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WO2022214340A1 - Procédé de détection de virus actifs - Google Patents

Procédé de détection de virus actifs Download PDF

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Publication number
WO2022214340A1
WO2022214340A1 PCT/EP2022/058045 EP2022058045W WO2022214340A1 WO 2022214340 A1 WO2022214340 A1 WO 2022214340A1 EP 2022058045 W EP2022058045 W EP 2022058045W WO 2022214340 A1 WO2022214340 A1 WO 2022214340A1
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WO
WIPO (PCT)
Prior art keywords
viruses
sample
filter
amplification
transport medium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2022/058045
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German (de)
English (en)
Inventor
Tanja Maucher
Christian GRUMAZ
Franz Laermer
Katrin Luckert
Eva WEIMER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Robert Bosch GmbH
Original Assignee
Robert Bosch GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Robert Bosch GmbH filed Critical Robert Bosch GmbH
Publication of WO2022214340A1 publication Critical patent/WO2022214340A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • PCR polymerase chain reaction
  • a transport medium can be understood to mean a container and/or a fluid in which the sample is received.
  • the purpose of the transport medium is to temporarily preserve the sample as undisturbed as possible.
  • Some transport media such as eNATTM from the company COPAN, also preprocess the sample, in particular a lysis of biological cells in the sample.
  • the invention relates to a method for detecting active viruses using a device.
  • a biological sample is flushed through a filter of the device, the filter being designed to hold back human or animal cells.
  • the cells retained on the filter are lysed to release genetic material contained therein, in particular genetic material of viruses resident in the cells.
  • at least some of the nucleic acids of the viruses are amplified before, in a fourth step, the viruses are detected by detecting the amplified parts.
  • the third step and the fourth step can also be carried out in parallel, in particular as part of a quantitative real-time polymerase chain reaction ( qPCR ).
  • Active viruses are to be understood in particular as those viruses which are able to cause an infection, ie in particular can penetrate human or animal body cells (host cells for short) and can therefore be referred to as infectious viruses.
  • the viruses can be corona viruses, for example SARS corona viruses such as SARS-CoV-2.
  • a biological sample can be understood in particular as a sample comprising a body fluid such as blood, sputum, urine or a smear comprising human or animal cells.
  • the device can in particular be a microfluidic device, in particular a lab-on-a-chip device, in particular based on a microfluidic cartridge as described, for example, in DE 10 2016 222 075 A1 or DE 10 2016 222 072 A1.
  • the filter is designed to retain human or animal body cells, preferably depending on the intended sample, and preferably to allow entities or particles smaller in size than the size of the intact host cells to pass through.
  • This can be a pore filter which has a pore size tailored to retain the desired host cells.
  • the pores have a size between 0.1 and 7 microns, preferably between 0.1 and 5 microns, in particular to retain host cells of a size of about 10 microns and larger.
  • nucleic acids in particular ribonucleic acids (RNA for short) or deoxyribonucleic acids (DNA for short).
  • RNA ribonucleic acids
  • DNA deoxyribonucleic acids
  • a part of the nucleic acids means in particular a section, also called a sequence, of a nucleic acid, for example a DNA or RNA section.
  • the invention thus advantageously provides a detection method for viruses which specifically enables detection of viruses located in human or animal cells. Since viruses are generally considered to be infectious when they are able to penetrate host cells, the invention has the particular advantage that infectious viruses in a sample can be detected with significantly increased reliability. Because of the filter according to the invention, intact host cells with viruses potentially contained therein are retained, while virions floating freely in the sample and fragments of virions are advantageously removed prematurely from the detection method.
  • the filter with the cells retained on it is rinsed, in particular with a non-lysing rinsing medium such as a phosphate-buffered saline solution (PBS for short) or UTM® from the company COPAN.
  • a non-lysing rinsing medium such as a phosphate-buffered saline solution (PBS for short) or UTM® from the company COPAN.
  • PBS phosphate-buffered saline solution
  • UTM® phosphate-buffered saline solution
  • This rinsing medium particularly advantageously contains a proportion of 20-100 g/l, preferably 50 g/l of ammonium citrate (binary ammonium citrate or particularly preferably ternary or tribasic ammonium citrate), as this prevents gelling and sticking of the gelatine contained in the UTM®.
