SAMPLE PREPARATION DEVICE

Abstract

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

Claims

1. A method for performing an isothermal nucleic acid amplification comprising: providing a raw liquid sample; processing the raw liquid sample to remove nucleic acid amplification inhibiting agents and/or fluorescent agents; performing an isothermal nucleic acid amplification on the processed sample; and monitoring for amplified nucleic acid.

2. The method according to claim 1 wherein the processing step comprises using size exclusion chromatography.

3. The method according to claim 1 further comprising contacting the raw liquid sample to a lytic agent prior to processing the raw liquid sample.

4. The method according to claim 1 further comprising buffering the raw liquid sample.

5. The method according to claim 1 wherein the raw liquid sample is selected from the group consisting of urine, blood, plasma, serum, saliva, cerebrospinal fluid, and tear fluid.

6. The method according to claim 1 wherein the raw liquid sample is an elute from a vaginal, nasal, throat, penile, anal, or skin swab.

7. The method of claim 1 further comprising buffering the raw liquid sample during processing the raw liquid sample.

8. The method according to claim 1 wherein processing the raw liquid sample comprises providing a manually actuated chromatography device comprising a chamber for receiving a liquid sample, a pump with a metering valve, and a chromatography element.

9. The method of claim 8 wherein the manually actuated chromatography device comprises a first part and a separate second part receivable in the first part.

10. The method of claim 9 further comprising receiving, in the first part, the second part.

11. The method of claim 10 comprising actuating the pump by the second part of the manually actuated chromatography device operably engaging the first part of manually actuated chromatography device.

12. The method of claim 8, wherein the pump moves a predetermined volume of liquid from the chamber to the chromatography element.

13. The method of claim 8, wherein the manually actuated chromatography device is a single-use device.

14. The method of claim 8, wherein the device is actuatable by a single movement.

15. The method of claim 14, wherein said single movement is a push or rotation.

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

17. The method of claim 1, wherein processing the raw liquid sample is completed in less than 3 minutes.

18. The method of claim 12, wherein said predetermined volume of liquid is 0.5 to 1.0 milliliters.

19. The method of claim 1, wherein said isothermal nucleic acid amplification is a recombinase polymerase amplification (RPA) or nicking and extension amplification reaction (NEAR).

20. The method of claim 1, wherein the method is performed at the point of care.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0105] FIG. 1 shows a prior art system.

[0106] FIG. 2 shows an exploded view of an exemplary device.

[0107] FIG. 3 shows an exemplary first part of the device before use.

[0108] FIG. 4 shows an exemplary first part of the device with the sample chamber cover removed.

[0109] FIG. 5 shows an exemplary second part of the device.

[0110] FIG. 6 shows the second part engaging the first part and metering a volume of the raw sample.

[0111] FIG. 7 shows the second part having displaced the metering member.

[0112] FIG. 8 shows the second part fully depressed.

[0113] FIG. 9 to FIG. 11 shows an exemplary device in-situ in a device for performing and monitoring isothermal nucleic acid amplification.

[0114] FIG. 12 shows prepared sample being transported to a reaction chamber.

[0115] FIG. 13a to FIG. 13e provide a schematic representation of the metering chamber during use.

DETAILED DESCRIPTION OF THE INVENTION

[0116] The present invention relates to the preparation of samples for use in assays and in particular in the preparation of samples for use in isothermal nucleic acid amplification. In particular, to a manually operated chromatography device, 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.

[0117] FIG. 1 shows a reaction vessel (200), sample collection reservoir (300) and liquid transfer device (100) suitable for use in the system illustrated in FIG. 13. Each subassembly can have a D-shaped or otherwise asymmetrical cross section (105, 205, 305) that is compatible with the other two subassemblies, such that the subassemblies may only be mated to each other in one orientation.

[0118] The reaction chamber 200 includes a microtube 220 held within an aperture in the bottom of the reaction vessel body.

