Sample preparation device

Abstract

A manually actuated chromatography device comprises: a chamber for receiving a liquid sample, a pump having a metering valve, and a chromatography element, wherein the pump moves a predetermined volume of liquid from the sample chamber to the chromatography element.

Description

Field of the Invention

[0001] The present invention relates to the preparation of samples for isothermal nucleic acid amplification. Specifically, the present invention relates to manually operated chromatography devices, compositions useful therein, devices for preparing samples for isothermal nucleic acid amplification, kits for performing isothermal nucleic acid amplification, and methods for performing isothermal nucleic acid amplification. The present invention also provides pumps and metering valves useful therein. Background of the Invention

[0002] Many diagnostic tests involving biological reactions must be performed in laboratories by skilled technicians and / or complex equipment. These laboratories may be subject to government regulation. The costs of complying with these regulations can increase the cost of diagnostic tests to patients and healthcare payers and exclude these tests from point-of-care facilities.

[0003] WO2013 / 041713 discloses a point-of-care system for performing isothermal nucleic acid amplification.

[0004] However, it has been discovered that in certain circumstances, in order to test certain fluids (such as urine, blood, plasma, serum, saliva, cerebrospinal fluid, tears, and sweat, or eluates from vaginal, nasal, throat, penis, anal, or skin swabs), the original sample may need to undergo multiple preparation steps prior to testing in known nucleic acid amplification assays.

[0005] Therefore, there is an unmet need for devices, methods, and kits that enable point-of-care preparation of samples for isothermal nucleic acid amplification and other testing. These devices need to be simple to use and inexpensive to produce.

[0006] The present invention solves these and other problems associated with the prior art. SUMMARY OF THE INVENTION

[0007] Therefore, in a first aspect, the present invention provides a chromatography device. The device is preferably manually actuated. The device comprises a chamber for receiving a liquid sample, a pump having a metering valve and a chromatography element. Preferably, the device is a size exclusion chromatography device and the chromatography element is a size exclusion chromatography element, with gel filtration chromatography elements being particularly preferred. In use, the pump moves a predetermined volume of liquid from the sample chamber to the chromatography element. Typically, the pump moves a predetermined volume of liquid from the sample chamber through the chromatography element. Preferably, the pump moves a predetermined volume of liquid from the sample chamber through the chromatography element to reach a sample collection container. Preferably, the device is single-use.

[0008] Alternative stationary phase chromatography elements can also be used in the device of the present invention. Suitable alternative stationary phase chromatography elements include, but are not limited to: ion exchange chromatography elements, including cation and anion exchange chromatography elements; reverse phase chromatography elements; and affinity chromatography elements. Thus, the device of the present invention can be used for ion exchange chromatography, reverse phase chromatography, and affinity chromatography.

[0009] Preferably, the device is actuable by a single movement, typically a single push or rotation.

[0010] Typically, in use, the pump and / or pumping is pneumatic. This is advantageous because it means that the separation performance of the device is substantially independent of the force and speed at which the device is actuated. In essence, the rate at which the liquid sample passes through the chromatographic element is substantially independent of the force applied by the user.

[0011] Preferably, the processing of the liquid sample is completed within the following predetermined time period: 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, at least about 10 minutes. Preferably, 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 about 5 minutes, preferably less than about 4 minutes. Preferably, the processing of the liquid sample is completed within a predetermined time period of about 1 minute to about 5 minutes, and a predetermined time period of about 1 minute to about 3 minutes is particularly preferred.

[0012] In an embodiment, the predetermined volume of fluid is about 0.1 ml to about 100 ml, preferably about 0.25 ml to about 10 ml, more preferably about 0.5 ml to about 1 ml. Preferably, the predetermined volume 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, at least about 5 mL.

[0013] In all aspects of the invention, the sample to be tested (i.e., the liquid or raw sample) will typically be a biological fluid such as urine, blood, plasma, serum, saliva, cerebrospinal fluid, tears, or eluate from a vaginal, nasal, throat, penis, anal, or skin swab.

[0014] It has been found that some fluids, such as those original samples noted above, may contain agents that have a negative impact on the performance of the assay in some cases, and specifically contain agents that have a negative interference with the performance of the isothermal nucleic acid amplification assay. Negative interference can take the form of an agent that inhibits nucleic acid amplification itself and / or fluoresces so that the amplification cannot be reliably detected. It has been found that these assay interfering agents tend to have a lower molecular weight than the target nucleic acid, and can therefore be removed by size exclusion chromatography. The present invention enables size exclusion chromatography to be performed in a simple one-step process in a point-of-care environment. Thus, it is possible to perform tests more quickly and to achieve any treatment required more quickly, thereby providing benefits to patients and healthcare professionals, etc. It has been recognized that not all assay / nucleic acid interfering agents are removed; however, sufficient amounts will be removed to enable the execution and measurement of nucleic acid amplification. Typically, substantially all nucleic acid amplification interfering agents are removed. Typically, nucleic acid amplification interfering agents will be salts and low molecular weight molecules such as proteins or lipids present in the liquid sample, and their molecular weights are typically less than about 5000kDa. Typically, the size exclusion chromatography element removes molecules with a molecular weight less than about 5000kDa. The skilled artisan will appreciate that the molecular weight cut-off may be increased or decreased by selecting a different chromatography resin.

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

[0016] Nick and extension amplification reactions are discussed in detail in WO2009 / 012246, which is incorporated herein by reference.

[0017] Recombinase polymerase amplification reactions are discussed in detail in WO2003 / 072805, WO2005 / 118853, WO2010 / 141940, WO2008 / 035205, WO2007 / 096702, WO2011 / 038197 and WO2012 / 138989, the contents of which are incorporated herein by reference.

[0018] In addition 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 an embodiment of the invention, the device comprises a first part and a preferably separate second part receivable in the first part. Typically, the first part and the second part are operably engaged to move a predetermined volume of fluid from the sample chamber to the chromatographic element. The pump can be actuated by the second part of the device being operably engaged with the first part of the device. Similarly, the metering valve can be actuated by the second part of the device being operably engaged with the first part of the device.

