WO2014111064A1

WO2014111064A1 - Method of manipulation with samples of biological material and a device for such manipulation

Info

Publication number: WO2014111064A1
Application number: PCT/CZ2013/000005
Authority: WO
Inventors: Jan WOLF; Jaroslav Beran; Jiri KOMAREK; Jozef KANIOK; Martin BILEK; Martin Konecny; Petr Zabka
Original assignee: Wolf & Danniel Sro; Technicka Univerzita v Liberci
Current assignee: Wolf & Danniel Sro; Technicka Univerzita v Liberci
Priority date: 2013-01-18
Filing date: 2013-01-24
Publication date: 2014-07-24
Anticipated expiration: 2015-07-18
Also published as: CZ201339A3; CZ304743B6; EP2945739A1

Abstract

Method of manipulation with samples of biological material, especially for molecular diagnostics or for isolation of nucleic acids and from them possible consequent detection of a certain genome, for example a pathogenic one, and/or diagnostics of genetically determined diseases of living organisms. The sample of biological material at the beginning of the process is inserted into the loading chamber (11) of the device for manipulation with samples of biological material, into whose storage chambers (12) all reagent chemicals necessary for the respective process separately one from another were deposited still before, while the following phases of the process are running inside the device without a direct manipulation of operating person with reagent chemicals, which are gradually mixed and react, at the same time the unwilling residues from reactions are deposited into the waste chamber (41), which is also situated inside the manipulation device and it is safely separated from other areas of the device, operating persons as well as environment.

Description

Method of manipulation with samples of biological material and a device for such manipulation

Technical field

The invention relates to the method of manipulation with samples of biological material, especially for molecular diagnostics or for isolation of nucleic acids and from them possible consequent detection of a certain genome, for example a pathogenic one, and/or diagnostics of genetically determined diseases of living organisms.

Next to this, the invention relates to the device for manipulation with samples of biological material, especially for molecular diagnostics or for isolation of nucleic acids and from them possible consequent detection of a certain genome, for example a pathogenic one, and/or diagnostics of genetically determined diseases of living organisms.

Background art

At present, molecular in vitro diagnostics based on a direct proof of the targeted DNA sequencing is one of the most progressively increasing segments in market of laboratory in vitro diagnostics. Traditionally it is being applied in diagnostics of tuberculosis, where it superseded a lengthy (3-9 weeks) and less sensitive (104 bacteria / 1ml of sample) microscopic examination, further in diagnostics of sexually transmitted diseases (chlamydial infections, syphilis) or in diagnostics of virus diseases with a low antibody response or too fast progression (viral hepatitis B and C, herpetic meningoencephalitis, cytomegalovirus). Yet more frequently the molecular diagnostics is utilised also in diagnostics of respiration infections (it was one of the first available tests for diagnostics of SARS) or nosocomial infections (methicillin resistant staphylococcus infection - MRSA) 2. One of shortcomings of molecular diagnostics is its considerable demand both as to personnel and devices. Though the methodics itself for molecular detection of infectious agents upon the present state-of-the-art lasts 2-4 hours, the methodics being so demanding, the laboratories are not able to optimise their workload, so that they provide the result within the specified period.

Today it is common (in the Czech Republic) that the results are delivered within 3-5 working days, which nevertheless does not enable the physicians a prompt and targeted intervention into a medical scheme and it results (among others) in an extreme use of antibiotics with its all negative consequences for the society, both economic in the form of high primary costs for a medical politics and epidemiological in the form of occurrence of resistance to common antibiotics at simultaneous occurrence of secondary costs for special antibiotics.

At present there are two types of molecular diagnostics available, so called closed type and opened type.

As a closed type such solutions are designated, at which the same producer delivers the diagnostic kits as well as the laboratory device itself. The laboratory device is designated for examination only with the diagnostic kits of the same producer, it cannot be used with diagnostic kits of other suppliers. An advantage of this solution for the customer is a greater responsibility which the producer assumes for the result of examination, a guarantee given by a good name of the producer's company, and in some cases a higher level of automatisation. Disadvantage are higher operational costs of such solution and a certain contractual dependence for the customer. A typical customer usually is a large laboratory with a high demand as to standardisation of methods and an automatic operation.-

An opened type of solution more meets the needs of smaller laboratories. It enables to buy some of laboratory devices freely available on the market and to combine it with a diagnostic kit of whatever other producer, possibly to develop own examination methods. The advantage is a contractual freedom and possibility to reduce the costs for one examination, disadvantage is a less quality customer's support from the part of producers and higher requirements as regards maintaining the required level in quality of the examinations performed.

Disadvantage of to date known solutions is the need of a trained personnel in laboratories, long periods in delivery of results, high price of device and high costs for one examination and especially risk of contamination of environment with hazardous substances, because dosing of reagent chemicals into the test tubes in laboratories is done manually using pipettes, etc.

The goal of the invention is to propose and create a method of manipulation and device for manipulation with samples of biological material, which would totally prevent a possible contact of an operating person with the sample or the reagent chemicals and would prevent whatever leakage of reactive agents outside the device for manipulation. Simultaneously the invention should enable transfer of molecular diagnostics from laboratories directly to the site where the patient is present, this is the Point of care testing (POCT) as a result of simplified manipulation with samples, when it is possible to process each sample independently upon preserving a laboratory quality and shortening of time till achieving the result to a possible minimum, which at present represents 2-4 hours.

Principle of the invention

The goal of the invention has been achieved by the method according to the invention, whose principle consists in that the sample of biological material at the beginning of the process is inserted into one chamber of a manipulation device, into whose other chambers all reagent chemicals necessary for the respective process separately one from another were deposited still before, while the following phases of the process are running inside the manipulation device without a direct manipulation of operating person with reagent chemicals, which are gradually mixed and react, at the same time the unwilling residues from reactions are deposited into the waste area, which is also situated inside the manipulation device and it is safely separated from other areas of manipulation device, operating persons as well as environment. This facilitates method of manipulation with samples of biological material and enables to perform such manipulation directly on site where the patient is present - point of care testing (POCT), instead in laboratories.

