Compositions, kits and methods for detection of campylobacter nucleic acid
View Patent ↗The disclosed invention is related to compositions, kits and methods comprising one or more oligomers targeting 16S rRNA target nucleic acid from Campylobacter species jejuni, coli and/or lari . Compositions include amplification oligomers, detection probe oligomers and/or target capture oligomers. Kits and methods comprise at least one of these oligomers.
1. A method for specifically detecting a Campylobacter target nucleic acid in a sample comprising the steps of:
(a) contacting a sample suspected of containing at least a Campylobacter target nucleic acid with at least two amplification oligomers that stably hybridize to a C. jejuni target nucleic acid and a C. coli , a C. lari , or a C. coli and a C. lari target nucleic acid, wherein a first of said amplification oligomers comprises SEQ ID NO:26 or SEQ ID NO:32 and wherein a second of said amplification oligomers comprises a target hybridizing sequence 15 to 45 nucleotides in length and configured to target a sequence in a region of a Campylobacter 16S rRNA gene corresponding to nucleotides 170 to 226 of GenBank Accession No.: AF393202.1, gi:20378208;
(b) performing an in vitro nucleic acid amplification reaction wherein any of a C. jejuni target nucleic acid, a C. coli target nucleic acid and a C. lari target nucleic acid present in said sample is used as a template for generating an amplification product; and
(c) performing a nucleic acid detection reaction that detects said amplification product to determine whether a Campylobacter target nucleic acid was present in said sample.
2. The method of claim 1 , wherein said second of said amplification oligomers comprises a target hybridizing sequence configured to target a sequence in a region of a Campylobacter 16S rRNA gene selected from the group consisting of: a region corresponding to nucleotides 170 to 212 of GenBank Accession No.: AF393202.1, gi:20378208; a region corresponding to nucleotides 184 to 212 of GenBank Accession No.: AF393202.1, gi:20378208; and a region corresponding to nucleotides 170 to 205 of GenBank Accession No.: AF393202.1, gi:20378208; or wherein said second amplification oligomer is selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO:12; SEQ ID NO:13; and SEQ ID NO:14.
3. The method of claim 1 , wherein said first of said amplification oligomers comprises SEQ ID NO:26.
4. The method of claim 1 , wherein said first of said amplification oligomers comprises SEQ ID NO:32.
5. The method of claim 1 , wherein said first amplification oligomer is SEQ ID NO:26 and said second amplification oligomer is selected from the group consisting of SEQ ID NOS:1 through 14.
6. The method of claim 1 , wherein said second amplification oligomer consists essentially of SEQ ID NO:7.
7. The method of claim 1 , wherein step b. further comprises contacting said sample with a blocker oligomer.
8. The method of claim 7 , wherein said blocker oligomer consists essentially of SEQ ID NO:50.
9. The method of claim 1 , wherein said detection reaction comprises contacting said amplification product with a detection probe oligomer configured to hybridize to a portion of said amplification product.
10. The method of claim 9 , wherein said detection is real-time detection.
11. The method of claim 9 , wherein said detection oligomer is selected from the group consisting of SEQ ID NOS:63 through 68.
12. The method of claim 9 , wherein said detection oligomer consists essentially of SEQ ID NO:67.
13. The method of claim 9 , wherein said detection oligomer consists essentially of SEQ ID NO:68.
14. The method of claim 9 , wherein said second amplification oligomer is SEQ ID NO:7.
15. The method of claim 14 , wherein said detection reaction comprises contacting said amplification product with a detection probe oligomer selected from the group consisting of SEQ ID NO:67 and SEQ ID NO:68.
16. The method of claim 15 , wherein said first amplification oligomer is SEQ ID NO:26, and wherein step b. further comprises contacting said sample with a blocker consisting essentially of SEQ ID NO:50.
17. The method of claim 15 , wherein said first amplification oligomer is SEQ ID NO:32, and wherein step b. further comprises contacting said sample with a blocker consisting essentially of SEQ ID NO:50.
18. The method of claim 1 , further comprising the step of contacting said sample suspected of containing a Campylobacter target nucleic acid with a target capture oligomer.
19. The method of claim 18 , wherein said target capture oligomer is selected from the group consisting of SEQ ID NOS:58, 71 and 73.
20. The method of claim 1 , wherein said sample further contains nucleic acid from one or more bacteria closely related to C. jejuni; C. coli , or C. lari and said target nucleic acid is specifically detected in said sample.
21. The method of claim 20 , wherein said one or more bacteria is at least one of: C. Fetus , ssp. fetus, C. fetus ssp venerealis, C upsaliensis, E. coli, C. fecalis, S. enteridis, H. pylori, E. vulnaris, E. hermannii, Enterobacter cloacae, Edwardsiella hoshinae, P. miribalis, Citrobacter brakii, Pseudomonas fluorescens, Shigella flexneri, Aeromonas hydrophila, A. butzleri, C. Fetus, ssp. fetus, C. fetus ssp venerealis , and C. upsaliensis.
22. The method of claim 1 , wherein at step c said target nucleic acid is specifically amplified in the presence of a nucleic acid from one or more bacteria closely related to C. jejuni; C. coli , or C. lari.
23. The method of claim 22 , wherein said one or more bacteria is at least one of: C. Fetus , ssp. fetus, C. fetus ssp venerealis, C upsaliensis, E. coli, C. fecalis, S. enteridis, H. pylori, E. vulnaris, E. hermannii, Enterobacter cloacae, Edwardsiella hoshinae, P. miribalis, Citrobacter brakii, Pseudomonas fluorescens, Shigella flexneri, Aeromonas hydrophila, A. butzleri, C. Fetus , ssp. fetus, C. fetus ssp venerealis , and C. upsaliensis.
24. A method for specifically detecting a Campylobacter target nucleic acid in a sample comprising the steps of:
(a) contacting a sample suspected of containing at least a Campylobacter target nucleic acid with at least two amplification oligomers that stably hybridize to a C. jejuni target nucleic acid and a C. coli , a C. lari , or a C. coli and a C. lari target nucleic acid, wherein a first of said amplification oligomers is a promoter based amplification oligomer comprising a target hybridizing sequence that is SEQ ID NO:27 or SEQ ID NO:33 and further comprising a 5′ promoter sequence that is SEQ ID NO:75 and wherein a second of said amplification oligomers comprises a target hybridizing sequence 15 to 45 nucleotides in length and configured to target a sequence in a region of a Campylobacter 16S rRNA gene corresponding to nucleotides 170 to 226 of GenBank Accession No.: AF393202.1, gi:20378208;
(b) performing an in vitro nucleic acid amplification reaction wherein any of a C. jejuni target nucleic acid, a C. coli target nucleic acid and a C. lari target nucleic acid present in said sample is used as a template for generating an amplification product; and
(c) performing a nucleic acid detection reaction that detects said amplification product to determine whether a Campylobacter target nucleic acid was present in said sample.