[0020] Typically, the metering valve comprises a metering chamber having an upper portion and a lower portion separated by a movable metering member. Thus, the volumes of the upper portion and the lower portion of the metering chamber are variable. Typically, the metering member and the metering chamber will have matching asymmetric cross-sections. Typically, the inner wall of the metering chamber will have a D-shaped cross-section. Similarly, the outer wall of the metering member will typically have a D-shaped cross-section that can be received in the metering chamber. Typically, the metering member will form an interference fit with the inner wall of the metering chamber, preferably a liquid-tight interference fit. Preferably, the metering member is a cup.

[0021] In an embodiment, the upper and lower portions of the metering chamber are selectively fluidly connected. That is, they may be in a configuration in which they are fluidly connected and in a configuration in which they are not fluidly connected. The metering valve may include, for example, a fluid bypass channel or a pressure release channel for providing fluid communication between the upper and lower portions of the metering chamber. Movement of the metering member relative to the fluid bypass channel may allow selective fluid communication between the upper and lower portions of the metering chamber. If the metering member is located above the fluid bypass channel, there is no fluid communication between the two portions of the metering chamber. However, the metering member may be lowered to expose the fluid bypass channel. Typically, the fluid bypass channel is a pressure release channel. Typically, the fluid bypass channel releases air from the metered lower portion to the upper portion of the chamber under pressure.

[0022] In embodiments, the device includes a lytic agent for treating the sample before the sample reaches the chromatographic element. Exposure of the sample to the lytic agent causes rapid lysis of any cells present in the liquid sample, thereby releasing intracellular nucleic acids for testing. Typically, the lytic agent is located in the metering chamber. The lytic agent will preferably be selected from the group consisting of a surfactant or a base. Preferred bases include potassium hydroxide and sodium hydroxide. At least one potassium hydroxide or sodium hydroxide pellet, typically held in place by a mesh, is particularly preferred. Preferred surfactants may be selected from the group consisting of: sodium dodecyl sulfate, Cetyltrimethylammonium bromide and combinations thereof. Typically, the dissolving agent will be dry.

[0023] Advantageously, the contents of the liquid sample are lysed prior to chromatography so that isothermal nucleic acid amplification inhibitors generated during cell lysis are also removed.

[0024] Typically, the chromatography element comprises a separation chamber for accommodating a chromatography substrate. Preferably, the size exclusion chromatography element comprises a separation chamber for accommodating a size exclusion chromatography gel suspension. Preferably, the chromatography element (preferably a size exclusion chromatography element) comprises a solution containing a buffer for determination (preferably for isothermal nucleic acid amplification), the buffer being preferably magnesium acetate. Preferably, the size exclusion chromatography element comprises a solution containing a buffer for isothermal nucleic acid amplification, in which gel filtration chromatography particles are suspended.

[0025] The concentration of the buffer in the size exclusion chromatography element is selected so that the buffer concentration in the treated sample is at a desired level. A typical concentration of the buffer in the size exclusion chromatography element is about 10 mM to about 200 mM. Preferably, the pH of the treated sample is about pH 6 to about pH 9. These preferred pH and concentrations are applicable to all aspects of the invention. The specific concentration and pH selected will depend on the application, e.g., RPA or NEAR.

[0026] Such an arrangement is advantageous because the processed sample collected in the collection container is then immediately ready for testing, thereby reducing the number of steps in the process.

[0027] Preferred gel filtration particles useful in all aspects of the invention have a 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 Da to 5000 Da for peptides and globular proteins. Preferably, the particles comprise dextran cross-linked with epichlorohydrin. Alternatively, the technician can select the chromatography gel based on the characteristics of the agent to be removed from the sample.

[0028] In an alternative aspect, the present invention provides a device for preparing a sample for determination, preferably for isothermal nucleic acid amplification, comprising a first part and a separate second part that can be received in the first part, wherein the first part includes a container for receiving the sample, and the second part includes a separation element for removing a determination interfering agent, preferably a nucleic acid amplification inhibitor and / or a fluorescent agent from the sample. When the second part is received in the first part, the first part and the second part are operably engaged to move the sample from the sample chamber to the separation element. Preferably, the device is single-use. Preferably, the device is manually actuated, preferably the device can be actuated by a single user-applied push or rotation.

[0029] Typically, the first part comprises a metering valve for metering a predetermined volume of sample from the sample chamber and which is actuatable by the second part of the device engaging the first part of the device.

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

[0031] The metering valve will typically comprise a metering chamber having an upper portion and a lower portion separated by a moveable metering member.

[0032] Typically, the metering valve comprises a metering chamber having an upper portion and a lower portion separated by a movable metering member. Thus, the size of the upper portion and the lower portion of the metering chamber is variable. Typically, the inner wall of the metering chamber will have a D-shaped cross section. Similarly, the outer wall of the metering member will typically have a D-shaped cross section, which can be received in the metering chamber. Typically, the metering member will have an interference fit with the inner wall of the metering chamber, preferably a liquid-tight interference fit. Preferably, the metering member is a cup.

[0033] In embodiments, the upper and lower portions of the metering chamber are selectively in fluid communication. That is, they may be in a configuration in which they are in fluid communication and a configuration in which they are not in fluid communication. The metering valve may include, for example, a fluid bypass channel for providing fluid communication between the upper and lower portions of the metering chamber. Movement of the metering member relative to the fluid bypass channel may allow selective fluid communication between the upper and lower portions of the metering chamber. Typically, the fluid bypass channel is a pressure relief channel.

[0034] In other embodiments, the device includes a dissolving agent for treating the sample before the sample moves to the separation element. Typically, the dissolving agent is located in the metering chamber. The dissolving agent will preferably be selected from the group consisting of a surfactant or a base. Preferred bases include potassium hydroxide and sodium hydroxide. The use of a mesh of material doped with potassium hydroxide or at least one potassium hydroxide or sodium hydroxide pellet preferably held in place by a mesh is particularly preferred. Preferred surfactants can be selected from the group consisting of: sodium lauryl sulfate, Cetyltrimethylammonium bromide and combinations thereof.

[0035] As discussed above, it is advantageous to perform lysis prior to separation, as assay interfering agents generated during lysis may also be removed.

[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 alternative embodiments, the separation element may comprise a filter or a suitable stationary phase chromatography element selected from the group consisting of: ion exchange chromatography elements, including cation and anion exchange chromatography elements; reverse phase chromatography elements; and affinity chromatography chromatography elements. Thus, the device of the present invention can be used for ion exchange chromatography, reverse phase chromatography and affinity chromatography.

