EP4301872A1

EP4301872A1 - Device, system and method for quantitative real-time pcr analysis (qpcr) and other microbiological analysis techniques

Info

Publication number: EP4301872A1
Application number: EP22719503.9A
Authority: EP
Inventors: Bernd Herzmann
Original assignee: Bionlyx GmbH
Current assignee: Bionlyx GmbH
Priority date: 2021-03-01
Filing date: 2022-03-01
Publication date: 2024-01-10
Also published as: DE102021104908B3; WO2022184210A1; DE112022001324A5

Abstract

The present invention relates to a device (2), a system (1) and a method for quantitative real-time PCR analysis (qPCR) and other molecular biological analysis techniques for determining the presence of certain genome sequences in a material. The device (2) according to the invention is characterised by at least one centrifuging and mixing unit (21) for receiving a sample in a sample tube (3), said unit being mounted such that it can rotate about an axis of rotation (212), and designed in such a way as to mix and to centrifuge the sample in the sample tube, and to heat and cool same in a controlled manner, as well as being designed to exchange control and/or measurement data with the control unit (24). The system (1) according to the invention consists of a device (2) as described and at least one sample tube (3), wherein a body (32) of the sample tube (3) is divided into two sections by at least one filter (33), and wherein the section of the body (32) located on the other side of the filter (33) with respect to a cover (31) of the sample tube comprises a test substance (5).

Description

5

DEVICE, SYSTEM AND METHOD FOR QUANTITATIVE REAL-TIME PCR ANALYSIS (QPCR) AND

OTHER MICROBIOLOGICAL ANALYTICAL TECHNIQUES

10 The present invention relates to a device for quantitative real-time PCR analysis (qPCR) and other molecular biological analysis techniques for determining the presence of certain genetic sequences in a material, in particular for reverse transcriptase qPCR analysis (qRT-PCR), for Carrying out a LAMP, a TMA and/or a CRISPR analysis, at least comprising 15 - at least one exposure means for exposing a sample in a sample tube to electromagnetic radiation;

- At least one analysis means for the quantitative analysis of a sample in a sample tube with respect to a content of DNA (sections); and

- A control unit, which is set up with the analysis means and with the

20 exposure means exchange control and / or measurement data.

The present invention also relates to a system for performing quantitative real-time PCR analysis (qPCR) and other molecular-biological analysis techniques for determining the presence of certain genetic material sequences in a material, in particular for reverse transcriptase qPCR (qRT-PCR). a LAMP, a TMA and/or a CRISPR analysis, consisting of such a device and at least one sample tube, wherein a body of the sample tube is divided into two sections by at least one filter and the one with respect to a lid of the sample tube is on the other side of the filter located section of the body comprises a test substance. 30

Finally, the present invention also relates to a method for quantitative real-time PCR analysis (qPCR) and other molecular-biological analysis techniques for determining Sensing the presence of certain genetic sequences in a material, in particular for reverse transcriptase qPCR (qRT-PCR), for carrying out a LAMP, a TMA and/or a CRISPR analysis, using a system as described above.

Quantitative real-time PCR analysis (qPCR) is used as an established and particularly precise analysis method in molecular laboratory diagnostics, for example to determine the viral load after infection or to identify hereditary diseases. It is an amplification method for nucleic acids, in particular for deoxyribonucleic acid (DNA), which is based on the principle of the polymerase chain reaction (PCR) and also offers the possibility of real-time quantification - usually by means of a during or at the end of a PCR Cycle carried out fluorescence analysis - offers. Since the enzyme DNA polymerase, which requires double-stranded DNA molecules or molecule fragments as a substrate, is used for the replication, it is used for the analysis of single-stranded ribonucleic acid (RNA) samples, for example for the investigation/quantitative detection of RNA viruses such as SARS-coronavirus-2 (SARS-CoV-2), another step upstream, namely the use of a reverse transcriptase (RT) to convert RNA into complementary DNA (cDNA). The corresponding analysis method is then referred to as reverse transcriptase qPCR analysis (qRT-PCR).

Especially since the beginning of the Covid-19 pandemic, the need for fast and reliable laboratory diagnostics for the detection of RNA viruses such as SARS-CoV-2 has increased significantly, since testing as many as possible, especially asymptomatic or very mildly symptomatic, but nevertheless infectious persons, an effective means in the fight against the virus is seen. A complete qRT-PCR analysis, including sample preparation, extraction of the nucleic acid and subsequent actual implementation of the qRT-PCR, takes an average of 3 to 5 hours in an examination laboratory and requires laboratory personnel trained in medical diagnostics. The really speed-determining step is usually not the sample preparation and analysis time in the laboratory, but the sampling from the person to be examined or from the patient and, above all, the transport of the sample to the laboratory and then the transmission time of the test results from the laboratory to the patient. In addition to qRT-PCR, other microbiological analysis techniques are constantly being established to determine the presence of certain genetic material sequences in a sample, which can differ in the analysis reagents used on the one hand and in the specified temperature protocol on the other. The term "genetic sequences" is understood to mean sections of both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules. The so-called LAMP analysis (loop-mediated isothermal amplification) is based, for example, on isothermal nucleic acid amplification using strand-shifted DNA polymerases as a test or analysis substance. The so-called TMA analysis (transcription mediated amplification) is also carried out at constant temperature (eg approx. 41 °C), but uses two enzymes as test reagents: an RNA polymerase and a reverse transcriptase. Finally, in the CRISPR analysis, a guide RNA (crRNA) is added to the sample as part of the test substance, which attaches itself to the gene to be examined, e.g. a virus gene. In a second step, the Casl3 enzyme, as another part of the test substance, cuts the gene to be examined at the attachment point and releases a so-called reporter RNA, which in turn carries a fluorescent marker. The reactions also take place isothermally, in this case, for example, at about 37 °C. All analysis methods briefly described here, qPCR, qRT-PCR, LAMP, TMA, CRISPR, have in common that the final detection step consists of a fluorescence-analytical evaluation of the reaction products.

