Nicking and extension amplification reaction (NEAR) of
View Patent ↗This invention relates to compositions, methods and kits for detecting the presence or absence of a bacterial species in a biological sample using isothermal nucleic acid amplification.
1. A composition comprising
i) a forward template comprising a nucleic acid sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of the Streptococcus pyogenes ( S. pyogenes ) cell envelope proteinase A (cepA) gene antisense strand; a nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site, the forward template comprising a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 1 (AGACTCCATATGGAGTCTAGCCAAACAGGAACA) or a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 8 (AGACTCCACACGGAGTCTAGCCAAACAGGAACA); and
ii) a reverse template comprises a nucleotide sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of the S. pyogenes cepA gene sense strand; a nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site, the reverse template comprising a nucleotide sequence having at least 80, 85 or 95% identity to SEQ ID NO: 2 (CGACTCCATATGGAGTCGAAAGCAATCTGAGGA) or a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 9 (GGACTCCACACGGAGTCCGCCAGCAATCUGAGG).
2. The composition of claim 1 , further comprising a probe oligonucleotide comprising a sequence complementary to the S. pyogenes cell envelope proteinase A (cepA) nucleotide sequence.
3. The composition of claim 2 , further comprising a probe oligonucleotide comprising a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 3 (ACAAGTATGTGAGGAGAGGCCATACTTGT).
4. The composition of claim 2 , wherein the probe is conjugated to a detectable label.
5. The composition of claim 4 , wherein the detectable label is selected from the group consisting of a fluorophore, an enzyme, a quencher, an enzyme inhibitor, a radioactive label, a member of a binding pair, and a combination thereof.
6. The composition of claim 1 , further comprising one or more of a DNA polymerase, one or more nicking enzymes, dNTPs, or a mixture of dNTPs and ddNTPs.
7. The composition of claim 6 , wherein the DNA polymerase is selected from the group consisting of Geobacillus bogazici DNA polymerase, Bst (large fragment), and exo-DNA Polymerase.
8. The composition of claim 6 , wherein the one or more nicking enzymes are selected from the group consisting of Nt.BspQI, Nb.BbvCi, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Bpu10I, Nt.Bpu10I, and N.BspD61.
9. The composition of claim 1 , further comprising a lytic agent.
10. The composition of claim 9 , wherein the lytic agent comprises a bacteriophage lysin.
11. The composition of claim 10 , wherein the bacteriophage lysin comprises streptococcal C 1 bacteriophage lysin (PlyC).
12. The composition of claim 1 , wherein the composition is lyophilized.
13. The composition of claim 1 , wherein the forward template and/or the reverse template comprises one or more modified nucleotides, spacers, or blocking groups.
14. The composition of claim 13 , wherein at least one modified nucleotide includes a 2′ modification.
15. The composition of claim 14 , wherein at least one modified nucleotide includes a 2′-O-methyl.
16. The composition of claim 13 , wherein the forward template and/or the reverse template comprises at least one phosphorothioate.
17. A method for the detection of the presence or absence of S. pyogenes in a biological sample, wherein the method comprises: contacting the biological sample with components comprising
i) a forward template comprising a nucleic acid sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of the Streptococcus pyogenes cell envelope proteinase A (cepA) gene antisense strand; a nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site, wherein the forward template comprises a nucleotide sequence having at least at least 80, 85, or 95% identity to SEQ ID NO: 1 (AGACTCCATATGGAGTCTAGCCAAACAGGAACA) or a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 8 (AGACTCCACACGGAGTCTAGCCAAACAGGAACA);
ii) a reverse template comprises a nucleotide sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of the Streptococcus pyogenes cepA gene sense strand; a nicking enzyme binding site and a nicking site upstream of said recognition region; and a stabilizing region upstream of said nicking site, wherein the reverse template comprises a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 2 (CGACTCCATATGGAGTCGAAAGCAATCTGAGGA) or a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 9 (GGACTCCACACGGAGTCCGCCAGCAATCUGAGG), and
(iii) a first nicking enzyme that is capable of nicking at the nicking site of said forward template and does not nick within said target sequence,
(iv) a second nicking enzyme that is capable of nicking at the nicking site of said reverse template and does not nick within said target sequence, and
(v) a DNA polymerase; amplifying the target nucleic acid sequence in a nucleic amplification reaction to provide an amplification product; and detecting the presence or absence of the amplification product.