[0038] In another aspect, the present invention provides a device for preparing a sample for isothermal nucleic acid amplification, comprising a lysing element and a separation element for removing a nucleic acid amplification inhibitor and / or a fluorescent agent from the lysate.

[0039] Typically, the dissolving element comprises a dissolving agent. The dissolving agent will preferably be selected from the group consisting of a surfactant or an alkali. Preferred alkalis include potassium hydroxide and sodium hydroxide. A mesh of material doped with potassium hydroxide is particularly preferred; more preferred is the use of at least one pellet of potassium hydroxide or sodium hydroxide, typically held in place by a mesh. Preferred surfactants may be selected from the group consisting of sodium lauryl sulfate, Cetyltrimethylammonium bromide and combinations thereof.

[0040] The present invention also contemplates a metering valve comprising a metering chamber including a dry solvent.

[0041] The dissolving agent will preferably be selected from the group consisting of a surfactant or a base. Preferred bases include potassium hydroxide and sodium hydroxide. A mesh of material doped with potassium hydroxide is particularly preferred; more preferred is the use of at least one pellet of potassium hydroxide or sodium hydroxide, typically held in place by the mesh. Preferred surfactants may be selected from the group consisting of sodium lauryl sulfate, Cetyltrimethylammonium bromide and combinations thereof.

[0042] In another aspect of the present invention, a kit for performing isothermal nucleic acid amplification on a sample is provided. The kit will generally include: a liquid transfer device, preferably including a housing with a pipette tip and a plunger assembly; a reaction chamber, the reaction chamber containing reagents for isothermal nucleic acid amplification reaction; a sample reservoir; and a sample preparation device, the sample preparation device including a separation element, the separation element is capable of removing nucleic acid amplification inhibitors and / or fluorescent agents from the sample before performing the isothermal nucleic acid amplification.

[0043] In the embodiment of the test kit, the reaction vessel holds reagents for recombinase polymerase amplification (RPA), such as recombinase, single-stranded binding protein and polymerase. The recombinase can be selected from T4 UvsX, T6 UvsX or RecA. The DNA polymerase can be selected from the group consisting of the following: Escherichia coli DNA polymerase I Klenow fragment, Bacillus stearothermophilus polymerase (Bst), Bacillus subtilis Phi-29 polymerase, Bacillus subtilis polymerase I (Bsu). The single-stranded binding protein is typically gp32.

[0044] Reagents for recombinase polymerase amplification generally also include crowding agents, ATP (adenosine triphosphate) or ATP analogs, dNTPs or T4 bacteriophage UvsY. Preferred crowding agents may be selected from the group comprising (preferably consisting of) polyethylene glycol (PEG), dextran, polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) or polysucrose.

[0045] When present, PEG is preferably PEG 1450, PEG 3000, PEG 8000 or PEG 10000. PEG will preferably have a molecular weight of between about 15,000 and about 20,000.

[0046] When present, dNTP is preferably selected from the group consisting of dATP, dGTP, dCTP and dTTP.

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

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

[0049] The reagents are usually in dry or lyophilized form, but may be in liquid form.

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

[0051] Preferably, the sample preparation device contains a lytic agent to which the sample is exposed, after which the separation element removes the nucleic acid amplification inhibitor and / or fluorescent agent from the lysate. The use of at least one potassium hydroxide or sodium hydroxide pellet, typically held in place by a mesh, is particularly preferred.

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

[0053] In alternative embodiments, the separation element may comprise a filter.

[0054] The sample preparation device may be a device according to any preceding aspect of the invention.

[0055] Specifically, the sample preparation device can be a manually actuated size exclusion chromatography device. The device comprises a chamber for receiving a liquid sample, a pump having a metering valve, and a size exclusion chromatography element. In use, the pump moves a predetermined volume of liquid from the sample chamber to the size exclusion chromatography element. Typically, the pump moves a predetermined volume of fluid from the sample chamber through the size exclusion chromatography element. Preferably, the pump moves a predetermined volume of fluid from the sample chamber through the size exclusion chromatography element to reach a sample collection container. Preferably, the device is single-use. Preferably, the device can be actuated by a single downward push or rotation. Typically, the pump is pneumatic.

[0056] Alternatively, the sample preparation device can be a device for preparing a sample for isothermal nucleic acid amplification, comprising a first part and a separate second part receivable in the first part, wherein the first part comprises a container for receiving the sample, and the second part comprises a separation element for removing nucleic acid amplification inhibitors and / or fluorescent agents from the sample, wherein when the second part is received in the first part, the first part and the second part are operably coupled to move the sample from the sample chamber to the separation element.

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

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

[0059] Preferably, the sample preparation device is removably engageable with the fluid reservoir. Preferably, the sample preparation device is actuated when positioned in the fluid reservoir, preferably by pushing the device against the fluid reservoir. Typically, the prepared sample is collected in the fluid reservoir.

[0060] The liquid transfer device can be configured to lockably engage with the reaction chamber, for example, without dispensing the liquid sample, before dispensing the liquid sample, and / or after dispensing the liquid sample.In some embodiments, the reaction chamber includes one or more components of a biological reaction.

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

[0062] Typically, movement of the housing relative to the plunger assembly results in a vacuum being formed within the pipette tip, and optionally, the plunger assembly can be configured to lock in a position resulting in the vacuum being formed. The housing can be configured to move relative to the plunger assembly by pushing the housing downward onto the fluid reservoir. The device can further be configured to provide an audible and / or visual indication that the plunger assembly is in a position resulting in the vacuum being formed.

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

[0064] The liquid transfer device may be configured to draw a sample from a fluid reservoir by pushing the device against the reservoir and a system including the liquid transfer device and one or both of a reaction chamber and a fluid reservoir.

[0065] In the above system, all four of the liquid transfer device, the reaction chamber, the sample preparation device, and the fluid reservoir may have compatible asymmetric cross-sections.

[0066] In another aspect, the present invention provides a system for performing isothermal nucleic acid amplification, which comprises a kit according to the aforementioned aspect of the present invention and a detection device.

[0067] The detection device will typically include a first station adapted to securely hold the sample collection chamber and a second station adapted to securely hold the reaction chamber. When in use, the sample preparation device is positioned in the sample collection chamber. Typically, the first portion of the device is placed in the sample collection chamber. Then, the original sample is placed in the metering chamber of the device. Then, the second portion of the device is inserted into the first portion of the device. Then, the second portion of the device is pushed into the first portion, typically until an audible or visual signal is emitted, and the prepared sample is collected in the sample collection container. The sample preparation device is then removed and disposed of.

