# US 2013/0157277 A1 · Device for capture and lysis of microorganisms from liquids and methods of use thereof

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- Number: US 2013/0157277 A1
- Document ID: US20130157277A1
- Status: Published application
- Kind: A1
- Application number: 13/819,240
- Filed: 2011-09-01
- Published: 2013-06-20
- Earliest claimed: 2010-09-01
- Assignees: Life Technologies Corporation
- Inventors: Kevin Hacker; Gregory Govoni; Nikolay Sergeev; Elena Bolchacova; Maxim Brevnov; Manohar Furtado; Johnie Young; Jim Nurse; Mareila Cuadras
- Web page: https://patentgrep.com/patent/US20130157277A1

## CPC

- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- B01D 29/13: Supported filter elements
- C12N 1/066: by physical processes
- C12Q 1/68: involving nucleic acids
- C12Q 1/689: for bacteria
- C12Q 1/6895: for plants, fungi or algae
- G01N 21/75: Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated (systems in which material is burnt in a flame or plasma G01N21/72, G01N21/73)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Devices and methods for detecting microbial contaminants, such as bacteria and fungi, in fluids such as drinking water, pharmaceutical solutions and tissue culture media are provided. More particularly, provided are filtration devices for capture and processing of microorganisms from fluids, and improved methods for recovery, lysis and detection of microorganisms based on a combination of physical disruption with small beads and lysis solutions.

## Claims

### Claim 1

A device for capture and lysis of microorganisms from a liquid comprising:
a) a filter housing consisting of a hollow body defining a fluid path, having an upper input end adapted for receiving a liquid, a lower outflow end, and a filter cavity therebetween;
b) a filter, having an input face and an output face, secured within the filter cavity in an orientation perpendicular to the fluid path such that fluid entering the input end of the housing passes through the filter before exiting the output end, wherein the filter divides the filter cavity into an upper input cavity between the input end of the housing and the input face of the filter, and a lower ouput cavity between the output face of the filter and the output end of the housing;
c) at least one bead disposed within the input cavity, wherein the bead can move freely within the input cavity when the device is agitated;
d) a first sealing means for removably sealing the input end; and
e) a second sealing means for removably sealing the output end.

### Claim 2 (depends on 1)

The device of claim 1, wherein the first and second sealing means are each independently selected from a cap and a plug.

### Claim 3 (depends on 1)

The device of claim 1, further comprising at least one ridge or baffle disposed on an inner surface of the input cavity.

### Claim 4 (depends on 1)

The device of claim 1, wherein the input end comprises a female luer fitting.

### Claim 5 (depends on 1)

The device of claim 1, wherein the output end comprises a male luer fitting.

### Claim 6 (depends on 1)

The device of claim 1, wherein the filter is a membrane made of a material selected from the group consisting of polyethersulfone, polysulfone, nylon, nitrocellulose, polyvinylidene fluoride, polytetrafluoroethylene, polypropylene, cellulose acetate, regenerated cellulose, acrylic copolymers, perfluoro alkoxy, fluorinated ethylene propylene, and Teflon®.

### Claim 7 (depends on 6)

The device of claim 6, wherein the membrane has a nominal pore size of 0.2 μm.

### Claim 8 (depends on 6)

The device of claim 6, wherein the membrane has a circular shape and is about 13 mm in diameter.

### Claim 9 (depends on 8)

The device of claim 8, wherein the input cavity has a generally cylindrical shape and is about 13 mm in diameter.

### Claim 10 (depends on 8)

The device of claim 8, wherein the depth of the input cavity is at least about 10 times the diameter of the at least one bead.

### Claim 11 (depends on 8)

The device of claim 8, wherein the depth of the input cavity is at least about 100 times the diameter of the at least one bead.

### Claim 12 (depends on 1)

The device of claim 1, further comprising a support disposed within the output cavity upon which the filter rests.

### Claim 13 (depends on 12)

The device of claim 12, wherein the support is a perforated element adjacent to the output face of the filter.

### Claim 14 (depends on 12)

The device of claim 12, wherein the support comprises at least one radial protrusion from an inner surface of the output cavity.

### Claim 15 (depends on 1)

The device of claim 1, wherein the at least one bead comprises glass or zirconium.

### Claim 16 (depends on 1)

The device of claim 1, wherein the bead is about 10 μm to about 100 μm in diameter.

### Claim 17 (depends on 1)

The device of claim 1, wherein the at least one bead consists of a plurality of beads.

### Claim 18 (depends on 17)

The device of claim 17, wherein the plurality of beads are about the same size.

### Claim 19 (depends on 17)

The device of claim 17, where in the plurality of beads range in size from about 10 μm to about 500 μm in diameter.

### Claim 20 (depends on 19)

The device of claim 19, wherein the volume of the input cavity is at least about 2 to about 20 times the volume occupied by the plurality of beds.

### Claim 21 (depends on 1)

A method for detecting a microbial contaminant in a liquid comprising:
a) filtering a sample of a liquid through the device of claim 1, wherein microorganisms in the liquid are captured by the filter within the input cavity;
b) sealing the output end of the device;
c) dispensing into the input cavity an amount of a lysis solution sufficient to cover the filter;
d) sealing the input end of the device;
e) agitating the device, wherein agitating causes the at least one bead to strike the membrane, at least one captured microorganism, an inner surface of the device or a combination thereof, thereby forming a lysate of the captured microorganisms;
f) recovering the lysate from the device; and
g) detecting the presence or absence of a nucleic acid of at least one of the microorganisms in the recovered lysate, wherein the presence of the nucleic acid indicates a microbial contaminant in the liquid.

### Claim 22 (depends on 21)

The method of claim 21, wherein the at least one bead is recovered with the lysate in step f), the method further comprising the step of agitating the recovered lysate and at the least one bead prior to detecting the nucleic acid.

### Claim 23 (depends on 21)

The method of claim 21, further comprising incubating the device at a temperature between 50° C. and 90° C. after step c).

### Claim 24 (depends on 21)

The method of claim 21, wherein the microorganisms are bacteria.

### Claim 25 (depends on 21)

The method of claim 21, wherein the microorganisms are fungi.

### Claim 26 (depends on 21)

The method of claim 21, wherein the microorganisms comprise a mixture of bacteria and fungi.

### Claim 27 (depends on 21)

The method of claim 21, wherein detecting the nucleic acid consists of performing a PCR reaction with primers that amplify the nucleic acid.

### Claim 28 (depends on 27)

The method of claim 27, wherein the lysis solution is a PCR-compatible lysis solution.

### Claim 29 (depends on 28)

The method of claim 28, wherein the PCR-compatible lysis solution comprises at least one detergent selected from the group consisting of n-lauroyl sarcosine sodium, sodium deoxycholate, and Tween-20.

### Claim 30 (depends on 27)

The method of claim 27, wherein the primers amplify a nucleic acid from a single species of bacteria or fungi.

### Claim 31 (depends on 27)

The method of claim 27, wherein the primers comprises a pan-bacterial or pan-fungal primer pair.

### Claim 32 (depends on 27)

The method of claim 27, wherein the PCR reaction is performed in the presence of a plurality primer pairs.

### Claim 33 (depends on 21)

The method of claim 21, wherein the liquid is selected from the group consisting of water, a cell culture medium, a pharmacological solution and a biological solution.