US 5,478,724 AGranted

Lentivirus-specific nucleotide probes and methods of use

AssigneeThe Rockefeller University

InventorsStephen S. Morse, Irwin H. Gelman, Hidesaburo Hanafusa

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Abstract

Nucleic acid oligonucleotide probes or primers that hybridize specifically and exclusively to two regions of the pol gene, identified as LV I and LV II, conserved among a wide range of lentiviruses, wherein the preferred amino acid sequences of the conserved regions are: PHPX.sub.1 GLX.sub.2 KX.sub.3 X.sub.3 X.sub.4 X.sub.5 T wherein X.sub.1 =A, P or G, X.sub.2 =K or A, X.sub.3 =K or R, X.sub.4 =S or R, and X.sub.5 =V or I; and WX.sub.1 GX.sub.2 X.sub.3 LX.sub.4 PX.sub.5 KWX.sub.6 wherein X.sub.1 =M, L or I; X.sub.2 =Y, F or T; X.sub.3 =E or Q; X.sub.4 =H or W; X.sub.5 =D or T; and X.sub.6 =T or K. Methods of use of the probes and primers in the detection of lentiviral genomic sequences in biological samples and the termination of replication of lentiviruses are also included.

Claims37

Dependent claims

A probe for detecting the presence of lentiviral nucleic acid comprising an oligonucleotide of at least seventeen nucleotides which hybridizes to a lentivirus nucleic acid sequence in the pol gene specific to and conserved among lentiviruses and does not hybridize to a nucleic acid sequence of retroviruses outside of the lentivirus family, selected from the group consisting of:

5'-X.sub.1 X.sub.2 NAX.sub.2 X.sub.3 X.sub.3 X.sub.4 AX.sub.3 AACCCAX.sub.5 CCA-3'
wherein X.sub.1 =T, U, C or I;
X.sub.2 =A, G or I;
N is any nucleotide;
X.sub.3 =T or U;
X.sub.4 =C, A, T, U or I;
and X.sub.5 =G, T, U or I;
3'-X.sub.1 X.sub.2 NX.sub.3 X.sub.2 AAX.sub.4 X.sub.3 AX.sub.3 X.sub.3 GGGX.sub.3 X.sub.5 GGX.sub.3 -5'
wherein X.sub.1 =A, G or I;
X.sub.2 =T, U, C or I;
N is any nucleotide;
X.sub.3 =T or U;
X.sub.4 =G, A, T, U or I;
and X.sub.5 =C, A or I;
3'-X.sub.1 X.sub.2 NAX.sub.2 X.sub.3 X.sub.3 X.sub.4 AX.sub.3 AACCCAX.sub.5 CCA-5'
wherein X.sub.1 =T, U, C or I;
X.sub.2 =A, G or I;
N is any nucleotide;
X.sub.3 =T or U;
X.sub.4 =C, A, T, U or I;
and X.sub.5 =G, T, U or I;
5'-X.sub.1 X.sub.2 NX.sub.3 X.sub.2 AAX.sub.4 X.sub.3 AX.sub.3 X.sub.3 GGGX.sub.3 X.sub.5 GGX.sub.3 -3'
wherein X.sub.1 =A, G or I;
X.sub.2 =T, U, C or I;
N is any nucleotide;
X.sub.3 =T or U;
X.sub.4 =G, A, T, U or I;
and X.sub.5 =C, A or I, and degenerate sequences thereof encoding the amino acid sequence:
WX.sub.1 GX.sub.2 X.sub.3 LX.sub.4 PX.sub.5 KWX.sub.6
wherein X.sub.1 =M, L or I;
X.sub.2 =Y, F or T;
X.sub.3 =E or Q;
X.sub.4 =H or W;
X.sub.5 =D or T;
and X.sub.6 =T or K and the sequence is designated SEQ ID NO:2 in the Sequence Listing.