[0068] The transfer device is movable between the collection chamber at the first station and the reaction chamber at the second station.

[0069] The detection device comprises a cover which can be closed when the detection device is in operation or for storage.

[0070] There may be a touch screen user interface for inputting data and displaying information about the assay. The second station may include a bar code reader or similar device to automatically detect a bar code or similar code present on the amplification chamber. The first station and the second station may be adapted to heat or cool the contents of the sample collection chamber and the reaction chamber. The second station may also be adapted to provide optical, fluorescent or other monitoring and / or agitation to the microtube.

[0071] In some embodiments, a liquid transfer device or pipette tip disclosed herein may be configured to collect and dispense volumes between 1 μl and 5 ml (e.g., volumes between any two of the following: 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 another aspect, the present invention provides a method for performing isothermal nucleic acid amplification. The method comprises the following steps: providing an original liquid sample for testing; treating the original liquid sample to remove nucleic acid amplification inhibitors and / or fluorescent agents; performing isothermal nucleic acid amplification on the treated sample; and monitoring the amplified nucleic acid.

[0073] In another aspect of the present invention, the present invention provides a composition for gel filtration chromatography, which comprises an aqueous solution containing a constant temperature nucleic acid amplification buffer and a gel filtration chromatography particle dispersion. Generally, the buffer is selected from the group consisting of magnesium acetate or Tris acetate; preferably magnesium acetate.

[0074] Preferred gel filtration particles have a 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 about 1000 Da to about 5000 Da for peptides and globular proteins. Preferably, the particles comprise dextran cross-linked with epichlorohydrin. Alternatively, the size exclusion chromatography gel may be selected by the skilled artisan.

[0075] The present invention also contemplates the use of such compositions in preparing samples for isothermal nucleic acid amplification.

[0076] The present invention also provides a metering valve for a pump. The metering valve will generally include: a metering chamber having an upper portion and a lower portion separated by a movable metering member; and a pressure relief passage. Preferably, the metering member and the pressure relief passage are arranged so that the metering member can be moved from an initial position, in which the metering member separates the upper portion from the lower portion, to a subsequent position, in which the pressure relief passage provides fluid communication between the lower portion of the metering chamber and the upper portion of the metering chamber.

[0077] Thus, as the metering member descends, thereby reducing the volume of the lower portion of the metering chamber, the pressure in the lower portion of the metering chamber increases. After the metering member has advanced a predetermined distance, the pressure relief passage is able to provide fluid communication between the lower portion and the upper portion of the metering chamber. Once in fluid communication, air from the lower portion of the metering chamber will move along the pressure relief passage and displace fluid in the upper portion of the metering chamber.

[0078] Typically, a fluid outlet passage is provided. Preferably, the fluid is discharged along the outlet passage and thereby exits the metering valve. In a preferred embodiment, the fluid outlet passage is located within a movable actuator for the metering valve. In such embodiments, the movable actuator is operably engaged with the metering member so that movement of the actuator meters a predetermined volume of sample and pumps the predetermined volume out of the outlet passage.

[0079] Typically, the metering member will have an interference fit, preferably a fluid-tight interference fit, with the inner wall of the metering chamber. The inner wall of the metering chamber may have an asymmetric cross-section, preferably a D-shaped cross-section. Similarly, the outer wall of the metering member may have an asymmetric cross-section that matches the cross-section of the metering chamber, typically a D-shaped cross-section. Preferably, the metering member is a cup. Typically, the movable actuator is operably engaged with an inner bottom plate of the cup.

[0080] Typically, the metering chamber will have a single opening for receiving the liquid sample to be metered and the movable actuator. Typically, when the movable actuator is received in the opening of the metering chamber, the periphery of the opening will form an interference fit, preferably a liquid-tight interference fit, with the outer wall of the movable actuator.

[0081] In another aspect of the present invention, a method of manufacturing a single-use metering valve is provided. The method comprises the steps of providing a metering chamber having a first opening and a second opening and a pressure relief passage extending partially along an inner wall of the chamber, generally in an axial direction; providing a metering member receivable in the metering chamber to divide the metering chamber into a first portion and a second portion; positioning the metering member in the metering chamber so that the pressure relief passage does not provide fluid communication between the first portion and the second portion; and sealing the opening of the metering chamber with a seal. Typically, the metering member is movable within the metering chamber. Preferably, the metering member is moved by sliding the opening to be sealed into position, and preferably, the other opening is not sealed.

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

[0083] Typically, the metering member enters the metering chamber through the second opening and advances along the metering chamber until the metering member engages an abutment, typically an annular abutment.

[0084] Preferably, the metering member forms an interference fit with the metering chamber, preferably a fluid-tight interference fit.Preferably, the metering member is a cup.

[0085] The present invention also provides an artificially actuated pump, which includes a first part and a separate second part that can be operably received in the first part. Typically, the first part and the second part are joined to form a metering valve, preferably, the first part includes a metering chamber for receiving a liquid sample, a movable metering member and a pressure release channel, and the second part includes an actuator, which is arranged to operably engage the metering member when the second part is received in the first part. In use, the second part is advanced into the first part and a predetermined volume of fluid leaves the pump through an outlet channel, and the predetermined volume of fluid will generally include a predetermined volume of liquid sample and a predetermined volume of air. Typically, the outlet channel is arranged in the second part. In a preferred embodiment, the fluid outlet channel is located in the actuator. The pump is typically single-use.

[0086] Typically, the metering chamber will have a single opening for receiving the liquid sample to be metered and the actuator. Typically, when the movable actuator is received in the opening of the metering chamber, the periphery of the opening will form an interference fit, preferably a liquid-tight interference fit, with the outer wall of the actuator.

[0087] Typically, the metering member will have an interference fit, preferably a fluid-tight interference fit, with the inner wall of the metering chamber. The metering member thereby divides the metering chamber into an upper part and a lower part. The inner wall of the metering chamber may have an asymmetric cross-section, preferably a D-shaped cross-section. Similarly, the outer wall of the metering member may have an asymmetric cross-section that matches the cross-section of the metering chamber, typically a D-shaped cross-section. Preferably, the metering member is a cup. Typically, the movable actuator is operably engaged with the inner bottom plate of the cup.