  • This rinsing has the advantage that further undesired components from the sample, in particular non-cell-bound
  • proteases such as proteinase K or surfactants/detergents such as Tween®, Triton®-X, and/or sodium lauryl sulfate (SDS) as well as further ammonium citrate (binary or preferably ternary NH4 citrate) can be used to prevent gelatin residues from the UTM® from gelling, be added. This aids in isolation of the nucleic acids from the disrupted host cells.
  • Parts of the nucleic acids can be amplified using a polymerase chain reaction, isothermal amplification, or a ligase chain reaction.
  • the duplicated parts can be detected in order to detect the viruses, in particular with the aid of fluorescence spectroscopy, with fluorescence probes (fluorophores) being able to be used to dock onto the duplicated parts.
  • the multiplication and the detection of the viruses can also be carried out in parallel, for example a quantitative real-time PCR.
  • the amplification can preferably be ended when a positive detection signal reaches a predetermined detection limit. This has the advantage that the process can be shortened in terms of time and resources can be saved.
  • the detection of the virus includes a detection of at least one virus-specific nucleic acid segment and at least one human- or animal-specific nucleic acid segment.
  • the part of the human or animal nucleic acids can preferably also be amplified before the detection, preferably in parallel with the amplification of the virus-specific nucleic acid parts.
  • the detection of genetic material of the host cell for example the detection of a "housekeeping gene", has the advantage Control function that actual host cells were included in the sample and retained on the filter.
  • the human-specific or animal-specific nucleic acid segment can be a nucleic acid segment induced by the virus, i.e. a nucleic acid segment which the infected cell produces or produces to a greater extent due to the infection by the active virus.
  • the induced nucleic acid segment is one or more segments of one or more messenger RNA (mRNA, German also messenger RNA), which can encode, for example, proteins, preferably of the interferon family or chemokines, which are produced by the infected host cells due to of infection are expressed more strongly, as for example in loannidis I, et al. Plasticity and virus specificity of the airway epithelial cell immune response during respiratory virus infection. J Virol. 2012 May;86(10):5422-36. doi:
  • the mRNA sections can be sections of the genes OASL2, RSAD2, CCL5, IFIT3 or IFI44L.
  • an at least 10-fold stronger expression can be expected relative to non-infected cells.
  • a detection signal for one or more of these induced nucleic acid sequences, which is at least 10 times stronger than in non-infected cells, can thus preferably serve as a strong indication of the presence of actually infected host cells.
  • the method can thus preferably provide three proofs, namely a first proof of a virus-specific nucleic acid segment as proof of viruses in the sample, a second proof of a first host cell-specific nucleic acid segment as proof of host cells in the sample and a third proof of a second host cell specific nucleic acid segment, wherein the second host cell-specific nucleic acid segment, corresponding to the virus-induced nucleic acid segment described above, is present in larger quantities in virus-infected host cells than in non-virus-infected host cells.
  • the third proof in particular by comparing the strength of the second proof with the strength of the third proof, a reliable indication for or against the presence of actually infected host cells in the sample can be obtained.
  • the three tests can preferably be carried out in parallel or (in part) one after the other, in particular with the aid of a (real-time) polymerase chain reaction with suitable primers for selecting the nucleic acid segments to be detected.
  • the sample is transferred into a non-lysing transport medium or mixed with a non-lysing transport medium before it is introduced into the microfluidic device or before it is rinsed through the filter.
  • the transport medium is a liquid such as phosphate buffered saline (PBS for short) or UTM®.
  • PBS phosphate buffered saline
  • UTM® Ultrathyroxine
  • Ammonium citrate can be added to the UTM transport medium in order to prevent the gelatin contained therein from gelling later in the process.
  • the non-lysing transport medium includes neutralizing bodies.
  • neutralizing bodies are to be understood as entities, in particular molecules, which can bind to surface features, in particular proteins, of virions and thus preferably impair or prevent their further function.
  • Surface features of virions mean parts of virions, in particular parts of an envelope, for example the capsid, or an outside of the virions, such as proteins or carbohydrates, which are located on a surface of the virions. Proteins of virions are generally to be understood as meaning proteins which are part of a virus particle.
  • this can be understood to mean proteins which are encoded by the genetic information of the virus or which are produced with the participation of the virus.
  • this means proteins which are arranged on or in a membrane or a capsid of the virion, hereinafter referred to as membrane proteins for short.
  • This has the advantage that binding sites of the virions are occupied by the neutralizing bodies, which preferably makes further interaction of these binding sites with other entities difficult or completely prevents them.
