EP3181229B1 - Method for carrying out a pcr reaction - Google Patents

Method for carrying out a pcr reaction Download PDF

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Publication number
EP3181229B1
EP3181229B1 EP15003599.6A EP15003599A EP3181229B1 EP 3181229 B1 EP3181229 B1 EP 3181229B1 EP 15003599 A EP15003599 A EP 15003599A EP 3181229 B1 EP3181229 B1 EP 3181229B1
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EP
European Patent Office
Prior art keywords
chamber
chambers
amplification
annealing
liquid sample
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EP15003599.6A
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German (de)
French (fr)
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EP3181229A1 (en
Inventor
Markus Cavalar
Ulf Steller
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Euroimmun Medizinische Labordiagnostika AG
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Euroimmun Medizinische Labordiagnostika AG
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Priority to EP15003599.6A priority Critical patent/EP3181229B1/en
Priority to DE102016013403.8A priority patent/DE102016013403A1/en
Publication of EP3181229A1 publication Critical patent/EP3181229A1/en
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    • 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
    • B01L7/525—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 with physical movement of samples between temperature zones
    • 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
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • 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
    • B01L2200/0621—Control of the sequence of chambers filled or emptied
    • 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
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/087—Multiple sequential chambers
    • 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
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/088—Channel loops

Definitions

  • PCR polymerase chain reaction
  • pathogens viruses, bacteria, fungi, parasites
  • MRSA methicillin-resistant Staphylococcus aureus
  • certain human nucleic acid sequences that are of diagnostic importance, for example because they have mutations associated with certain diseases, can be detected in patient samples.
  • PCR is also important outside of medical applications, for example when examining soil or food samples for certain microorganisms, in forensics or in the synthetic production of complex nucleic acids.
  • PCR requires considerable effort with regard to reagents, equipment and workload, especially in diagnostic applications.
  • a reaction batch has to be put together that contains a whole series of specific, partly thermo-unstable reagents.
  • the finished reaction batch has to be incubated cyclically at different temperatures for a long time. A single error or inactivation of a reagent prevents the reaction from proceeding.
  • reaction must be protected against contaminations through which the nucleic acids falsifying the result can be introduced, as well as against substances that can accelerate the decay of essential reagents.
  • reaction product requires considerable equipment, for example a device that allows sensitive fluorescence measurements.
  • Samples to be analyzed must then be packaged and sent to a special laboratory where a large number of samples are processed. This delays the analysis and increases the costs. During transport, there is a risk that a sample will be lost, spoiled or become unusable due to damage.
  • EP 1769848 A2 discloses a chemical reaction cartridge comprising three or more chambers.
  • DE 102014200509 A1 discloses an analysis unit for performing a PCR, the analysis unit comprising three or more chambers.
  • an object on which the present invention is based is to provide a system for the implementation of PCR reactions which is as user-friendly as possible and which is easy to use and in particular does not require the preparation of complex reaction batches.
  • the system should function as quickly as possible and save resources (time, energy, reagent and sample consumption), demand no special conditions and be as little susceptible to faults, damage and operating errors as possible.
  • the system should allow the parallel processing of as many samples as possible.
  • the system should enable the analysis of any type of sample or any interesting analysis with the largest possible spectrum of parameters, flexibly and at short notice. It should be possible to examine a sample, for example from a patient, for several diagnostic parameters with as little effort and time as possible.
  • the system should combine the advantages mentioned with protection against external contamination, which could be entered, for example, by the personnel entrusted with the operation.
  • the device further comprises an analytical reagent which is soluble in the liquid sample, preferably a lyophilized reagent.
  • a mixing chamber is provided downstream of the receiving chamber and upstream of the denaturing chamber, preferably directly upstream, which preferably has the analytical reagent.
  • the analytical device comprises at least two sets of chambers, each comprising a denaturing chamber, an amplification chamber and an annealing chamber, whereby preferably a mixing chamber is provided downstream of the receiving chamber and upstream of the denaturing chamber, particularly preferably directly upstream, which preferably contains the analytical reagent and a part of a liquid sample can be transferred from the mixing chamber into a chamber of the respective set.
  • the diagnostic device has at least one heating zone, the denaturing chamber, preferably additionally the amplification chambers, more preferably additionally the annealing chambers of the at least two sets each being arranged on a heating zone.
  • the device has a readout chamber into which the liquid sample from the denaturing chamber, from the amplification chamber or from the annealing chamber can be transferred.
  • the readout chamber comprises at least one immobilized nucleic acid.
  • the diagnostic device is a laboratory chip.
  • an analysis unit suitable for receiving the analytical device preferably comprising the analytical device, is disclosed herein. comprising a heating device which is designed such that the required temperature can be set in the denaturing chamber, amplification chamber and annealing chamber and / or in the heating zone for the denaturing chambers, amplification chamber and annealing chambers and / or comprising means for reading a liquid sample in the reading chamber of the analytical device.
  • the device comprises a nucleic acid-containing liquid sample.
  • the present invention relates to a method comprising an analytical device with at least one set of chambers, each comprising a denaturing chamber, an amplification chamber and an annealing chamber.
  • the analytical device allows an externally obtained sample to be introduced into the denaturing chamber, optionally via another chamber.
  • the sample is a liquid sample or a solid sample, provided the latter is taken up in a liquid inside the analytical device, so that the sample is also available in liquid form for further processing.
  • a suitable device for transferring a solid sample into a liquid and a method is, for example, in US Pat EP15002317.4 described.
  • the sample is preferably a sample containing nucleic acid or a sample which is to be examined for whether it contains a nucleic acid, wherein the sample can be in a largely unprocessed form, for example a sample obtained directly from a patient or a soil sample.
  • the sample can be prepared, for example by isolating and / or enriching the nucleic acid contained therein.
  • the nucleic acid can be any nucleic acid or a derivative thereof that can be amplified using a polymerase chain reaction (PCR), preferably DNA or RNA.
  • the device comprises a system with chambers and connecting channels that can be locked, preferably closed, from the environment.
  • “lockable” means that the inner system can be opened temporarily for the introduction of a sample; in the closed state, it is not possible to introduce the sample or contaminating material.
  • the system can have an opening that can be closed with a lid.
  • a liquid sample controlled by the user or a control unit, can be moved in a compact, coherent form, in particular by transfer from a first chamber to a second chamber.
  • the connecting channels can include valves that allow the control of a liquid flow. The isolation from the environment prevents external contamination and loss of liquid through evaporation.
  • the term "chamber" as used herein means an area within the device in which the entire liquid sample can be incubated in a compact, coherent form under essentially defined, uniform conditions.
  • a chamber can be a cavity which is geometrically delimited by its shape, for example has a widened diameter and a correspondingly larger volume compared to a connecting channel.
  • a chamber can be created in such a way that the liquid sample continuously occupies a delimited area of a connecting channel, the surface of the liquid forming the boundary of the chamber relative to the gas phase surrounding it.
  • the chambers can be designed to be lockable to prevent liquid sample from escaping, for example from a chamber at high temperature.
  • laboratory chip as used herein is understood to mean a closed system which contains chambers and connecting channels in which all steps from the introduction of an unprocessed sample through its preparation and processing to analysis can be carried out ,
  • the device has at least one set comprising a denaturing chamber, an amplification chamber and an annealing chamber, for example 2, 3, 4, 5, 6, 8 or 12 sets.
  • the denaturing chamber is designed and has suitable conditions such that a double-stranded nucleic acid contained in the liquid sample can be denatured, ie the double strands dissolve with the release of corresponding single strands.
  • the temperature can be set to a suitable value or is set to it, preferably between 90 ° C. and 105 ° C., more preferably between 90 ° C. and 99 ° C.
  • suitable conditions in particular to determine a suitable temperature for denaturing a given double-stranded nucleic acid.
  • the annealing chamber is designed and has suitable conditions such that primers contained in the liquid sample specifically anneal to the single strands of the nucleic acid in the liquid sample, i.e. hybridize specifically with them.
  • the temperature can be set to a suitable value or is set to it, preferably between 40 ° C and 67 ° C.
  • suitable conditions in particular a suitable temperature, for the annealing of given primers to the single strands of a given nucleic acid with sufficient specificity.
  • the amplification chamber is designed and has suitable conditions such that the single strands of the nucleic acid are synthesized from the annealed primers into a double strand by a polymerase present. As a result, the part of a double-stranded nucleic acid delimited by the primers becomes the liquid introduced into the device Sample amplified.
  • the person skilled in the art is able to determine suitable conditions as part of routine calculations and preliminary tests.
  • the temperature can be set to a suitable value or is set to it, preferably between 60 ° C and 80 ° C, more preferably between 65 ° C and 74 ° C.
  • the amplification chamber is connected downstream of the denaturation chamber.
  • the term means that a second chamber is "downstream" of a first, as used herein, that a liquid sample introduced into the device, which passes through the device in the shortest possible way, first passes the first chamber and then the second Chamber.
  • the fact that a second chamber is “directly downstream” of a first means that the sample is transferred from the first chamber directly into the second chamber without passing through further chambers in between.
  • the device to which at least one set comprising a denaturing chamber, an amplification chamber and an annealing chamber is connected upstream, preferably directly upstream, has a mixing chamber.
  • the mixing chamber is designed such that a volume of the liquid sample suitable for the detection reaction can be transferred therefrom into the at least one set. If the device has more than one set, the mixing chamber allows the liquid sample to be portioned in a suitable manner, so that a part in each set the liquid sample is transferred with a volume sufficient for the detection reaction.
  • the device preferably further comprises an analytical reagent.
  • the analytical reagent comprises all substances which are necessary for carrying out the detection reaction, preferably the PCR reaction, provided that these are not already present in the liquid sample or their presence in the liquid sample is to be detected.
  • the liquid sample comprises the nucleic acid to be amplified or is to be examined for whether it contains the nucleic acid to be amplified
  • the analytical reagent comprises, in addition to a nucleic acid suitable as a template, all other substances which are necessary for carrying out the PCR reaction.
  • the analytical reagent is preferably in a dry, preferably lyophilized, form. It is such that it dissolves in contact with the liquid sample so that the substances required for the detection reaction, preferably the PCR reaction, are present in sufficient concentration.
  • the device optionally has a means or a suitable method step is provided to promote the dissolution of the analytical reagent in the sample. For example, the liquid sample can be contacted several times with the reagent, or the analytical reagent is mixed with the liquid sample by a micro stirrer.
  • the analytical reagent preferably comprises a polymerase and / or reverse transcriptase, dNTPs, a buffer, magnesium chloride, at least one primer, preferably at least one pair of primers, optionally labeled, a detergent, preferably from the group comprising betaine, tween or dimethyl sulfoxide, and a polypeptide such as bovine serum albumin.
  • the analytical reagent comprises several substances, these can be located separately from one another in the device.
  • the analytical reagent is preferably in a compact form which comprises all substances.
  • the analytical reagent can be located in any part of the device, provided that it is ensured that it comes into contact with the liquid sample and is mixed before the detection reaction is carried out. It is preferably located in the mixing chamber.
  • each set of chambers can contain at least one specific reagent, in particular one or more than one primer, which dissolves in the part of the liquid sample which is transferred into this set.
  • the device has more than one set of chambers, it preferably comprises at least one heating zone.
  • This is a device for continuous heating of an area on the device in which at least two chambers are located, which are preferably set to the same temperature. If the device comprises two sets, for example, then both denaturing chambers can be set to a temperature of 95 ° C. with the same heating zone.
  • the temperature of all denaturing chambers can be set by a heating zone for the denaturing chambers or is set for them, so that the same temperature prevails in the denaturing chambers.
  • the temperature of all amplification chambers can be set by a heating zone for the amplification chambers or is set for them, so that the same temperature prevails in the amplification chambers.
  • the temperatures of the respective annealing chambers can be set individually.
  • the temperature of all annealing chambers can be set by a heating zone for the annealing chambers or is set for them, so that the same temperature prevails in the annealing chambers.
  • the temperature of all denaturation chambers and all amplification chambers can be set or is set for them by a heating zone for all denaturation chambers or by a heating zone for all amplification chambers, so that the same temperature prevails in all denaturation chambers and so that in all Amplification chambers have the same temperature.
  • the temperatures of the respective annealing chambers and / or denaturing chambers can be set individually.
  • the temperature of all denaturing chambers, all amplification chambers and all annealing chambers can be set or is set for them by a heating zone for all denaturing chambers or by a heating zone for all amplification chambers or by a heating zone for all annealing chambers all denaturation chambers have the same temperature, all amplification chambers have the same temperature and all annealing chambers have the same temperature.
  • the analytical device is a disposable item which is discarded in its entirety after use.
  • the disposable article is adapted to an analysis unit to be used several times, each with a new disposable article, in such a way that the latter has at least one heating device with which the required denaturation chamber, amplification chamber and annealing chamber and / or in the heating zone for the denaturation chambers, amplification chamber and annealing chambers Temperature can be set or is set.
  • the analysis unit preferably contains a means for reading out a liquid sample, which is located in the reading chamber of the analytical device, which in turn has been introduced into the analysis unit.
  • This can be a fluorescence spectroscope if the amplified nucleic acid contains a fluorescence label or a UV / vis spectrophotometer if it contains a chromophore.
  • the analysis unit can also include further units, for example for the internal transport of disposable articles introduced into the analysis unit and for processing data which are obtained when analyzing a liquid sample in a disposable article.
  • the method according to the invention is preferably a method for the detection of a nucleic acid sequence, more preferably a diagnostic method for the detection of a nucleic acid sequence.
  • the nucleic acid sequence can be a human sequence or the sequence of a pathogen.
  • the method requires the introduction of a liquid sample into the device.
  • the sample is first introduced into the open receiving chamber. If it is a non-liquid sample, it can first be transferred to a liquid phase. There is also the possibility of having the sample already in the Mixing chamber or previously with analytical reagent. The access of the receiving chamber to the surface of the device can then be closed.
  • the liquid sample is then transferred to the denaturing chamber; optionally, it can be contacted with analytical reagent beforehand in a mixing chamber and / or portioned into chambers for transfer to several sets.
  • the denaturation is carried out in the denaturation chamber (step a).
  • the liquid sample from step a) is then transferred into the annealing chamber, and the annealing is carried out therein (step b).
  • the liquid sample from step b) is then transferred into the amplification chamber, and the amplification is carried out (step c).
  • reaction cycle comprising steps a), b), and c) is repeated at least 10 times. In total, at least 15, 20, 30, 40 or 45 reaction cycles are preferably carried out.
  • the liquid sample preferably from step c) carried out last, is transferred to the readout chamber and read out there. After reading, it can be discarded by transferring it to the waste chamber. If at least two detection reactions are carried out in at least two sets of chambers, these are preferably carried out simultaneously or at least overlapping in order to minimize the overall duration of the method. The parts of the liquid sample from these at least two detection reactions are then successively transferred to the read-out chamber and read out there and then discarded by transfer to the waste chamber, at least all parts of the liquid sample except for the last part that can remain in the read-out chamber.
  • the device contains two or more readout chambers.
  • Parts of the liquid sample from different sets can each be analyzed comprising a denaturing chamber (5), an amplification chamber (6) and an annealing chamber (7) in different readout chambers, for example with microarrays that differ in the probes and / or primers they contain , Alternatively, a sample or part of a sample can be analyzed in different readout chambers.
  • Fig. 1 shows a preferred embodiment of the analytical device (1).
  • This has a receiving chamber (2) which can be closed with a cover (13) and into which a sample can be introduced.
  • the sample can be transferred in liquid form into a mixing chamber (3) from the receiving chamber.
  • the analytical reagent When it arrives in the mixing chamber, it comes into contact with an analytical reagent (4), which dissolves in the liquid sample.
  • the analytical reagent preferably has all the reagents required for a PCR reaction, except for the nucleic acid to be amplified, so that - provided that the sample contains the nucleic acid - all the reagents required for the reaction are present after dissolution.
  • the liquid sample can then be divided, and in each case a part enters a denaturing chamber (5), which is part of a set comprising a denaturing chamber (5), an amplification chamber (6) and an annealing chamber (7).
  • All denaturing chambers are arranged in a heating zone for the denaturing chambers (8), analogously all amplification chambers and all annealing chambers in the heating zone for the amplification chambers (9) and heating zone for the annealing chambers (10).
  • the different sets can contain specific reagents for different detection reactions.
  • a first set can contain a first pair of primers and a second set can contain another second pair of primers.
  • Non-specific reagents required for all reactions can be added to the sample beforehand, for example in the mixing chamber (3).
  • a PCR reaction can be carried out in such a way that the liquid sample is first introduced into the denaturation chamber under suitable conditions, is present there and double strands of nucleic acid dissolve and single strands are released.
  • the sample is then transferred to the annealing chamber, comprising the individual strands, and is present there under conditions which allow the primers to be annealed to the single strands.
  • the liquid sample with single strands and primers attached to them is then transferred into the amplification chamber and is present there under conditions which, starting from the primers, permit the amplification, ie the synthesis of new single strands which are complementary to the existing single strands.
  • the sample can then be transferred to the denaturation chamber again for the start of a new amplification cycle comprising steps a), b) and c).
  • the part of the sample which was contained in one of the sets comprising a denaturing chamber (5), an amplification chamber (6) and an annealing chamber (7) can be transferred to the readout chamber (11).
  • a suitable detection reaction takes place there, for example a fluorescence measurement, with which nucleic acid strands can be detected which contain at least one fluorescence-labeled primer and in this way differ from nucleic acids which were already contained in the sample originally introduced into the device.
  • the part of the sample contained in the readout chamber can be discarded by transferring it to the waste chamber (12).
  • the readout chamber is thus empty and is ready for a part of the sample from another set to be transferred to it and to be examined with a detection reaction. In this way, several parts of the liquid sample can be examined in the same readout chamber.
  • Fig. 2 shows a further preferred embodiment of the analytical device (1). It differs from that in Fig. 1 demonstrated device in particular in that it contains an annular channel in which the denaturing chamber, amplification chamber and annealing chamber are not provided in the form of geometrically delimited chambers, but in that the liquid sample (14) in the form of a compact, coherent drop along the ring is transferred into the heating zone of the denaturing chamber, amplification chamber or annealing chamber (8, 9 or 10). Together with the walls of the annular channel, the boundary surfaces of this drop form the boundaries of the chamber, for example the denaturing chamber (5), as shown here.
  • a plurality of annular channels can be arranged one above the other perpendicular to the paper plane; the heating zones then preferably also run perpendicular to the plane of the paper, so that in each case one of the heating zones can heat or heats a plurality of ring-shaped channels at the corresponding point to the temperature desired for the denaturing chamber, amplification chamber or annealing chamber.
  • the liquid sample never leaves the annular channel during the PCR reaction. It is transmitted to the point of the ring-shaped channel that is currently required, at that point for the step to be carried out a), b) or c) there is a suitable temperature. In this way, there is no need to go through a bottleneck, ie a particularly narrow point, and the speed of the sample transfer between the different chambers can be maximized.

