# US 11,999,936 B2 · Extraction system and method for extracting microorganisms contained in a sample

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- Number: US 11,999,936 B2
- Document ID: US11999936B2
- Status: Granted
- Kind: B2
- Application number: 16/461,007
- Filed: 2017-11-15
- Published: 2019-09-05
- Granted: 2024-06-04
- Earliest claimed: 2016-11-15
- Assignees: Biomerieux
- Applicants: BioméRieux
- Inventors: Patrick Broyer; Marie-Hélène Charles; Jérôme Blaze; Nadine Perrot; Frédéric Pinston; Hervé Rostaing
- Web page: https://patentgrep.com/patent/US11999936B2

## CPC

- C12M 33/14: {with filters, sieves or membranes}
- B01L 3/502: {with fluid transport, e.g. in multi-compartment structures}
- C12M 23/12: {Well or multiwell plates (C12M25/04 takes precedence)}
- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- G16B 20/00: ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- B01L 9/06: Test-tube stands; Test-tube holders
- G01N 1/4005: {by transferring a selected component through a membrane}
- G01N 2035/0436: {with pre-packaged reagents, i.e. test-packs}
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

An extraction system includes a support bar including a sample well, a reagent well, at least one washing well, and at least one protection and retention housing; a pipetting element removably mounted on the support bar and extending at least partially into the at least one protection and retention housing; and a filtering and transfer device configured to capture and accumulate microorganisms contained in the sample and transfer the captured and accumulated microorganisms, the filtering and transfer device being removably mounted on the support bar and extending at least partially into the at least one protection and retention housing. The filtering and transfer device includes a hollow body having an elongated shape and including a proximal end portion and a distal end portion; and a filtering element fixed to the distal end portion of the hollow body.

## Claims

### Claim 1

A method for identifying and/or characterizing microorganisms contained in a sample, the method comprising:
providing an extraction system comprising:
a support bar comprising a sample well, a reagent well containing a selective lysis buffer, at least one washing well containing a washing reagent, and at least one protection and retention housing,
a pipetting element removably mounted on the support bar and extending at least partially into the at least one protection and retention housing, and
a filtering and transfer device removably mounted on the support bar and extending at least partially into the at least one protection and retention housing, said filtering and transfer device comprising:
a hollow body having an elongated shape and comprising a proximal end portion and a distal end portion, and
a filtering element fixed to the distal end portion of the hollow body,
introducing a predetermined amount of a sample containing microorganisms in the sample well,
withdrawing a predetermined amount of the selective lysis buffer contained in the reagent well by the pipetting element,
placing the withdrawn selective lysis buffer in the sample well by the pipetting element,
lysing cells and/or particles that are not micro-organic contained in the sample and/or lysing predetermined microorganisms contained in the sample so as to supply a lysed sample,
at least partially immersing the filtering and transfer device in the lysed sample contained in the sample well,
filtering at least part of the lysed sample through the filtering element under aspiration,
capturing and accumulating microorganisms in the filtering element,
at least partially immersing the filtering and transfer device in the washing reagent contained in the at least one washing well,
washing the filtering element and the captured and accumulated microorganisms in the washing reagent contained in the at least one washing well,
transferring, by the filtering and transfer device, the washed microorganisms onto an analysis support or into a receiver, the transferring including depositing, by the filtering and transfer device, at least one part of the washed microorganisms onto one or more predetermined spots of a MALDI-TOF MS analysis plate, and
analyzing the transferred microorganisms so as to identify and/or characterize the transferred microorganisms, the analyzing including performing a mass spectrometry analysis of the MALDI-TOF type on the microorganisms deposited on the MALDI-TOF MS plate,
wherein the filtering element has a cylindrical shape with a diameter adapted to the diameter of a spot of the MALDI-TOF MS analysis plate, and has a flat or hemispherical distal portion that protrudes from the distal end portion of the hollow body.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the transferring further comprises resuspending at least part of the washed microorganisms in a saline solution contained in a release well.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the resuspending comprises:
at least partially immersing the filtering and transfer device in the saline solution contained in the release well, and
repeatedly contacting the filtering element with an inner layer of crosslinked silicone at least partly covering the inside surface of the release well for a predetermined time.

### Claim 4 (depends on 3)

The method according to claim 3, further comprising:
measuring the optical density of the saline solution contained in the release well and containing at least part of the washed microorganisms,
adjusting the concentration of microorganisms in the saline solution contained in the release well by adding saline solution to the release well,
withdrawing, from the release well, a predetermined amount of the saline solution containing the microorganisms, and
introducing the withdrawn saline solution into an analysis container containing a predetermined amount of saline solution.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the analyzing comprises:
transferring a predetermined amount of a consumable contained in the analysis container and containing the transferred microorganisms, into an antibiogram analyzer, and
performing an antibiogram analysis on the consumable transferred.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the protection and retention housing is arranged between the sample well and the reagent well along a longitudinal direction of the support bar.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the microorganisms are captured and accumulated in the filtering element and other elements contained in the sample including formed elements of blood that have been lysed with the lysis buffer pass through the filtering element.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the distal end portion of the hollow body and the filtering element extend into the at least one protection and retention housing.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the filtering and transfer device comprises an absorbent material arranged in the hollow body.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the support bar further comprises a release well.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the reagent well and the at least one washing well are each sealed by a sealing element.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the extraction system further comprises a sealing device movable between a sealing position in which the sealing device seals the sample well and a release position in which the sealing device releases the sample well at least partly.

### Claim 13 (depends on 1)

The method according to claim 1, wherein the filtering element has a pore size in a range of from 0.1 and 10 μm.

### Claim 14 (depends on 1)

The method according to claim 1, wherein the filtering element has a pore size in a range of from 0.1 to 0.8 μm.

### Claim 15 (depends on 1)

The method according to claim 1, wherein the filtering element has a pore size in a range of from 0.1 to 0.45 μm.

### Claim 16 (depends on 1)

The method according to claim 1, wherein the filtering element comprises a material selected from the group consisting of polyether sulfone, glass fibers, cellulose acetate, and regenerated cellulose.

### Claim 17 (depends on 1)

The method according to claim 1, wherein the distal portion of the filtering element has a hemispherical shape.