Methods for diagnosing bacterial vaginosis

1. A method for diagnosing Bacterial Vaginosis (BV) in a subject, the method comprising:

(a) providing a sample from a subject suspected of having BV; and

(b) performing a nucleic-acid-based detection assay for the detection of select bacterial species in each of the genera Eggerthella, Prevotella , and Lactobacillus in the sample, wherein

the assay detects 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 detect other Eggerthella species,

the assay detects P. amnii, P. disiens , and P. bivia but does not detect other Prevotella species, and

the assay detects select Lactobacillus species but does not detect L. iners ,

wherein the assay targets (i) an Eggerthella 16S rRNA region corresponding to nucleotide positions 615 to 679 of SEQ ID NO:1, (ii) a Prevotella 16S rRNA region corresponding to nucleotide positions 954 to 1037 of SEQ ID NO:2, and (iii) a Lactobacillus 16S rRNA region corresponding to nucleotide positions 837 to 944 of SEQ ID NO:3,

wherein if Lactobacillus is not detected, then the detection of at least one of Eggerthella and Prevotella indicates BV in the subject, and if Lactobacillus is detected, then the detection of both Eggerthella and Prevotella indicates BV in the subject, and

wherein the nucleic-acid-based detection assay is a cleavage-based assay comprising

(i) contacting the sample with

(A) an Eggerthella -specific primer that specifically hybridizes to a target sequence within nucleotide positions 615 to 679 of SEQ ID NO:1,

(B) a Prevotella -specific primer that specifically hybridizes to a target sequence within nucleotide positions 954 to 1037 of SEQ ID NO:2, and

(C) a Lactobacillus -specific primer that specifically hybridizes to a target sequence within nucleotide positions 837 to 944 of SEQ ID NO:3,

wherein said contacting is performed under reaction conditions whereby each primer specifically hybridizes to its respective 16S rRNA target sequence within an Eggerthella target 16S rRNA, a Prevotella target 16S rRNA, or a Lactobacillus target 16S rRNA, if present;

(ii) providing reaction conditions whereby the 3′ end of each hybridized primer is extended, thereby generating a single-stranded cDNA having a sequence complementary to a region of the Eggerthella, Prevotella , or Lactobacillus target 16S rRNA, said region located 5′ to the respective primer target sequence;

(iii) contacting any Eggerthella, Prevotella , or Lactobacillus cDNA from step (ii) with

(A) a first Eggerthella probe oligonucleotide having a 3′ portion that specifically hybridizes to a first target sequence within the Eggerthella cDNA and a 5′ portion that does not specifically hybridize to the Eggerthella cDNA,

(B) a first Prevotella probe oligonucleotide having a 3′ portion that specifically hybridizes to a first target sequence within the Prevotella cDNA and a 5′ portion that does not specifically hybridize to the Prevotella cDNA,

(C) a first Lactobacillus probe oligonucleotide having a 3′ portion that specifically hybridizes to a first target sequence within the Lactobacillus cDNA and a 5′ portion that does not specifically hybridize to the Lactobacillus cDNA,

(D) a second Eggerthella probe oligonucleotide having a 5′ portion that specifically hybridizes to a second target sequence with the Eggerthella cDNA, wherein the second Eggerthella cDNA target sequence is located 3′ and adjacent to the first Eggerthella cDNA target sequence,

(E) a second Prevotella probe oligonucleotide having a 5′ portion that specifically hybridizes to a second target sequence with the Prevotella cDNA, wherein the second Prevotella cDNA target sequence is located 3′ and adjacent to the first Prevotella cDNA target sequence, and

(F) a second Lactobacillus probe oligonucleotide having a 5′ portion that specifically hybridizes to a second target sequence with the Lactobacillus cDNA, wherein the second Lactobacillus cDNA target sequence is located 3′ and adjacent to the first Lactobacillus cDNA target sequence,

wherein said contacting is performed under reaction conditions whereby

if the Eggerthella cDNA is present, the first and second Eggerthella probe oligonucleotides stably hybridize to the Eggerthella cDNA so as to form an Eggerthella linear duplex cleavage structure,

if the Prevotella cDNA is present, the first and second Prevotella probe oligonucleotides stably hybridize to the Prevotella cDNA so as to form a Prevotella linear duplex cleavage structure, and

if the Lactobacillus cDNA is present, the first and second Lactobacillus probe oligonucleotides stably hybridize to the Lactobacillus cDNA so as to form a Lactobacillus linear duplex cleavage structure;

