WO2018138471A2 - Systèmes d'agitation - Google Patents

Systèmes d'agitation Download PDF

Info

Publication number
WO2018138471A2
WO2018138471A2 PCT/GB2018/000011 GB2018000011W WO2018138471A2 WO 2018138471 A2 WO2018138471 A2 WO 2018138471A2 GB 2018000011 W GB2018000011 W GB 2018000011W WO 2018138471 A2 WO2018138471 A2 WO 2018138471A2
Authority
WO
WIPO (PCT)
Prior art keywords
vessel
reaction vessel
reaction
reagents
contents
Prior art date
Application number
PCT/GB2018/000011
Other languages
English (en)
Other versions
WO2018138471A3 (fr
Inventor
Nelson Nazareth
David Edge
Adam Tyler
Original Assignee
Bg Research Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bg Research Ltd filed Critical Bg Research Ltd
Publication of WO2018138471A2 publication Critical patent/WO2018138471A2/fr
Publication of WO2018138471A3 publication Critical patent/WO2018138471A3/fr

Links

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/45—Magnetic mixers; Mixers with magnetically driven stirrers
    • B01F33/452—Magnetic mixers; Mixers with magnetically driven stirrers using independent floating stirring elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F35/00—Accessories for mixers; Auxiliary operations or auxiliary devices; Parts or details of general application
    • B01F35/71—Feed mechanisms
    • B01F35/713—Feed mechanisms comprising breaking packages or parts thereof, e.g. piercing or opening sealing elements between compartments or cartridges
    • B01F35/7137—Piercing, perforating or melting membranes or closures which seal the compartments
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • B01L3/50825—Closing or opening means, corks, bungs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0689—Sealing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/12—Specific details about manufacturing devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/047—Additional chamber, reservoir
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0848—Specific forms of parts of containers
    • B01L2300/0858—Side walls
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C45/00—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor
    • B29C45/14—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor incorporating preformed parts or layers, e.g. injection moulding around inserts or for coating articles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C45/00—Injection moulding, i.e. forcing the required volume of moulding material through a nozzle into a closed mould; Apparatus therefor
    • B29C45/16—Making multilayered or multicoloured articles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/0332—Cuvette constructions with temperature control

