GB2574046A

GB2574046A - Improved container

Info

Publication number: GB2574046A
Application number: GB1808553.0A
Authority: GB
Inventors: Moysey Ruth; Keenan Jemma
Original assignee: Oxford Nanopore Technologies PLC
Current assignee: Oxford Nanopore Technologies PLC
Priority date: 2018-05-24
Filing date: 2018-05-24
Publication date: 2019-11-27
Anticipated expiration: 2038-05-24
Also published as: GB2574046B; GB201808553D0

Abstract

A container for receiving a sample having cells in a fluid via a closable inlet 110 at a proximal end 104 and an outlet 118 having a breakable seal 120 for controlled dispensing of a lysed portion of the sample at a distal end 106, the container having at least one movable mechanical bead 122 located within the container between the inlet and outlet for mixing the sample when the container is manually moved, a reagent configured to lyse a cell and release nucleic acids from within the cell into the sample and at least one chemical bead 124 located within the container between the inlet and outlet for binding released nucleic acid, wherein said outlet, when exposed, is configured to controllably release at least the chemical bead through the outlet for deposition in a flowcell. The outlet is an end cap with a seal which is manually breakable by applying a torque force to the end cap. The outlet may comprise a funnel 114. The container may comprise a mechanical barrier (130;fig 7) to prevent the mechanical bead locating in the outlet.

Description

IMPROVED CONTAINER

The invention relates to an improved container that is configured to collect, prepare or process and deliver a sample to a device for analysis. Generally, the invention relates to a container for collecting a sample, using the container to isolate elements of said sample for analysis and dispensing a processed sample for analysis. In particular, the invention relates to a container for collecting an organic sample, such as a blood sample, lysing nucleic acid, such as DNA or RNA, from said sample and controllably outputting nucleic acid extracted or captured from the sample for application in a flowcell for nucleic acid analysis.

Known vials resemble miniature pots or jars for collecting samples. They have a sealable lid for preserving the sample for analysis. Figure 1 shows a known vial 2 having an inlet 4, an outlet 6, a cavity 8 and a filter 10 disposed in the cavity of a column 12 between the inlet and outlet, for depositing a filtered sample via the outlet 6. After placing a sample inside the column 12 a closure 14 can secure the sample within. A twist-off bottom 16 keeps the outlet closed and is removed before an extract of the sample can be ejected from the outlet. The vial 2 as shown is typical of a spin column currently available from ThermoFischer Scientific and is branded a Pierce™ Centrifuge Column.

Known vials or lysing tubes are designed for specific applications and are not suitable for all sample collection and dispensing requirements. Moreover, known vials are configured for use in a laboratory environment and require processing using an electronic or mechanical machine or device. Moreover, the sample collected in the vial often requires preparation before and/or after collection. The known vial in Figure 1 requires a centrifuge.

The changes that a sample experiences as it passes in to and out of a vial e.g. filtering, and the nature and volume of the sample required can influence the subsequent analysis performed on the sample. Known vials and processes cater for specific or particular analysis methods and are not interchangeable or compatible with other methods of analysis. Although the concept of collecting and treating a sample in a vial is known, the inventors have been unable to identify a vial having the functions required or desired. The inventors have created a bespoke solution for their needs. It is therefore an aim of the present invention to provide an improved container

Generally, the invention resides in a container for receiving a sample having fluid and for controllably dispensing a lysed portion of said sample to the inlet of a flowcell. The container is configured to lyse a sample, preferably through during manual agitation or shaking of the container - shaking is not essential but speeds up the processing of a sample. The container has an outlet, closed with a seal, said being breakable to expose the outlet, said outlet configured to controllably release at least a portion of the contents of the container through the outlet for deposition in a device for analysis, such as a flowcell. The container contains a breaker, which can be a mechanical bead, chemical bead and reagent for mechanically mixing and dispersing and selectively binding with nucleic acid, such as DNA, contained in the sample.

According to one aspect the invention resides in a container for receiving a sample having cells in a fluid via a releasably closable inlet at a proximal end, which can be the input port. The container is suitable for controllably dispensing a lysed portion of said sample via an outlet having a breakable seal, said seal breakable to expose the outlet, at a distal end. The container is adapted for dispensing a lysed sample in to an analysis device, such as a flowcell.

