# US 2016/0257987 A1 · Method for isolating microorganisms from a complex sample

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- Number: US 2016/0257987 A1
- Document ID: US20160257987A1
- Status: Published application
- Kind: A1
- Application number: 15/032,523
- Filed: 2014-10-09
- Published: 2016-09-08
- Earliest claimed: 2013-10-30
- Assignees: Merck Patent GmbH
- Applicants: Merck Patent GmbH
- Inventors: Marisa HOHNADEL; Sebastien JOUETTE
- Web page: https://patentgrep.com/patent/US20160257987A1

## CPC

- C12Q 1/24: Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
- C12N 1/06: Lysis of microorganisms
- C12N 13/00: Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- 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)}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to a method for isolating and optionally detecting microorganisms from a sample comprising mammalian cells, comprising the use of a lysis buffer comprising Benzonase® Nuclease. The invention further relates to a lysis buffer comprising Benzonase® Nuclease and its use.

## Claims

### Claim 1

Method for isolating microorganisms from a sample comprising mammalian cells, comprising
a) contacting the sample with a lysis buffer comprising Benzonase® Nuclease,
b) obtaining the microorganisms by removing the mammalian cells.

### Claim 2

Method for isolating and detecting microorganisms from a sample comprising mammalian cells, comprising
a) contacting the sample with a lysis buffer comprising Benzonase® Nuclease,
b) obtaining the microorganisms by removing the mammalian cells,
c) performing a universal lysis on the microorganisms,
d) performing a specific or universal detection method for detecting the microorganisms.

### Claim 3 (depends on 1)

Method according to claim 1, characterized in that the sample is a bioreactor sample.

### Claim 4 (depends on 1)

Method according to claim 1, characterized in that the quantity of the mammalian cells in the sample is at least 106 cells.

### Claim 5 (depends on 1)

Method according to claim 1, characterized in that the volume of the sample is at least 5 ml.

### Claim 6 (depends on 1)

Method according to claim 1, characterized in that the lysis buffer comprises Benzonase® Nuclease in a concentration of 2.5 U to 175 U per sample.

### Claim 7 (depends on 1)

Method according to claim 1, characterized in that the mammalian cells are selected from the group consisting of cells growing in suspension culture, cells growing on microcarriers and/or hybridoma cell lines.

### Claim 8 (depends on 1)

Method according to claim 1, characterized in that the mammalian cells are selected from the group of CRL-8018 cells, CF-10H5 cells, CHO cells, NSO cells, Sp2/0 cells, HT-1080 cells, MDCK cells, HeLa cells or Vero cells.

### Claim 9 (depends on 1)

Method according to claim 1, characterized in that the microorganisms are bacteria, yeast, fungi and/or combinations thereof.

### Claim 10 (depends on 1)

Method according to claim 1, characterized in that in step b) centrifugation is employed.

### Claim 11 (depends on 2)

Method according to claim 2, characterized in that in step c) sonication is employed.

### Claim 12 (depends on 2)

Method according to claim 2, characterized in that in step d) PCR, preferably real-time PCR, is employed.

### Claim 13

Lysis buffer comprising the following ingredients:
0.05 to 0.5% by weight SDS
0.5 M to 4 M urea
0.5 mM to 10 mM MgCl2
2.5 to 175 U Benzonase® Nuclease
PBS 1×-PBS×⅛.

### Claim 14 (depends on 1)

Method according to claim 1 wherein the lysis buffer comprises the following ingredients:
0.05 to 0.5% by weight SDS
0.5 M to 4 M urea
0.5 mM to 10 mM MgCl2
2.5 to 175 U Benzonase® Nuclease
PBS 1×-PBS×⅛.