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 reaction mixture 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 reaction mixture comprising:
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, wherein said first target region is a region of SEQ ID NO:129 from about nucleotide position 133 to about nucleotide position 259, 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 second Candida sp. nucleic acid target region, wherein said second target region is 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 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 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;
a Candida -specific detection probe comprising a target-hybridizing sequence that is complementary to the first Candida sp. target region and a C. glabrata -specific detection probe comprising a target-hybridizing sequence that is complementary to the second Candida sp. target region, wherein each of the Candida -specific and C. glabrata -specific detection probes is a molecular torch and comprises a fluorescent label and a quencher; and
an RNA polymerase.
2 . The reaction mixture 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.
3 . The reaction mixture of claim 1 , wherein the second C. glabrata -specific target-hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:12.
4 . The reaction mixture of claim 1 , wherein the promoter sequence is a T7 promoter sequence.
5 . The reaction mixture of claim 4 , wherein the promoter sequence has the nucleotide sequence of residues 1-27 of SEQ ID NO:9.
6 . The reaction mixture of claim 1 , wherein the first Candida -specific amplification oligomer has the nucleotide sequence of SEQ ID NO:9.
7 . The reaction mixture of claim 1 , wherein the first C. glabrata -specific amplification oligomer has the nucleotide sequence of SEQ ID NO:14.
8 . The reaction mixture 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.
9 . The reaction mixture of claim 1 , wherein 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.
10 . The reaction mixture 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.
11 . The reaction mixture of claim 1 , wherein 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.
12 . A kit 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 kit comprising:
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, wherein said first target region is a region of SEQ ID NO:129 from about nucleotide position 133 to about nucleotide position 259, 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 second Candida sp. nucleic acid target region, wherein said second target region is 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 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 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;
a Candida -specific detection probe comprising a target-hybridizing sequence that is complementary to the first Candida sp. target region and a C. glabrata -specific detection probe comprising a target-hybridizing sequence that is complementary to the second Candida sp. target region, wherein each of the Candida -specific and C. glabrata -specific detection probes is a molecular torch and comprises a fluorescent label and a quencher; and
an RNA polymerase.
13 . The kit of claim 12 , wherein the first C. glabrata -specific target-hybridizing sequence consists of the nucleotide sequence of residues 28-49 of SEQ ID NO:14.
14 . The kit of claim 12 , wherein the second C. glabrata -specific target-hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:12.
15 . The kit of claim 12 , wherein the promoter sequence is a T7 promoter sequence.
16 . The kit of claim 15 , wherein the promoter sequence has the nucleotide sequence of residues 1-27 of SEQ ID NO:9.
17 . The kit of claim 12 , wherein the first Candida -specific amplification oligomer has the nucleotide sequence of SEQ ID NO:9.
18 . The kit of claim 12 , wherein the first C. glabrata -specific amplification oligomer has the nucleotide sequence of SEQ ID NO:14.
19 . The kit of claim 12 , 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.
20 . The kit of claim 12 , 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.