DEVICE FOR THE LYSIS OF MICROORGANISMS PRESENT IN AN ENVIRONMENTAL OR CLINICAL SAMPLE AND THE EXTRACTION OF NUCLEIC ACIDS FROM SAID MICROORGANISMS FOR ANALYSIS

• US 20100075313A1
• Filed: 06/06/2008
• Published: 03/25/2010
• Est. Priority Date: 06/07/2007
• Status: Active Grant

• Current Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Inventors: Verwimp, Emiel Gerebern Maria, Kreuwel, Hermanus Johannes Maria
• Granted Patent: US 10,119,114 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12M 25/14 Scaffolds; Matrices in general C12N5/0068
• CPC Class Codes: 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
• CPC Class Codes: C12Q 1/68 involving nucleic acids

Abstract

The present invention relates to a cartridge which can be positioned inside an air collection means and receive a means for recovering nucleic acids, said cartridge being substantially cylindrical and comprising a microorganism retaining zone, said retaining zone comprising microorganism lysis means. The invention also relates to a device for collecting microorganisms contained in the air and a device for microorganism lysis.

Claims

1. A cartridge which can be positioned inside an air collection means and receive a means for recovering nucleic acids, said cartridge being substantially cylindrical and comprising a microorganism retaining zone, said retaining zone comprising microorganism lysis means.

2. The cartridge as claimed in claim 1, in which the microorganism retaining zone comprises a material capable of retaining the microorganisms, of keeping the lysis means in place and of dissolving in the presence of a liquid.

3. The cartridge as claimed in claim 2, in which the material is a gelled material.

4. The cartridge as claimed in claim 3, in which the gelled material is a microorganism culture medium.

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

6. The cartridge as claimed in claim 5, in which the lysis means are constituted of beads.

7. The cartridge as claimed in claim 1, in which the diameter of the beads is between 200 and 600 μ

m.

8. A device for collecting microorganisms contained in the air, said device comprising:

an air collection means, 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 means;

the cartridge as claimed in claim 1 being positioned inside said air collection means.

9. The device as claimed in claim 8, in which the air collection means is capable of being connected to an air recycling circuit.

10. A device for microorganism lysis, with the aim of isolating the nucleic acids from said microorganisms, 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.

11. The device as claimed in claim 10, in which the means for recovering nucleic acids comprises a means for drawing up/delivering liquid.

12. The device as claimed in claim 10, in which the means for recovering nucleic acids also comprise a liquid storage zone.

13. The device as claimed in claim 10, 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.

14. A method for concentrating microorganisms contained in the air, said method comprising:

a) placing the cartridge as claimed in claim 1 inside the air collection means, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection means,

b) causing air to enter said air collection means by any appropriate means,

c) concentrating the microorganisms contained in the air in the retaining zone of the cartridge.

15. The method of concentration as claimed in claim 14, which also comprises a step d) consisting in growing the microorganisms in the retaining zone.

16. The method of concentration as claimed in claim 14, in which the microorganisms are retained on the lysis means present in the retaining zone.

17. A method for the lysis of microorganisms contained in the air, said method comprising:

a) placing the cartridge as claimed in claim 1 inside the air collection means, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection means,

b) causing air to enter said air collection means by any appropriate means,

c) concentrating the microorganisms contained in the air, in the retaining zone of the cartridge,

d) removing the cartridge from the air collection means,

e) placing the means for recovering nucleic acids in the cartridge,

f) introducing a liquid of interest 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.

18. A method for the lysis of microorganisms contained in the air, said method comprising:

a) placing the cartridge as claimed in claim 1 inside the air collection means, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection means,

b) causing air to enter said air collection means by any appropriate means,

c) concentrating the microorganisms contained in the air, in the retaining zone of the cartridge,

d) removing the cartridge from the air collection means,

e) fitting the means for recovering nucleic acids into the cartridge,

f) causing the delivery of a liquid of interest previously placed in the storage zone of the means for recovering nucleic acids, said delivery being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the liquid thus delivered filling the 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 the vertical internal wall of the cartridge and the vertical external wall of the means for recovering nucleic acids, 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.

19. 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 the air collection means, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection means,

b) causing air to enter said air collection means by any appropriate means,

c) concentrating the microorganisms contained in the air, in the retaining zone of the cartridge,

d) removing the cartridge from the air collection means,

e) placing the means for recovering nucleic acids in the cartridge,

f) introducing a liquid of interest 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, and

h) drawing up the liquid of interest containing the nucleic acids of said microorganisms, released during the lysis.

20. 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 the air collection means, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection means,

b) causing air to enter said air collection means by any appropriate means,

c) concentrating the microorganisms contained in the air, in the retaining zone of the cartridge,

d) removing the cartridge from the air collection means,

e) fitting the means for recovering nucleic acids into the cartridge,

f) causing the delivery of a liquid of interest previously placed in the storage zone of the means for recovering nucleic acids, said delivery being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the liquid thus delivered filling the 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 the vertical internal wall of the cartridge and the vertical external wall of the means for recovering nucleic acids,

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, thus releasing the nucleic acids from said microorganisms, and

h) causing the liquid of interest in the storage zone of the means for recovering nucleic acids to be drawn up, said drawing up being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the liquid of interest thus drawn up containing the nucleic acids of said microorganisms, released during the lysis.

21. The method as claimed in claim 17, comprising an additional step d′

) consisting in growing the concentrated microorganisms in the retaining zone of the cartridge.

22. The method as claimed in claim 21, 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.

23. 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 of interest previously placed in the storage zone of the means for recovering nucleic acids, said delivery being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the liquid thus delivered filling the 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 the vertical internal wall of the cartridge and the vertical external wall of the means for recovering nucleic acids, and

d) 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.

24. 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 of interest 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 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.

25. 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 of interest previously placed in the storage zone of the means for recovering nucleic acids, said delivery being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the liquid thus delivered filling the 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 the vertical internal wall of the cartridge and the vertical external wall of the means for recovering nucleic acids,

d) 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, thus releasing the nucleic acids from said microorganisms, and

e) causing the liquid of interest in the storage zone of the means for recovering nucleic acids to be drawn up, said drawing up being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the liquid of interest thus drawn up containing the nucleic acids of said microorganisms, released during the lysis.

26. A method for the extraction of nucleic acids from microorganisms, said method comprising:

a) obtaining the cartridge, as claimed in claim 1, a in which microorganisms are concentrated in the retaining zone,

b) placing the means for recovering nucleic acids in the cartridge,

c) introducing a liquid of interest 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 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, and

e) drawing up the liquid of interest containing the nucleic acids of said microorganisms, released during the lysis.

27. 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 the 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 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.

28. 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 the 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 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,

d) drawing up the liquid of interest containing the nucleic acids of said microorganisms, released during the lysis.

29. 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 8,

identifying the microorganism(s) thus isolated.

30. The method of identification as claimed in claim 29, also comprising an intermediate step consisting in purifying the nucleic acids.

31. The method of identification as claimed in claim 29, in which the identification step further comprises:

specifically amplifying the isolated nucleic acids,

detecting the nucleic acids thus amplified.

32. The method of identification as claimed in claim 29, 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.

33. The method of identification as claimed in claim 32, 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.

34. The method of identification as claimed in claim 33, in which the transfer of the nucleic acids is obtained by delivery of the liquid of interest containing the nucleic acids, said liquid of interest being contained in the storage zone of the means for recovering nucleic acids, by means of the drawing up/delivering means of the means for recovering nucleic acids.