  • the surface features are preferably saturated by the neutralizing bodies. This is particularly advantageous when the surface features are parts that the virus needs to infect a human or animal, for example for docking and then for penetrating a human or animal cell.
  • An inventive addition of the neutralizing bodies to the transport medium, in particular in a soluble form thus has the advantage that the risk of the viruses present in the sample being significantly reduced for the user of the sample and handling the sample is thus significantly safer.
  • the subject matter of the invention is therefore also a transport medium for a sample containing human or animal cells, the transport medium comprising such neutralizing bodies for binding to surface features, in particular proteins of virions.
  • the term "transport medium” generally means a medium or means suitable for the transport of samples.
  • the transport medium can be a container, in particular a container made of glass or plastic.
  • the container is preferably suitable for transporting biological samples, in particular body fluids such as blood, sputum, urine or a smear.
  • the container is a microreaction vessel, for example a so-called Eppendorf tube, Eppi for short.
  • the transport medium can be a fluid, in particular a liquid.
  • the transport medium can comprise a phosphate buffered saline solution (PBS for short) or UTM® from the company COPAN.
  • PBS phosphate buffered saline solution
  • UTM® from the company COPAN.
  • the transport medium is preferably a non-lysing medium, ie a medium which in particular does not lyse human or animal cells and preferably stabilizes them.
  • UTM® also contains gelatine, which is obtained by adding ammonium citrate to the transport medium or subsequently to the lab-on- chip process can be rendered "harmless". This has the advantage that the cells remain in the sample and viruses contained in the cells are therefore not affected by the neutralizing bodies, while virions swimming freely in the sample are preferably inactivated by the neutralizing bodies.
  • the neutralizing bodies include peptides, in particular polypeptides or proteins, in particular also recombinant proteins, which bind to surface features, in particular proteins of virions.
  • the transport medium includes angiotensin-converting enzyme 2 (ACE2 for short) to simulate binding of corona viruses, in particular SARS-CoV or SARS-CoV2, to human cells and to block the spike protein receptor of the virions.
  • ACE2 angiotensin-converting enzyme 2
  • the neutralizing bodies comprise antibodies.
  • the antibodies can in particular be antibodies against surface features or surface molecules of virions, for example antibodies which bind to the spike protein of a corona virion, in particular to a spike protein of Sars-CoV2.
  • they can be antibodies of the isotope G (IgG), for example the monoclonal antibodies casirivimab or imdevimab or other therapeutic antibodies.
  • the neutralizing bodies comprise nanobodies.
  • Nanobodies also known as nanobodies, can be understood as meaning fragments of antibodies, in particular single-domain antibodies with a size between 12 and 15 kilodaltons, which are advantageous for use against Sars-Cov2, for example.
  • antibody fragments as described in Esparza, T.J., Martin, N.P., Anderson, G.P. et al.
  • High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme. See Rep 10, 22370 (2020). https://doi.org/10.1038/s41598-020-79036-Q.
  • the neutralizing bodies include organic or inorganic sulfur compounds, which can dock onto proteins of the virions.
  • the sulfur compounds can be compounds which release nanoscale sulfur in particular in aqueous solution, for example thiosulfates or thiocyanates in non-host cell lysing concentrations, in particular nanoscale sulfur clusters. It can also be elementary colloidal sulfur, which is formed, for example, from the decomposition of thiocyanates or thiosulfates in the form of sulfur nanoparticles.
  • the neutralizing bodies can comprise other organic or inorganic compounds which can dock onto proteins of the virions and preferably inactivate them.
  • the neutralizing bodies can preferably be attached to an inner wall of the vessel, for example to a side wall and/or to the bottom of the vessel, for example in the form of a coating. This has the advantage that virions in the sample are also fixed to the inner wall by binding to the bodies and are thus separated from the rest of the sample. Furthermore, it is advantageous that the neutralizing bodies can be reliably stored in advance in the vessel until a sample is introduced.
  • the neutralizing bodies can be immobilized covalently or non-covalently on the vessel.
  • the vessel can have a carboxylated surface, with the neutralizing bodies, in particular the bodies based on proteinaceous structures, being bound by means of peptide bonding via proteinaceous amino groups (NH2 groups).
  • further proteins can be immobilized on the vessel, for example in the form of a coating, with the further proteins having an affinity for the neutralizing bodies.
