Nicking and extension amplification reaction (NEAR) of respiratory syncytial virus species
This invention relates to methods and compositions for detecting the presence or absence of a Respiratory Syncytial Virus target nucleic acid in a biological sample using isothermal nucleic acid amplification.
1. A composition comprising a forward template comprising a nucleic acid sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of a Respiratory Syncytial Virus (RSV) nonstructural NS2 gene antisense strand; a nicking enzyme binding site and a nicking site upstream of the recognition region; and a stabilizing region upstream of the nicking site, wherein the nucleic acid sequence of the forward template comprises a nucleotide sequence having at least 80%, at least 85%, or at least 95% sequence identity to SEQ ID NO: 1 (CGACTCACACGAGTCGAAAACTTGATGAAAGA); and
a reverse template comprising a nucleic acid sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of a RSV nonstructural NS2 gene sense strand; a nicking enzyme binding site and a nicking site upstream of the recognition region; and a stabilizing region upstream of the nicking site, wherein the nucleic acid sequence of the reverse template comprises a nucleotide sequence having at least 80%, at least 85%, or at least 95% sequence identity to SEQ ID NO: 2 (AGACTCCACACGGAGTCTAGTTGACCAGGAATG).
2. The composition of claim 1 , wherein the composition further comprises a probe oligonucleotide conjugated to a detectable label, wherein said probe oligonucleotide comprises a nucleotide sequence complementary to the RSV nonstructural NS2 gene nucleotide sequence.
3. The composition of claim 2 , wherein the detectable label of the probe oligonucleotide 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 any combination thereof.
4. The composition of claim 1 , wherein the composition further comprises a probe oligonucleotide comprising a nucleotide sequence having at least 80%, at least 85%, or at least 95% sequence identity to SEQ ID NO: 3 (ACCAGGAATGTAAATGTGGCCTGGT).
5. 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.
6. The composition of claim 5 , wherein the DNA polymerase is selected from the group consisting of Geobacillus bogazici DNA polymerase, Bst (large fragment), exo-DNA Polymerase, and Manta 1.0 DNA Polymerase.
7. The composition of claim 5 , wherein the one or more nicking enzymes is/are independently selected from the group consisting of Nt.BspQI, Nb.BbvCi, Nb.Bsml, Nb.BsrDI, Nb.Btsl, Nt.Alwl, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Bpu10l, Nt.Bpu10l, and N.BspD61.
8. The composition of claim 1 , wherein the forward template and/or the reverse template comprises one or more modified nucleotides, spacers, or blocking groups.
9. A kit comprising:
a pair of template oligonucleotides selected from the group consisting of:
(a) a first pair of template oligonucleotides comprising a first forward template comprising a nucleotide sequence having at least 80%, at least 85%, or at least 95% sequence identity to SEQ ID NO: 1; and
a first reverse template comprising a nucleotide sequence having at least 80%, at least 85%, or at least 95% sequence identity to SEQ ID NO 2; and
(b) a second pair of template oligonucleotides comprising a second forward template comprising a nucleotide sequence having at least 80%, at least 85%, or at least 95% sequence identity to SEQ ID NO: 6; and
a second reverse template comprising a nucleotide sequence having at least 80%, at least 85%, or at least 95% sequence identity to SEQ ID NO: 7.
10. A composition comprising a forward template comprising a nucleic acid sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of a RSV nucleocapsid gene N antisense strand; a nicking enzyme binding site and a nicking site upstream of the recognition region;
and a stabilizing region upstream of the nicking site, wherein the nucleic acid sequence of the forward template comprises a nucleotide sequence having at least 80%, at least 85%, or at least 95% sequence identity to SEQ ID NO: 6 (GACTCGCACACGAGTCACGTAGTACAGGAGATAA); and
a reverse template comprising a nucleic acid sequence comprising a recognition region at the 3′ end that is complementary to the 3′ end of a RSV nucleocapsid gene N sense strand; a nicking enzyme binding site and a nicking site upstream of the recognition region; and a stabilizing region upstream of the nicking site, wherein the nucleic acid sequence of the reverse template comprises a nucleotide sequence having at least 80%, at least 85%, or at least 95% sequence identity to SEQ ID NO 7: (GACTCCACACGGAGTCGCTTTTGCACATCATAA).
11. The composition of claim 10 , wherein the composition further comprises a probe oligonucleotide conjugated to a detectable label, wherein said probe oligonucleotide comprises a nucleotide sequence complementary to the RSV nucleocapsid gene N nucleotide sequence.
12. The composition of claim 11 , wherein the detectable label of the probe oligonucleotide 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 any combination thereof.
13. The composition of claim 10 , wherein the composition further comprises a probe oligonucleotide comprising a nucleotide sequence having at least 80%, at least 85%, or at least 95% sequence identity to SEQ ID NO: 8 (TGACACATCATAATTGGGAGTGTCA).
14. The composition of claim 10 , further comprising one or more of a DNA polymerase, one or more nicking enzymes, dNTPs, or a mixture of dNTPs and ddNTPs.
15. The composition of claim 14 , wherein the DNA polymerase is selected from the group consisting of Geobacillus bogazici DNA polymerase, Bst (large fragment), exo-DNA Polymerase, and Manta 1.0 DNA Polymerase.
16. The composition of claim 14 , wherein the one or more nicking enzymes is/are independently selected from the group consisting of Nt.BspQI, Nb.BbvCi, Nb.Bsml, Nb.BsrDI, Nb.Btsl, Nt.Alwl, Nt.BbvCI, Nt.BstNBI, Nt.CviPII, Nb.Bpu10l, Nt.Bpu10l, and N.BspD61.
17. The composition of claim 10 , wherein the forward template and/or the reverse template comprises one or more modified nucleotides, spacers, or blocking groups.