Optimized oligonucleotides and methods of using same for the detection, isolation, amplification, quantification, monitoring, screening and sequencing of clostridium difficile genes encoding toxin b, and/or toxin a and/or binary toxin
AssigneeIntelligent Medical Devices, Inc.
InventorsDavid L. DOLINGER, James R. HULLY, Alice A. JACOBS, Chesley LESLIN, Heinz R. REISKE, Chunyang ZHENG
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Abstract
Described herein are oligonucleotides useful for detecting, isolating, amplifying, quantitating, monitoring, screening and sequencing the C. Difficile genes encoding toxin B, and/or toxin A, and/or binary toxin, and methods of using the described oligonucleotides.
Claims61
An isolated nucleic acid sequence comprising a sequence selected from the group consisting of:
- SEQ ID NOS:
- 1-69 and 138.
A method of hybridizing one or more isolated nucleic acid sequences comprising a sequence selected from the group consisting of:
- SEQ ID NOS:
- 1-69 and 138 to a C. Difficile sequence, comprising contacting one or more isolated nucleic acid sequences to a sample comprising the C. Difficile sequence under conditions suitable for hybridization.
A primer set comprising at least one forward primer selected from the group consisting of SEQ ID NOS:
- 1, 4, 6, 8, 10, 12, 13, 18, 21, 23, 24, 26, 28, 30, 35, 36, 37, 40, 43, 45, 48, 51, 53, 55, 58, 63, 66, and 68, and at least one reverse primer selected from the group consisting of SEQ ID NOS:
- 3, 5, 7, 9, 11, 15, 17, 20, 25, 32, 33, 34, 39, 42, 44, 47, 50, 52, 54, 57, 60, 62, 65, 67 and 138.
A method of producing a nucleic acid product, comprising contacting one or more isolated nucleic acid sequences selected from the group consisting of SEQ ID NOS:
- 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 20, 21, 23, 24, 25, 26, 28, 30, 32, 33, 34, 35, 36, 37, 39, 40, 42, 43, 44, 45, 47, 48, 50, 51, 52, 53, 54, 55, 57, 58, 60, 62, 63, 65, 66, 67, 68 and 138 to a sample comprising a C. Difficile sequence under conditions suitable for nucleic acid polymerization.
A method for detecting a C. Difficile sequence in a sample, comprising:
- a) contacting the sample with at least one forward primer comprising a sequence selected from the group consisting of:
- SEQ ID NOS:
- 1, 4, 6, 8, 10, 12, 13, 18, 21, 23, 24, 26, 28, 30, 35, 36, 37, 40, 43, 45, 48, 51, 53, 55, 58, 63, 66, and 68, and at least one reverse primer comprising a sequence selected from the group consisting of:
- SEQ ID NOS:
- 3, 5, 7, 9, 11, 15, 17, 20, 25, 32, 33, 34, 39, 42, 44, 47, 50, 52, 54, 57, 60, 62, 65, 67 and 138 under conditions such that nucleic acid amplification occurs to yield an amplicon;
- and
- b) contacting the amplicon with one or more probes comprising one or more sequences selected from the group consisting of:
- SEQ ID NOS:
- 2, 14, 16, 19, 22, 27, 29, 31, 38, 41, 46, 49, 56, 59, 61, 64, and 69 under conditions such that hybridization of the probe to the amplicon occurs;
- wherein hybridization of the probe is indicative of C. Difficile in the sample.
A kit for detecting a C. Difficile sequence in a sample, comprising one or more probes comprising a sequence selected from the group consisting of:
- SEQ ID NOS:
- 2, 14, 16, 19, 22, 27, 29, 31, 38, 41, 46, 49, 56, 59, 61, 64, and 69.
A method of diagnosing a C. Difficile-associated colonization, condition, syndrome or disease, comprising:
- a) contacting a sample with at least one forward and reverse primer set selected from the group consisting of:
- Groups 1-129 and 184 of Table 4, Groups 130-138 of Table 5, and Groups 139-145 of Table 6;
- b) conducting an amplification reaction, thereby producing an amplicon;
- and
- c) detecting the amplicon using one or more probes selected from the group consisting of:
- SEQ ID NOS:
- 2, 14, 16, 19, 22, 27, 29, 31, 38, 41, 46, 49, 56, 59, 61, 64, and 69;
- wherein the detection of an amplicon is indicative of the presence of C. Difficile in the sample.