[0119] FIG. 1 shows the transfer device 100 and reaction vessel 200 as described above with one pipette tip 120 and one microtube 220. The transfer device may, however, have two or more pipette tips, and the reaction vessel may have two or more microtubes.

[0120] Such assemblies are discussed in detail in WO2013/041713 which is incorporated herein by reference.

[0121] FIG. 2 an exploded view of a device (20) according to the invention. The device comprises a first part (20a) and a second part (20b). The second part (20b) is receivable in the first part (20a). Also shown is a sample reservoir (210). The first part (20a) is receivable in the sample collection reservoir (210).

[0122] The first part (20a) comprises a main body (21). The main body (21) comprises a sample chamber (not shown) and a metering chamber (211). The metering chamber (211) and sample chamber are in fluid communication. The metering member (22) is in the form of a cup-shaped member with a D-shaped cross-section. The metering chamber (211) also has a D-shaped cross-section. The metering member (22) will typically contain a dehydrated lytic agent. Typically at least one pellet of lytic agent (221), typically potassium hydroxide or sodium hydroxide, held in place by a gauze (222). The potassium hydroxide/sodium hydroxide is present to cause rapid lysis of cellular material in the sample fluid, thereby releasing intracellular nucleic acid that is to be detected by isothermal nucleic acid amplification.

[0123] The metering member (22) is movably receivable in the metering chamber (211). In use, the metering member (22) divides the metering chamber into an upper portion and a lower portion. The metering member (22) forms a fluid-tight interference fit with the inner wall of the metering chamber (211). A gas tight membrane (23) closes the end of the metering chamber. In manufacture, the metering member (22) is inserted into the base of the metering chamber (211) and pushed upwards until it sits just beneath the annular seal (not visible) separating the metering chamber from the sample receiving chamber. A gas tight membrane (23) is then heat sealed over the base of the metering chamber (211).

[0124] The second part (20b) comprises a main body (24) comprising an actuator (25) receivable in the sample chamber of the first part (20a). In use, the actuator (25) operably engages with the metering member (22). The actuator (25) has a distal end (215) and a proximal end (216). An aperture (214) is located in the distal end of the actuator (25). The aperture (214) is in fluid communication with a separation chamber (not shown) containing an aqueous solution comprising an isothermal nucleic acid amplification buffer and a dispersion of gel-filtration chromatography particles. Suitable gel filtration particles are sold under the trade name Sephadex G-25 Superfine by GE Healthcare, other suitable chromatography substrates are known to the skilled person. A microfluidic channel (not shown) provides fluid communication between the separation chamber and the exit aperture (217) at the distal end of the return leg (26). An insert (27) is inserted in the return leg (26) and closes the separation chamber. The outer wall of the actuator (25) comprises a shoulder (213) for engaging the first part. A channel (212) is present in the shoulder (213). In use, the channel (213) allows excess liquid to escape the metering chamber before it is sealed.

[0125] Pealable seals (223, 224) are provided on the first part and second part.

[0126] FIG. 3 shows the first part of the device (32) before use. A pealable seal (31) covers the sample chamber (not shown). The pealable seal (31) protects the contents of the first part (32) from contamination, and the dry lytic agent from moisture, before use. An intact pealable seal (31) also indicates to the user that the device has not been used before. The first part of the device (32) is engaged with a sample reservoir (33).

[0127] FIG. 4 shows the first part of the device (42) with the pealable seal removed. The sample chamber (41) is now accessible. Again, the first part of the device (42) is engaged with a sample reservoir (43).

[0128] FIG. 5 shows a second part of the device (60). The second part of the device (60) comprises a first actuator leg (63) and a second return leg (62). A stopper (61) is fixed in place. A pealable seal (64) covers apertures located at the distal ends of the actuator leg and return leg to prevent contamination and/or leakage of the fluid within the actuator.

