GB2443243A

GB2443243A - Device, system and method for processing a sample

Info

Publication number: GB2443243A
Application number: GB0615110A
Authority: GB
Inventors: Helen Hwai-An Lee; Magda Anastassova Dineva; Craig Alan Wisniewski; Phillip John Stankus
Original assignee: Cambridge Enterprise Ltd; Diagnostics for the Real World Ltd
Current assignee: Cambridge Enterprise Ltd; Diagnostics for the Real World Ltd
Priority date: 2006-07-28
Filing date: 2006-07-28
Publication date: 2008-04-30
Anticipated expiration: 2026-07-28
Also published as: GB0615110D0; GB2443243B; CN101610845A

Abstract

A device for the processing of a sample comprises a location apparatus, a processing chamber for receiving the sample and a plurality of reagent chambers. The reagent chambers have openings defined in the location apparatus. The processing chamber is movable relative to the reagent chambers to enable sequential communication with each reagent chamber in turn such that each reagent chamber communicates with the procesing chamber when its associated opening is disposed in overlapping relationship with the processing chamber opening; methods and systems for processing a sample using such a device. Also claimed is a device for processing and analysing biological samples using lyophilised reagents comprising at least one processing chamber which is sealed from the external environment, wherein the lyophilised reagents are preloaded into the device prior to despatch to a user.

Description

-1-2443243 * Device. System and Method for Processin a Saflnple The

invention relates to a device, system and method for the processing of a sample. In particular the invention relates to in-the-field and on-site testing of nucleic acid in a biological sample.

Background

The importance of nucleic acid testing (NAT) has become increasingly evident during the last decade for many purposes such as screening and diagnosis of io infectious diseases and genetic disorders, testing for disease susceptibility, therapy monitoring, and improving the safety of blood supplies. NAT combines the advantages of direct and highly sequence-specific detection of the genome of an infectious agent with an analytic sensitivity that is several orders of magnitude greater than that of immuno-based tests, or virus isolation and cell is culture methods. Due to the high sensitivity of NAT, its use in blood banks reduces the risk of infectious agent transmission during the period between infection and seroconversion, of infection with immunovariant viruses, of immunosilent or occult carnage. NAT-based assays consist of three basic steps: extraction of nucleic acid, genome amplification mediated by procedures o such as (RT)-PCR; strand-displacement amplification (SDA) and transcription-based amplification system lAS (Guatelli et a!., Proc. Nati. Acad. Sci. 87: 1874-1878 (1990); Compton, Nature 350: 91-92 (1991)), and amplicon detection.

Currently available NAT assays are complex and entail multi-step procedures that require highly trained personnel and specialised facilities. They require cold-chain transport and storage of reagents, a high investment cost for instruments, high running costs for reagents, and regular maintenance support.

All of these restrict the use of NAT only to specialized well-equipped and technically advanced laboratories. Correspondingly, current NAT assays design is unsuitable for near-patient and field- testing e.g. physician's office, community-based clinics, emergency rooms, battlefield surgery units or point-of care health centres, district hospitals and inner-city clinics in the resource-p47251gb01 SG 27/07/2006 limited settings of developing countries. These include predominantly countries of Africa, Asia, and Latin America with a high prevalence of infectious diseases.

An essential requirement for assays based on nucleic acid amplification is protection from amplicon contamination, currently solved by working in specialized laboratories using dedicated spaces for sample preparation, amplification and detection. This approach is not applicable for field-testing, near-patient testing and in resource-limited settings.

to Summary of Invention

The invention provides a device, a system, a couplable reagent chamber and a method according to the appended independent claims, to which reference should now be made. Preferred or advantageous features of the invention are defined in dependent sub-claims. is

The invention may therefore advantageously provide an apparatus and method suitable for processing a sample, in particular suitable for amplifying nucleic acids from a sample in conditions where there is a lack of facilities and a limited supply of skilled personnel.

Accordingly, in a first aspect the invention provides a device or apparatus for the processing of a sample. The device comprises a processing chamber for receiving the sample and a plurality of reagent chambers suitable for containing processing reagents.

Preferably, the device also comprises an analyser chamber for containing an analyser or suitable analysis means, and a location apparatus or body for bringing the processing chamber sequentially into communication with the reagent chambers and with the analyser chamber to mix reagents with the sample and so implement a processing protocol or method.

p47251gb01 SG 27/07/2006 Preferably, the device also comprises a sealing apparatus for sealing the processing chamber from the external environment. Such a sealing device may help prevent contamination of the sample during processing and may also, advantageously, prevent contamination of the point-of-use site, for example a clinic, with the processed sample.

The processing chamber advantageously has a processing-chamber opening for introduction of the sample and for communication with the reagent chambers as described below. In an alternative embodiment, the processing io chamber may optionally have separate openings for these two functions.

In a preferred embodiment, the reagent chambers each have an associated reagent-chamber opening which is defined in the location apparatus of the device such that each reagent chamber can communicate with the processing is chamber when its respective associated opening is disposed in overlapping relationship with the processing-chamber opening. The processing chamber is movable relative to the reagent-chamber openings such that a sequential communication is provided between the processing chamber and each reagent chamber in turn.