In order to establish near-patient testing (point-of-care laboratory diagnostics, POC), various compact test systems have been developed for some time, some of which can deliver test results in less than an hour. However, for the preparation of the samples, for the operation of the respective test system and for the evaluation of the analytical results, specialist personnel trained in medicine and/or laboratory diagnostics are necessary. In this context, DE 102012 219491 A1, DE 102010 003 223 A1 and DE 690 17 454 T2 have become known.

Since the PCR analysis of RNA viruses and the variants described above represent the most accurate and reliable methods to date for quantifying any viral load and thus also enable statements to be made about acute infectiousness, it would be desirable to also carry out such analyzes outside of medical facilities by personnel not trained in medical/laboratory diagnostics, quickly and safely to be able to have it carried out, for example to test the guests of a restaurant or the visitors of a cultural or sporting event directly before/during their visit.

Proceeding from this, the present invention has the object of providing an improved device or an improved system and method for quantitative real-time PCR analysis (qPCR) and other molecular-biological analysis techniques for determining the presence of certain genetic material sequences in a material compared to the prior art. in particular for reverse transcriptase qPCR analysis (qRT-PCR), for carrying out a LAMP, a TMA and/or a CRISPR analysis, which or which is a fast, simple and inexpensive but nevertheless reliable implementation of such Allows analyzes and its operation requires no relevant medical and / or laboratory diagnostic knowledge. In addition, the handling of the device or the system and the method carried out with it should be safe for the person carrying out the analysis, ie the risk of becoming infected should be as small as possible.

This object is first achieved by a device having the features of independent patent claim 1, by a system having the features of the independent claim 11, and by a method having the features of claim 14.

The device according to the invention is distinguished from devices of the prior art in that it comprises at least one centrifugation and mixing unit for receiving a sample in a sample tube, which is rotatably mounted with respect to an axis of rotation; and which is set up to mix, centrifuge, heat and cool the sample in a controlled manner in the sample tube, and wherein the centrifugation and mixing unit is also set up to exchange control and/or measurement data with the control unit.

The system according to the invention consists of a device as described above and at least one sample tube, wherein a body of the sample tube is divided into two sections by at least one filter and the section of the body beyond the filter with respect to a lid of the sample tube comprises a test substance. The device according to the invention and the system according to the invention advantageously enable all steps of a quantitative real-time PCR analysis (qPCR) and other molecular-biological analysis techniques to determine the presence of certain genetic material sequences in a material, in particular a reverse transcriptase qPCR analysis (qRT PCR), to carry out a LAMP, a TMA and/or a CRISPR analysis, and to evaluate the analysis results directly, in particular with the help of the control unit. The system of the device and sample tube according to the invention advantageously also makes it possible to carry out the sample preparation, in particular the so-called lysis, i.e. the detachment of the RNA from a sample swab with the aid of a solvent and the filtration of the sample mixture obtained in this way, in an automated manner. The sampling that can be carried out by a user—ideally by the person to be examined himself—can then advantageously only be that the user carries out a nasal and/or throat swab on himself (possibly also under guidance), the swab himself on a Breaking point breaks off in the sample tube, for example using an already filled disposable syringe with solvent and then closes the lid of the sample tube and places the tube through the insertion opening in the centrifugation and mixing unit of the device. After starting the device according to the invention, the rest of the quantitative real-time PCR analysis (qPCR) or other molecular-biological analysis techniques for determining the presence of certain genetic material sequences in a material, in particular the reverse transcriptase qPCR analysis (qRT-PCR), LAMP, TMA and/or CRISPR analysis, including output of the analysis results fully automatically, so that no third party has to come into direct contact with the potentially infectious person to be tested. To select the specific type of analysis carried out (PCR, LAMP, TMA, or CRISPR, etc.), it is advantageous to use only one sample tube with a suitable test substance and to select the respective temperature program on the control unit.

Further advantageous refinements and developments, which can be used individually or in combination with one another, are the subject matter of the dependent claims.

In a first embodiment of the invention, it has proven useful if the centrifugation and mixing unit comprises at least one contacting element which is at least one correspondingly designed, electrically current-carrying contact point of the device can be reversibly brought into contact and which is set up to supply electronic components, which are arranged on the centrifugation and mixing unit, with electric current. The at least one contacting element can be designed in particular as at least one metal contact point on the upper side of the centrifugation and mixing unit, which can be connected via cable to electronic components such as preferably a heating element, a light barrier and/or various sensors for temperature and/or speed measurement, which can be arranged on the centrifugation and mixing unit, in particular in at least one channel of the centrifugation and mixing unit, is electrically conductively connected. If the centrifugation and mixing unit is in a stationary measuring position while the method according to the invention is being carried out, the contacting element can then advantageously be brought into reversible contact with the at least one correspondingly designed, electrically current-carrying contact point of the device and the electronic component mentioned supply with electricity. To carry out a centrifugation and/or mixing step within the method according to the invention, the contact between the contacting element and the contact point of the device can be released again, so that advantageously free rotation of the centrifugation and mixing unit is ensured.

In a further embodiment of the invention, it has proven useful if at least one channel is formed in the centrifugal and mixing unit, in which a sample tube can be held in a defined position by insertion via an insertion opening. An insertion opening for inserting a sample tube into a channel of a centrifugation and mixing unit advantageously enables the sample tube to be quickly and easily attached in the device for an upcoming analysis. In this case, the channel can advantageously function as a type of guideway and thus ensure correct positioning of the sample in relation to the respective analysis means. The position of the sample tube can preferably be determined via a position sensor, such as a light barrier, and/or by forming a mechanical stop in the channel.

It is advantageous if the at least one channel is aligned obliquely with respect to its longitudinal axis to the axis of rotation of the centrifugation and mixing unit, wherein the end of the channel facing away from the axis of rotation is preferably arranged closer to the bottom of the device than the end of the channel facing the axis of rotation. Such a channel advantageously enables, on the one hand, the use of gravity when the centrifugation and mixing unit is at rest or at low rotational speeds, in order to fix the sample, a solvent and/or a test substance in the sample tube, and, on the other hand, during a centrifugation step, i.e. at high hen rotation speeds to allow relatively dense (physical density) sample components to move away from the axis of rotation under the influence of centrifugal/centripetal force over a longer section of the body and to separate at the end of the sample tube remote from the axis of rotation.

