# US 2025/0146090 A1 · Compositions and Methods for Detecting Nucleic Acids of Epstein-Barr Virus

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- Number: US 2025/0146090 A1
- Document ID: US20250146090A1
- Status: Published application
- Kind: A1
- Application number: 19/014,660
- Filed: 2025-01-09
- Published: 2025-05-08
- Earliest claimed: 2018-08-01
- Assignees: Gen-Probe Incorporated
- Applicants: Gen-Probe Incorporated
- Inventors: Ankur SHAH; Jian Yu FUNG
- Web page: https://patentgrep.com/patent/US20250146090A1

## CPC

- C12Q 1/705: {for herpetoviridae, e.g. herpes simplex, varicella zoster}
- C12Q 2600/158: Expression markers
- 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

## Abstract

Disclosed are compositions, methods, and kits that can be used to Epstein-Barr virus (EBV) in a sample undergoing testing. Nucleic acids of EBV can be isolated, amplified and detected with specificity, and without interference from non-EBV organisms. In some embodiments, nucleic acids used for amplification are isolated from human blood, or blood products. Nucleic acid isolation, amplification and detection steps can all be carried out using an automated instrument.

## Claims

### Claim 1

A reaction mixture for determining the presence or absence of Epstein-Barr virus (EBV) in a sample, said reaction mixture comprising:
a detection probe oligomer for detecting EBV nucleic acids,
wherein the detection probe oligomer is up to 30 nucleotides in length and comprises the base sequence of SEQ ID NO:22 or the complement thereof, allowing for substitution of RNA and DNA equivalent bases; and
a pair of amplification oligomers,
wherein a first amplification oligomer of the pair comprises 18-25 contiguous bases of SEQ ID NO:3, allowing for substitution of RNA and DNA equivalent bases, and
wherein a second amplification oligomer of the pair comprises 18-25 contiguous bases of SEQ ID NO:4, allowing for substitution of RNA and DNA equivalent bases.

### Claim 2 (depends on 1)

The reaction mixture of claim 1, wherein the detection probe oligomer further comprises a detectable label.

### Claim 3 (depends on 2)

The reaction mixture of claim 2, wherein the detectable label comprises an interactive label pair comprising a fluorophore moiety and a quencher moiety.

### Claim 4 (depends on 1, 2, 3)

The reaction mixture of any one of claims 1 to 3, wherein the detection probe oligomer further comprises at least one nucleotide analog.

### Claim 5 (depends on 4)

The reaction mixture of claim 4, wherein the at least one nucleotide analog of the detection probe oligomer comprises at least one 5-methyl cytosine base.

### Claim 6 (depends on 1, 2, 3, 4, 5)

The reaction mixture of any one of claims 1 to 5, wherein the detection probe oligomer is 22 nucleotides in length and comprises the base sequence of SEQ ID NO:9.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The reaction mixture of any one of claims 1 to 6, wherein each of the first and second amplification oligomers is up to 25 nucleotides in length.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The reaction mixture of any one of claims 1 to 7, wherein the first amplification oligomer is 20 nucleotides in length, and wherein the base sequence of the first amplification oligomer consists of 20 contiguous bases of SEQ ID NO:3.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The reaction mixture of any one of claims 1 to 8, wherein the base sequence of the first amplification oligomer is SEQ ID NO:5.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The reaction mixture of any one of claims 1 to 9, wherein the second amplification oligomer is 20 nucleotides in length, and wherein the base sequence of the second amplification oligomer consists of 20 contiguous bases of SEQ ID NO:4.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The reaction mixture of any one of claims 1 to 10, wherein the base sequence of the second amplification oligomer is SEQ ID NO:7.

### Claim 12 (depends on 7)

The reaction mixture of claim 7, wherein the base sequence of the first amplification oligomer is either SEQ ID NO:5 or SEQ ID NO:6, and wherein the base sequence of the second amplification oligomer is either SEQ ID NO:7 or SEQ ID NO:8.

### Claim 13 (depends on 12)

The reaction mixture of claim 12, wherein the base sequence of the first amplification oligomer is SEQ ID NO:5.

### Claim 14 (depends on 12)

The reaction mixture of claim 12, wherein the base sequence of the second amplification oligomer is SEQ ID NO:7.

### Claim 15 (depends on 13)

The reaction mixture of claim 13, wherein the base sequence of the second amplification oligomer is SEQ ID NO:7.