In different implementations the location apparatus may employ different geometries. For example the processing chamber may move in a circular path between its different positions, or stations, or it may move in a linear path or in any other suitable manner.

Advantageously, the device may be a non-reusable, one-shot disposable device: In this circumstance the device may be constructed from cheap materials and simply be thrown away after use. This may be an advantage when the device is used to conduct processing and analysis in regions of the world with limited resources, for instance if the device is used for medical testing in third world countries.

p47251gb01 SG 27/07/2006 The device may be advantageously employed in the processing and analysis of biological samples, for instance blood samples or samples of genetic materials.

Such biological testing is difficult to perform at present without using expensive equipment and experienced personnel.

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It is preferred that, during processing of a sample, the contents of the processing chamber are sealed from the external environment, i.e. the device provides a closed system and the sample cannot escape from the device. The closed system helps eliminate contamination from external sources, and at the io same time protects the external environment from contamination with the amplified product of the processed sample, which may produce false results.

This is particularly important where processing of the sample involves the amplification of nucleic acid, as a small quantity of rogue nucleic acid could easily be amplified to provide a false result. As a result, it is also preferred that the sealing apparatus acts to seal the reagent chambers from the external environment.

The sealing apparatus may comprise any suitable means for sealing the processing chamber from the external environment while still alt owing processing reagents contained in the various reagent chambers access to the processing chamber, for example when the processing chamber is moved to a predetermined position within the device in which it is in communication with a particular reagent chamber. In preferred embodiments of the invention the sealing apparatus may include elements from a body, frame, guide or supporting element of the device or a housing for the reagent chamber.

Sealing in such cases may be simply achieved by the abutment of the processing chamber opening and a portion of the location apparatus or body, the seal optionally incorporating a seal element.

Although sealing of the contents of the processing chamber is important, the sealing apparatus need only act to seal the chamber during processing of the sample, when the sample is most likely to be affected by contamination.

p47251gb01 SG 27/07/2006 Preferably, however, the sealing apparatus also acts to seal the processing chamber from the external environment during any analysis step.

It is preferred that the device further comprises an access port which can open into or be aligned with the processing-chamber opening for providing initial access to the processing chamber. This is advantageous as the sample may then be introduced to the processing chamber without the need for removing the processing chamber from the device or opening the device. Such an arrangement further reduces the risks of contamination of the sample and io corruption of the processing step on the sample.

Advantageously, any access port may be protected by a removable seal, for example a removable foil seal. This helps ensure that the device is free of contamination prior to introduction of the sample. The sample may be introduced to the processing chamber after removal of the seal and the processing chamber then moved such that the sealing apparatus seals the processing chamber from the environment.

Advantageously, the processing chamber may be movable between a plurality of discrete positions, or stations. In some of these positions the processing-chamber opening is advantageously disposed in overlapping relationship with a reagent-chamber opening. This allows communication between the chambers and thus the transfer of reagent from the reagent chamber to the processing chamber.

It may be desirable that there are positions for the processing chamber in which the processing chamber opening is not in overlapping relationship with any of the reagent chamber openings. Such positions may be advantageously used for incubation or mixing stages during processing, if required for a processing protocol for which the device is designed.

p47251gb01 SG 27107/2006 Reagent chamber openings may be defined in or through the device body or location apparatus and it may be advantageous for the reagent chambers themselves to be formed integrally with any such body or location apparatus.

Reagents may be pie-loaded into the reagent chambers in such a construction, s in a clean facility for exaniple, thereby minimising opportunity for contamination prior to use. Each such chamber could be formed by a blister or bubble extending from the body or may be a more complex structure such as a tube rising up from the body. An integral chamber may be particularly advantageous for use with dried reagents, for example freeze-dried or lyophilised reagents.

It may be preferred that at least one of the reagent chambers is couplable to the body or location apparatus at its associated reagent-chamber opening.

Such a construction for the device may be advantageous where liquid reagents are used as it may be more difficult to load liquid reagents into integral chambers and to store liquid reagents in integral chambers before use of the device. Removable chambers may also be particularly advantageous where the device is designed to be used for a wide range of different tests, each of which requires different reagents. In such a situation the specifically required reagents could be added to the device by coupling the appropriate reagent zo chamber(s) to the body.

It is preferred that any couplable chambers form a seal when coupled to the device. Coupling may be achieved by any suitable means, for example by use of a screw or bayonet fitting aligning the reagent chamber with its associated opening, or may be a press fit.

It may be particularly advantageous for the device to comprise both one or more integral chambers for containing dried reagents and one or more couptable chambers for containing liquid reagents, if the device is designed for a test protocol involving both solid and liquid reagents.

Preferably, during use of the device the reagents or any analyser are introduced into the processing chamber under the influence of gravity. This p47251gb01 SG 27O7/2OD6 minimises the mechanical elements required in the device and simplifies its use. An example of this would be where a reagent is maintained within its chamber by a bamer and the barrier is temporarily removed as the processing chamber passes beneath the opening leading from the reagent chamber.

