Sample preparation device

Abstract

To provide a method of manufacturing a single use metering valve, and to provide a device for preparing a liquid sample for isothermal nucleic acid amplification.SOLUTION: Provided is a method for manufacturing a single use metering valve, comprising: a. providing a metering chamber 211 having a first opening and a second opening, and a pressure release channel extending partially along the inner wall of the chamber; b. providing a metering member 22 that can be fitted into the metering chamber to divide the metering chamber into a first portion 20a and a second portion 20b; c. positioning the metering member within the metering chamber such that the pressure release channel does not provide fluid communication between the first portion and the second portion; and d. sealing the second opening of the metering chamber with a seal.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to the preparation of samples for isothermal nucleic acid amplification. In particular, it relates to a manual operation chromatography device, useful compositions related thereto, a device for preparing a sample for isothermal nucleic acid amplification, a kit for performing isothermal nucleic acid amplification, and a method for performing isothermal nucleic acid amplification. The present invention also provides useful pumps and metering valves related thereto.

Background Art

[0002] Many diagnostic tests involved in biological reactions need to be performed in a laboratory by skilled technicians and / or complex equipment. Such laboratories can be subject to government regulations. The cost of complying with such regulations can increase the cost of diagnostic tests for patients and healthcare payers and may exclude such tests from point-of-care facilities.

[0003] WO 2013 / 041713 discloses a point-of-care system useful for performing isothermal nucleic acid amplification.

[0004] However, in known nucleic acid amplification assays, in certain situations, in order to test certain body fluids, such as urine, blood, plasma, serum, saliva, cerebrospinal fluid, tears and sweat, or to extract from vaginal swabs, nasal swabs, throat swabs, penile swabs, anal swabs or skin swabs, it has been found that biological samples may need to go through several preparation steps before those tests.

[0005] Therefore, there is still a need for devices, methods and kits that enable point-of-care preparation of samples for isothermal nucleic acid amplification and other tests. Such devices need to be easy to use and can be manufactured at low cost.

[0006] The present invention addresses these and other problems in the prior art.

Summary of the Invention

[0007] Accordingly, in a first embodiment, the present invention provides a chromatography apparatus. The apparatus is preferably manually operated. The apparatus includes a chamber for containing a liquid sample, a pump with a metering valve, and a chromatography element. Preferably, the apparatus is a size exclusion chromatography apparatus, and the chromatography element is a size exclusion chromatography element, with a gel filtration chromatography element being particularly preferred. During use, the pump moves a predetermined amount of liquid from the sample chamber to the chromatography element. Generally, the pump moves a predetermined amount of liquid from the sample chamber through the chromatography element. Preferably, the pump moves a predetermined amount of liquid from the sample chamber through the chromatography element to a sample collection container. Preferably, the apparatus is for single use.

[0008] Alternative stationary phase chromatography elements may also be used in the apparatus of the present invention. Suitable alternative stationary phase chromatography elements include, but are not limited to, ion exchange chromatography elements, reversed phase chromatography elements, and affinity chromatography elements, including cation / anion exchange chromatography elements. Therefore, the apparatus of the present invention can be used for ion exchange chromatography, reversed phase chromatography, and affinity chromatography.

[0009] Preferably, the device is operable in a single action, generally with a single push or a single rotation.

[0010] Generally, during use, the pump and / or pumping is pneumatic. This is advantageous because it means the separation performance of the device is substantially independent of the force and speed operating the device. Essentially, the rate at which the liquid sample passes through the chromatographic elements is substantially independent of the force applied by the user.

[0011] Preferably, the processing of the liquid sample is completed within a predetermined time of at least about 30 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, or at least about 10 minutes. Preferably, it is completed in less than about 10 minutes, preferably less than about 8 minutes, preferably less than about 7 minutes, preferably less than about 6 minutes, preferably less than 5 minutes, or preferably less than 4 minutes. It is preferable that the processing of the liquid sample is completed within a predetermined time of about 1 to about 5 minutes, and particularly preferably between about 1 and about 3 minutes.

[0012] In the embodiment, the predetermined amount of fluid is about 0.1 to about 100 ml, preferably about 0.25 ml to about 10 ml, and more preferably about 0.5 ml to about 1 ml. Preferably, the predetermined amount of fluid is at least about 100 μL, at least about 200 μL, at least about 300 μL, at least about 400 μL, at least about 500 μL, at least about 600 μL, at least about 700 μL, at least about 800 μL, at least about 900 μL, at least about 1 mL, at least about 2 mL, at least about 3 mL, at least about 4 mL, and at least about 5 mL.

[0013] In all embodiments of the present invention, the sample to be tested, i.e., the liquid or raw sample, is generally a biological fluid, such as urine, blood, plasma, serum, saliva, cerebrospinal fluid, or tears, or is extracted from a vaginal swab, nasal swab, throat swab, penile swab, anal swab, or skin swab.

[0014] It has been found that certain fluid samples, such as those described above, may, in some circumstances, contain substances that negatively affect the performance of the assay, specifically substances that negatively interfere with the performance of isothermal nucleic acid amplification assays. This negative interference can take the form of inhibiting the nucleic acid amplification itself and / or the fluorescence-emitting substances, resulting in the amplification not being reliably detected. It has been found that these assay interfering substances tend to have lower molecular weights than the target nucleic acid and can therefore be removed by size exclusion chromatography. This invention allows size exclusion chromatography to be performed as a simple, single-step procedure in a point-of-care setting. This enables faster testing and more rapid execution of any necessary processing, benefiting both patients and healthcare professionals. It is recognized that not all assay / nucleic acid interfering substances are removed, but a sufficient amount is removed to allow the nucleic acid amplification to be performed and measured. Generally, substantially all nucleic acid amplification interfering substances are removed. Generally, nucleic acid amplification interfering substances are salts and low molecular weight molecules, such as proteins or lipids with molecular weights typically less than approximately 5000 kDa, present in liquid samples. Generally, size exclusion chromatography elements remove molecules with a molecular weight of less than approximately 5000 kDa. Those skilled in the art will understand that the molecular weight cutoff can be increased or decreased by selecting different chromatography resins.

[0015] Isothermal nucleic acid amplification assays that can be performed using the present invention include recombinase polymerase amplification (RPA), nicking and extension amplification (NEAR), strand substitution amplification, and loop-mediated isothermal amplification.

[0016] Nicking and extensional amplification reaction methods are discussed in detail in International Publication No. 2009 / 012246, which is incorporated herein by reference.

[0017] Recombinase polymerase amplification reactions are discussed in detail in International Publications 2003 / 072805, 2005 / 118853, 2010 / 141940, 2008 / 035205, 2007 / 096702, 2011 / 038197, and 2012 / 138989, the contents of which are incorporated herein by reference.

