# US 10,119,114 B2 · Device for the lysis of microorganisms present in an environmental or clinical sample and the extraction of nucleic acids from said microorganisms for analysis

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- Number: US 10,119,114 B2
- Document ID: US10119114B2
- Status: Granted
- Kind: B2
- Application number: 12/451,486
- Filed: 2008-06-06
- Published: 2010-03-25
- Granted: 2018-11-06
- Earliest claimed: 2007-06-07
- Assignees: Biomerieux
- Applicants: Hermanus Johannes Maria Kreuwel; Emiel Gerebern Maria Verwimp
- Inventors: Hermanus Johannes Maria Kreuwel; Emiel Gerebern Maria Verwimp
- Web page: https://patentgrep.com/patent/US10119114B2

## CPC

- C12M 47/06: {Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material (lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003)}
- C12M 25/14: {Scaffolds; Matrices (in general C12N5/0068)}
- C12Q 1/68: involving nucleic acids
- 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}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A cartridge that can be positioned inside an air collection component and receive a component for recovering nucleic acids is described, the cartridge being substantially cylindrical and containing a microorganism retaining zone, the retaining zone containing a microorganism lysis mechanism. Also described is a device for collecting microorganisms contained in the air and a device for microorganism lysis.

## Claims

### Claim 1

A cartridge which can be positioned inside an air collection device and receive a means for recovering nucleic acids, said cartridge being substantially cylindrical and having an upper end and lower end, said cartridge comprising a microorganism retaining zone, said retaining zone comprising microorganism lysis means for lysing the microorganisms, wherein
the upper end of the cartridge is open,
the lower end is constituted of a wall, having, in its center, an orifice, there being, along an extension of said orifice, a duct extending, inside said cartridge, and
the microorganism retaining zone comprises a material configured to retain the microorganisms, configured to keep the lysis means in place, and configured to dissolve in the presence of a liquid to release the lysis means from the material.

### Claim 2 (depends on 1)

The cartridge as claimed in claim 1, in which the material is a gelled material.

### Claim 3 (depends on 2)

The cartridge as claimed in claim 2, in which the gelled material is a microorganism culture medium.

### Claim 4 (depends on 1)

The cartridge as claimed in claim 1, also comprising a means for connection of an analyzing device.

### Claim 5 (depends on 4)

The cartridge as claimed in claim 4, in which the lysis means comprises beads as the lysis means.

### Claim 6 (depends on 5)

The cartridge as claimed in claim 5, in which the diameter of the beads is between 200 and 600 μm.

### Claim 7 (depends on 1)

A device for collecting microorganisms contained in the air, said device comprising:
an air collection device, comprising an upper element comprising an air inlet duct and a lower element comprising an air outlet duct, it being possible for said upper and lower elements to be interlocked with one another such that a current of air can be created inside said air collection device;
the cartridge as claimed in claim 1 being positioned inside said air collection device.

### Claim 8 (depends on 7)

The device as claimed in claim 7, in which the air collection device is capable of being connected to an air recycling circuit.

### Claim 9 (depends on 1)

A device for microorganism lysis, said device comprising:
the cartridge as claimed in claim 1, said cartridge comprising microorganisms placed in the microorganism retaining zone;
a substantially cylindrical means for recovering nucleic acids, that can be placed in the cartridge, said recovering means cooperating with the microorganism lysis means in order to lyze said microorganisms and enable release of the nucleic acids.

### Claim 10 (depends on 9)

The device as claimed in claim 9, in which the means for recovering nucleic acids comprises a means for drawing up/delivering liquid.

### Claim 11 (depends on 9)

The device as claimed in claim 9, in which the means for recovering nucleic acids also comprise a liquid storage zone.

### Claim 12 (depends on 9)

The device as claimed in claim 9, in which the internal diameter of the cartridge is greater than the external diameter of the means for recovering nucleic acids, such that, when the means for recovering nucleic acids is fitted into the cartridge, the distance separating the internal wall of the cartridge from the external wall of the means for recovering nucleic acids is sufficiently large to allow the lysis means to sit in this interstitial space and sufficiently small for the lysis means to be in contact with one or other of said walls.

### Claim 13 (depends on 1)

A method for concentrating microorganisms contained in the air, said method comprising:
a) placing the cartridge as claimed in claim 1 inside an air collection device, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection device,
b) causing air to enter said air collection device, and
c) concentrating the microorganisms contained in the air in the retaining zone of the cartridge.