[0088] In use, the volume of fluid, typically air, displaced by the actuator as it advances into the metering chamber causes a pressure increase within the upper portion of the metering chamber and thereby moves the predetermined volume of fluid through the outlet passage. Selecting the correct volume displacement is within the capabilities of the skilled person.

[0089] The pressure relief passage 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. When the metering member is moved along the metering chamber by the actuator, the pressure relief passage is exposed and fluid communication between the upper and lower parts of the metering chamber is achieved. In a preferred embodiment, the pressure relief passage is located in the metering chamber wall. The pressure relief passage can be in the form of an open groove or a closed conduit. Typically, the pressure relief passage will extend from the base of the metering chamber or its vicinity to a position below the top of the metering member in the initial position.

[0090] Preferably, the metering chamber comprises a dissolving agent. Suitable dissolving agents have been discussed earlier in this disclosure.

[0091] Another aspect of the present invention provides a pump for metering a predetermined volume of liquid, preferably a manually driven pump, comprising: a container, the container comprising a sample chamber and a metering chamber; an actuator, the actuator comprising a fluid outlet channel, the fluid outlet channel having an opening in a distal portion of the actuator, wherein the actuator is movable 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 metering chamber, and wherein when the actuator is in the first position, the sample chamber is in fluid communication with the metering chamber, and when the actuator is in the first position, the sample chamber is in fluid communication with the metering chamber, and when the actuator is in the second position, the sample chamber is in fluid communication with the metering chamber. When the actuator is in the second position, the sample chamber is separated from the metering chamber; wherein the metering chamber is divided into an upper part and a lower part by a movable metering member, and wherein the metering chamber also includes a pressure release channel; and wherein the metering member, the pressure release channel and the actuator are arranged so that when the actuator moves from its first position to its second position, the metering member moves from an initial position to a subsequent position, in which the metering member separates the upper part from the lower part, and in which the pressure release channel provides fluid communication between the lower part of the metering chamber and the upper part of the metering chamber.

[0092] Typically, the metering member will have an interference fit, preferably a fluid-tight interference fit, with the inner wall of the metering chamber. The metering member thereby divides the metering chamber into an upper part and a lower part. The inner wall of the metering chamber may have an asymmetric cross-section, preferably a D-shaped cross-section. Similarly, the outer wall of the metering member may have an asymmetric cross-section that matches the cross-section of the metering chamber, typically a D-shaped cross-section. Preferably, the metering member is a cup. Typically, the movable actuator is operably engaged with the inner bottom plate of the cup.

[0093] In use, the volume of fluid, typically air, displaced by the actuator as the actuator advances into the metering chamber causes an increase in pressure within the upper portion of the metering chamber and thereby moves the predetermined volume of fluid through the fluid outlet passage. The volume of the upper portion of the metering chamber displaced by the actuator should preferably be greater than the volume of the liquid sample to be moved, so that when the metering member is fully displaced by the actuator, all of the liquid sample in the upper portion of the metering chamber is forced to leave the metering chamber. The excess volume ensures that a certain volume of air also passes through the second portion of the device to prevent dripping and ensure dose uniformity. It is within the skill of the technician to select the correct volume of the metering chamber to be displaced by the actuator.

[0094] In a preferred embodiment, the pressure relief passage is located in the metering chamber wall. The pressure relief passage may be in the form of an open recess or a closed conduit. Typically, the pressure relief passage will extend from at or near the base of the metering chamber to a position below the top of the metering member in the initial position.

[0095] Preferably, the metering chamber and / or the sample chamber comprises a lytic agent. Suitable lytic agents have been disclosed earlier in this disclosure.

[0096] The pump is usually single-use. For the purpose of the present invention, single-use means that in normal use, the pump cannot be reset and reused.

[0097] The pumps and metering valves according to these aspects of the invention are particularly contemplated for use in the apparatus according to the preceding aspects of the invention.

[0098] For the purposes of the present invention, manual actuation has its normal meaning. That is, the device, pump and metering valve can be actuated by hand. Although it is contemplated that certain aspects and embodiments of the present invention can also be actuated by alternative means, all devices, pumps and metering valves of the present invention can be manually actuated.

[0099] The details of one or more embodiments of the invention are set forth in the drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 A prior art system is shown.

[0101] Figure 2 An exploded view of an exemplary device is shown.

[0102] Figure 3 An exemplary first portion of the device is shown before use.

[0103] Figure 4 An exemplary first portion of a device for removing a sample chamber cover is shown.

[0104] Figure 5 An exemplary second portion of the device is shown.

[0105] Figure 6 The second portion is shown joining the first portion and metering the volume of the original sample.

[0106] Figure 7 The second portion is shown having displaced the metering member.

[0107] Figure 8 The second part is shown fully depressed.

[0108] Figures 9 to 11 An exemplary device is shown in situ in an apparatus for performing and monitoring isothermal nucleic acid amplification.

[0109] Fig.12 The prepared sample is shown being transferred to the reaction chamber.

[0110] Fig.13 a to Fig.13 e provides a schematic illustration of the metering chamber during use. DETAILED DESCRIPTION OF THE INVENTION

[0111] The present invention relates to the preparation of samples for determination, and in particular to the preparation of samples for isothermal nucleic acid amplification. In particular, the present invention relates to manually operated chromatography devices, compositions useful therein, devices for preparing samples for isothermal nucleic acid amplification, kits for performing isothermal nucleic acid amplification, and methods for performing isothermal nucleic acid amplification. The present invention also provides pumps and metering valves.

[0112] Figure 1 Shown for Fig.13The reaction vessel (200), sample collection reservoir (300) and liquid transfer device (100) of the system shown. Each subassembly can have a D-shaped or other asymmetric cross-section (105, 205, 305) that is compatible with the other two subassemblies, so that the subassemblies can only mate with each other in one direction.

[0113] Reaction chamber 200 includes a microtube 220 held within an orifice in the bottom of the reaction vessel body.

[0114] Figure 1 The transfer device 100 and the reaction vessel 200 described above are shown to have one pipette tip 120 and one microtube 220. However, the transfer device may have two or more pipette tips, and the reaction vessel may have two or more microtubes.