Gravity could then act on the reagent, if the device is held in an appropriate orientation, to urge it into the processing chamber. Alternatively, in the case of liquid reagents, the reagent chamber may be provided with a bung or valve, which when removed or opened allows the reagent to flow into the processing chamber. If gravity is the means by which a reagents is introduced to the io processing chamber it is important that the device is used in the correct orientation.

Optionally, at least one dispenser, such as a plunger, may be used in the device to facilitate introduction of a reagent from its reagent chamber into the is processing chamber. The use of plungers may be particularly applicable to the

introduction of liquid reagents.

Although it may be possible to load reagents into the appropriate reagent chambers at the point of use, it is preferred that the device is loaded or charged with the correct reagents prior to arriving at the point of use. This helps prevent contamination, which may provide false results in any analysis, and it also removes the need for a skilled technician to handle and measure out correct quantities of reagents at the point of use.

The analyser or analysis means, when present, may itself be contained in a chamber with an associated opening. Such a chamber may be termed an analyser chamber, and may function on the same principle as the reagent chambers described above. Where contact is required between the analyser and the processed sample it may not matter whether the analyser passes into the processing chamber, or whether the contents of the processing chamber pass into the analyser chamber.

p47251gb01 SG 27/07/2006

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It is particularly advantageous for the analyser to be a test strip or dipstick providing a visual result. The test strip may be dropped into the processing chamber to contact the processed biological sample. The processed sample may then be wicked up the test strip to provide the required analysis.

A test strip commonly has a greater length dimension than thickness and width, and thus may be housed in a similarly-dimensioned analyser chamber. In such a case it may be preferred that the test strip is positioned in a chamber lying horizontally on the surface of the device in use, for example in order to make the device more compact, in which case the entire device may need to be rotated to allow communication between the test strip and the processed sample. Advantageously, where the analyser gives a visual result the wall of the analysis chamber may be substantially transparent so that the result of the analysis can be seen without any need to open the device.

Alternatively, the analyser may comprise a reflectometer or a densitometer.

The device may comprise a ratchet apparatus or indexing means to aid the location of the processing chamber. Such an apparatus or means may enable the processing chamber to be moved to discrete, fixed, positions within the device and may also advantageously prevent the processing chamber from moving in a reverse direction through the device.

In a further aspect the invention may provide a system for the processing of a biological sample comprising a device as previously described or as defined in any claim and an external heat source or heating means adapted to engage with the device. Many biological processing steps require carefully controlled thermal conditions and thus a heat source adapted to engage with the apparatus may be desirable for the accurate use of the device. Preferably the heat source is adapted to engage with the processing chamber of the device, thus it may be advantageous for the outer portion of the processing chamber to project from the device so as to be accessible.

p47251gb01 SG 27/0712006 To facilitate mixing, the system may further comprise a vibrator or vibration means for vibrating the device, or the external heat source may incorporate a vibrator. Preferably the external heat source is a simple heating block shaped to receive the device, or at least to receive the processing chamber.

The system may additionally comprise one or more couplable reagent chambers. Any such chambers may be pre-loaded with reagent and can advantageously be stored separately from the device, for example in a refrigerator if necessary. I0

In a further aspect the invention provides a method of processing a sample in a device having a processing chamber, a location apparatus and a plurality of reagent chambers. The method comprises the steps of loading the sample into the processing chamber and operating the location apparatus first to seal the is processing chamber from the external environment, and then to move the processing chamber relative to the plurality of reagent chambers so as to introduce in sequence, a corresponding plurality of reagents into the processing chamber from the reagent chambers.

2o Each reagent may be added or introduced to the processing chamber by the action of gravity, or a dispenser such a plunger may be used.

The resulting processed sample may be analysed using an analyser. Any such analyser may be advantageously contained in an analyser chamber of the device.

Advantageously, the above-described method can be applied to a device with any number of reagent chambers, the steps of moving the processing chamber and adding reagents being modified for any number of reagent chambers and associated reagents.

p47251gb01 SG 27/07/2006 -10 -Advantageously, the processing chamber moves sequentially past each of a number of reagent chambers in turn. The number and contents of the reagent chambers can be tailored to any processing required for analysis of the sample.

In the field, the end user need only follow a simple set of instructions and need not be concerned with the details of the science involved at each step. Thus the processing of the sample need not be carried out by a skilled user.

Optionally, additional steps may be added both prior to and subsequent to each addition of reagent to the processing chamber. Such steps may include mixing io and incubation steps and such additional steps would depend on the type of processing desired for the sample.

It may be advantageous for the device to be disposed of after completing analysis on the processed sample.

Where the processing protocol involves amplification and detection of nucleic acid it may be advantageous to treat the device and the used sample post-analysis to help prevent contamination of the point-of-use site. For example, to help prevent amplicon carryover contamination, the amplicon left in the device after a detection step could be treated with nucleic acid modifying or hydrolysing agents that prevent priming of further amplification reactions.

Decontamination may be particularly desirable where batches of samples are to be tested on the same site.