[0129] In use, the pealable seal (64) is removed before the second part is engaged with the first part. The pealable seal (64) prevents contamination and leakage. An intact pealable seal (64) indicates to the user that the device has not been used before.

[0130] A microfluidic pathway (not visible) runs from the top of the actuator leg (63) and down the inside of the return leg (62), through which, in use, the processed liquid flows.

[0131] The second part of the device (60) is made in two pieces by injection moulding: an outer wall (65) and an insert (66). The insert fills the majority of the return leg (62) and includes the circular part (66) visible on the top surface of the second part (60). There is a groove in the return leg portion of the insert (not shown) that forms a closed channel when inserted in the return leg (62). As better shown in FIG. 2, in the actuator leg (25), the insert (27) compresses the top frit (218) down onto the matrix (219) and bottom frit (220) and includes an opening and groove (not shown) that permits processed fluid to move across to the return leg (26).

[0132] FIG. 6a shows the device of the invention with the second part (71) inserted in the first part (72). Again the first part of the device (72) is engaged with a sample collection reservoir (73).

[0133] FIG. 6b shows a section through the first (72) and second (71) parts of the device and the sample reservoir (73) shown in FIG. 7a.

[0134] The cross-section in FIG. 6b reveals the separation chamber (76) containing size exclusion chromatography matrix (75). The chamber (76) that contains the matrix, is fitted with frits top (77) and bottom (78) to keep the matrix (75) in place. There are also cut out features (cross shapes) in the moulded plastic that permit fluid to spread and enter the separation chamber (75).

[0135] In use, the user is instructed to introduce the second part (71) into the first part (72). The device is shaped such that it is apparent the actuator leg (79) is the one to be introduced into the chamber (711) into which raw sample is added.

[0136] Provided sufficient liquid sample has been added to fill the upper portion (712) of the metering chamber, when in its original position, and preferably allow liquid to sit above the annular seal (713) separating the metering chamber from the sample chamber (711), the desired volume of processed sample will be achieved.

[0137] As the second part (71) is inserted, the narrower portion of the actuator leg initially protrudes through the annular seal (713) and into the metering chamber (714). The diameter of the actuator leg is initially smaller than that of the annular seal (713), thus liquid can escape around the edges of the actuator leg (79) into the sample chamber (711) above as the actuator (79) displaces liquid from the upper portion of the metering chamber (712).

[0138] In FIGS. 6a and 6b the second part (71) has been pushed, by hand, into the first part (72) such that that the shoulder (715) is engaged with an annular seal (713) positioned between the sample chamber (711) and the metering chamber (714). In this position, the 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 has just engaged the metering member (717). The volume of the upper portion of the metering chamber surrounding the actuator defines the volume of raw sample that will pass through the size-exclusion chromatography gel (75). The size-exclusion chromatography gel (75) is suspended in a solution comprising a buffer suitable for an isothermal nucleic acid amplification, typically magnesium acetate. The concentration of the buffer in the separation chamber (76) will be such that it is present in the correct concentration in the processed sample.

[0139] In the position shown in FIG. 6b, the metering member (717) is above the upper end of the pressure release channel (719). This means that the pressure release channel (719) is not providing fluid communication between the upper (712) and lower (720) portions of the metering chamber (714). Thus, as the actuator descends further, it advances the metering member (717) and raises the pressure of the air in the lower chamber (720).

[0140] FIGS. 7a and 7b show the device with the second part (81) having been pushed further into the first part (82). In this position the actuator shoulder (84) has descended below the annular seal (85) which is now engaged with 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 such 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 upper portion (812) of the metering chamber (87) and because the pressure is higher in the lower portion (811) of the chamber that 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 forming a high pressure region above the cup-shaped metering member (89) containing the liquid sample as a result of a contraction in the internal volume of the upper portion (812) caused by the presence of the actuator (86) therein. In turn, liquid sample is forced through the orifice (814) in the distal end of the actuator (86) and into the size-exclusion chromatography/separation chamber (815) for treatment.