Description

Die vorliegende Erfindung betrifft ein Verfahren umfassend eine analytische Vorrichtung umfassend eine Denaturierungskammer, eine Amplifikationskammer und eine Annealingkammer,
wobei die Temperatur der Denaturierungskammer auf eine Temperatur zwischen 90°C und 105 °C eingestellt werden kann, bevorzugt eingestellt ist,
wobei die Temperatur der Amplifikationskammer auf eine Temperatur zwischen 60°C und 80 °C eingestellt werden kann, bevorzugt eingestellt ist,
wobei die Temperatur der Annealingkammer auf eine Temperatur zwischen 40°C und 67 °C eingestellt werden kann, bevorzugt eingestellt ist,
und wobei die Vorrichtung so ausgestaltet ist, dass eine flüssige Probe in die Denaturierungskammer eingebracht werden kann und zwischen der Denaturierungskammer, der Amplifikationskammer, der Annealingkammer und optional weiteren Kammern hin und her übertragen werden kann,
dadurch gekennzeichnet, dass die analytische Vorrichtung weiter eine Aufnahmekammer mit einem Zugang zur Oberfläche der diagnostischen Vorrichtung, über den die Probe in die Aufnahmekammer eingebracht werden und anschließend zu weiteren Kammern übertragen werden kann, umfasst und
wobei die Amplifikationskammer der Denaturierungskammer nachgeschaltet ist und die Annealingkammer der Amplifikationskammer,
wobei das Verfahren die Schritte umfasst:

  1. a) Einbringen der flüssigen Probe in die Denaturierungskammer der analytischen Vorrichtung, anschließend Denaturieren der Probe,
  2. b) Übertragen der flüssigen Probe aus Schritt a) in die Annealingkammer, anschließend Annealing,
  3. c) Übertragen der flüssigen Probe aus Schritt b) in die Amplifikationskammer, anschließend Amplifizieren,
  4. d) optional Wiederholen der Schritte a), b) und c),
  5. e) optional: bertragen der flüssigen Probe aus Schritt c) oder d) in eine Auslesekammer, anschließend Auslesen der flüssigen Probe.
The present invention relates to a method comprising an analytical device comprising a denaturing chamber, an amplification chamber and an annealing chamber,
wherein the temperature of the denaturing chamber can be set to a temperature between 90 ° C and 105 ° C, is preferably set,
wherein the temperature of the amplification chamber can be set to a temperature between 60 ° C and 80 ° C, is preferably set,
wherein the temperature of the annealing chamber can be set to a temperature between 40 ° C and 67 ° C, is preferably set,
and the device is designed such that a liquid sample can be introduced into the denaturing chamber and can be transferred back and forth between the denaturing chamber, the amplification chamber, the annealing chamber and optionally further chambers,
characterized in that the analytical device further comprises a receiving chamber with access to the surface of the diagnostic device, via which the sample can be introduced into the receiving chamber and then transferred to further chambers, and
the amplification chamber being connected downstream of the denaturing chamber and the annealing chamber of the amplification chamber,
the method comprising the steps of:
  1. a) introducing the liquid sample into the denaturing chamber of the analytical device, then denaturing the sample,
  2. b) transferring the liquid sample from step a) into the annealing chamber, then annealing,
  3. c) transferring the liquid sample from step b) into the amplification chamber, then amplifying,
  4. d) optionally repeating steps a), b) and c),
  5. e) optional: transfer the liquid sample from step c) or d) into a readout chamber, then read out the liquid sample.