18. The method of claim 17 , wherein the components of a nucleic amplification reaction further comprise a probe oligonucleotide comprising a sequence complementary to the S. pyogenes cell envelope proteinase A (cepA) gene nucleotide sequence.
19. The method of claim 18 , wherein the probe is conjugated to a detectable label.
20. The method of claim 19 , wherein the detectable label is selected from the group consisting of a fluorophore, an enzyme, a quencher, an enzyme inhibitor, a radioactive label, a member of a binding pair, and a combination thereof.
21. The method of claim 17 , wherein amplification is performed under isothermal conditions.
22. The method of claim 17 , wherein amplification is performed by multiple cycles of said polymerase extending said forward and reverse templates along said target sequence producing a double-stranded nicking site, and said nicking enzymes nicking at said nicking sites, and, following a first round of amplification, at amplified copies of said sites, producing an amplification product, thereby providing a product.
23. The method of claim 18 , wherein the probe oligonucleotide comprises a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 3 (ACAAGTATGTGAGGAGAGGCCATACTTGT).
24. The method of claim 17 , wherein the components of a nucleic amplification reaction further comprise dNTPs or a mixture of dNTPs and ddNTPs.
25. The method of claim 17 , wherein the DNA polymerase is selected from the group consisting of Geobacillus bogazici DNA polymerase, Bst (large fragment), and exo-DNA polymerase.
26. The method of claim 17 , wherein said forward and reverse templates comprise nicking enzyme binding sites recognized by the same nicking enzyme and said first and said second nicking enzymes are the same.
27. The method of claim 17 , wherein the first and second nicking enzymes are selected from the group consisting of Nt.BspQI, Nb.BbvCi, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Bpu10I, Nt.Bpu10I, and N.SBspD61.
28. The method of claim 17 , wherein the first and second nicking enzymes nick downstream of the nicking enzyme binding site.
29. The method of claim 17 , optionally comprising extracting or isolating nucleic acid from the sample prior to amplification.
30. The method of claim 17 , wherein the biological sample is selected from feces, swabs of the oral cavity/throat, saliva, pus, sputum, blood, and urine, or wherein the biological sample is from infected or non-infected tissues.
31. The method of claim 17 , wherein the forward template and/or the reverse template comprises one or more modified nucleotides, spacers, or blocking groups.
32. The method of claim 31 , wherein at least one modified nucleotide includes a 2′ modification.
33. The method of claim 32 , wherein at least one modified nucleotide includes a 2′-O-methyl.
34. The method of claim 31 , wherein the forward template and/or the reverse template comprises at least one phosphorothioate.
35. A kit comprising: a forward template oligonucleotide comprising a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 1 (AGACTCCATATGGAGTCTAGCCAAACAGGAACA) and/or
a forward template oligonucleotide comprising a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 8 (AGACTCCACACGGAGTCTAGCCAAACAGGAACA); and
a reverse template oligonucleotide comprising a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 2 (CGACTCCATATGGAGTCGAAAGCAATCTGAGGA) and/or a reverse template oligonucleotide comprising a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 9 (GGACTCCACACGGAGTCCGCCAGCAATCUGAGG); and a polymerase.
36. The kit of claim 35 , further comprising a probe oligonucleotide comprising a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 3 (ACAAGTATGTGAGGAGAGGCCATACTTGT).
37. The kit of claim 35 , further comprising a swab.
38. The kit of claim 35 , further comprising a lytic agent and one or more of dNTPs or a mixture of dNTPs and ddNTPs.
39. The kit of claim 35 , optionally comprising reagent for extracting or isolating nucleic acid from a biological sample.
40. The kit of claim 35 , wherein one or more of the template oligonucleotides comprise one or more spacers, and/or one or more blocking groups, and/or one or more modified nucleotides.
41. The kit of claim 40 , wherein at least one modified nucleotide includes a 2′ modification.
42. The kit of claim 41 , wherein at least one modified nucleotide includes a 2′-O-methyl.