The container has: a mechanical bead located within the container between the inlet and outlet, for mixing the sample when the container is manually moved; a reagent configured to lyse a cell and release nucleic acids from within a cell into the sample; and a chemical bead located within the container between the inlet and outlet, and for binding released nucleic acid, wherein said outlet, when exposed, is configured to controllably release at least the chemical bead through the outlet for deposition in a device for analysis. The device can be a flowcell.

The mechanical bead can be movably configured to mechanically lyse a cell within the container. It can be shaken or agitated. The reagent can be configured adapted to lyse cells and release the nucleic acids from within the nucleic acid analyte of interest in the sample and attach to the bead. In other words, the reagent can selectively bind with the nucleic acid analyte of interest.

A releasably securable or screwable lid can be provided for enclosing a sample received via the inlet. The lid can form an airtight seal. The lid and/or inlet has a seal. The seal can have a washer or O-ring. An end-cap can be provided to seal or enclose the outlet, said seal breakable to expose the outlet.

A wall extending between the inlet and outlet can define an enclosable space between the proximal and distal ends. Overall, the container forms a cylindrical volume of space defined by a lid at the proximal end, a floor at the distal end and walls in between. The outlet can be in the form of a funnel and extend from the floor of the container.

The outlet, in cross-section, can narrow or taper towards the distal end. The outlet can taper linearly. The outlet can be configured to inhibit a lysed sample from dripping from the outlet. The aperture can be circular in cross-section. The aperture can be elliptical in cross-section. The outlet can be enclosed by a cap or seal such that after the seal is broken the size and shape of the outlet is unchanged. The cap or seal at the outlet can, alternatively, define the size and shape of the aperture when removed. The cap or seal at the outlet can be configured with a base that enables the container to stand on a surface with container extending vertically from the distal end to the proximal end such that a lysed sample and bead is gravitationally biased towards the outlet.

The mechanical bead is type of a breaker. The breaker can be spherical. The breaker can be asymmetrical, irregular or granular. A combination is breakers having different shapes can be used. The mechanical bead or breaker can have an uneven surface. The breaker can have inert properties. A plurality of mechanical beads or breakers can be provided, such that multiple contacts between breakers and other between other surfaces in the container during manual agitation. The number of breakers typically used can be between 1 and 30, or between 1 and 5. The number of beads used is influenced by the size of the breaker and the size of the container. In light of the teaching herein an appropriate number of breakers and appropriate size or mix of breakers can be selected according to the size of the container and the sample type to be mixed. The beads can have an uneven surface to improve the mixing effect. The mechanical bean can be shaped to optimise agitation and lysing of the sample, e.g. it can be the shape of a prism, e.g. a five-sided prism. It is to be noted that the size of a breaker can be selected to inhibit entry to the outlet, or funnel, of the container. The mechanical bead is preferably of a high density relative to the liquid such that it can efficiently move through and mix the sample when manually moved. The mechanical bead may be metallic such as stainless steel. The beads may typically have a width or diameter cross section of between 1 and 10mm.

The chemical beads can be a bead or matrix with a ligand that will preferably be pH switchable for the charge to capture nucleic acid. The chemical bead may be silica. The chemical bead can comprise 2-(2-Pyridyl)ethyl Silica Gel.

The beads and reagent can be provided on the interior surface of the walls of the container. The beads can be provided in a liquid. The liquid can be prefilled in the container.

The beads and reagent can be provided or coated on the exterior surface of the mechanical bead and releasable therefrom. By coating the surface of the mechanical bead or breaker can increased the rate of lysing and/or bonding to the nucleic acid of the sample. This can also apply to other surfaces on the interior of the container.

The container can have a mechanical barrier, which can be a rod, configured to prevent a mechanical bead from locating in the outlet or passage. The rod can inhibit a breaker from blocking the flow of a lysed sample from the container to the outlet. The mechanical bead, or breaker, can be optional. The mixing function can alternatively be achieved by a barrier or a rod configured within the container.

The mechanical barrier or rod can extend non-tangentially to an axis defined by the proximal and distal ends of the container. A rod can be located adjacent the outlet to permit maximum movement of a breaker within the container. The rod, which can be a barrier can extend perpendicularly from the wall at one side of the container to the contact the other side of the container. The barrier can be a rod. The rod can have a circular cross-section. The cross-section 3 of the rod can, alternatively, have a non-circular profile to increase the level of agitation and lysing of a sample when the container is mechanically shaken. The rod can be located in the chamber by means of a compression fit. In other words, a barrier, such as a rod, is cut to a size larger than the maximum diameter of the container and when placed inside can be pressed downwards at the distal end until both ends of the rod contact the wall and the force applied to the insertion of the rod squeezes the rod into a fixed position. This can provide a simple and low-cost barrier. The barrier can inhibit the mechanical bead from inhibiting the flow of lysed sample into the outlet. Additionally or alternatively, a barrier can be configured in the opening of the outlet or funnel.

