# US 10,655,122 B2 · Method, lysis solution and kit for selectively depleting animal nucleic acids in a sample

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- Number: US 10,655,122 B2
- Document ID: US10655122B2
- Status: Granted
- Kind: B2
- Application number: 15/568,098
- Filed: 2015-04-20
- Published: 2018-05-24
- Granted: 2020-05-19
- Earliest claimed: 2015-04-20
- Assignees: Qiagen GmbH
- Applicants: QIAGEN GmbH
- Inventors: Johann Kubicek; Thorsten Singer; Antje-Katrin Sander; Eva Hänssler; Dominic O'Neil
- Web page: https://patentgrep.com/patent/US10655122B2

## CPC

- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12Q 2521/301: Endonuclease
- C12Q 2521/537: Protease
- C12Q 2523/113: Denaturating agents
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

The present invention relates to a method for selectively depleting animal nucleic acids from non-animal nucleic acids in a sample, which comprises animal cells and at least one further type of cells, selected from microbial cells and plant cells or a combination thereof, to a lysis solution A to be used in and to a kit to carry out said method as well as to the use of a particular silica membrane as both, a filtration matrix for separating essentially intact microbial and/or plant cells from lysed animal cells and an adsorption matrix for nucleic acids, in particular in a method according to the present invention.

## Claims

### Claim 1

A method for selectively depleting animal nucleic acids from non-animal nucleic acids in a sample, the sample comprising animal cells and at least one further type of cells selected from microbial cells and plant cells or a combination thereof, the method comprising the steps of:
i) selectively lysing the animal cells by contacting the sample with a lysis solution to obtain a mixture comprising intact microbial cells and/or plant cells as well as the animal nucleic acids released from the lysed animal cells into solution, said lysis solution being essentially chaotrope-free and comprising 0.01 to 15% (w/v) of a non-ionic surfactant, a water soluble salt selected from the group consisting of acetate, sulfate, glutamate, and mixtures thereof, and a sugar as a viscosity modifier,
ii) digesting both DNA and RNA of the animal nucleic acids in the presence of the intact microbial and/or plant cells, and
iii) separating the microbial and/or plant cells from the liquid part of the sample, including the digested animal nucleic acids.

### Claim 2 (depends on 1)

The method according to claim 1, wherein in step ii) both DNA and RNA of the animal nucleic acids are digested in one step.

### Claim 3 (depends on 1)

The method according to claim 1, wherein in step ii) both DNA and RNA of the animal nucleic acids are digested in one step with an endonuclease that degrades both DNA and RNA.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the nonionic surfactant is selected from the group consisting of a sorbitan ester of fatty acids, a polyoxyethylene sorbitan ester of fatty acids, a polyoxyalkylene ether of fatty alcohols, a polyoxyalkylene etherof alkylphenols, a poloxamer, a saponin, or a mixture of any thereof.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the sugar is glucose or sucrose.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the lysis solution further comprises a polyanionic sulfonate.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the animal cells comprise mammalian cells, and/or wherein the further cells comprise microbial cells that are bacterial cells, archae cells, fungal cells, or any combination thereof, and/or wherein the sample comprises a tissue, tissue homogenate, cell culture, bone marrow aspirate, bone homogenate, swab, swab rinsate, or body fluid.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the body fluid is amniotic fluid, aqueous humour, bile, bladder lavage, blood, breast exudates, bronchoalveolar lavage, cerebrospinal fluid, chyle, chyme, cytosol, feces, gastric contents, in particular gastric acid, interstitial fluid, joint fluid, lymph, menses, mucus, plasma, pleural fluid, pus, saliva, sebum, semen, serum, sputum, sweat, synovial fluid, tears, urine, vaginal secretions, or vitreous humour.

### Claim 9 (depends on 1)

The method according to claim 1, further comprising the steps of:
i) removing a liquid part of the sample from the remaining cells, and/or
ii) removing and/or inactivating any nuclease present along with the remaining cells.

### Claim 10 (depends on 9)

The method according to claim 9, wherein removing and/or inactivating any nuclease present along with the remaining cells comprises digesting the nuclease with a proteinase.

### Claim 11 (depends on 1)

The method according to claim 1, further comprising after step iii), analyzing the microbial and/or plant cells or any parts thereof.

### Claim 12 (depends on 11)

The method according to claim 11, wherein analyzing the microbial and/or plant cells or any parts thereof comprises culturing, cytometry, microscopy, immunodetecting, and/or lysing the microbial or plant cells; and/or concentrating, isolating, purifying, amplifying and/or analyzing the microbial and/or plant nucleic acids.

### Claim 13 (depends on 1)

The method according to claim 1, wherein step iii) comprises separating the microbial cells or plant cells by centrifugation.

### Claim 14 (depends on 1)

The method according to any claim 1, wherein step iii) comprises filtering the sample through a solid filter to retain the microbial and/or plant cells as retentate on the filter while a liquid part of the sample, including the digested animal nucleic acids, passes the filter as a filtrate.

### Claim 15 (depends on 14)

The method according to claim 14, wherein the solid filter fulfills at least one of the following conditions:
i) the filter comprises at least one part having a pore size being in the range of 0.1-1.5 μM;
ii) the filter comprises polyester, polyethersulfone (PES), cellulose, cellulose acetate (CA), mixed cellulose esters (MCE), polytetrafluoroethylene (PTFE), polyamide (PA), Nylon, polypropylene (PP), ceramics, glass fibers, silica, or any combination or mixture thereof.