Amplification of HIV-1 sequences for detection of sequences associated with drug-resistance mutations
View Patent ↗Sequences of nucleic acid oligonucleotides for amplifying different portions of gag and pol genes of HIV-1 and for detecting such amplified nucleic acid sequences are disclosed. Methods of amplifying and detecting HIV-1 nucleic acid in a biological sample using the amplification oligonucleotides specific for gag and pol target sequences are disclosed.
1. A mixture of nucleic acid oligomers for specifically amplifying a plurality of nucleotide sequences in distinct regions of the HIV-1 genome in pol and gag gene sequences, comprising:
oligomers specific for a region in the pol sequence, referred to as a RT1 sequence, that include an oligonucleotide in a size range of 18 to 23 nucleotides that contains the sequence consisting of SEQ ID NO:14 or an oligonucleotide that contains a 5′ promoter sequence covalently attached to a sequence in a size range of 18 to 23 nucleotides that contains the sequence consisting of SEQ ID NO:14, and an oligonucleotide in a size range of 19 to 20 nucleotides that contains the sequence consisting of SEQ ID NO:64,
oligomers specific for a region in the pol sequence, referred to as a RT4 sequence, that include an oligonucleotide consisting of SEQ ID NO:16 or an oligonucleotide that contains a 5′ promoter sequence covalently attached to the sequence consisting of SEQ ID NO:16, and an oligonucleotide consisting of SEQ ID NO:22, and
oligomers specific for a region in the gag sequence, referred to as a Gag2 sequence, that include an oligonucleotide consisting of SEQ ID NO:12 or an oligonucleotide that contains a 5′ promoter sequence covalently attached to the sequence consisting of SEQ ID NO:12, and an oligonucleotide consisting of SEQ ID NO:18.
2. The mixture of nucleic acid oligomers according to claim 1 , wherein at least one oligomer comprises one or more 2′-methoxy linkages, peptide nucleic acid linkages, phosphorothioate linkages, methylphosphonate linkages or any combination of these linkages in nucleic acid backbone structure of the oligomer.
3. The mixture of nucleic acid oligomers according to claim 1 , wherein the oligonucleotide that contains a 5′ promoter sequence covalently attached to a sequence in a size range of 18 to 23 nucleotides that contains the sequence consisting of SEQ ID NO:14 is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41, which each include a 5′ promoter sequence for T7 RNA polymerase.
4. The mixture of nucleic acid oligomers according to claim 1 , wherein the oligonucleotide in a size range of 19 to 20 nucleotides that contains the sequence consisting of SEQ ID NO:64 is selected from the group consisting of SEQ ID NO:20and SEQ ID NO:64.
5. The mixture of nucleic acid oligomers according to claim 1 , wherein the oligonucleotide that contains a 5′ promoter sequence covalently attached to the sequence consisting of SEQ ID NO:12 consists of SEQ ID NO:6.
6. The mixture of nucleic acid oligomers according to claim 1 , wherein the oligonucleotide that contains a 5′ promoter sequence covalently attached to the sequence consisting of SEQ ID NO:16 consists of SEQ ID NO:10.
7. The mixture of nucleic acid oligomers according to claim 1 , wherein the mixture further comprises a labeled oligonucleotide that hybridizes specifically to a sequence amplified from the RT1 sequence consisting of SEQ ID NO:70 and a label that results in a detectable signal.
8. The mixture of nucleic acid oligomers according to claim 7 , wherein the labeled oligonucleotide includes in its nucleic acid backbone one or more 2′-methoxy linkages, peptide nucleic acid linkages, phosphorothioate linkages, methylphosphonate linkages or any combination of these linkages.
9. The mixture of nucleic acid oligomers according to claim 7 , wherein the label is a compound that produces a luminescent signal that can be detected in a homogeneous detection system.
10. The mixture of nucleic acid oligomers according to claim 9 , wherein the label is an acridinium ester (AE) compound.
11. The mixture of nucleic acid oligomers according to claim 1 , further comprising a labeled oligonucleotide that hybridizes specifically to a sequence amplified from the Gag2 sequence consisting of SEQ ID NO:69 and a label that results in a detectable signal.