[0018] Similar to isothermal nucleic acid amplification assays, the present invention can also be used to prepare liquid samples for immunoassays, mass spectrophotometric assays, and polymerase chain reaction assays.

[0019] In embodiments of the present invention, the apparatus includes a first part and a separate second part, which can preferably be housed within the first part. Generally, the first and second parts are operably engaged to move a predetermined amount of fluid from the sample chamber to the chromatography element. A pump can be actuated by the second part of the apparatus, which is operably engaged with the first part of the apparatus.

[0020] Generally, a metering valve includes a metering chamber having an upper and lower section separated by a movable metering member. Thus, the upper and lower sections of the metering chamber have variable volumes. Generally, the metering member and the metering chamber have matching asymmetrical cross-sections. Generally, the inner wall of the metering chamber has a D-shaped cross-section. Similarly, the outer wall of the metering member usually has a D-shaped cross-section that can fit within the metering chamber. Generally, the metering member, together with the inner wall of the metering chamber, forms an interference fit, preferably a liquid-tight interference fit. Preferably, the metering member is a cup.

[0021] In embodiments, the upper and lower parts of the metering chamber are selectively fluid-connected. That is, there can be configurations where the upper and lower parts are fluid-connected, and other configurations where the two parts are not fluid-connected. The metering valve may include, for example, a fluid bypass or pressure release channel to bring fluid communication between the upper and lower parts of the metering chamber. The movement of the metering member in relation to the fluid bypass channel may allow selective fluid communication between the upper and lower parts of the metering chamber. When the metering member is above the fluid bypass channel, there is no fluid communication between the upper and lower parts of the metering chamber. However, the metering member can be lowered until the fluid bypass channel is exposed. Generally, the fluid bypass channel is a pressure release channel. Generally, the fluid bypass channel releases pressurized air from the lower part of the meter to the upper part of the chamber.

[0022] In embodiments, the apparatus includes a lysate for processing the sample before it reaches the chromatographic elements. Exposure of the sample to the lysate rapidly dissolves any cells present in the liquid sample, releasing intracellular nucleic acids for analysis. Generally, the lysate is located in a weighing chamber. The lysate is preferably selected from the group consisting of surfactants or bases. Preferred bases include potassium hydroxide and sodium hydroxide. Generally, at least one pellet of potassium hydroxide or sodium hydroxide held in place by a mesh is particularly preferred. Preferred surfactants can be selected from the group consisting of sodium dodecyl sulfate, Triton®, Tween®, Brij®, cetyltrimethylammonium bromide, and combinations thereof. Generally, the lysate is dry.

[0023] Dissolving the contents of the liquid sample before chromatography is advantageous, and therefore isothermal nucleic acid amplification inhibitors generated during cell lysis are also removed.

[0024] Generally, a chromatography element includes a separation chamber containing a chromatography substrate. Preferably, a size exclusion chromatography element includes a separation chamber containing a size exclusion chromatography gel suspension. Preferably, a chromatography element, preferably a size exclusion chromatography element, includes a solution containing a buffer for assays, preferably for isothermal nucleic acid amplification, preferably magnesium acetate. Preferably, a size exclusion chromatography element includes a solution containing a buffer for isothermal nucleic acid amplification, along with the gel filtration chromatography particles suspended therein.

[0025] The concentration of buffer in the size exclusion chromatography element is selected so that the concentration of buffer in the processed sample reaches a desired level. Typical buffer concentrations in size exclusion chromatography elements are approximately 10 mM to 200 mM. Preferably, the pH of the processed sample is approximately pH 6 to pH 9. These preferred pH values ​​and concentrations apply to all embodiments of the present invention. The specific concentration and pH selected are determined by the application, e.g., RPA or NEAR.

[0026] Subsequently, the processed samples collected in the collection container can be immediately tested, and such preparation is advantageous because it reduces several steps in the processing.

[0027] Preferred gel filtration particles useful in all embodiments of the present invention have a particle size range of about 10 μm to about 100 μm, more preferably about 15 μm to about 88 μm, and preferably have a fractionation range of 1000 to 5000 Da for peptides and globular proteins. Preferably, the particles contain dextran crosslinked with epichlorohydrin. Alternative chromatographic gels can be selected by those skilled in the art depending on the properties of the substance to be removed from the sample.

[0028] In another aspect, the present invention provides an apparatus for preparing a sample for an assay, preferably for isothermal nucleic acid amplification, comprising a first part and a separate second part that can fit within the first part, the first part including a container for containing the sample, and the second part including a separation element for removing from the sample substances that interfere with the assay, preferably nucleic acid amplification inhibitors and / or fluorescent substances. When the second part fits within the first part, the first and second parts are operatively engaged to move the sample from the sample chamber to the separation element. Preferably, the apparatus is for single use. Preferably, the apparatus is manually operated, and preferably, the apparatus is operable by a push or rotation applied by a single user.

[0029] Generally, the first part includes a metering valve for metering a predetermined amount of sample from the sample chamber and can be actuated by the second part of the apparatus engaged with the first part of the apparatus.

[0030] In use, the apparatus typically pumps a predetermined amount of fluid from the sample chamber through the separation element. Preferably, the pump moves a predetermined amount of fluid from the sample chamber through the separation element to a sample collection container. Preferably, the predetermined amount of fluid is from about 0.1 to about 100 ml, preferably from about 0.25 to about 10 ml, more preferably from about 0.5 to about 1 ml. Preferably, the pump is pneumatic.

[0031] The metering valve typically includes a metering chamber having an upper and a lower part separated by a movable metering member.

[0032] Generally, the metering valve includes a metering chamber having an upper and a lower part separated by a movable metering member. Thus, the upper and lower parts of the metering chamber are variable in size. Generally, the inner wall of the metering chamber has a D-shaped cross-section. Similarly, the outer wall of the metering member typically has a D-shaped cross-section that can fit within the metering chamber. Generally, the metering member has an interference fit, preferably a liquid-tight interference fit, with the inner wall of the metering chamber. Preferably, the metering member is a cup.

[0033] In embodiments, the upper and lower parts of the metering chamber are selectively fluid-connected; that is, selectively fluid-connected. This means that there can be configurations where the upper and lower parts are fluid-connected, and other configurations where the two parts are not fluid-connected. The metering valve may include, for example, a fluid bypass channel to provide fluid communication between the upper and lower parts of the metering chamber. The movement of the metering member in relation to the fluid bypass channel may enable selective fluid communication between the upper and lower parts of the metering chamber. Generally, the fluid bypass channel is a pressure relief channel.

[0034] In further embodiments, the apparatus includes a dissolving agent for processing the sample before it is transferred to the separation element. Generally, the dissolving agent is located in the weighing chamber. The dissolving agent is preferably selected from the group consisting of surfactants or bases. Preferred bases include potassium hydroxide and sodium hydroxide. The use of a material mesh doped with potassium hydroxide, or preferably at least one pellet of potassium hydroxide or sodium hydroxide held in place by the mesh, is particularly preferred. Preferred surfactants can be selected from the group consisting of sodium dodecyl sulfate, Triton®, Tween®, Brij®, cetyltrimethylammonium bromide, and combinations thereof.