One such decontamination treatment described in US Patent No 5,035,996 (Hartley. Life Technologies, lnc) involves incorporation into the amplified product of a nbo-or deoxy-nucleoside triphosphate (rNTP or dNTP) base that is not generally found in the sample to be analyzed: for example dUTP in the case of DNA analysis. The amplified product will thus have a sequence that has Uracil in multiple positions. The enzyme uracil DNA glycosylase (UDG) is added to the sample prior to amplification. This will cause enzyme hydrolysis of any contaminating reaction product (containing Uracil) without affecting the natural DNA in the sample.

p47251gb01 SG 27/07/2006 -11 -Preferably decontamination is a chemical treatment or reagent that not only modifies, but also degrades nucleic acid e.g. non-enzymatic degradation of nucleic acid with chemical nucleases. Examples of chemical nucleases are known in the art e.g. divalent metal chelate complexes, such as copper Phenantroline-Cu (II) or Ascorbate-Cu (II) cleavage as described by Sigman D.S. et al (J. Biol. Chem (1979) 254, 12269-12272) and Chiou S. (J. Biocheni (1954) 96, 1307-1310).

io A decontamination reagent could be conveniently delivered into the processing chamber of the device, after analysis of the sample, using a couplable reagent chamber, as described above. The device may, therefore, be preloaded with both processing reagents and a post-analysis treatment, or decontamination, reagent. Alternative methods for delivery of decontamination reagents include is delivery by luer lock syringe or through a septum.

Where the processing protocol uses a liquid reagent it may be advantageous to supply the device to the end user in two parts. One part of the device may comprise the processing chamber and reagent chambers loaded with lyophilised dry reagents and an analysis means, for example a test strip. This first part of the device may be hermetically sealed with desiccant. The second part may be a couplable reagent chamber, for example as described above, containing a liquid reagent. The two parts of the device would be clipped together before use.

p47251gb01 SG 27/07/2006 -12 -Specific Embodiments Specific embodiments of the invention will now be described by way of example, with reference to the drawings in which; Figure IA is a device according to a first embodiment of the invention viewed from above, Figure 1 B is a device according to Figure IA viewed from below, io Figure 1C is a plan view of the device of Figure 1A, Figure 1D is a section view along the line A-A as shown in Figure 1C, Figure 1 E is a projection view of a seal element used in the device of IS Figure 1A, viewed showing v-ring profile sealing ridges, Figure 1 F is a plan view of the seal element of Figure 1 E, Figure 1 G is a section view along the tine D-D as shown in Figure IF, Figure 2 is an exploded view of the device according to a first embodiment of the invention, Figure 3 is a flow chart illustrating the method steps involved in performing an assay using a device according to the invention, Figure 4A is a three-quarter view of a device according to a second embodiment of the invention with its processing chamber in a position to receive a sample, Figure 4B shows the device according to the second embodiment of the invention with the processing chamber sealed within the device housing, p47251gb01 SG 27/07/2006 Figure 4C shows the device according to the second embodiment of the invention with the processing chamber positioned beneath an opening of a first reagent chamber, S Figure 4D shows the device according to the second embodiment of the invention with the processing chamber positioned in an incubation position between the first and second reagent chambers, Figure 4E shows the device according to the second embodiment of the io invention with a test strip analysis means coming into contact with the sample in the processing chamber, Figure 5A illustrates a couplable reagent chamber suitable for containing a liquid reagent,

IS

Figure 5B illustrates the couplable reagent chamber of Figure 5A after actuation to release its contents, Figure 6A is a three-quarter view of a device according to a third embodiment of the invention, Figure 6B is an exploded view of the device of Figure 6A.

A preferred embodiment of a device according to the invention is illustrated by Figures 1A, 1B, 1C, 1D, 1E, iF, 1G and 2.

The device 10 comprises a substantially circular body, or location apparatus 20.

The location apparatus comprises two portions, an upper portion 21 and a lower portion 22, both of which are circular and rotatably engagable with each other about a common central point.

The device further comprises first 30, second 40, and third 50 reagent chambers and an analyser chamber 100 depending from the upper portion of the location apparatus, and a processing chamber 60 depending from the lower portion of the location apparatus.

p47251gb01 SG 27/07/2006 -14 -The tower portion has a downwardly-extending circumferential circular lip 23, whose lower edge acts as a stand for the device during processing. The processing chamber is positioned the circular lip at a fixed radius from the s central point.

The upper portion has a slightly greater diameter than the circular lip of the lower portion. The upper portion has a downwardly depending skirt around its entire circumference that fits over and engages with the seal element and the io circular lip of the tower portion, this engagement enabling the upper portion to be rotated relative to the lower portion about the common central point of both upper and lower portions.

The processing chamber has an opening 62 defined by an entrance 63. The entrance to the processing chamber 63 is arranged to lie in the same plane as, i.e. flush with, the upper edge of the circular lip. The processing chamber itself depends from the lower portion and is defined by processing chamber walls.

A seal element is arranged such that it is fixed relative to the upper portion and, thus, is moveable relative to the lower portion.

The upper portion supports the first, second and third reagent chambers and the analyser chamber. Each of these chambers is associated with a respective opening defined in the upper portion at a fixed radius from the centre of the upper portion such that each opening may, when the upper portion has been rotated appropriately relative to the lower portion, overlap with the processing chamber opening. This allows communication between the processing chamber and each of the reagent chambers and the analyser chamber to be effected in turn.

