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US 2026/0234736 A1Published application
Compositions and methods for detecting gastrointestinal parasites
AssigneeGen-Probe Incorporated
InventorsBrian A. DOCKTER, Joshua K. MOBERLY, Brett W. KIRKCONNELL, Marcella A. CARVALLO PINTO
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Abstract
Disclosed are nucleic acid oligomers, including amplification oligomers and detection probes, for detection of Cryptosporidium spp., Entamoeba histolytica, Giardia lamblia, and Cyclospora cayetanensis target nucleic acid. Also disclosed are methods of specific nucleic acid amplification and detection using the disclosed oligomers, as well as corresponding formulations, reaction mixtures, and kits. Methods of synthesizing the nucleic acid oligomers are also disclosed.
Claims25
A composition or kit for determining the presence or absence of at least one enteric parasite in a sample, wherein the at least one enteric parasite is selected from the group consisting of Cryptosporidium spp., Entamoeba histolytica, Giardia lamblia, and Cyclospora cayetanensis, said composition or kit comprising a set of oligonucleotides comprising at least one of (a)-(d):
- (a) a Cryptosporidium-specific amplification oligomer set capable of amplifying a target region of a Cryptosporidium spp. target nucleic acid, wherein the Cryptosporidium-specific amplification oligomer set comprises first and second Cryptosporidium-specific amplification oligomers respectively comprising target-hybridizing sequences as shown in (i) SEQ ID NO:8 and SEQ ID NO:37, including from 0 to 16 nucleotide analogs;
- (ii) SEQ ID NO:33 and SEQ ID NO:28, including from 0 to 16 nucleotide analogs;
- (iii) SEQ ID NO:4 and SEQ ID NO:37, including from 0 to 16 nucleotide analogs;
- (iv) SEQ ID NO:11 and SEQ ID NO:49, including from 0 to 16 nucleotide analogs;
- (v) SEQ ID NO:4 and SEQ ID NO:49, including from 0 to 16 nucleotide analogs;
- (vi) SEQ ID NO:11 and SEQ ID NO:37, including from 0 to 16 nucleotide analogs;
- or (vii) SEQ ID NO:8 and SEQ ID NO:49, including from 0 to 16 nucleotide analogs;
- (b) an Entamoeba-specific amplification oligomer set capable of amplifying a target region of an Entamoeba histolytica target nucleic acid, wherein the Entamoeba-specific amplification oligomer set comprises first and second Entamoeba-specific amplification oligomers respectively comprising target-hybridizing sequences as shown in (i) SEQ ID NO:46 and SEQ ID NO:19, including from 0 to 16 nucleotide analogs;
- (ii) SEQ ID NO:21 and SEQ ID NO:20, including from 0 to 16 nucleotide analogs;
- (iii) SEQ ID NO:36 and SEQ ID NO:43, including from 0 to 16 nucleotide analogs;
- or (iv) SEQ ID NO:42 and SEQ ID NO:19, including from 0 to 16 nucleotide analogs;
- (c) a Giardia-specific amplification oligomer set capable of amplifying a target region of a Giardia lamblia target nucleic acid, wherein the Giardia-specific amplification oligomer set comprises first and second Giardia-specific amplification oligomers respectively comprising target-hybridizing sequences as shown in (i) SEQ ID NO:25 and SEQ ID NO:2, including from 0 to 16 nucleotide analogs;
- (ii) SEQ ID NO:5 and SEQ ID NO:29, including from 0 to 16 nucleotide analogs;
- (iii) SEQ ID NO:6 and SEQ ID NO:29, including from 0 to 16 nucleotide analogs;
- or (iv) SEQ ID NO:25 and SEQ ID NO:3, including from 0 to 16 nucleotide analogs;
- and
- (d) a Cyclospora-specific amplification oligomer set capable of amplifying a target region of a Cyclospora cayetanensis target nucleic acid, wherein the Cyclospora-specific amplification oligomer set comprises first and second Cyclospora-specific amplification oligomers respectively comprising target-hybridizing sequences as shown in (i) SEQ ID NO:26 and SEQ ID NO:14, including from 0 to 16 nucleotide analogs;
- (ii) SEQ ID NO:23 and SEQ ID NO:18, including from 0 to 16 nucleotide analogs;
- (iii) SEQ ID NO:31 and SEQ ID NO:22, including from 0 to 16 nucleotide analogs;
- (iv) SEQ ID NO:35 and SEQ ID NO:13, including from 0 to 16 nucleotide analogs;
- (v) SEQ ID NO:38 and SEQ ID NO:51, including from 0 to 16 nucleotide analogs;
- or (vi) SEQ ID NO:1 and SEQ ID NO:9, including from 0 to 16 nucleotide analogs.
