Methods for the diagnosis of bacterial vaginosis
The present invention relates to methods for the diagnosis of bacterial vaginosis based on an analysis of a patient sample. For example, patient test samples are analyzed for the presence or absence of one or more lactobacilli and two or more pathogenic organisms. The presence or absence of one or more lactobacilli and two or more pathogenic organisms may be detected using PCR analysis of nucleic acid segments corresponding to each target organism. The quantity of the target organisms can then be used to determine a score which is indicative of a diagnosis of bacterial vaginosis.
1 . A master mix for detecting bacterial vaginosis in a female subject by amplifying fragments of ribosomal RNA nucleic acid from no more than five bacteria, the master mix comprising:
(a) a primer pair and a probe for amplifying and detecting a fragment of a ribosomal RNA nucleic acid from Atopobium vaginae;
(b) a primer pair and a probe for amplifying and detecting a fragment of a ribosomal RNA nucleic acid from Gardnerella vaginalis;
(c) a primer pair and a probe for amplifying and detecting a fragment of a ribosomal RNA nucleic acid from Lactobacillus jensenii;
(d) a primer pair and a probe for amplifying and detecting a fragment of a ribosomal RNA nucleic acid from Lactobacillus crispatus;
(e) a primer pair and a probe for amplifying and detecting an internal control nucleic acid; and
(f) the internal control nucleic acid;
wherein each said probe is detectably labeled to allow for quantitation of amplification products, and
wherein all primer pairs and probes for amplifying and detecting said fragments of ribosomal RNA nucleic acid from no more than five bacteria in the master mix are specific for organisms associated with bacterial vaginosis or for Lactobacilli species.
2 . The master mix of claim 1 , wherein each ribosomal RNA nucleic acid is 16S ribosomal RNA.
3 . The master mix of claim 2 , further comprising an RNA polymerase for performing transcription-mediated amplification.
4 . The master mix of claim 3 , wherein the master mix further comprises a reverse transcriptase.
5 . The master mix of claim 1 , wherein at least one primer of at least one of said primer pairs comprises a promoter sequence for an RNA polymerase.
6 . The master mix of claim 5 , wherein the promoter sequence is a T7 promoter sequence.
7 . The master mix of claim 1 , wherein each primer pair comprises a forward primer and a reverse primer, and wherein the forward primer for amplifying the fragment from Lactobacillus jensenii and the forward primer for amplifying the fragment from Lactobacillus crispatus are different.
8 . The master mix of claim 1 , wherein each probe comprises a fluorescent label.
9 . The master mix of claim 8 , wherein each probe comprises a quencher dye.
10 . The master mix of claim 9 , wherein each probe is a hydrolysis probe.
11 . A reaction mixture for detecting bacterial vaginosis in a female subject, the reaction mixture comprising reagents for nucleic acid amplification of fragments of ribosomal RNA nucleic acid from no more than five bacteria, the reagents comprising:
(a) a primer pair and a probe for amplifying and detecting a fragment of a ribosomal RNA nucleic acid from Atopobium vaginae;
(b) a primer pair and a probe for amplifying and detecting a fragment of a ribosomal RNA nucleic acid from Gardnerella vaginalis;
(c) a primer pair and a probe for amplifying and detecting a fragment of a ribosomal RNA nucleic acid from Lactobacillus jensenii;
(d) a primer pair and a probe for amplifying and detecting a fragment of a ribosomal RNA nucleic acid from Lactobacillus crispatus;
(e) a primer pair and a probe for amplifying and detecting an internal control nucleic acid;
(f) the internal control nucleic acid; and
(g) a plurality of nucleic acids obtained from a sample from the female subject;
wherein each said probe is detectably labeled to allow for quantitation of amplification products; and
wherein all primer pairs and probes for amplifying and detecting said fragments of ribosomal RNA nucleic acid from no more than five bacteria in the reaction mixture are specific for organisms associated with bacterial vaginosis or for Lactobacilli species.
12 . The reaction mixture of claim 11 , wherein each ribosomal RNA nucleic acid is 16S ribosomal RNA.
13 . The reaction mixture of claim 11 , wherein the reagents further comprise an RNA polymerase for performing transcription-mediated amplification.
14 . The reaction mixture of claim 11 , wherein the reaction mixture further comprises a reverse transcriptase.
15 . The reaction mixture of claim 14 , wherein at least one primer of at least one of said primer pairs comprises a promoter sequence for an RNA polymerase.
16 . The reaction mixture of claim 15 , wherein the promoter sequence is a T7 promoter sequence.
17 . The reaction mixture of claim 11 , wherein each primer pair comprises a forward primer and a reverse primer, and wherein the forward primer for amplifying the fragment from Lactobacillus jensenii and the forward primer for amplifying the fragment from Lactobacillus crispatus are different.
18 . The reaction mixture of claim 11 , wherein each detectably labeled probe comprises a fluorescent label.
19 . The reaction mixture of claim 11 , wherein each probe comprises a quencher dye.
20 . The reaction mixture of claim 19 , wherein each probe is a hydrolysis probe.
21 . A kit for detecting the presence of bacterial vaginosis in a sample from a female subject, the kit comprising reagents for nucleic acid amplification of fragments of ribosomal RNA nucleic acid from no more than five bacteria, the reagents comprising:
(a) forward primers for amplifying fragments of ribosomal RNA nucleic acid from:
(i) Atopobium vaginae;
(ii) Gardnerella vaginalis;
(iii) Lactobacillus jensenii ; and
(iv) Lactobacillus crispatus;
(b) forward primers for amplifying an internal control nucleic acid;
(c) reverse primers for amplifying said fragments of ribosomal RNA nucleic acid from said no more than five bacteria;
(d) reverse primers for amplifying said internal control nucleic acid;
(e) detectably labeled probes for detecting said amplified fragments;
(f) a detectably labeled probe for detecting said amplified internal control nucleic acid;
(g) enzymes for performing transcription-mediated amplification; and
(h) said internal control nucleic acid;
wherein all forward primers and reverse primers in the kit for amplifying said fragments of ribosomal RNA nucleic acid from said no more than five bacteria, and all probes in the kit for detecting said fragments of ribosomal RNA nucleic acid from said no more than five bacteria are specific for organisms associated with bacterial vaginosis or for Lactobacilli species.
22 . The kit of claim 21 , wherein each ribosomal RNA nucleic acid is 16S ribosomal RNA.
23 . The kit of claim 21 , wherein the forward primer for amplifying fragments of ribosomal RNA nucleic acid for Lactobacillus jensenii and the forward primer for amplifying fragments of ribosomal RNA nucleic acid for Lactobacillus crispatus are different.
24 . The kit of claim 21 , wherein at least one of said forward primers comprises a promoter sequence for an RNA polymerase.
25 . The kit of claim 24 , wherein the promoter sequence is a T7 promoter sequence.
26 . The kit of claim 21 , wherein each detectably labeled probe comprises a fluorescent label.
27 . The kit of claim 26 , wherein each detectably labeled probe comprises a quencher dye.
28 . The kit of claim 27 , wherein each detectably labeled probe is a hydrolysis probe.