Additionally the upper portion defines an access opening, or access port, 70 at a fixed radius from the centre of the upper portion such that it may overlap with the processing chamber opening. This access opening or access port is covered with a removable foil seal 80 to prevent contamination of the device by the external environment prior to use. When the device is ready for use the p47251gb01 SG 27/07/2006

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-15 -access opening in the upper portion is aligned with the processing chamber in the lower portion.

A seal element 90 comprises a disk of resilient material, e.g. rubber, having an s upper and a lower surface. Six circular holes are defined through the thickness of the seal element and each hole is outlined on the upper surface by a square profile locating ridge 91 and on the lower surface by a v-profile sealing ridge, or v-ring 92. The entire circumference of the lower surface of the seal element is also bounded by a v-ring 92. I0

The v-ring abuts a planar surface of the lower portion, thereby forming a seal.

Thus, the reagent chamber openings are accessible through associated openings in the sealing element and closed, or blocked, by the planar surface of the lower portion.

Rotation of the lower portion relative to the upper portion allows the processing chamber to move into overlapping relationship with each opening in turn.

In doing so, communication is provided between each reagent chamber and the processing chamber in turn.

The seal element has holes defined through it that align with the respective openings in the upper portion. The ridges on the upper side of the seal element mate with recesses defined in the upper portion to locate the seal element such that its holes align with the openings in the upper portion.

The seal element may have a different design to that illustrated in Figures 1A to 2. For example, the seal element may only define a single through-hole, which locates over the opening to the processing chamber and may, in this case, be fixed relative to the lower portion and movable relative to the upper portion.

p47251gb01 SG 27/07/2006 -16 -In this case the seal element would act to block each of the openings in the upper portion until the upper and lower portions are rotated such that a particular opening is aligned with the processing chamber opening. As an example, if the processing chamber is brought into alignment with the first reagent chamber opening, the hole in the seal element also aligns with the opening of the first reagent chamber and the contents of the first reagent chamber, previouslymaintained in the first reagent chamber by the seal element, fall into the processing chamber.

io The illustrated seal element utilises v-ring type seal profiles, however, other seal profiles such as 0-ring profiles or a combination of different profiles could be used; for example, a v-ring could be used for the seal around the circumference of the seal element which acts to seal the device from the external environment and 0-rings could be used for the internal sealing of the individual chambers within the device.

Other sealing mechanisms and methods could be used, for example based on variations of the Luer-lock, frit and bayonet, screw threads or plunger seals.

Rotation of the lower portion of the body, or locating apparatus, relative to the upper portion thus moves the processing chamber between six positions, or stations, each enabling a step in a processing protocol for which the device is designed. In a first position, the processing chamber is opposite the access port 70 for receiving a sample. In a second position it is opposite a blank section 25 of the upper portion, which acts to seal the processing chamber without adding any reagent, for an incubation processing step. In third, fourth and fifth positions the processing chamber aligns with the first 30, second 40, and third 50 reagent chambers for the delivery of reagents and in a sixth position it aligns with the analyser chamber 100. A ratchet apparatus (not shown) acts between the upper and lower portions of the locating apparatus to prevent rotation in a reverse direction and to locate the location apparatus in position during processing at each position or station. In alternative embodiments, any suitable number and arrangements may be defined in the p47251gb01 SC 27107/2006 -17 * upper portion of the locating apparatus depending on the processing protocol for which the device is designed.

The first reagent chamber 30 is in the form of a blister or cell defined by walls extending from the upper portion of the location apparatus, and contains a dried processing reagent. The processing reagent is contained in the reagent chamber by the reagent chamber's walls and the seal element, which blocks the opening associated with the first reagent chamber.

to The second reagent chamber 40 is a separately couplable chamber that contains a liquid reagent. The second reagent chamber couples to the upper portion at its associated opening by means of a bayonet fit. When coupled to the upper portion of the location apparatus, the liquid reagent within the second reagent chamber can be dispensed through its associated opening. As with the first reagent chamber, the seal element acts to block the opening until the opening is aligned with the processing chamber, at which point liquid from the second reagent chamber may be dispensed through the opening and through the processing chamber opening into the processing chamber.

Figures 5A and 5B illustrate a separately couplable reagent chamber 900 suitable for containing liquid reagents in a device according to a further embodiment of the invention. The couplable chamber defines an internal space 910 for containing a liquid reagent. A lower portion of the removably couplable chamber is adapted to enable a push-fit with the device at the chamber's associated opening defined in the device. (This is an alternative construction to the bayonet fit described in the first embodiment.) A stopper arrangement 930 includes a spigot 940 that extends through the internal space 910 and seals a hole 950 at the bottom of the internal space. When the stopper arrangement is lifted, as illustrated in Figure 5B, the spigot 940 is removed from the hole 950. A vent 960 near an upper portion of the internal space allows air into the internal space, thus displacing any liquid contained in the internal space through the hole 950. The vent is arranged so that the air is drawn from within the sealed device and not from the external environment, to reduce any risk of contamination dunng processing.

p47251gb01 SG 27/07/2006 -18 -Alternative methods for liquid reagent delivery could be used, for example by syringe attached to the device via a Luer-lock or bayonet system.