According to another aspect the invention resides in a kit have container as claimed.

According to another aspect the invention resides in a method of preparing a sample for deposition in a measuring device, which can be a flowcell, using the container or kit as claimed.

The method includes: removing a cap from the container, placing a sample to be lysed therein and then securing the cap on the container to seal the sample therein; optionally shaking the container for a period to agitate the sample to mechanically and chemically lysed the sample; allowing the lysed sample to gravitate toward the distal end of the container; removing the endcap; and placing the tip of the container in contact with the input port of an analysis device, such as a flowcell. The method can further comprise loosening the cap to at least partially open the proximal end and/or applying a manual pressure to the walls of the container.

Reference herein to a nucleic acid or a polynucleotide include both naturally occurring nucleic acids, such as DNA or RNA and synthetic polynucleotides. The polynucleotide may be oxidized or methylated. The polynucleotide may be damaged. The polynucleotide may be single or double stranded.

The container of the invention has been adapted for use with a flowcell and other such analysis devices manufacture by Oxford Nanopore Technologies Ltd. In light of the teaching herein the invention is suitable for use with other devices.

A known vial has already been described in relation to Figure 1 and now the invention is discussed below, by way of example only, with reference to the following figures of cross-sections of containers for processing a sample in which:

Figure 2a is a container having two mechanical breakers and chemical beads and reagent for mixing and breaking a sample deposited therein, while Figure 2b is a view of the distal end of the container alongside a view from within the container looking towards the outlet, and Figure 2c shows the accumulation of beads at the distal end of the container;

Figures 3 a and 3b are containers having beads and reagent and at least a rod for mixing a sample deposited therein;

Figure 4 is a container having a breaker, beads, reagent and a rod for mixing and lysing a sample deposited therein;

Figure 5 is the container of Figure 3 having five breakers;

Figure 6 shows the container of Figure 5 in which the rod is aligned non-tangentially in the container;

Figure 7 shows the container of Figure 6 in which the end-cap is configured as a stand; and Figure 8 shows a container having a lid having a protrusion manually operable to break a sample.

The container 100 of Figures 2a and 2b is defined by a body 102 having a proximal end 104 and a distal end 106. The container is preferably cylindrical with the side defined by a wall 108, the proximal end wall defined by a cap 110 and a base 112 defining the distal end. The cap can releasably seal a sample within the container. The seal is preferably airtight. The cap can be a screw-on type cap with O-ring or other sealing surface, or a compression fit.

A funnel 114 extends from the base and narrows to a tip 116 having an aperture 118 defined by the distal wall of the funnel. An end-cap 120 is secured to the wall of the funnel. A seal (not shown) is provided between the wall of the funnel and the end-cap to protect and enclose the tip 116. The seal is manually breakable by applying a torque force to the end-cap. The various components of the container 100 are made of a plastic material, such as polypropylene. The container is configured for disposal after a single use and have low material cost and low manufacturing cost.

Within the body 102 two breakers 122 are provided and shown located at the base 112. Breakers can be mechanical beads. The breakers are suitable for mechanically dispersing a sample and made of an inert material. They are preferably heavy to have momentum when shaken in the container and can be made of metal, glass or ceramic material. The breakers can be stainless steel ball bearings.

The breakers 122 function to mix a sample of the container, when sealed and shaken; the breakers can shear genomic nucleic acid, such as DNA or RNA, within a sample in to fragments. Chemical beads 124, indicated by fine dots, are provided within the container and can be bead coated on at least one of the internal walls of the body 102, the base 112 and the breakers 122. The beads are a silica matrix and, by way of example, the beads are a 2-(2-Pyridyl)ethyl silica gel having an anion-exchange retention mechanism compatible with aqueous solutions. The beads can be spherical or granular. Additionally or alternatively the beads can be made of at least one of ceramic or metal. The density of the beads is preferably heavier than the sample or solution to allow them to sediment.

