Compositions and methods to detect
View Patent ↗The disclosed invention include nucleic acid oligomers that may be used as amplification oligomers, including primers, as capture probes for sample preparation, and detection probes for detection of 16S rRNA from Atopobium vaginae in samples by using methods of specific nucleic acid amplification and detection.
1. A method for detecting in a sample a 16S rRNA of A. vaginae or a gene encoding a 16S rRNA of A. vaginae , comprising the steps of:
a. providing a sample, wherein said sample is suspected of containing an Atopobium vaginae bacterium;
b. contacting said sample with at least two amplification oligomers, wherein a first amplification oligomer comprises a target hybridizing sequence that consists of SEQ ID NO:2, and wherein a second amplification oligomer comprises a target hybridizing sequence that consists of SEQ ID NO:27, and wherein said first and second amplification oligomers are configured to specifically hybridize to a target sequence within a 16S rRNA of A. vaginae or a gene encoding a 16S rRNA of A. vaginae in the presence of one or more of Gardnerella vaginalis, Prevotella sp, anaerobic gram positive cocci, Mobiluncus sp, Mycoplasma hominis, Eggerthella hongkongensis, Megasphaera sp, and/or Leptotrichia sanguinegens;
c. performing an in vitro isothermal nucleic acid amplification reaction wherein any target nucleic acid present in said sample is used as a template for generating an amplification product; and
d. contacting said in vitro nucleic acid amplification reaction with a detection probe oligomer that comprises a nucleotide sequence consisting of SEQ ID NO:4 or SEQ ID NO:17 and providing conditions for a detection reaction to determine the presence or absence of an amplification product;
wherein the detection of the presence of an amplification product indicates the presence of at least 100 CFU/ml of Atopobium vaginae bacterium in the sample.
2. The method of claim 1 , wherein said second amplification oligomer further comprises at its 5′ end a promoter sequence.
3. The method of claim 2 , wherein said promoter sequence is selected from the group consisting of RNA polymerase promoter sequences, T7 RNA polymerase promoter sequences and SEQ ID NO:25.
4. The method of claim 1 , wherein said second amplification oligomer nucleotide sequence consists of SEQ ID NO:3.
5. The method of claim 1 , wherein said detection reaction step is performed in real-time.
6. A composition comprising amplification and detection probe oligomers of claim 1 , wherein: (i) a first amplification oligomer comprises a target hybridizing sequence consisting of SEQ ID NO:2; (ii) a second amplification oligomer comprises a target hybridizing sequence consisting of SEQ ID NO:27; and (iii) a detection probe oligomer comprises a chemiluminescent detection label and a nucleotide sequence consisting of SEQ ID NO:4 or SEQ ID NO:17.
7. The amplification and detection probe oligomers of claim 6 , wherein the second amplification oligomer further comprises a promoter sequence attached to its 5′ end.
8. The amplification and detection probe oligomers of claim 7 , wherein said second amplification oligomer nucleotide sequence consists of SEQ ID NO:3.
9. The method of claim 1 , wherein said contacting step at d) is performed after the amplification step at c).
10. The method of claim 1 , wherein said contacting step at d) is performed simultaneously with the amplification step at c).
11. The method of claim 1 , wherein said detection reaction step is performed with a detection probe oligomer further comprising a detectable label selected from the group consisting of: an acridinium ester label, a fluorescent label, or a combination thereof.
12. The method of claim 1 , wherein the detection probe oligomer nucleotide sequence consists of SEQ ID NO:4.
13. The amplification and detection probe oligomers of claim 6 , wherein said oligomers are part of a kit.