In a further preferred embodiment of the device according to the invention, the at least one channel can comprise at least one window transparent to electromagnetic radiation, the window or windows preferably being arranged at one end of the channel facing away from the axis of rotation of the centrifugation and mixing unit. A window that is permeable to electromagnetic radiation, in particular at an end of the channel facing away from the axis of rotation of the centrifugation and mixing unit, can advantageously enable interference-free and loss-free detection of the electromagnetic radiation emitted by the sample, in particular the emitted fluorescence radiation, through the enable at least one means of analysis. The window or windows can be formed by one or more mere recesses, ie by one or more openings in the channel, but the window or windows can also be formed by a pane material that is transparent for certain areas of the electromagnetic spectrum. In this case, the channel can then advantageously be designed as a component that is closed except for the insertion opening.

In addition, it has proven useful if the at least one channel comprises at least one heating element, with the at least one heating element preferably being arranged at one end of the channel facing away from the axis of rotation of the centrifugation and mixing unit. A heating element advantageously allows a sample in the sample tube, in particular a part of a sample which is located in a section of a sample tube facing away from a cover of the sample tube, to be heated or cooled in a controlled manner. The heating element or elements can preferably be configured as a Peltier element, which can be connected to the control unit for control and/or measurement data exchange/exchange. Several heating elements, which are distributed over the length of at least one channel, advantageously allow the sample in the sample tube to be heated to different temperatures at different points.

In a further embodiment, it is preferred that the device, in particular the centrifugation and mixing unit, comprises a motor which is set up to rotate the centrifugation and mixing unit both clockwise and counterclockwise about the axis of rotation. A rotary movement in a single direction of rotation about the axis of rotation, be it clockwise or counterclockwise, advantageously enables a sample to be centrifuged as a function of the respective rotational speed. A rotary movement with changing direction of rotation, particularly at short time intervals, can produce a thorough mixing of a sample in the sample tube and thus advantageously replace the use of a so-called vortex mixer.

It is advantageous if the axis of rotation of the centrifugation and mixing unit is operatively connected to the motor at one end and is rotatably mounted in a cover of the device at the opposite end when the device is closed with the cover. A rotatable mounting of the end of the axis of rotation opposite the motor in a cover of the device advantageously increases the centering of the rotation and the stability of the rotational movement of the centrifugation and mixing unit, in particular at high rotational speeds and/or with a rapid change in direction of rotation Process step of mixing.

In addition, an embodiment of the invention is advantageous in which the exposure means emits electromagnetic radiation with wavelengths in a range from 480 to 560 nm or preferably in a range from 485 to 540 nm or particularly preferably with a wavelength of 488 nm. Electromagnetic radiation in a wavelength range from 480 to 560 nm advantageously enables the excitation of the most common fluorescent dyes that are used in qPCR analysis. In particular, electromagnetic radiation with a wavelength in the range from 485 to 540 nm and particularly preferably with a wavelength of 488 nm enables the excitation of two particularly frequently used fluorescent dyes for, among other things, DNA sequencing rang and the qPCR analysis, namely 5-(and 6)-carboxylfluorescein (5(6)-FAM, absorption maximum at 495 nm) and hexachloro-fluorescein (HEX, absorption maximum at 535 nm).

In another embodiment, it has proven useful if the exposure means emits electromagnetic radiation with wavelengths in the range from 610 to 680 nm or preferably from 640 to 675 nm or particularly preferably with a wavelength of 650 nm. Electromagnetic radiation with a wavelength in the range from 610 to 680 nm, preferably in the range from 640 to 675 nm, particularly preferably with a wavelength of 650 nm, advantageously enables the excitation of another frequently used fluorescent dye for, among other things, DNA sequencing and qPCR analysis of the non-sulfonated cyanine dye indodicarbocyanine (Cy5™) (absorption maximum at 650 nm).

The exposure means or means can be designed in particular as light-emitting diodes with different emission wavelengths (preferably in the green, blue and/or violet spectral range). In a preferred embodiment of the invention, the light emitted by the exposure means(s) can also be limited to a desired wavelength range with the aid of transmission filters.

In a further embodiment of the invention, it has proven useful if the analysis means is designed as a detector for electromagnetic radiation, preferably as a fluorescence detector. Such a detector advantageously enables the quantitative detection of an electromagnetic signal, preferably a fluorescence signal, of a change in wavelength and/or intensity, with the fluorescence increasing in proportion to the quantity of products formed during the PCR, or the wavelength and/or intensity increasing proportional to the amount of products formed during the PCR. Such an analysis means can also include a reception filter, which advantageously limits the detected wavelength range to predetermined values. In addition, such an analysis means enables, in particular, the detection of wavelength changes in the light scattered by the sample and/or, for example, the measurement of a change in intensity of the scattered light due to turbidity of the sample solution. Finally, an embodiment of a device according to the invention is preferred which comprises at least one means for data transmission, the means for data transmission being a means for data transmission via cable, such as in particular a USB connection, and/or as a means for wireless data transmission How, in particular, a Bluetooth interface or a WLAN interface can be designed. With the help of such a means of data transmission, the analysis results evaluated by the control unit can be displayed immediately on an output device and/or forwarded to the patient or to a treating doctor, etc., e.g. using a smartphone application. This advantageously enables infectious persons to be informed immediately and can help to quickly break chains of infection.

In a further embodiment of the system according to the invention consisting of device and sample tube, it has also proven itself that the body of the sample tube is impermeable to electromagnetic radiation, in particular to fluorescence radiation, except for a window area in the section of the body beyond the filter with respect to the lid of the sample tube . Said impermeability to electromagnetic radiation can be achieved either by choosing a radiation-impermeable material for the body outside the window area or by coloring the outer wall of the body outside the window area, in particular black. Such a configuration of the sample tube advantageously prevents reflection of the electromagnetic radiation, in particular the fluorescence radiation.

