EP4118201A1 - Procédé de traitement d'un échantillon biologique et dispositif pour isoler des cellules d'un milieu de transport - Google Patents

Procédé de traitement d'un échantillon biologique et dispositif pour isoler des cellules d'un milieu de transport

Info

Publication number
EP4118201A1
EP4118201A1 EP21710470.2A EP21710470A EP4118201A1 EP 4118201 A1 EP4118201 A1 EP 4118201A1 EP 21710470 A EP21710470 A EP 21710470A EP 4118201 A1 EP4118201 A1 EP 4118201A1
Authority
EP
European Patent Office
Prior art keywords
transport medium
membrane filter
complexing agent
ammonium
ions
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.)
Pending
Application number
EP21710470.2A
Other languages
German (de)
English (en)
Inventor
Franz Laermer
Eva WEIMER
Tanja Maucher
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 EP4118201A1 publication Critical patent/EP4118201A1/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes

Definitions

  • the present invention relates to a method for treating a biological sample in a transport medium, for example in an Amies medium.
  • the present invention also relates to a device for isolating cells from the transport medium.
  • transport media for biological samples, for example for bacterial samples, have established themselves as clinical standards. These make it possible to transport patient swabs or patient samples to a diagnostic laboratory without damaging the bacteria in the transport medium or changing the bacterial composition in a falsified manner. Transport media create a controlled “feel-good atmosphere” for the pathogens so that they can, for example, be introduced into a special nutrient medium at their destination and can continue to multiply.
  • the bacterial multiplication in a bacterial culture is an important diagnostic parameter that makes statements about the identity, viability, proliferation rate and also about the antibiotic sensitivities of such germs removed from the patient.
  • the transport medium must therefore ensure that the later diagnosis is not falsified at this point, for example in that particularly sensitive, but possibly quite dangerous germs die prematurely and can then no longer be detected in the microbiological culture.
  • a widely used transport medium is the so-called Amies medium. Disclosure of the invention
  • the transport medium is in particular an Amies medium. Since Amies media exist in numerous variants, all media are understood as Amies media for the purposes of this process, which contain calcium ions, magnesium ions, hydrogen phosphate ions and dihydrogen phosphate ions in a composition in which at least one of the solubility products of calcium hydrogen phosphate, calcium dihydrogen phosphate, magnesium hydrogen phosphate and magnesium dihydrogen phosphate is exceeded will.
  • the method is particularly advantageously suitable for treating biological samples which are contained in an Amies medium in which all four solubility products are exceeded.
  • the Amies medium thus contains a suspension of alkaline earth metal hydrogen phosphates and / or alkaline earth metal dihydrogen phosphates, some of which are present undissolved as suspended matter.
  • the problem with these compounds is the fact that they are emulsifiers which are insoluble or only sparingly soluble in water themselves, but can very well bind water-like substances in their molecular structure.
  • Such salts are therefore often used as emulsifiers for water in fatty phases.
  • Calcium salts of this type in particular are used in foods, for example for emulsifying water in sausage products.
  • this effect leads to an undesired binding of free nucleic acids to the undissolved calcium salt and magnesium salt molecule clusters when the biological sample is lysed, so that the nucleic acids are subsequently no longer available for a PCR detection reaction and, since they can no longer be detected, must be considered lost.
  • the addition of the complexing agent has the effect that the poorly soluble alkaline earth metal hydrogen phosphates and / or alkaline earth metal hydrogen phosphates are converted into a dissolved form which no longer has the undesirable binding properties with respect to free nucleic acids.
  • the complexing agent is preferably added immediately after the Amies medium has been entered into a lab-on-chip system, which is provided for the analysis of the biological sample.
  • the complexing agent is particularly preferably stored upstream in the lab-on-chip system at a suitable location and in a suitable form, for example as a solid or as a liquid reagent.
  • a complexing agent presented as a solid can, for example, be dissolved on contact with the Amies sample and then convert the alkaline earth phosphates into a soluble form by complex formation.
  • the complexing agent preferably contains [NR 1 R 2 R 3 R 4 ] + ions, where R 1 , R 2 , R 3 and R 4 are independently selected from hydrogen and alkyl groups.
  • the complexing agent particularly preferably contains ammonium ions (NH4 + ). Ammonium ions form readily soluble diamminocalcium complexes and diamminomagnesium complexes with calcium ions and magnesium ions.
