# US 10,119,172 B2 · Methods and reagents for detecting ebola virus

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- Number: US 10,119,172 B2
- Document ID: US10119172B2
- Status: Granted
- Kind: B2
- Application number: 15/552,157
- Filed: 2016-02-18
- Published: 2018-02-15
- Granted: 2018-11-06
- Earliest claimed: 2015-02-20
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Applicants: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Inventors: Ute Stroeher; Tara Sealy; Jonathan Towner; Stuart Nichol; César Albariño
- Web page: https://patentgrep.com/patent/US10119172B2

## CPC

- C12Q 1/701: {Specific hybridization probes}
- A61K 38/02: Peptides of undefined number of amino acids; Derivatives thereof
- C12Q 1/686: Polymerase chain reaction [PCR]
- 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
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

Probes and primers are disclosed for detecting EBOV nucleic acid in a sample. Methods are also disclosed that utilize these probes and primers, wherein the methods can be used to detect an EBOV in a sample to identify a subject with an EBOV infection.

## Claims

### Claim 1

A method for detecting an EBOV NP nucleic acid in a biological sample from a subject, comprising:
amplifying a DNA template produced from a reverse transcription reaction performed on nucleic acid containing RNA isolated from the biological sample by:
contacting the DNA template under conditions suitable for amplification with a set of forward and reverse primers that can be used to amplify an EBOV nucleic acid molecule comprising the sequence set forth as SEQ ID NO: 1, and
performing a polymerase chain reaction amplification to form an amplified DNA product; and
contacting the amplified DNA product with a probe that can hybridize to the amplified DNA product;
wherein hybridization of the probe to the amplified DNA product indicates that EBOV NP nucleic acid is present in the sample.

### Claim 2 (depends on 1)

The method of claim 1, wherein the polymerase chain reaction amplification comprises a real-time polymerase chain reaction (RT-PCT).

### Claim 3 (depends on 1)

The method of claim 1, wherein the set of primers comprises a forward primer comprising, consisting essentially of, or consisting of the nucleic acid sequence set forth as SEQ ID NO: 2 (NP2-F).

### Claim 4 (depends on 1)

The method of claim 1, wherein the set of primers comprises a reverse primer comprising, consisting essentially of, or consisting of the nucleic acid sequence set forth as SEQ ID NO: 3 (NP2-R).

### Claim 5 (depends on 1)

The method of claim 1, wherein the set of primers comprises a forward primer and a reverse primer comprising, consisting essentially of, or consisting of the nucleic acid sequences set forth as SEQ ID NO: 2 (NP2-F) and SEQ ID NO: 3 (NP2-R), respectively.

### Claim 6 (depends on 1)

The method of claim 1, wherein the set of primers comprises a pair of reverse primers comprising, consisting essentially of, or consisting of the nucleic acid sequences set forth as SEQ ID NOs: 15 (NP2-R1) and 16 (NP2-R2).

### Claim 7 (depends on 1)

The method of claim 1, wherein the set of primers comprises:
a forward primer comprising, consisting essentially of, or consisting of the nucleic acid sequence set forth as SEQ ID NO: 2 (NP2-F), and a pair of reverse primers comprising, consisting essentially of, or consisting of the nucleic acid sequences set forth as SEQ ID NOs: 15 (NP2-R1) and 16 (NP2-R2).

### Claim 8 (depends on 1)

The method of claim 1, wherein the probe comprises or consists of the nucleotide sequence set forth as SEQ ID NO: 4 (NP2-P), or the complement thereof.

### Claim 9 (depends on 1)

The method of claim 1, wherein the set of primers comprises a forward primer comprising, consisting essentially of, or consisting of the nucleic acid sequence set forth as SEQ ID NO: 2 (NP2-F), and a pair of reverse primers comprising, consisting essentially of, or consisting of the nucleic acid sequences set forth as SEQ ID NOs: 15 (NP2-R1) and 16 (NP2-R2); and the probe comprises or consists of the nucleotide sequence set forth as SEQ ID NO: 4 (NP2-P), or the complement thereof.

### Claim 10 (depends on 1)

The method of claim 1, wherein the set of primers comprises a forward primer consisting of the nucleic acid sequence set forth as SEQ ID NO: 2 (NP2-F), and a pair of reverse primers consisting of the nucleic acid sequences set forth as SEQ ID NOs: 15 (NP2-R1) and 16 (NP2-R2); and the probe consists of the nucleotide sequence set forth as SEQ ID NO: 4 (NP2-P), or the complement thereof.

### Claim 11

A method for detecting an EBOV VP40 nucleic acid in a biological sample from a subject, comprising:
amplifying a DNA template produced from a reverse transcription reaction performed on nucleic acid containing RNA isolated from the biological sample by:
contacting the DNA template under conditions suitable for amplification with a set of forward and reverse primers that can be used to amplify an EBOV nucleic acid molecule comprising the sequence set forth as SEQ ID NO: 5, and
performing a polymerase chain reaction amplification to form an amplified DNA product; and
contacting the amplified DNA product with a probe that can hybridize to the amplified DNA product;
wherein hybridization of the probe to the amplified DNA product indicates that EBOV NP nucleic acid is present in the sample.

### Claim 12 (depends on 1)

The method of claim 1, further comprising detecting the amplified DNA product hybridized to the probe, wherein detecting hybridization of the probe to the amplified DNA product indicates that EBOV NP nucleic acid is present in the sample.

### Claim 13 (depends on 1)

The method of claim 1, further comprising providing the DNA template.

### Claim 14 (depends on 1)

The method of claim 1, wherein the probe is labeled with a detectable moiety that provides a detectable signal.

### Claim 15 (depends on 14)

The method of claim 14, wherein the probe is labeled with a terminally-linked fluorophore and a terminally-linked non-fluorescent quencher for use in a RT-PCR assay.

### Claim 16 (depends on 1)

The method of claim 1, wherein:
detecting the amplified DNA product hybridized to the probe comprises determining a threshold cycle (Ct) of the test amplification; and
determining if the probe is hybridized to the amplified DNA product comprises determining that the Ct of the test amplification is less than the Ct of a negative control RT-PCR amplification performed using the forward and reverse primers and the probe on a sample lacking EBOV nucleic acid.

### Claim 17 (depends on 16)

The method of claim 16, wherein the threshold cycle of the test amplification is 38 cycles or fewer.

### Claim 18 (depends on 1)

The method of claim 1, wherein the DNA template is produced by:
isolating the nucleic acid containing RNA from the biological sample; and
performing the reverse transcription reaction on the isolated nucleic acid to produce the DNA template.

### Claim 19 (depends on 1)

The method of claim 1, wherein the biological sample is a blood, serum, plasma, or urine sample.

### Claim 20 (depends on 1)

The method of claim 1, wherein the subject has or is at risk of an EBOV infection.

### Claim 21 (depends on 1)

The method of claim 1, wherein the method detects EBOV nucleic acid from at least 95% of EBOV isolates.

### Claim 22 (depends on 1)

The method of claim 1, further comprising identifying the subject as a subject with an EBOV infection if EBOV nucleic acid is detected in the biological sample.

### Claim 23 (depends on 22)

The method of claim 22, further comprising treating EBOV infection in the subject.

### Claim 24 (depends on 23)

The method of claim 23, comprising administering a therapeutically effective amount of an anti-Ebola agent to the subject.