[0035] As already mentioned, performing dissolution before separation is advantageous because it can also remove assay interfering substances that are generated during dissolution.

[0036] The separation element typically comprises a size exclusion chromatography suspension. Preferably, the separation element comprises a solution containing a buffer for isothermal nucleic acid amplification, preferably magnesium acetate, Tris, or phosphate buffer.

[0037] In other embodiments, the separation element may include a filtration or suitable stationary-phase chromatography element selected from ion-exchange chromatography elements, reverse-phase chromatography elements, and affinity chromatography elements, including cation / anion exchange chromatography elements. Thus, the apparatus of the present invention can be used for ion-exchange chromatography, reverse-phase chromatography, and affinity chromatography.

[0038] In a further embodiment, the present invention provides an apparatus for preparing a sample for isothermal nucleic acid amplification, comprising a dissolution element and a separation element for removing nucleic acid amplification inhibitors and / or fluorescent substances from a lysate.

[0039] Generally, the dissolving element includes a dissolving substance. The dissolving substance is preferably selected from the group consisting of surfactants or bases. Preferred bases include potassium hydroxide and sodium hydroxide. A material mesh doped with potassium hydroxide is particularly preferred, and more preferably, at least one pellet of potassium hydroxide or sodium hydroxide held in place by the mesh. Preferred surfactants can be selected from the group consisting of sodium dodecyl sulfate, Triton®, Tween®, Brij®, cetyltrimethylammonium bromide, and combinations thereof.

[0040] The present invention also considers a metering valve containing a metering chamber containing a dried dissolved substance to be possible.

[0041] The dissolving agent is preferably selected from the group consisting of surfactants or bases. Preferred bases include potassium hydroxide and sodium hydroxide. A material mesh doped with potassium hydroxide is particularly preferred, and more generally, the use of at least one pellet of potassium hydroxide or sodium hydroxide held in place by the mesh is preferred. Preferred surfactants can be selected from the group consisting of sodium dodecyl sulfate, Triton®, Tween®, Brij®, cetyltrimethylammonium bromide, and combinations thereof.

[0042] In a further embodiment of the present invention, a kit is provided for performing isothermal nucleic acid amplification on a sample. The kit typically, preferably, includes a housing comprising a pipette tip and a plunger assembly; a reaction chamber containing reagents for the isothermal nucleic acid amplification reaction; a liquid transfer device comprising a sample reservoir; and a sample preparation device comprising a separation element capable of removing nucleic acid amplification inhibitors and / or fluorescent substances from the sample before performing isothermal nucleic acid amplification.

[0043] In the kit embodiment, the reactor contains reagents for recombinase polymerase amplification (RPA), such as a recombinase, a single-strand binding protein, and a polymerase. The recombinase can be selected from T4 UvsX, T6 UvsX, or RecA. The DNA polymerase can be selected from the group consisting of Escherichia coli (E. coli) DNA polymerase IKlenow fragment, thermophilic bacterium (Bacillus stearothermophilus) polymerase (Bst), Bacillus subtilis (Bacillus subtilis) Phi-29 polymerase, and Bacillus subtilis (Bsu). The single-strand binding protein is generally gp32.

[0044] Generally, reagents for recombinase polymerase amplification include a crowding agent, ATP (adenosine triphosphate) or an ATP analog, dNTP, or T4 bacteriophage UvsY. Preferred crowding agents can be selected from the group including (preferably) polyethylene glycol (PEG), dextran, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or Ficoll.

[0045] If present, PEG is preferably PEG1450, PEG3000, PEG8000, or PEG10000. PEG preferably has a molecular weight between about 15000 and about 20000.

[0046] If present, dNTPs are preferably selected from the group consisting of dATP, dGTP, dCTP, and dTTP.

[0047] If present, ATP or an ATP analogue is typically selected from ATP, ATP-γ-S, ATB-β-S, ddATP, or a combination thereof.

[0048] Alternatively, the reactor may contain reagents for nicking and extension amplification (NEAR) reactions. NEAR reagents typically include a nicking enzyme, a forward template nucleic acid, a reverse template nucleic acid, and a polymerase.

[0049] Reagents are usually in dry or lyophilized form, but they can also be in liquid form.

[0050] The kit may further include a patient collection container and a Pasteur pipette for transferring the fluid from the patient collection container to a sample preparation device.

[0051] Preferably, the sample preparation apparatus contains a lysate to which the sample is exposed before the separation element removes nucleic acid amplification inhibitors and / or fluorescent substances from the lysate. Generally, the use of at least one pellet of potassium hydroxide or sodium hydroxide, which is held in place by a mesh, is particularly preferred.

[0052] Generally, the separation element includes a separation chamber containing a size exclusion chromatography gel suspension. Preferably, the size exclusion chromatography element includes a solution containing a buffer for isothermal nucleic acid amplification, preferably magnesium acetate. Preferably, the size exclusion chromatography element includes a solution containing a buffer for isothermal nucleic acid amplification and a suspension of gel filtration chromatography particles.

[0053] In other embodiments, the separation element may include a filter.

[0054] The sample preparation apparatus may be an apparatus according to any of the above-described embodiments of the present invention.

[0055] Specifically, the sample preparation apparatus may be a manually operated size exclusion chromatography apparatus. The apparatus includes a chamber for containing a liquid sample, a pump with a metering valve, and a size exclusion chromatography element. During use, the pump moves a predetermined amount of liquid from the sample chamber to the size exclusion chromatography element. Generally, the pump moves a predetermined amount of fluid from the sample chamber through the size exclusion chromatography element. Preferably, the pump moves a predetermined amount of fluid from the sample chamber through the size exclusion chromatography element to the sample collection container. Preferably, the apparatus is for single use. Preferably, the apparatus is activated by a single push or rotation. Generally, the pump is pneumatic.

[0056] Alternatively, the sample preparation apparatus may be a device for preparing a sample for isothermal nucleic acid amplification, comprising a first part and a separate second part that can be housed within the first part, wherein the first part includes a container for containing the sample, and the second part includes a separation element for removing nucleic acid amplification inhibitors and / or fluorescent substances from the sample, and when the second part is housed within the first part, the first and second parts operably engage to move the sample from the sample chamber to the separation element.

[0057] Alternatively, the sample preparation apparatus may include a dissolving element and a separation element for removing nucleic acid amplification inhibitors and / or fluorescent substances from the lysate.

[0058] In some embodiments, the housing of the liquid transfer device is configured to engage sealably with the reaction chamber. In some embodiments, the housing of the liquid transfer device may include a sealing component configured to engage sealably with the reaction chamber. In some embodiments, the reaction chamber may include a sealing component configured to engage sealably with the liquid transfer device. The system may further include a fluid reservoir, and the reaction chamber may optionally be configured to engage lockably with the fluid reservoir.

