Compositions, kits and related methods for the detection and/or monitoring of
View Patent ↗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 method for detecting Listeria in a sample, the method comprising:
performing a nucleic acid amplification assay using a set of oligonucleotides comprising two or more T7 provider oligonucleotides and one or more primer oligonucleotides,
wherein the two or more T7 provider oligonucleotides and the one or more primer oligonucleotides are configured such that nucleic acids from L. monocytogenes, L. innocua, L. grayi, L. ivanovii, L. welshimeri, L. murrayi , and L. seeligeri are amplifiable under nucleic acid amplification assay conditions without substantial amplification of nucleic acid from Brochothrix thermosphacta or Erysipelothrix rhusiopathiae,
wherein at least one of the T7 provider oligonucleotides targets a sequence in a region of Listeria nucleic acid corresponding to nucleotide positions of about 49-68 of SEQ ID NO: 103, and
wherein at least one of the one or more primer oligonucleotides targets a sequence in a region of Listeria nucleic acid corresponding to nucleotide positions of about 90-156 of SEQ ID NO: 103; and
detecting presence or absence of an amplification product, wherein presence of the amplification product indicates the presence of Listeria in the sample.
2. The method of claim 1 , wherein a first of the T7 provider oligonucleotide has a sequence comprising SEQ ID NO: 13, and a second of the T7 provider oligonucleotides has a sequence comprising SEQ ID NO: 14.
3. The method of claim 2 , wherein at least one of the one or more primer oligonucleotides targets a sequence in a region of Listeria nucleic acid corresponding to nucleotide positions of about 131-152 of SEQ ID NO: 103.
4. The method of claim 3 , wherein at least one of the one or more primer oligonucleotides has a sequence comprising SEQ ID NO: 23.
5. The method of claim 1 , wherein the two or more T7 provider oligonucleotides comprise a first and a second T7 provider oligonucleotide and the second T7 provider oligonucleotide has a sequence overlapping the first T7 provider oligonucleotide, and the first and second T7 provider oligonucleotides differ in one or more bases at a position where there is a mismatch between base sequences of one or more Listeria species.
6. The method of claim 5 , wherein the first T7 provider oligonucleotide comprises an adenine at a nucleotide position that is complementary to a nucleotide position in a Listeria nucleic acid sequence corresponding to nucleotide position 58 of SEQ ID NO: 103, and the second T7 provider oligonucleotide comprises a guanine at the nucleotide position that is complementary to the nucleotide position in a Listeria nucleic acid sequence corresponding to nucleotide position 58 of SEQ ID NO: 103.
7. The method of claim 5 , wherein at least one of the T7 provider oligonucleotides has a sequence selected from the group consisting of SEQ ID NOS: 13 and 14.
8. The method of claim 5 , wherein one of the T7 provider oligonucleotides has a sequence comprising SEQ ID NO: 13.
9. The method of claim 5 , wherein one of the of the T7 provider oligonucleotides has a sequence comprising SEQ ID NO: 14.
10. The method of claim 6 , wherein the first T7 provider oligonucleotide has a sequence comprising SEQ ID NO: 13 and the second T7 provider oligonucleotide has a sequence comprising SEQ ID NO: 14 .
11. The method of claim 1 , wherein at least one of the one or more primer oligonucleotides targets a sequence in a region of Listeria nucleic acid corresponding to nucleotide positions of about 131-152 of SEQ ID NO: 103.
12. The method of claim 1 , wherein at least one of the one or more primer oligonucleotides has a sequence selected from the group consisting of SEQ ID NOS: 16, 21, or and 23.
13. The method of claim 12 , wherein one of the one or more primer oligonucleotides has a sequence comprising SEQ ID NO: 23.
14. The method of claim 1 , wherein one of the two or more T7 provider oligonucleotides has a sequence comprising SEQ ID NO: 13, and one of the one or more primer oligonucleotides has a sequence comprising SEQ ID NO: 23.
15. The method of claim 5 , wherein one of the two or more T7 provider oligonucleotides has a sequence comprising SEQ ID NO: 13, and one of the one or more primer oligonucleotides has a sequence comprising SEQ ID NO: 23.
16. The method of claim 1 , wherein one of the two or more T7 provider oligonucleotides has a sequence comprising SEQ ID NO: 14, and one of the one or more primer oligonucleotides has a sequence comprising SEQ ID NO: 23.
17. The method of claim 10 , wherein one of the one or more primer oligonucleotides has a sequence comprising SEQ ID NO: 23.
18. The method of claim 1 , further comprising a detection oligonucleotide.
19. The method of claim 18 , wherein the detection oligonucleotide is a torch oligonucleotide, and the torch oligonucleotide has a sequence selected from the group consisting of SEQ ID NOS: 24, 25, 26, 27, 28 and the full complements thereof.
20. The method of claim 1 , further comprising a blocker oligonucleotide, wherein the blocker oligonucleotide has a sequence selected from the group consisting of SEQ ID NOS: 1, 5, 6, and 7.
21. The method of claim 1 , further comprising a target capture oligonucleotide, wherein the target capture oligonucleotide has a sequence selected from the group consisting of SEQ ID NOS: 29, 30, 31, 32, 33, 34, 35, and 36.
22. The method of claim 18 , further comprising a helper oligonucleotide.