In addition, it is advantageous if the sample tube is designed in multiple parts, with at least one upper section and at least one lower section that can be reversibly fastened to the upper section. Preferably, the lower section can have a conically tapering funnel area in its interior, the larger diameter of which is arranged near the filter or can be divided into a collection area for a prepared sample and an analysis area, with the collection area communicating with the analysis area via at least one feed opening connection.

Finally, the present invention also relates to a method for quantitative real-time PCR analysis (qPCR) and other molecular-biological analysis techniques to determine the presence of certain genetic sequences in a material, in particular for reverse transcriptase qPCR (qRT-PCR) to carry out one LAMP, one TMA and/or a CRISPR analysis, with a system as described above. A sample is introduced unprocessed into a sample tube, the sample is mixed with a solvent from a solvent already in the sample tube or by adding solvent from the outside into the sample tube and then sealed with a lid. The sample tube is then picked up by a centrifugation and mixing unit of the device and the sample in the sample tube is mixed with the solvent by the centrifugation and mixing unit. The sample-solvent mixture obtained in this way is then centrifuged through the centrifugation and mixing unit, with the sample solution migrating via at least one filter into a section of a body of the sample tube beyond the filter with respect to the lid of the sample tube and being filtered as a result. Using the centrifugation and mixing unit, the sample solution filtered in this way is now mixed with a test substance located in the section of the body beyond the filter in relation to the lid of the sample tube and then subjected to thermocycling or thermostatting and fluorescence analysis in the context of a quantitative real-time PCR analysis (qPCR) or another molecular biological analysis technique to determine the presence of certain genetic sequences in a material, in particular as part of a reverse transcriptase qPCR (qRT-PCR), LAMP, TMA and/or CRISPR analysis , subjected.

It is particularly preferred if, in a further method step, the analysis result is transmitted to a smartphone application using a means for data transmission, with the means for data transmission being a means for data transmission via cable, such as in particular a USB connection and/or or as a means for wireless data transmission, such as a Bluetooth interface or a WLAN interface in particular.

The device according to the invention and the system according to the invention enable a rapid and automated implementation of quantitative real-time PCR analyzes (qPCR) and other molecular-biological analysis techniques for determining the presence of certain genetic material sequences in a material, in particular reverse transcriptase qPCR. (qRT-PCR), LAMP, TMA and/or CRISPR analyses, and can also be used by users who have not been trained in medicine and/or laboratory diagnostics. The compact and inexpensive design of the device or of the system also advantageously allow corresponding PCR tests to be carried out at very different, in particular "non-medical" locations, such as for example in old people's homes, authorities, catering establishments and at airports, etc. These, as well as additional details and further advantages of the invention hereinafter described with reference to preferred embodiments, to which the present invention is not limited, and in conjunction with the accompanying drawings.

It shows schematically:

1 shows an embodiment of a device according to the invention without a cover in a side view;

FIG. 2 shows an embodiment of a system according to the invention without a cover, consisting of a device of the embodiment from FIG. 1 and a small sample tube, in a side view;

FIG. 3 shows the device from FIG. 1 in a plan view; FIG. FIG. 4 shows the system from FIG. 2 in a plan view; FIG.

5 in sub-figures a to d, each embodiment of a sample tube in the various phases of sample preparation; and

7a, b show two further configurations of a sample tube, each in a longitudinal section.

In the following description of preferred embodiments of the present invention, the same reference symbols denote the same or comparable components. 1 shows an embodiment of a device 2 according to the invention without a cover in a side view. FIG. 3 shows the device 2 from FIG. 1 in a plan view.

A device 2 according to the invention for quantitative real-time PCR analysis (qPCR) and other molecular-biological analysis techniques to determine the presence of a specific genetic sequence in a material, in particular for reverse transcription tase qPCR analysis (qRT-PCR) and to carry out a LAMP, a TMA and/or a CRISPR analysis comprises at least one exposure means 23 for exposing a sample in a sample tube 3 to electromagnetic radiation; at least one analysis means 22 for quantitatively analyzing a sample in a sample tube 3 for a content of DNA (sections); and a control unit 24 which is set up to exchange control and/or measurement data with the analysis means 22 and with the exposure means 23 . FIGS. 1 to 4 each show configurations of a device 2 according to the invention without a cover. The device 2 can, however, preferably comprise at least one cover to protect the technical components from damage and/or contamination. Such a cover can in turn have at least one access opening to one or more of the insertion openings 213 of a centrifugation and mixing unit 21, and preferably a display for displaying control and measurement data of the control unit 24. However, such a display can also be on one of the Walls 25 of the device 2 can be arranged. The cover and/or the walls of the device 2 can also have one or more operating elements for operating the control unit 24 .

As can also be seen in FIG. 1, the device 2 according to the invention is characterized by at least one centrifugation and mixing unit 21 for receiving a sample in a sample tube 3, which is rotatably mounted with respect to an axis of rotation 212; and which is set up to both mix, centrifuge, heat and cool the sample in a controlled manner in the sample tube 3 , and wherein the centrifugation and mixing unit 21 is also set up to exchange control and/or measurement data with the control unit 24 .

The centrifugation and mixing unit 21 can preferably, as shown in FIGS. 1 to 4, comprise at least one contacting element 215 which is nem correspondingly designed, electrically current-carrying contact point of the device 2 can be reversibly brought into contact and which is set up to supply electronic components, which are arranged on the centrifugation and mixing unit 21, with electricity. 1 to 4 are examples of two contacting elements 215 shown, which gations- and mixing unit 21 are arranged on top of the centrifuge. Corresponding to these contacting elements 215, electrical current-carrying contact points of the device 2 can be arranged in a preferred embodiment of the invention in the cover described above and in the closed operating state of the device 2 with the help of a servomotor arranged on the cover automatically, preferably by moving the contact points of the device 2 along the axis of rotation 212, which is preferably mounted on one side in the cover, towards the centrifugation and mixing unit 21, with the contacting elements 215 when the centrifugation and mixing unit 21 is in a stationary position measuring position is located. After the measurement has taken place, the contact can then be released again with the aid of the servomotor, so that advantageously free rotation of the centrifugation and mixing unit 21 about the axis of rotation 212 is ensured.