Bei vielen diagnostischen, labormedizinischen oder sonstigen analytischen Untersuchungen bedient man sich chemischer Reaktionen und/oder physikalischer Nachweisverfahren in einer flüssigen Phase. Besondere analytische und diagnostische Bedeutung kommt der Polymerase-Kettenreaktion (PCR) zu, mit der Nukleinsäuresequenzen spezifisch vervielfältigt und nachgewiesen werden können, sofern sie in einer bestimmten Probe vorhanden sind. Damit können beispielsweise Krankheitserreger (Viren, Bakterien, Pilze, Parasiten) wie gegen Methicillin resistente Staphylococcus aureus (MRSA) oder bestimmte menschliche Nukleinsäuresequenzen, die diagnostisch von Bedeutung sind, beispielsweise, weil sie mit bestimmten Krankheiten zusammenhängende Mutationen aufweisen, in Patientenproben nachgewiesen werden.Many diagnostic, laboratory medical or other analytical examinations use chemical reactions and / or physical detection methods in a liquid phase. The polymerase chain reaction (PCR), with which nucleic acid sequences can be specifically reproduced and detected if they are present in a specific sample, is of particular analytical and diagnostic importance. For example, pathogens (viruses, bacteria, fungi, parasites) such as methicillin-resistant Staphylococcus aureus (MRSA) or certain human nucleic acid sequences that are of diagnostic importance, for example because they have mutations associated with certain diseases, can be detected in patient samples.

Auch außerhalb medizinischer Anwendungen ist die PCR von Bedeutung, beispielsweise bei der Untersuchung von Boden- oder Lebensmittelproben auf bestimmte Mikroorganismen, in der Forensik oder bei der synthetischen Herstellung von komplexen Nukleinsäuren.PCR is also important outside of medical applications, for example when examining soil or food samples for certain microorganisms, in forensics or in the synthetic production of complex nucleic acids.

Die PCR erfordert in der herkömmlichen Form einen erheblichen Aufwand mit Hinblick auf Reagenzien, Geräte und Arbeitsanfall, insbesondere bei diagnostischen Anwendungen. Für jede einzelne Probe muss ein Reaktionsansatz zusammengestellt werden, der eine ganze Reihe spezifischer, teilweise thermoinstabiler Reagenzien enthält. Der fertige Reaktionsansatz muss über längere Zeit zyklisch bei verschiedenen Temperaturen inkubiert werden. Schon ein einzelner Fehler oder eine Inaktivierung eines Reagenz verhindern den Ablauf der Reaktion.In its conventional form, PCR requires considerable effort with regard to reagents, equipment and workload, especially in diagnostic applications. For each individual sample, a reaction batch has to be put together that contains a whole series of specific, partly thermo-unstable reagents. The finished reaction batch has to be incubated cyclically at different temperatures for a long time. A single error or inactivation of a reagent prevents the reaction from proceeding.

Kann die Zuverlässigkeit der Reaktion nicht garantiert werden, so bleibt das Ergebnis ohne Aussagekraft. Die Reaktion muss vor Kontaminationen geschützt werden, über die das Ergebnis verfälschende Nukleinsäuren eingebracht werden können, ebenso vor Stoffen, die den Zerfall von essentiellen Reagenzien beschleunigen können.If the reliability of the reaction cannot be guaranteed, the result remains meaningless. The reaction must be protected against contaminations through which the nucleic acids falsifying the result can be introduced, as well as against substances that can accelerate the decay of essential reagents.

Die Analyse des Reaktionsproduktes erfordert eine erhebliche apparative Ausstattung, beispielsweise ein Gerät, das empfindliche Fluoreszenzmessungen gestattet.The analysis of the reaction product requires considerable equipment, for example a device that allows sensitive fluorescence measurements.

Für viele Anwender mit begrenzter Expertise und Infrastruktur ist die Verwendung der PCR nicht mit der erforderlichen Zuverlässigkeit durchführbar. Insbesondere bei geringem Probenaufkommen lohnt der Aufwand der Etablierung eines dafür geeigneten Labors nicht.For many users with limited expertise and infrastructure, the use of PCR cannot be carried out with the required reliability. The effort of establishing a suitable laboratory is not worth it, especially if the sample volume is low.

Zu analysierende Proben müssen dann verpackt und zu einem Speziallabor versandt werden, bei dem eine große Zahl von Proben abgearbeitet wird. Dies verzögert die Analyse und erhöht die Kosten. Beim Transport besteht die Gefahr, dass eine Probe verloren geht, verdirbt oder durch Beschädigung unbrauchbar wird.Samples to be analyzed must then be packaged and sent to a special laboratory where a large number of samples are processed. This delays the analysis and increases the costs. During transport, there is a risk that a sample will be lost, spoiled or become unusable due to damage.

EP 1769848 A2 offenbart eine Katusche für chemische Reaktionen, die drei oder mehr Kammern umfasst. EP 1769848 A2 discloses a chemical reaction cartridge comprising three or more chambers.

DE 102014200509 A1 offenbart eine Analyseeinheit zum Druchführen einer PCR, wobei die Analyseeinheit drei oder mehr Kammern umfasst. DE 102014200509 A1 discloses an analysis unit for performing a PCR, the analysis unit comprising three or more chambers.

Vor diesem Hintergrund besteht eine der vorliegenden Erfindung zu Grunde liegende Aufgabe darin, ein möglichst anwenderfreundliches System für die Durchführung von PCR-Reaktionen zu schaffen, das einfach zu bedienen ist, insbesondere nicht die Zubereitung komplexer Reaktionsansätze verlangt.Against this background, an object on which the present invention is based is to provide a system for the implementation of PCR reactions which is as user-friendly as possible and which is easy to use and in particular does not require the preparation of complex reaction batches.

Das System soll möglichst schnell und Ressourcen (Zeit, Energie, Reagenzien- und Probenverbrauch) sparend funktionieren, keine besonderen Bedingungen verlangen und gegenüber Störungen, Beschädigungen und Bedienungsfehlern möglichst wenig anfällig sein.The system should function as quickly as possible and save resources (time, energy, reagent and sample consumption), demand no special conditions and be as little susceptible to faults, damage and operating errors as possible.

Gleichzeitig soll das System die parallele Abarbeitung möglichst vieler Proben gestatten. Je nach Bedarf soll das System die Untersuchung möglichst jeder Art von Probe bzw. jede interessierende Untersuchung, mit einem möglichst großen Spektrum von Parametern, flexibel und kurzfristig ermöglichen. Es soll möglich sein, eine Probe, beispielsweise von einem Patienten, mit möglichst geringem Aufwand und Zeitbedarf auf mehrere diagnostische Parameter zu untersuchen.At the same time, the system should allow the parallel processing of as many samples as possible. Depending on the requirements, the system should enable the analysis of any type of sample or any interesting analysis with the largest possible spectrum of parameters, flexibly and at short notice. It should be possible to examine a sample, for example from a patient, for several diagnostic parameters with as little effort and time as possible.

Nicht zuletzt soll das System die genannten Vorteile mit einer Sicherung gegen Kontaminationen von außen vereinen, die beispielsweise durch das mit der Bedienung betraute Personal eingetragen werden könnten.Last but not least, the system should combine the advantages mentioned with protection against external contamination, which could be entered, for example, by the personnel entrusted with the operation.

Diese und weitere Aufgaben werden durch den Gegenstand der vorliegenden Anmeldung und insbesondere auch durch den Gegenstand der beigefügten unabhängigen Ansprüche gelöst, wobei sich Ausführungsformen aus den Unteransprüchen ergeben.These and other tasks are covered by the subject matter of the present application and in particular by the subject matter of the attached one independent claims solved, embodiments result from the subclaims.

Die der Erfindung zu Grunde liegende Aufgabe wird in einem ersten Aspekt gelöst durch ein Verfahren umfassend eine analytische Vorrichtung umfassend eine Denaturierungskammer, eine Amplifikationskammer und eine Annealingkammer,
wobei die Temperatur der Denaturierungskammer auf eine Temperatur zwischen 90 °C und 105 °C eingestellt werden kann, bevorzugt eingestellt ist,
wobei die Temperatur der Amplifikationskammer auf eine Temperatur zwischen 60 °C und 80 °C eingestellt werden kann, bevorzugt eingestellt ist,
wobei die Temperatur der Annealingkammer auf eine Temperatur zwischen 40°C und 67 °C eingestellt werden kann, bevorzugt eingestellt ist,
und wobei die Vorrichtung so ausgestaltet ist, dass eine Probe in die Denaturierungskammer eingebracht werden kann und zwischen der Denaturierungskammer, der Amplifikationskammer, der Annealingkammer und optional weiteren Kammern hin und her übertragen werden kann,
dadurch gekennzeichnet, dass die analytische Vorrichtung weiter eine Aufnahmekammer mit einem Zugang zur Oberfläche der diagnostischen Vorrichtung, über den die Probe in die Aufnahmekammer eingebracht werden und anschließend zu weiteren Kammern übertragen werden kann, umfasst und
wobei die Amplifikationskammer der Denaturierungskammer, nachgeschaltet ist und die Annealingkammer der Amplifikationskammer,
wobei das Verfahren die Schritte umfasst:

  1. a) Einbringen der flüssigen Probe in die Denaturierungskammer der analytischen Vorrichtung, anschließend Denaturieren der Probe,
  2. b) Übertragen der flüssigen Probe aus Schritt a) in die Annealingkammer, anschließend Annealing,
  3. c) Übertragen der flüssigen Probe aus Schritt b) in die Amplifikationskammer, anschließend Amplifizieren,
  4. d) optional Wiederholen der Schritte a), b) und c),
  5. e) optional: Übertragen der flüssigen Probe aus Schritt c) oder d) in eine Auslesekammer, anschließend Auslesen der flüssigen Probe.
The object on which the invention is based is achieved in a first aspect by a method comprising an analytical device comprising a denaturing chamber, an amplification chamber and an annealing chamber,
wherein the temperature of the denaturing chamber can be set to a temperature between 90 ° C and 105 ° C, is preferably set,
wherein the temperature of the amplification chamber can be set to a temperature between 60 ° C and 80 ° C, is preferably set,
wherein the temperature of the annealing chamber can be set to a temperature between 40 ° C and 67 ° C, is preferably set,
and wherein the device is designed such that a sample can be introduced into the denaturing chamber and can be transferred back and forth between the denaturing chamber, the amplification chamber, the annealing chamber and optionally further chambers,
characterized in that the analytical device further comprises a receiving chamber with access to the surface of the diagnostic device, via which the sample can be introduced into the receiving chamber and then transferred to further chambers, and
the amplification chamber being connected downstream of the denaturing chamber and the annealing chamber of the amplification chamber,
the method comprising the steps of:
  1. a) introducing the liquid sample into the denaturing chamber of the analytical device, then denaturing the sample,
  2. b) transferring the liquid sample from step a) into the annealing chamber, then annealing,
  3. c) transferring the liquid sample from step b) into the amplification chamber, then amplifying,
  4. d) optionally repeating steps a), b) and c),
  5. e) optional: transfer the liquid sample from step c) or d) into a readout chamber, then read out the liquid sample.

In einer bevorzugten Ausführungsform umfasst die Vorrichtung weiter ein analytisches Reagenz, das in der flüssigen Probe löslich ist, bevorzugt ein lyophilisiertes Reagenz.In a preferred embodiment, the device further comprises an analytical reagent which is soluble in the liquid sample, preferably a lyophilized reagent.

In einer weiteren bevorzugten Ausführungsform ist der Aufnahmekammer nachgeschaltet und der Denaturierungskammer vorgeschaltet, bevorzugt direkt vorgeschaltet, eine Mischkammer vorgesehen, die bevorzugterweise das analytische Reagenz aufweist.In a further preferred embodiment, a mixing chamber is provided downstream of the receiving chamber and upstream of the denaturing chamber, preferably directly upstream, which preferably has the analytical reagent.

In einer weiteren bevorzugten Ausführungsform umfasst die analytische Vorrichtung wenigstens zwei Sets von Kammern, jeweils umfassend eine Denaturierungskammer, eine Amplifikationskammer und eine Annealingkammer,
wobei bevorzugt der Aufnahmekammer nachgeschaltet und der Denaturierungskammer vorgeschaltet, besonders bevorzugt direkt vorgeschaltet, eine Mischkammer vorgesehen ist, die bevorzugterweise das analytische Reagenz aufweist, und aus der Mischkammer jeweils ein Teil einer flüssigen Probe in eine Kammer des jeweiligen Sets übertragen werden kann.In a further preferred embodiment, the analytical device comprises at least two sets of chambers, each comprising a denaturing chamber, an amplification chamber and an annealing chamber,
whereby preferably a mixing chamber is provided downstream of the receiving chamber and upstream of the denaturing chamber, particularly preferably directly upstream, which preferably contains the analytical reagent and a part of a liquid sample can be transferred from the mixing chamber into a chamber of the respective set.

In einer weiteren bevorzugten Ausführungsform weist die diagnostische Vorrichtung wenigstens eine Heizzone auf, wobei die Denaturierungskammer, bevorzugt zusätzlich die Amplifikationskammern, noch bevorzugter zusätzlich die Annealingkammern der wenigstens zwei Sets jeweils auf einer Heizzone angeordnet sind.In a further preferred embodiment, the diagnostic device has at least one heating zone, the denaturing chamber, preferably additionally the amplification chambers, more preferably additionally the annealing chambers of the at least two sets each being arranged on a heating zone.

In einer bevorzugten Ausführungsform weist die Vorrichtung eine Auslesekammer auf, in die die flüssige Probe aus der Denaturierungskammer, aus der Amplifikationskammer oder der Annealingkammer übertragen werden kann.In a preferred embodiment, the device has a readout chamber into which the liquid sample from the denaturing chamber, from the amplification chamber or from the annealing chamber can be transferred.

In einer weiteren bevorzugten Ausführungsform umfasst die Auslesekammer wenigstens eine immobilisierte Nukleinsäure.In a further preferred embodiment, the readout chamber comprises at least one immobilized nucleic acid.

In einer weiteren bevorzugten Ausführungsform handelt es sich bei der diagnostischen Vorrichtung um einen Laborchip.In a further preferred embodiment, the diagnostic device is a laboratory chip.

Ferner wird hierin offenbart eine Analyseeinheit geeignet zur Aufnahme der analytischen Vorrichtung, bevorzugt umfassend die analytische Vorrichtung,
umfassend eine Heizvorrichtung, die so beschaffen ist, dass in der Denaturierungskammer, Amplifikationskammer und Annealingkammer und/oder in der Heizzone für die Denaturierungskammern, Amplifikationskammer bzw. Annealingkammern die erforderliche Temperatur eingestellt werden kann bzw. eingestellt ist
und/oder umfassend ein Mittel zum Auslesen einer flüssigen Probe in der Auslesekammer der analytischen Vorrichtung.Furthermore, an analysis unit suitable for receiving the analytical device, preferably comprising the analytical device, is disclosed herein.
comprising a heating device which is designed such that the required temperature can be set in the denaturing chamber, amplification chamber and annealing chamber and / or in the heating zone for the denaturing chambers, amplification chamber and annealing chambers
and / or comprising means for reading a liquid sample in the reading chamber of the analytical device.

Hierin wird ferner offenbart eine Verwendung der oben beschriebenen analytischen Vorrichtung zur Untersuchung einer flüssigen Probe mittels Polymerase-Kettenreaktion.This also discloses a use of the analytical device described above for examining a liquid sample by means of the polymerase chain reaction.

In einer weiteren bevorzugten Ausführungsform umfasst die Vorrichtung eine nukleinsäurehaltige flüssige Probe.In a further preferred embodiment, the device comprises a nucleic acid-containing liquid sample.

Die vorliegende Erfindung betrifft ein Verfahren umfassend eine analytische Vorrichtung mit wenigstens einem Set an Kammern, jeweils umfassend eine Denaturierungskammer, eine Amplifikationskammer und eine Annealingkammer. Die analytische Vorrichtung gestattet es, eine extern gewonnene Probe in die Denaturierungskammer einzubringen, optional über eine andere Kammer.The present invention relates to a method comprising an analytical device with at least one set of chambers, each comprising a denaturing chamber, an amplification chamber and an annealing chamber. The analytical device allows an externally obtained sample to be introduced into the denaturing chamber, optionally via another chamber.

Bei der Probe handelt es sich um eine flüssige Probe oder aber um eine feste Probe, sofern letztere im Inneren der analytischen Vorrichtung in eine Flüssigkeit aufgenommen wird, so dass die Probe für die weitere Prozessierung ebenfalls in flüssiger Form zur Verfügung steht. Eine geeignete Vorrichtung zum Überführen einer festen Probe in eine Flüssigkeit und ein Verfahren ist beispielsweise in der EP15002317.4 beschrieben.The sample is a liquid sample or a solid sample, provided the latter is taken up in a liquid inside the analytical device, so that the sample is also available in liquid form for further processing. A suitable device for transferring a solid sample into a liquid and a method is, for example, in US Pat EP15002317.4 described.

Bei der Probe handelt es sich bevorzugt um eine nukleinsäurehaltige Probe oder eine Probe, die darauf zu untersuchen ist, ob sie eine Nukleinsäure enthält, wobei die Probe in einer weitgehend unprozessierten Form vorliegen kann, beispielsweise einer direkt von einem Patienten gewonnen Probe oder eine Bodenprobe. Alternativ kann die Probe aufbereitet sein, beispielsweise durch Isolieren und/oder Anreichern der darin enthaltenen Nukleinsäure. Bei der Nukleinsäure kann es sich um jegliche Nukleinsäure oder um ein Derivat davon handeln, die bzw. das mit Hilfe einer Polymerase-Kettenreaktion (PCR) amplifiziert werden kann, bevorzugt um DNA oder RNA.The sample is preferably a sample containing nucleic acid or a sample which is to be examined for whether it contains a nucleic acid, wherein the sample can be in a largely unprocessed form, for example a sample obtained directly from a patient or a soil sample. Alternatively, the sample can be prepared, for example by isolating and / or enriching the nucleic acid contained therein. The nucleic acid can be any nucleic acid or a derivative thereof that can be amplified using a polymerase chain reaction (PCR), preferably DNA or RNA.

Die Vorrichtung umfasst ein gegenüber der Umgebung abschließbares, bevorzugt abgeschlossenes System mit Kammern und Verbindungskanälen. In einer bevorzugten Ausführungsform bedeutet "abschließbar", dass das innere System zeitweilig zum Einbringen einer Probe geöffnet werden kann, im abgeschlossenen Zustand ein Einbringen von Probe oder kontaminierendem Material nicht möglich ist. Beispielsweise kann das System eine Öffnung aufweisen, die mit einem Deckel verschlossen werden kann.The device comprises a system with chambers and connecting channels that can be locked, preferably closed, from the environment. In a preferred embodiment, “lockable” means that the inner system can be opened temporarily for the introduction of a sample; in the closed state, it is not possible to introduce the sample or contaminating material. For example, the system can have an opening that can be closed with a lid.

In diesem System kann eine flüssige Probe, vom Anwender bzw. einer Steuerungseinheit gesteuert, in kompakter, zusammenhängender Form bewegt werden, insbesondere durch Übertragung von einer ersten Kammer zu einer zweiten Kammer. Dem Fachmann sind zahlreiche Wege bekannt, wie dies bewerkstelligt werden kann, beispielsweise mit Hilfe von Druckunterschieden, die über eine Pumpe oder Spritze erzeugt werden. Die Verbindungskanäle können Ventile umfassen, die die Lenkung eines Flüssigkeitsstromes gestatten. Der Abschluss gegenüber der Umgebung verhindert Kontaminationen von außen und den Verlust von Flüssigkeit durch Verdampfen.In this system, a liquid sample, controlled by the user or a control unit, can be moved in a compact, coherent form, in particular by transfer from a first chamber to a second chamber. Numerous ways are known to the person skilled in the art of how this can be accomplished, for example with the aid of pressure differences which are generated by means of a pump or syringe. The connecting channels can include valves that allow the control of a liquid flow. The isolation from the environment prevents external contamination and loss of liquid through evaporation.