Definitions

  • the present invention relates to apparatus and process for amplifying nucleic acid targets. It is particularly concerned with Nucleic Acid Amplification (NAA) including Polymerase Chain Reaction (PCR), RT-QPCR and QPCR as set out in European Patent Application EP2585581 , and most particularly with direct amplification from crude biological samples such as blood, swabs and sputum.
  • NAA Nucleic Acid Amplification
  • PCR Polymerase Chain Reaction
  • RT-QPCR Real-QPCR
  • QPCR Polymerase Chain Reaction
  • European Patent Application EP 2585581 describes a method for cell disruption performed by multiple cycles of freezing and thawing a sample whereby the ice crystals formed physically disrupt cell membranes or viral capsids and subsequent thawing induces massive osmotic shock, releasing cellular contents.
  • direct detection of the released nucleic acids is obtained by combining in a single closed tube the freezing and thawing process and followed by nucleic acid amplification such as real-time PGR.
  • PCT patent application PCT/GB2016/000178 describes apparatus and process wherein a relatively large NAA reaction volume can be processed and even a very low concentration of a target pathogen nucleic acid can be detected, and that within the desired short time. This however incurs a challenge also, that of ensuring that the target nucleic acids have been fully accessed by the reagents.
  • a first aspect of the present invention addresses this challenge.
  • Single closed tube processing including freezing, thawing, then amplification is extremely valuable when confirming the presence of a dangerous pathogen because, on the one hand contamination of and by the sample can be minimised and on the other the confirmation can be obtained within one hour.
  • a problem can exist in some cases with the preferred single closed tube method. This is that in order to lyse target pathogens efficiently there are either required chemical modifications to the buffer or thermal excursions which can mean heating constituent enzymes, particularly proteinaceous enzymes, above their survival temperature. Either option is likely to be deleterious to the process.
  • One solution can be to add the reagents after the freeze/thaw lysis step has been completed. However this risks the undesired contamination by or of the sample, especially if the lysis step has included boiling. It also loses precious time and risks exposure of the operative to the pathogen to be detected.
  • apparatus for effecting in a closed tube reaction vessel identification of a blood-borne pathogen comprises stirring means for mixing the contents of the reaction vessel, the vessel having a capacity greater than 200 ⁇ and less than 1001 ⁇
  • the stirring means may comprise a mechanical or electro-mechanical shaker or vibrator or an ultrasonic stimulator.
  • a stirring device within the reaction chamber of the reaction vessel, the stirring device comprising a ferrous member, and being adapted for movement within the reaction chamber under the influence of a shuttle magnet system outside the chamber, thus to mix the vessel contents
  • the ferrous member may be spherical, for example a ball bearing.
  • the preferred ferrous member is elongated, for example sausage or rod shaped.
  • the arrangement may then be one in which one end of the member can be retained by an external stationary magnet and the other end thereof oscillated by a shuttle magnet system.
  • the shuttle magnet system may comprise a magnet moved between two stations. Movement between two stations may be accomplished with a cam device or a linear actuator such as a solenoid or a stepper motor.
  • the two magnets, preferable neodymium may be matched and typically have a pull of the order of 3.5Kg (at full closure) for the moving magnet and 1 Kg (at full closure) for the magnet running on a common vertical axis.
  • a further advantage of this mixing process is more rapid equilibration of temperatures during thermal cycling, facilitated by the mechanical mixing.
  • a major advantage of this approach and applicable even in the absence of the reagents being added from the cap, is that the stirring , methodology increases the efficiency of reverse transcription reactions at low copy numbers of target nucleic acid (provide data).
  • the mixing of the reaction maximises the reverse transcription efficiency such that all replicates present identical real-time PCR curves and Ct values. Therefore, mixing can be applied to any RT-QPCR methodology to improve the low level sensitivity.
  • the ferrous member may be rendered biocompatible to biological processing by being coated, for example with parylene.
  • a nucleic acid amplification (NAA) reaction vessel may accordingly comprise: two opposing major walls; a minor wall system attaching the major walls and thus defining a reaction chamber having a base, the major and minor walls being formed of a thermally conductive material; an inlet port permitting the introduction of fluid into the reaction vessel; a cap arranged for sealing the inlet port; and a light transmissive window.
  • NAA nucleic acid amplification
  • the vessel may have any, some or all of the following elements:
  • the reaction chamber has a base somewhat narrower than the remainder and may even be substantially pointed.
  • the shape may be round or, more likely oval with the major axis vertical, or it may be rhomboid or square. It may be shaped like an opened letter envelope with the apex at the reaction chamber base, or a shield;
  • the light transmissive window comprises a part, perhaps a major part of the minor wall.
  • the site of the optics may be variable or determined to be at any location from the base of the vessel to an upper side;
  • a pierceable station preferably in an upper region of a minor wall, arranged for ready access to transfer vessel contents to an electrophoresis device;