The centrifugation and mixing unit 21 can include at least one insertion opening 213 through which a sample tube 3 can be inserted into at least one channel 214 and can thus be fixed in the centrifugation and mixing unit 21 . 1 to 4 are configurations of the device 2 with two insertion openings 213 and two channels 214 each. The provision of two channels 214 for accommodating a total of two sample tubes 3 at the same time not only advantageously increases the sample throughput, but also enables a balanced loading of the centrifugation and mixing unit 21, which ensures a uniform rotational movement particularly in a centrifugation process step. As shown, the at least one channel 214, or here the two channels 214, can preferably run obliquely with respect to their longitudinal axis relative to the axis of rotation 212 of the centrifugation and mixing unit 21, with the end of the channel 214 facing away from the axis of rotation 212 preferably being closer to the bottom 251 of the device 2 is arranged as the end of the channel 214 facing the axis of rotation 212. The channel or channels 214 preferably include at least one window 2141 which is permeable to electromagnetic radiation. and mixing unit 21 facing away from the end or the channel 214 can be arranged. The opening of the window or windows 2141 is preferably aligned at the end of the respective channels 214 in such a way that the electromagnetic radiation emitted by the sample can reach the analysis means 22 with as little impediment as possible. The channel(s) 214 can also include at least one heating element 2142, with the heating element(s) 2142 preferably being arranged at one end of the channel 214 facing away from the axis of rotation 212 of the centrifugation and mixing unit 21. If a sample tube 3 is inserted into a channel 214 via an insertion opening 213, the section of a body 32 of the sample tube 3 which is located beyond the filter 33 with respect to a cover 31 of the sample tube 3 and in which a mixture is present after filtration is removed in this way of sample solution and a test substance 5 is in contact with the heating element(s) 2142 and can be heated or cooled in a controlled manner. Peltier elements can preferably be used as heating elements 2142 , which can be controlled in particular via the control unit 24 . A plurality of heating elements 2142, which are arranged distributed over the length of the at least one channel 214, also advantageously make it possible to control the temperature of the sample in the sample tube 3 at different points. In this context, an embodiment of the device 2 according to the invention has proven particularly useful, in which, in addition to the heating element 2142 at one end of the channel 214 facing away from the axis of rotation 212 of the centrifugation and mixing unit 21, a further heating element 2142 is installed in the middle of the channel 214, is arranged approximately at the level of the filter 33 of a sample tube 3 introduced into the channel 214 . A device 2 configured in this way is shown in FIG. 6 by way of example. The heating element or elements 2142 can also be cooled outwards with respect to the channel 214, ie in the direction of the interior of the device 2, by a cooler, in particular by a passive heat sink (not shown).

The device 2, in particular the centrifugation and mixing unit 21, can also preferably include a motor 211 which is set up to rotate the centrifugation and mixing unit 21 both clockwise and counterclockwise about the axis of rotation 212. The motor 211, controlled by the control unit 24, can implement different movement modes of the centrifugation and mixing unit 21: for example, a rapid rotation in the same direction about the axis of rotation 212 to implement a centrifugation step within an operating method of the system 1; a moderate to fast, uniform and/or non-uniform change in direction of the rotational movement, so that mixing of a sample located in a sample tube 3, which is located in one of the channels 214, is induced and a mixing step within an operating process (in the sense of a mixing by a vortex mixer).

The exposure means 23, which is arranged on the bottom 251 of the respective device 2 in the figures shown, preferably emits electromagnetic radiation with wavelengths in a range from 480 to 560 nm, preferably in a range from 485 to 540 nm, particularly preferably with a Wavelength of 488 nm. Alternatively or in addition to this, the exposure means 23 can also emit electromagnetic radiation with wavelengths in the range from 610 to 680 nm, preferably from 640 to 675 nm, particularly preferably with a wavelength of 650 nm. The exposure means 23 can also be arranged on one or more of the walls 25 of the device 2 or on an inside of a cover (not shown). In addition, the exposure means 23 can be arranged centrally within the device 2 or also in an edge area. In particular when the cover is closed and the inner walls of the device 2 are of a suitable, particularly reflective design, the exposure means 23--because of the advantageously compact design of the device 2 according to the invention--enables optimal exposure regardless of its or their positioning within the device 2 of the sample in the sample tube, in particular via the window 2141 of the channel or channels 214. The analysis means 22 can be designed as a detector for electromagnetic radiation, preferably as a fluorescence detector. It is controlled by the control unit 24, records in particular fluorescence signals during a PCR cycle or after each individual PCR cycle and, in cooperation with the control unit 24, advantageously evaluates the measurement data obtained directly. Using at least one means for data transmission (not shown here), the measurement data and/or the evaluation results can then be transmitted within device 2 from analysis means 22 to control unit 24 and vice versa, and from device 2 to an external receiver, e.g. an application on a external computer, a cloud or a smartphone. The means for data transmission can be designed as a means for data transmission via cable, such as in particular a USB connection, and/or preferably as a means for wireless data transmission, such as in particular a Bluetooth interface or a WLAN interface . FIG. 2 shows an embodiment of a system 1 according to the invention without a cover consisting of a device 2 of the embodiment from FIG. 1 and a sample tube 3 in a side view. FIG. 4 shows the system 1 from FIG. 2 in a plan view.

A system 1 according to the invention for quantitative real-time PCR analysis (qPCR), in particular for reverse transcriptase qPCR (qRT-PCR), consists of a device 2 as previously described and at least one sample tube 3, with a body 32 of the sample tube 3 is divided into two sections by at least one filter 33, and the section of the body 32 located beyond the filter 33 with respect to a cover 31 of the sample tube 3 comprises a test substance 5 (cf. also the illustrations of the sample tube 3 in Fig. 5a to d). The test substance 5 is preferably, depending on the application, ie depending on the RNA or DNA sample to be examined, a commercially manufactured test assay approved by the relevant approval authorities, including a fluorescence marker. The test substance 5 can be present in the form of a liquid, but also as freeze-dried granules or powder.