(iv) contacting the sample with a flap endonuclease capable of cleaving any cleavage structure from step (iii) under reaction conditions whereby

if the Eggerthella cleavage structure is present, cleavage of the Eggerthella cleavage structure occurs to generate a Eggerthella cleavage product comprising the 5′ portion of the first Eggerthella probe oligonucleotide,

if the Prevotella cleavage structure is present, cleavage of the Prevotella cleavage structure occurs to generate a Prevotella cleavage product comprising the 5′ portion of the first Prevotella probe oligonucleotide, and

if the Lactobacillus cleavage structure is present, cleavage of the Lactobacillus cleavage structure occurs to generate a Lactobacillus cleavage product comprising the 5′ portion of the first Lactobacillus probe oligonucleotide; and

(v) detecting the presence or absence of the Eggerthella, Prevotella , and Lactobacillus cleavage products.

2. The method of claim 1 , wherein the detection of Eggerthella, Prevotella , and Lactobacillus is performed using a homogeneous detection reaction.

3. The method of claim 2 , wherein the detection of Eggerthella, Prevotella , and Lactobacillus is performed in real time.

4. The method of claim 1 ,

wherein the Eggerthella -specific primer comprises the sequence shown in SEQ ID NO:6, and/or

wherein the Prevotella -specific primer comprises the sequence shown in SEQ ID NO:9, and/or

wherein the Lactobacillus -specific primer comprises the sequence shown in SEQ ID NO:13, and/or

wherein the 3′ portion of the first Eggerthella probe oligonucleotide comprises the sequence shown in residues 11-27 of SEQ ID NO:4, and/or

wherein the 3′ portion of the first Prevotella probe oligonucleotide comprises the sequence shown in residues 11-25 of SEQ ID NO:7, and/or

wherein the 3′ portion of the first Lactobacillus probe oligonucleotide comprises the sequence shown in residues 11-27 of SEQ ID NO:10, and/or

wherein the 5′ portion of the second Eggerthella probe oligonucleotide comprises the sequence shown in residues 1-20 of SEQ ID NO:5, and/or

wherein the 5′ portion of the second Prevotella probe oligonucleotide comprises the sequence shown in residues 1-24 of SEQ ID NO:8, and/or

wherein the 5′ portion of the second Lactobacillus probe oligonucleotide comprises a sequence selected from the group consisting of (1) the sequence shown in residues 1-27 of SEQ ID NO:11 and (2) the sequence shown in residues 1-32 of SEQ ID NO:12.

5. The method of claim 4 , wherein detecting the Eggerthella, Prevotella , and Lactobacillus cleavage products comprises

contacting the Eggerthella cleavage product with a first FRET cassette comprising a first fluorescent label and a first quencher, contacting the Prevotella cleavage product with a second FRET cassette comprising a second fluorescent label and a second quencher, and contacting the Lactobacillus cleavage product with a third FRET cassette comprising a third fluorescent label and a third quencher, optionally wherein the first, second, and third quenchers are the same,

wherein each FRET cassette hybridizes with the respective cleavage product so as to form a second Eggerthella, Prevotella , or Lactobacillus cleavage structure capable of being cleaved by the flap endonuclease,

wherein if the Eggerthella cleavage product is present, the first fluorescent label is released from the first FRET cassette comprising the first quencher,

wherein if the Prevotella cleavage product is present, the second fluorescent label is released from the second FRET cassette comprising the second quencher, and

wherein if the Lactobacillus cleavage product is present, the third fluorescent label is released from the third FRET cassette comprising the third quencher; and

detecting the released first, second, or third fluorescent label.

6. The method of claim 5 ,

(i) wherein the Eggerthella cleavage product comprises the sequence shown in residues 1-11 of SEQ ID NO:4, wherein residue 11 of SEQ ID NO:4 corresponds to the 3′ terminal end of said cleavage product and optionally wherein the first FRET cassette comprises the sequence shown in SEQ ID NO:14, and/or

(ii) wherein the Prevotella cleavage product comprises the sequence shown in residues 1-11 of SEQ ID NO:7, wherein residue 11 of SEQ ID NO:7 corresponds to the 3′ terminal end of said cleavage product and optionally wherein the second FRET cassette comprises the sequence shown in SEQ ID NO:15, and/or

(iii) wherein the Lactobacillus cleavage product comprises the sequence shown in residues 1-11 of SEQ ID NO:10, wherein residue 11 of SEQ ID NO:10 corresponds to the 3′ terminal end of said cleavage product, and optionally wherein the third FRET cassette comprises the sequence shown in SEQ ID NO:16.

7. The method of claim 1 , wherein the method includes the detection of no more than ten bacterial genera associated with BV.