In einer bevorzugten Ausführungsform wird unter dem Begriff "Kammer", wie hierin verwendet, ein Bereich innerhalb der Vorrichtung verstanden, in dem die gesamte flüssige Probe in kompakter, zusammenhängender Form unter im Wesentlichen definierten, gleichförmigen Bedingungen inkubiert werden kann. Eine Kammer kann ein Hohlraum sein, der geometrisch durch seine Form abgegrenzt ist, beispielsweise gegenüber einem Verbindungskanal einen verbreiterten Durchmesser und ein entsprechend größeres Volumen aufweist. Alternativ kann eine Kammer dadurch geschaffen werden, dass die flüssige Probe zusammenhängend einen abgegrenzten Bereich eines Verbindungskanals einnimmt, wobei die Oberfläche der Flüssigkeit gegenüber der sie umgebenden Gasphase die Grenze der Kammer bildet. Die Kammern können abschließbar ausgeführt sein, um ein Entweichen von flüssiger Probe zu verhindern, beispielsweise aus einer Kammer mit hoher Temperatur.In a preferred embodiment, the term "chamber" as used herein means an area within the device in which the entire liquid sample can be incubated in a compact, coherent form under essentially defined, uniform conditions. A chamber can be a cavity which is geometrically delimited by its shape, for example has a widened diameter and a correspondingly larger volume compared to a connecting channel. Alternatively, a chamber can be created in such a way that the liquid sample continuously occupies a delimited area of a connecting channel, the surface of the liquid forming the boundary of the chamber relative to the gas phase surrounding it. The chambers can be designed to be lockable to prevent liquid sample from escaping, for example from a chamber at high temperature.

In einer bevorzugten Ausführungsform wird unter dem Begriff "Laborchip", wie hierin verwendet, ein abgeschlossenes System verstanden, das Kammern und Verbindungskanäle enthält, in denen alle Schritte von der Einbringung einer unprozessierten Probe über deren Vorbereitung und Prozessierung bis hin zur einer Analyse ausgeführt werden können.In a preferred embodiment, the term "laboratory chip" as used herein is understood to mean a closed system which contains chambers and connecting channels in which all steps from the introduction of an unprocessed sample through its preparation and processing to analysis can be carried out ,

Die Vorrichtung weist wenigstens ein Set umfassend eine Denaturierungskammer, eine Amplifikationskammer und eine Annealingkammer auf, beispielsweise 2, 3, 4, 5, 6, 8 oder 12 Sets.The device has at least one set comprising a denaturing chamber, an amplification chamber and an annealing chamber, for example 2, 3, 4, 5, 6, 8 or 12 sets.

Die Denaturierungskammer ist so ausgestaltet und weist geeignete Bedingungen auf, dass eine in der flüssigen Probe enthaltene doppelsträngige Nukleinsäure denaturiert werden kann, d. h. die Doppelstränge lösen sich unter Freisetzung entsprechender Einzelstränge. Insbesondere kann die Temperatur auf einen geeigneten Wert eingestellt werden bzw. ist darauf eingestellt, bevorzugt zwischen 90 °C und 105 °C, noch bevorzugter zwischen 90 °C und 99 °C. Dem Fachmann ist es im Rahmen routinemäßiger Berechnungen und Vorversuche möglich, geeignete Bedingungen, insbesondere eine geeignete Temperatur, zur Denaturierung einer gegebenen doppelsträngigen Nukleinsäure zu ermitteln.The denaturing chamber is designed and has suitable conditions such that a double-stranded nucleic acid contained in the liquid sample can be denatured, ie the double strands dissolve with the release of corresponding single strands. In particular, the temperature can be set to a suitable value or is set to it, preferably between 90 ° C. and 105 ° C., more preferably between 90 ° C. and 99 ° C. In the context of routine calculations and preliminary tests, the person skilled in the art is able to determine suitable conditions, in particular to determine a suitable temperature for denaturing a given double-stranded nucleic acid.

Die Annealingkammer ist so ausgestaltet und weist geeignete Bedingungen auf, dass in der flüssigen Probe enthaltene Primer spezifisch an die Einzelstränge der Nukleinsäure in der flüssigen Probe annealen, d.h. spezifisch mit ihnen hybridisieren. Insbesondere kann die Temperatur auf einen geeigneten Wert eingestellt werden bzw. ist darauf eingestellt, bevorzugt zwischen 40 °C und 67 °C. Dem Fachmann ist es im Rahmen routinemäßiger Berechnungen und Vorversuche möglich, geeignete Bedingungen, insbesondere eine geeignete Temperatur, für das Annealen gegebener Primer an die Einzelstränge einer gegebenen Nukleinsäure mit ausreichender Spezifität zu ermitteln.The annealing chamber is designed and has suitable conditions such that primers contained in the liquid sample specifically anneal to the single strands of the nucleic acid in the liquid sample, i.e. hybridize specifically with them. In particular, the temperature can be set to a suitable value or is set to it, preferably between 40 ° C and 67 ° C. Within the framework of routine calculations and preliminary tests, the person skilled in the art is able to determine suitable conditions, in particular a suitable temperature, for the annealing of given primers to the single strands of a given nucleic acid with sufficient specificity.

Die Amplifikationskammer ist so ausgestaltet und weist geeignete Bedingungen auf, dass durch eine anwesende Polymerase die Einzelstränge der Nukleinsäure ausgehend von den annealten Primer zu einem Doppelstrang aufsysnthetisiert werden, Im Ergebnis wird der durch die Primer eingegrenzte Teil einer doppelsträngigen Nukleinsäure aus der in die Vorrichtung eingebrachten flüssigen Probe amplifiziert. Dem Fachmann ist es im Rahmen routinemäßiger Berechnungen und Vorversuche möglich, geeignete Bedingungen zu ermitteln. Insbesondere kann die Temperatur auf einen geeigneten Wert eingestellt werden bzw. ist darauf eingestellt, bevorzugt zwischen 60 °C und 80 °C, noch bevorzugter zwischen 65 °C und 74 °C.The amplification chamber is designed and has suitable conditions such that the single strands of the nucleic acid are synthesized from the annealed primers into a double strand by a polymerase present. As a result, the part of a double-stranded nucleic acid delimited by the primers becomes the liquid introduced into the device Sample amplified. The person skilled in the art is able to determine suitable conditions as part of routine calculations and preliminary tests. In particular, the temperature can be set to a suitable value or is set to it, preferably between 60 ° C and 80 ° C, more preferably between 65 ° C and 74 ° C.

Die Amplifikationskammer ist der Denaturierungskammer nachgeschaltet. In einer bevorzugten Ausführungsform bedeutet der Begriff, dass eine zweite Kammer einer ersten "nachgeschaltet" ist, wie hierin verwendet, dass eine in die Vorrichtung eingebrachte flüssige Probe, die die Vorrichtung auf kürzest möglichem Wege durchläuft, zuerst die erste Kammer passiert und anschließend die zweite Kammer. Dass eine zweite Kammer einer ersten "direkt nachgeschaltet" ist, bedeutet bevorzugt, dass die Probe dabei aus der ersten Kammer direkt in die zweite Kammer übertragen wird, ohne dazwischen weitere Kammern zu passieren.The amplification chamber is connected downstream of the denaturation chamber. In a preferred embodiment, the term means that a second chamber is "downstream" of a first, as used herein, that a liquid sample introduced into the device, which passes through the device in the shortest possible way, first passes the first chamber and then the second Chamber. The fact that a second chamber is “directly downstream” of a first means that the sample is transferred from the first chamber directly into the second chamber without passing through further chambers in between.

Optional weist die Vorrichtung, dem wenigstens ein Set umfassend eine Denaturierungskammer, eine Amplifikationskammer und eine Annealingkammer vorgeschaltet, bevorzugt direkt vorgeschaltet, eine Mischkammer auf. Die Mischkammer ist so ausgestaltet, dass ein für die Nachweisreaktion geeignetes Volumen der flüssigen Probe daraus in das wenigstens eine Set übertragen werden kann. Sofern die Vorrichtung mehr als ein Set aufweist, gestattet die Mischkammer, dass die flüssige Probe in geeigneter Weise portioniert wird, dass in jedes Set ein Teil der flüssigen Probe mit einem jeweils für die Nachweisreaktion ausreichenden Volumen übertragen wird.Optionally, the device, to which at least one set comprising a denaturing chamber, an amplification chamber and an annealing chamber is connected upstream, preferably directly upstream, has a mixing chamber. The mixing chamber is designed such that a volume of the liquid sample suitable for the detection reaction can be transferred therefrom into the at least one set. If the device has more than one set, the mixing chamber allows the liquid sample to be portioned in a suitable manner, so that a part in each set the liquid sample is transferred with a volume sufficient for the detection reaction.

Bevorzugt umfasst die Vorrichtung weiterhin ein analytisches Reagenz. Das analytische Reagenz umfasst alle Stoffe, die für die Durchführung der Nachweisreaktion, bevorzugt der PCR-Reaktion, erforderlich sind, sofern diese nicht ohnehin in der flüssigen Probe vorhanden sind oder ihre Anwesenheit in der flüssigen Probe nachgewiesen werden soll. Beispielsweise umfasst die flüssige Probe die zu amplifizierende Nukleinsäure oder soll darauf untersucht werden, ob sie die zu amplifizierende Nukleinsäure enthält, und das analytische Reagenz umfasst außer einer als Template geeigneten Nukleinsäure alle anderen Stoffe, die für die Durchführung der PCR-Reaktion erforderlich sind.The device preferably further comprises an analytical reagent. The analytical reagent comprises all substances which are necessary for carrying out the detection reaction, preferably the PCR reaction, provided that these are not already present in the liquid sample or their presence in the liquid sample is to be detected. For example, the liquid sample comprises the nucleic acid to be amplified or is to be examined for whether it contains the nucleic acid to be amplified, and the analytical reagent comprises, in addition to a nucleic acid suitable as a template, all other substances which are necessary for carrying out the PCR reaction.