A reagent is also provided in the container, although is not shown because it too appears as a fine powder. The reagent can, alternatively, be provided in an aqueous liquid. Reference to 5 the beads 124, as shown, throughout the description includes the reagent. The reagent is a dry material and can be rehydrated. The reagent is configured to capture nucleic acids polynucleotides, such as DNA or RNA. The beads 124 and reagent are complimentary because the bead has a positive charge in the lysis environment, which has a lower pH value than the pKa of the ligand, which in this example is 2-(2-Pyridyl)ethyl. Nucleic acid has a negative charge. Therefore, when a portion of the reagent has captured a polynucleotide it is attracted to a bead.

The depth of the fine dots representing the beads 124 and reagent, as shown in the Figures, is for illustrative purposes only. In a physical example of the invention the beads and reagent appear like a fine powder, or dust, which covers at least a portion of the interior surface of the container, rod or breaker. It can be present with an aqueous solution.

The base 112 has a passage 126 that corresponds to the proximal end of the funnel 114, which provides fluid communication between the container and funnel. The fluid communication between the volume defined by the body 102 and the funnel 114 is unfiltered and direct. To be clear, no filter is provided at the entrance to the funnel and beads can gravitate towards the aperture 118. A strut 128 is provided across the passage to prevent a breaker 122, when included, from blocking the passage or the funnel. The strut 128 does not function as a filter.

The aperture 118 formed in the tip 116 of the funnel is configured to inhibit a fluid sample from forming a drip. The formation of a drip at the tip is inhibited when the cap is screwed on because no air can enter at the proximal end and the aperture 118 is sized to withhold liquid. This is achievable because the size of the aperture is such that the surface tension of the contents of the tip 116 hold back the contents. The aperture 118 in the tip 116 is configured to release a bead. A plurality of these beads will have captured nucleic acid, such as DNA or RNA, if present in a sample. The beads that accumulate at the tip can be referred to as DNA loaded beads. When the layer of beads that accumulate at the tip, and are held under surface tension, come in to contact with another liquid, such as the liquid at the input port of a measurement device such as a flowcell, then the surface tension is broken and beads are released through at least one of capillary action, wicking or gravity. In this way, the beads can be drawn out of the container for sampling. The body 102 of the container 100 can be deformable to bias a portion of the sample towards the aperture 118 to initiate deposition through contact or wicking.

Figure 2b shows a portion of the distal end 106 of the container 100 alongside a view from within the container looking towards the distal end. The position of a breaker 122 is shown positioned partially over the passage 126 through which a lysed sample will pass towards the aperture 118 in the tip 116 of the funnel 114. The strut 128 forms a barrier to inhibit the spherical breaker and/or large portions of the sample, from substantially entering and/or blocking the funnel. Beads 124 and reagent are shown covering the interior surface of the base. Additionally or 6 alternatively a protrusion extending from the peripheral edge of the passage 126 can inhibit a breaker 122 from resting in, and blocking, the passage. In the example shown in Figures 2 to 7, the height of the container from proximal to distal ends is typically between 30mm and 60mm. The diameter of the body is typically between about 7mm and about 12mm. Although the Figures are not to scale the components of the container are shown proportional to these dimensions.

The aperture 118 in the tip 116 is fine, and preferably circular, having an internal diameter of between 0.1mm and 1mm. The aperture can have an internal diameter of about 0.6mm.

The container 102 defines an enclosable space for holding a sample. In use, the cap 110 or lid is removed and a sample to be analysed is placed in the container and the cap replaced and secured. With the sample secured inside, the container is manually shaken. The sample can be lysed manually and without external powered means, such as sonication, electronics, pressure, heating or freezing. When shaken, the breakers, which function as mechanical mixing beads 122 are manually shakeable within the container to at least one of grind, break up, mix, disperse and resuspend the sample. Lysis of the cells within the sample may also take place to some extent. When shaken the breakers also shear or fragment the nucleic acid, such as DNA or RNA, reducing the length of the genomic nucleic acid to a size that can interact with at least one bead. The number of breakers and/or rods configured in a container can be adapted according to the type of sample in order that after shearing stands of DNA are length that inhibits interaction with so many beads that a clump of beads forms at the aperture in the tip. Preferably the DNA is fragmented to lengths of between about lOOOOkb to about lOOOOOkb, but can be smaller and does not exclude cell free DNA or shorter RNA. The DNA can be fragmented to lengths of about 5bp up to about lOOOOkb.

By shearing the DNA, the accumulation of long strands or coiled DNA nucleic acid that is combined into a clump with the binding beads or matrix is inhibited because blocking the tip is to be avoided. Moreover, long genomic DNA is inhibited from multiple contact with multiple beads.

