Nicking and extension amplification reaction (near) of
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;
ii) a reverse template comprising 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; and
iii) a probe oligonucleotide comprising a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 3
(ACAAGTATGTGAGGAGAGGCCATACTTGT).
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 wherein said probe oligonucleotide comprising a sequence complementary to the S. pyogenes cell envelope proteinase A (cepA) nucleotide sequence is conjugated to a detectable label.
4. The composition of claim 3 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.
5. The composition of claim 1 wherein the portion of the nucleic acid sequence that is complementary to the 3′ end of the target antisense strand is 8-30 nucleotides in length.
6. The composition of claim 1 wherein the portion of the nucleic acid sequence that is complementary to the 3′ end of the target sense strand is 8-30 nucleotides in length.
7. The composition of claim 1 further comprising one or more of; a) a DNA polymerase; b) one or more nicking enzymes; and c) dNTPs or a mixture of dNTPs and ddNTPs.
8. The composition of claim 7 wherein the DNA polymerase is selected from the group consisting of Geobacillus bogazici DNA polymerase, Bst (large fragment), and exo-DNA Polymerase.
9. The composition of claim 7 wherein the one or more nicking enzymes is/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.
10. The composition of claim 1 further comprising a lytic agent.
11. The composition of claim 10 wherein the lytic agent comprises a bacteriophage lysin.
12. The composition of claim 11 wherein the bacteriophage lysin comprises streptococcal C1 bacteriophase lysin (PlyC).
13. The composition of claim 1 wherein the composition is lyophilized.
14. The composition of claim 1 wherein the forward template and/or the reverse template comprises one or more modified nucleotides, spacers, or blocking groups.
15. The composition of claim 14 wherein at least one modified nucleotide includes a 2′ modification.
16. The composition of claim 15 wherein at least one modified nucleotide includes a 2′-O-methyl.
17. The composition of claim 14 wherein the forward template and/or the reverse template comprises at least one phosphorothioate.
18. A kit 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;
ii) a reverse template comprising 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; and
iii) a probe oligonucleotide comprising a nucleotide sequence having at least 80, 85, or 95% identity to SEQ ID NO: 3
(ACAAGTATGTGAGGAGAGGCCATACTTGT).
19. The kit of claim 18 further comprising a swab.
20. The kit of claim 18 further comprising a lytic agent and one or more of; 1) dNTPs; or 2) a mixture of dNTPs and ddNTPs.
21. The kit of claim 18 optionally comprising reagent for extracting and/or isolating nucleic acid from a biological sample.
22. The kit of claim 18 wherein the template oligonucleotide comprises one or more modified nucleotides, spacers, or blocking groups.
23. The kit of claim 22 wherein at least one modified nucleotide includes a 2′ modification.
24. The kit of claim 23 wherein at least one modified nucleotide includes a 2′-O-methyl.
25. The kit of claim 18 further comprising a polymerase.