Das analytische Reagenz liegt bevorzugt in trockener, bevorzugt lyophilisierter Form vor. Es ist derart beschaffen, dass es sich bei Kontakt mit der flüssigen Probe darin auflöst, so dass die für die Nachweisreaktion, bevorzugt die PCR-Reaktion, notwendigen Stoffe in ausreichender Konzentration vorhanden sind. Optional weist die Vorrichtung ein Mittel auf oder es wird ein geeigneter Verfahrensschritt vorgesehen, um das Lösen des analytischen Reagenzes in der Probe zu fördern. Beispielsweise kann die flüssige Probe mehrfach mit dem Reagenz kontaktiert werden, oder das analytische Reagenz wird durch einen Mikrorührer mit der flüssigen Probe vermischt. Das analytische Reagenz umfasst bevorzugt eine Polymerase und/oder reverse Transkriptase, dNTPs, einen Puffer, Magnesiumchlorid, wenigstens einen Primer, bevorzugt wenigstens ein Primerpaar, optional markiert, ein Detergens, bevorzugt aus der Gruppe umfassend Betain, Tween oder Dimethylsulfoxid, sowie ein Polypeptid wie Rinderserumalbumin.The analytical reagent is preferably in a dry, preferably lyophilized, form. It is such that it dissolves in contact with the liquid sample so that the substances required for the detection reaction, preferably the PCR reaction, are present in sufficient concentration. The device optionally has a means or a suitable method step is provided to promote the dissolution of the analytical reagent in the sample. For example, the liquid sample can be contacted several times with the reagent, or the analytical reagent is mixed with the liquid sample by a micro stirrer. The analytical reagent preferably comprises a polymerase and / or reverse transcriptase, dNTPs, a buffer, magnesium chloride, at least one primer, preferably at least one pair of primers, optionally labeled, a detergent, preferably from the group comprising betaine, tween or dimethyl sulfoxide, and a polypeptide such as bovine serum albumin.

Umfasst das analytische Reagenz mehrere Stoffe, so können diese getrennt voneinander in der Vorrichtung lokalisiert sein. Bevorzugt liegt das analytische Reagenz in einer kompakten Form vor, die alle Stoffe umfasst.If the analytical reagent comprises several substances, these can be located separately from one another in the device. The analytical reagent is preferably in a compact form which comprises all substances.

Das analytische Reagenz kann in jedem Teil der Vorrichtung lokalisiert sein, sofern sichergestellt ist, dass es vor Durchführung der Nachweisreaktion mit der flüssigen Probe in Kontakt kommt und durchmischt wird. Bevorzugt ist es in der Mischkammer lokalisiert.The analytical reagent can be located in any part of the device, provided that it is ensured that it comes into contact with the liquid sample and is mixed before the detection reaction is carried out. It is preferably located in the mixing chamber.

Weist die Vorrichtung mehrere Sets an Kammern auf und sollen in diesen verschiedene PCR-Reaktionen ablaufen, so müssen für die jeweilige Reaktion spezifische Reagenzien, insbesondere Primer, dem jeweiligen Teil der Probe in dem jeweiligen Set spezifisch zugeführt werden, statt dass das analytische Reagenz in einer kompakten Form alle Stoffe umfasst. Die spezifischen Reagenzien, insbesondere ein oder mehr als ein Primer, können dem für die jeweilige Reaktion bestimmten Teil der Probe zugeführt werden, ohne dass sie mit den Teilen der Probe für die anderen Reaktionen in Kontakt geraten. Zu diesem Zweck kann jedes Set an Kammern wenigstens ein spezifisches Reagenz, insbesondere einen oder mehr als einen Primer, enthalten, das sich in dem Teil der flüssigen Probe löst, der in dieses Set übertragen wird.If the device has several sets of chambers and if different PCR reactions are to take place in them, then specific reagents, in particular primers, must be specifically supplied to the respective part of the sample in the respective set instead of the analytical reagent in one compact shape includes all fabrics. The specific reagents, especially one or more than one primer can be added to the part of the sample intended for the respective reaction without contacting the parts of the sample for the other reactions. For this purpose, each set of chambers can contain at least one specific reagent, in particular one or more than one primer, which dissolves in the part of the liquid sample which is transferred into this set.

Weist die Vorrichtung mehr als ein Set an Kammern auf, so umfasst sie bevorzugt wenigstens eine Heizzone. Dabei handelt es sich um eine Vorrichtung zum durchgehenden Beheizen eines Bereichs auf der Vorrichtung, in dem wenigstens zwei Kammern lokalisiert sind, die auf die gleiche Temperatur eingestellt werden können, bevorzugt eingestellt sind. Umfasst die Vorrichtung beispielsweise zwei Sets, so können beide Denaturierungskammern mit der gleichen Heizzone auf eine Temperatur von 95 °C eingestellt werden.If the device has more than one set of chambers, it preferably comprises at least one heating zone. This is a device for continuous heating of an area on the device in which at least two chambers are located, which are preferably set to the same temperature. If the device comprises two sets, for example, then both denaturing chambers can be set to a temperature of 95 ° C. with the same heating zone.

Umfasst die Vorrichtung wenigstens zwei Sets und wenigstens eine Heizzone, so kann in einer bevorzugten Ausführungsform die Temperatur aller Denaturierungskammern durch eine Heizzone für die Denaturierungskammern eingestellt werden bzw. wird für sie eingestellt, so dass in den Denaturierungskammern die gleiche Temperatur herrscht.If the device comprises at least two sets and at least one heating zone, in a preferred embodiment the temperature of all denaturing chambers can be set by a heating zone for the denaturing chambers or is set for them, so that the same temperature prevails in the denaturing chambers.

In einer weiteren bevorzugten Ausführungsform kann die Temperatur aller Amplifikationskammern durch eine Heizzone für die Amplifikationskammern eingestellt werden bzw. wird für sie eingestellt, so dass in den Amplifikationskammern die gleiche Temperatur herrscht. Alternativ können die die Temperaturen der jeweiligen Annealingkammern einzeln eingestellt werden.In a further preferred embodiment, the temperature of all amplification chambers can be set by a heating zone for the amplification chambers or is set for them, so that the same temperature prevails in the amplification chambers. Alternatively, the temperatures of the respective annealing chambers can be set individually.

In einer weiteren bevorzugten Ausführungsform kann die Temperatur aller Annealingkammern durch eine Heizzone für die Annealingkammern eingestellt werden bzw. wird für sie eingestellt, so dass in den Anneaiingkammern die gleiche Temperatur herrscht.In a further preferred embodiment, the temperature of all annealing chambers can be set by a heating zone for the annealing chambers or is set for them, so that the same temperature prevails in the annealing chambers.

In einer weiteren bevorzugten Ausführungsform kann die Temperatur aller Denaturierungskammern und aller Amplifikationskammern durch eine Heizzone für alle Denaturierungskammern bzw. durch eine Heizzone für alle Amplifikationskammern eingestellt werden bzw. wird für sie eingestellt, so dass in allen Denaturierungskammern die gleiche Temperatur herrscht und so dass in allen Amplifikationskammern die gleiche Temperatur herrscht.In a further preferred embodiment, the temperature of all denaturation chambers and all amplification chambers can be set or is set for them by a heating zone for all denaturation chambers or by a heating zone for all amplification chambers, so that the same temperature prevails in all denaturation chambers and so that in all Amplification chambers have the same temperature.

Alternativ können die die Temperaturen der jeweiligen Annealingkammern und/oder Denaturierungskammern einzeln eingestellt werden.Alternatively, the temperatures of the respective annealing chambers and / or denaturing chambers can be set individually.

In einer weiteren bevorzugten Ausführungsform kann die Temperatur aller Denaturierungskammern, aller Amplifikationskammern und aller Annealingkammern durch eine Heizzone für alle Denaturierungskammern bzw. durch eine Heizzone für alle Amplifikationskammern bzw. durch eine Heizzone für alle Annealingkammern eingestellt werden bzw. wird für sie eingestellt, so dass in allen Denaturierungskammern die gleiche Temperatur herrscht, in allen Amplifikationskammern die gleiche Temperatur herrscht und in allen Annealingkammern die gleiche Temperatur herrscht.In a further preferred embodiment, the temperature of all denaturing chambers, all amplification chambers and all annealing chambers can be set or is set for them by a heating zone for all denaturing chambers or by a heating zone for all amplification chambers or by a heating zone for all annealing chambers all denaturation chambers have the same temperature, all amplification chambers have the same temperature and all annealing chambers have the same temperature.

In einer bevorzugten Ausführungsform ist die analytische Vorrichtung ein Einwegartikel, der nach dem Gebrauch in seiner Gesamtheit verworfen wird. Der Einwegartikel ist an eine mehrfach, mit jeweils einem neuen Einwegartikel zu benutzende Analyseeinheit derart angepasst, dass letztere wenigstens eine Heizvorrichtung aufweist, mit der in der Denaturierungskammer, Amplifikationskammer und Annealingkammer und/oder in der Heizzone für die Denaturierungskammern, Amplifikationskammer bzw. Annealingkammern die erforderliche Temperatur eingestellt werden kann bzw. eingestellt ist.In a preferred embodiment, the analytical device is a disposable item which is discarded in its entirety after use. The disposable article is adapted to an analysis unit to be used several times, each with a new disposable article, in such a way that the latter has at least one heating device with which the required denaturation chamber, amplification chamber and annealing chamber and / or in the heating zone for the denaturation chambers, amplification chamber and annealing chambers Temperature can be set or is set.

Bevorzugt enthält die Analyseeinheit ein Mittel zum Auslesen einer flüssigen Probe, die sich in der Auslesekammer der analytischen Vorrichtung befindet, welche wiederum in die Analyseeinheit eingebracht wurde. Dabei kann es sich um ein Fluoreszenzspektroskop handeln, wenn die amplifizierte Nukleinsäure Fluoreszenzlabel enthält oder um ein UV/vis-Spektrophotometer, wenn sie ein Chromophor enthält. Die Analyseeinheit kann darüber hinaus weitere Einheiten umfassen, beispielsweise zum internen Transport von in die Analyseeinheit eingebrachten Einwegartikeln sowie zur Verarbeitung von Daten, die bei der Analyse einer flüssigen Probe in einem Einwegartikel erhalten werden.The analysis unit preferably contains a means for reading out a liquid sample, which is located in the reading chamber of the analytical device, which in turn has been introduced into the analysis unit. This can be a fluorescence spectroscope if the amplified nucleic acid contains a fluorescence label or a UV / vis spectrophotometer if it contains a chromophore. The analysis unit can also include further units, for example for the internal transport of disposable articles introduced into the analysis unit and for processing data which are obtained when analyzing a liquid sample in a disposable article.