19 more elements
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A method for determining the presence or absence of at least one enteric parasite in a sample, wherein the at least one enteric parasite is selected from the group consisting of Cryptosporidium spp., Entamoeba histolytica, Giardia lamblia, and Cyclospora cayetanensis, the method comprising:
- (1) contacting a sample, said sample suspected of containing the at least one enteric parasite, with an oligomer combination capable of amplifying a target region of Cryptosporidium spp., Entamoeba histolytica, Giardia lamblia, and Cyclospora cayetanensis target nucleic acid, said oligomer combination comprising
- (a) a Cryptosporidium-specific amplification oligomer set capable of amplifying a target region of a Cryptosporidium spp. target nucleic acid, wherein the Cryptosporidium-specific amplification oligomer set comprises first and second Cryptosporidium-specific amplification oligomers respectively comprising target-hybridizing sequences as shown in (i) SEQ ID NO:8 and SEQ ID NO:37, including from 0 to 16 nucleotide analogs;
- (ii) SEQ ID NO:33 and SEQ ID NO:28, including from 0 to 16 nucleotide analogs;
- (iii) SEQ ID NO:4 and SEQ ID NO:37, including from 0 to 16 nucleotide analogs;
- (iv) SEQ ID NO:11 and SEQ ID NO:49, including from 0 to 16 nucleotide analogs;
- (v) SEQ ID NO:4 and SEQ ID NO:49, including from 0 to 16 nucleotide analogs;
- (vi) SEQ ID NO:11 and SEQ ID NO:37, including from 0 to 16 nucleotide analogs;
- or (vii) SEQ ID NO:8 and SEQ ID NO:49, including from 0 to 16 nucleotide analogs;
- and/or
- (b) an Entamoeba-specific amplification oligomer set capable of amplifying a target region of an Entamoeba histolytica target nucleic acid, wherein the Entamoeba-specific amplification oligomer set comprises first and second Entamoeba-specific amplification oligomers respectively comprising target-hybridizing sequences as shown in (i) SEQ ID NO:46 and SEQ ID NO:19, including from 0 to 16 nucleotide analogs;
- (ii) SEQ ID NO:21 and SEQ ID NO:20, including from 0 to 16 nucleotide analogs;
- (iii) SEQ ID NO:36 and SEQ ID NO:43, including from 0 to 16 nucleotide analogs;
- or (iv) SEQ ID NO:42 and SEQ ID NO:19, including from 0 to 16 nucleotide analogs;
- and/or
- (c) a Giardia-specific amplification oligomer set capable of amplifying a target region of a Giardia lamblia target nucleic acid, wherein the Giardia-specific amplification oligomer set comprises first and second Giardia-specific amplification oligomers respectively comprising target-hybridizing sequences as shown in (i) SEQ ID NO:25 and SEQ ID NO:2, including from 0 to 16 nucleotide analogs;
- (ii) SEQ ID NO:5 and SEQ ID NO:29% including from 0 to 16 nucleotide analogs;
- (iii) SEQ ID NO:6 and SEQ ID NO:29% including from 0 to 16 nucleotide analogs;
- or (iv) SEQ ID NO:25 and SEQ ID NO:3, including from 0 to 16 nucleotide analogs;
- and/or
- (d) a Cyclospora-specific amplification oligomer set capable of amplifying a target region of a Cyclospora cayetanensis target nucleic acid, wherein the Cyclospora-specific amplification oligomer set comprises first and second Cyclospora-specific amplification oligomers respectively comprising target-hybridizing sequences as shown in (i) SEQ ID NO:26 and SEQ ID NO:14, including from 0 to 16 nucleotide analogs;
- (ii) SEQ ID NO:23 and SEQ ID NO:18, including from 0 to 16 nucleotide analogs;
- (iii) SEQ ID NO:31 and SEQ ID NO:22, including from 0 to 16 nucleotide analogs;
- (iv) SEQ ID NO:35 and SEQ ID NO:13, including from 0 to 16 nucleotide analogs;
- (v) SEQ ID NO:38 and SEQ ID NO:51, including from 0 to 16 nucleotide analogs;
- (vi) SEQ ID NO:1 and SEQ ID NO:9, including from 0 to 16 nucleotide analogs;
- (2) performing an in vitro nucleic acid amplification reaction, wherein any Cryptosporidium spp., Entamoeba histolytica, Giardia lamblia, and/or Cyclospora cayetanensis target nucleic acid present in the sample is used as a template for generating one or more amplification products corresponding to the Cryptosporidium spp., Entamoeba histolytica, Giardia lamblia, and/or Cyclospora cayetanensis target regions;
- and
- (3) detecting the presence or absence of the one or more amplification products, thereby determining the presence or absence of the at least one enteric parasite in the sample.
25 more elements
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A method for synthesizing a pair of oligonucleotides, comprising synthesizing a first oligonucleotide and synthesizing a second oligonucleotide,
- wherein each of the synthesizing the first oligonucleotide and the synthesizing the second oligonucleotide comprises the steps of:
- (a) obtaining a solid support comprising at least one nucleobase residue, wherein the at least one nucleobase residue is covalently bound at a 3′ position to the solid support;
- (b) coupling a 5′ position of the nucleobase residue furthest from the solid support to a 3′ position of another nucleobase residue;
- (c) repeating step (b) at least 15 additional times, thereby generating at least 17 contiguous nucleobase residues coupled to the solid support;
- and
- (d) cleaving the at least 17 contiguous nucleobase residues generated in step (c), thereby obtaining the oligonucleotide, and
- wherein the first oligonucleotide and the second oligonucleotide respectively comprise the nucleotide sequences of any one of
- SEQ ID NO:8 and SEQ ID NO:37, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:33 and SEQ ID NO:28, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:4 and SEQ ID NO:37, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:11 and SEQ ID NO:49, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:4 and SEQ ID NO:49, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:11 and SEQ ID NO:37, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:8 and SEQ ID NO:49, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:46 and SEQ ID NO:19, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:21 and SEQ ID NO:20, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:36 and SEQ ID NO:43, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:42 and SEQ ID NO:19, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:25 and SEQ ID NO:2, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:5 and SEQ ID NO:29, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:6 and SEQ ID NO:29, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:25 and SEQ ID NO:3, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:26 and SEQ ID NO:14, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:23 and SEQ ID NO:18, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:31 and SEQ ID NO:22, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:35 and SEQ ID NO:13, including from 0 to 16 nucleotide analogs;
- SEQ ID NO:38 and SEQ ID NO:51, including from 0 to 16 nucleotide analogs;
- or
- SEQ ID NO:1 and SEQ ID NO:9, including from 0 to 16 nucleotide analogs.
26 more elements
Dependencies are inferred from the claim text.