# US 8,541,171 B2 · Assay for detection of human parvovirus B19 nucleic acid

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- Number: US 8,541,171 B2
- Document ID: US8541171B2
- Status: Granted
- Kind: B2
- Application number: 13/712,789
- Filed: 2012-12-12
- Published: 2013-04-18
- Granted: 2013-09-24
- Earliest claimed: 2001-08-31
- Assignees: Gen-Probe Incorporated
- Applicants: Gen-Probe Incorporated
- Inventors: Steven T. Brentano; Margarita Batranina-Kaminsky; Cynthia S. Hasselkus-Light; Daniel P. Kolk
- Web page: https://patentgrep.com/patent/US8541171B2

## CPC

- C12Q 1/701: {Specific hybridization probes}
- C12Q 1/6865: Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
- 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

Nucleic acid oligomers specific for human parvovirus B19 genomic DNA are disclosed. An assay for amplifying and detecting human parvovirus B19 nucleic acid in biological specimens is disclosed. Compositions for detecting the presence of parvovirus B19 genomic DNA in human biological specimens are disclosed.

## Claims

### Claim 1

A reaction mixture containing a combination of at least two separate nucleic acid oligomers, wherein the first oligomer is selected from the group consisting of:
a) SEQ ID NO:6,
b) the complete complement of SEQ ID NO:6, and
c) the RNA equivalent of the sequence of a) or b),
wherein the first oligomer optionally includes a promoter sequence joined to the 5′ terminus of the sequence;
and wherein the second oligomer is selected from the group consisting of:
d) SEQ ID NO:13,
e) the complete complement of SEQ ID NO:13, and
f) the RNA equivalent of the sequence of d) or e).

### Claim 2 (depends on 1)

The combination of oligomers of claim 1 wherein the oligomer that consists of SEQ ID NO:6 and a promoter sequence joined to the 5′ terminus of SEQ ID NO:6 consists of SEQ ID NO:5.

### Claim 3 (depends on 1)

The combination of oligomers of claim 1 wherein the promoter sequence joined to the 5′ terminus of the sequence is the promoter sequence of SEQ ID NO:19.

### Claim 4 (depends on 1)

The combination of oligomers of claim 1, further comprising a third nucleic acid oligomer that contains a target complementary sequence of 27 to 33 nucleotides and which includes a target specific sequence consisting of a) SEQ ID NO:41, b) the complete complement of SEQ ID NO:41, or c) the RNA equivalent of the sequence of a) or b); and optionally a 3′ tail portion that is nonspecific for a parvovirus target sequence, and wherein the target specific sequence optionally includes at least one 2′-methoxy substituted RNA group.

### Claim 5 (depends on 4)

The combination of oligomers of claim 4 wherein the third oligomer contains the target specific sequence consisting of a) SEQ ID NO:20, b) the complete complement of SEQ ID NO:20, or c) the RNA equivalent of the sequence of a) or b); and optionally a 3′ tail portion that is nonspecific for a parvovirus target.

### Claim 6 (depends on 4)

The combination of oligomers of claim 4 wherein the third oligomer contains the target specific sequence consisting of a) SEQ ID NO:21, b) the complete complement of SEQ ID NO:21, or c) the RNA equivalent of the sequence of a) or b); and optionally a 3′ tail portion that is nonspecific for a parvovirus target.

### Claim 7 (depends on 4)

The combination of oligomers of claim 4, wherein the target specific sequence of the third oligomer includes at least one 2′-methoxy substituted RNA group.

### Claim 8 (depends on 1)

The combination of oligomers of claim 1, further comprising a third nucleic acid oligomer consisting of a target specific sequence of at least 25 contiguous bases contained in a) SEQ ID NO:1, b) the complete complement of SEQ ID NO:1, or c) the RNA equivalent of the sequence of a) or b); and optionally a 3′ tail portion that is nonspecific for a parvovirus target.

### Claim 9 (depends on 1)

The combination of oligomers of claim 1, further comprising a third nucleic acid oligomer with a sequence consisting of a) SEQ ID NO:36, b) the complete complement of SEQ ID NO:36, or c) the RNA equivalent of the sequence of any a) or b).

### Claim 10 (depends on 9)

The combination of oligomers of claim 9 wherein the third oligomer includes at least one 2′-methoxy substituted RNA group.