Das erfindungsgemäße Verfahren ist bevorzugt ein Verfahren zum Nachweis einer Nukleinsäuresequenz, noch bevorzugter ein diagnostisches Verfahren zum Nachweis einer Nukleinsäuresequenz. Bei der Nukleinsäuresequenz kann es sich um eine humane Sequenz oder die Sequenz eines Pathogens handeln.The method according to the invention is preferably a method for the detection of a nucleic acid sequence, more preferably a diagnostic method for the detection of a nucleic acid sequence. The nucleic acid sequence can be a human sequence or the sequence of a pathogen.

Das Verfahren erfordert das Einbringen einer flüssigen Probe in die Vorrichtung. Optional wird die Probe zunächst in die offene Aufnahmekammer eingebracht. Sofern es sich um eine nicht flüssige Probe handelt, kann diese zunächst in eine flüssige Phase überführt werden. Es besteht auch die Möglichkeit, die Probe bereits in der Aufnahmekammer oder zuvor mit analytischem Reagenz zu vermischen. Anschließend kann der Zugang der Aufnahmekammer zur Oberfläche der Vorrichtung verschlossen werden.The method requires the introduction of a liquid sample into the device. Optionally, the sample is first introduced into the open receiving chamber. If it is a non-liquid sample, it can first be transferred to a liquid phase. There is also the possibility of having the sample already in the Mixing chamber or previously with analytical reagent. The access of the receiving chamber to the surface of the device can then be closed.

Danach wird die flüssige Probe in die Denaturierungskammer übertragen, optional kann sie zuvor in einer Mischkammer mit analytischem Reagenz kontaktiert und/oder für die Übertragung in mehrere Sets an Kammern portioniert werden. In der Denaturierungskammer wird die Denaturierung durchgeführt (Schritt a). Anschließend wird die flüssige Probe aus Schritt a) in die Annealingkammer übertragen, und es wird darin das Annealing durchgeführt (Schritt b). Anschließend wird die flüssige Probe aus Schritt b) in die Amplifikationskammer übertragen, und es wird die Amplifikation durchgeführt (Schritt c).The liquid sample is then transferred to the denaturing chamber; optionally, it can be contacted with analytical reagent beforehand in a mixing chamber and / or portioned into chambers for transfer to several sets. The denaturation is carried out in the denaturation chamber (step a). The liquid sample from step a) is then transferred into the annealing chamber, and the annealing is carried out therein (step b). The liquid sample from step b) is then transferred into the amplification chamber, and the amplification is carried out (step c).

Der Reaktionszyklus umfassend die Schritte a), b), und c) wird wenigstens 10-mal wiederholt. Insgesamt werden bevorzugt wenigstens 15, 20, 30, 40 oder 45 Reaktionszyklen durchgeführt.The reaction cycle comprising steps a), b), and c) is repeated at least 10 times. In total, at least 15, 20, 30, 40 or 45 reaction cycles are preferably carried out.

Schließlich wird die flüssige Probe, bevorzugt aus dem zuletzt durchgeführten Schritt c), in die Auslesekammer übertragen und dort ausgelesen. Nach dem Auslesen kann sie durch Übertragen in die Abfallkammer verworfen werden. Sofern wenigstens zwei Nachweisreaktionen in wenigstens zwei Sets Kammern durchgeführt werden, werden diese bevorzugt zeitgleich oder wenigstens zeitüberlappend durchgeführt, um die Gesamtdauer des Verfahrens zu minimieren. Die Teile der flüssigen Probe aus diesen wenigstens zwei Nachweisreaktionen werden anschließend nacheinander in die Auslesekammer übertragen und dort ausgelesen und anschließend durch Übertragen in die Abfallkammer verworfen, wenigstens alle Teile der flüssigen Probe bis auf den letzten Teil, der in der Auslesekammer verbleiben kann.Finally, the liquid sample, preferably from step c) carried out last, is transferred to the readout chamber and read out there. After reading, it can be discarded by transferring it to the waste chamber. If at least two detection reactions are carried out in at least two sets of chambers, these are preferably carried out simultaneously or at least overlapping in order to minimize the overall duration of the method. The parts of the liquid sample from these at least two detection reactions are then successively transferred to the read-out chamber and read out there and then discarded by transfer to the waste chamber, at least all parts of the liquid sample except for the last part that can remain in the read-out chamber.

Die Vorrichtung enthält zwei oder mehr Auslesekammern.The device contains two or more readout chambers.

Dabei können Teile der flüssigen Probe aus unterschiedlichen Sets jeweils umfassend eine Denaturierungskammer (5), eine Amplifikationskammer (6) und eine Annealingkammer (7) in unterschiedlichen Auslesekammern analysiert werden, beispielsweise mit Mikroarrays, die sich durch die darin enthaltenen Sonden und/oder Primer unterscheiden. Alternativ kann eine Probe oder ein Teil einer Probe in verschiedenen Auslesekammern analysiert werden.Parts of the liquid sample from different sets can each be analyzed comprising a denaturing chamber (5), an amplification chamber (6) and an annealing chamber (7) in different readout chambers, for example with microarrays that differ in the probes and / or primers they contain , Alternatively, a sample or part of a sample can be analyzed in different readout chambers.

Im Folgenden wird die Erfindung unter Bezugnahme auf die Figuren anhand von Ausführungsbeispielen erläutert. Die beschriebenen Ausführungsformen sind in jeder Hinsicht lediglich beispielhaft und nicht als einschränkend zu verstehen, und verschiedene Kombinationen der angeführten Merkmale sind vom Umfang der Erfindung umfasst.The invention is explained below with reference to the figures using exemplary embodiments. The described embodiments are to be considered in all respects only as examples and not as restrictive, and various combinations of the features listed are within the scope of the invention.

Fig. 1 zeigt eine bevorzugte Ausführungsform der analytischen Vorrichtung (1). Diese weist eine mit einem Deckel (13) verschließbare Aufnahmekammer (2) auf, in die eine Probe eingebracht werden kann. Aus der Aufnahmekammer kann die Probe in flüssiger Form in eine Mischkammer (3) überführt werden. Fig. 1 shows a preferred embodiment of the analytical device (1). This has a receiving chamber (2) which can be closed with a cover (13) and into which a sample can be introduced. The sample can be transferred in liquid form into a mixing chamber (3) from the receiving chamber.

Bei ihrer Ankunft kommt sie in der Mischkammer mit einem analytischen Reagenz (4) in Kontakt, das sich dabei in der flüssigen Probe auflöst. Das analytische Reagenz weist bevorzugt alle für eine PCR-Reaktion erforderlichen Reagenzien bis auf die zu amplifizierende Nukleinsäure auf, so dass - vorausgesetzt, dass die Probe die Nukleinsäure enthält - nach dem Auflösen alle für die Reaktion erforderlichen Reagenzien anwesend sind.When it arrives in the mixing chamber, it comes into contact with an analytical reagent (4), which dissolves in the liquid sample. The analytical reagent preferably has all the reagents required for a PCR reaction, except for the nucleic acid to be amplified, so that - provided that the sample contains the nucleic acid - all the reagents required for the reaction are present after dissolution.

Anschließend kann die flüssige Probe aufgeteilt werden, und jeweils ein Teil gelangt in eine Denaturierungskammer (5), die jeweils ein Teil eines Sets umfassend eine Denaturierungskammer (5), eine Amplifikationskammer (6) und eine Annealingkammer (7) ist. Sämtliche Denaturierungskammern sind in einer Heizzone für die Denaturierungskammern (8) angeordnet, analog sämtliche Amplifikationskammern und sämtliche Annealingkammern in der Heizzone für die Amplifikationskammern (9) bzw. Heizzone für die Annealingkammern (10). Die unterschiedlichen Sets können für unterschiedliche Nachweisreaktionen spezifische Reagenzien enthalten. Beispielsweise kann ein erstes Set ein erstes Primerpaar enthalten und ein zweites Set ein anderes zweites Primerpaar. Für alle Reaktionen benötigte unspezifische Reagenzien können der Probe zuvor zugeführt werden, beispielswese in der Mischkammer (3).The liquid sample can then be divided, and in each case a part enters a denaturing chamber (5), which is part of a set comprising a denaturing chamber (5), an amplification chamber (6) and an annealing chamber (7). All denaturing chambers are arranged in a heating zone for the denaturing chambers (8), analogously all amplification chambers and all annealing chambers in the heating zone for the amplification chambers (9) and heating zone for the annealing chambers (10). The different sets can contain specific reagents for different detection reactions. For example, a first set can contain a first pair of primers and a second set can contain another second pair of primers. Non-specific reagents required for all reactions can be added to the sample beforehand, for example in the mixing chamber (3).

In der Folge kann eine PCR-Reaktion in der Weise ausgeführt werden, dass die flüssige Probe zunächst bei geeigneten Bedingungen in die Denaturierungskammer eingebracht wird, dort anwesend ist und sich Nukleinsäure-Doppelstränge lösen und Einzelstränge freigesetzt werden. Anschließend wird die Probe umfassend die Einzelstränge in die Annealingkammer übertragen und ist dort unter Bedingungen anwesend, die das Annealen der Primer an die Einzelstränge gestatten. Anschließend wird die flüssige Probe mit Einzelsträngen und daran annealten Primern in die Amplifikationskammer übertragen und ist dort unter Bedingungen anwesend, die ausgehend von den Primern das Amplifizieren, d.h. die Synthese neuer Einzelstränge gestatten, die zu den vorhandenen Einzelsträngen komplementär sind. Anschließend kann die Probe für den Beginn eines neuen Amplifizierungszyklus umfassend die Schritte a), b) und c) erneut in die Denaturierungskammer übertragen werden.As a result, a PCR reaction can be carried out in such a way that the liquid sample is first introduced into the denaturation chamber under suitable conditions, is present there and double strands of nucleic acid dissolve and single strands are released. The sample is then transferred to the annealing chamber, comprising the individual strands, and is present there under conditions which allow the primers to be annealed to the single strands. The liquid sample with single strands and primers attached to them is then transferred into the amplification chamber and is present there under conditions which, starting from the primers, permit the amplification, ie the synthesis of new single strands which are complementary to the existing single strands. The sample can then be transferred to the denaturation chamber again for the start of a new amplification cycle comprising steps a), b) and c).

Ist eine ausreichende Zahl von Amplifizierungszyklen abgelaufen, so kann der Teil der Probe, der in einem der Sets umfassend eine Denaturierungskammer (5), eine Amplifikationskammer (6) und eine Annealingkammer (7) enthalten war, in die Auslesekammer (11) übertragen werden. Dort erfolgt eine geeignete Nachweisreaktion, beispielsweise eine Fluoreszenzmessung, mit der Nukleinsäurestränge nachgewiesen werden können, die wenigstens einen fluoreszenzmarkierten Primer enthalten und sich auf diese Weise von Nukleinsäuren unterscheiden, die bereits in der ursprünglich in die Vorrichtung eingebrachten Probe enthalten waren.When a sufficient number of amplification cycles has expired, the part of the sample which was contained in one of the sets comprising a denaturing chamber (5), an amplification chamber (6) and an annealing chamber (7) can be transferred to the readout chamber (11). A suitable detection reaction takes place there, for example a fluorescence measurement, with which nucleic acid strands can be detected which contain at least one fluorescence-labeled primer and in this way differ from nucleic acids which were already contained in the sample originally introduced into the device.