  • the width of the vessel between the major wall is 2 - 3mm, preferably 2.4mm;
  • the major walls are 0.2 - 0.6 mm, preferably 0.4mm thick, thus making the overall width of the order of 3.2mm;
  • the overall dimensions of the vessel are up to 40mm tall by 33mm broad;
  • the vessel may be manufactured in a two-step moulding process, the first step comprising forming a frame of transparent material, the frame comprising a part of the vessel side wall including a clear window, and an entry manifold, then moulding on to the frame the remaining side wall portion and two faces comprising a highly carbon loaded thermally conductive compound.
  • the transparent plastic window may be formed of polypropylene and the remaining side wall portion and the faces being formed of polypropylene loaded with 25 - 70% carbon, preferably 40% to 65%. Other transparent materials could be employed but polypropylene is particularly suitable for integration with the remaining vessel material.
  • the frame may have a T-section so that the thermally conductive portions may be moulded to the flanges of the T.
  • the value of the vessel having effectively a reaction chamber which tapers down, either by being triangular (such as shield shaped), oval or circular is that the vessel is thus capable of being used with very small samples, for example of blood, sputum or swab, as well as much larger samples.
  • the ferrous member may have an overall breadth or diameter of 0.8 to 1 .5mm, optimally 1 mm.
  • a rod may thus have a clearance of about 0.7mm either side thereof and 1 mm at the end thereof.
  • the length of the rod may be of the order of 10 to 12mm.
  • a closed tube reaction vessel adapted for the performance therein of a two-step process comprising lysis, for example by freeze/thaw, followed by nucleic acid amplification may be arranged ab initio to contain the reagents in lyophilised form such as HawkZ05 (Roche), or any other thermostable enzymes.
  • a reaction vessel may be supplied as a consumable already containing the reagents, preferably in lyophilised form.
  • the reagent may be added following the thermal excursion, for example by there being a hollow cap closure to the vessel, the cap closure having a chamber adapted to contain the reagents, there being also provided a device configured to enable the release of the reagents into the lysed target in the vessel upon completion of the freeze/thaw step.
  • the vessel can be reduced in temperature to a temperature below the deleterious temperature and then the reagent pellet can be added into the vessel, preferably by an automated means.
  • Delivery of a lyophilised pellet can ensure delivery of the full reagent amount. Ensuring dissolution and uniform distribution of the reagents into the sample can be effected using the stirring facility of the first aspect of the present invention.
  • the vessel lid/closure may comprise a housing with a proboscis, perhaps having a fir tree profile, and a pierceable membrane upon which the lyophilisation of the reagents into pellet form can be effected. Following this lyophilisation the fir tree lid closure mechanism can be placed into an opening on the top of the cap closure mechanism. Then, in due time the proboscis can be depressed in order to pierce the membrane and drive the pellet such that it falls into the reaction chamber below.
  • a preferred fir tree lid closure has 3 different sealing points to minimise the risk of virus escaping the vessel, that is for example, sealing at the interface between the proboscis and the orifice into which it fits, at an o-ring in the ceiling of the lid, where the vessel will abut, and at the base rim of the lid.
  • the lyophilised reagents can be held dry in this cap closure.
  • An example of the value of this is when reverse transcriptases are involved. These are known to be thermally labile.
  • viral targets such as viruses are present then it would not be possible to heat the contents for example in excess of 60C.
  • Standard methodology for making blood products safe involves heating stored blood to in excess of 60C for a period in excess of 30 minutes, which naturally means that blood borne viral pathogens are thus rendered inactive.
  • the efficiency of genomic release for some viral pathogens can be improved by then heating the sample above these temperatures with the benefit that the pathogen will additionally be rendered non-infectious.
  • these temperatures are in excess of 60C for a shorter period of time.
  • the Applicants have discovered that following 4 cycles of freeze/thaw, 5 seconds at 62C is sufficient to lyse Dengue and Zika viruses (include real-time PCR data).
  • the temperature required to render them inactive has proven to be in excess of 70C.
  • Sindbis virus requires a period of 5 seconds at 75C to release the viral RNA.
  • enzymes such as HawkZ05 is recommended.
  • these can be lyophilised directly in the reaction vessel without needing to be stored in the lid thereof as they are able to survive the thermal excursion.
  • a nucleic acid amplification reaction apparatus constructed to receive a reaction vessel according to the first aspect of the invention; the apparatus comprising: at least one reaction vessel receiving station; two heater guard plates, one to be each side of the vessel and contiguous with the major walls thereof; a Peltier cell having a working face contiguous with each heater guard plate on the face of the guard plate destined to be remote from the reaction vessel, the Peltier cell having also a base face; a reference temperature unit contiguous with the base face of each Peltier cell; and a mobile magnet facility arranged for agitating the stirring device.