By mechanically shearing or fragmenting the genomic nucleic acid it is broken in to components small enough such that when they attach to the beads the formation of large clumps is inhibited. Large clumps have been observed to form between multiple chemical beads and very long polynucleotide strands. They are desirably avoided because they are inclined to block the aperture. This can be achieved by the impacting of portions of the sample between breakers and/or between a breaker and at least one or the cap 110, wall 108 and base 112. In addition to the mechanical breakers, or balls 122, the beads 124 and reagent capture DNA from the sample. It is to be noted that the mechanical mixing and shearing of the sample can be achieved, additionally or alternatively, with a rod (described in other Figures) such that the breakers 122 can be optional, depending on the sample to be lysed.

With the sample lysed - mechanically and chemically - and with DNA captured on the beads 124 using the reagent, the container is configured to enable the beads to drop under gravity when the container extends vertically. The density of the beads is selected to be heavier than the solution such that they drop towards the tip 118 when the container is orientated vertically. The beads are adapted to form a sediment rather than be held in suspension. The beads are configured, after agitation, to drop in to the funnel 114 and flow past the strut 128 and through the passage 126. The breakers 122 are prevented from closing the passage by the strut.

As shown in Figure 2c, after dropping down in to the tip 118, the beads and reagent substantially displace liquid solution (indicated by lines in the funnel 114) in the tip such that the beads accumulate in the tip. The beads and reagent accumulate in the tip in a similar manner to grains of sand in an hour-glass, except that the aperture and beads are sized such that surface tension inhibits dripping or flow from the tip. When the surface tension is broken it is predominately beads, with the reagent and captured DNA, that initially flow from the time. The volume of fluid that flows is out of the tip is minimised because the beads have displaced most of the fluid, which resides only in the gaps between the beads.

After shaking and lysing the sample, the end-cap 120 can be removed by breaking its seal with the funnel such that the tip 116 is accessed or revealed. Beads 124 in the funnel 114 adjacent the aperture 118 in the tip can be drawn out by touching the surface of the device that is used to analyse the lysed sample. Additionally or alternatively, the walls 108 of the container can be squeezed to apply a pressure to the space within the container and bias a portion of the lysed sample on the beads towards the aperture. The tip is configured to limit the volume of lysed sample dispensed from the aperture. Dripping is inhibited. The tip of funnel is configured to encourage beads to pass out from the aperture. The mixing action by the breakers and/or rods, together with the reagent is configured to capture at least a portion of any nucleic acid, or DNA, from the sample. The dimension of the aperture 118 inhibits larger portions or volumes of the lysed sample from passing therethrough and is configured to permit small volumes of beads, to pass through. The aperture is sized to permit between about 0. lul and about lOul of beads to pass through, or egress from the aperture, when contacted with a fluid in the measuring device, or flowcell. Preferably, between about lul and about 5ul of beads can pass through. After shaking the sample, beads fill up most of the volume of the tip adjacent the aperture such that liquid is inhibited from occupying this space. Liquid can, however, reside in the gaps between the beads.

The beads can be spherical or asymmetrically shaped, like grains of sand. Beads are sized to pass through the aperture but be held within the tip under surface tension. The beads and tip are configured such that they pass through the aperture only when the surface tension is broken or released. The material used to make the funnel, in particular the tip of the funnel, can have 8 properties that encourage the retention of beads, under surface tension, that have accumulated in the tip of the funnel. The material can be hydrophobic and/or the interior surface of the funnel can be provided with a hydrophobic coating.

The container and funnel 114 are configured to lyse a sample and encourage the beads and reagent with captured DNA to drop to the aperture 118. Unlike known containers for sample analysis, which are designed to hold beads in suspension, the container and beads 124 are encouraged to sediment. The beads are sized smaller than the aperture and drop efficiently because of their density yet remain small enough to have a greater binding surface area and form a compact bead plug at the aperture, such that a controllable volume of lysed sample can be dispensed from the aperture on the beads.

The size and quantity of the breakers 122, and the size and quantity of the beads 124, are selected to permit a sample to be shaken, lysed and deposit a sample of the captured DNA on the beads, typically in a time-period of between 1 to 60 mins and preferably in around 5 minutes.

