Compositions, kits and related methods for the detection and/or monitoring of
View Patent ↗Provided are compositions, kits, and methods for the identification of Salmonella . In certain aspects and embodiments, the compositions, kits, and methods may provide improvements in relation to specificity, sensitivity, and speed of detection.
1. A method for detecting Salmonella in a sample, said method comprising a detecting step of detecting the presence or absence of a Salmonella target nucleic acid using a fluorescently labeled molecular torch oligonucleotide, wherein the molecular torch oligonucleotide comprises the nucleotide sequence consisting essentially of SEQ ID NO: 66, 67, 68, 69, or 70, and wherein the detecting step is performed below 60° C.
2. The method of claim 1 , wherein the Salmonella target nucleic acid is an amplicon that comprises a sequence corresponding to bases from about 164 to about 209 of SEQ ID NO: 150.
3. The method of claim 2 , wherein the method further comprises contacting the Salmonella target nucleic acid with a T7 provider oligonucleotide and a primer oligonucleotide to generate the amplicon, wherein the T7 provider oligonucleotide comprises a nucleotide sequence that is SEQ ID NO: 1, 3, 5, or 6, and wherein the primer oligonucleotide has a sequence that is SEQ ID NO: 35, 37, 38, 39, 40, 42, 43, 45, or 46.
4. The method of claim 1 , wherein the sample is a bioprocess stream where Salmonella is a known or suspected contaminant.
5. The method of claim 1 , further comprising purifying or enriching the Salmonella target nucleic acid from the sample prior to performing the step of detecting the Salmonella target nucleic acid.
6. The method of claim 5 , wherein the purifying or enriching step comprises contacting the sample with a target capture oligonucleotide comprising (i) a sequence-binding region that specifically hybridizes to a target sequence in the Salmonella target nucleic acid and (ii) an immobilized probe-binding region, wherein the immobilized probe-binding region joins to a binding partner on an immobilized probe to capture any Salmonella target nucleic acid hybridized to said sequence-binding region to a solid support.
7. The method of claim 6 , wherein said target capture oligonucleotide has a sequence that is SEQ ID NO: 71-76 or 77.
8. A composition for use in detecting Salmonella in a sample, said composition comprising (i) a molecular torch oligonucleotide comprising the nucleotide sequence consisting essentially of SEQ ID NO: 66, 67, 68, 69, or 70, (ii) an RNA polymerase, and (iii) a promoter oligonucleotide, wherein the molecular torch specifically hybridizes to a sequence within a Salmonella target nucleic acid.
9. The composition of claim 8 , wherein the Salmonella target nucleic acid is an amplicon that comprises a sequence corresponding to bases from about 164 to about 209 of SEQ ID NO: 150.
10. The composition of claim 9 , wherein the compositions further comprises a T7 provider oligonucleotide comprising a nucleotide sequence that is SEQ ID NO: 1, 3, 5, or 6, and further comprises a primer oligonucleotide comprising a nucleotide sequence that is SEQ ID NO: 35, 37, 38, 39, 40, 42, 43, 45, or 46.
11. The composition of claim 8 , further comprising a target capture oligonucleotide comprising (i) a sequence-binding region that specifically hybridizes to a target sequence in the Salmonella target nucleic acid and (ii) an immobilized probe-binding region.
12. The composition of claim 11 , wherein said target capture oligonucleotide has a sequence that is SEQ ID NO: 71-76 or 77.
13. A kit for use in detecting Salmonella in a sample, said kit comprising (i) a molecular torch oligonucleotide comprising the nucleotide sequence consisting essentially of SEQ ID NO: 66, 67, 68, 69, or 70 wherein the molecular torch specifically hybridizes to a sequence within a Salmonella target nucleic acid, (ii) an RNA polymerase, and (iii) a promoter oligonucleotide for isothermally amplifying the Salmonella target nucleic acid.
14. The kit of claim 13 , wherein the Salmonella target nucleic acid is an amplicon that comprises a sequence corresponding to bases from about 164 to about 209 of SEQ ID NO: 150.
15. The kit of claim 13 , wherein the kit further comprises T7 provider oligonucleotide comprising a nucleotide sequence that is SEQ ID NO: 1, 3, 5, or 6, and further comprises a primer oligonucleotide comprising a nucleotide sequence that is SEQ ID NO: 35, 37, 38, 39, 40, 42, 43, 45, or 46.
16. The kit of claim 13 , further comprising a target capture oligonucleotide comprising (i) a sequence-binding region that specifically hybridizes to a target sequence in the Salmonella target nucleic acid and (ii) an immobilized probe-binding region.
17. The kit of claim 16 , wherein said target capture oligonucleotide is selected from the sequences of SEQ ID NOs: 71-76 or 77.
18. The method of claim 1 , wherein the detecting step is performed in a reaction mixture comprising the molecular torch oligonucleotide, an RNA polymerase, and a promoter oligonucleotide.
19. The method of claim 1 , wherein the detecting step is performed as part of a real time amplification and detection method.
20. The method of claim 1 , wherein the detecting step is performed at about 42° C.