It is preferred, if the sample of biological material inside the manipulation device in its mixing chamber mixes with lysing enzyme and simultaneously or subsequently from the respective storage chamber of the manipulation device the lysis buffer is added and after the next mixing the mixture in the mixing chamber is subject to incubation, during which the solution of mixture is repeatedly mixed and lysis in the solution is running till a lysed mixture is created, subsequently to the lysed mixture from another storage chamber of the manipulation device a precipitation agent is brought, preferably ethanol or isopropanol, after which the lysed mixture with precipitation agent is subject to homogenisation and during reaction with precipitation agent the free nucleic acid agglutinates, i.e. its precipitation occurs. After precipitation the mixture is brought to a separating membrane arranged inside the manipulation device, through which it is filtrated into the waste area, created inside the manipulation device, where the separating membrane retains residues of cell structures and the precipitate nucleic acid, after which the residues of cell structures from separating membrane are removed by flushing with a washing buffer, being brought from another storage chamber of the manipulation device, after then the waste area is closed and to separating membrane from another storage chamber of the manipulation device the elution buffer is brought, which is left to act to the separating membrane and subsequently passes through the separating membrane into the reaction chamber of the manipulation device, while in the solution the corresponding nucleic acid from the examined sample of biological material is isolated. That device, that all reactions necessary to isolate the corresponding nucleic acid from the examined sample of biological material are running inside the manipulation device.

To speed up the process, the incubation of the mixture is running at a temperature higher than the ambient temperature, preferably 70 °C for a period of 10 to 30 minutes.

Further speeding up and improvement of quality of the process is achieved by increasing the temperature of separating membrane above the ambient temperature before the eluting buffer is brought, preferably to 70 °C for a period of 3 minutes.

After the process is initiated, to speed up the start and to reduce the number of storage chambers, the sample of biological material is brought into a mixing chamber, into which the lysing enzyme was situated before. Of the same reasons it is preferred, if the lysing enzyme is a part of the lysis buffer.

Accelerated passage of mixture through the separating membrane is achieved by action of a centrifugal force.

In the preferred embodiment the separating membrane is formed of an ion exchange membrane.

After isolation of nucleic acid in the reaction chamber a polymerase chain reaction may be running, while its results can be easily detected through the wall of reaction chamber, which is preferably transparent. In case of a non- transparent wall of reaction chamber the detection can be performed using radioactive radiation.

The goal of the invention has also been achieved through the device for manipulation with samples of biological material according to the invention, whose principle consists in that, in one enclosed unit it contains the storage chambers for mutually separated releaseable deposition of reagent chemicals, loading chamber for inserting the sample of biological material, to which there is assigned the mixing chamber, to whose outlet the filtration chamber can be assigned, to whose outlet through the separating membrane the waste chamber or reaction chamber can be assigned, while only the loading chamber is provided with openable and lockable lid for operating person. Taking into account, that all reactions are running in one enclosed unit without possible access to reagent chemicals and without necessity of such an access, such device enables to perform the molecular diagnostics directly on site of POCT without requirement for special space and specially skilled personnel.

In a general embodiment the storage and loading chambers are arranged in the storage section, which extends to the mixing section equipped with mixing chamber, to whose inlet individual chambers of the storage section may be optionally assigned, which can be coupled with opening means for releasing of its content into the mixing chamber, while to outlet of mixing chamber there can be assigned the inlet of filtration chamber of filtration section, to whose outlet there can be optionally assigned the waste chamber or reaction chamber of reaction section, the storage section, while the mixing section, filtration section and reaction section are formed of independent bodies, which are mutually connected, at the same time the connection enables mutual motion and secures tightness.

At this general embodiment two possibilities of particular embodiment are feasible, when the individual sections are connected either rotatably or slidably, while at least one of them is provided with means for coupling with drive, at least one of them is provided with a means for fixation of its position, the remaining sections are provided with means for transfer of motion from the section coupled with the drive.

In one of preferred embodiments the separating membrane is formed of ion exchange membrane.

For an easy opening the storage chambers and the loading chamber in the lower part are equipped with protrusions, to which there is assigned the opening means, which is preferably formed of knife-edge firmly mounted in the mixing chamber. Cutting of the lower part of the protrusion of corresponding chamber secures releasing of its content and its transport into the mixing chamber and in a case this is already opened, transport into further parts of the device.

Description of the drawing

The device for manipulation with samples of biological material according to the invention is schematically represented in the attached drawings, where Fig. 1 shows a longitudinal section through the first exemplary embodiment of device for manipulation with samples of biological material, Fig. 2 section A-A from the Fig. 1 , Fig. 3 section B-B from the Fig. 1 , Fig. 4 section C-C from the Fig. 1 , Fig. 5 view to the first exemplary embodiment of the device and Fig. 6 section through the second exemplary embodiment of the device.

Examples of embodiment

The device for manipulation with samples of biological material, especially for molecular diagnostics or for isolation of nucleic acids and from them possible consequent detection of a certain genome, for example a pathogenic one, and/or diagnostics of genetically determined diseases of living organisms will be described on examples of embodiment, which serve to explain the structure and function of the device, not restricting the solution to such embodiments only. The device for manipulation is illustrated and described in a vertical position, which does not preclude a possibility to be in a different position during its function. The device for manipulation comprises four basic sections, which in the represented embodiment are mutually rotatably connected. In the upper section there is created the storage section 1, which in direction downwards extends to the mixing section 2, which in direction downwards extends to the filtration section 3, under which the waste and reaction section 4 is positioned. Individual sections 1, 2, 3, 4 are mutually rotatably connected by means of locks 5, formed of protrusion 51 on the inner from sections being connected and a groove 52 in the outer from sections being connected. Individual sections 1 , 2, 3, 4 are simultaneously mutually sealed, e.g. by means of o-rings 6 represented in Fig. 1 or by means of other suitable manner. This prevents the reaction mixture to leak outside the specified space of the device or to surroundings and unwilling mixing of residues from reactions and a result of reactions.

