Compositions and methods for detecting nucleic acid from mollicutes
View Patent ↗Compositions, reaction mixtures, kits and methods used in amplifying and detecting nucleic acids from various species of the class Mollicutes. Particular regions of the 23S rRNA or its gene have been identified as preferred targets for nucleic acid amplification reactions of a sample suspected containing at least one species of Mollicutes. Some oligomers comprise tag regions, target closing regions, promoter sequences, and/or binding moieties. Samples can be from any source suspected of containing a species of the class Mollicutes. Preferred sample sources include bioreactors, cell lines, cell culture wares and pharmaceutical manufacturing wares.
1. A method for the in vitro amplification of a nucleic acid in a sample, said nucleic acid being from one or more species in the class Mollicutes, comprising the steps of:
(a.) contacting a sample with four tagged amplification oligomers, each of which individually comprises a target hybridizing region and a tag region, wherein said four tagged amplification oligomers are each individually selected from the group consisting of:
(i) a tagged amplification oligomer comprising a target hybridizing region that is from 15 to 50 nucleobases in length and contains a nucleotide sequence that is configured to specifically hybridize to all or a portion of a region of a target nucleic acid corresponding to residues 5065 to 5088 of SEQ ID NO:1;
(ii) a tagged amplification oligomer comprising a target hybridizing region that is from 15 to 50 nucleobases in length and contains a nucleotide sequence that is configured to specifically hybridize to all or a portion of a region of a target nucleic acid corresponding to residues 4752 to 4798 of SEQ ID NO:2;
(iii) a tagged amplification oligomer comprising a target hybridizing region that is from 15 to 50 nucleobases in length and contains a nucleotide sequence that is configured to specifically hybridize to all or a portion of a region of a target nucleic acid corresponding to residues 1954 to 2006 of SEQ ID NO:3; and
(iv) a tagged amplification oligomer comprising a target hybridizing region that is from 15 to 50 nucleobases in length and contains a nucleotide sequence that is configured to specifically hybridize to all or a portion of a region of a target nucleic acid corresponding to residues 1994 to 2036 of SEQ ID NO:4; and
(b.) providing suitable conditions for performing an in vitro amplification reaction.
2. The method of claim 1 , wherein one of said four tagged amplification oligomers comprises a target hybridizing region that is from 20 to 25 nucleobases in length and contains a nucleotide sequence that is configured to specifically hybridize to all or a portion of a region of a target nucleic acid corresponding to residues 1994 to 2036 of SEQ ID NO:4.
3. The method of claim 2 , wherein said tag region is from 18 to 24 nucleobases in length.
4. The method of claim 3 , wherein said tag region contains a nucleotide sequence selected from the group consisting of: a sequence with at least 95% identity to SEQ ID NO:43 or complement thereof; a sequence with at least 95% identity to SEQ ID NO:44 or complement thereof; a sequence with 100% identity to SEQ ID NO:43, SEQ ID NO:44, the complement of SEQ ID NO:43, and the complement of SEQ ID NO:44.
5. The method of claim 2 , wherein said tagged amplification oligomer further comprises a tag-closing region that is 3 to 20 nucleobases in length and is substantially complementary to a portion of said target hybridizing region so as to hybridize under a set of conditions to all or a portion of said target hybridizing region and thereby place the tagged amplification oligomer into the closed inactive configuration.
6. The method of claim 1 , wherein the amplification reaction of step (b) is an isothermal amplification reaction.
7. The method of claim 1 , further comprising the steps of (c) providing at least one additional amplification oligomer selected from the group consisting of a tag-targeting amplification oligomer, a primer oligomer and a promoter-based amplification oligomer; and (d) providing suitable conditions for performing a second amplification reaction using the at least one amplification oligomer from step (c) and using the initial amplification product generated in step (b) as a target nucleic acid.
8. The method of claim 7 , wherein said at least one additional amplification oligomer is a tag-targeting amplification oligomer comprising a target hybridizing region that contains a nucleotide sequence configured to specifically hybridize to a tag sequence selected from the group consisting of: a sequence with at least 95% identity to SEQ ID NO:43 or complement thereof; a sequence with at least 95% identity to SEQ ID NO:44 or complement thereof; a sequence with 100% identity to SEQ ID NO:43 or complement thereof; and a sequence with 100% identity to SEQ ID NO:44 or complement thereof; optionally, wherein said target hybridizing region of said tag-targeting amplification oligomer is 18 to 24 nucleobases in length.
9. The method of claim 7 , wherein said additional amplification oligomer is a promoter-based amplification oligomer comprising a target hybridizing region that is 10 to 50 nucleobases in length and contains a nucleotide sequence that is configured to specifically hybridize to all or a portion of a region of a target nucleic acid corresponding to residues 5016 to 5045 of SEQ ID NO:1; or wherein said target hybridizing region of said promoter-based amplification oligomer has a sequence that is at least 95% identical to SEQ ID NO:42; or wherein said target hybridizing region of said promoter-based amplification oligomer is a sequence consisting of SEQ ID NO:42, or wherein said promoter-based amplification oligomer is SEQ ID NO:15.
10. The method of claim 7 , wherein the amplification reaction of step (d) is an isothermal amplification reaction.
11. The method of claim 7 , further comprising a detection step to identify an initial amplification product generated in step (b), an amplification product generated in step (d) or the amplification products generated in steps (b) and (d).
12. The method of claim 11 , wherein said detection step is a probe-based detection step.
13. The method of claim 12 , comprising a detection probe that is SEQ ID NO:14.