  • the guard plates The function of the guard plates is to locate and mount the Peltier cells and retain them in position, as well as to protect them from wear or damage afflicted by repeated insertion and removal of vessels. Moreover, the guard plates can contain a recess at their working faces whereby the reaction vessels are more or less completely encapsulated, except for optics access. This goes with making the vessels with the same thermally conductive materials, that is to say major and minor walls, substantially throughout except where the (transparent) sections are required for optical access. Thus the guard plates may be recessed on both sides, that is to say that they preferably have edge walls within which the Peltier cells nestle According to features of this third aspect of the invention the apparatus may have a longitudinal clamp and incorporate elastic pads, e.g. rubber O-rings to accommodate expansion and contraction of the Peltier cells. It may also comprise:
  • a retainer arranged for urging the reaction vessel within its station in the apparatus, thus to maintain contiguity between the vessel exterior walls and the heater guard plates;
  • the retainer may be an overhead device arranged to bear on the vessel caps, thus ensuring they too remain in place.
  • the reference temperature unit may comprise a thermally conductive, preferably metal, even sintered metal case having fluid flow ducts formed therethrough, which ducts may be connected in a circuit comprising also a fluid pump, to a heat exchanger arranged to keep fluid, usually water, at a constant temperature.
  • the reference temperature is such that the ⁇ T (Delta T) of the Peltier is able to encompass both freezing and boiling. Typically this may be 18 to 26°C when active cooling is used, but in the case of it being passively cooled then it should be maintained below 45°C and in conjunction with a peltier device having a delta T of in excess of 65°C such that a freezing temperature of -20°C can be achieved.
  • the ducts may incorporate a chicane system to maximize heat transfer and ensure mixing of the temperature control medium such that they are at a substantially even temperature from entrance to exit from the unit.
  • the temperature reference unit may incorporate crenellations to nestle the base face of the Peltier cell.
  • the Peltier cell will normally be arranged to be operated by reversible direct current whereby the working face can at one instance be a heater and at another a cooler.
  • the Peltier cell and the reaction vessel major walls are generally arranged to be substantially coterminous one with the other, with the Peltier cell being usually square.
  • a preferred optical array is a reflectance probe arrangement employing two core optical fibres, one core arranged to transmit the excitation light into the reaction chamber from a laser diode or LED or other high powered light source and the other emerging light to a spectrophotometer which may be incorporated in the apparatus.
  • a shutter may be incorporated between the vessel and the fibre to minimize optical interference between one station and another when a station is not being employed.
  • a preferred arrangement for a multi-station apparatus is for the apparatus to be arranged for individual station operation in a random access fashion.
  • This being the case an arrangement of solenoid switches may be built into the optic fibre array such that the light from any one reaction vessel can be imaged on the shared spectrophotometer without interference from any other of the vessels or indeed environmental light in the case of one or more reaction stations being empty.
  • An alternate approach is to use a dedicated optical sensor for each reaction position such that the requirement for a shuttering system is removed.
  • An example is the Pixelsensor by Pixelteq which has eight individual photodiodes each coated in a bandpass filter and as such can measure fluorescence output at multiple wavelengths and remove the requirement for a shared spectrophotometer.
  • a process for the amplification and detection of nucleic acid comprises:
  • nucleic acid amplification (NAA) reagents including primers/probes and fluorescently labelled probes;
  • freeze/thaw temperatures may be of the order of -20 and +20°C respectively; ⁇ there may be a heating stage following the freeze/thaw stage, with a temperature of the order of 60-75°C. Optionally this temperature excursion will be to just above the melting point of the primers/probes;
  • the probes may be labelled with dyes such as fluorescein, HEX and TET.
  • the NAA may be PCR or isothermal amplification methods and the reagents may comprise lyophilized reagents which will be activated upon contact with liquid in the reaction vessel.
  • the various aspects of the invention permit the detection of target pathogen nucleic acids from crude samples, with the necessary two steps being completed in a hands off process within a single closed tube. This is especially valuable in the detection of highly dangerous pathogens such as Ebola from whole blood, when re-opening the vessel between the first and second steps for example could expose the user to the pathogen.
  • the preferred embodiment addresses this risk by means of the reagents being lyophilised within the reaction vessel prior to use; or for thermolabile enzymes, retaining the reagents as a lyophilised pellet either within the vessel closure such that this pellet can be added in an automated fashion following a viral high temperature kill step, or within the reaction vessel itself.
  • NAA is complete, that is the step of identifying a plurality of pathogens in the one sample, the virus will be rendered non-infectious and so the vessel can be safely transported back to a laboratory for subsequent processing such as next- generation sequencing analysis