In example of embodiment represented in Fig. 1 to 5 the storage section i is positioned in a sleeve created in the mixing section 2, so that the protrusion 51 of the lock 5 is created on circumference of the storage section 1_ and the groove 52 of the lock is created in the mixing section 2. The mixing section 2 on its outlet side is provided with a sleeve in which the filtration section 3 is positioned, so that the protrusion 51 of the lock is created on circumference of the filtration section 3 and the groove 52 of the lock is created in the mixing section 2. Filtration section 3 on its outlet side is provided with a sleeve, in which the waste and reaction section 4 is positioned, so that the protrusion 5j of the lock 5 is created on circumference of the waste and reaction section 4 and the groove 52 of the lock is created in the filtration section 3.

Especially if the device for manipulation with samples of biological material during manipulation are subjected to action of centrifugal force in direction from the storage section λ towards the waste and reaction section 4, advantageous is an opposite mutual arrangement of individual sections 2, 3, 4. That means that upon connection of the filtration section 3 of the waste and reaction section 4 the sleeve is created in waste and reaction section 4 and in this sleeve the filtration section 3 is positioned, so that the protrusion 51 of the lock is created on circumference of the filtration section 3 and the groove 52 of the lock is created in the waste and reaction section 4. Filtration section 3 on its inlet side is provided with the sleeve, in which the mixing section 2 is positioned, so that the protrusion 51 of the lock is created on circumference of the mixing section 2 and the groove 52 of the lock is created in the filtration section 3. Connection of the mixing section 2 and the storage section in this manner is already performed in the represented embodiment. Nevertheless this connection may be created also in opposite manner, that means the sleeve may be created on the storage section 1 in which the mixing section 2 is positioned. Protrusion 51 of the lock is then created on circumference of the mixing section 2 and the groove 52 of the lock is created in the storage section 2.

Above mentioned description of locks may be mutually combined as the need may be.

The storage section 1 comprises a loading chamber 11 for inserting of the examined sample of biological material, which is provided with an openable lid 110. The storage section 1 further comprises the storage chambers 12, of which in the represented embodiment are seven pieces and which serve for mutually separated releaseable deposition of reagent chemicals and after inserting reagent chemicals they are closed with a common fixed lid 120. The storage section 1_ in the represented embodiment is formed of upper cylindrical section, which downwards narrows into a lower cylindrical section, under which the loading chamber H is terminated with a drop-like protrusion 111 and individual storage chambers 12 are terminated with drop-like protrusions 1210 to 1270, whose wall in the preferable embodiment is made thinner. On circumference of the storage section 1. two positioning protrusions 13 are performed, which serve for positioning the device for manipulation with samples of biological material during diagnostics, for example in centrifuge.

As a result of its mounting the storage section Λ with its lower cylindrical section extends into inlet area of the mixing section 2. Inlet area of the mixing section 2 is formed of a cylindrical cavity 21 , in which there is positioned a sieve ^•22, in which the knife 23 is positioned extending with its edge between the droplike protrusions 112, 122 of chambers 11, 12 of the storage section 1 and serving for cutting their ends. Around the cylindrical cavity 21 on circumference of the mixing section the pinion 24 is performed. Under the cylindrical cavity 21 in the mixing section 2 the mixing chamber 25 is performed, which in direction downwards narrows up to outlet opening 26, which is performed in a bottom 27 of the mixing section 2. Around the outlet opening 26 in the lower side of the bottom 27 there is mounted a sealing, in the represented embodiment formed of an o-ring 6, which fits the lid 31 of filtration section 3 and prevents leakage of reagents from the mixing chamber 25, until its outlet opening 26 upon mutual rotating the mixing section 2 and filtration section 3 gets above the inlet opening 321 of filtration chamber 32.

Filtration section 3 in its principle features a cylindrical shape, while the filtration chamber 32 in the represented embodiment is formed of a cylindrical cavity and the remaining inner space is formed of a lightening cavity 33. In filtration chamber 32 at least one separating membrane 322 is positioned. In the represented embodiment in the filtration chamber 32 two separating membranes 322 are positioned, among which a separating annulus 323 is mounted. Separating annulus 323 according to the represented embodiment is also arranged above the upper separating membrane 322. In bottom of filtration chamber 32 an outlet opening 324 is performed.

Waste and reaction section 4 comprise a waste chamber 41 , which in the represented example of embodiment features a shape of an enlarged crescent, and the reaction chamber 42, which is formed of a cylindrical cavity narrowing in downwards direction, while the lower part of reaction chamber 42 protrudes from a system of the device for manipulation downwards, its wall is made thinner and from all sides is freely accessible both for optical and for other evaluation means.