The third reagent chamber is in the form of a blister defined by walls extending from the upper portion in the same way as the first reagent chamber defined above. The third reagent chamber contains dried reagents.

The analyser chamber is defined in and extends vertically from the upper io portion. This analyser chamber is a tall, thin chamber for containing a test strip.

The test strip is maintained in the analyser chamber by the sealing element in the same way as described above for dried processing reagents in the first and third reagent chambers.

is The analyser chamber has a transparent wall to enable the test strip to be visually inspected.

The device of the embodiment is designed for on-site nucleic acid testing.

In such a test, a blood sample must be processed by a number of steps to amplify the nucleic acid after which the processed sample is tested for the presence of a particular nucleic acid by use of a test strip. The closed system of the present invention is particularly advantageous to prevent contamination with rogue nucleic acids.

The following method for using the device relates to a method of amplifying and detecting a nucleic acid and refers to Figure 3, a flow diagram illustrating the steps involved in nucleic acid testing.

A sample is collected from a patient and, in steps 1 to 3, is pre-processed prior to introduction into the device. The pre-processing steps can be any suitable pre-processing steps such as those currently known in the art for use with commercially available kits for nucleic acid extraction.

p47251gb01 SG 27/07/2006 -19 -Further more simple procedures involve sample lysis by heat or chemical treatment and sample dilution prior to amplification. These are especially applicable for sample types that have high copy numbers of target nucleic acids e.g. ribosomal RNA present in thousands copies/cell.

The sample is added to a lysis buffer (step 1) and incubated (step 2). The sample is then diluted with a suitable buffer solution (step 3).

The device is prepared by coupling the separately couplable reagent io chamber 40 containing a detection buffer to the upper portion of the location apparatus 20.

The foil seal 80 sealing the access port 70 is removed and pre-processed sample is introduced through the access port into the processing chamber 60 (step 4). The processing chamber contains a pre-loaded first freeze-dried reagent. The upper portion of the location apparatus is then rotated relative to lower portion and the processing chamber so that the processing chamber moves away from the access port and seals within the body, aligned with the blank section 25 of the upper portion, and the device is then shaken to mix the o first freeze-dried reagent with the sample.

The device is then positioned on a heat source comprising a heating block shaped to receive the base of the processing chamber, and the sample within the chamber is incubated (step 5).

The device is removed from the heat source and the upper and lower portions are rotated relative to each other until the processing chamber opening overlaps with the opening associated with the first reagent chamber 30, which contains a second freeze-dried reagent. The second freeze-dried reagent falls into the processing chamber (step 6).

p47251gb01 SG 27/07/2006 -20 -The device is again positioned on the heat source and incubated before being removed from the heat source (step 7).

The upper and lower portions of the location device are rotated further until the s opening of the processing chamber aligns with the opening associated with the second reagent chamber. The couplable second reagent chamber has a stopper arrangement that needs to be removed so that its liquid detection buffer contents can flow into the processing chamber. The stopper is removed and the detection buffer is added to the processing chamber (step 8).

The upper and lower portions of the location device are rotated further until the opening of the processing chamber aligns with the opening associated with the third reagent chamber, containing third and fourth freeze-dried reagents.

These reagents are added to the processing chamber (step 9).

The upper and lower portions of the location device are rotated to a final position in which the processing chamber opening overlaps with the opening associated with the analyser chamber containing a test strip. The test strip drops into the processing chamber so that its end is in contact with the processed sample (step 10).

The processed sample is wicked up by the test strip (step 11).

The results of the test are obtained by reading a visual signal on the test zs strip (step 12).

There may be further steps involved such as a step to treat the sample after analysis to prevent contamination of the environment around the device and/or a step to dispose of the device.

p47251gb01 SG 27/07/2006 -21 -Figures 4A -4E illustrate a second embodiment of a device according to the invention.

The device 200 has a location apparatus or body 270, within which a passage s of rectangular cross-section is defined. Along an upper wall of the passage are positioned an access port 280, three reagent chambers depending from the location apparatus 220, 230, and 240, and an analysis chamber 250 also depending from the location apparatus. The analysis chamber contains a test-strip 255 for analysis of the processed sample. Between the access port and io the first reaction chamber, and between the reaction chambers, blank sections of the upper wall of the passage provide mixing and incubating positions, or stations. The device further comprises a processing chamber 210 set or moulded within a rubber block, which fits sealingly within the passage with the processing chamber opening abutting the upper wall of the location apparatus, so that it is sealed from the external environment when within the location apparatus.

A push-rod or end-plunger 260 enables a user to propel the processing chamber along the passage within the location apparatus 270. A plunger-type dispenser 251 is also utilised to retain the test-strip within the analysis chamber until it is required. A ratchet apparatus could be used to prevent the push-rod from being withdrawn and to aid location of the processing chamber at any one of a number of positions or stations.