  • the complexing agent preferably contains at least one salt selected from the group consisting of ammonium hydrogen citrate (“binary ammonium citrate”), ammonium citrate (“ternary ammonium citrate”), ammonium thioglycolate, ammonium chloride, ammonium acetate, tetramethylammoniumthiamethylammonium acetate, tetramethylammoniumthiamethylammonium. While ammonium chloride can serve as a source of ammonium ions in a simple and inexpensive manner, the other ammonium salts and tetramethylammonium salts contained in the group provide additional anions which form a buffer system in the transport medium.
  • the transport medium is preferably adjusted to a pFI value in the range from 5 to 6 by virtue of the additives mentioned. This further promotes the dissolution of the poorly soluble alkaline earth salts, because at acidic pH there is a shift in the plyrogen phosphates to the more soluble dihydrogen phosphates, as the following reaction equilibrium shows:
  • the bacteria contained in the biological sample for example, still remain intact in this pPI range. You will be in this pPI area though possibly severely impaired in their vitality, but for subsequent molecular biological processes it is only important that the cells with undamaged cell membranes remain intact.
  • the setting of the pH is preferably carried out simultaneously with the addition of the complexing agent or in the form of the addition of the complexing agent, since with a suitable selection this can already have the above-mentioned buffer effect on its own.
  • the complexing agent also functions as a buffer. This applies, for example, to ammonium hydrogen citrate, the so-called “binary ammonium citrate" (NH ⁇ H-citrate, which automatically sets a pH value of 5 to 6.
  • ternary ammonium citrate which is (NH4) 3- citrate
  • the result is an approximately neutral pH value of about 7.
  • citric acid allows the desired pH range between 5 and 6 to be set in the combination as a buffer system.
  • a citric acid / citrate buffer and / or an acetic acid / acetate buffer and / or a thioglycolic acid / thioglycolate buffer is added to the transport medium.
  • These buffers are particularly suitable for setting a pH value in the range from 5 to 6.
  • the complexing agent is preferably ammonium hydrogen citrate, ammonium citrate or
  • Tetramethylammonium citrate and citric acid is added to this.
  • the buffer is to be an acetic acid / acetate buffer
  • the complexing agent is preferably ammonium acetate and acetic acid is added to this.
  • a thioglycolic acid / thioglycolate buffer is to be used, the complexing agent is preferably ammonium thioglycolate or tetramethylammonium thioglycolate and thioglycolic acid, which is also referred to as mercaptoacetic acid, is added to this.
  • the complexing agent contains only one of the salts suitable for buffer formation, i.e.
  • a hydrogen citrate, a citrate, an acetate or a thioglycolate, and the corresponding acid is then released from this by adding another organic acid.
  • Mandelic acid is particularly suitable for this.
  • the addition of a further organic acid such as mandelic acid to an already existing system of salt and corresponding acid can also be provided. It is very difficult to produce sufficiently chaotropic conditions in transport media, as would be required for binding nucleic acids to a filter frit, without diluting the sample very strongly.
  • the transport medium is first filtered through a porous volume filter after the complexing agent has been added. It is then filtered through a membrane filter with a pore size in the range from 0.2 ⁇ m to 2.0 ⁇ m.
  • the two filters are preferably combined in the form of a single filter stack stacked one on top of the other.
  • the pore size of the membrane filter is particularly preferably in the range from 0.4 ⁇ m to 1.2 ⁇ m and very particularly preferably in the range from 0.8 ⁇ m to 1.0 ⁇ m.
  • the specification of a numerical value for a pore size always relates to the membrane filter and not to the upstream porous volume filter. Due to this pore size, the membrane filter, in conjunction with the upstream porous volume filter, can hold back cells such as bacterial cells without clogging themselves. Other substances such as dissolved mucus or blood components or other impurities from the biological sample pass through the filter arrangement or are already retained by the volume filter without clogging the membrane filter.
  • the porous volume filter connected upstream of the membrane filter can in particular have a constant porosity or a sequence of porosity values that decrease towards the membrane filter. It enables suspended matter contained in the transport medium, such as activated carbon or sample contaminants, to be retained so that these cannot clog the membrane filter and only the cells from the biological sample collect on the membrane filter.
  • cells remaining on the membrane filter are freed from alkaline earth metal hydrogen phosphates, alkaline earth metal dihydrogen phosphates and impurities in the transport medium. Since these have been converted into a soluble form by the addition of the complexing agent, this can be achieved in a simple manner by washing using a washing buffer. In particular, a polysorbal / water mixture can be used for this purpose. Other residues of the transport medium and remaining small impurities such as mucus are washed down from the biological sample.
  • Suitable methods of lysing are, in particular, heating to a temperature of preferably more than 60 ° C. and / or the addition of a lysis medium such as, in particular, Octoxinol 9 and / or mechanical stress, in particular through the action of ultrasound.