On circumference of waste and reaction section 4 two protrusions 13 are created which serve for positioning the device for manipulation with samples of biological material during diagnostics, for example in a centrifuge. For control of mutual position of individual sections 1 , 2, 3,. 4 of the device during manipulation and diagnostics there serves the pinion 24 on the mixing section, which in a known manner may be coupled with a not represented control tooth wheel or worm gear, while the storage section 1_ and waste and reaction section 4 feature a stable position, in the represented embodiment given by engaging their circumferential protrusions 13 into corresponding recesses in the not represented bushing. The mixing section 2 is provided with protrusion 28, in embodiment according to Fig. 1 to 5 with a circumferential protrusion, which extends into the groove 34 finished with a stop 341 , which specifies angle of rotation of the mixing section 2 towards the filtration section 3. In embodiment according to Fig. 1 to 5 the groove 34 is created on circumference of the filtration section 3. A basic position of filtration section 3 towards the waste and reaction section 4 is specified by the protrusion 35, which extends into a recess between a pair of stops 44, which at the embodiment according to Fig. 1 to 5 are created on circumference of the waste and reaction section 4 similarly like between them extending protrusion 35 created on circumference of filtration section 3. In direction of rotation of filtration section 3 behind a pair of stops 44 in a track of protrusion 35 of filtration section on the waste and reaction section 4 the end stop 45 is situated, which after engagement of protrusion 35 of filtration section 3 finishes a mutual rotation of filtration section 3 and the waste and reaction section 4 in the end position, in which the outlet opening 324 of filtration section 3 is to be found above the reaction chamber 42.

Embodiment of positioning protrusions, grooves and stops represented in Fig. 1 to 5 is exemplary and serves to explain the structure and function of the device, and not to its restriction. Protrusions, grooves and stops may be performed in any other suitable manner.

In second example of embodiment represented in the Fig. 6 the device for manipulation with samples of biological material differs from the previous embodiment by another particular connection of the mixing section 2, filtration section 3 and waste and reaction section 4. The mixing section 2 on its outlet side is provided with a sleeve, in whose cylindrical cavity the filtration section 3 is rotatably mounted by its inlet section. Outlet side of filtration section 3 is rotatably mounted in a sleeve created on inlet side of the waste and reaction section 4, while this sleeve of filtration section 3 outreaches up to the mixing section 2, which it embraces and which is mounted inside it with its outer circumference. Protrusion 51_ of the lock is created on circumference of the mixing section 2 and the groove 62 of the lock is created in the waste and reaction section 4. The filtration section 3 is mounted inside the mixing section 2 and the waste and reaction sections 4 without locks. This embodiment is more simple and less expensive as to manufacture.

Moreover, the waste and reaction section 4 is provided with auxiliary waste groove 43, which serves for trapping of possible residues from reactions, in case they get between bottom of the filtration section 3 and upper surface of waste and reaction section 4.

The device for manipulation with samples of biological material has been designed for carrying out a quick and effective diagnostics of infectious and genetically determined diseases of a human being in the POCT (Point of care testing) mode in premises with fully automatic operation in all phases of diagnostics, simple attendance and high safeness for operating personnel as well as environment. The device for manipulation with samples of biological material is in advance filled with all necessary reagents, which are enclosed in individual storage chambers 12 of the storage section Λ . The below mentioned example describes a device for manipulation with samples of biological material with seven storage chambers 12 and one loading chamber 1_1 in the storage section

The operating person inserts a sample of biological material into the loading chamber 11. and closes the loading chamber with the openable lid 111.

In the first storage chamber 121 the lysing enzyme, e.g. proteinase K is stored.

In the second storage chamber 22 there is stored the lysis buffer, that is formed of sodium dodecylsulphate and chelating agents.

In the third storage chamber 23 a precipitation agent, in this particular case, ethanol is stored. The fourth storage chamber 124 in the described example of embodiment is empty.

In the fifth storage chamber 125 there is stored the first washing buffer, in the described exemplary embodiment formed of seventy percent ethanol and stabilising ions.

In the sixth storage chamber 126 there is stored the second washing buffer, in the described exemplary embodiment formed of seventy percent ethanol and stabilising ions.

In the seventh storage chamber 127 there is stored elution buffer, which is formed of solution with a low concentration of ions Tris- hydroxymethylaminomethane + chelating agent EDTA (ethylenediaminetetraacetic acid).

In a basic initial position the filtration chamber 32_ with its outlet opening 321 is above the waste chamber 41 , while the inlet opening 321 of filtration chamber 32 is outside the outlet opening 26 of mixing chamber 25, so that the mixing chamber 25 is closed and sealed with sealing, which in the represented embodiment is o-ring 6. Track of the knife 23 intersects the drop-like protrusions 1210 to 1270 and in the basic initial position in rotation direction it is to be found before the drop-like protrusion 111 of loading chamber 1JL Position of the device for manipulation with samples of biological material is given by position of positioning protrusions 13 of the storage section and positioning protrusions 13 of waste and reaction section into a respective recesses in the not represented centrifuge, while in the represented and described embodiment the positioning protrusions 13 prevent rotation of the storage section 1 and rotation of the waste and reaction section 4. The mixing section 2 and filtration section 3 upon manipulation with samples of biological material rotate, at the same time the torque is transferred to them by means of a pinion 24.

Once the sample of biological material is inserted into the loading chamber 1J. of the storage section 1_ and the openable lid 111 is closed, the device for manipulation with samples if biological material is positioned into an apparatus, in which the whole diagnostic process is running and which usually is centrifuge. Once inserted, the pinion 24 is coupled with a known driving member of a not represented drive, e.g. with worm gear or toothed wheel. Individual sections of the device for manipulation with samples of biological material are in initial position, in which the knife is to be found before the protrusion 111 of loading chamber 11_, the outlet opening 26 of mixing chamber 25 is closed by the lid 3J. of filtration section 3, and the outlet opening 324 of filtration chamber 32 is to be found above the waste chamber 41.

In embodiments represented in Fig. 1 and 6 all drop-like protrusions 111 , 1210 to 1270 are illustrated when they are not cut off and the knife 23 in Fig. 1 is represented schematically before one of the drop-like protrusions. It is a schematic illustration and individual steps of manipulation with samples will be described below including a gradual cutting off the drop-like protrusions.