  • the affinity of the further proteins is preferably designed in such a way that the neutralizing bodies binding therein have an advantageous orientation after binding, in which a binding site remains accessible for the surface features of the virions.
  • the other proteins based on antibody-binding proteins known in the prior art can have, for example, a binding site for the Fc part of the antibody, see above that the antigen-binding parts (Fab parts) of the antibodies are freely accessible for binding to the surface features of the virions.
  • a non-covalent immobilization of the neutralizing bodies, in particular the bodies based on protein structures, can take place, for example, via adsorption.
  • the neutralizing antibodies can be partially or completely dissolved in the sample when the sample is added to the vessel.
  • the immobilization can be designed to be water-soluble, particularly in the case of non-covalent binding. In this way, free-swimming virions can be neutralized both on the walls of the vessel and directly in the sample.
  • the neutralizing bodies are bound to magnetic particles.
  • the magnetic particles can be nanoparticles with an iron-containing core and a plastic coating, such as in DOI: 10.1039/C7AN01424D (Paper) Analyst, 2017, 142, 4247-4256, which are described with capture molecules in the form of, for example, antibodies or are functionalized on membrane proteins of the virion-binding proteins (ACE2 for example in the case of corona viruses).
  • ACE2 virion-binding proteins
  • the invention also relates to a method for transporting a sample containing human or animal cells using a transport medium, the transport medium comprising neutralizing bodies for binding and preferably saturating surface features, in particular proteins of virions.
  • a magnet is used to separate the neutralizing bodies bound to magnetic particles from the rest of the sample.
  • FIG. 1 shows an exemplary embodiment of a device for carrying out the detection method according to the invention
  • FIG. 2 shows an embodiment of the invention
  • FIG. 3 shows a flowchart of an exemplary embodiment of the detection method according to the invention
  • FIG. 4 shows a flow chart of an exemplary embodiment of the transport method according to the invention.
  • FIG. 1 shows an exemplary embodiment of the device 200
  • FIG. 3 shows a flowchart for an exemplary embodiment of the method 500 according to the invention, which this device 200 can use for implementation.
  • the device 500 can in particular be based on a microfluidic cartridge, for example on a cartridge as described in DE 102016 222 075 A1 or DE 102016222 072 A1.
  • the device 500 has an input chamber 210 for inputting a sample 10 .
  • the sample 10 can be a body fluid with human or animal cells. For example, it is a smear from the nose or throat to detect infection with a virus, particularly infection with a coronavirus such as SARS-CoV-2.
  • the sample 10 may include a non-lysing transport medium such as UTM® or PBS to preserve cells contained therein.
  • the input chamber 210 is fluidically connected to a processing chamber 220 , the processing chamber 220 having a filter 221 through which the sample 10 can be rinsed in a first step 501 of the method 500 .
  • Filter 221 is designed to retain human or animal cells 11 (abbreviated as host cells above) as shown in Figure 1, while allowing a remainder of sample 10 to pass through filter 221 and be disposed of in a waste chamber (not shown).
  • the filter 221 has, for example, pores 222 with a pore size between 0.1 and 5 micrometers, since host cells have a size of about 10 to about 20 micrometers.
  • the filter 221 is a silica filter that is typically used in microfluidics and has an exemplary pore size of 1 micrometer.
  • the preparation chamber 220 can be connected to a buffer chamber 230, with washing buffers being stored upstream in the buffer chamber 230, for example.
  • the buffer chamber 230 can include a rinsing medium such as UTM® or PBS that does not lyse the host cells 11 in order to leave the retained host cells 11 intact and to remove all other components of the sample 10 from the filter 221 in the course of a post-rinse. If the UTM® medium is present, ammonium citrate (binary or particularly preferably ternary NH citrate) should also be added to the washing buffer to neutralize the gelatine contained in the UTM.
  • a second step 502 of the method 500 the retained host cells 11 are lysed, for example chemically using a conventional lysis buffer with added surfactants, and the nucleic acids of the viruses located in the host cells are released in the process.
  • proteases such as proteinase K can also be used.
  • parts, in particular sections, of nucleic acids of the viruses are duplicated, in particular via a (quantitative real-time) polymerase chain reaction or an isothermal amplification.