Nach Ablauf der Nachweisreaktion kann der in der Auslesekammer enthaltene Teil der Probe durch Übertragen in die Abfallkammer (12) verworfen werden. Die Auslesekammer ist damit leer und steht bereit dafür, dass ein Teil der Probe aus einem anderen Set in sie übertragen und mit einer Nachweisreaktion untersucht werden kann. Auf diese Weise können in der gleichen Auslesekammer mehrere Teile der flüssigen Probe untersucht werden.After the detection reaction has elapsed, the part of the sample contained in the readout chamber can be discarded by transferring it to the waste chamber (12). The readout chamber is thus empty and is ready for a part of the sample from another set to be transferred to it and to be examined with a detection reaction. In this way, several parts of the liquid sample can be examined in the same readout chamber.

Fig. 2 zeigt eine weitere bevorzugte Ausführungsform der analytischen Vorrichtung (1). Sie unterscheidet sich von der in Fig. 1 bezeigten Vorrichtung insbesondere dadurch, dass sie einen ringförmigen Kanal enthält, in dem die Denaturierungskammer, Amplifikationskammer und Annealingkammer nicht in Form von geometrische abgegrenzten Kammern bereitgestellt werden, sondern dadurch, dass die flüssige Probe (14) in Form eines kompakten, zusammenhängenden Tropfens entlang des Ringes in die Heizzone der Denaturierungskammer, Amplifikationskammer bzw. Annealingkammer (8, 9 bzw. 10) übertragen wird. Zusammen mit den Wänden des ringförmigen Kanals bilden die Grenzflächen dieses Tropfens die Grenzen der Kammer aus, beispielsweise, wie hier abgebildet, die Denaturierungskammer (5). Fig. 2 shows a further preferred embodiment of the analytical device (1). It differs from that in Fig. 1 demonstrated device in particular in that it contains an annular channel in which the denaturing chamber, amplification chamber and annealing chamber are not provided in the form of geometrically delimited chambers, but in that the liquid sample (14) in the form of a compact, coherent drop along the ring is transferred into the heating zone of the denaturing chamber, amplification chamber or annealing chamber (8, 9 or 10). Together with the walls of the annular channel, the boundary surfaces of this drop form the boundaries of the chamber, for example the denaturing chamber (5), as shown here.

In einer bevorzugten Ausführungsform können mehrere ringförmige Kanäle senkrecht zur Papierebene übereinander angeordnet sein; die Heizzonen verlaufen dann bevorzugt ebenfalls senkrecht zur Papierebene, so dass jeweils eine der Heizzonen mehrere ringförmige Kanäle jeweils an der entsprechenden Stelle auf die für die Denaturierungskammer, Amplifikationskammer bzw. Annealingkammer gewünschte Temperatur heizen kann bzw. heizt.In a preferred embodiment, a plurality of annular channels can be arranged one above the other perpendicular to the paper plane; the heating zones then preferably also run perpendicular to the plane of the paper, so that in each case one of the heating zones can heat or heats a plurality of ring-shaped channels at the corresponding point to the temperature desired for the denaturing chamber, amplification chamber or annealing chamber.

Bei dieser bevorzugten Ausführungsform verlässt die flüssige Probe während der PCR-Reaktion niemals den ringförmigen Kanal. Sie wird an die jeweils gerade benötigte Stelle des ringförmigen Kanals übertragen, an der die für den auszuführenden Schritt a), b) oder c) geeignete Temperatur herrscht. Auf diese Weise muss kein Flaschenhals, d. h. eine besonders enge Stelle, durchlaufen werden und die Geschwindigkeit der Übertragung der Probe zwischen den unterschiedlichen Kammern kann maximiert werden.In this preferred embodiment, the liquid sample never leaves the annular channel during the PCR reaction. It is transmitted to the point of the ring-shaped channel that is currently required, at that point for the step to be carried out a), b) or c) there is a suitable temperature. In this way, there is no need to go through a bottleneck, ie a particularly narrow point, and the speed of the sample transfer between the different chambers can be maximized.

Bezugszeichenliste:LIST OF REFERENCE NUMBERS

11
Analytische VorrichtungAnalytical device
22
Aufnahmekammerreceiving chamber
33
Mischkammermixing chamber
44
Analytisches ReagenzAnalytical reagent
55
DenaturierungskammerDenaturierungskammer
66
Amplifikationskammeramplification chamber
77
AnnealingkammerAnnealingkammer
88th
Heizzone für die DenaturierungskammernHeating zone for the denaturing chambers
99
Heizzone für die AmplifikationskammernHeating zone for the amplification chambers
1010
Heizzone für die AnnealingkammernHeating zone for the annealing chambers
1111
Auslesekammerelite chamber
1212
Abfallkammerwaste chamber
1313
Deckelcover
1414
Flüssige ProbeLiquid sample

Claims (10)

  1. Method comprising
    an analytical device (1) comprising a denaturation chamber (5), an amplification chamber (6) and an annealing chamber (7),
    wherein the temperature of the denaturation chamber (5) is set to a temperature between 90 °C and 105 °C,
    wherein the temperature of the amplification chamber (6) is set to a temperature between 60 °C and 80 °C,
    wherein the temperature of the annealing chamber (7) is set to a temperature between 40 °C and 67 °C,
    and wherein the device (1) is adapted such that a liquid sample (14) can be introduced into the denaturation chamber (5) and transferred back and forth between the denaturation chamber (5), the amplification chamber (6), the annealing chamber (7) and optionally further chambers (11, 12),
    characterized in that the analytical device (1) further comprises a receiving chamber (2) with access to the surface of the diagnostic device, via which the sample (14) can be introduced into the receiving chamber (2) and subsequently transferred to further chambers (5, 6, 7),
    wherein the amplification chamber (6) is downstream of the denaturation chamber (5) and the annealing chamber (7) is downstream of the amplification chamber (6),
    said method comprising the steps of:
    a) introducing the liquid sample (14) into the denaturation chamber (5) of the analytical device (1), subsequently denaturing the sample,
    b) transferring the liquid sample (14) from step a) into the annealing chamber (7), subsequently annealing,
    c) transferring the liquid sample from step b) into the amplification chamber (6), subsequently amplifying,
    d) repeating steps a), b) and c),
    e) transferring the liquid sample (14) from d) into two or more read-out chambers (11), subsequently reading out the liquid sample (14),
    wherein the reaction cycle comprising steps a), b) and c) is repeated at least 10 times,
    wherein the analytical device (1) comprises at least two sets of chambers, each comprising a denaturation chamber (5), an amplification chamber (6) and an annealing chamber (7), and
    the two or more read-out chambers differ by the probes contained therein.
  2. Method according to claim 1, wherein the amplification chamber (6) is directly downstream of the denaturation chamber (5) and the annealing chamber (7) is directly downstream to the amplification chamber (6).
  3. Method according to any one of claims 1 or 2, further comprising an analytical reagent (4) which is soluble in the liquid sample (14), preferably a lyophilized reagent.
  4. Method according to any one of claims 1 to 3, wherein a mixing chamber (3) is downstream of the receiving chamber (2) and upstream of the denaturing chamber (5), preferably directly upstream, which preferably comprises the analytical reagent (4).
  5. Method according to any one of claims 1 to 4, wherein the diagnostic device (1) comprises at least two sets of chambers, each comprising a denaturation chamber (5), an amplification chamber (6) and an annealing chamber (7),
    and wherein preferably downstream of the receiving chamber (2) and upstream of the denaturing chamber (5), more preferably directly upstream, is a mixing chamber (3), which preferably comprises the analytical reagent (4), and from said mixing chamber (3) a part of a liquid sample (14) can be transferred into a chamber (5, 6, 7) of the different sets, respectively.
  6. Method according to claim 5, wherein the analytical device (1) comprises at least one heating zone (8, 9, 10), wherein the denaturation chamber (5), preferably in addition the amplification chambers (6), more preferably in addition the annealing chambers (7) of the at least two sets are each arranged on a heating zone.
  7. Method according to any one of claims 1 to 6, wherein the device (1) comprises the read-out chamber (11) into which the liquid sample (14) can be transferred from the denaturation chamber (5), the amplification chamber (6) or the annealing chamber (7).
  8. Method according to any one of claims 1 to 7, wherein the read-out chamber (11) comprises at least one immobilized nucleic acid.
  9. Method according to any one of claims 1 to 8, wherein the diagnostic device (1) is a laboratory chip.
  10. Method according to any one of claims 1 to 9, comprising a nucleic acid-containing liquid sample.
EP15003599.6A 2015-12-17 2015-12-17 Method for carrying out a pcr reaction Active EP3181229B1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP15003599.6A EP3181229B1 (en) 2015-12-17 2015-12-17 Method for carrying out a pcr reaction
DE102016013403.8A DE102016013403A1 (en) 2015-12-17 2016-11-11 Analytical device for carrying out a PCR reaction

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
EP15003599.6A EP3181229B1 (en) 2015-12-17 2015-12-17 Method for carrying out a pcr reaction

Publications (2)

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EP3181229A1 EP3181229A1 (en) 2017-06-21
EP3181229B1 true EP3181229B1 (en) 2020-02-26

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DE (1) DE102016013403A1 (en)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002081729A2 (en) * 2001-04-06 2002-10-17 California Institute Of Technology Nucleic acid amplification utilizing microfluidic devices
JP4830432B2 (en) * 2005-09-30 2011-12-07 横河電機株式会社 Chemical reaction cartridge and method of use thereof
US9387478B2 (en) * 2012-08-17 2016-07-12 Lexmark International, Inc. Micro-fluidic modules on a chip for diagnostic applications
DE102014200509A1 (en) * 2014-01-14 2015-07-16 Robert Bosch Gmbh Analysis unit for performing a nested polymerase chain reaction, analysis device, method for operating such an analysis unit and method for producing such an analysis unit
EP3127613B1 (en) 2015-08-04 2019-07-24 Euroimmun Medizinische Labordiagnostika AG Device for the transfer of sample material into a liquid

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
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DE102016013403A1 (en) 2017-06-22
EP3181229A1 (en) 2017-06-21

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