In the first step, using the pinion 24 the mixing section 4 is turned by 45°, while the other sections are not moving. Thereby the lower part of the drop-like protrusion 110 of loading chamber 11. is cut off and its content formed of an examined sample flows into the mixing chamber 25, while the cut off lower part of the drop-like protrusion 110 is caught on the sieve 22. The outlet opening 26 has been shifted by 45° above the full part of the lid 31 of filtration section 3_and it is still closed. For a perfect transfer of the whole content of the loading chamber V\ into the mixing chamber 25 the centrifuge is shortly brought to rotation to cca 500 g.

In the second step, using the pinion 24 the mixing section 2 is turned by another 45°, while the other sections are not moving. Thereby the drop-like protrusion 1210 of the first storage chamber 121 in which the lysing enzyme is positioned, e.g. proteinase K, is cut off. Outlet opening 26 is shifted by another 45° above the full part of the lid 31 of filtration section 3_and its is still closed. Lysing enzyme, e.g. proteinase K is transferred into the mixing chamber 25, preferably under action of centrifugal force, and the device for manipulation with samples of biological material is heated to temperature higher than the ambient temperature, preferably to 70 °C, through which possibly precipitated components are dissolved.

In the third step, using the pinion 24 the mixing section 2 is turned by another 45°, while the other sections are not moving. Total turning of the mixing section 2 is 135°. Thereby the drop-like protrusion 1220 of second storage chamber 122 in which the lysis buffer is positioned, is cut off. Outlet opening 26 is shifted by another 45° above the full part of the lid 31 of filtration section 3 and it is still closed. Lysis buffer is transferred into the mixing chamber 25, preferably under action of centrifugal force, and after a complete mixing of examined sample of biological material with enzyme and lysis buffer the lysis process is started, this is degradation of cell walls and releasing of DNA into a solution.

During the following incubation of mixture at temperature higher than ambient temperature, preferably 70 °C for a period of 10 to 30 min, the solution is repeatedly mixed.

After incubation the temperature, at least in the middle section of the device for manipulation with samples of biological material, is reduced to laboratory temperature (20 to 25 °C).

In the fourth step, the mixing section 2 is turned by another 45° to total 180°. Thereby the drop-like protrusion 1230 of the third storage chamber is cut off and the precipitation agent, in the described example of embodiment ethanol, is transported from the third storage chamber 1230. Quality and speed for transporting the precipitation agent into the mixing chamber 25 is increased by rotating the centrifuge to cca 500 g. The mixture is thoroughly mixed. Subsequently a thorough homogenisation and mixing secures a correct bond of DNA to separating membrane 322, in described exemplary embodiment to ion exchange membrane. Outlet opening 26 has been shifted by another 45° above the full part of the lid 31 of filtration section 3_and it is still closed.

In the fifth step the mixing section turns by another 45°to total 225° and the knife cuts off the drop-like protrusion 1240 of the fourth storage chamber 124, which in the exemplary embodiment is empty. Outlet opening 26 of the mixing chamber 25 has been shifted by another 45° and it is positioned above the inlet opening 321 of filtration chamber 32, whose outlet opening 324 still is positioned above the waste chamber 4_1 of waste and reaction section 4, as the filtration section 3 was not moving till this time. Protrusion 28 on the mixing section 2 engaged the stop 341 at the end of the groove 34 in filtration section 3, through which these sections are connected and then they will be turning together. Lysing mixture homogenised with ethanol transfers from the mixing chamber 25 into filtration chamber to the separating membrane 322, under which the waste chamber 41 is to be found.

At subsequent centrifugation of 5000 to 15000 g, the lysing mixture is filtered through a separating membrane 322 into the waste chamber 41 of waste and reaction section 4, while the separating membrane 322 retains residues of cell structures and DNA.

In the sixth step by means of pinion 24 the mixing section 2 is turned again by another 45° to total displacement of 270°, and due to this the filtration section 3 turns by 45°, as it is carried away by the protrusion 28 and by the stop 341. At the same time the protrusion 35 on filtration section overcomes a force of a pair of stops 44, which till this time specified its position. Outlet opening 26 of the mixing chamber 25 is still to be found above the inlet opening 321 of filtration chamber 32 and the outlet opening of filtration chamber 32 is above the waste chamber 41 of waste and reaction section 4. The knife 23 cuts off the drop-like protrusion 1250 of the fifth storage chamber 125 and from it into the mixing chamber 25 the first washing buffer runs out, which flows through into the filtration chamber 32 above the separating membrane 322.

At centrifugation of 5000 to 15000 g the first washing buffer flows through the separating membrane 322 into the waste chamber 41 of waste and reaction section 4. Thereby the separating membrane 322 is washed and residues of cell structures except for DNA are eliminated.

In the seventh step by means of a pinion 24 the mixing section 2 is turned by another 45°, simultaneously the mixing section turns also the filtration section 3 by 45°. Outlet opening 26 of the mixing chamber 25 is still above the inlet opening 321 of filtration chamber 32, because the filtration section 3 is carried away by the mixing section 2^ Outlet opening 324 of filtration chamber 32 though turned by 45°still is to be found above the waste chamber 4JL The knife 23 cuts off the drop-like protrusion 1260 of the sixth storage chamber 126, from which the second washing buffer is released, which also flows through into the filtration chamber 32 above the separating membrane 322. The second washing buffer flows through the separating membrane 322 upon centrifugation of 5000 to 15000 g. Thereby the separating membrane 322 is washed once again, and the residues of cell structures except for DNA are definitely eliminated.

After then, at least the middle section of the device for manipulation with samples of biological material is heated above the ambient temperature, preferably to the temperature of 70 °C.