  • these are preferably eluted from the membrane filter. This can take place both during the lysing and after the lysing. If the elution is to take place at the same time as the lysing, the lysis medium functions at the same time as the eluent. In a subsequent elution, the nucleic acids are rinsed out of the membrane filter by means of a suitable eluent after the completion of the lysis. After mixing the eluate with a PCR master mix, for example in the form of a lyophilized PCR bead presented in a lab-on-chip, a PCR amplification and detection reaction can then take place.
  • a PCR master mix for example in the form of a lyophilized PCR bead presented in a lab-on-chip
  • a device for isolating cells from a transport medium which has, one behind the other, a porous volume filter and a membrane filter with a pore size in the range from 0.2 ⁇ m to 2.0 ⁇ m.
  • the pore size of the membrane filter is preferably in the range from 0.4 to 1.2 ⁇ m and particularly preferably in the range from 0.8 to 1.0 ⁇ m.
  • FIG. 1 shows a flow chart of an exemplary embodiment of the method according to the invention.
  • Figure 2 shows a schematic representation of an embodiment of the device according to the invention.
  • a biological sample which is a bacterial sample in the form of a patient swab, is introduced into an Amies medium as a transport medium.
  • the Amies medium contains in the present embodiment
  • the Amies medium with the biological sample has been transported to a diagnostic laboratory, it is treated there by means of an exemplary embodiment of the method according to the invention.
  • the Amies medium with the biological sample is introduced 11 into a lab-on-chip system.
  • the complex-forming buffer mixture contains ammonium hydrogen citrate and citric acid in a mixing ratio by means of which the Amies medium is buffered to a pFI value in the range from 5 to 6.
  • the device 20 has a porous volume filter 21. Arranged immediately downstream of this is a porous membrane filter 22 which, in the present exemplary embodiment, has a pore size of 0.9 ⁇ m.
  • the Amies medium is introduced into the device 20 at an introduction position 23 on the volume filter 21.
  • the Amies medium is first filtered 13 through the volume filter 21. Agar and larger particles or sample contaminants are retained in the volume filter 21, while bacterial cells hold them back Easily pass through the volume filter and thus reach the membrane filter 22.
  • the Amies medium is then filtered 14 further through the membrane filter 22.
  • the bacterial cells of the biological sample remain on the membrane filter 22, while all soluble components of the Amies medium pass through the device 20 through the membrane filter 22 and a subsequent outlet position 24 as aqueous Exit solution.
  • a washing is carried out of the cells on the membrane filter 22 by a Tween ® water mixture (Polysorbal / water mixture) is introduced into the device 20 through the inlet point 23 /.
  • the cells are then lysed 16 by introducing a 0.1% strength by weight solution of Triton® X-100 (Octoxinol 9) into the device 20 through the introduction position 23.
  • the eluate is mixed with a PCR master mix in the form of a lyophilized PCR bead and a PCR detection reaction carried out 17. The process is then ended 18.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de traitement d'un échantillon biologique qui est contenu dans un milieu de transport. Au moins un agent complexant, qui forme des complexes avec des ions alcalino-terreux, est ajouté au milieu de transport. Le procédé peut faire intervenir un dispositif (20) qui présente successivement un filtre à volume poreux (21) et un filtre à membrane (22) ayant une taille de pores de l'ordre de 0,2 µm à 2,0 µm.
EP21710470.2A 2020-03-10 2021-03-08 Procédé de traitement d'un échantillon biologique et dispositif pour isoler des cellules d'un milieu de transport Pending EP4118201A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102020203035.9A DE102020203035A1 (de) 2020-03-10 2020-03-10 Verfahren zum Behandeln einer biologischen Probe und Vorrichtung zum Isolieren von Zellen aus einem Transportmedium
PCT/EP2021/055744 WO2021180628A1 (fr) 2020-03-10 2021-03-08 Procédé de traitement d'un échantillon biologique et dispositif pour isoler des cellules d'un milieu de transport

Publications (1)

Publication Number Publication Date
EP4118201A1 true EP4118201A1 (fr) 2023-01-18

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ID=74859934

Family Applications (1)

Application Number Title Priority Date Filing Date
EP21710470.2A Pending EP4118201A1 (fr) 2020-03-10 2021-03-08 Procédé de traitement d'un échantillon biologique et dispositif pour isoler des cellules d'un milieu de transport

Country Status (5)

Country Link
US (1) US20230357750A1 (fr)
EP (1) EP4118201A1 (fr)
CN (1) CN115190913A (fr)
DE (1) DE102020203035A1 (fr)
WO (1) WO2021180628A1 (fr)

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102016209904A1 (de) 2016-06-06 2017-12-07 Robert Bosch Gmbh Verfahren und mikrofluidische Vorrichtung zum Verarbeiten einer Probe biologischen Materials
WO2018213641A1 (fr) * 2017-05-17 2018-11-22 T2 Biosystems, Inc. Méthodes et systèmes de rmn pour une détection rapide d'espèces de candida

Also Published As

Publication number Publication date
WO2021180628A1 (fr) 2021-09-16
CN115190913A (zh) 2022-10-14
US20230357750A1 (en) 2023-11-09
DE102020203035A1 (de) 2021-09-16

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