An exemplary sequence of a method according to the invention for quantitative real-time PCR analysis (qPCR), in particular for reverse transcriptase qPCR (qRT-PCR), with a system 1 as described above, will now be described with reference to FIG. 5, among others. However, the procedure described here as an example can be transferred to other molecular-biological analysis techniques for determining the presence of certain genetic sequences in a material, such as LAMP, TMA and/or CRISPR tests, since these analysis methods are only affected by the test substance 5 and used distinguish the temperature program to be carried out from each other.

Fig. 5 shows in the sub-figures a to d, each embodiment of a sample tube 3 in the various phases of sample preparation.

To carry out a quantitative real-time PCR analysis (qPCR) and other molecular biological analysis techniques to determine the presence of specific genetic material sequences in a material, in particular a reverse transcriptase qPCR (qRT-PCR), to carry out a LAMP, a TMA and/or a CRISPR analysis, with a system 1 according to the invention, the sample is initially unprocessed in a Introduced sample tube 3, mixed with a solvent 6 and then closed with a lid 31. 5a to 5c illustrate these steps schematically. The sample can be taken with the help of a sterile swab 4 by the subject himself or by a third party via a nose and/or throat swab. The swab 4 preferably has a predetermined breaking point just above a sample receiving area, for example a wad of cotton. After the sample has been taken from the nose and/or throat, the sample receiving area of the swab 4 is then broken off into the sample tube 3 and falls there onto a filter 33, which can be either removable or fixed in the body 32 of the sample tube 3. 5 shows a removable filter 33 in the form of a so-called filter basket. The solvent 6 can simply be dripped onto the sample using a syringe and/or a pipette, as shown in FIGS. 5b and 5c. As an alternative to this, the solvent 6 can also already be in the sample tube 3 in the area of the filter 33 (not shown here). For this purpose, an embodiment has proven itself, for example, in which the solvent 6 is placed in a capsule in the area of the filter 33 and caused to burst when the sample tube 3 is closed with the lid 31 and/or by the insertion of the swab 4 into the sample tube 3 is, so that the solvent can occur from 6. The pore size of the filter 33 is preferably selected in such a way that the liquid does not drip through the filter 33 due to gravity alone. After the sample tube 3 has been closed with the lid 31 , it is picked up by a centrifugation and mixing unit 21 of the device 2 . For this purpose, the sample tube 3 can advantageously simply be pushed into a channel 214 by the user via an insertion opening 213 .

All the method steps that now follow are carried out automatically by the device 2 according to the invention and advantageously do not require any further manual intervention by the user.

After pressing a start button, the device 2 checks the correct positioning of the sample tube 3 within the channel 214, for example by the signal of a light barrier or by the contact of the sample tube 3 with a mechanical stop in the channel 214, and then mixes the sample in the sample tube 3 through the centrifugation and mixing unit 21 with the solvent 6. For this purpose, the motor 211 of the centrifugation and mixing unit 21 preferably moves it back and forth (by quickly changing the direction of rotation). The solvent volume 6 located in the area of the swab 4 on the filter 33 then dissolves the sample from the sample receiving area of the swab 4 , but preferably does not drip through the filter 33 yet. The sample-solvent mixture obtained in this way is now centrifuged by the centrifugation and mixing unit 21, with the sample solution due to the centrifugal force acting through at least one filter 33 in a section of a body 32 located beyond the filter 33 with respect to the cover 31 of the sample tube 3 of the sample tube 3 migrates and is thereby filtered. For this purpose, the motor 211 of the centrifugation and mixing unit 21 preferably moves it rapidly in one direction of rotation.

7a and 7b show two further configurations of a sample tube 3, each in a longitudinal section.

The sample tubes 3 shown in FIGS. 7a and 7b are in these configurations in several parts (here in two parts), with at least one upper section 321 and at least one lower section 322 that can be reversibly fastened to the upper section 321. The reversible connection of the upper 321 and lower 322 sections can be, for example, a plug-in and/or screw connection.

As shown in FIG. 7a, the lower section 322 can have a tapered funnel area 323 in its interior, the larger diameter of which is arranged close to the filter 33. FIG. However, said funnel area 323 can also be designed (with and without filter 33) as a separate adapter, so that the body 32 of the sample tube can then consist of three sections that can preferably be reversibly fastened to one another.

Such a funnel area 323 advantageously prevents the formation of bubbles in the sample solution (solvent 6 with sample dissolved therein) when said solution flows down into the section of the body 32 beyond the filter 33 with respect to the lid 31 of the sample tube 3, in which the test substance is 5 is located. A reduction in the number of bubbles in the sample mixture examined (solvent 6, sample and test substance 5) advantageously increases the accuracy of the measurement of the electromagnetic radiation, in particular the fluorescence measurement. The sample solution filtered in this way is then mixed by means of the centrifugation and mixing unit 21 with a test substance 5 located in the section of the body 32 beyond the filter 33 with respect to the cover 31 of the sample tube 3, again preferably by a rapid process initiated by the motor 211 Changing the direction of rotation of the centrifugation and mixing unit 21.

Alternatively, the interior of the lower section 322 can also be divided into a collection area 325 for a prepared sample 7 and an analysis area 326, with the collection area 325 being connected to the analysis area 326 via at least one feed opening 327 (cf. Fig. 7b ). 7b shows a situation in which a prepared sample 7, consisting of the solvent 6 and the sample already dissolved out of the swab 4, was filtered through the filter 33 and then flowed into the collection area 325. An upper partition wall 329 can advantageously prevent the processed sample 7 from flowing directly into the analysis area 326 via the feed opening 327 . The test substance 5 is located in the analysis area 326. As shown, the collection area 325 can preferably be formed in that part of the interior of the lower section 322 of the sample tube 3 has at least one lower partition 330 and at least one feed wall 328 apart from a feed opening 327 from the Analysis area 326 is separated. The supply wall 328 can also run approximately cylindrically inside the lower portion 322 of the sample tube 3 and thus form a type of tube through which the collecting area 325 is connected to the analysis area 326 via the feed opening 327 .