  • One particular example of the value of the present invention is in the detection of Ebola from whole human blood.
  • apparatus according to the present invention it is possible to perform direct RTQPCR and detect as few as 60 filovirus (ZEBOV) particles in a crude blood sample.
  • ZBOV filovirus
  • Other pathogens which may be identified in the process of the invention include Lassa, Marburg, Zika, Chikungunya, Dengue, Yellow fever, Rickettsia, HIV, Crimea Congo fever, Blue tongue, and PPRV, all of which are blood borne.
  • a disposable reaction vessel in accordance with the first aspect of the invention may be packaged in a kit, the package containing the reaction vessel, a container of extraction buffer, water to resuspend the reagents and a container of lyophilized reagents.
  • a finger pricking device and a capillary such as a microsafe device for taking blood.
  • a large reaction vessel as herein described allows the examination of a reasonable quantity of blood sample whilst minimizing the concentration of that blood in the reaction, given that the blood also inhibits PCR.
  • a larger reaction vessel when used in the two step process of the invention provides (in-tube lysis then PCR), allows adding extraction buffer at high concentration in the first step and then diluting it out via the additional PCR reagents in the second step.
  • a 62.5 ⁇ assay has 5 ⁇ whole blood component, this being 8% volume by volume.
  • this assay can detect 10 4 targets per ml so that doubling the amount of blood should double the sensitivity.
  • this ignores the inhibitory effect of blood on both amplification and fluorescent signals.
  • apparatus and process according to the present invention it is possible to show that a larger reaction with the same amount of target added and hence reducing the final blood concentration may actually be ten times more sensitive even though the amount of target remains the same.
  • a high capacity reaction vessel with high thermal conductivity and a high aspect ratio that is capable of performing rapid freezing/thawing.
  • a larger volume reaction vessel according to the invention makes possible a two step process still contained within a single vessel. The process can then for example have a very low or high pH in the first step and then, due to the greater space above that of the first step reaction have this buffered out ready for the second step. Likewise, the first step could include thermal excursions that would denature enzymes which might have been included ab initio to be ready for the second step. Therefore a two step method performed entirely within a larger capacity vessel of the invention actually increases the sensitivity over and above existing (ower volume methods providing benefits specific to direct detection methodology.
  • each reaction position may have its own associated optical fibre terminating in a ferule situated just below the optical window at the base of the vessel.
  • the fibre may be bifurcated with one member terminating at an excitation source, and the other at a spectrophotometer.
  • the excitation source could comprise a laser diode in a housing.
  • the other fibre member may terminate at the entrance slit of a spectrophotometer.
  • the excitation and/or observation may be effected at any point along that edge.
  • the emission from these multiple wells may still be focused on a single spectrophotometer.
  • individual and separate readings can be generated by the illumination of single laser diodes, and hence reaction vessels, sequentially and in a manner which ensures that only emission light from a single reaction vessel is captured at any one time, thus avoiding cross-talk between vessel data.
  • the optical fibre below the reaction vessel may be placed and arranged such that its cone of light acceptance captures light from the greater majority of the vessel contents.
  • Figure 1 is a face section elevation of a first embodiment tube
  • Figure 2 is an isometric view of a first embodiment tube assembly
  • Figure 3 is a face section elevation of a second embodiment tube assembly
  • Figure 4 is a part sectional exploded view of the second embodiment tube assembly
  • Figure 5 is a side elevation of the second embodiment tube assembly
  • Figure 6 is sectioned side elevation of the second embodiment tube assembly
  • Figure 7 is an isometric view of the second embodiment tube assembly
  • Figures 8a and 8b are part sectional face views of an apparatus reaction station with a second embodiment tube assembly in situ;
  • Figure 9 is an isometric view of an apparatus reaction station
  • Figure 10 is an isometric plan view of part of a reaction station with a second embodiment tube assembly in situ;
  • Figure 1 1 is an exploded view of a reaction station
  • Figure 12 is a part exploded view of a reaction station
  • Figure 13 is a sketch of an optical monitoring arrangement
  • Figure 14 is an isometric view of a closed tube reaction apparatus having four reaction stations.
  • FIGS 1 and 2 show a first embodiment of a reaction vessel 10.
  • the vessel 10 is essentially shield shaped with major face walls 10a and minor side and base walls 10b and a ceiling 10c defining a reaction chamber.
  • the vessel 10 is formed mainly of carbon loaded polypropylene but has an optically clear window 10d at the base and extending around one edge thereof.
  • the vessel 10 is sealed at the top by a cap closure 12 containing a "fir tree" insert 13 and incorporating a pierceable membrane 14.
  • the cap closure 12 is recessed at 12a to reduce the likelihood of the insert 13 being accidently depressed.
  • the drawing shows a pellet of lyophilised reagents 15 above the membrane 14. The construction is such that manual depression of the insert 13 pushes the pellet 15 through the membrane 14 and comes to rest with its base flush with the reaction chamber ceiling 10c.
