# US 2019/0376124 A1 · Apparatus and method for specific detection and quantization of nucleic acid

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2019/0376124 A1
- Document ID: US20190376124A1
- Status: Published application
- Kind: A1
- Application number: 16/306,314
- Filed: 2017-06-01
- Published: 2019-12-12
- Earliest claimed: 2016-06-02
- Assignees: Takena Technologies, Inc.
- Applicants: Takena Technologies, Inc.
- Inventors: David Clayton Roberts; Gregory Allen Peek; John Enrique Mata
- Web page: https://patentgrep.com/patent/US20190376124A1

## CPC

- C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer
- C12Q 1/701: {Specific hybridization probes}
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- C12Q 2600/112: Disease subtyping, staging or classification
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- 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

A method for detecting and quantifying a target nucleic acid sequence comprises combining a first phosphorodiamidate morpholino oligomer (PMO) probe with a donor dye, the first probe having a sequence complementary to a first portion of the of the target nucleic acid sequence, a second PMO probe comprising a receptor dye, the second probe having a sequence complementary to a second portion of the target nucleic acid sequence, in near proximity to the first portion; and a sample material, exposing the combined solution to incident light to cause the first fluorescent dye to donate energy to the second fluorescent, causing the second fluorescent dye to fluoresce, and detecting and measuring fluorescence of the second fluorescent dye.

## Claims

### Claim 1

A method for detecting and quantifying a target nucleic acid sequence in a sample, comprising the steps of:
(a) combining in a single solution:
i. a first phosphorodiamidate morpholino oligomer (PMO) probe comprising a first fluorescent dye actionable under exposure to incident light to act as a donor, the first probe having a sequence complementary to a first portion of the of the target nucleic acid sequence;
ii. a second PMO probe comprising a second fluorescent dye actionable as a receptor, the second probe having a sequence complementary to a second portion of the target nucleic acid sequence, the second portion in near proximity to the first portion; and
iii. a sample material suspected of comprising strands of the target nucleic acid sequence;
(b) exposing the combined solution to incident light at a specific wavelength known to cause the first fluorescent dye of the first PMO probe to donate energy in a non-radiative manner to the second fluorescent dye of the second PMO probe, causing the second fluorescent dye to fluoresce as a result; and
(c), detecting and measuring fluorescence of the second fluorescent dye by a light detection device, detection indicating presence of the target nucleic acid sequence in the sample.

### Claim 2 (depends on 1)

The method of claim 1 wherein the first and second PMO probes are added together or sequentially to a solution containing the sample material suspected of comprising strands of the target nucleic acid sequence.

### Claim 3 (depends on 1)

The method of claim 1 wherein the sample material suspected of comprising strands of the target nucleic acid sequence is added to a solution containing the first and second PMO probes

### Claim 4 (depends on 1)

The method of claim 1 wherein the light detection device is enabled to record intensity of the emitted photons as an indication of the quantity of the target nucleic acid present.

### Claim 5 (depends on 1)

The method of claim 1 further comprising adding a quencher dye having substantially lower binding affinity than the target nucleic acid, which quencher dye binds to the acceptor dye in solution, absorbing energy from the acceptor dye when the second PMO probe is free in solution, and disassociates when and if the second PMO probe binds to the target nucleotide.

### Claim 6 (depends on 1)

The method of claim 1 wherein the first and second PMO probes contain multiple donor or acceptor dyes, enabling multiplexed amplification of signal from more than two oligomers.

### Claim 7 (depends on 1)

The method of claim 1 wherein the first and second PMO probes are prepared complementary to one of the nucleic acid sequences Ebola virus, Zika virus, tuberculosis virus, salmonella virus, HIV virus, or HCV virus.

### Claim 8 (depends on 1)

The method of claim 1 further comprising a step for comparing result of measurement of light emission of the second fluorescent dye to prepared standards, to determine quantity of target nucleic acid sequence in the sample.