[0141] A portion of raw sample remains sealed in the sample chamber (88). This can be disposed of with the device.

[0142] As the second part (81) of the device is pushed further into the first part (82), the actuator (86) moves the metering member (89) further down within the metering chamber (87), forcing air from the lower portion (811) to the upper portion (812) along the pressure release channel (810) and, thereby, advancing the sample through the size exclusion chromatography gel in the separation chamber (815), along the microfluidic channel (not shown) in the horizontal upper portion of the second part (81) of the device, and along a channel in the return leg (817) of the second part (81), before exiting the second part (81) and dripping into the sample collection reservoir (818).

[0143] The size-exclusion chromatography gel removes isothermal nucleic acid amplification inhibiting agents and/or fluorescent agents from the sample. Thus, the treated sample which is collected in the sample collection reservoir (818) is sufficiently free from said inhibiting/fluorescent agents that an isothermal nucleic acid amplification can be successfully performed on nucleic acid present in the sample and then detected. Furthermore, because the size exclusion chromatography gel is suspended in a solution comprising a buffer for performing an isothermal nucleic acid amplification. The treated sample collected in the sample collection reservoir (818) is at the correct pH for performing an isothermal nucleic acid amplification. Typically, the pH is from about 6 to about 9. This avoids the need for any further sample preparation steps.

[0144] FIGS. 8a and 8b show the second part fully inserted (91) into the first part (92). An audible prompt, typically a click, indicates to the user that full insertion has been achieved and therefore that the correct volume of sample will be treated. In use, the user will typically perform a single push with a finger or thumb until the click is heard. The member responsible for the audible click will typically be a latch, which also locks the second part (91) in the first part (92). This also means that unprocessed raw sample is safely contained for disposal. Preferably a fluid tight seal is created between the top of the second part (91) and the sample chamber (94) to thereby contain excess raw sample fluid (93) and prevent spillage of raw sample when the device is disposed of.

[0145] 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. As shown in FIG. 8b, the no raw sample remains in the metering chamber (96). Further, a small volume of air travels through the second part (91) of the device and exits through orifice (98) at the end of the return leg (99). This forces out the liquid present in the channel and prevents any dripping. It also allows any residual compressed gas on the sample side of the device to dissipate. It further prevents residual fluid being pushed through the column after the device has been removed from the sample collection vessel (910) to prevent excess fluid potentially dripping out of the return leg (99) and contaminating the work area.

[0146] This is achieved by ensuring that when the actuator fully inserted the volume of the metering chamber displaced by the actuator is greater than the volume of raw sample which is metered for treatment.

[0147] There will typically be a delay between the audible click and all of the treated sample arriving in the sample collection reservoir (910). This is caused by a damping effect from compressing the air in the lower portion (95) of the metering chamber (96), and that pressure being released through the device. This damping effect, caused by fluidic resistance, is advantageous because it slows the flow rate of sample being processed and ensures that the sample is properly treated by the size exclusion chromatography gel. If the sample travelled through the gel too quickly, insufficient removal of the nucleic acid amplification inhibiting/fluorescent agents would occur and the device would not achieve its desired function. Achieving the correct level of damping is within the competence of the skilled person.

[0148] In the position shown in FIG. 8b, the sample collection reservoir (910) will contain a treated sample ready for use in an isothermal nucleic acid amplification. The treated sample is buffered at the require pH and sufficiently free from nucleic acid amplification inhibiting agents and fluorescent agents for amplification to be performed and detected. A liquid transfer device (not shown) is used to pipette a portion of the treated sample from the sample collection vessel (910) to a testing device for performance of an isothermal nucleic acid amplification assay.

[0149] FIGS. 9 to 12 show a device (10) according to the invention in-situ in a sample processing device (101) for performing an isothermal nucleic acid amplification. In FIG. 9 a first part (102) of the device according to the invention is located in a sample collection vessel (103), which is, in turn, received in the sample processing device (101). Suitable sample processing devices are available from Alere Inc. under the brand name Alere i.

