Compositions, kits and related methods for the detection and/or monitoring of
Provided are compositions, kits, and methods for the identification of Listeria . In certain aspects and embodiments, the compositions, kits, and methods may provide improvements in relation to specificity, sensitivity, and speed of detection.
1. A set of oligonucleotides configured to amplify in an amplification reaction a Listeria nucleic acid sequence from L. monocytogenes, L. innocua, L. grayi, L. ivanovii, L. welshimeri, L. murrayi , and L. seeligeri without substantial amplification of a Brochothrix thermosphacta nucleic acid or an Erysipelothrix rhusiopathiae nucleic acid comprising a first T7 provider oligonucleotide and a first primer oligonucleotide,
wherein the first T7 provider oligonucleotide targets a sequence in a first Listeria nucleic acid region corresponding to nucleotide positions of about 15-91 of SEQ ID NO: 103; and
wherein the first primer oligonucleotide targets a sequence in a second Listeria nucleic acid region corresponding to nucleotide positions of about 90-156 of SEQ ID NO: 103.
2. The set of oligonucleotides of claim 1 , wherein the first T7 provider oligonucleotide comprises a hybridizing sequence joined at its 5′ end to a T7 promoter sequence, wherein the hybridizing sequence is 19-24 nucleobases in length and comprises the hybridizing sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.
3. The set of oligonucleotides of claim 2 , wherein the first T7 provider oligonucleotide has a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14,-SEQ ID NO: 15, or SEQ ID NO: 12 having a guanine at a position corresponding to nucleotide position 58 of SEQ ID NO: 103.
4. The set of oligonucleotides of claim 2 , wherein the primer oligonucleotide has a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 23, SEQ ID NO: 21, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 22.
5. The set of oligonucleotides of claim 1 , further comprising a second T7 provider oligonucleotide wherein (a) the first T7 provider oligonucleotides has a nucleobase sequence comprising a T7 promoter sequence and the hybridization sequence of SEQ ID NO: 12 or SEQ ID NO: 13 and the second T7 provider oligonucleotide has a nucleobase sequence comprising a T7 promoter sequence and the hybridization sequence of SEQ ID NO: 14.
6. The set of oligonucleotides of claim 5 , wherein the primer oligonucleotide has a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 23, SEQ ID NO: 21, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID: 19.
7. The set of oligonucleotides of claim 6 , wherein the primer oligonucleotide has a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 23, SEQ ID NO: 21, or SEQ ID NO: 19.
8. The set of oligonucleotides of claim 1 , further comprising a second T7 provider oligonucleotide having a nucleobase sequence that overlaps the nucleobase sequence of the first T7 provider oligonucleotide, wherein the first and second T7 provider oligonucleotides differ in one or more nucleobases at a position where there is a mismatch between the nucleobase sequences of one or more Listeria species.
9. The set of oligonucleotides of claim 8 , wherein the first or second T7 provider oligonucleotide has a nucleobase sequence comprising a T7 promoter sequence and the hybridization sequence of SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.
10. The set of oligonucleotides of claim 8 , wherein the first or second T7 provider oligonucleotide has a nucleobase sequence consisting of the nucleobase sequence of SEQ ID NO: 12 or SEQ ID NO: 13.
11. The set of oligonucleotides of claim 8 , wherein the first or second T7 provider oligonucleotides has a nucleobase sequence consisting of the nucleobase sequence of SEQ ID NO: 14 or SEQ ID NO: 12 having a guanine at the position corresponding to nucleotide position 58 of SEQ ID NO: 103.
12. The set of oligonucleotides of claim 8 , wherein the primer oligonucleotide has a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 23, SEQ ID NO: 21, or SEQ ID NO: 19.
13. The set of oligonucleotides of claim 8 , wherein the first T7 provider oligonucleotide comprises an adenine at a nucleobase position that is complementary to a nucleobase position in a Listeria nucleic acid sequence corresponding to nucleobase position 58 of SEQ ID NO: 103, and the second T7 provider oligonucleotide comprises a guanine at the nucleobase position that is complementary to the nucleobase position in a Listeria nucleic acid sequence corresponding to nucleobase position 58 of SEQ ID NO: 103.
14. The set of oligonucleotides of claim 13 , wherein the first T7 provider oligonucleotide has a nucleobase sequence comprising a T7 promoter sequence and the hybridization sequence of SEQ ID NO: 12 or SEQ ID NO: 13 and the second T7 provider oligonucleotide has a nucleobase sequence comprising a T7 promoter sequence and the hybridization sequence of SEQ ID NO: 14.
15. The set of oligonucleotides of claim 14 , wherein the primer oligonucleotide has a nucleobase sequence comprising the nucleobase sequence of SEQ ID NO: 23, SEQ ID NO: 21, or SEQ ID NO: 19.
16. A set of oligonucleotides for detecting a Listeria nucleic acid sequence in a sample comprising a T7 provider oligonucleotide, a primer oligonucleotide, and a detection oligonucleotide, wherein the T7 provider oligonucleotide and the primer oligonucleotide are configured amplify in an amplification reaction a nucleic acid sequence from L. monocytogenes, L. innocua, L. grayi, L. ivanovii, L. welshimeri, L. murrayi , and L. seeligeri without substantial amplification of a Brochothrix thermosphacta nucleic acid or an Erysipelothrix rhusiopathiae nucleic acid, wherein the first T7 provider oligonucleotide targets a sequence in a first Listeria nucleic acid region corresponding to nucleotide positions of about 15-91 of SEQ ID NO: 103, and wherein the first primer oligonucleotide targets a sequence in a second Listeria nucleic acid region corresponding to nucleotide positions of about 90-156 of SEQ ID NO: 103.
17. The set of oligonucleotides of claim 16 , wherein the detection oligonucleotide is a molecular torch oligonucleotide having a nucleobase sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, and a complement thereof.
18. The set of oligonucleotides of claim 1 , further comprising a blocker oligonucleotide having a nucleobase sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
19. The set of oligonucleotides of claim 1 , further comprising a target capture oligonucleotide having a nucleobase sequence selected from the group consisting of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.
20. The set of oligonucleotides of claim 16 , further comprising a helper oligonucleotide.