Reaction mixtures for diagnosing bacterial vaginosis
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.
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.
2. 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.
3. 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.
4. 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.
5. The reaction mixture of claim 3 , wherein the reaction mixture comprises the Lactobacillus -specific oligonucleotide.
6. 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.
7. 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.
8. 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.
9. The reaction mixture of claim 1 , wherein the reaction mixture comprises the Lactobacillus -specific oligonucleotide.