### Claim 9

A kit for detecting and quantifying a target nucleic acid sequence in a test sample, comprising:
a first container holding a sample solution possibly comprising the target nucleic acid;
a second container holding, in solution, a first phosphorodiamidate morpholino oligomer (PMO) probe comprising a first fluorescent dye actionable under exposure to incident light to act as a donor, the first PMO probe having a sequence complementary to a first portion of the of the target nucleic acid sequence;
a third container holding, in solution, a second PMO probe comprising a second fluorescent dye actionable as a receptor, the second probe having a sequence complementary to a second portion of the target nucleic acid sequence, the second portion in near proximity to the first portion;
a light source enabled to illuminate contents of the first container at a wavelength inducing the first fluorescent dye to donate energy in a non-radiative manner to the second fluorescent dye; and
a device to collect and measure fluorescent light emissions from the acceptor probe dye.

### Claim 10 (depends on 9)

The kit of claim 9 further comprising a fourth container holding, in solution, a quencher dye having substantially lower binding affinity than the target nucleic acid, which binds to the acceptor dye in solution, absorbing energy from the acceptor dye when the second oligomer probe is free in solution, and disassociates when and if the second oligomer probe binds to the target nucleotide.

### Claim 11 (depends on 9)

The kit of claim 9 wherein the light detection device is enabled to record intensity of the emitted photons as an indication of the quantity of the target nucleic acid present.

### Claim 12 (depends on 9)

The method of claim 9 wherein the first and second PMO probes contain multiple donor or acceptor dyes, enabling multiplexed amplification of signal from more than two probes.

### Claim 13 (depends on 9)

The kit of claim 9 wherein the first and second PMO probes in the second and third containers, respectively, are implemented to be complementary to one of nucleic acid sequence of Ebola virus,

### Claim 14 (depends on 9)

The kit of claim 9 wherein the first and second oligomer probes in the second and third containers, respectively, are prepared to be complementary to one of the nucleic acid sequences Ebola virus, Zika virus, tuberculosis virus, salmonella virus, HIV virus, or HCV virus.

### Claim 15

A portable detection system for detecting and quantifying a target nucleic acid sequence in a sample, comprising:
a hand-held, computerized appliance having a cartridge holder for holding a sample cartridge, a laser for illuminating a sample in a cartridge in the cartridge holder, a light sensor for detecting light emission from the sample in the cartridge, and wireless circuitry for communication with a network-connected communication device; and
a cartridge configured to insert and withdraw from the cartridge holder, the cartridge comprising a donor probe chamber, an acceptor probe chamber, a sample chamber, and a target reaction chamber, with interconnecting internal channels;
wherein a cartridge implemented with donor and acceptor probes specific to a particular target nucleotide in respective chambers is loaded with a sample in the sample chamber, and inserted into the cartridge holder, where the sample and probes are mixed in the reaction chamber and illuminated by the laser, and intensity determined by the light detector is communicated wirelessly to the network-connected communication device.

### Claim 16 (depends on 15)

The system of claim 15 further comprising an application executing on the network-connected communication device, enabling digital storage of results of assays and communication with a remote, network-connected database.

### Claim 17 (depends on 15)

The system of claim 15 further comprising the cartridge having machine readable information thereon, identifying at least a unique serial number for tracking, and further comprising apparatus in the hand-held, computerized appliance, enabling reading of the machine-readable indicia.

### Claim 18 (depends on 17)

The system of claim 17 wherein the machine-readable information is in the nature of a bar code or Quick Response (QR) code, and the reading apparatus is optical in nature.

### Claim 19 (depends on 17)

The system of claim 17 wherein the machine-readable indicia is electronic in nature, and the reading apparatus comprises wireless circuitry for accessing the data on the cartridge.

### Claim 20 (depends on 19)

The system of claim 19 wherein the reading apparatus comprises electronic contacts between the cartridge and circuitry in the hand-held device.

### Claim 21 (depends on 17)

The system of claim 17, wherein, in addition to a unique serial number, the information comprises one or more of an assay name, date of manufacture and lot number, and one or more data tables.