[0059] Preferably, the sample preparation device can be detachably engaged with the fluid reservoir. Preferably, the sample preparation device is operated by pushing the device toward the fluid reservoir while it is positioned within the fluid reservoir. Generally, the prepared sample is collected in the fluid reservoir.

[0060] The liquid transfer device may be configured to engage with the reaction chamber in a lockable manner, for example, before and / or after dispensing the liquid sample, without dispensing it. In some embodiments, the reaction chamber contains one or more components of a biological reactant.

[0061] The liquid transfer device may include a housing with a pipette tip and a plunger assembly disposed within the housing and pipette tip, wherein a portion of the plunger assembly is configured to engage with the fluid reservoir so that the plunger assembly remains stationary relative to the fluid reservoir and the housing moves relative to the plunger assembly.

[0062] Generally, the movement of the housing relative to the plunger assembly creates a vacuum inside the pipette tip, and optionally the plunger assembly can be configured to lock into a position that produces the vacuum. The housing can be configured to move relative to the plunger assembly by pressing it down onto a fluid reservoir. The device can further be configured to indicate, by auditory and / or visual, that the plunger assembly is in a position that produces a vacuum.

[0063] The kit may include a liquid transfer device, one or more fluid reservoirs, and a reaction chamber. When the liquid transfer device and the reaction chamber are connected, the reaction chamber may be configured to unlock the plunger assembly.

[0064] The liquid transfer device may be configured to draw a sample from the fluid reservoir by pushing the device toward the reservoir, and the system which includes either the liquid transfer device itself or one or both of the reaction chamber and the fluid reservoir.

[0065] In the system described above, all four components—the liquid transfer device, the reaction chamber, the sample preparation device, and the fluid reservoir—may have a compatible asymmetrical cross-section.

[0066] In a further embodiment, the present invention provides a system comprising a kit according to the above-described embodiment of the present invention for performing isothermal nucleic acid amplification, and a detection device.

[0067] The detection device generally includes a first station adapted to securely hold the sample collection chamber and a second station adapted to securely hold the reaction chamber. During use, the sample preparation device is placed inside the sample collection chamber. Generally, the first part of the device is placed inside the sample collection chamber. The raw sample is then placed inside the weighing chamber of the device. The second part of the device is then inserted into the first part of the device. The second part of the device is then pushed into the first part, usually until an audible or visible signal is emitted, and the prepared sample is then collected in the sample collection container. The sample preparation device is then removed and discarded.

[0068] The mobile device can move between the collection chamber of the first station and the reaction chamber of the second station.

[0069] The detection device includes a lid that can be closed when the detection device is in operation or for storage.

[0070] A touchscreen user interface may be provided for inputting assay data and displaying information. A second station may include a barcode reader or similar device for automatically detecting barcodes or similar codes present on the amplification chamber. The first and second stations may be adapted to heat or cool the contents of the sample collection chamber and reaction chamber. The second station may also be adapted for optical, fluorescent or other monitoring and / or stirring of microcentrifuge tubes.

[0071] In some embodiments, the liquid transfer devices or pipette tips disclosed herein can be configured to collect and dispense volumes between 1 μl and 5 ml (for example, between two of 1 μl, 2 μl, 5 μl, 10 μl, 20 μl, 50 μl, 100 μl, 200 μl, 500 μl, 1 ml, 2 ml, and 5 ml).

[0072] In a further embodiment, the present invention provides a method for performing isothermal nucleic acid amplification. The method includes the steps of: providing a raw liquid sample for testing; treating the raw liquid sample to remove nucleic acid amplification inhibitors and / or fluorescent substances; performing isothermal nucleic acid amplification on the treated sample; and monitoring the amplified nucleic acid.

[0073] In a further embodiment of the present invention, the present invention provides a composition comprising an aqueous solution containing an isothermal nucleic acid amplification buffer for use in gel filtration chromatography and dispersed gel filtration chromatography particles. Generally, the buffer is selected from the group consisting of magnesium acetate or tris acetate, and is preferably magnesium acetate.

[0074] Preferred gel-filtered particles have a particle size range of about 10 μm to about 100 μm, more preferably about 15 μm to about 88 μm, and a fractionation range of about 1000 to about 5000 Da for peptides and globular proteins. Preferably, the particles contain dextran crosslinked with epichlorohydrin. Those skilled in the art may select size exclusion chromatography gels as an alternative.

[0075] The present invention also considers that such compositions may be used in sample preparation for isothermal nucleic acid amplification.

[0076] The present invention further provides a metering valve for a pump. The metering valve typically includes a metering chamber having an upper and lower part separated by a movable metering member, and a pressure relief channel. Preferably, the metering member and the pressure relief channel are arranged so that the metering member can move from an initial position where the metering member separates the upper part from the lower part to a subsequent position where the pressure relief channel provides fluid interlocking between the lower and upper parts of the metering chamber.

[0077] Thus, as the measuring element descends, the volume at the bottom of the measuring chamber decreases, and the pressure within the bottom of the measuring chamber increases. After the measuring element has traveled a predetermined distance, the pressure release channel can create fluid communication between the bottom and top of the measuring chamber. Once fluid communication is established, air from the bottom of the measuring chamber moves along the pressure release channel, moving fluid into the top of the measuring chamber.

[0078] Generally, a fluid outlet channel is provided. Preferably, the fluid moves along the outlet channel and thereby exits the metering valve. In a preferred embodiment, the fluid outlet channel is located within a movable actuator for the metering valve. In such an embodiment, the movable actuator is operably engaged with a metering member, and as a result, the movement of the actuator meters a predetermined amount of sample and then pumps a predetermined amount out of the outlet channel.

[0079] Generally, the measuring member has an interference fit, preferably a liquid-tight interference fit, with the inner wall of the measuring chamber. The inner wall of the measuring chamber may have an asymmetrical cross-section, preferably a D-shaped cross-section. Similarly, the outer wall of the measuring member may have an asymmetrical cross-section, generally a D-shaped cross-section that matches the cross-section of the measuring chamber. Preferably, the measuring member is a cup. Generally, a movable actuator operably engages with the inner floor of the cup.

[0080] Generally, a metering chamber has a single opening for containing the liquid sample to be measured and a movable actuator. Generally, the periphery of the opening of the metering chamber forms an interference fit, preferably a liquid-tight interference fit, with the outer wall of the movable actuator when the actuator is positioned within the opening.

