# US 9,434,980 B2 · Method for concentrating sample constituents and for multiplying nucleic acids from a biological sample which are contained in the sample constituents

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- Number: US 9,434,980 B2
- Document ID: US9434980B2
- Status: Granted
- Kind: B2
- Application number: 13/283,279
- Filed: 2011-10-27
- Published: 2012-05-31
- Granted: 2016-09-06
- Earliest claimed: 2010-10-27
- Assignees: Robert Bosch GmbH
- Applicants: Peter Rothacher; Jan Weile; Susanne Muench; Martina Daub
- Inventors: Peter Rothacher; Jan Weile; Susanne Muench; Martina Daub
- Web page: https://patentgrep.com/patent/US9434980B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- B01L 2200/10: Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
- 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
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

## Abstract

A method is disclosed for concentrating sample constituents and for multiplying nucleic acids from a biological sample which are containing in the sample constituents. The nucleic acids are amplified on the same filter on which the sample constituents are also separated off.

## Claims

### Claim 1

A method for concentrating sample constituents and for multiplying nucleic acids from a biological sample which are contained in the sample constituents, comprising:
1) providing a biological sample in liquid form;
2) introducing the sample into a separating system having at least one filter;
3) accumulating the sample constituents contained in the sample by separating off the sample constituents on the filter;
4) adding amplification reagents to the separating system; and
5) directly amplifying at least one nucleic acid present in the sample constituents;
wherein the amplification in step 5) is carried out by PCR and comprises at least:
5.1) extracting nucleic acids without using chemical lysing agents and denaturing the at least one nucleic acid;
5.2) annealing primers; and
5.3) extending primers.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the filter is modified in step 5.1) by the addition of filter-blocking polymers in step 4), selected from the group consisting of soluble proteins, albumins, and synthetic polymers.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the filter in step 3) is perfused laterally.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the filter is selected from the group consisting of
fiber filters,
membrane filters, and
ion exchangers.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the nucleic acid amplified in step 5) is subsequently eluted from the filter.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the nucleic acid amplified in step 5) is rinsed off the filter with a precipitation solution and the eluate is mixed in a microfluidic system and applied again to the filter.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the precipitation solution perfuses the filter against the earlier direction of perfusion and the mixed eluate perfuses the filter in the earlier direction of perfusion.

### Claim 8 (depends on 2)

The method according to claim 2, wherein the albumin is BSA.

### Claim 9 (depends on 2)

The method according to claim 2, wherein the synthetic polymer is PEG or PPG.

### Claim 10 (depends on 4)

The method according to claim 4, wherein the fiber filter is a silica-containing fiber filter or a glass fiber filter.

### Claim 11 (depends on 4)

The method according to claim 4, wherein the ion exchanger is an anion exchanger.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the step of extracting nucleic acids is accomplished by the denaturation step of the PCR.

### Claim 13 (depends on 12)

The method according to claim 12, wherein the denaturation step of the PCR takes place at about 95° C.