# US 10,697,002 B2 · Amplification and detection of nucleic acids in a biological sample

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- Number: US 10,697,002 B2
- Document ID: US10697002B2
- Status: Granted
- Kind: B2
- Application number: 15/529,343
- Filed: 2015-11-24
- Published: 2017-09-21
- Granted: 2020-06-30
- Earliest claimed: 2014-11-25
- Assignees: Quest Diagnostics Investments Incorporated
- Applicants: Quest Diagnostics Investments Incorporated
- Inventors: Lucien Jacky; Albert Castro; Peter Lee; Jose Matud; Michelle Tabb
- Web page: https://patentgrep.com/patent/US10697002B2

## CPC

- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- C12N 1/06: Lysis of microorganisms
- C12N 15/1003: {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor}
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C12Q 1/6853: using modified primers or templates
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 2523/32: Centrifugation
- C12Q 2565/629: being a microfluidic device
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Disclosed are methods for identifying the presence or absence of a target nucleic acid from one or more organisms in a biological sample, said method comprising: (a) spinning the sample at a rotational velocity sufficient to pellet cellular debris and fluorescence inhibitors present within the sample and reduce fluorescence interference or quenching in the sample; and (b) directly amplifying and detecting the target nucleic acid in the sample.

## Claims

### Claim 1

A method for identifying the presence or absence of a target nucleic acid in a biological sample, the method comprising:
(a) directly amplifying and detecting a target nucleic acid in a biological sample containing whole blood using a real-time polymerase chain reaction (PCR) assay, wherein the target nucleic acid is not extracted from the biological sample prior to amplifying; and
(b) rotating a biological sample during one or more steps of the real-time PCR assay at a rotational velocity of greater than 140×g and less than or about 1500×g, wherein the rotational velocity is sufficient to pellet cellular debris and fluorescence inhibitors present within the sample to reduce fluorescence interference or quenching in the sample.

### Claim 2

The method of 1 claim, wherein the biological sample is placed in a microfluidic disc capable of being subjected to centrifugal force.

### Claim 3 (depends on 1)

The method of claim 1, wherein the detecting step comprises detecting a visible signal emitted through an optical path in the rotated sample.

### Claim 4 (depends on 1)

The method of claim 1, wherein the detectable signal is a fluorescent emission.

### Claim 5 (depends on 1)

The method of claim 1, wherein the target nucleic acid is DNA.

### Claim 6 (depends on 1)

The method of claim 1, wherein the target nucleic acid is RNA.

### Claim 7 (depends on 1)

The method of claim 1, wherein the target nucleic acid is from one or more organisms found in blood.

### Claim 8 (depends on 7)

The method of claim 7, wherein the target nucleic acid is a human nucleic acid.

### Claim 9 (depends on 7)

The method of claim 7, wherein the target nucleic acid is from a microorganism.

### Claim 10 (depends on 9)

The method of claim 9, wherein the microorganism is a virus.

### Claim 11 (depends on 10)

The method of claim 10, wherein the virus is one or more of an Ebola virus, Marburg virus, an influenza virus, a respiratory syncytial virus, a varicella zoster virus, a herpes simplex virus, an enterovirus, a Dengue virus, or any combination thereof.

### Claim 12 (depends on 9)

The method of claim 9, wherein the microorganism is a bacterium.

### Claim 13 (depends on 12)

The method of claim 12, wherein the bacterium is a gram-negative or a gram-positive bacterium.

### Claim 14 (depends on 13)

The method of claim 13, wherein the bacterium is one or more of Bacillus, Bordetella, Borrelia, Listeria, Escherichia, Salmonella, Campylobacter, Clostridium, Helicobacter, Mycobacterium, Staphylococcus, Campylobacter, Enterococcus, Neisseria, Shigella, Streptococcus, Vibrio, Yersinia, and Pseudomonas, or any combination thereof.

### Claim 15 (depends on 14)

The method of claim 14, wherein the bacterium is Bacillus anthracis.

### Claim 16 (depends on 9)

The method of claim 9, wherein the microorganism is a fungus.

### Claim 17 (depends on 1)

The method of claim 1, wherein cellular debris comprises lysed and intact red blood cells and denatured proteins that can physically block or quench fluorescence.

### Claim 18 (depends on 1)

The method of claim 1, wherein the biological sample is rotated at a rotational velocity of about 1500×g.