[0081] In a further embodiment of the present invention, a method for manufacturing a single-use metering valve is provided. The method includes the steps of: preparing a metering chamber having a first opening and a second opening, and a pressure relief channel along the inner wall of the chamber, partly extending typically axially; preparing a metering member that can be housed in the metering chamber in order to divide the metering chamber into a first and a second part; positioning the metering member in the metering chamber such that the pressure relief channel does not result in fluid communication between the first and second parts; and sealing the openings of the metering chamber with a seal. Generally, the metering member is movable within the metering chamber. Preferably, the metering member slides into position through the sealed opening, and preferably, the other openings are not sealed.

[0082] In this embodiment of the method, the measuring member is preferably prevented from moving through the first opening by an abutment, preferably an annular abutment, and more preferably by an annular abutment at the first opening.

[0083] Generally, the weighing member enters the weighing chamber through a second opening and travels along the weighing chamber until it engages with an abutment, usually an annular abutment.

[0084] Preferably, the measuring member is an interference fit by a measuring chamber, preferably forming a liquid-tight interference fit. Preferably, the measuring member is a cup.

[0085] The present invention further provides a manually operated pump comprising a first part and a separate second part that can be operably housed within the first part. Generally, the first and second parts engage to form a metering valve, preferably the first part includes a metering chamber for housing a liquid sample, a movable metering member and a pressure relief channel, and the second part includes an actuator arranged to operably engage with the metering member when the second part is housed within the first part. During use, the second part moves into the first part, and a predetermined amount of fluid exits the pump through an outlet channel. The predetermined amount of fluid typically includes a predetermined amount of liquid sample and a predetermined amount of air. Generally, the outlet channel is located within the second part. In a preferred embodiment, the fluid outlet channel is located within the actuator. The pump is generally for single use.

[0086] Generally, a metering chamber has a single opening for containing the liquid sample to be measured and an actuator. Generally, the periphery of the opening of the metering chamber forms an interference fit, preferably a liquid-tight interference fit, with the outer wall of the actuator when the actuator is positioned within the opening.

[0087] Generally, the measuring member has an interference fit, preferably a liquid-tight interference fit, with the inner wall of the measuring chamber. The measuring member thereby divides the measuring chamber into upper and lower sections. The inner wall of the measuring chamber may have an asymmetrical cross-section, preferably a D-shaped cross-section. Similarly, the outer wall of the measuring member may have an asymmetrical cross-section, generally a D-shaped cross-section, matching the cross-section of the measuring chamber. Preferably, the measuring member is a cup. Generally, a movable actuator operably engages with the inner floor of the cup.

[0088] During use, the amount of fluid, typically air, moved by the actuator moving through the metering chamber results in a pressure increase in the upper part of the metering chamber, thereby causing a predetermined amount of fluid to move through the outlet channel. Selecting an appropriate amount of displacement is within the capabilities of those skilled in the art.

[0089] The pressure relief channel provides selective fluid communication between the upper and lower parts of the metering chamber. When the metering member is in its initial position, the upper and lower parts of the metering chamber are separated, i.e., not in fluid communication. As the metering member is moved along the metering chamber by the actuator, the pressure relief channel is exposed, and fluid communication is established between the upper and lower parts of the metering chamber. In a preferred embodiment, the pressure relief channel is located in the wall of the metering chamber. The pressure relief channel may be in the form of an open groove or a sealed conduit. Generally, the pressure relief channel extends from the bottom of the metering chamber, or near the bottom, to a position below the upper part of the metering member in its initial position.

[0090] Preferably, the weighing chamber contains the dissolved substance. Suitable dissolved substances are discussed previously in this disclosure.

[0091] A further aspect of the present invention provides a pump for measuring a predetermined amount of liquid, preferably a manually operated pump, the pump comprising a container including a sample chamber and a measuring chamber, and an actuator including a fluid outlet channel having an opening at the distal end of the actuator, wherein the actuator can move from a first position in which the distal end of the actuator is located in the sample chamber to a second position in which the opening of the fluid outlet channel is located in the measuring chamber, and when the actuator is in the first position, the sample chamber is in fluid communication with the measuring chamber, and when the actuator is in the second position, the sample chamber is separated from the measuring chamber; the measuring chamber is divided into upper and lower parts by a movable measuring member, and the measuring chamber further includes a pressure release channel; and the measuring member, pressure release channel and actuator are arranged such that when the actuator is moved from its first position to its second position, the measuring member moves from an initial position in which the measuring member separates the upper part from the lower part to a subsequent position in which the pressure release channel provides fluid interlocking between the lower part and the upper part of the measuring chamber.

[0092] Generally, the measuring member has an interference fit, preferably a liquid-tight interference fit, with the inner wall of the measuring chamber. The measuring member thereby divides the measuring chamber into upper and lower sections. The inner wall of the measuring chamber may have an asymmetrical cross-section, preferably a D-shaped cross-section. Similarly, the outer wall of the measuring member may have an asymmetrical cross-section, generally a D-shaped cross-section, matching the cross-section of the measuring chamber. Preferably, the measuring member is a cup. Generally, a movable actuator operably engages with the inner floor of the cup.

[0093] During use, the amount of fluid, generally air, moved by the actuator moving through the measuring chamber results in a pressure increase in the upper part of the measuring chamber, thereby moving a predetermined amount of fluid through the fluid outlet channel. The volume of the upper part of the measuring chamber moved by the actuator should preferably be greater than the amount of liquid sample being moved, so that when the measuring member is fully moved by the actuator, all liquid sample in the upper part of the measuring chamber is pushed out of the measuring chamber. The excess volume ensures that a certain amount of air also passes through the second part of the device, preventing dripping and ensuring uniformity of the dose. Selecting an appropriate volume for the measuring chamber moved by the actuator is within the capabilities of those skilled in the art.

[0094] In a preferred embodiment, the pressure relief channel is located within the wall of the metering chamber. The pressure relief channel may be in the form of an open groove or a sealed conduit. Generally, the pressure relief channel extends from the bottom of the metering chamber, or near the bottom, to a position below the upper part of the metering member in its initial position.

[0095] Preferably, the weighing chamber and / or sample chamber contain a dissolving agent. Suitable dissolving agents are previously disclosed in this disclosure.

[0096] Pumps are typically single-use. For the purposes of this invention, single-use means that, under normal use, the pump is reset and cannot be reused.

[0097] Pumps and metering valves according to these embodiments of the present invention can be specifically used in the apparatus according to the aforementioned embodiments of the present invention.

[0098] For the purposes of this invention, "operated manually" has its usual meaning. That is, the devices, pumps, and metering valves are manually operable; all of the devices, pumps, and metering valves of this invention can be operated manually, however, it is considered that certain aspects and embodiments of this invention can also be operated by alternative means.