  • FIGS 3 to 7 show a second embodiment of a reaction vessel.
  • the vessel 20 defines a shield shaped reaction chamber portion with major face walls 20a and minor side and base walls 20b and a ceiling 20c. This portion is formed mainly of carbon loaded polypropylene but has an optically clear window 20d in the minor wall 20b at the base and to one side thereof. Sonically welded to the reaction chamber portion is an entry manifold 21 in barrel form defining a funnel leading into the reaction chamber (The letters A and B in figure 4 indicate where entry manifold 21 and the reaction chamber portion are joined).
  • the vessel 20 is sealable at the top by a screw cap closure 22 having lugs 22a.
  • the lid 22 includes a proboscis 22b formed to fit sealably in a mating recess 21 b in the manifold 21. As shown in figures 3 and 6 a taper 22b on the proboscis 22b is constructed for contiguity with a shoulder 21 c in the manifold 21. Elsewhere in the funnel, as indicated by the double lines, contact between the proboscis 22b and the manifold 21 may or may not occur, having regard to manufacturing tolerances.
  • the vessel contains a stirrer 30, described in detail below.
  • the reaction chamber portion 10, 20 of the reaction vessel has a taper in the minor walls 10b, 20b downward from top to bottom. This taper is 2° in total.
  • the width of the vessel between the major walls 10a, 20a is 2.4mm.
  • the major walls are 0.4mm thick, thus making the overall width of the order of 3.2mm.
  • the overall dimensions of the vessel are 40mm tall by 33mm broad. It has a capacity of 536 ⁇ .
  • the reaction vessel is manufactured in a two-step moulding process.
  • a frame is moulded of transparent material, the frame comprising a part of the vessel side wall including a clear window and having a T cross section, and an entry manifold,
  • the remaining side wall portion and two faces are moulded on to the frame.
  • the transparent plastic window is formed of polypropylene.
  • the remaining side wall portion and the faces are formed of polypropylene loaded with 40% to 65% carbon.
  • the reaction vessel is a consumable. In the normal course of events, after a full closed tube reaction, including amplification and identification, the contents are no longer pathogenic though careful disposal will nonetheless normally be required.
  • the disposable reaction vessel is packaged in a kit, the package containing the reaction vessel, a container of extraction buffer, water to resuspend the reagents and a container of Iyophilized reagents, a finger pricking device and a microsafe capillary device for taking blood.
  • Figures 8a and 8b show a reaction apparatus reaction station.
  • a reaction vessel 20 with a cap closure 22 having lugs 22a.
  • a parylene or Teflon coated ferrous rod 30 within the vessel 20 is a parylene or Teflon coated ferrous rod 30 while the apparatus has a stationary magnet 31 proximate one edge of the reaction chamber and a shuttle magnet 32 proximate the other edge.
  • the magnets 31 , 32 are placed with opposite poles across the vessel.
  • the fixed magnet 31 has its north pole directed towards the reaction vessel 20 and the movable magnet 32 has its south pole presented towards the vessel.
  • the fixed magnet 31 is proximal but not actually in contact with the vessel for thermal isolation reasons.
  • the movable magnet 32 is placed for stowage yet further away from the vessel 20.
  • the ferrous rod 30 when the shuttle magnet 32 is stowed the ferrous rod 30 will be retained against a side wall 20b adjacent the fixed magnet 31 and not obscure the field of view of the optics (described below).
  • the shuttle magnet 32 is operated by a solenoid against a spring 32a.
  • a frame 50 provides a base to which are mounted various apparatus elements.
  • the ferrous rod 30 has a 1 mm diameter and is 25mm long.
  • the neodymium magnets are matched and have a pull of 3.5Kg (at full closure) for the moving magnet and a 1 Kg (at full closure) running on a common vertical axis.
  • the station receptor 34 having an external screw thread 34a.
  • the station receptor 34 is arranged for attachment to the apparatus (frame 50, see fig 12) once the sealed reaction vessel 20 has been loaded into a reaction station.
  • the receptor 34 serves to locate the vessel 20 correctly and act as a base for a location maintenance device 35 which screws into place over the receptor 34 via the screw threads 34a.
  • a spring 36 in the location maintenance device 35 serves to urge the vessel 20 downward into the station.
  • the apparatus has a temperature sensor located in a recess in the guards 45, 45'.
  • the station shown in figure 9 is in a prior-to-loading configuration and shows a frame 50 to which is mounted a cramp jaw 40.
  • Springs 41 and associated bolts 42 maintain lateral pressure on temperature reference unit 43, peltiers 47 (see fig 12) and a reaction vessel 20 to ensure maximum thermal conduction.
  • the temperature reference unit 43 has liquid entry and exit manifolds 43a, 43b.
  • the receptor 34 with its screw thread 34a is more clearly seen, with the cap 22 nestling thereinside.
  • the springs 41 and bolts 42 serving to maintain lateral pressure on the reaction vessel 20, together with the temperature reference liquid entry and exit manifolds 43a, 43b of the temperature reference unit 43.
  • the springs have a stiffness such that once the bolts 42 have been initially tightened, the reaction vessel can be inserted and the lateral pressure required for contiguity automatically maintained.
  • figure 11 can be seen an exploded view of most of the principal components of a reaction station. These include the frame 50 and, from left to right, a cramp plate 40, a temperature reference unit 43 with liquid entry manifold 43a, thermoelectric cell mounts 45, temperature reference unit 43' with liquid entry and exit manifolds 43a' and 43b', and cramp plate 40'.