### Claim 14 (depends on 13)

The method of concentration as claimed in claim 13, which also comprises a step d) comprising growing the microorganisms in the retaining zone.

### Claim 15 (depends on 13)

The method of concentration as claimed in claim 13, in which the microorganisms are retained on the lysis means present in the retaining zone.

### Claim 16 (depends on 1)

A method for the lysis of microorganisms contained in the air, said method comprising:
a) placing the cartridge as claimed in claim 1 inside an air collection device, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection device,
b) causing air to enter said air collection device,
c) concentrating the microorganisms contained in the air, in the retaining zone of the cartridge,
d) removing the cartridge from the air collection device,
e) placing the means for recovering nucleic acids in the cartridge,
f) introducing a liquid into the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, and
g) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained.

### Claim 17 (depends on 1)

A method for the lysis of microorganisms contained in the air, said method comprising:
a) placing the cartridge as claimed in claim 1 inside an air collection device, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection device,
b) causing air to enter said air collection device by,
c) concentrating the microorganisms contained in the air, in the retaining zone of the cartridge,
d) removing the cartridge from the air collection device,
e) fitting the means for recovering nucleic acids into the cartridge,
f) causing the delivery of a liquid previously placed in a storage zone of the device for recovering nucleic acids, said delivery being obtained by a drawing up/delivering means for recovering nucleic acids, the liquid thus delivered filling an interstitial space located between the means for recovering nucleic acids and the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, said lysis means coming to sit between a vertical internal wall of the cartridge and a vertical external wall of the means for recovering nucleic acids, and
g) mechanically lysing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained.

### Claim 18 (depends on 1)

A method for the extraction of nucleic acids from microorganisms contained in the air, said method comprising:
a) placing the cartridge as claimed in claim 1 inside an air collection device, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection device,
b) causing air to enter said air collection device,
c) concentrating the microorganisms contained in the air, in the retaining zone of the cartridge,
d) removing the cartridge from the air collection device,
e) placing the means for recovering nucleic acids in the cartridge,
f) introducing a liquid into the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, and
g) mechanically lysing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained, and
h) drawing up the liquid containing the nucleic acids of said microorganisms, released during the lysis.

### Claim 19 (depends on 1)

A method for the extraction of nucleic acids from microorganisms contained in the air, said method comprising:
a) placing the cartridge as claimed in claim 1 inside an air collection device, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection device,
b) causing air to enter said air collection device,
c) concentrating the microorganisms contained in the air, in the retaining zone of the cartridge,
d) removing the cartridge from the air collection device,
e) fitting the means for recovering nucleic acids into the cartridge,
f) causing the delivery of a liquid previously placed in a storage zone of the device for recovering nucleic acids, said delivery being obtained by a drawing up/delivering means for recovering nucleic, acids, the liquid thus delivered filling an interstitial space located between the means for recovering nucleic acids and the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, said lysis means coming to sit between a vertical internal wall of the cartridge and a vertical external wall of the means for recovering nucleic acids,
g) mechanically lysing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained, thus releasing the nucleic acids from said microorganisms, and
h) causing the liquid in the storage zone to be drawn up, said drawing up being obtained by the drawing up/delivering means for recovering nucleic acids, the liquid of interest thus drawn up containing the nucleic acids of said microorganisms, released during the lysis.

### Claim 20 (depends on 16)

The method as claimed in claim 16, comprising an additional step d′) comprising growing the concentrated microorganisms in the retaining zone of the cartridge.

### Claim 21 (depends on 20)

The method as claimed in claim 20, in which the growing is obtained by incubation of the cartridge in an incubator for a period of time ranging from 2 to 24 hours.

### Claim 22 (depends on 1)

A method for the lysis of microorganisms, said method comprising:
a) obtaining the cartridge, as claimed in claim 1, in which microorganisms are concentrated in the retaining zone,
b) fitting the means for recovering nucleic acids into the cartridge,
c) causing the delivery of a liquid previously placed in a storage zone of the means for recovering nucleic acids, said delivery being obtained by a drawing up/delivering means for recovering nucleic acids, the liquid thus delivered filling an interstitial space located between the means for recovering nucleic acids and the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, said lysis means coming to sit between a vertical internal wall of the cartridge and a vertical external wall of the means for recovering nucleic acids, and
d) mechanically lysing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained.