[0150] In FIG. 9 a reaction vessel (103) is in-situ in the sample processing device. The sample processing device may be used with reaction vessels configured to perform NEAR and/or RPA isothermal nucleic acid amplifications. Accordingly, the reaction chambers may contain reagents necessary for performing NEAR and/or RPA on samples introduced into said chambers. Suitable reaction vessels (105) are available from Alere Inc.

[0151] In FIG. 9 the protective pealable film covering sample chamber (104) has been removed. In this position, the raw sample is introduced into the sample chamber using a pastette, typically 1.5 ml of raw sample will be introduced.

[0152] FIG. 10 shows a system of the invention (11) with the second part (113) of the device partially inserted into the first part (112) of the device (111). Whilst FIG. 11 shows a system of the invention (12) with the second part (123) of the device (121) fully depressed into the first part (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 liquid transfer device is then used to transfer a portion of the processed sample to the reaction vessel (125) for testing.

[0153] FIG. 12 shows a liquid transfer device (100), reaction vessel (200) and sample collection reservoir (300), along with a sample processing device (400). In use, the screen (440) provides step-by-step instructions to the user and displays the results of the isothermal nucleic acid amplification test. The invention contemplates kits comprising a reaction vessel (200), liquid transfer device (100), sample collection chamber (300) and sample preparation device, and systems including the kit and a sample processing device (400).

[0154] FIG. 12 shows the system with an exemplary detection device (400). The detection device (400) includes a first station (410) adapted to securely hold the sample collection vessel (300) and a second station (420) adapted to securely hold the reaction chamber (200). When in use, the transfer device (100) is moved between the sample collection vessel (300) at the first station (410) and the reaction chamber (200) at the second station (420). The detection device includes a lid (430) that can be closed when the detection device (400) is in operation or for storage. A touchscreen user interface (440) is present for inputting data and displaying information regarding the assay. The second station (420) can 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 (410) and second (420) stations can be adapted to heat or cool the contents of the sample collection vessel (300) and reaction chamber (200). The second station (420) can also be adapted to provide optical, fluorescence, or other monitoring and/or agitation of the microtube (220).

[0155] FIG. 13a to FIG. 13e provide a schematic representation of the device during use.

[0156] FIG. 13a shows the device before use with the second part (142) separated from the first part (141). The cup-shaped metering member (143) is at the top of the metering chamber (144) abutted against the annular seal (145) separating the sample chamber (146) from the metering chamber (144).

[0157] In FIG. 13b raw liquid sample (147) has been introduced into the sample chamber (146) and the cup-shaped metering member (143) in the metering chamber (144). Preferably, the level of the liquid is above annular seal (145).

[0158] In FIG. 13c the movable actuator (148) has been lowered through the sample chamber (146) and into the metering chamber (144), such that a distal end thereof has engaged the metering member (143) and the shoulder (1410) of the moveable actuator (148) is just about to engage 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 actuator (148) distal to the shoulder (1410) is the predetermined volume of liquid metered for treatment (1412) as in FIG. 13d.

[0159] In FIG. 13d the movable actuator (148) has moved the metering member (143) along the metering chamber (144), 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 in fluid communication by means of a pressure release channel (1415) in the wall of the metering chamber. As the moveable 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) out of the metering chamber (144) through an exit channel (not shown) located in the movable actuator (148).

[0160] When the moveable actuator (148) is fully depressed, as illustrated in FIG. 13e, the volume of air in the metering chamber (144) displaced by the moveable actuator (148) is greater than the volume of the metered sample of liquid so that substantially all of the liquid is forced out of the metering chamber (144) through the exit channel (not shown). Unprocessed raw sample (1416) is stored in the sample chamber (146) for safe disposal.