The chemical beads 124 are configured to bind DNA under the conditions used for chemical lysis. Chemical lysis may be carried out using a suitable lysis reagent such as guanidine thiocyanate or guanidine hydrochloride. The reagent may be added to the container along with the sample or optionally provided in the container in a dry form, for example as a coating on the inside of the container. The reagent may optionally comprise a surfactant such as Triton, Brij, or Tween, available from ThermoFisher Scientific that acts to release soluble proteins and solubilize membrane proteins and lipids. The surfactant may also reduce clumping of beads. Typical concentrations of guanidine that may be used may be between about 0.1M and about 3M, or between about 0.5M to about 1.5M, and can be 0.8M. Guanidine also serves to denature any DNAse or RNAse enzymes that may otherwise digest any DNA or RNA released from the cells. The reagent may also contain a suitable lysing buffer such as NP-40, RIP A, SDS (sodium dodecyl sulfate) or ACK (Ammonium-Chloride-Potassium) lysis buffer.

The chemical lysis may be carried out on various different types of cells, such as bacterial, fungal, plant and animal tissue cells. It may also be used to lyse virus particles.

The chemical beads serve to bind polynucleotides released from the cells or particles and may comprise a surface coating of a binding ligand. Following binding of the polynucleotide to the beads and removal from the container, for example for measurement, the polynucleotides may thereafter be removed from the beads. The pKa of the beads may be chosen such that the beads are positively charged in one pH range whereas they become negatively charged in another pH range. The pH conditions may be selected such that the polynucleotides, which are negatively charged, bind to the positively charged beads in the container. The polynucleotides may thereafter 9 be released by changing the pH conditions such that the beads become negatively charged. By way of example, the beads may have pKa of 6. In this particular case, the pH level of the liquid in the container is sleeted to be less than 6.

The container is optimised for use in the field and away from laboratory conditions when there are no special devices, such as powered devices, available to lyse or process the sample through sonication, voltage stimulation, bead beating, pressure, heating or freezing.

The container is compatible with receiving and lysing a sample for loading into a flowcell without transferring detergents, proteases or other chemistry that would be detrimental to the performance of an analysis device, such as a flowcell.

It is to be noted that the container does not selectively lyse a sample and can result in the release of DNA and RNA from virus particles.

A further restriction on lysis chemistry is the requirement to control or prevent DNAses or RNAses from digesting released DNA or RNA. These enzymes require relatively harsh denaturing conditions as these enzymes are known to be robust. DNAses digest the cellular DNA released, which are ubiquitously present in biological fluids and certain white cells. The lysis conditions can be chosen such that any DNAses released are denatured thus inhibiting them from digesting nucleic acid released from a cell in a sample.

The nature of the mechanical breaking makes the container suitable for samples containing a mixture of solids and liquids, including viscous samples such as colony pick or saliva sample, liquid sample such as bacterial broth, urine or blood, tears, phlegm, snot, faecal matter, semen, gut samples, swab sample or sampling stick device. The container can be used to process any organic matter such as a sample from a human, plant or animal. For example, samples of food, soft tissue and environmental pond water can be received in the container for processing.

The configuration of the tip 116 to inhibit dripping and encourage or prioritise the deposition of beads from the container results in a limited volume of lysed sample entering a flowcell, in use. A limited volume is preferred when delivering sample to a flowcell to avoid adding substances that interfere with flowcell chemistry and with the osmotic balance within the flowcell. The fine aperture and sedimentation of the beads excludes liquid from the area that comes into contact with the flowcell and can inhibit residue from being carried through the aperture and in to a port of a flowcell.

Overall, the purpose of the container is to minimise the number of steps required for sample preparation prior to analysis in a device such as a flowcell. The container, its ability to mechanically and chemically lyse a sample and deposit beads holding DNA(or RNA) extracted from a sample in a controlled manner for analysis in a flowcell, simplifies the procedural steps required to analyse a sample. This is especially important in the field i.e. outside laboratory environments, when sample preparation is difficult, and no electro-mechanical devices are available for lysing or processing a sample. The container provides a simple, low-cost, ‘one-pot’ solution that can be manually processed without ancillary devices or chemicals.

The container, it’s operation and functions have been described above in relation to the example shown in Figures 2a to 2c, which defines an enclosable space for holding a sample, which can then be mechanically and chemically lysed before a portion of the lysed sample is controllably deposited through the tip. Like numerals in the Figures represent like features and Figures 3 to 7 show alternative examples of containers with different configurations. The functions of the various features of the examples are appreciable by the skilled person in light of the teaching herein and he would recognise that the features of Figures 2 to 7 were compatible and/or interchangeable to optimise one or more functions of the invention as described and claimed.