### Claim 23 (depends on 1)

A method for the lysis of microorganisms, said method comprising:
a) obtaining the cartridge, as claimed in claim 1, in which microorganisms are concentrated in the retaining zone,
b) placing the means for recovering nucleic acids in the cartridge,
c) introducing a liquid into the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, and
d) mechanically lysing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained.

### Claim 24 (depends on 1)

A method for the extraction of nucleic acids from microorganisms, said method comprising:
a) obtaining the cartridge, as claimed in claim 1, in which microorganisms are concentrated in the retaining zone,
b) fitting the means for recovering nucleic acids into the cartridge,
c) causing the delivery of a liquid previously placed in a storage zone of the means for recovering nucleic acids, said delivery being obtained by the drawing up/delivering means for recovering nucleic acids, the liquid thus delivered filling an interstitial space located between, the means for recovering, nucleic acids and the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, said lysis means coming to sit between a vertical internal wall of the cartridge and a vertical external wall of the means for recovering nucleic acids,
d) mechanically lysing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering, nucleic acids rotating the lysis means on which the microorganisms are retained, thus releasing the nucleic acids from said microorganisms, and
e) causing the liquid in the storage zone of the means for recovering nucleic acids to be drawn up, said drawing up being obtained by the drawing up/delivering means recovering nucleic acids, the liquid of interest thus drawn up containing the nucleic acids of said microorganisms, released during the lysis.

### Claim 25 (depends on 1)

A method for the extraction of nucleic acids from microorganisms, said method comprising:
obtaining the cartridge, as claimed in claim 1, in which microorganisms are concentrated in the retaining zone,
b) placing the means for recovering nucleic acids in the cartridge,
c) introducing a liquid into the cartridge, which leads to the lysis means located in t e microorganism retaining zone of the cartridge being placed in suspension, and
d) mechanically lysing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained, and
e) drawing up the liquid containing the nucleic acids of said microorganisms, released during the lysis.

### Claim 26 (depends on 1)

A method for the lysis of microorganisms, said method comprising:
a) introducing a liquid sample containing said microorganisms into the cartridge as claimed in claim 1, in a vicinity of the retaining zone, such that said liquid sample leads to the lysis means located in said microorganism retaining zone of the cartridge being placed in suspension,
b) placing the means for recovering nucleic acids in the cartridge,
c) mechanically lysing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained.

### Claim 27 (depends on 1)

A method for the extraction of nucleic acids from microorganisms, said method comprising:
a) introducing a liquid sample containing said microorganisms into the cartridge as claimed in claim 1 in a vicinity of the retaining zone, such that said liquid sample leads to the lysis means located in said microorganism retaining zone of the cartridge being placed in suspension,
b) placing the means for recovering nucleic acids in the cartridge,
c) mechanically lysing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained,
d) drawing up the liquid containing the nucleic acids of said microorganisms, released during the lysis.

### Claim 28 (depends on 7)

A method for identifying one or more microorganisms contained in a sample, the method comprising:
isolating the nucleic acids from the microorganisms contained in said sample by means of the device as claimed in claim 7,
identifying the microorganism(s) thus isolated.

### Claim 29 (depends on 28)

The method of identification as claimed in claim 28, also comprising an intermediate step consisting in purifying the nucleic acids.

### Claim 30 (depends on 28)

The method of identification as claimed in claim 28, in which the identification step further comprises:
specifically amplifying the isolated nucleic acids, and
detecting the nucleic acids thus amplified.

### Claim 31 (depends on 28)

The method of identification as claimed in claim 28, in which the identification step is carried out in an identification device in fluid communication with the cartridge, the cartridge comprising microorganisms placed in the microorganism retaining zone.

### Claim 32 (depends on 31)

The method of identification as claimed in claim 31, in which the isolated nucleic acids are transferred from a substantially cylindrical means for recovering nucleic acids, that can be placed in the cartridge, to the identification device.

### Claim 33 (depends on 32)

The method of identification as claimed in claim 32, in which the transfer of the nucleic acids is obtained by delivery of a liquid containing the nucleic acids, said liquid being contained in a storage zone of the device for recovering nucleic acid, by a drawing up/delivering means for recovering nucleic acids.

### Claim 34 (depends on 1)

The cartridge as claimed in claim 1, in which the material is agarose.