[0099] Details of one or more embodiments of the present invention are described in the accompanying drawings and the following description. Other features, purposes, and advantages of the present invention will become apparent from the description and drawings and from the claims. [Brief explanation of the drawing]

[0100] [Figure 1] This shows a conventional system. [Figure 2] An exploded view of an exemplary device is shown. [Figure 3] The first part of the device before use is shown as an example. [Figure 4] The first exemplary part of the apparatus is shown with the sample chamber cover removed. [Figure 5] The second part of the apparatus is shown as an example. [Figure 6] The first part engages with the second part, which measures the amount of raw sample. [Figure 7] This shows the second part where the weighing component has been moved. [Figure 8] This shows the second part, which has been completely pushed down. [Figure 9] This shows an exemplary in-situ apparatus for performing and monitoring isothermal nucleic acid amplification. [Figure 10] This shows an exemplary in-situ apparatus for performing and monitoring isothermal nucleic acid amplification. [Figure 11]This shows an exemplary in-situ apparatus for performing and monitoring isothermal nucleic acid amplification. [Figure 12] The prepared sample is shown being transferred to the reaction chamber. [Figure 13] Figures 13a to 13e show schematic diagrams of the weighing chamber in use. [Modes for carrying out the invention]

[0101] The present invention relates to the preparation of samples used in assays, particularly the preparation of samples used in isothermal nucleic acid amplification. In particular, it relates to a manually operated chromatography apparatus, useful compositions related thereto, an apparatus for preparing samples for isothermal nucleic acid amplification, a kit for performing isothermal nucleic acid amplification, and a method for performing isothermal nucleic acid amplification. The present invention also provides a pump and a metering valve.

[0102] Figure 1 shows a reactor (200), sample collection reservoir (300), and liquid transfer device (100) suitable for use in the system illustrated in Figure 13. Each subassembly can have a D-shaped or other asymmetrical cross-section (105, 205, 305) and be compatible with the other two subassemblies. Thus, the subassemblies can only be engaged with each other in one orientation.

[0103] The reaction chamber 200 includes a microtube 220 held within an opening at the bottom of the reactor body.

[0104] Figure 1 shows the aforementioned mobile device 100 and reactor 200, each comprising one pipette tip 120 and one microtube 220. However, the mobile device may be equipped with two or more pipette tips, and the reactor may be equipped with two or more microtubes.

[0105] Such assemblies are described in detail in International Publication No. 2013 / 041713, which is incorporated herein by reference.

[0106] Figure 2 is an exploded view of the apparatus (20) according to the present invention. The apparatus includes a first part (20a) and a second part (20b). The second part (20b) can be housed within the first part (20a). A sample reservoir (210) is also shown. The first part (20a) can be housed within the sample collection reservoir (210).

[0107] The first part (20a) includes a main body (21). The main body (21) includes a sample chamber (not shown) and a weighing chamber (211). The weighing chamber (211) and the sample chamber are in fluid communication. The weighing member (22) is in the shape of a cup-shaped member having a D-shaped cross-section. The weighing chamber (211) also has a D-shaped cross-section. The weighing member (22) usually contains a dry soluble substance. Generally, the soluble substance (221) of at least one pellet, usually potassium hydroxide or sodium hydroxide, is held in place by gauze (222). The potassium hydroxide / sodium hydroxide is present to cause rapid dissolution of cell-forming components in the sample fluid, thereby releasing intracellular nucleic acids that will be detected by isothermal nucleic acid amplification.

[0108] The measuring element (22) can be movably housed within the measuring chamber (211). During use, the measuring element (22) divides the measuring chamber into upper and lower sections. The measuring element (22), together with the inner wall of the measuring chamber (211), forms a liquid-tight interference fit. The airtight membrane (23) closes the end of the measuring chamber. In manufacturing, the measuring element (22) is inserted into the bottom of the measuring chamber (211) and pushed upward until it is positioned directly below the annular seal (not shown) separating the measuring chamber from the sample chamber. The airtight membrane (23) is then heat-sealed over the bottom of the measuring chamber (211).

[0109] The second part (20b) includes a body (24) equipped with an actuator (25) that can be housed within the sample chamber of the first part (20a). During use, the actuator (25) operably engages with a metering member (22). The actuator (25) has a distal end (215) and a proximal end (216). An opening (214) is located at the distal end of the actuator (25). The opening (214) is in fluid communication with a separation chamber (not shown) containing an aqueous solution of isothermal nucleic acid amplification buffer and dispersed gel filtration chromatography particles. Suitable gel filtration particles are sold by GE Healthcare under the trademark name Sephadex G-25 Superfine, and other suitable chromatography substrates are known to those skilled in the art. A microfluidic channel (not shown) provides fluid communication between the separation chamber and an outlet opening (217) at the distal end of a return leg (26). An insertion portion (27) is inserted into the return leg (26) to close the separation chamber. The outer wall of the actuator (25) includes a shoulder portion (213) for engaging the first part. The channel (212) is located within the shoulder portion (213). During use, the channel (213) allows excess liquid to drain before the metering chamber is sealed.

[0110] Peelable seals (223, 224) are provided on the first and second parts.

[0111] Figure 3 shows the first part of the apparatus (32) before use. The peelable seal (31) covers the sample chamber (not shown). Before use, the peelable seal (31) protects the contents of the first part (32) from contamination and protects dry dissolved substances from moisture. Also, an undamaged peelable seal (31) informs the user that the apparatus has not been used previously. The first part of the apparatus (32) engages with the sample reservoir (33).

[0112] Figure 4 shows the first part of the apparatus (42) with the peelable seal removed. The sample chamber (41) is now accessible. The first part of the apparatus (42) is again engaged with the sample reservoir (43).

[0113] Figure 5 shows a second part of the device (60). The second part of the device (60) includes a first actuator leg (63) and a second return leg (62). A stopper (61) is fixed in place. To prevent contamination and / or leakage of fluid within the actuator, a peelable seal (64) covers openings located at the distal ends of the actuator leg and the return leg.

[0114] During use, the peelable seal (64) is removed before the second part engages with the first part. The peelable seal (64) prevents contamination and leakage. Additionally, an undamaged peelable seal (64) informs the user that the device has not been used previously.

[0115] A microfluidic pathway (not shown) descends from the top of the actuator leg (63) to the inside of the return leg (62). During use, the treated liquid flows through this pathway.

[0116] The second part of the apparatus (60) is made by injection molding of two parts: an outer wall (65) and an insert portion (66). The insert portion fills most of the return leg (62) and includes a circular portion (66) visible on the upper surface of the second part (60). When inserted into the return leg (62), the insert portion has a groove in the return leg portion (not shown) that forms a sealed channel. As shown better in Figure 2, the insert portion (27) within the actuator leg (25) includes an opening and a groove (not shown) that compresses the upper frit (218) over the matrix (219) and bottom frit (220), and allows the processed fluid to move into the return leg (26).

[0117] Figure 6a shows the apparatus of the present invention, including a second part (71) inserted into a first part (72). Here again, the first part (72) of the apparatus engages with a sample collection reservoir (73).

