# US 8,785,125 B2 · Compositions, reaction mixtures and methods for detecting nucleic acids from type A1 and/or type C1 human papillomavirus

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- Number: US 8,785,125 B2
- Document ID: US8785125B2
- Status: Granted
- Kind: B2
- Application number: 14/040,399
- Filed: 2013-09-27
- Published: 2014-01-30
- Granted: 2014-07-22
- Earliest claimed: 2004-12-08
- Assignees: Gen-Probe Incorporated
- Applicants: Gen-Probe Incorporated
- Inventors: Sylvia A. Norman; Jennifer J. Bungo; William L. Hanna; Neeraj P. Rao
- Web page: https://patentgrep.com/patent/US8785125B2

## CPC

- C12Q 1/708: {for papilloma}
- C07K 14/005: from viruses
- C12N 2710/20022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12Q 2600/156: Polymorphic or mutational markers
- C12Q 2600/158: Expression markers
- C12Q 2600/16: Primer sets for multiplex assays
- 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
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Nucleic acid oligonucleotide sequences are disclosed which include amplification oligomers and probe oligomers which are useful for detecting multiple types of human papillomaviruses (HPV) associated with cervical cancer. Methods for detecting multiple HPV types in biological specimens by amplifying HPV nucleic acid sequences in vitro and detecting the amplified products are disclosed.

## Claims

### Claim 1

A multiplex method of detecting the presence of type A1 and type C1 human papillomavirus (HPV) nucleic acids present in a biological sample, comprising the steps of:
(a) treating a biological sample suspected of containing HPV type A1 or HPV type C1 to release intracellular components;
(b) separating an HPV nucleic acid away from a component of the biological sample;
(c) contacting the separated HPV nucleic acid with a combination of amplification oligonucleotides that amplify a HPV type A1 target sequences and a combination of amplification oligomers that amplify HPV type C1 target sequences in an E6/E7 target region sequence, wherein the combination of amplification oligomers configured to amplify HPV type A1 comprises amplification oligomers comprising nucleic acid sequences consisting essentially of SEQ ID NOS:18, 20, 22, and 38;
(d) amplifying a HPV target sequence by using the amplification oligonucleotides and a nucleic acid polymerase in vitro to produce an HPV amplified product; and
(e) detecting the amplified product to indicate the presence in the sample of at least one of HPV types A1 or C1.

### Claim 2 (depends on 1)

The method of claim 1, wherein the step of separating an HPV nucleic acid from components of the sample uses at least two capture oligonucleotides, wherein a capture oligonucleotide sequence for separating HPV type A1 nucleic acid is SEQ ID NO:2 joined to a ligand moiety or SEQ ID NO:1.

### Claim 3 (depends on 1)

The method of claim 1, wherein the amplifying step uses an amplification process that is substantially isothermal.

### Claim 4 (depends on 1)

The method of claim 1, wherein the detecting step uses a combination of probe oligonucleotides configured to detect amplification product generated in step (d) and thereby indicate the presence of at least one of HPV types 16, 18, 31, 35, 45, and 59 in the biological sample, and wherein the combination of probe oligomers includes one or more probes having a sequence of SEQ ID NO:11, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46.

### Claim 5 (depends on 4)

The method of claim 4, wherein each probe oligonucleotide is joined directly or indirectly to a label.

### Claim 6 (depends on 1)

The method of claim 1, wherein the combination of amplification oligomers configured to amplify HPV type C1 comprises a first amplification oligomer nucleic acid sequences that is SEQ ID NO:29, joined at its 5′ end to a promoter sequence, or SEQ ID NO:31, joined at its 5′ end to a promoter sequence, and a second amplification oligomer that is SEQ ID NO:39.

### Claim 7 (depends on 1)

The method of claim 1, wherein the combination of amplification oligomers configured to amplify HPV type C1 comprise sequences that are SEQ ID NOS:28 and 39; SEQ ID NOS:30 and 39 or SEQ ID NOS:28, 30 and 39.

### Claim 8 (depends on 1)

The method of claim 1, wherein the biological sample is treated to physically disrupt the sample to release the intracellular components.

### Claim 9 (depends on 1)

The method of claim 1, wherein the biological sample is treated with an enzyme to release intracellular components.

### Claim 10

An amplification reaction mixture for use in a multiplex method of amplifying type A1 and type C1 human papillomavirus (HPV) nucleic acids present in a biological sample, wherein the reaction mixture contains (i) amplification oligomers configured to generate an amplification product from HPV type A1, wherein the amplification oligomers comprise sequences that that consist essentially of SEQ ID NOS:18, 20, 22, and 38 and (ii) amplification oligomers for generating an amplification product from HPV type C1.

### Claim 11 (depends on 10)

The reaction mixture of claim 10, wherein the amplification oligomers configured to generate an amplification product from HPV type C1 generate a type C1 amplification product that can be detected by one or more of detection probes SEQ ID NOS:44, 45 or 46.

### Claim 12

A method of detecting the presence of type A1 and type C1 human papillomavirus (HPV) nucleic acids present in a biological sample, comprising the steps of:
(a) treating a biological sample suspected of containing HPV type A1 or HPV type C1 to release an HPV nucleic acid;
(b) separating the HPV nucleic acid away from a component of the biological sample;
(c) contacting the separated HPV nucleic acid with:
(i) a combination of amplification oligonucleotides that amplify an HPV type A1 target sequence, the amplification oligonucleotides comprising sequences consisting of SEQ ID NOS:18, 20, 22, and 38; and
(ii) a combination of amplification oligonucleotides that amplify an HPV type C1 target sequence, the amplification oligonucleotides comprising sequences consisting essentially of SEQ ID NOS:28 or 30 and SEQ ID NO:39;
(d) amplifying a HPV target sequence by using the amplification oligonucleotides from step (c) and a nucleic acid polymerase to produce an HPV amplification product; and
(e) detecting the amplification product to indicate the presence in the sample of at least one of HPV types A1 or C1.

### Claim 13 (depends on 12)

The method of claim 12, wherein the treating step physically disrupts the sample to release the nucleic acid.

### Claim 14 (depends on 12)

The method of claim 12, wherein the separating step uses one or more capture oligonucleotides.

### Claim 15 (depends on 12)

The method of claim 12, wherein the amplification step is a transcription-associated amplification.

### Claim 16 (depends on 12)

The method of claim 12, wherein the amplification step amplifies the HPV type A1 and the HPV type C1 nucleic acids in the same reaction well.

### Claim 17 (depends on 12)

The method of claim 12, wherein the detection reaction uses probe oligonucleotides.

### Claim 18 (depends on 12)

The method of claim 12, wherein the detection reaction uses two or more probe oligonucleotides comprising sequences selected from the group consisting of SEQ ID NOS:11, 44, 45 and 46.