To feed the prepared sample 7 to the analysis area 326, a targeted force can then be exerted on the prepared sample 7 by means of the centrifugation and mixing unit 21 by means of a rotational movement at a predetermined rotational speed, so that a controlled amount of the prepared sample 7 can flow through the feed opening 327 is transferred to the analysis area 326. This is indicated by the two small arrows inside the lower section 322 in FIG. 7b. In this way, the volume of prepared sample 7 used for a subsequent analysis in the analysis area 326 can advantageously be metered.

Finally, depending on the desired type of analysis, the sample is subjected to thermal cycling or thermostatting and then to a fluorescence analysis as part of a quantitative ative real-time PCR analysis (qPCR) or a reverse transcriptase qPCR analysis (qRT-PCR) or another molecular biological analysis technique to determine the presence of certain genetic material sequences in a material (e.g. LAMP, TMA, or CRISPR test).

5

In Fig. 7a it is indicated by different hatching of the sample tube 3 that the body 32 of the sample tube 3 is preferably impermeable to electromagnetic radiation, in particular with the exception of a window area 324 in the section of the body 32 located beyond the filter 33 with respect to the cover 31 of the sample tube 3 for fluorescence fO zenz radiation. Said impermeability to electromagnetic radiation can be achieved either by selecting a radiation-impermeable material for the body 32 outside the window area 324 or by coloring the outer wall of the body 32 outside the window area 324, in particular black. The window area 324 can preferably extend in the lower section 322 over 15 2 to 4 mm, preferably over 3 mm, the longitudinal extent of the body 32 and include the entire circumference of the body 32 or only a certain angular section of the circumference of the body 32. A Such a configuration of the sample tube 3 advantageously prevents a reflection of the electromagnetic radiation, in particular the fluorescent radiation, and can also be used in the configurations of a sample tube 3 without a funnel area 323 shown in FIGS. 5a to 5d and 7b.

If the test substance 5 also contains a reverse transcriptase, in particular for carrying out a reverse transcriptase qPCR analysis (qRT-PCR), then thermocycling can include the following steps, for example: In a further method step, the analysis result can be transmitted to a smartphone application using a means for data transmission, the means for data transmission being a means for data transmission via cable, such as in particular a USB connection, and/or a means for wireless data transmission How, in particular, a Bluetooth interface or a WLAN interface can be designed.

If one of the isothermal examination methods is to be carried out, instead of the thermocycling process shown as an example, the sample can also be thermostated for a certain period of time before the analysis of the electromagnetic radiation is carried out, in particular as part of a fluorescence analysis. The term "thermostatization" is understood here to mean the creation of defined, constant temperature conditions before, during and/or after the analysis.

The present invention relates to a device 2, a system 1 and a method for quantitative real-time PCR analysis (qPCR) and other molecular-biological analysis techniques for determining the presence of specific DNA sequences in a material, in particular for reverse transcriptase qPCR analysis ( qRT-PCR) to perform a LAMP, a TMA and/or a CRISPR analysis. The device 2 according to the invention is characterized by at least one centrifugation and mixing unit 21 for receiving a sample in a sample tube 3, which is rotatably mounted with respect to an axis of rotation 212; and which is set up to mix, centrifuge, heat and cool the sample in a controlled manner in the sample tube 3 , and which is also set up to exchange control and/or measurement data with the control unit 24 . The system 1 according to the invention consists of a device 2 as described and at least one sample tube 3, wherein a body 32 of the sample tube 3 is divided into two sections by at least one filter 33 and the one located beyond the filter 33 with respect to a cover 31 of the sample tube 3 Section of the body 32 includes a test substance 5. The device 2 according to the invention and the system 1 according to the invention advantageously enable quantitative real-time PCR analyzes (qPCR) and other molecular-biological analysis techniques to be carried out quickly and automatically to determine the presence of certain genetic material sequences in a material, in particular reverse trans scriptase qPCR (qRT-PCR), LAMP, TMA and/or CRISPR analyses, and can also be used by users who are not trained in medical and/or laboratory diagnostics will. The compact and inexpensive design of the device 2 or the system 1 also advantageously enables the corresponding PCR test to be carried out at very different, in particular “non-medical” locations, such as, for example, in old people’s homes, authorities, restaurants and at airports , Etc.

List of reference symbols System for the qPCR analysis device

21 centrifugation and mixing unit

211 engine

212 axis of rotation

213 insertion opening

214 channel 2141 window

2142 heating element

215 contacting element

22 means of analysis

221 detector

222 fans

23 means of exposure

24 control unit

25 wall

251 floor 3 sample tubes

31 cover

32 body

321 upper section

322 lower section

323 funnel area

324 pane

325 containment area

326 analysis area

327 feed opening

328 feeder wall

329 upper partition

330 lower partition

33 filters

4 Swabs for nose/throat swab 5 Test substance (“assay”)

6 solvent (“lysis”)

7 prepared sample in solvent (6)

Claims

patent claims

1. Device (2) for quantitative real-time PCR analysis (qPCR) and other molecular biological analysis techniques to determine the presence of certain genetic sequences in a material, in particular for reverse transcriptase qPCR analysis (qRT-PCR), for performing a LAMP, a TMA and/or a CRISPR analysis, at least comprising

- At least one exposure means (23) for exposing a sample in a sample tube (3) with electromagnetic radiation;

- At least one analysis means (22) for the quantitative analysis of a sample in a sample tube (3) with regard to a content of DNA (sections); and

- A control unit (24) which is set up to exchange control and/or measurement data with the analysis means (22) and with the exposure means (23); characterized in that the device (2) - comprises at least one centrifugation and mixing unit (21) for receiving a sample in a sample tube (3),

- which is rotatably mounted with respect to an axis of rotation (212); and

- which is set up to mix, centrifuge, heat and cool the sample in the sample tube (3) in a controlled manner,

- And which is also set up to exchange control and/or measurement data with the control unit (24).

