METHODS AND KITS FOR ISOLATING MICROORGANISMS FROM CULTURE

• US 20130045532A1
• Filed: 07/20/2012
• Published: 02/21/2013
• Est. Priority Date: 07/22/2011
• Status: Active Grant

• Current Assignee: Biomerieux Incorporated (Compagnie Merieux Alliance SAS)
• Original Assignee: Boimerieux Incorporated
• Inventors: Walsh, John D., Robinson, Ronnie J., Ronsick, Christopher, Hyman, Jones M.
• Granted Patent: US 10,774,300 B2
• Time in Patent Office: Days
• US Class Current: 435/297.1
• CPC Class Codes: C12M 47/02 Separating microorganisms from the culture medium; Concentration of biomass separating microorganisms from their culture media C12N1/02
• CPC Class Codes: C12N 1/02 Separating microorganisms from their culture media
• CPC Class Codes: C12N 1/06 Lysis of microorganisms
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C12Q 1/045 Culture media therefor
• CPC Class Codes: C12Q 1/24 Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae

Abstract

The present invention is directed to methods and kits for separating, accumulating, characterizing and/or identifying microorganisms known to be present or that may be present in a test sample. The method of the invention comprises optional lysing non-microorganism cells and/or particulates that may be present in a test sample, followed by a subsequent filtration step for isolation and/or accumulation of microorganisms. The kit of the present invention may comprise at least one filter membrane or an integrated filtration and sample transfer device for isolation and/or accumulation of microorganisms.

Claims

1. A kit for the isolation, accumulation and/or purification of microorganisms from a test sample comprising, in a packaged combination:

(a) optionally a selective lysis buffer for the selective lysis of non-microorganism known to be present or that may be present in a test sample;

(b) at least one filter material having a pore size which is small enough to prevent passage of microorganisms through the filter but allows other components in the sample, (e.g., lysed non-microorganism cells and/or debris) to pass through the filter; and

(c) at least one wash buffer for washing the isolated, accumulated and/or purified microorganism sample.

2. The kit of claim 1, wherein the kit comprises a selective lysis buffer and wherein said selective lysis buffer comprising one or more detergents.

3. The kit of claim 2, wherein said wherein said one or more detergents is selected from the group consisting of Triton®

X-100, Triton®

X-100-R, Triton®

X-114, NP-40, Genapol®

C-100, Genapol®

X-100, Igepal®

CA 630, Arlasolve™

200, Brij®

96/97, CHAPS, octyl β

-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), sodium dodecyl sulfate, N-laurylsarcosine, sodium deoxycholate, bile salts, hexadec yltrimethyl ammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, C7BzO, Brij®

98, Brij®

58, Brij®

35, Tween®

80, Tween®

20, Pluronic®

L64, Pluronic®

P84, non-detergent sulfobetaines (NDSB

     201), amphipols (PMAL-C8), and methyl-β

-cyclodextrin.

4. The kit of claim 2, wherein said detergent is a polyoxyethylene detergent comprising the structure Cx/Ey, wherein x lies within the range of 10-20 and y lies within the range of 8-12.

5. The kit of claim 1, wherein said lysis solution further comprises an enzymes composition comprising one or more proteinases and/or one or more nucleases.

6. The kit of claim 1, wherein said filter material has a pore size of from about 0.1 μ

m to about 30.0 μ

m and wherein said filter material is selected from the group consisting of polyethersulfone (PES) membranes (e.g., Express PLUS, Supor®

200, Supor®

450, Supor®

MachV), HT Tuffryn®

(polysulfone), GN Metricel®

(mixed cellulose ester), Nylaflo®

FP Verticel (PVDF), Nuclepore (polycarbonate), glass fiber type GF/F, GF/C and GMF150, Metrigard®

AP15, GD/X combination filters (Whatman), spin filters (e.g., VectaSpin, Whatman), syringless filters (e.g., Mini-UniPrep, Whatman), cellulose, polyester, polyproplyene and other fiber or particulate filters.

7. The kit of claim 1, wherein said filter material comprises a disposable filtration device having a built in filtration membrane.

8. The kit of claim 1, wherein said wash buffer is selected from the group consisting phosphate buffer, MOPS and TRIS buffer.