  • the bolts 42 are surrounded by plastic sleeves 42a and there are likewise insulating plastic washers 42c.
  • the bolts have nuts 42b.
  • the shuttle magnet 32 with its spring 32a are also visible in the figure 11 .
  • Figure 12 is a further exploded view of a reaction station with a reaction vessel 20 poised for insertion.
  • the receptor 34 (not shown) is attached to the frame and, with the cramp jaws 40 and the temperature reference units 43, 43' maintained in situ in the reaction station the guard plates 45, 45'.
  • the guard plates 45, 45' are constructed of highly thermally conductive metal, for example copper, to surround quite contiguously the reaction vessel portion of a reaction vessel 10, 20. There are dowels and mating hollows between these guard plates to retain their mutual assembly, and they are also formed to locate the peltier cells 47.
  • Holes 50a in the frame 50 receive the bolts 42 and sleeves 42a.
  • a recess in the guard plates 45, 45' carries a temperature sensor 48
  • each reaction station has its own associated optical fibre 60 terminating in the ferule 37.
  • the fibre is bifurcated with one member 60a terminating at an excitation source 61 , and the other member 60b terminating at the entrance slit of a spectrophotometer 62.
  • the excitation source 61 comprises a laser diode in a housing.
  • the emission from these multiple wells may still be focused on a single spectrophotometer.
  • individual and separate readings can be generated by the illumination of single laser diodes, and hence reaction vessels, sequentially and in a manner which ensures that only emission light from a single reaction vessel is captured at any one time, thus avoiding cross-talk between vessel data.
  • the optical fibre 60 below the reaction vessel is placed and arranged such that its cone of light acceptance captures light from the greater majority of the vessel contents.
  • the apparatus depicted in figure 14 has a housing 70 with four reaction stations whereon can be seen receptors 34 and location maintenance devices 35.
  • a fan vent 71 serves to permit environmental air to be drawn in to a heat exchanger (not shown) arranged to supply liquid at a constant temperature to the temperature reference units 43, 43'.
  • Magnets 72 under the top , of the housing 70 serve for temporary retention of the devices 35 during loading of the reaction stations.
  • 25 ⁇ of blood suspected to contain the target pathogenic virus is then removed from a patient via fingerprick or veinous draw and pipetted into the reaction chamber.
  • the reaction vessel is then immediately closed with the cap 22 (540° turn) and the receptor 34 emplaced followed by the location maintenance device 35.
  • the springs 41 act to grip the reaction vessel laterally and ensure contiguity and thermal conductivity between the temperature reference units 43, 43' and the base faces of the peltiers 47 and between the working faces of the peltiers 45 and the reaction vessel 20.
  • the shuttle magnet 32 can now be operated to oscillate the ferrous rod 30 and assist the dissolving of the reagent pellet and the mixing of the vessel contents. 0.5 hertz oscillation frequency has been found to be adequate.
  • Reverse transcription is now engendered, assisted by the stirring device, followed by nucleic acid amplification, these being effected again by the judicious application of alternate positive and negative direct current to the peltiers 47.
  • a reaction vessel 10 as described with reference to figures 1 and 2 is used.
  • the vessel still contains a ferrous rod stirring device 30 and the apparatus the stationary (31 ) and shuttle (32) magnets.
  • the reagents pellet 15 is located within the cap closure 12 and this pellet will have been created by drying (lyophilising) the reagents on top of the piercable membrane 14.
  • a volume of buffer is placed in the reaction vessel 10 and a small (in the range of 1-20% by volume) amount of crude sample (whole blood) added.
  • the plastic fir tree lid closure device 13 is then placed into the opening in the top of the cap closure 12.
  • An alternative embodiment has the magnets equidistant but that either of the magnets may differ in size and hence field strength such that the ferrous rod can be retained against the magnet of interest.
  • the ferrous rod (1 1 ) preferably coated in an inert material such as Teflon, is held horizontally between the poles of the magnets.
  • the movable magnet (10) is driven vertically up and down such that the ferrous rod pivots around the fixed magnet and a vertical movement is induced in the rod by moving between positions 1 1 and 13.
  • Live virus testing has been carried out on apparatus as described above. It was able to detect viral pathogens down to 3,000 virions per millilitre of whole blood added directly to the reaction vessel. Tests took less than 50 minutes, thus reducing the cost per test. Moreover multiple fluorophores were detected concurrently in the presence of blood. For example the instrument detected Rift Valley Fever down to ⁇ 1 pfu/ml in an infected mouse model.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Analytical Chemistry (AREA)
  • Hematology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé et un appareil de détection d'acides nucléiques pathogènes cibles à partir d'échantillons bruts dans un seul récipient de réaction fermé ayant une capacité s'inscrivant dans une plage allant de 200 µl à 1001 µl, l'appareil incorporant un moyen d'agitation.
PCT/GB2018/000011 2017-01-26 2018-01-18 Systèmes d'agitation WO2018138471A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1701336.8 2017-01-26
GBGB1701336.8A GB201701336D0 (en) 2017-01-26 2017-01-26 Stirring systems