### Claim 11

A method of detecting human parvovirus B19 nucleic acid in a biological sample, comprising the steps of:
amplifying in vitro a portion of a parvovirus B19 nucleic acid contained in a biological sample by using at least one nucleic acid polymerase activity and at least one first amplification oligomer and one second amplification oligomer, selected from the group consisting of
a first amplification oligomer consisting of SEQ ID NO:5 or SEQ ID NO:6 optionally including a promoter sequence joined to a 5′ terminus of SEQ ID NO:6, and
a second amplification oligomer consisting of SEQ ID NO:13; and
detecting an amplified product of the parvovirus B19 nucleic acid by using a labeled detection probe comprising: a nucleic acid sequence consisting of SEQ ID NO:17 with an acridinium ester compound (“AE”) linked between positions 7/8 or 11/12, a nucleic acid sequence consisting of SEQ ID NO:27 with an AE linked between positions 7/8 or 9/10 or 12/13 or 13/14, a nucleic acid sequence consisting of SEQ ID NO:28 with an AE linked between positions 12/13, a nucleic acid sequence consisting of SEQ ID NO:30 with an AE linked between positions 5/6 or 13/14, a nucleic acid sequence consisting of SEQ ID NO:31 with an AE linked between positions 9/10 or 10/11, a nucleic acid sequence consisting of SEQ ID NO:32 with an AE linked between positions 8/9 or 12/13, a nucleic acid sequence consisting of SEQ ID NO:34 with an AE linked between positions 7/8 or 9/10 or 11/12 or 13/14 or 16/17 or 17/18, a nucleic acid sequence consisting of SEQ ID NO:36 with an AE linked between positions 6/7 or 7/8 or 10/11 or 11/12 or 14/15 or 15/16, and a nucleic acid sequence consisting of SEQ ID NO:37 with an AE linked between positions 7/8 or 8/9 or 9/10 or 11/12 or 12/13 or 14/15 or 16/17 or 17/18 or 18/19, thereby indicating presence of parvovirus B19 nucleic acid in the biological sample.

### Claim 12 (depends on 11)

The method of claim 11 wherein the amplifying step uses the oligomer consisting of the sequence of SEQ ID NO:6 including a promoter sequence joined to the 5′ terminus of SEQ ID NO:6, wherein the promoter sequence is a T7 promoter sequence.

### Claim 13 (depends on 12)

The method of claim 12 wherein the amplifying step uses the oligomer consisting of the sequence of SEQ ID NO:6 including a promoter sequence joined to the 5′ terminus of SEQ ID NO:6, wherein the T7 promoter sequence is the sequence of SEQ ID NO:19.

### Claim 14 (depends on 11)

The method of claim 11, further comprising a step of separating the parvovirus B19 nucleic acid from the sample before the amplifying step by using a nucleic acid oligomer selected from the group consisting of:
a target complementary sequence of 27 to 33 nucleotides that contains the target specific sequence consisting of a) SEQ ID NO:41, b) the complete complement of SEQ ID NO:41, or c) the RNA equivalent of the sequence of a) or b); and optionally a 3′ tail sequence that is nonspecific for a parvovirus sequence;
the target specific sequence consisting of d) SEQ ID NO:20, e) the complete complement of SEQ ID NO:20, or f) the RNA equivalent of the sequence of d) or e); and optionally a 3′ tail sequence that is nonspecific for a parvovirus sequence;
the target specific sequence consisting of g) SEQ ID NO:21, h) the complete complement of SEQ ID NO:21, or i) the RNA equivalent of the sequence of g) or h); and optionally a 3′ tail portion that is nonspecific for a parvovirus sequence; and
a target complementary sequence of at least 25 contiguous bases contained in j) SEQ ID NO:1, k) the complete complement of SEQ ID NO:1, or l) the RNA equivalent of the sequence of j) or k); and optionally a 3′ tail sequence that is nonspecific for a parvovirus sequence.

### Claim 15 (depends on 11)

The method of claim 11 wherein the amplifying step uses an amplification reaction that is substantially isothermal.

### Claim 16 (depends on 11)

The method of claim 11, wherein the detecting step uses a labeled detection probe comprising at least one 2′-methoxy substituted RNA group.