2. Device (2) according to claim 1, characterized in that the centrifugation and mixing unit (21) comprises at least one contacting element (215),

- Which can be reversibly brought into contact with at least one correspondingly designed, electric current-carrying contact point of the device (2). - And which is set up to supply electronic components which are arranged on the centrifugation and mixing unit (21) with electrical power.

3. Device (2) according to claim 1 or 2, characterized in that in the centrifugation and mixing unit (21) at least one channel (214) is formed, in which a sample tube (3) is inserted through an insertion opening (213) can be held in a defined position, with the longitudinal axis of the at least one channel (214) preferably being aligned at an angle to the axis of rotation (212) of the centrifugation and mixing unit (21), the end of the channel (214 ) is preferably arranged closer to the bottom (251) of the device (2) than the axis of rotation (212) facing the end of the channel (214).

4. The device (2) according to claim 3, characterized in that the at least one channel (214) comprises at least one window (2141) which is transparent to electromagnetic radiation, the window or windows (2141) preferably being on one of the axes of rotation (212) the end of the channel (214) facing away from the centrifugation and mixing unit (21).

5. Device (2) according to one of Claims 3 or 4, characterized in that the at least one channel (214) comprises at least one heating element (2142), the at least one heating element (2142) preferably being mounted on one of the axes of rotation (212 ) the end of the channel (214) facing away from the centrifugation and mixing unit (21).

6. Device (2) according to one or more of the preceding claims, characterized in that the device (2), in particular the centrifugation and mixing unit (21), comprises a motor (211) which is set up to centrifuge rifugation and mixing unit (21) to rotate both clockwise and counterclockwise about the axis of rotation (212).

7. Device (2) according to claim 6, characterized in that the axis of rotation (212) of the centrifugation and mixing unit (21) is operatively connected at one end to the motor (211) and at the opposite end rotatable in a cover of the device (2) is stored when the device (2) is closed with the cover.

8. Device (2) according to one or more of the preceding claims, characterized in that the exposure means (23) electromagnetic radiation with wavelengths in a range from 480 to 560 nm or preferably in a range from 485 to 540 nm or particularly preferably emitted with a wavelength of 488 nm and/or that the exposure means (23) emits electromagnetic radiation with wavelengths in the range from 610 to 680 nm or preferably from 640 to 675 nm or particularly preferably with a wavelength of 650 nm.

9. Device (2) according to one or more of the preceding claims, characterized in that the analysis means (22) is designed as a detector for electromagnetic radiation, preferably as a fluorescence detector.

10. Device (2) according to one or more of the preceding claims, characterized in that the device (2) comprises at least one means for data transmission, wherein the means for data transmission as a means for data transmission via cable, such as in particular a USB port, and / or as a means for wireless data transmission, such as in particular a Bluetooth interface or a WLAN interface is formed.

11. System (1) for quantitative real-time PCR analysis (qPCR) and other molecular biological analysis techniques for determining the presence of certain genetic sequences in a material, in particular for reverse transcriptase qPCR (qRT-PCR), for implementation a LAMP, a TMA and/or a CRISPR analysis consisting of

- A device (2) according to one or more of the preceding claims 1 to 10

- And at least one sample tube (3), wherein a body (32) of the pro benröhrchens (3) by at least one filter (33) in two sections is divided and with respect to a lid (31) of the sample tube (3) beyond the filter (33) located section of the body (32) comprises a test substance (5).

12. System (1) according to claim 11, characterized in that the body (32) of the sample tube (3) except for a window area (324) in relation to the cover (31) of the sample tube (3) beyond the filter (33) located Section of the body (32) is opaque to electromagnetic radiation, in particular to fluorescence radiation.

13. System (1) according to claim 11 or 12, characterized in that the sample tube (3) is multi-part, with at least one upper section (321) and at least one lower section (322 ), - wherein preferably the lower section (322) has a conically tapering funnel area (323) in its interior, the larger diameter of which is arranged close to the filter (33); or

- Preferably, the interior of the lower section (322) is divided into a collection area (325) for a prepared sample (7) and an analysis area (326), the collection area (325) being connected to the analysis area (326) over at least a feed opening (327) communicating with each other.

14. Methods for quantitative real-time PCR analysis (qPCR) and other molecular biological analysis techniques to determine the presence of certain genetic material sequences in a material, in particular for reverse transcriptase qPCR (qRT-PCR), to carry out a LAMP , a TMA and/or a CRISPR analysis, with a system (1) according to claim 11, in which

- a sample is introduced unprocessed into a sample tube (3),

- the sample

- by a solution already in the sample tube (3) (6) or - by adding a solvent (6) from the outside into the sample tube (3) with a solvent (6) and then closed with a lid (31),

- the sample tube (3) is received by a centrifugation and mixing unit (21) of the device (2),

- the sample in the sample tube is mixed with the solvent (6) by the centrifugation and mixing unit (21),

- the sample-solvent mixture thus obtained is centrifuged through the centrifugation and mixing unit (21),

- the sample solution migrating via at least one filter (33) into a section of a body (32) of the sample tube (3) located beyond the filter (33) with respect to the cover (31) of the sample tube (3) and being filtered as a result,

- The sample solution filtered in this way is now mixed by means of the centrifugation and mixing unit (21) with a test substance (5th ) is mixed,

- and then thermal cycling or thermostatting and fluorescence analysis as part of a quantitative real-time PCR analysis (qPCR) or another molecular-biological analysis technique to determine the presence of certain genetic material sequences in a material, in particular as part of a reverse transcriptase qPCR (qRT-PCR) LAMP, TMA and/or CRISPR analysis.

Method according to claim 14, in which the analysis result is transmitted to a smartphone application using a means for data transmission, the means for data transmission being a means for data transmission via cable, such as in particular a USB connection, and/or as a means for Wireless data transmission, in particular a Bluetooth interface or a WLAN interface is designed.