[0115] Such components are discussed in detail in WO 2013 / 041713, which is incorporated herein by reference.

[0116] Figure 2 is an exploded view of a device (20) according to the present invention. The device comprises a first portion (20a) and a second portion (20b). The second portion (20b) is receivable in the first portion (20a). A sample reservoir (210) is also shown. The first portion (20a) is receivable in the sample collection reservoir (210).

[0117] The first part (20a) includes a body (21). The body (21) includes a sample chamber (not shown) and a metering chamber (211). The metering chamber (211) is in fluid communication with the sample chamber. The metering member (22) is in the form of a cup-shaped member having a D-shaped cross section. The metering chamber (211) also has a D-shaped cross section. The metering member (22) will typically accommodate a dehydrated dissolving agent. Typically, at least one dissolving agent (typically potassium hydroxide or sodium hydroxide) pellet (221) is held in place by a wire mesh (222). Potassium hydroxide / sodium hydroxide is present to cause rapid dissolution of cellular material in the sample fluid, thereby releasing intracellular nucleic acids to be detected by isothermal nucleic acid amplification.

[0118] The metering member (22) can be movably received in the metering chamber (211). In use, the metering member (22) divides the metering chamber into an upper part and a lower part. The metering member (22) forms a liquid-tight interference fit with the inner wall of the metering chamber (211). The airtight film (23) closes the end of the metering chamber. In manufacturing, the metering member (22) is inserted into the base of the metering chamber (211) and pushed upward until the metering member (22) is just below the annular seal (not visible) that separates the metering chamber from the sample receiving chamber. The airtight film (23) is then heat-sealed over the base of the metering chamber (211).

[0119] The second part (20b) includes a body (24), which includes an actuator (25) that can be received in the sample chamber of the first part (20a). In use, the actuator (25) is operably engaged with the metering member (22). The actuator (25) has a distal end (215) and a proximal end (216). The orifice (214) is located in the distal end of the actuator (25). The orifice (214) is in fluid communication with a separation chamber (not shown) containing an aqueous solution, the aqueous solution comprising a dispersion of a constant temperature nucleic acid amplification buffer and gel filtration chromatography particles. Suitable gel filtration particles are sold by GE Healthcare under the trade name Sephadex G-25Superfine, and other suitable chromatography substrates are known to technicians. A microfluidic channel (not shown) provides fluid communication between the separation chamber and the exit orifice (217) at the distal end of the return leg (26). An insert (27) is inserted into the return leg (26) and closes the separation chamber. The outer wall of the actuator (25) includes a shoulder (213) for engaging the first portion. A channel (212) is present in the shoulder (213). In use, the channel (213) allows excess liquid to escape from the metering chamber before the metering chamber is sealed.

[0120] Peelable seals (223, 224) are provided on the first portion and the second portion.

[0121] Figure 3 The first part (32) of the device is shown before use. A peelable seal (31) covers a sample chamber (not shown). Prior to use, the peelable seal (31) protects the contents of the first part (32) from contamination and protects the dry dissolving agent from moisture. An intact peelable seal (31) also indicates to the user that the device has not been used before. The first part (32) of the device is engaged with a sample reservoir (33).

[0122] Figure 4 The first part (42) of the device is shown with the peelable seal removed. The sample chamber (41) is now accessible. Again, the first part (42) of the device is engaged with the sample reservoir (43).

[0123] Figure 5 The second part (60) of the device is shown. The second part (60) of the device includes a first actuator leg (63) and a second return leg (62). A stopper (61) is fixed in place. A peelable seal (64) covers the orifices at the distal ends of the actuator and return legs to prevent contamination and / or leakage of fluid within the actuator.

[0124] In use, the peelable seal (64) is removed before joining the second part to the first part. The peelable seal (64) prevents contamination and leakage. An intact peelable seal (64) indicates to the user that the device has not been used before.

[0125] A microfluidic path (not visible) extends from the top of the actuator leg (63) and down the inside of the return leg (62) through which, in use, treated liquid flows.

[0126] The second part (60) of the device is made by injection molding in two pieces: an outer wall (65) and an insert (66). The insert fills most of the return leg (62) and includes a rounded portion (66) visible on the top surface of the second part (60). There is a groove (not shown) in the return leg portion of the insert that forms a closed channel when inserted into the return leg (62). Figure 2 As better shown in FIG. 1 , in the actuator leg ( 25 ), the insert ( 27 ) compresses the top frit ( 218 ) downwardly onto the substrate ( 219 ) and the bottom frit ( 220 ), and includes openings and grooves ( not shown ) that allow the treated fluid to traverse to the return leg ( 26 ).

[0127] Figure 6 a shows the device of the invention with the second part (71) inserted into the first part (72). Again, the first part (72) of the device engages with the sample collection reservoir (73).

[0128] Figure 6 b shows the passage through Figure 7 a shows a cross section of the first part (72) and the second part (71) of the device and the sample reservoir (73).

[0129] Figure 6 The cross section in b reveals the separation chamber (76) that holds the size exclusion chromatography matrix (75). The chamber (76) that holds the matrix is ​​equipped with a top frit (77) and a bottom frit (78) to hold the matrix (75) in place. There is also a cutout feature (cross shape) in the molded plastic that allows fluid to diffuse and enter the separation chamber (75).

[0130] In use, the user is guided to introduce the second part (71) into the first part (72). The device is shaped so that the actuator leg (79) is clearly the leg to be introduced into the chamber (711) to which the original sample is added.

[0131] Assuming that enough liquid sample has been added to fill the upper portion of the metering chamber (712) while in the original position, and preferably the liquid is allowed to reside above the annular seal (713) separating the metering chamber from the sample chamber (711), the desired volume of processed sample will be obtained.

[0132] When the second portion (71) is inserted, the narrower portion of the actuator leg first protrudes through the annular seal (713) and 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) displaces liquid from the upper portion (712) of the metering chamber, the liquid can escape upward around the edge of the actuator leg (79) into the sample chamber (711).

[0133] exist Figure 6 a and Figure 6 In Figure 1, the second part (71) has been pushed into the first part (72) by hand so that the shoulder (715) engages with the annular seal (713) positioned between the sample chamber (711) and the metering chamber (714). In this position, a groove (not shown) in the shoulder (715) provides fluid communication between the metering chamber (714) and the sample chamber (711). The distal end (718) of the actuator just engages the metering member (717). The volume of the upper part of the metering chamber around the actuator defines the volume of the original sample that will pass through the size exclusion chromatography gel (75). The size exclusion chromatography gel (75) is suspended in a solution containing a buffer (usually magnesium acetate) suitable for isothermal nucleic acid amplification. The concentration of the buffer in the separation chamber (76) should be such that it is present in the processed sample at the correct concentration.