Figure 3a shows a container 100 having a rod 130 configured to extend between the walls 108 of the body 102 of the container, while Figure 3b shows a container having a plurality of rods - nominally five. The rod is circular in cross-section, although can have different profiles. The rod can be provided with a helical surface or a roughened surface. The number of rods and/or their cross-sectional profiles can vary depending at least one of: the level or rate of agitation required when mechanically lysing a sample through shaking; the type of sample; the surface area of the rods; and the quantity or number of beads and/or reagent to be coated on the rods. The rod in Figure 3a is shown extending perpendicularly from the wall 108 on one side of the body to the opposite side, such that it extends parallel to the base 112. The rods in Figure 3b extend in various directions. The rod is formed of a plastic, such as polyethylene, but can be made of a metal. During manufacture the rod can be sized such that it is held in compression between the walls. Additionally or alternatively the rod can be glued in to place, or secured using vibration welding. As shown in other Figures the rod can be angled with respect to the base 116. The rod is located above the surface of the base 116 such that a sample within the body can be shaken against a rod and flow around the rod to aid mixing and lysing of the sample. The profile of the rod can be shaped to aid mixing or lysing.

Figure 4 shows the container of Figure 3a having a breaker 122. As described in relation to Figure 2a and 2b, the breaker functions to mix and break down a sample. The rod 130 compliments the lysing action by providing an additional different surface for the breaker to contact during shaking of the container 100 and enhance lysing by encouraging mixing of the sample. The rod and breaker can operate in a complimentary manner. Moreover, the rod 130 can be provided in addition to, or alternatively to, the strut 128 and prevent a breaker from entering the passage 126. In light of the teaching herein a skilled person would select the size and position of the rod and size of a breaker, or breakers, to prevent a breaker blocking the passage. For 11 example, the size of the rod and the breaker is such that the smallest breaker cannot pass the rod closest to the base 112 to contact the base. Moreover, the size of the rod and the breaker is configured to inhibit a breaker from being jammed between a rod and the wall of the container. If a plurality of rods and/or breakers are provided they can have different sizes and shapes.

To be clear, the rod implements at least one of several functions: - it aids mixing by interacting with a sample during manual lysing and mixing; it interacts with a breaker during manual lysing and mixing; and prevents a breaker from blocking the passage.

It is to be noted that the breaker of Figure 4 is coated with beads 124 and reagent and that it is feasible for all examples of the invention to have a breakers and/or rod coated with beads and/or reagent prior to adding a sample, which can improve lysing.

Figure 5 shows the container of Figure 4 having five breakers 122. When manually shaken the increased number of breakers accelerate the lysing of a sample because there are more collisions or impacts between the sample and the breakers, rod and interior surfaces of the container.

Figure 6 shows the container of Figure 5 having five breakers 122 and a rod 130 configured at an angle with respect to the base 112 of the container 100. Overall, the rods can provide a surface for a breaker to contact but can additionally create turbulence in the sample during shaking and this can be encouraged by the asymmetric or off-set position of the rod to aid mixing and lysing of the sample.

As described above, the container is configured to mix and lyse a sample and capture DNA on the beads 124 and reagent, which then drop under gravity to the distal end 106 of the container when it is arranged to extend vertically.

Figure 7 shows the container of Figure 6 having the end-cap 120 configured as a stand 132. After manually shaking a sample in the container it can be placed on a flat surface to allow beads to drop to the tip of the funnel. Not only does the stand allow a user to arrange the container to allow the beads to drop, but the larger end-cap or stand 132 allows a user to apply a greater torque to the seal between the stand and the funnel making it easier to separate them.

Figure 8 is a schematic view of the cap 110 of the container 100 having a protrusion 134 or stick having an irregular or roughened surface at its end. The stick is configured to extend between the walls 108 of the container and be manually operable to mechanically breakdown a sample in the container. The stick 134 can extend to contact a rod 130.

In addition to mechanical means that function to break or grind a sample, such as the breakers 122 and the rod 130; the beads 124 and reagent that function together to lyse the sample, the container can contain an enzyme.