Initial access is provided to the processing chamber by the access port 280 after removal of a foil seal (not shown).

Each reagent chamber has an associated opening defined in the location apparatus 222, 232, and 242 through which reagent contained in the reagent chamber can pass.

p47251gb01 SG 27/0712006

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-22 -The processing chamber is movable within the location apparatus relative to the openings associated with the reagent chambers. In the example illustrated in Figure 4A reagent chambers 220 and 240 contain freeze-dried balls of reagent 221 and 241, and reagent chamber 230 contains a liquid reagent 231.

Each reagent chamber comprises a hollow tube with an opening at one end leading through the upper wall of the location apparatus. At the opposite end of each reagent chamber a plunger 225, 235, and 245 seals the opposite end of the chamber and is actuatable to introduce the respective reagent into the io processing chamber through the reagent chamber opening, when the processing chamber opening is disposed in overlapping relationship with the particular reagent chamber opening.

In use, a sample is loaded into the processing chamber through the processing is chamber access port. The push-rod is used to slide the processing chamber within the location apparatus to an incubation position 290, illustrated in Figure 4B. In this position the processing chamber is sealed from the external environment.

After an incubation step, the processing chamber is moved into a position directly underneath the opening associated with the first processing chamber 220, in which its opening is in overlapping relationship with the first reagent chamber opening 222.

The plunger on the first processing chamber is pushed to deliver the ball of reagent 221 into the processing chamber (Figure 4C).

The plunger is moved to a second incubation position 295 illustrated in Figure 40.

After the second incubation the processing chamber is moved directly beneath the opening 232 associated with the second reagent chamber 230. The p47251gb01 SG 27/07/2006 -23 -plunger on the second reagent chamber is pushed to deliver the reagent contained within it 231 to the processing chamber.

The processing chamber is then moved directly beneath the third reagent chamber opening 242 and the plunger is pushed to deliver the reagent 241 contained in the third reagent chamber to the processing chamber.

The processed reagent is then moved, within the processing chamber, to a position directly beneath the analysis chamber 250 containing the test strip 255.

io The plunger on the analysis chamber 251 is pushed to allow the test strip to drop into the processing chamber and contact the processed sample (Figure 4E).

A third embodiment of the invention is illustrated in Figures 6A and 6B and the same reference numerals are used for components as were used for the first embodiment illustrated in Figures IA to 2 and described above. This third embodiment is the same as the first embodiment in all regards except that the analyser chamber is defined in a horizontal aspect on the upper portion of the location apparatus in order to help make the whole device more compact.

The device of the third embodiment is used in the same way as described above for the first embodiment except that the entire device must be rotated by degrees to enable the processed sample to contact the test strip contained in the analyser chamber after the processing chamber opening has been brought into register or overlapping relationship with the opening associated with the analyser chamber.

p47251gb01 SG 27/07/2006

Claims

-24 -Claims 1. A device for the processing of a sample, comprising; a

location apparatus, a processing chamber for receiving the sample having an opening, and a plurality of reagent chambers having associated openings defined in to the location apparatus, the processing chamber being movable relative to the reagent chambers to enable sequential communication between the processing chamber and each reagent chamber, such that each reagent chamber communicates with the processing chamber when its associated opening is disposed in overlapping relationship with the processing chamber opening.
2. A device according to Claim 1 further comprising a sealing apparatus for sealing the processing chamber from the external environment.
3. A device according to Claim 2 in which the location apparatus comprises the sealing apparatus.
4. A device according to Claims 2 or 3 in which the sealing apparatus seals the reagent chambers from the external environment.
5. A device according to any preceding claim, further comprising an analyser chamber for containing an analyser for analysing the sample after processing, the analyser chamber having an associated opening defined in the device such that it communicates with the processing chamber when its associated opening is disposed in overlapping relationship with the processing chamber opening.
6. A device according to any preceding claim in which the device is non-reusable.

p47251gb01 SG 27/07/2006 -25 -
7. A device according to any preceding claim for processing a biological sample.
8. A device according to any preceding claim further comprising an access port providing initial external access to the processing chamber opening

to allow introduction of the sample.
9. A device according to Claim 8 in which the access port is protected, prior to introduction of the sample, by a removable seal. I0
10. A device according to any preceding claim, in which the processing chamber is movable between a plurality of discrete positions or stations and in at least some of these positions the processing chamber opening is disposed in overlapping relationship with a reagent chamber opening.
11. A device according to any preceding claim in which at least one of the reagent chambers is couplable to the device.
12. A device according to Claim 11 in which the, or each, couplable chamber forms a seal when coupled to the device.
13. A device according to any preceding claim in which gravity acts to introduce at least one reagent and/or the analysis means into the processing chamber.
14. A device according to any preceding claim having at least one dispenser actuatable to introduce a reagent from its reagent chamber into the processing chamber.
15. A device according to any preceding claim suitable for use with a freeze-dried or lyophilised reagent.
16. A device according to any preceding claim suitable for use with a liquid reagent.