In the eighth step by means of pinion 23 the mixing section is turned by another 45° to total 360° and further turning of filtration section 3 with respect to the waste and reaction section 4 is realised. Outlet opening 26 of the mixing chamber 25 still is to be found above inlet opening 321 of filtration chamber 32. Outlet opening 324 of filtration chamber 32 through this turning by 45° shifts above the wall 40 between the waste chamber 41 and reaction chamber 42, on which it is sealed with the sealing ring 6. Upon turning the mixing section 2 the drop-like protrusion 1270 of the seventh storage chamber 127 is cut off, out of which after rotating the centrifuge to cca 500 g the elution buffer flows into the filtration chamber 32 to separating membrane 322. At incubation for a period of 3 minutes at temperature of 70 °C from the separating membrane 322 DNA is released into elution buffer. Incubation may be running also at a lower temperature, but for a longer time period.

In the ninth step another turning of the mixing chamber 2 by 45° is realised, and its total turning again is 45°. Simultaneously also the filtration section 3 turns by 45° so that the outlet opening 324 of filtration chamber 32 shifts above the reaction chamber 42. By centrifugation at 5000 to 15000 g the elution buffer with isolated DNA flows through the separating membrane 322 into the space of reaction chamber 42.

By further turning of the mixing chamber 2, in the tenth step, the outlet opening 324 of filtration chamber shifts above the waste chamber 41 and the reaction chamber 41 closes, so that the polymerase chain reaction (PCR) may run inside the chamber or the isolated DNA may be taken out of it for another use. Polymerase chain reaction runs in a standard mechanism of cyclic cooling and heating the mixture in reaction space.

In the same method isolation of RNA as well as general molecular diagnostics is performed. Lysing enzyme may be added into the loading chamber 11_ together with sample of biological material or vice versa the sample may be added into the loading chamber H to lysing enzyme. After then the first storage chamber is empty. Thus, the knife 23 in one step turns by 90°, so as to cut off protrusion of the loading chamber V\ with lysing enzyme and a sample and simultaneously a protrusion of empty storage chamber. This embodiment is obvious, therefore it will not be described hereinafter.

For increasing of accuracy it is suitable to remove from the separating membrane 322 residual content of liquids after washing the separating membrane 322 with a washing buffer and before closing the waste chamber 41 , so that any undesirable substances cannot get into the reaction chamber 42. This can be achieved by washing the separating membrane 322 with inert liquid and/or elution buffer in a quantity corresponding to a known residual content of liquids in separating membrane 322, which causes expulsion of these liquids from the separating membrane. At this embodiment the arrangement of content of loading chamber 11. and individual storage chambers is following:

Operating person inserts into the loading chamber 11 a sample of biological material together with lysing enzyme, e.g. proteinase K, and closes the loading chamber with the openable lid 111.

In the first storage chamber 121 lysis buffer is stored, which is formed of a mixture of sodium dodecylsulphate and chelating agents.

In the second storage chamber 122 the precipitation agent is stored, in particular case ethanol.

The third storage chamber 123 in the described exemplary embodiment is empty (in this embodiment the carrying away details on mixing chamber and mixing section are modified, they secure a joint turning of these two sections by one step earlier = 45 degrees).

In the fourth storage chamber 124 the first washing buffer is stored, in the described exemplary embodiment formed of seventy percent ethanol and stabilising ions. In the fifth storage chamber 125 the second washing buffer is stored, in the described exemplary embodiment formed of seventy percent ethanol and stabilising ions.

In the sixth storage chamber 126 there is stored inert liquid and/or elution buffer in a quantity corresponding to residual content of liquids in separating membrane.

In the seventh storage chamber 127 there is stored elution buffer, which is formed of a solution with low concentration of ions Tris- hydroxymethylaminomethane + chelating agent EDTA (ethylenediaminetetraacetic acid).

In the first step by means of pinion 24 the mixing section 4 is turned by 45°, while the other sections are not moving. Thereby the lower part of drop-like protrusion 110 of loading chamber H is cut off and its content formed of an examined sample and proteinase K flows into the mixing chamber 25. The outlet opening 26 of mixing chamber 25 has been shifted by 45° above a full part of the lid 3_1 of filtration section 3_and it is still closed. For a perfect transfer of the whole content of the loading chamber 11_ into the mixing chamber 25 the centrifuge is shortly brought to rotation to cca 500 g. The device for manipulation with samples of biological material is heated to temperature higher than the ambient temperature, preferably to 70 °C, through which possibly precipitated components are dissolved.

In the second step, using the pinion 24 the mixing section 2 is turned by another 45°, while the other sections are not moving. Thereby the drop-like protrusion 1210 of the first storage chamber 121 in which the lysis buffer is stored, is cut off. The outlet opening 26 is still closed. Lysis buffer is transferred into the mixing chamber 25, preferably under action of centrifugal force, and after a complete mixing of examined sample of biological material with lysing enzyme and lysis buffer the lysis process is started, this is degradation of cell walls and releasing of DNA into a solution.

During the following incubation of mixture at temperature higher than ambient temperature, preferably 70 °C for a period of 10 to 30 min, the solution is repeatedly mixed. After incubation the temperature, at least in the middle section of the device for manipulation with samples of biological material, is reduced to laboratory temperature (20 to 25 °C).

In the third step, using the pinion 24 the mixing section 2 is turned by another 45°, while the other sections are not moving. Total turning of the mixing section 2 is 135°. Thereby the drop-like protrusion 1220 of second storage chamber 22 in which the precipitation agent is stored, is cut off. Outlet opening 26 is shifted by another 45° above the full part of the lid 31 of filtration section 3 and it is still closed. Quality and speed of transport of the precipitation agent into the mixing chamber 25 is increased by rotating the centrifuge to cca 500 g. The mixture is thoroughly mixed. Subsequently a thorough homogenisation and mixing secures a correct bond of DNA to separating membrane 322, in described exemplary embodiment to ion exchange membrane. Outlet opening 26 has been shifted by another 45° above the full part of the lid 3J. of filtration section 3_and it is still closed.