9. The kit of claim 1, wherein said kit further comprises one or more containers within which the lysis step can be carried out.

10. The kit of claim 1, wherein said kit further comprises a filtration device and wherein said filtration device comprises side-arm vacuum flasks with reusable filter holder or wherein said filtration device comprises a manifold with a filtrate reservoir and a plurality of reusable filter holders.

11. The kit of claim 1, wherein said kit further comprises a vacuum source.

12. The kit of claim 1, wherein said kit further comprises a device for harvesting or collecting microorganisms from the filter material and wherein said device for harvesting or collecting microorganisms comprises a swab or scraper.

13. The kit of claim 1, wherein said kit is configured from processing from about 1 to about 500 test samples.

14. A kit for the isolation, accumulation and/or purification of microorganisms from a test sample comprising, in a packaged combination:

(a) optionally a selective lysis buffer for the selective lysis of non-microorganism known to be present or that may be present in a test sample;

(b) at least one integrated filtration and sample transfer device for the isolation, accumulation and/or purification of microorganisms from a test sample, and for the subsequent harvesting and transfer of microorganisms; and

(c) at least one wash buffer for washing the isolated, accumulated and/or purified microorganism sample.

15. The kit of claim 14, wherein said integrated filtration and sample transfer device comprises a hollow shaped body having a first end that is provided with, or capped with a filtration material and a second end adapted for connection to a vacuum system or device.

16. The kit of claim 14, wherein said integrated filtration and sample transfer device comprises a hollow shaped body having a first end that is provided with, or capped with a filtration material and a second end, and wherein said device further comprises an adsorbent material contained within said hollow shaped body, wherein said adsorbent provides passive filtration by capillary action or wicking force.

17. The kit of claim 14, wherein the kit comprises a selective lysis buffer and wherein said selective lysis buffer comprising one or more detergents.

18. The kit of claim 17, wherein said wherein said one or more detergents is selected from the group consisting of Triton®

X-100, Triton®

X-100-R, Triton®

X-114, NP-40, Genapol®

C-100, Genapol®

X-100, Igepal®

CA 630, Arlasolve™

200, Brij®

96/97, CHAPS, octyl β

-D-glucopyranoside, saponin, nonaethylene glycol monododecyl ether (C12E9, polidocenol), sodium dodecyl sulfate, N-laurylsarcosine, sodium deoxycholate, bile salts, hexadecyltrimethylammonium bromide, SB3-10, SB3-12, amidosulfobetaine-14, C7BzO, Brij®

98, Brij®

58, Brij®

35, Tween®

80, Tween®

20, Pluronic®

L64, Pluronic®

P84, non-detergent sulfobetaines (NDSB

     201), amphipols (PMAL-C8), and methyl-β

-cyclodextrin.

19. The kit of claim 14, wherein said filter material is selected from the group consisting of polyethersulfone (PES) membranes (e.g., Express PLUS, Supor®

200, Supor®

450, Supor®

MachV), HT Tuffryn®

(polysulfone), GN Metricel®

(mixed cellulose ester), Nylaflo®

FP Verticel (PVDF), Nuclepore (polycarbonate), glass fiber type GF/F, GF/C and GMF150, Metrigard®

AP15, GD/X combination filters (Whatman), spin filters (e.g., VectaSpin, Whatman), syringless filters (e.g., Mini-UniPrep, Whatman), cellulose, polyester, polyproplyene and other fiber or particulate filters.

20. The kit of claim 14, wherein said wash buffer is selected from the group consisting phosphate buffer, MOPS and TRIS buffer.

21. The kit of claim 14, wherein said kit further comprises one or more containers within which the lysis step can be carried out.

22. The kit of claim 14, wherein said kit further comprises a filtration device and wherein said filtration device comprises side-arm vacuum flasks with reusable filter holder or wherein said filtration device comprises a manifold with a filtrate reservoir and a plurality of reusable filter holders.

23. The kit of claim 14, wherein said kit further comprises a vacuum source.

24. The kit of claim 14, wherein said kit further comprises a device for harvesting or collecting microorganisms from the filter material and wherein said device for harvesting or collecting microorganisms comprises a swab or scraper.

25. The kit of claim 14, wherein said kit is configured from processing from about 1 to about 500 test samples.