[0118] Figure 6b shows cross-sections of the first part (72) and the second part (71) of the apparatus, as well as the sample reservoir (73) shown in Figure 7a.

[0119] The cross-sectional view in Figure 6b shows a separation chamber (76) containing a size exclusion chromatography matrix (75). The chamber (76) containing the matrix is ​​equipped with an upper frit (77) and a bottom frit (78) to hold the matrix (75) in place. There is also a cutout shape configuration (cross-shaped) of molded plastic that allows the fluid to spread and enter the separation chamber (75).

[0120] During use, the user is instructed to introduce the second part (71) into the first part (72). The apparatus is shaped such that it is clear that the actuator leg (79) is introduced into the chamber (711) to which the raw sample is added.

[0121] When in its original position, if a sufficient amount of liquid sample is added to fill the upper part (712) of the measuring chamber, and preferably the liquid is located above the annular seal (713) separating the measuring chamber from the sample chamber (711), then the processed sample reaches the desired volume.

[0122] When the second portion (71) is inserted, the narrow portion of the actuator leg is first pushed through the annular seal (713) and then into the metering chamber (714). The diameter of the actuator leg is initially smaller than the diameter of the annular seal (713), so as the actuator (79) moves the liquid from the top of the metering chamber (712), the liquid can flow out from around the edge of the actuator leg (79) into the sample chamber (711).

[0123] In Figures 6a and 6b, the second portion (71) was manually pushed into the first portion (72) so that the shoulder portion (715) engaged with a ring-shaped seal (713) located between the sample chamber (711) and the metering chamber (714). In this position, a groove (not shown) in the shoulder portion (715) provides fluid communication between the metering chamber (714) and the sample chamber (711). The distal end (718) of the actuator is now engaged with the metering member (717). The volume of the upper part of the metering chamber surrounding the actuator determines the amount of raw sample passing through the size exclusion chromatography gel (75). The size exclusion chromatography gel (75) is suspended in a solution containing a buffer suitable for isothermal nucleic acid amplification, typically magnesium acetate. The concentration of the buffer in the separation chamber (76) is such that it is present in appropriate concentrations in the processed sample.

[0124] At the position shown in Figure 6b, the metering member (717) is above the upper end of the pressure release channel (719). This means that the pressure release channel (719) does not provide fluid communication between the upper (712) and lower (720) parts of the metering chamber (714). Therefore, as the actuator moves further down, it advances the metering member (717) and increases the air pressure in the lower chamber (720).

[0125] Figures 7a and 7b show the apparatus having a second part (81) further pushed into the first part (82). In this position, the actuator shoulder (84) is lowered below the annular seal (85), which is now engaged with the outer wall (86) of the actuator, thereby sealing the metering chamber (87) from the sample chamber (88). The upper end of the pressure release channel (810) is now exposed above the metering member (89), and thus the actuator (86) is pushing the metering member (89) down to a lower position. The pressure release channel (810) provides fluid communication between the lower (811) and upper (812) parts of the metering chamber (87), and because the pressure is higher in the lower (811) part of the chamber than in the upper (812) part, air moves along the pressure release channel (810) from the lower (811) part of the chamber to the upper (812) part of the chamber, and as a result of the reduction in the internal volume of the upper (812) caused by the actuator (86) present therein, a high-pressure region is formed above the cup-shaped metering member (89) containing the liquid sample. The liquid sample is then pushed through the orifice (814) at the distal end of the actuator (86) into the size exclusion chromatography / separation chamber (815) for processing.

[0126] A portion of the raw sample remains sealed within the sample chamber (88). This can be disposed of by the device.

[0127] As the second part (81) of the apparatus is further pushed into the first part (82), the actuator (86) further pushes the metering member (89) into the metering chamber (87), moving air from the bottom (811) to the top (812) along the pressure release channel (810), thereby causing the sample to pass through the size exclusion chromatography gel in the separation chamber (815), along the microfluidic channels (not shown) in the horizontal upper part of the second part (81) of the apparatus, then along the channels in the return leg (817) of the second part (81), and then exit the second part (81) and drop into the sample collection reservoir (818).

[0128] The size exclusion chromatography gel removes isothermal nucleic acid amplification inhibitors and / or fluorescent substances from the sample. Thus, the treated sample collected in the sample collection reservoir (818) is sufficiently free of the aforementioned inhibitors / fluorescent substances, allowing for efficient isothermal nucleic acid amplification on the nucleic acids present in the sample, and subsequently, detection. Furthermore, the size exclusion chromatography gel is suspended in a solution containing a buffer for isothermal nucleic acid amplification. The pH of the treated sample collected in the sample collection reservoir (818) is appropriate for isothermal nucleic acid amplification. Generally, the pH is approximately 6 to 9. This eliminates the need for any further sample preparation steps.

[0129] Figures 8a and 8b show the second part (91) fully inserted into the first part (92). An audible prompt, typically a click, informs the user that full insertion has been achieved and therefore the appropriate amount of sample has been processed. During use, the user typically presses with a finger or thumb until a click is heard. The component involved in the audible click is usually a latch that also locks the second part (91) within the first part (92). This also means that the unprocessed raw sample is safely contained for disposal. Preferably, a fluid seal is created between the top of the second part (91) and the sample chamber (94), thereby containing excess raw sample fluid (93) and preventing the raw sample from spilling out when the device is disposed of.

[0130] Once fully inserted, air from the lower part (95) of the metering chamber (96) flows into the upper part (97) of the metering chamber (96), and this flow continues until the pressures in both the upper and lower chambers are equal. As shown in Figure 8b, no raw sample remains in the metering chamber (96). Furthermore, a small amount of air moves through the second part (91) of the device and exits through the orifice (98) at the end of the return leg (99). This pushes out any liquid present in the channel, preventing any dripping. It also allows any residual compressed gas on the sample side of the device to dissipate. To prevent excess fluid from dripping from the return leg (99) and potentially contaminating the work area, residual fluid is further prevented from being pushed through the column after the device is removed from the sample collection container (910).

[0131] This is achieved by ensuring that when the actuator is fully inserted, the volume of the weighing chamber moved by the actuator is greater than the amount of raw sample to be weighed for processing.

[0132] Generally, there is a delay between the audible click and the arrival of all processed sample into the sample collection reservoir (910). This is caused by the damping effect from compressing the air in the lower part (95) of the weighing chamber (96), and the pressure is released from the instrument. The damping effect caused by fluid resistance is advantageous because it slows down the flow rate of the sample being processed and ensures that the sample is properly and reliably processed by the size exclusion chromatography gel. If the sample moves too rapidly through the gel, nucleic acid amplification inhibitors / fluorescent substances may not be sufficiently removed, and the instrument will not achieve its desired function. Achieving an appropriate level of damping is within the capabilities of those skilled in the art.