In the fourth step, the mixing section 2 is turned by another 45° to total 180°. Thereby the drop-like protrusion 1230 of the third storage chamber is cut off, in the exemplary embodiment this chamber is empty. Outlet opening 26 of the mixing chamber 25 has been shifted by another 45° and it is to be found above the inlet opening 321 of filtration chamber 32, whose outlet opening 324 is still positioned above the waste chamber 41 of waste and reaction section 4, as the waste and reaction section 4 was not moving till this time. Protrusion 28 on the mixing section 2 engaged the stop 341 at the end of the groove 34 in filtration section 3, through which these sections are connected and then they will be turning together. Lysing mixture homogenised with ethanol transfers from the mixing chamber 25 into filtration chamber to the separating membrane 322, under which the waste chamber 41 is to be found.

In the fifth step the mixing section turns by another 45°to total 225° due to which the filtration section 3 turns by 45°, as it is carried away by the protrusion 28 and by the stop 341. Outlet opening 26 of the mixing chamber 25 is still to be found above the inlet opening 321 of filtration chamber 32 and the outlet opening of filtration chamber 32 is above the waste chamber 41 of waste and reaction section 4. The knife 23 cuts off the drop-like protrusion 1240 of the fourth storage chamber 124 and from it into the mixing chamber 25 the first washing buffer runs out, which flows through into the filtration chamber 32 above the separating membrane 322. At centrifugation of 5000 to 15000 g the first washing buffer flows through the separating membrane 322 into the waste chamber 41 of waste and reaction section 4. Thereby the separating membrane 322 is washed and residues of cell structures except for DNA are eliminated.

In the sixth step by means of pinion 24 the mixing section 2 is again turned by another 45° to total displacement of 270° and the mixing section turns also the filtration section 3 by 45°. Outlet opening 26 of the mixing chamber 25 is still above the inlet opening 321 of filtration chamber 32, because the filtration section 3 is carried away by the mixing section 2. Outlet opening 324 of filtration chamber 32 though turned by 45°still is to be found above the waste chamber 41. The knife 23 cuts off the drop-like protrusion 1250 of the fifth storage chamber 125, from which the second washing buffer is released, which also flows through into the filtration chamber 32 above the separating membrane 322. The second washing buffer flows through the separating membrane 322 upon centrifugation of 5000 to 15000 g. Thereby the separating membrane 322 is washed once again, and the residues of cell structures except for DNA are definitely eliminated.

In the seventh step by means of a pinion 24 the mixing section 2 is turned by another 45°, simultaneously the mixing section turns also the filtration section 3 by 45°. Outlet opening 26 of the mixing chamber 25 is still above the inlet opening 321 of filtration chamber 32, because the filtration section 3 is carried away by the mixing section 2. Outlet opening 324 of filtration chamber 32 though turned by 45°still is to be found above the waste chamber 41. The knife 23 cuts off the drop-like protrusion 1260 of the sixth storage chamber 126, from which inert liquid and/or elution buffer in a quantity corresponding to residual content of liquids in the separating membrane 322 flows out and flows through into the filtration chamber 32 above the separating membrane 322. This inert liquid and/or elution buffer flows through the separating membrane 322 at centrifugation of 5000 to 15000 g and expels the residual liquids in separation membrane 322, which after then cannot affect further reactions. Thus residues of cell structures except for DNA are completely eliminated.

The next steps, eight to ten, are performed in the same manner as at the earlier described method.

Industrial applicability

Method and device for manipulation with samples of biological material according to the invention are designed for molecular diagnostics or for isolation of nucleic acids and from them possible consequent detection of a certain genome, for example a pathogenic one, and/or diagnostics of genetically determined diseases of living organisms, for example for automatic isolation of DNA/RNA from a sample, automatic detection by qPCR method and automatic evaluation of the result of reaction outside specialised laboratories, this is on site directly - Point of care testing (POCT).

List of referential markings

storage section

I I loading chamber

110 drop-like protrusion of loading chamber 1 1 openable lid of loading chamber

12 storage chambers

120 fixed lid of storage chambers

121 first storage chamber

122 second storage chamber

123 third storage chamber

124 fourth storage chamber

125 fifth storage chamber

126 sixth storage chamber

127 seventh storage chamber

1210-1270 drop-like protrusions of storage chambers

13 positioning protrusions

2 mixing section

21 cylindrical cavity

22 sieve

23 knife

24 pinion

25 mixing chamber

26 outlet opening of mixing chamber

27 bottom of mixing section

28 protrusion

3 filtration section

31 lid of filtration section

32 filtration chamber

321 inlet opening of filtration chamber

322 separating membrane

323 separating annulus

324 outlet opening of filtration chamber

33 lightening cavity

34 groove

341 stop

35 protrusion

4 waste and reaction section

41 waste chamber

42 reaction chamber

43 auxiliary waste groove

44 pair of stops

45 end stop

5 locks

51 protrusion of lock

52 groove of lock

6 o-ring

Claims

1. Method of manipulation with samples of biological material, especially for molecular diagnostics or for isolation of nucleic acids and from them possible consequent detection of a certain genome, for example a pathogenic one, and/or diagnostics of genetically determined diseases of living organisms, characterised in that, the sample of biological material at the beginning of the process is inserted into the loading chamber (11 ) of the device for manipulation with samples of biological material, in whose storage chambers (12) all reagent chemicals necessary for the respective process separately one from another were deposited still before, whereas the following phases of the process are running inside the devices without a direct manipulation of operating person with reagent chemicals, which are gradually mixed and react, whereas the unwilling residues from reactions are deposited into the waste chamber (41), which is also situated inside the manipulation device and it is safely separated from other areas of the device, operating persons as well as environment.