12. The mixture of nucleic acid oligomers according to claim 1 , further comprising a labeled oligonucleotide that hybridizes specifically to a sequence amplified from the RT4 sequence consisting of SEQ ID NO:29 and a label that results in a detectable signal.
13. A method of detecting HIV-1 in a biological sample, comprising the steps of:
providing a biological sample containing HIV-1 nucleic acid;
mixing the sample with a mixture of amplification oligomers that specifically amplify a plurality of distinct regions in HIV-1 target sequences contained within a pol gene sequence, referred to as a RT1 sequence and a RT4 sequence, and contained in a gag sequence, referred to as a Gag2 sequence, under conditions that allow amplification of nucleic acid, wherein the amplification oligomers comprise
oligomers specific for the RT1 sequence that include an oligonucleotide in a size range of 18 to 23 nucleotides that contains the sequence consisting of SEQ ID NO:14 or an oligonucleotide that contains a 5′ promoter sequence covalently attached to a sequence in a size range of 18 to 23 nucleotides that contains the sequence consisting of SEQ ID NO:14, and an oligonucleotide in a size range of 19 to 20 nucleotides that contains the sequence consisting of SEQ ID NO:64,
oligomers specific for the RT4 sequence that include an oligonucleotide consisting of SEQ ID NO:16 or an oligonucleotide that contains a 5′ promoter sequence covalently attached to the sequence consisting of SEQ ID NO:16, and an oligonucleotide consisting of SEQ ID NO:22, and
oligomers specific for the Gag2 sequence that include an oligonucleotide consisting of SEQ ID NO:12 ; or an oligonucleotide that contains a 5′ promoter sequence covalently attached to the sequence consisting of SEQ ID NO:12, and an oligonucleotide consisting of SEQ ID NO:18 to permit amplification of the RT1, RT4 and Gag2 sequences in the HIV-1 nucleic acid to produce amplified products of the HIV-1 nucleic acid; and
detecting the presence of the amplified products of the HIV-1 nucleic acid to indicate the presence of HIV-1 in the biological sample.
14. The method of claim 13 , wherein the amplifying step uses a transcription-mediated amplification method which is conducted in substantially isothermal conditions.
15. The method of claim 13 , wherein the detecting step uses;
a labeled oligomer consisting of SEQ ID NO:70 and a label, to hybridize specifically to the amplified products of the HIV-1 nucleic acid from the RT1 sequence,
a labeled oligomer consisting of SEQ ID NO:29 and a label, to hybridize specifically to the amplified products of the HIV-1 nucleic acid from the RT4 sequence, and
a labeled oligomer consisting of SEQ ID NO:69 and a label, to hybridize specifically to the amplified products of the HIV-1 nucleic acid from the Gag2 sequence.
16. The method of claim 15 , wherein the detecting step detects hybridization of the labeled oligomer to the amplified products of the HIV-1 nucleic acid in a homogeneous detection system.
17. The method of claim 13 , wherein the detecting step detects hybridization of the amplified products of the HIV-1 nucleic acid to an array of nucleic acid probes.
18. The method of claim 13 , further comprising the step of contacting the sample containing HIV-1 nucleic acid with at least one capture oligomer having a sequence that hybridizes specifically to the HIV-1 nucleic acid, thus forming a hybridization complex that includes the HIV-1 nucleic acid and separating the hybridization complex from other sample components.
19. The method of claim 13 , wherein at least one amplification oligomer includes a nucleic acid backbone that comprises one or more 2′-methoxy linkages.
20. The method of claim 13 , wherein the oligonucleotide specific for the RT1 sequence that contains a 5′ promoter sequence covalently attached to a sequence in a size range of 18 to 23 nucleotides that contains the sequence consisting of SEQ ID NO:14 is selected from the group consisting of SEQ ID NO:8, SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41.
21. The method of claim 13 , wherein the oligonucleotide specific for the RT4 sequence that contains a 5′ promoter sequence covalently attached to the sequence consisting of SEQ ID NO:16 consists of SEQ ID NO:10, and wherein the oligonucleotide specific for the Gag2 sequence that contains a 5′ promoter sequence covalently attached to the sequence consisting of SEQ ID NO:12 consists of SEQ ID NO:6.