### Claim 16

A method of determining the presence or absence of Epstein-Barr virus (EBV) in a sample, said method comprising the steps of:
(a) contacting a sample to be tested for the presence of EBV with an oligomer combination that comprises,
a first amplification oligomer that comprises 18-25 contiguous bases of SEQ ID NO:3, allowing for substitution of RNA and DNA equivalent bases,
a second amplification oligomer that comprises 18-25 contiguous bases of SEQ ID NO:4, allowing for substitution of RNA and DNA equivalent bases, and
a detection probe oligomer of up to 30 nucleotides in length that comprises the base sequence of SEQ ID NO:22 or the complement thereof, allowing for substitution of RNA and DNA equivalent bases;
(b) performing an in vitro nucleic acid amplification reaction using the oligomer combination, wherein any EBV target nucleic acid, if present in the sample, is a template for generating an amplification product; and
(c) detecting, with the detection probe oligomer, the presence or absence of the amplification product, thereby determining the presence or absence of EBV in the sample.

### Claim 17 (depends on 16)

The method of claim 16, wherein the detection probe oligomer further comprises a detectable label.

### Claim 18 (depends on 17)

The method of either claim 17, wherein the detectable label of the detection probe oligomer comprises an interactive label pair comprising a fluorophore moiety and a quencher moiety.

### Claim 19 (depends on 16, 17, 18)

The method of any one of claims 16 to 18, wherein the detection probe oligomer further comprises at least one nucleotide analog.

### Claim 20 (depends on 19)

The method of claim 19, wherein the at least one nucleotide analog comprises at least one 5-methyl cytosine base.

### Claim 21 (depends on 16, 17, 18, 19, 20)

The method of any one of claims 16 to 20, wherein the detection probe oligomer is 22 nucleotides in length and comprises the base sequence of SEQ ID NO:9.

### Claim 22 (depends on 16, 17, 18, 19, 20, 21)

The method of any one of claims 16 to 21, wherein the sample comprises nucleic acids isolated from any of human blood, human plasma, or human serum.

### Claim 23 (depends on 16, 17, 18, 19, 20, 21, 22)

The method of any one of claims 16 to 22, wherein the in vitro nucleic acid amplification reaction comprises Taq DNA polymerase.

### Claim 24 (depends on 16, 17, 18, 19, 20, 21, 22, 23)

The method of any one of claims 16 to 23, wherein steps (b) and (c) take place concurrently, the in vitro nucleic acid amplification reaction being a real-time nucleic acid amplification reaction.

### Claim 25 (depends on 16, 17, 18, 19, 20, 21, 22, 23, 24)

The method of any one of claims 16 to 24, wherein the in vitro nucleic acid amplification reaction in step (b) is a multiplex in vitro nucleic acid amplification reaction that amplifies and detects, in addition to any nucleic acid of EBV that may be present in the sample, any nucleic acid of cytomegalovirus (CMV) that may be present in the sample.

### Claim 26 (depends on 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)

The method of any one of claims 16 to 25, wherein the in vitro nucleic acid amplification reaction in step (b) is a PCR amplification reaction that comprises a DNA polymerase with a 5′ to 3′ exonuclease activity.

### Claim 27 (depends on 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)

The method of any one of claims 16 to 26, wherein before step (a) there is the step of isolating nucleic acids, and wherein all of the steps are performed using a single automated instrument.

### Claim 28

A kit of reagents, comprising in one or more vials:
a detection probe oligomer for detecting Epstein-Barr virus (EBV) nucleic acids,
wherein the detection probe oligomer is up to 30 nucleotides in length and comprises the base sequence of SEQ ID NO:22 or the complement thereof, allowing for substitution of RNA and DNA equivalent bases; and
a pair of amplification oligomers,
wherein a first amplification oligomer of the pair comprises 18-25 contiguous bases of SEQ ID NO:3, allowing for substitution of RNA and DNA equivalent bases, and
wherein a second amplification oligomer of the pair comprises 18-25 contiguous bases of SEQ ID NO:4, allowing for substitution of RNA and DNA equivalent bases.

### Claim 29 (depends on 28)

The kit of claim 28, wherein the detection probe oligomer further comprises a detectable label.

### Claim 30 (depends on 29)

The kit of claim 29, wherein the detectable label of the detection probe oligomer comprises an interactive label pair comprising a fluorophore moiety and a quencher moiety.

### Claim 31 (depends on 28, 29, 30)

The kit of any one of claims 28 to 30, wherein the detection probe oligomer further comprises at least one nucleotide analog.

### Claim 32 (depends on 31)

The kit of claim 31, wherein the at least one nucleotide analog of the detection probe oligomer comprises at least one 5-methyl cytosine base.

### Claim 33 (depends on 28, 29, 30, 31, 32)

The kit of any one of claims 28 to 32, wherein the detection probe oligomer is 22 nucleotides in length and comprises the base sequence of SEQ ID NO:9.

### Claim 34 (depends on 28, 29, 30, 31, 32, 33)

The kit of any one of claims 28 to 33, wherein each of the first and second amplification oligomers is up to 25 nucleotides in length.