A kit for binding, amplifying and sequencing a C. Difficile sequence in a sample, comprising:
- a) at least one forward primer comprising the sequence selected from the group consisting of:
- SEQ ID NOS:
- 1, 4, 6, 8, 10, 12, 13, 18, 21, 23, 24, 26, 28, 30, 35, 36, 37, 40, 43, 45, 48, 51, 53, 55, 58, 63, 66, and 68;
- b) at least one reverse primer comprising the sequence selected from the group consisting of:
- SEQ ID NOS:
- 3, 5, 7, 9, 11, 15, 17, 20, 25, 32, 33, 34, 39, 42, 44, 47, 50, 52, 54, 57, 60, 62, 65, 67 and 138;
- and
- c) reagents for the sequencing of amplified DNA fragments.
A method of diagnosing a C. Difficile-associated colonization, condition, syndrome or disease, comprising contacting a denatured target from a sample with one or more probes comprising a sequence selected from the group consisting of:
- SEQ ID NOS:
- 2, 14, 16, 19, 22, 27, 29, 31, 38, 41, 46, 49, 56, 59, 61, 64, and 69 under conditions for hybridization to occur;
- wherein hybridization of the one or more probes to a denatured target is indicative of the presence of C. Difficile in the sample.
A method for identifying the causative agent of watery diarrhea by detecting one or more C. Difficile strains in a sample based on its gene(s)) coding for toxin(s), the method comprising:
- a) contacting the sample with at least one forward primer comprising the sequence selected from the group consisting of:
- SEQ ID NOS:
- 1, 4, 6, 8, 10, 12, 13, 18, 21, 23, 24, 26, 28, 30, 35, 36, 37, 40, 43, 45, 48, 51, 53, 55, 58, 63, 66, and 68, and at least one reverse primer comprising the sequence selected from the group consisting of:
- SEQ ID NOS:
- 3, 5, 7, 9, 11, 15, 17, 20, 25, 32, 33, 34, 39, 42, 44, 47, 50, 52, 54, 57, 60, 62, 65, 67 and 138 under conditions such that nucleic acid amplification occurs to yield an amplicon;
- and
- b) contacting the amplicon with one or more probes comprising one or more sequences selected from the group consisting of:
- SEQ ID NOS:
- 2, 14, 16, 19, 22, 27, 29, 31, 38, 41, 46, 49, 56, 59, 61, 64, and 69 under conditions such that hybridization of the probe to the amplicon occurs;
- wherein the hybridization of the probe is indicative of C. Difficile in the sample.
A method for identifying the causative agent of colitis by detecting one or more C. Difficile strains in a sample based on its gene(s) coding for toxin(s), the method comprising:
- a) contacting the sample with at least one forward primer comprising the sequence selected from the group consisting of:
- SEQ ID NOS:
- 1, 4, 6, 8, 10, 12, 13, 18, 21, 23, 24, 26, 28, 30, 35, 36, 37, 40, 43, 45, 48, 51, 53, 55, 58, 63, 66, and 68, and at least one reverse primer comprising the sequence selected from the group consisting of:
- SEQ ID NOS:
- 3, 5, 7, 9, 11, 15, 17, 20, 25, 32, 33, 34, 39, 42, 44, 47, 50, 52, 54, 57, 60, 62, 65, 67 and 138 under conditions such that nucleic acid amplification occurs to yield an amplicon;
- and
- b) contacting the amplicon with one or more probes comprising one or more sequences selected from the group consisting of:
- SEQ ID NOS:
- 2, 14, 16, 19, 22, 27, 29, 31, 38, 41, 46, 49, 56, 59, 61, 64, and 69 under conditions such that hybridization of the probe to the amplicon occurs;
- wherein the hybridization of the probe is indicative of C. Difficile in the sample.
A screening kit for binding, amplifying and sequencing a C. Difficile sequence, comprising:
- a) at least one forward primer comprising the sequence selected from the group consisting of:
- SEQ ID NOS:
- 1, 4, 6, 8, 10, 12, 13, 18, 21, 23, 24, 26, 28, 30, 35, 36, 37, 40, 43, 45, 48, 51, 53, 55, 58, 63, 66, and 68;
- b) at least one reverse primer comprising the sequence selected from the group consisting of:
- SEQ ID NOS:
- 3, 5, 7, 9, 11, 15, 17, 20, 25, 32, 33, 34, 39, 42, 44, 47, 50, 52, 54, 57, 60, 62, 65, 67 and 138;
- and
- c) reagents for the sequencing of amplified DNA fragments.
Dependencies are inferred from the claim text.