Compositions and methods for detecting bv-associated bacterial nucleic acid

Abstract

Disclosed are nucleic acid oligomers, including amplification oligomers, capture probes, and detection probes, for detection of a 16S rRNA or its encoding gene from bacterial species associated with bacterial vaginosis. Also disclosed are methods of specific nucleic acid amplification and detection using the disclosed oligomers, as well as corresponding reaction mixtures and kits.

What is claimed is: 
     
         1 . A combination of at least two oligomers for detecting in a sample a  Gardnerella vaginalis  (GV) 16S rRNA or a gene encoding said 16S rRNA, said oligomer combination comprising:
 first and second amplification oligomers for amplifying a GV nucleic acid target region corresponding to the GV 16S rRNA or the 16S-rRNA-encoding gene, wherein   (a) the first amplification oligomer comprises a first target-hybridizing sequence that is from about 15 to about 27 contiguous nucleotides contained in the sequence of SEQ ID NO:75 and that includes at least the sequence of at least one of SEQ ID NO:72 and SEQ ID NO:73; and   (b) the second amplification oligomer comprises a second target-hybridizing sequence that is from about 15 to about 27 contiguous nucleotides contained in the sequence of SEQ ID NO:71 and that includes at least the sequence of SEQ ID NO:70.   
     
     
         2 .- 30 . (canceled) 
     
     
         31 . The combination of at least two oligomers as in  claim 1 , wherein the at least two oligomers are in a kit for amplification of a  Gardnerella vaginalis  target nucleic acid. 
     
     
         32 . (canceled) 
     
     
         33 . A method for detecting in a sample a  Gardnerella vaginalis  (GV) target nucleic acid, wherein the target nucleic acid is a GV 16S rRNA or a gene encoding said 16S rRNA, said method comprising:
 (a) contacting a sample, said sample suspected of containing a GV bacterium, with at least two oligomers for amplifying a GV nucleic acid target region corresponding to the target nucleic acid, said oligomer combination comprising
 (i) a first amplification oligomer comprising a first target-hybridizing sequence that is from about 15 to about 27 contiguous nucleotides contained in the sequence of SEQ ID NO:75 and that includes at least the sequence of at least one of SEQ ID NO:72 and SEQ ID NO:73; and 
 (ii) a second amplification oligomer comprising a second target-hybridizing sequence that is from about 15 to about 27 contiguous nucleotides contained in the sequence of SEQ ID NO:71 and that includes at least the sequence of SEQ ID NO:70; 
   (b) performing an in vitro nucleic acid amplification reaction, wherein any GV target nucleic acid present in said sample is used as a template for generating an amplification product; and   (c) detecting the presence or absence of the amplification product, thereby indicating the presence or absence of GV in said sample.   
     
     
         34 . The method of  claim 33 , wherein the first target-hybridizing sequence is selected from the group consisting of:
 a target hybridizing sequence contained in the sequence of SEQ ID NO:76;   a target hybridizing sequence contained in the sequence of SEQ ID NO:76 and that includes at least the sequence of SEQ ID NO:72;   a target hybridizing sequence contained within the sequence of SEQ ID NO:78;   a target hybridizing sequence contained in the sequence of SEQ ID NO:78 and that includes at least the sequence of SEQ ID NO:77;   a target hybridizing sequence contained within the sequence of SEQ ID NO:79;   a target hybridizing sequence contained within the sequence of SEQ ID NO:79 and that includes at least the sequence of SEQ ID NO:73;   a target hybridizing sequence contained within the sequence of SEQ ID NO:81; and   a target hybridizing sequence contained within the sequence of SEQ ID NO:81 and that includes at least the sequence of SEQ ID NO:80;   a target hybridizing sequence of SEQ ID NOs:6-10.   
     
     
         35 .- 45 . (canceled) 
     
     
         46 . The method of  claim 34 , wherein the second target-hybridizing sequence has a sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, a sequence contained within the sequence of SEQ ID NO:86, and a sequence contained within the sequence of SEQ ID NO:86 and includes at least the sequence of SEQ ID NO:85. 
     
     
         47 . The method of  claim 33 , wherein the second target-hybridizing sequence is contained in the sequence of SEQ ID NO:86. 
     
     
         48 . The method of  claim 47 , wherein the second target-hybridizing sequence includes at least the sequence of SEQ ID NO:85. 
     
     
         49 . The method of  claim 48 , wherein the second target-hybridizing sequence is selected from the group consisting of SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:39. 
     
     
         50 . The method of  claim 33 , wherein the second amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located 5′ to the target-hybridizing sequence. 
     
     
         51 . (canceled) 
     
     
         52 . The method of  claim 50 , wherein the T7 promoter sequence has the sequence shown in SEQ ID NO:87. 
     
     
         53 . The method of  claim 52 , wherein the second amplification oligomer has a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. 
     
     
         54 . The method of  claim 33 , further comprising purifying the GV target nucleic acid from other components in the sample before step (a), wherein the purifying step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein said target-hybridizing sequence is selected from the group consisting of SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46. 
     
     
         55 .- 56 . (canceled) 
     
     
         57 . The method of  claim 33 , wherein the detecting step (c) comprises contacting said in vitro nucleic acid amplification reaction with a detection probe oligomer configured to specifically hybridize to the amplification product under conditions whereby the presence or absence of the amplification product is determined, thereby indicating the presence or absence of GV in said sample. 
     
     
         58 . The method of  claim 57 , wherein the detection probe oligomer is selected from the group consisting of
 a detection probe oligomer comprising a target-hybridizing sequence that is from about 14 to about 35 nucleotides in length and is configured to specifically hybridize to a target sequence contained within SEQ ID NO:88 from about nucleotide position 164 to about nucleotide position 205;   a detection probe oligomer comprising a target hybridizing sequence contained in the sequence of SEQ ID NO:84 and includes at least the sequence of SEQ ID NO:83; and   a detection probe oligomer comprising a target hybridizing sequence that is SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.   
     
     
         59 .- 62 . (canceled) 
     
     
         63 . The method of  claim 58 , wherein the detection probe comprises a label selected from the group consisting of
 (a) a chemiluminescent label;   (b) a fluorescent label;   (c) a quencher; and   (d) a combination of one or more of (a), (b), and (c).   
     
     
         64 . The method of any of  claim 58 , wherein the detecting step (c) occurs during the amplifying step (b). 
     
     
         65 .- 66 . (canceled) 
     
     
         67 . The method of any of  claim 58 , wherein the detection probe further comprises a non-target-hybridizing sequence. 
     
     
         68 . The method of  claim 67 , wherein the detection probe is a hairpin detection probe a TaqMan detection probe, a molecular beacon, or a molecular torch. 
     
     
         69 . (canceled) 
     
     
         70 . The method of  claim 33 , wherein the amplification reaction at step (b) is an isothermal amplification reaction, or is a PCR amplification reaction. 
     
     
         71 . The method of  claim 70 , wherein the isothermal amplification reaction is a transcription-mediated amplification (TMA) reaction or wherein the isothermal amplification reaction is a reverse TMA reaction. 
     
     
         72 .- 74 . (canceled) 
     
     
         75 . A detection probe oligomer for detecting a  Gardnerella vaginalis  target nucleic acid, wherein said detection probe oligomer comprises a target-hybridizing sequence that is from about 14 to about 35 nucleotides in length and is configured to specifically hybridize to a target sequence contained within SEQ ID NO:88 from about nucleotide position 164 to about nucleotide position 205. 
     
     
         76 .- 83 . (canceled)