Methods and compositions for detecting
Disclosed are methods utilizing specific amplification of Candida sp. target nucleic acid for detecting the presence or absence of Candida sp. in a sample. Also disclosed are corresponding oligomers, including amplification oligomers, capture probes and detection probes, and combinations thereof, as well as corresponding reaction mixtures and kits.
1. A method for determining the presence or absence of Candida species (sp.) in a sample, wherein the Candida sp. is one or more of C. albicans, C. parapsilosis, C. dubliniensis, C. tropicalis , and C. glabrata , the method comprising:
(1) contacting a sample, said sample suspected of containing Candida sp., with a first amplification oligomer combination and a second amplification oligomer combination, wherein
(a) the first amplification oligomer combination comprises first and second Candida -specific amplification oligomers for amplifying a first Candida sp. nucleic acid target region or a second Candida sp. nucleic acid target region, wherein said first target region corresponds to a region of SEQ ID NO:129 from about nucleotide position 133 or 161 to about nucleotide position 259 and said second region corresponds to a region of SEQ ID NO:130 from about nucleotide position 202 to about nucleotide position 308, and wherein the first and second Candida -specific amplification oligomers respectively comprise first and second Candida -specific target-hybridizing sequences, wherein
the first Candida -specific target-hybridizing sequence consists of the nucleotide sequence of residues 28-46 of SEQ ID NO:9; and
the second Candida -specific target-hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:26; and
(b) the second amplification oligomer combination comprises first and second C. glabrata -specific amplification oligomers for amplifying a third Candida sp. nucleic acid target region, wherein said third target region corresponds to a region of SEQ ID NO:131 from about nucleotide position 355 to about nucleotide position 554, and wherein the first and second C. glabrata-specific amplification oligomers respectively comprise first and second C. glabrata -specific target-hybridizing sequences, wherein
the first C. glabrata -specific target-hybridizing sequence is a sequence of from 15 to 24 contiguous nucleotides contained in the sequence of SEQ ID NO:134 and that includes at least the sequence of SEQ ID NO:135; and/or
the second C. glabrata -specific target-hybridizing sequence is a sequence of from 16 to 21 contiguous nucleotides contained in the sequence of SEQ ID NO:136 and that includes at least the sequence of SEQ ID NO:137;
wherein each of the first Candida -specific amplification oligomer and the first C. glabrata -specific amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located 5′ to the respective target-hybridizing sequence, and
wherein each of the second Candida -specific amplification oligomer and the second C. glabrata -specific amplification oligomer is a non-promoter primer;
(2) performing a transcription-mediated amplification (TMA) reaction, wherein any Candida sp. target nucleic acid, if present in the sample, is used as a template for generating one or more amplification products corresponding to at least one of the first, second, and third target regions; and
(3) detecting in real time the presence or absence of the one or more amplification products, thereby determining the presence or absence of Candida sp. in the sample, wherein said detection comprises (i) contacting one or more amplification products with a Candida -specific detection probe that specifically hybridizes to the first or second Candida sp. target region and a C. glabrata -specific detection probe that specifically hybridizes to the third Candida sp. target region, wherein each of the Candida -specific and C. glabrata -specific detection probes is a molecular torch, and (ii) detecting the presence or absence of any hybridized Candida -specific or C. glabrata -specific detection probe.
2. The method of claim 1 , wherein
the first C. glabrata -specific target-hybridizing sequence consists of the nucleotide sequence of residues 28-49 of SEQ ID NO:14; and/or
the second C. glabrata -specific target-hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:12.
3. The method of claim 1 , wherein the promoter sequence is a T7 promoter sequence.
4. The method of claim 1 , wherein
the first Candida -specific amplification oligomer has the nucleotide sequence of SEQ ID NO:9; and/or
the first C. glabrata -specific amplification oligomer has the nucleotide sequence of SEQ ID NO:14.
5. The method of claim 1 , wherein
the Candida -specific detection probe target-hybridizing sequence comprises a sequence selected from the group consisting of
the sequence of residues 1-22 of SEQ ID NO:27,
the DNA equivalent or an RNA/DNA chimeric thereof, and
the full complement of any of the foregoing; and/or
the C. glabrata -specific detection probe target-hybridizing sequence comprises a sequence selected from the group consisting of
the sequence of residues 1-17 of SEQ ID NO:60,
the sequence of residues 1-23 of SEQ ID NO:45,
the sequence of residues 1-17 of SEQ ID NO:18,
the sequence of residues 1-20 of SEQ ID NO:21,
the DNA equivalent or an RNA/DNA chimeric of any of the foregoing, and
the full complement of any of the foregoing.
6. The method of claim 1 , wherein
the Candida -specific detection probe has a sequence selected from the group consisting of the sequence of SEQ ID NO:27, the DNA equivalent or an RNA/DNA chimeric thereof, and the full complement of any of the foregoing; and/or
the C. glabrata -specific detection probe target-hybridizing sequence has a sequence selected from the group consisting of SEQ ID NO:60, SEQ ID NO:45, SEQ ID NO:18, SEQ ID NO:21, the DNA equivalent or an RNA/DNA chimeric of any of the foregoing, and the full complement of any of the foregoing.