  • the amplification can take place in an amplification chamber 240 of the device 200 connected to the processing chamber 220, in which substances required for the reaction can be stored upstream, in particular a PCR bead including primers for the targeted amplification of genetic material of the viruses sought. Simultaneously (particularly in the case of a real-time PCR) or subsequently, the parts sought can be detected in a fourth step 504 of the method 500, in particular with the aid of fluorescence techniques, provided that the host cells 11 were actually infected with the viruses sought. In the case of simultaneous amplification and detection, as described above, the amplification can be terminated prematurely if a sufficiently strong detection signal is achieved.
  • the sample 10 can be transferred to a non-lysing transport medium via the filter before rinsing, for example before or during the transport of the sample 10 to the device 200.
  • Figure 2 shows an exemplary embodiment of the transport medium 100 according to the invention
  • Container 110 with a liquid 120 the sample 10 are introduced.
  • neutralizing bodies 130 for binding to proteins of virions are located in the vessel.
  • the transport medium 100 can be understood to mean the container 110 and/or the liquid 120 , both of which comprise the neutralizing bodies 130 .
  • the container 110 is a glass container or a plastic (polypropylene) container having a capacity between 0.2 and 10 milliliters (mL), such as an Eppendorf tube.
  • the liquid 120 can in particular be a non-lysing buffer, for example PBS or UTM®.
  • the neutralizing bodies 130 can be mixed with the liquid and/or, as described above, be covalently or non-covalently bound to a wall 111 of the container 110, for example as a water-soluble coating 131, which can be dried up after being dispensed as an aqueous solution has been raised.
  • the neutralizing bodies 130 can be peptides, proteins, antibodies, nanobodies or sulfur compounds which bind to surface features of the virions, in particular to membrane proteins, and thus preferably render the virions swimming in the sample harmless.
  • the neutralizing bodies 130 can also be bound to magnetic particles 140 in order to isolate them from the sample 10 with the aid of a magnet 40 .
  • a preferred number or concentration of neutralizing bodies depends on a typical virus concentration in the sample and a typical number of surface features of the virions to be bound. With an order of magnitude of 100 surface features per virion, which corresponds to a typical number of spike proteins on a SARS-CoV-2 virion, at least 10 12 bodies per milliliter can be used assuming a virus concentration between 10 8 and 10 10 per milliliter.
  • FIG. 4 shows a flowchart 600 of an exemplary embodiment of the method 600 according to the invention, which can be carried out with the exemplary embodiment of the transport medium 100 described above.
  • the transport medium 100 is provided.
  • the sample 10 is introduced into the transport medium 100 .
  • the neutralizing bodies 130 are mixed with the sample 10, in particular by adding the bodies 130 to the sample 10, by dissolving the bodies 130 from the coating 131 in the sample 10 or by dissolving the bodies stored upstream in the transport medium 100 130 when sample 10 is added.

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Abstract

L'invention concerne un procédé (500) de détection de virus actifs, notamment de coronavirus actifs, comprenant un dispositif (200), notamment un dispositif microfluidique (200). Dans une première étape (501), un échantillon biologique (10) est rincé à travers un filtre (221) du dispositif (200), le filtre (221) étant conçu pour retenir des cellules humaines ou animales (11). Dans une deuxième étape (502), les cellules (11) retenues sur le filtre (221) sont lysées pour libérer les acides nucléiques viraux présents dans les cellules (11). Dans une troisième étape (503), au moins des parties des acides nucléiques viraux sont copiées. Dans une quatrième étape (504), les virus sont détectés par détection des parties copiées.
PCT/EP2022/058045 2021-04-07 2022-03-28 Procédé de détection de virus actifs Ceased WO2022214340A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102021203409.8A DE102021203409A1 (de) 2021-04-07 2021-04-07 Verfahren zum Nachweis aktiver Viren
DE102021203409.8 2021-04-07

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WO2022214340A1 true WO2022214340A1 (fr) 2022-10-13

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DE102023208916A1 (de) * 2023-09-14 2025-03-20 Robert Bosch Gesellschaft mit beschränkter Haftung Verfahren und mikrofluidische Vorrichtung zur Aufbereitung einer Probe für eine Analyse biologischer Zellen, insbesondere zirkulierender Tumorzellen

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DE102016222075A1 (de) 2016-11-10 2018-05-17 Robert Bosch Gmbh Prozessiersystem und Verfahren zur Prozessierung einer mikrofluidischen Kartusche mit einer Prozessiereinheit
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