Methods and kits for isolating microorganisms from culture

• US 10,774,300 B2
• Filed: 07/20/2012
• Issued: 09/15/2020
• Est. Priority Date: 07/22/2011
• Status: Active Grant

• Current Assignee: Biomerieux Incorporated (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux Incorporated (Compagnie Merieux Alliance SAS)
• Inventors: Hyman, Jones M., Walsh, John D., Robinson, Ronnie J., Ronsick, Christopher
• Primary Examiner(s): Turk, Neil N
• Application Number:
  US13/554,704
• Publication Number:
  US 20130045532A1
• Time in Patent Office: 2,979 Days
• Field of Search: 435 61
• 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) a selective lysis buffer comprising Brij and CAPS for the selective lysis of non-microorganism known to be present or that may be present in a test sample;

(b) a filtration device comprising a hollow elongated body having a first end and a second end, wherein the first end is capped with a 0.45 μ

m filtration membrane to prevent passage of microorganisms through the filtration membrane but allows other components in the test sample to pass through the filtration membrane, wherein the filtration membrane extends from the first end in a direction opposite the second end;

(c) a wash buffer consisting of a Brij 97 detergent in a sodium phosphate buffer containing sodium chloride for washing the isolated, accumulated and/or purified microorganism sample;

(d) a vacuum system configured to connect to the second end of the filtration device;

(e) at least one micro-swab for harvesting or collecting microorganisms from the filtration membrane;

(f) at least one disposable transfer pipette adapted to transfer the lysed sample to the filtration membrane; and

(g) at least one MALDI-TOF target plate,

wherein the swab is adapted for transferring the microorganisms from the filtration membrane to the MALDI-TOF target plate.

2. 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.

3. The kit of claim 1, wherein said filtration membrane is selected from the group consisting of polyethersulfone (PES) membranes, polysulfone, mixed cellulose ester, polycarbonate, glass fiber type GF/F, GF/C and GMF150, GD/X combination filters, spin filters, syringless filters, cellulose, polyester, polyproplyene and other fiber or particulate filters.

4. The kit of claim 1, wherein said filtration device is disposable.

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

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