity and exerts pressure on the non-porous sheet against the PCR valve to thereby cut off liquid flow through the PCR valve.

14. The cartridge of claim 13, wherein the PCR valve is at least one of: an inlet valve of the PCR chamber and an outlet valve of the PCR chamber.

15. The cartridge of any one of the preceding claims, wherein the reaction chamber is provided with a venting port configured for release of extra air from the reaction chamber during loading of reagents into the reaction chamber while preventing contaminants from entering the reaction chamber.

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