[0134] exist Figure 6 In the position shown in b, the metering member (717) is located 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 portion (712) and the lower portion (720) of the metering chamber (714). Therefore, when the actuator is further lowered, it advances the metering member (717) and increases the pressure of the air in the lower chamber (720).

[0135] Figure 7 a and Figure 7b shows the device in which the second part (81) has been pushed further into the first part (82). In this position, the actuator shoulder (84) has dropped below the annular seal (85), which now engages the outer wall of the actuator (86), thereby sealing the metering chamber (87) from the sample chamber (88). The actuator (86) has pushed the metering member (89) to a lower position, so that the upper end of the pressure release channel (810) is now exposed above the metering member (89). The pressure release channel (810) provides fluid communication between the lower portion (811) of the metering chamber (87) and the upper portion (812) of the metering chamber (87), and because the pressure in the lower portion (811) of the chamber is higher than the pressure in the upper portion (812) of the chamber, air travels along the pressure release channel (810) from the lower portion (811) of the chamber to the upper portion (812) of the chamber, thereby forming a high pressure area above the cup-shaped metering member (89) containing the liquid sample as a result of the internal volume of the upper portion (812) contracting due to the presence of the actuator (86) in the upper portion (812). In turn, the liquid sample is forced to pass through the hole (814) in the distal end of the actuator (86) and enter the size exclusion chromatography / separation chamber (815) for processing.

[0136] A portion of the original sample remains sealed in the sample chamber (88). This can be disposed of with the device.

[0137] When the second part (81) of the device is pushed further into the first part (82), the actuator (86) causes the metering member (89) to move further downward in the metering chamber (87), thereby forcing the air along the pressure release channel (810) from the lower part (811) to the upper part (812), and thereby causing the sample to travel along the microfluidic channel (not shown) in the horizontal upper part of the second part (81) of the device and along the channel in the return leg (817) of the second part (81) through the size exclusion chromatography gel in the separation chamber (815), and then leave the second part (81) and drip into the sample collection reservoir (818).

[0138] The size exclusion chromatography gel removes constant temperature nucleic acid amplification inhibitors and / or fluorescent agents from the sample. Therefore, the processed sample collected in the sample collection reservoir (818) is fully free of the inhibitors / fluorescent agents, so that the nucleic acid present in the sample can be successfully subjected to constant temperature nucleic acid amplification and then detected. In addition, because the size exclusion chromatography gel is suspended in a solution containing a buffer for performing constant temperature nucleic acid amplification. The processed sample collected in the sample collection reservoir (818) is at the correct pH for performing constant temperature nucleic acid amplification. Typically, the pH is about 6 to about 9. This avoids the need for additional sample preparation steps.

[0139] Figure 8 a and Figure 8 b shows the second part (91) fully inserted into the first part (92). An audible prompt (usually a click) indicates to the user that full insertion has been achieved and the correct amount of sample will therefore be processed. In use, the user will typically perform a single push with a finger or thumb until a click is heard. The member causing the audible click will typically be a latch, which also locks the second part (91) in the first part (92). This also means that the unprocessed original sample is safely contained for disposal. Preferably, a liquid-tight seal is formed between the top of the second part (91) and the sample chamber (94), thereby accommodating excess original sample fluid (93) and preventing the original sample from overflowing when the device is disposed.

[0140] When fully inserted, air from the lower portion (95) of the metering chamber (96) continues to flow into the upper portion (97) of the metering chamber (96) until the pressure in the two chambers is equal. Figure 8 As shown in Fig. 1, no original sample is retained in the metering chamber (96). In addition, a small amount of air travels through the second portion (91) of the device and exits through the hole (98) at the end of the return leg (99). This pushes out the liquid present in the channel and prevents dripping. This also allows any residual compressed gas on the sample side of the device to be dissipated. This further prevents residual fluid from being pushed through the column after the device has been removed from the sample collection container (910) to prevent excess fluid from potentially dripping out of the return leg (99) and contaminating the working area.

[0141] This is achieved by ensuring that, when the actuator is fully inserted, the volume of the metering chamber displaced by the actuator is greater than the volume of the original sample metered for processing.

[0142] There is usually a delay between the audible click and all processed samples arriving at the sample collection reservoir (910). This is due to the damping effect of the air in the lower portion (95) of the compression metering chamber (96) and the release of this pressure by the device. This 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 treated by the size exclusion chromatography gel. If the sample travels through the gel too quickly, insufficient removal of nucleic acid amplification inhibitors / fluorescers will occur and the device will not be able to achieve its desired function. Achieving the correct level of damping is within the capabilities of the technician.

[0143] exist Figure 8b, the sample collection reservoir (910) will contain the processed sample ready for isothermal nucleic acid amplification. The processed sample is buffered to the required pH and is sufficiently free of nucleic acid amplification inhibitors and fluorescent agents to perform and detect amplification. A liquid transfer device (not shown) is used to pipette a portion of the processed sample from the sample collection container (910) to the test device for performing an isothermal nucleic acid amplification assay.

[0144] Figures 9 to 12 The device (10) according to the invention is shown in situ in a sample processing device (101) for performing isothermal nucleic acid amplification. Fig. 9 In the embodiment of the present invention, the first part (102) of the device according to the invention is located in a sample collection container (103), which in turn is received in a sample processing device (101). Suitable sample processing devices are commercially available from Alere Ltd. under the trade name Alere i.

[0145] exist Fig. 9 In the embodiment of the present invention, the reaction vessel (103) is in situ in the sample processing device. The sample processing device can be used with a reaction vessel configured to perform NEAR and / or RPA isothermal nucleic acid amplification. Thus, the reaction chamber can contain the reagents necessary to perform NEAR and / or RPA on the sample introduced into the chamber. Suitable reaction vessels (105) are available from Alere Ltd.

[0146] exist Fig. 9 In the embodiment of the invention, the protective peelable film covering the sample chamber (104) has been removed. In this position, the original sample is introduced into the sample chamber using a pipette, typically 1.5 ml of the original sample will be introduced.