2. Method according to the claim 1 , characterised in that, the sample of biological material are inside the device for manipulation with samples of biological material in its mixing chamber (25) mixed with lysing enzyme and simultaneously or subsequently from the respective storage chamber of the manipulation device the lysis buffer is added and after the next mixing the mixture in the mixing chamber (25) is subject to incubation, during which the solution of mixture is repeatedly mixed and lysis in the solution is running till a lysed mixture is created, subsequently to the lysed mixture from another storage chamber of the manipulation device a precipitation agent is brought, after which the lysed mixture with precipitation agent is subject to homogenisation and during reaction with precipitation agent the free nucleic acid precipitates, after precipitation the mixture is brought to a separating membrane (322) arranged inside the device, through which it is filtrated into the waste chamber (41) created inside the device, whereas the separating membrane (322) retains residues of cell structures and the precipitate nucleic acid, after which the residues of cell structures from separating membrane (322) are removed by flushing with a washing buffer being brought from another storage chamber of the device, after then the waste chamber (41) is closed and to separating membrane (322) from another storage chamber of the device the elution buffer is brought, which is left to act to the separating membrane (322) and subsequently passes through the separating membrane (322) into the reaction chamber (42) of the manipulation device, whereas in the solution the corresponding nucleic acid from the examined sample of biological material is isolated.

3. Method according to the claim 2, characterised in that, after flushing the separating membrane (322) with washing buffer and before closing the waste chamber (41) the separating membrane (322) is washed with inert liquid and/or elution buffer in a quantity corresponding to a known residual content of liquids in separating membrane (322).

4. Method according to the claim 2 or 3, characterised in that, the incubation of mixture in the mixing chamber (25) is running at a temperature higher than the ambient temperature, preferably at a temperature of 70 °C for a period of 10 to 30 minutes.

5. Method according to any of the claims 2 to 4, characterised in that, before the elution buffer is brought the separating membrane (322) is warmed to a temperature higher than the ambient temperature, preferably to 70 °C for a period of 3 minutes.

6. Method according to any of the claims 2 to 5, characterised in that, the sample of biological material, after the process is started, is brought into the mixing chamber (25) of the device in which the lysing enzyme was positioned already in advance.

7. Method according to any of the claims 2 to 5, characterised in that, the lysing enzyme is a part of the lysis buffer.

8. Method according to any of the claims 2 to 7, characterised in that, the passage of the mixture through the separating membrane (322) is facilitated by action of a centrifugal force.

9. Method according to any of the claims 2 to 8, characterised in that, the separating membrane (322) is an ion exchange membrane.

10. Method according to any of the claims 2 to 9, characterised in that, in the reaction chamber (42) there is running a polymerase chain reaction.

11. Device for manipulation with samples of biological material, especially for molecular diagnostics or for isolation of nucleic acids and from them possible consequent detection of a certain genome, for example a pathogenic one, and/or diagnostics of genetically determined diseases of living organisms, characterised in that, in one enclosed unit it contains the storage chambers (12) for mutually separated releaseable deposition of reagent chemicals, the loading chamber (11) for inserting the sample of biological material, to which there is assigned the mixing chamber (25), to whose outlet the filtration chamber (32) is assignable, to whose outlet through the separating membrane (322) the waste chamber (41) or reaction chamber (42) is assignable, whereas only the loading chamber (11 ) is provided for an operating person with openable and lockable lid (11 1).

12. Device according to the claim 11 , characterised in that, the storage chambers (12) and loading chamber (11 ) are arranged in the storage section (1), which extends to the mixing section (2) equipped with mixing chamber (25), to whose inlet individual chambers (11 , 12) of the storage section (1) are optionally assignable, which are able to be coupled with opening means for releasing of its content into the mixing chamber (25), whereas to outlet of mixing chamber (25) there is assignable the inlet of filtration chamber (32) of filtration section (3), to whose outlet there is optionally assignable the waste chamber (41) or reaction chamber (42) of reaction section (4), whereas the storage section (1), the mixing section (2), filtration section (3) and reaction section (4) are formed of independent bodies, which are mutually connected, at the same time the connection enables mutual motion and secures tightness.

13. Device according to the claim 12, characterised in that, the storage section (1), mixing section (2), filtration section (3) and reaction section (4) are mutually rotatably connected, whereas at least one of them is provided with means for coupling with a drive, at least one of them is provided with a means for fixation of its position, and the remaining sections are provided with means for transfer of rotation motion from the section coupled with the drive.

14. Device according to the claim 12 or 13, characterised in that, the mixing section (2) oh its outlet side is provided with a sleeve, in whose cylindrical cavity by its inlet section the filtration section (3) is rotatably mounted, whose outlet side is rotatably mounted in a sleeve created on inlet side of the waste and reaction section (4), whereas this sleeve of filtration section (3) outreaches up to the mixing section (2), which it embraces and which is mounted inside it with its outer circumference.

15. Device according to any of the claims 11 to 14, characterised in that, in the waste and reaction section (4) there is performed a waste groove (43) for trapping of possible residues from reactions in case they get between bottom of the filtration section (3) and upper surface of the waste and reaction section (4).

16. Device according to any of the claims 11 to 15, characterised in that, the separating membrane (322) is an ion exchange membrane.

17. Device according to any of the claims to 16, characterised in that, the storage chambers (12) and the loading chamber (11) in the lower part are provided with drop-like protrusions, to which the opening means is assigned.

18. Device according to the claim 17, characterised in that, the opening means is the knife (23) firmly mounted in the mixing chamber (25).