Kit 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 kit for determining the presence or absence of one or more of Candida ( C .) albicans, C. parapsilosis, C. dubliniensis , and C. tropicalis in a sample, the kit comprising:
(a1) a target capture reagent comprising first and second Candida -specific target capture oligomers (TCOs) and a Candida -specific T7 primer, wherein
(i) the first Candida -specific TCO comprises a first Candida -specific TCO target-hybridizing sequence consisting of the nucleotide sequence of residues 1-20 of SEQ ID NO:24,
(ii) the second Candida -specific TCO comprises a second Candida -specific TCO target-hybridizing sequence consisting of the nucleotide sequence of residues 1-17 of SEQ ID NO:66, and
(iii) the Candida -specific T7 primer comprises (A) a Candida -specific T7 primer target-hybridizing sequence consisting of the nucleotide sequence of residues 28-46 of SEQ ID NO:9 and (B) a T7 promoter sequence located 5′ to the Candida -specific T7 primer target-hybridizing sequence;
(b1) an amplification reagent comprising first and second Candida -specific non-T7 primers, wherein
(i) the first Candida -specific non-T7 primer comprises a first Candida -specific non-T7 primer target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:26, and
(ii) the second Candida -specific non-T7 primer comprises a second Candida -specific non-T7 primer target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:34; and
(c1) a promoter reagent comprising the Candida -specific T7 primer defined in (a1) (iii) and a Candida -specific detection probe, wherein the Candida -specific detection probe comprises a Candida -specific detection probe target-hybridizing sequence consisting of the nucleotide sequence of residues 1-22 of SEQ ID NO:27.
2 . The kit of claim 1 , wherein the kit is for further determining the presence or absence of C. glabrata in the sample, wherein
(a2) the target capture reagent further comprises a C. glabrata -specific target capture oligomer (TCO) and a C. glabrata -specific T7 primer, wherein
(i) the C. glabrata -specific TCO comprises a C. glabrata -specific TCO target-hybridizing sequence consisting of the nucleotide sequence of residues 1-26 of SEQ ID NO:48, and
(ii) the C. glabrata -specific T7 primer comprises (A) a C. glabrata -specific T7 primer target-hybridizing sequence consisting of the nucleotide sequence of residues 28-49 of SEQ ID NO:14 and (B) a T7 promoter sequence located 5′ to the C. glabrata -specific T7 primer target-hybridizing sequence;
(b2) the amplification reagent further comprises a C. glabrata -specific non-T7 primer comprising a C. glabrata -specific non-T7 primer target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:12; and
(c2) the promoter reagent further comprises the C. glabrata -specific T7 primer defined in (a2)(ii) and a C. glabrata -specific detection probe, wherein the C. glabrata -specific detection probe comprises a C. glabrata -specific detection probe target-hybridizing sequence consisting of the nucleotide sequence of residues 1-17 of SEQ ID NO:60.
3 . The kit of claim 1 , wherein
the first Candida -specific TCO has the nucleotide sequence of SEQ ID NO:24, and/or
the second Candida -specific TCO has the nucleotide sequence of SEQ ID NO:66.
4 . The kit of claim 1 , wherein the Candida -specific T7 primer has the nucleotide sequence of SEQ ID NO:9.
5 . The kit of claim 1 , wherein the Candida -specific detection probe further comprises a label, a substitution at the 2′ position of at least one ribose moiety, and/or a blocking group at or near the 3′ end of the detection probe.
6 . The kit of claim 5 , wherein the Candida -specific detection probe comprises the label.
7 . The kit of claim 6 , wherein the label is a fluorescent label and the Candida -specific detection probe further comprises a quencher.
8 . The kit of claim 7 , wherein the Candida -specific detection probe further comprises a non-target-hybridizing sequence.
9 . The kit of claim 8 , wherein the Candida -specific detection probe is a molecular torch or a molecular beacon.
10 . The kit of claim 8 , wherein the Candida -specific detection probe is a molecular torch and has the nucleotide sequence of SEQ ID NO:27 or SEQ ID NO:155.
11 . The kit of claim 2 , wherein the C. glabrata -specific TCO has the nucleotide sequence of SEQ ID NO:48.
12 . The kit of claim 2 , wherein the C. glabrata -specific T7 primer has the nucleotide sequence of SEQ ID NO:14.
13 . The kit of claim 2 , wherein the C. glabrata -specific detection probe further comprises a label, a substitution at the 2′ position of at least one ribose moiety, and/or a blocking group at or near the 3′ end of the detection probe.
14 . The kit of claim 13 , wherein the C. glabrata -specific detection probe comprises the label.
15 . The kit of claim 14 , wherein the label is a fluorescent label and the C. glabrata -specific detection probe further comprises a quencher.
16 . The kit of claim 15 , wherein the C. glabrata -specific detection probe further comprises a non-target-hybridizing sequence.
17 . The kit of claim 16 , wherein the C. glabrata -specific detection probe is a molecular torch or a molecular beacon.
18 . The kit of claim 16 , wherein the C. glabrata -specific detection probe is a molecular torch and has the nucleotide sequence of residues 1-17 of SEQ ID NO:60.
19 . The kit of claim 1 , wherein the kit further comprises an enzyme solution comprising a mixture of an MMLV reverse transcriptase and a T7 RNA polymerase.
20 . The kit of claim 2 , wherein the kit further comprises an enzyme solution comprising a mixture of an MMLV reverse transcriptase and a T7 RNA polymerase.