Publications (2)

Publication Number Publication Date
WO2018138471A2 true WO2018138471A2 (fr) 2018-08-02
WO2018138471A3 WO2018138471A3 (fr) 2018-09-27

Family

ID=58462890

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2018/000011 WO2018138471A2 (fr) 2017-01-26 2018-01-18 Systèmes d'agitation

Country Status (2)

Country Link
GB (1) GB201701336D0 (fr)
WO (1) WO2018138471A2 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2585581A2 (fr) 2010-06-15 2013-05-01 BG Research Ltd Fractionnement cellulaire

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4214874A (en) * 1979-02-08 1980-07-29 American Hospital Supply Corporation Combination and method for mixing the contents of a blood collection tube and thereafter removing the mixing element
US20040132082A1 (en) * 1995-02-14 2004-07-08 Bio101 Method for isolating DNA
US6635492B2 (en) * 1996-01-25 2003-10-21 Bjs Company Ltd. Heating specimen carriers
EP1419820A1 (fr) * 2002-11-14 2004-05-19 F. Hoffmann-La Roche Ag Méthode, dispositif et enceinte de reaction pour le traitement d'échantillons biologiques
US7446288B2 (en) * 2005-05-06 2008-11-04 Applied Biosystems Inc. Device including inductively heatable fluid retainment region, and method

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2585581A2 (fr) 2010-06-15 2013-05-01 BG Research Ltd Fractionnement cellulaire

Also Published As

Publication number Publication date
GB201701336D0 (en) 2017-03-15
WO2018138471A3 (fr) 2018-09-27

Similar Documents

Publication Publication Date Title
US10427162B2 (en) Systems and methods for molecular diagnostics
US20180214879A1 (en) Nucleic acid amplification
CN107338180B (zh) 样品制备、处理和分析系统
WO2003033740A2 (fr) Appareil et procede pour isoler un acide nucleique
US20140186841A1 (en) Sensing and identifying biological samples on microfluidic devices
JP2013520204A (ja) 一体化された試料調製、反応、及び検出のための装置及び方法
ES2661402T3 (es) Aparato para aplicación de sonicación
US20210077993A1 (en) Methods
US11447767B2 (en) Apparatus for and methods of the disruption of a biological cell
KR20240013129A (ko) 분석물 검출 카트리지 및 그 사용 방법
US11608521B2 (en) Method of detecting genetic material in a biological sample and a device for its implementation
WO2018138471A2 (fr) Systèmes d'agitation
KR102177634B1 (ko) 분자진단용 전처리 키트
US10473566B2 (en) Blood sampling, storage and treatment apparatus
JP7334363B2 (ja) 検査容器、検査装置及び核酸検査方法
Bao et al. A simple, pipette-free, and power-free device for virus nucleic acid detection
RU2800583C2 (ru) Система для полимеразной цепной реакции
US20220347672A1 (en) Systems, methods, and apparatus for automated self-contained biological analysis
US20220081730A1 (en) Method, system and apparatus for respiratory testing
WO2014127805A1 (fr) Méthode de diagnostic de pathogène
WO2014127806A1 (fr) Kit d'amplification d'adn
JPWO2003004596A1 (ja) 反応容器及び反応装置

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18712637

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18712637

Country of ref document: EP

Kind code of ref document: A2