# US 8,785,176 B2 · Post protein hydrolysis removal of a potent ribonuclease inhibitor and the enzymatic capture of DNA

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- Number: US 8,785,176 B2
- Document ID: US8785176B2
- Status: Granted
- Kind: B2
- Application number: 13/734,367
- Filed: 2013-01-04
- Published: 2013-05-23
- Granted: 2014-07-22
- Earliest claimed: 2002-08-15
- Assignees: University of South Florida
- Applicants: University of South Florida
- Inventors: Matt Ewert; Philip Amuso; Andrew Cannons
- Web page: https://patentgrep.com/patent/US8785176B2

## CPC

- C07K 17/10: the carrier being a carbohydrate
- C07C 51/42: Separation; Purification; Stabilisation; Use of additives
- C07C 65/40: containing singly bound oxygen-containing groups
- C12M 33/00: {Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus (chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00)}
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12N 15/1006: {by means of a solid support carrier, e.g. particles, polymers}
- C12N 15/101: {by chromatography, e.g. electrophoresis, ion-exchange, reverse phase}
- 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 1/24: Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
- C12Q 1/56: involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- G01N 1/34: Purifying; Cleaning {(processes or apparatus for extracting or separating nucleic acids from biological samples C12N15/1003)}
- G01N 33/86: involving blood coagulating time {or factors, or their receptors}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- 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
- 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

The present invention concerns compositions and methods of extracting infectious pathogens from a volume of blood. In one embodiment, the method includes the steps of creating a fibrin aggregate confining the pathogens and introducing a fibrin lysis reagent to expose the pathogens for analysis. The present invention also concerns materials and methods for removing aurintricarboxylic acid (ATA) from a sample.

## Claims

### Claim 1

A method for capturing a particle from a biological sample including the particle, the method comprising:
(a) contacting the biological sample with:
(i) 2 to 10 U plasminogen and 5,000 to 10,000 U streptokinase or plasmin produced by combining 2 to 10 U plasminogen and 5,000 to 10,000 U streptokinase,
(ii) 200 to 3,650 U DNase,
(iii) 1,500 to 4,500 KU phospholipase A2, and
(iv) a detergent; and
(b) capturing the particle,
wherein the particle is a prion, a toxin, a metabolic marker, cancerous matter, a disease state marker, a bacterium, a virus, or a fungus.

### Claim 2 (depends on 1)

The method of claim 1, wherein the particle is a bacterium.

### Claim 3 (depends on 1)

The method of claim 1, wherein the particle is a fungus.

### Claim 4 (depends on 1)

The method of claim 1, wherein the DNase is an endonuclease.

### Claim 5 (depends on 1)

The method of claim 1, wherein the method further comprises contacting the biological sample with 10,000 to 100,000 U lipase.

### Claim 6 (depends on 1)

The method of claim 1, wherein the capturing the particle comprises filtering the biological sample to extract the particle.

### Claim 7 (depends on 6)

The method of claim 6, wherein the filtering comprises filtering the biological sample through a 0.22 to 0.45 mm filter unit.

### Claim 8 (depends on 7)

The method of claim 7, wherein the filter unit is a Polyethersulfone (PES) filter unit.

### Claim 9 (depends on 1)

The method of claim 1, wherein the biological sample is blood.

### Claim 10 (depends on 1)

The method of claim 1, wherein the biological sample is a fibrin aggregate.

### Claim 11 (depends on 6)

The method of claim 6, wherein the method further comprises:
contacting the extracted particle with proteinase K, thereby producing a lysate;
eluting the lysate from the filter surface; and
extracting a nucleic acid from the lysate.

### Claim 12 (depends on 11)

The method of claim 11, wherein the method further comprises:
amplifying the nucleic acid by polymerase chain reaction (PCR).

### Claim 13 (depends on 1)

The method of claim 1, wherein the capturing the particle comprises centrifuging the biological sample to extract the particle.