The enzyme can be a protease that performs proteolysis. By way of example the enzyme can be a pepsin. The pepsin can have a low pH value of about 2 to about 3, which is suitable for ‘pick’ activity. The enzyme can function to chop up proteins within the sample under lysis conditions. The enzyme can be active up to about 3M guanidine. The enzyme can be loaded on magnetic beads such that they can be retained by, for example, a magnet in the cap to inhibit the magnetic beads from passing in to the funnel to reach the aperture. Additionally or alternatively the enzyme can be retained, fixed or immobilised onto the inner surface of the container or other matrix. The enzyme can be free in a solution. The enzyme can pull part virus particles and guanidine can denature the DNA or RNA in proteins.

The inventor has found guanidine compliments pepsin - the guanidine will denature or partially denature the proteins allowing the pepsin or protease to have increased access and therefore work faster to chop them up, for capture and analysis. The pepsin (or other protease) enzyme can work to digest the proteins that form the virus capsid. Guanidine can function alone and pepsin can function alone but have been found to work faster together. Once the capsid is broken apart the nucleic acid of the virus is released (DNA/RNA, ssDNA, dsDNA etc).

Pepsin is a preferred example, as too are proteases that are not detrimental to an analysis device and chemistry therein e.g. flowcell chemistry, such as a motor protein. Although the introduction of pepsin in to flowcell is not considered problematic because it is not active/deactivated above pH8 - it is, however, preferable only to introduce the material to be analysed.

The features in Figures of the invention are interchangeable and compatible in light of the teaching herein. The present invention has been described above purely by way of example, and modifications can be made within the spirit and scope of the invention, which extends to equivalents of the features described and combinations of one or more features described herein. The invention also consists in any individual features described or implicit herein.

List of features:

2	Vial	110	Cap
4	Inlet	112	Base
6	Outlet	114	Funnel
8	Cavity	116	Tip
10	Filter	118	Aperture
12	Column	120	End-cap
14	Closure	122	Breaker
16	Twist-off bottom	124	Beads
100	Container	126	Passage
102	Body	128	Strut
104	Proximal end	130	Rod
106	Distal end	132	Stand
108	Wall	134	Protrusion

Claims

1. A container for receiving a sample having cells in a fluid via a releasably closable inlet at a proximal end and for controllably dispensing a lysed portion of said sample via an outlet having a breakable seal, said seal breakable to expose the outlet, at a distal end, said container having:

a mechanical bead located within the container between the inlet and outlet, the mechanical bead movably configured to mechanically mix the sample when the container is manually moved;

a reagent configured to lyse a cell and release nucleic acids from within a cell into the sample; and a chemical bead located within the container between the inlet and outlet, and for binding released nucleic acid, wherein said outlet, when exposed, is configured to controllably release at least the chemical bead through the outlet for deposition in a flowcell.

2. A container according to claim 1, wherein the outlet, in cross-section, narrows towards the distal end.

3. A container according to claim 2, wherein the outlet is configured to inhibit a lysed sample from dripping from the outlet.

4. A container according to any preceding claim, wherein the outlet is enclosed by the seal such that after the seal is broken the size and shape of the outlet is unchanged.

5. A container according to any preceding claim, wherein the seal is configured with a base that enables the container to stand on a surface with container extending vertically from the distal end to the proximal end such that a lysed sample and bead is gravitationally biased towards the outlet.

6. A container according to any preceding claim, wherein the mechanical bead is spherical.

7. A container according to any preceding claim, wherein the mechanical bead has an uneven surface.

8. A container according to any preceding claim, wherein the chemical bead is a bead or matrix with a ligand that will be pH switchable for the charge.

9. A container according to any preceding claim, wherein the beads and reagent are provided on the interior surface of the walls of the container.

10. A container according to any preceding claim, wherein the beads and reagent are provided or coated on the exterior surface of the mechanical bead and releasable therefrom.

11. A container according to any preceding claim, further having a mechanical barrier configured to prevent a mechanical bead from locating in the outlet or passage and/or inhibiting flow of a lysed sample from the container to the outlet.

12. A container according to any preceding claim, wherein the mechanical barrier is a rod that extends non-tangentially to an axis defined by the proximal and distal ends of the container.

13. A kit having a container according to any preceding claim.

14. A method of preparing a sample for deposition in a flowcell using the container according to any of claims 1 to 12 or the kit according to claim 13, the method including: removing a cap from the container, placing a sample to be lysed therein and then securing the cap on the container to seal the sample therein; allowing the lysed sample to gravitate toward the distal end of the container; removing the end-cap; and placing the tip of the container in contact with the input port of an analysis device, such as a flowcell.

15. The method of claim 14, wherein the method further comprises loosening the cap to at least partially open the proximal end and/or applying a manual pressure to the walls of the container.