### Claim 35 (depends on 28, 29, 30, 31, 32, 33, 34)

The kit of any one of claims 28 to 34, wherein the first amplification oligomer is 20 nucleotides in length, and wherein the base sequence of the first amplification oligomer consists of 20 contiguous bases of SEQ ID NO:3.

### Claim 36 (depends on 28, 29, 30, 31, 32, 33, 34, 35)

The kit of any one of claims 28 to 35, wherein the base sequence of the first amplification oligomer is SEQ ID NO:5.

### Claim 37 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36)

The kit of any one of claims 28 to 36, wherein the second amplification oligomer is 20 nucleotides in length, and wherein the base sequence of the second amplification oligomer consists of 20 contiguous bases of SEQ ID NO:4.

### Claim 38 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)

The kit of any one of claims 28 to 37, wherein the base sequence of the second amplification oligomer is SEQ ID NO:7.

### Claim 39 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 37)

The kit of any one of claims 28 to 35 or claim 37, wherein the base sequence of the first amplification oligomer is either SEQ ID NO:5 or SEQ ID NO:6, and wherein the base sequence of the second amplification oligomer is either SEQ ID NO:7 or SEQ ID NO:8.

### Claim 40 (depends on 39)

The kit of claim 39, wherein the base sequence of the first amplification oligomer is SEQ ID NO:5.

### Claim 41 (depends on 39)

The kit of claim 39, wherein the base sequence of the second amplification oligomer is SEQ ID NO:7.

### Claim 42 (depends on 40)

The kit of claim 40, wherein the base sequence of the second amplification oligomer is SEQ ID NO:7.

### Claim 43 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)

The kit of any one of claims 28 to 42, wherein the first and second amplification oligomers are packaged together in one vial, and wherein the probe oligonucleotide is packaged in a separate vial.

### Claim 44

A detection probe oligomer for detecting Epstein-Barr virus (EBV) nucleic acids, wherein the detection probe oligomer is up to 30 nucleotides in length and comprises the base sequence of SEQ ID NO:22 or the complement thereof, allowing for substitution of RNA and DNA equivalent bases.

### Claim 45 (depends on 44)

The detection probe oligomer of claim 44, further comprising a detectable label.

### Claim 46 (depends on 45)

The detection probe oligomer of claim 45, wherein the detectable label comprises an interactive label pair comprising a fluorophore moiety and a quencher moiety.

### Claim 47 (depends on 44, 45, 46)

The detection probe oligomer of any one of claims 44 to 46, further comprising at least one nucleotide analog.

### Claim 48 (depends on 47)

The detection probe oligomer of claim 47, wherein the at least one nucleotide analog comprises at least one 5-methyl cytosine base.

### Claim 49 (depends on 44, 45, 46, 47, 48)

The detection probe oligomer of any one of claims 44 to 48, wherein the detection probe oligomer is up to 26 nucleotides in length and comprises a base sequence fully contained within the sequence of SEQ ID NO:17 or the complement thereof.

### Claim 50 (depends on 44, 45, 46, 47, 48, 49)

The detection probe oligomer of any one of claims 44 to 49, wherein the detection probe oligomer is up to 22 nucleotides in length.

### Claim 51 (depends on 44, 45, 46, 47, 48, 49)

The detection probe oligomer of any one of claims 44 to 49, wherein the detection probe oligomer is 22 nucleotides in length and comprises the base sequence of SEQ ID NO:9 or the complement thereof.

### Claim 52 (depends on 51)

The detection probe oligomer of claim 51, wherein the detection probe oligomer is 22 nucleotides in length and comprises the base sequence of SEQ ID NO:9.

### Claim 53 (depends on 52)

The detection probe oligomer of claim 52, further comprising at least nine nucleotide analogs.

### Claim 54 (depends on 52)

The detection probe oligomer of claim 52, further comprising nine 5-methyl cytosine nucleotide analogs.

### Claim 55 (depends on 44, 45, 46)

The detection probe oligomer of any one of claims 44 to 46, wherein the detection probe oligomer is 22 nucleotides in length with a base sequence consisting of SEQ ID NO:9.

### Claim 56 (depends on 55)

The detection probe oligomer of claim 55, comprising a plurality of 5-methyl cytosine nucleotide analogs.

### Claim 57 (depends on 55)

The detection probe oligomer of claim 55, wherein every cytosine base in the sequence of SEQ ID NO:9 is the 5-methyl cytosine nucleotide analog.

### Claim 58 (depends on 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57)

The detection probe oligomer of any one of claims 46 to 57, wherein the detection probe oligomer is a dual-labeled hydrolysis probe.

### Claim 59 (depends on 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

The detection probe oligomer of any one of claims 44 to 58, wherein the probe is hybridized to a complementary nucleic acid strand in the presence of a DNA polymerase that comprises a 5′ to 3′ exonuclease activity.