# US 10,865,433 B2 · Reaction mixtures for diagnosing bacterial vaginosis

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- Number: US 10,865,433 B2
- Document ID: US10865433B2
- Status: Granted
- Kind: B2
- Application number: 15/543,944
- Filed: 2016-01-08
- Published: 2018-06-21
- Granted: 2020-12-15
- Earliest claimed: 2015-01-09
- Assignees: Gen-Probe Incorporated
- Applicants: Gen-Probe Incorporated
- Inventors: Michael Graham; Jeff G. Hall; Joseph J. King
- Web page: https://patentgrep.com/patent/US10865433B2

## CPC

- C12Q 1/04: Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)}
- C12Q 1/6883: for diseases caused by alterations of genetic material
- C12Q 1/689: for bacteria
- G01N 33/533: with fluorescent label
- G01N 33/6893: {related to diseases not provided for elsewhere}
- G01N 2800/26: Infectious diseases, e.g. generalised sepsis
- G01N 2800/36: Gynecology or obstetrics
- 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

Disclosed are methods for diagnosing Bacterial Vaginosis (BV). The disclosed methods generally include detecting select species of Eggerthella and/or Prevotella, and optionally detecting select species of Lactobacillus. Also disclosed are nucleic acid oligomers and related compositions for detection of a 16S rRNA or its encoding gene from select species of Eggerthella, Prevotella, or Lactobacillus.

## Claims

### Claim 1

A reaction mixture for specific detection of an Eggerthella target nucleic acid and a Prevotella target nucleic acid in the presence of other nucleic acids that may be in a sample, the reaction mixture comprising:
an Eggerthella-specific oligonucleotide that specifically hybridizes to a target sequence within a target nucleic acid of an Eggerthella species characterized by the presence of a 16S rRNA gene having a nucleobase sequence that is at least 98% identical to the sequence shown in SEQ ID NO:1, but does not specifically hybridize to a sequence within a nucleic acid from other Eggerthella species, wherein the Eggerthella-specific oligonucleotide comprises a 3′ portion that specifically hybridizes to the target sequence within the Eggerthella target nucleic acid and a 5′ portion that does not specifically hybridize to the Eggerthella target nucleic acid, wherein the 3′ portion of the Eggerthella-specific oligonucleotide is a target-hybridizing sequence consisting of the sequence shown in residues 11-27 of SEQ ID NO:4, and
a Prevotella-specific oligonucleotide that specifically hybridizes to a target sequence within a target nucleic acid of P. amnii, P. disiens, and P. bivia, but does not specifically hybridize to a sequence within a nucleic acid from other Prevotella species, wherein the Prevotella-specific oligonucleotide comprises a 3′ portion that specifically hybridizes to the target sequence within the Prevotella target nucleic acid and a 5′ portion that does not specifically hybridize to the Prevotella target nucleic acid, wherein the 3′ portion of the Prevotella-specific oligonucleotide is a target-hybridizing sequence consisting of the sequence shown in residues 11-25 of SEQ ID NO:7,
optionally wherein the reaction mixture further comprises a Lactobacillus-specific oligonucleotide that specifically hybridizes to a target sequence within a target nucleic acid of Lactobacillus species, but does not specifically hybridize to a sequence within a nucleic acid from L. iners, wherein the Lactobacillus-specific oligonucleotide comprises a 3′ portion that specifically hybridizes to the target sequence within the Lactobacillus target nucleic acid and a 5′ portion that does not specifically hybridize to the Lactobacillus target nucleic acid.

### Claim 2 (depends on 1)

The reaction mixture of claim 1, wherein the Eggerthella and Prevotella target nucleic acids are 16S rRNAs of Eggerthella and Prevotella, respectively, and wherein if the reaction mixture further comprises the Lactobacillus-specific oligonucleotide, then the Lactobacillus target nucleic acid is a 16S rRNA of Lactobacillus.

### Claim 3 (depends on 2)

The reaction mixture of claim 2, wherein
the 3′ portion of the Lactobacillus-specific oligonucleotide, if present, is a target-hybridizing sequence consisting of the sequence shown in residues 11-27 of SEQ ID NO:10.

### Claim 4 (depends on 3)

The reaction mixture of claim 3, wherein the reaction mixture comprises the Lactobacillus-specific oligonucleotide, and wherein the reaction mixture further comprises
(i) second and third Eggerthella-specific oligonucleotides that target a sequence within an Eggerthella 16S rRNA region corresponding to nucleotide positions 615 to 679 of SEQ ID NO: 1; and/or
(ii) second and third Prevotella-specific oligonucleotides that target a sequence within a Prevotella 16S rRNA region corresponding to nucleotide positions 954 to 1037 of SEQ ID NO:2; and/or
(iii) second and third Lactobacillus-specific oligonucleotides that target a sequence within a Lactobacillus 16S rRNA region corresponding to nucleotide positions 837 to 944 of SEQ ID NO:3.

### Claim 5 (depends on 3)

The reaction mixture of claim 3, wherein the reaction mixture comprises the Lactobacillus-specific oligonucleotide.

### Claim 6 (depends on 4)

The reaction mixture of claim 4, wherein the second Eggerthella-specific oligonucleotide comprises a target-hybridizing sequence corresponding to the sequence shown in residues 1-20 of SEQ ID NO:5, and wherein the third Eggerthella-specific oligonucleotide comprises a target-hybridizing sequence corresponding to the sequence shown in SEQ ID NO:6.

### Claim 7 (depends on 4)

The reaction mixture of claim 4, wherein the second Prevotella-specific oligonucleotide comprises a target-hybridizing sequence corresponding to the sequence shown in residues 1-24 of SEQ ID NO:8, and wherein the third Prevotella-specific oligonucleotide comprises a target-hybridizing sequence corresponding to the sequence shown in SEQ D NO:9.

### Claim 8 (depends on 4)

The reaction mixture of claim 4, wherein the second Lactobacillus-specific oligonucleotide is selected from the group consisting of (i) an oligonucleotide comprising a target-hybridizing sequence corresponding to the sequence shown in residues 1-27 of SEQ ID NO: 11 and (ii) an oligonucleotide comprising a target-hybridizing sequence corresponding to the sequence shown in residues 1-32 of SEQ ID NO: 12, and wherein the third Lactobacillus-specific oligonucleotide comprises a target-hybridizing sequence corresponding to the sequence shown in SEQ ID NO: 13.

### Claim 9 (depends on 1)

The reaction mixture of claim 1, wherein the reaction mixture comprises the Lactobacillus-specific oligonucleotide.