[0133] At the position shown in Figure 8b, the sample collection reservoir (910) contains a processed sample ready for use in isothermal nucleic acid amplification. The processed sample is buffered to the required pH and is sufficiently free of nucleic acid amplification inhibitors and fluorescent substances for amplification to be performed and detected. A portion of the processed sample is pipetted from the sample collection container (910) to the assay apparatus for performing the isothermal nucleic acid amplification assay using a liquid transfer device (not shown).

[0134] Figures 9-12 show the in situ apparatus (10) according to the present invention in a sample processing apparatus (101) for isothermal nucleic acid amplification. In Figure 9, the first part (102) of the apparatus according to the present invention is located in a sample collection container (103) and then fits into the sample processing apparatus (101). A suitable sample processing apparatus is available from Alere Inc. under the trademark name Alere i.

[0135] In Figure 9a, reactor (103) is an in-situ sample processing device. The sample processing device can be used with a reactor configured for NEAR and / or RPA isothermal nucleic acid amplification. Thus, the reaction chamber may contain the reagents necessary for performing NEAR and / or RPA on the sample introduced into the chamber. A suitable reactor (105) is available from Alere Inc.

[0136] In Figure 9, the protective, peelable film covering the sample chamber (104) has been removed. At this point, the raw sample is introduced into the sample chamber using a Pasteur pipette, typically 1.5 ml of raw sample.

[0137] Figure 10 shows system (11) of the present invention, which includes a second part (113) of the apparatus partially inserted into a first part (112) of the apparatus (111). On the other hand, Figure 11 shows system (12) of the present invention, which includes a second part (123) of the apparatus (121) fully pushed down into a first part (122) of the apparatus (121). Once the sample has been processed and collected in the sample collection reservoir (124), the sample preparation apparatus (121) can be removed and disposed of. A portion of the processed sample is then transferred to a reactor (125) for testing using a liquid transfer device.

[0138] Figure 12 shows the sample processing device (400), as well as the liquid transfer device (100), reactor (200), and sample collection reservoir (300). During use, a screen (440) provides step-by-step instructions to the user and displays the results of the isothermal nucleic acid amplification test. The present invention intends a kit comprising a reactor (200), a liquid transfer device (100), a sample collection chamber (300), and a sample preparation device, as well as a system comprising the kit and the sample processing device (400).

[0139] Figure 12 shows a system including an exemplary detection device (400). The detection device (400) includes a first station (410) adapted to securely hold a sample collection container (300) and a second station (420) adapted to securely hold a reaction chamber (200). When in use, a mobile device (100) moves between the sample collection container (300) of the first station (410) and the reaction chamber (200) of the second station (420). The detection device includes a lid (430) that can be closed while the detection device (400) is in operation or stored. A touchscreen user interface (440) is present for entering data and displaying information about the assay. The second station (420) may include a barcode reader or similar device for automatically detecting barcodes or similar codes present on the reaction chamber (200). The first station (410) and the second station (420) may be adapted for heating or cooling the contents of the sample collection container (300) and the reaction chamber (200). The second station (420) may also be adapted for optical, fluorescent or other monitoring and / or stirring of the microtube (220).

[0140] Figures 13a to 13e show schematic diagrams of the equipment in use.

[0141] Figure 13a shows the apparatus before use, including the second part (142) separated from the first part (141). The cup-shaped measuring member (143) is located at the top of the measuring chamber (144), in contact with a ring-shaped seal (145) that separates the sample chamber (146) from the measuring chamber (144).

[0142] In Figure 13b, the raw liquid sample (147) is introduced into the sample chamber (146) and the cup-shaped measuring member (143) in the measuring chamber (144). Preferably, the liquid level is above the ring-shaped seal (145).

[0143] In Figure 13c, the movable actuator (148) is lowered into the measuring chamber (144) through the sample chamber (146), so that its distal end engages with the measuring member (143), and the shoulder portion (1410) of the movable actuator (148) is almost engaged with the annular seal (145). When the annular seal (145) engages with the outer wall (1411) of the actuator (148) distal to the shoulder portion (1410), the amount of liquid in the cup-shaped measuring member (143) is a predetermined amount of liquid (1412) measured for processing, as shown in Figure 13d.

[0144] In Figure 13d, the movable actuator (148) moves the measuring member (143) along the measuring chamber (144), reducing the volume of the lower part (1413) of the measuring chamber (144) and increasing the volume of the upper part (1414). The lower and upper parts of the measuring chamber (144) are fluidly connected by a pressure release channel (1415) within the wall of the measuring chamber. As the movable actuator (148) moves into the measuring chamber (144), the internal volume of the measuring chamber (144) decreases, thereby increasing the pressure of the air contained within it. This increases the air pressure in the measuring chamber (144), which then pushes the metered liquid sample (1412) out of the measuring chamber (144) through an outlet channel (not shown) located within the movable actuator (148).

[0145] As shown in Figure 13e, when the movable actuator (148) is fully depressed, the amount of air in the metering chamber (144) moved by the movable actuator (148) exceeds the amount of liquid sample being metered, and as a result, substantially all of the liquid is pushed out of the metering chamber (144) through an outlet channel (not shown). The untreated raw sample (1416) is stored in the sample chamber (146) for safe disposal.

Claims

1. A method for manufacturing a single-use metering valve included in a chromatography apparatus, a. A step of preparing a measuring chamber having a first opening and a second opening, and a pressure release channel that partially extends along the inner wall of the chamber, b. A step of preparing a measuring member that can be housed within the measuring chamber in order to divide the measuring chamber into a first part and a second part configured to be positioned above the first part when in operation. c. The step of positioning the measuring member within the measuring chamber such that the pressure release channel does not result in fluid communication between the first and second portions, and d. The step of sealing the second opening of the measuring chamber with a seal. A method including, The aforementioned metering valve is The aforementioned measuring chamber, The aforementioned measuring member, The pressure release channel, Includes, The measuring member and the pressure release channel are arranged such that the measuring member can move relative to the pressure release channel from an initial position where the first portion separates the second portion to a subsequent position where the pressure release channel provides fluid communication between the first portion of the measuring chamber and the second portion of the measuring chamber. A method in which the metering member slides against the pressure release channel through the sealed second opening.

2. The method according to claim 1, wherein when the measuring member moves from its initial position toward a subsequent position, the pressure in the second portion of the measuring chamber initially increases.

3. The method according to claim 1 or 2, wherein once the first portion and the second portion are in fluid communication, air moves under pressure along the pressure release channel, moving a fluid, typically a liquid, into the first portion of the metering chamber.

4. The method according to any one of claims 1 to 3, wherein the measuring member is interference-fitted with the inner wall of the measuring chamber, preferably having a liquid-tight interference fit.

5. The method according to any one of claims 1 to 4, wherein the measuring member is a cup.

6. The method according to claim 3, wherein the metering valve further includes a fluid outlet channel, and during use, the fluid moves along the fluid outlet channel and thereby exits the metering valve.

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