p47251gb01 SG 27/07/2006 -26 -
17. A device according to any preceding claim in which the reagent chambers are pre-loaded with reagents.
18. A device according to any of Claims 5 to 17 in which the analyser chamber is pre-loaded with an analyser.
19. A device according to any of Claims 5 to 18, in which the analyser is a test strip.

io
20. A device according to any of Claims 5 to 19 in which a wall of the analyser chamber is substantially transparent.
21. A device according to any preceding claim, in which the location apparatus comprises a bottom portion and a top portion movable Is relative to the bottom portion.
22. A device according to Claim 21 in which the processing chamber is connected to the bottom portion and the reagent chamber associated openings are defined in the top portion.
23. A device according to Claim 21 or 22 in which the top portion and the bottom portion are substantially disc-shaped and rotatable relative to each other about a common central point.

z
24. A device according to Claim 21, 22 or 23 further comprising a seal element disposed between the top and bottom portions.
25. A device according to any preceding claim in which the reagent chamber associated openings are disposed in a substantially circular arrangement.
26. A device according to any of Claims I to 24 in which the reagent chamber associated openings are disposed in a substantially linear arrangement.

p47251gb01 SG 27/07/2006 -
27 - 27. A device according to any preceding claim comprising three reagent chambers, each pre-loaded with a reagent.
28. A device according to any preceding claim comprising four reagent chambers, each pre-loaded with a reagent.
29. A device according to any preceding claim in which the processing chamber is pre-loaded with a reagent prior to introduction of the biological sample. I0
30. A device according to any preceding claim in which movement of the processing chamber is regulated by a ratchet apparatus.
31. A system for the processing of a biological sample comprising a device is according to any of Claims 1 to 30, and an external heat source adapted to engage with the device.
32. A system according to Claim 31, in which the heat source is adapted to engage with the processing chamber.
33. A system according to Claim 31 or 32 further comprising at least one couplable reagent chamber, the couplable reagent chamber pre-loaded with a reagent.
34. A couplable reagent chamber for use with a device according to any of Claims 1 to 30 or for use with a system according to Claims 31 to 33.
35. A couplable reagent chamber according to Claim 34, which couples to the device by a screw fit or a bayonet fit or a push fit.
36. A method of processing a sample in a device having a processing chamber and a plurality of reagent chambers, comprising the steps of: loading the sample into the processing chamber, p47251gb01 SG 27/07/2006 -28 -moving the processing chamber relative to the plurality of reagent chambers and introducing a first reagent into the processing chamber from a first reagent chamber, moving the processing chamber relative to the plurality of reagent chambers and introducing a second reagent into the processing chamber from a second reagent chamber.
37. A method according to Claim 36, further comprising a sealing apparatus and the step of moving the processing chamber after loading the sample such that the sealing apparatus seals the sample from the external environment.
38. A method according to Claim 36 or Claim 37, in which the resulting Is processed sample is analysed by an analyser.
39. A method according to Claim 38, in which the analyser is contained in an analyser chamber of the device.
40. A method according to any of Claims 36 to 39 further comprising the step of disposing of the device after analysing the sample.
41. A method according to any of Claims 38 to 40, which is a method of processing and analysing a biological sample.
42. A method according to any of Claims 36 to 41 in which there are between two and five reagent chambers, each containing a reagent that is introduced into the processing chamber during processing.
43. A method according to Claim 42 in which there are three or four reagent chambers.
44. A method according to any of Claims 36 to 43 in which the processing chamber is pre-loaded with a reagent.

p47251gb01 SG 27/07/2006 -29 -
45. A method according to any of Claims 36 to 44 additionally comprising at least one step of incubating the sample in the processing chamber at predetermined temperature for a predetermined period of time.
46. A method according to any of Claims 36 to 45 additionally comprising mixing steps subsequent to each introduction of processing reagent.
47. A method according to any of Claims 36 to 46 in which the analyser is a test-strip that contacts the processed sample to conduct the analysis. I0
48. A method according to any of Claims 36 to 47 in which the processing involves an amplification step to amplify a nucleic acid in the sample.
49. A method according to any of Claims 38 to 48 in which the analysis step is involves the detection of a nucleic acid present in the sample.
50. A method according to any of claims 38 to 49 in which a post analysis treatment is carried out on the sample to neutralise previous processing reactions.
51. A device substantially as described herein and with reference to the drawings.
52. A system substantially as described herein and with reference to the drawings.
53. A couplable reagent chamber substantially as described herein and with reference to the drawings.

3o
54. A method substantially as described herein and with reference to the drawings.
55. A device for the processing and analysis of a biological sample using lyophilised processing reagents, p47251gb01 SG 27/07/2006

I

-30 -the device comprising at least one processing chamber which, in use, is sealed from the external environment, wherein the lyophilised reagents are preloaded into the device prior to s its despatch to a user.
56. A device according to Claim 55 used for the amplification and detection of nucleic acid in a biological sample.

io
57. A device for the processing and analysis of a biological sample, the device comprising at least one processing chamber which, in use, is sealed from the external environment, wherein the device is adapted to use both solid and liquid processing reagents.
58. A device according to Claim 57 used for the amplification and detection of nucleic acid in a biological sample.

p47251gb01 SG 27/07/2006