[0147] Fig.10 The system (11) of the present invention is shown in which the second part (113) of the device is partially inserted into the first part (112) of the device (111). Fig.11 The system (12) of the present invention is shown in which the second portion (123) of the device (121) is fully depressed into the first portion (122) of the device (121). Once the sample has been processed and collected in the sample collection reservoir (124), the device (121) for preparing the sample can be removed and disposed of. A portion of the processed sample is then transferred to a reaction vessel (125) for testing using a liquid transfer device.

[0148] Fig.12A liquid transfer device (100), a reaction vessel (200) and a sample collection reservoir (300) are shown, as well as a sample processing device (400). In 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 contemplates a test kit including a reaction vessel (200), a liquid transfer device (100), a sample collection chamber (300) and a sample preparation device, and a system including the test kit and the sample processing device (400).

[0149] Fig.12 A system with an exemplary detection device (400) is shown. 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). In use, the transfer device (100) is moved between the sample collection container (300) at the first station (410) and the reaction chamber (200) at the second station (420). The detection device includes a cover (430) that can be closed when the detection device (400) is in operation or for storage. There is a touch screen user interface (440) for entering data and displaying information about the assay. The second station (420) may include a bar code reader or similar device to automatically detect a bar code or similar code present on the reaction chamber (200). The first station (410) and the second station (420) may be adapted to heat or cool the contents of the sample collection container (300) and the reaction chamber (200). The second station (420) may also be adapted to provide optical, fluorescent or other monitoring and / or agitation of the microtube (220).

[0150] Fig.13 a to Fig.13 e provides a schematic illustration of the device during use.

[0151] Fig.13 a shows the device before use with the first part (141) separated from the second part (142). A cup-shaped metering member (143) is located at the top of the metering chamber (144), abutting against an annular seal (145) separating the sample chamber (146) from the metering chamber (144).

[0152] exist Fig.13 In b, the original liquid sample (147) has been introduced into the cup-shaped metering member (143) in the sample chamber (146) and the metering chamber (144). Preferably, the liquid level is above the annular seal (145).

[0153] exist Fig.13c, the movable actuator (148) has been lowered through the sample chamber (146) and into the metering chamber (144) such that the distal end of the movable actuator (148) has engaged the metering member (143) and its shoulder (1410) has almost engaged the annular seal (145). The volume of liquid in the cup-shaped metering member (143) when the annular seal (145) engages the outer wall (1411) of the actuator (148) distal to the shoulder (1410) is the predetermined volume of liquid (1412) to be metered for processing, as shown in FIG. Fig.13 As shown in d.

[0154] exist Fig.13 d, the movable actuator (148) has moved the metering member (143) along the metering chamber (144), thereby reducing the volume of the lower portion (1413) of the metering chamber (144) and increasing the volume of the upper portion (1414). The lower and upper portions of the metering chamber (144) are fluidly connected through a pressure release channel (1415) in the wall of the metering chamber. When the movable actuator (148) advances into the metering chamber (144), the internal volume of the metering chamber (144) is reduced, thereby increasing the pressure of the air contained therein. This increase in air pressure within the metering chamber (144) in turn forces the metered liquid sample (1412) to leave the metering chamber (144) through an outlet channel (not shown) located in the movable actuator (148).

[0155] When the movable actuator (148) is fully depressed, as Fig.13 As shown in Figure 1, the volume of air displaced by the movable actuator (148) in the metering chamber (144) is greater than the volume of the liquid sample being metered, so that substantially all of the liquid is forced to leave the metering chamber (144) through an outlet passage (not shown). The unprocessed raw sample (1416) is stored in the sample chamber (146) for safe disposal.

Claims

1. A method for performing isothermal nucleic acid amplification, comprising: a) provide the original liquid sample for testing; b) treating the original liquid sample to remove nucleic acid amplification inhibitors and / or fluorescent agents; c) performing isothermal nucleic acid amplification on the processed sample; and d) monitoring the amplified nucleic acid, wherein the processing step is performed using an artificially actuated chromatography device comprising a first portion and a separate second portion receivable in the first portion, wherein the first portion comprises a sample chamber for receiving the liquid sample and a pump having a metering valve for metering a predetermined volume of liquid from the sample chamber, and the second portion comprises a chromatography element for removing nucleic acid amplification inhibitors and / or fluorescent agents from the liquid sample, and wherein the pump is actuated by the second portion of the artificially actuated chromatography device being operably engaged with the first portion of the artificially actuated chromatography device to move the predetermined volume of liquid from the chamber to the chromatography element, The isothermal nucleic acid amplification is recombinase polymerase amplification (RPA), nicking and extension amplification reaction (NEAR), strand displacement amplification or loop-mediated isothermal amplification.

2. The method of claim 1, wherein the processing step comprises size exclusion chromatography.

3. The method of claim 1 or 2, wherein the processing step comprises exposing the original liquid sample to a lytic agent followed by size exclusion chromatography of the lysate.

4. The method of claim 1 or 2, wherein the processing step comprises buffering the original liquid sample before, during or after removing the nucleic acid amplification inhibitor and / or fluorescent agent.

5. The method according to claim 1 or 2, wherein the processing step includes buffering the original liquid sample during the removal of the nucleic acid amplification inhibitor and / or fluorescent agent.

6. The method of claim 1 or 2, wherein the raw liquid sample is selected from the group consisting of urine, blood, plasma, serum, saliva, cerebrospinal fluid, tears, or eluate from a vaginal, nasal, throat, penis, anal, or skin swab.

7. The method of claim 1 or 2, wherein the manually actuated chromatographic device is a single-use device.

8. The method of claim 1 or 2, wherein the manually actuated chromatographic device is actuated by a single movement.

9. The method of claim 8, wherein the single movement is a push or a rotation.

10. The method according to claim 1 or 2, wherein the predetermined volume of liquid is 0.5 ml to 1 ml.

11. The method of claim 1 or 2, wherein processing the raw liquid sample is completed in less than 10 minutes.

12. The method of claim 11, wherein processing the raw liquid sample is completed in less than 4 minutes.

13. The method according to claim 11, wherein processing the raw liquid sample is completed within a predetermined time period of 1 minute to 3 minutes.

14. The method of claim 1 or 2, wherein the method is performed at the point of care.

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