32 more elements

A probe for detecting the presence of lentiviral nucleic acid comprising an oligonucleotide of at least seventeen consecutive nucleotide bases which hybridizes to a lentivirus nucleic acid sequence in the pol gene specific to and conserved among lentiviruses and does not hybridize to a nucleic acid sequence of retroviruses outside of the lentivirus family, wherein the lentivirus nucleic acid sequence is:

5'-CCX.sub.1 CAX.sub.2 CCNGX.sub.3 AGGAX.sub.2 X.sub.4 AX.sub.5 AA-3'
wherein X.sub.1 =G, T, U, A or I;
X.sub.2 =T, U, C or I;
N is any nucleotide;
X.sub.3 =G, C or I;
X.sub.4 =T or U;
and X.sub.5 =C, A or I, designated SEQ ID NO:5 and SEQ ID NO:7 in the Sequence Listing;
3'-GGX.sub.1 GTX.sub.2 GGNCX.sub.3 TCCTX.sub.2 ATX.sub.4 TT-5'
wherein X.sub.1 =C, T, U, A or I;
X.sub.2 =A, G or I;
N is any nucleotide;
X.sub.3 =G, C or I;
and X.sub.4 =G, T, U or I, a sequence complementary to SEQ ID NO:5 and SEQ ID NO:7 in the Sequence Listing;
3'-CCX.sub.1 CAX.sub.2 CCNGX.sub.3 AGGAX.sub.2 X.sub.4 AX.sub.5 AA-5'
wherein X.sub.1 =G, T, U, A or I;
X.sub.2 =T, U, C or I;
N is any nucleotide;
X.sub.3 =G, C or I;
X.sub.4 =T or U;
and X.sub.5 =C, A or I designated SEQ ID NO:6 and SEQ ID NO:8 in the Sequence Listing;
and,
5'-GGX.sub.1 GTX.sub.2 GGNCX.sub.3 TCCTX.sub.2 ATX.sub.4 TT-3'
wherein X.sub.1 =C, T, U, A or I;
X.sub.2 =A, G or I;
N is any nucleotide;
X.sub.3 =G, C or I;
and X.sub.4 =G, T, U or I, a sequence complementary to SEQ ID NO:6 and SEQ ID NO:8 in the Sequence Listing.

24 more elements

A pair of degenerate oligonucleotide primers for amplification and detection of lentivirus in a biological sample comprising a first oligonucleotide which hybridizes to a nucleotide sequence conserved in the lentivirus genome identified as LV I and a second oligonucleotide which hybridizes to a nucleotide sequence conserved in the lentivirus genome identified as LV II, and neither oligonucleotide hybridizes to a nucleic acid sequence of retroviruses outside of the lentivirus family, wherein the first oligonucleotide has a nucleotide sequence selected from the group consisting of:

5'-CCX.sub.1 CAX.sub.2 CCNGX.sub.3 AGGAX.sub.2 X.sub.4 AX.sub.5 AA-3'
wherein X.sub.1 =G, T, U, A or I;
X.sub.2 =T, U, C or I;
N is any nucleotide;
X.sub.3 =G, C or I;
X.sub.4 =T or U;
and X.sub.5 =C, A or I;
designated SEQ ID NO:5 and SEQ ID NO:7 in the Sequence Listing;
3'-GGX.sub.1 GTX.sub.2 GGNCX.sub.3 TCCTX.sub.2 ATX.sub.4 TT-5'
wherein X.sub.1 =C, T, U, A or I;
X.sub.2 =A, G or I;
N is any nucleotide;
X.sub.3 =G, C or I;
and X.sub.4 =G, T, U or I;
a complementary sequence of SEQ ID NO:5 and SEQ ID NO:7;
3'-CCX.sub.1 CAX.sub.2 CCNGX.sub.3 AGGAX.sub.2 X.sub.4 AX.sub.5 AA-5'
wherein X.sub.1 =G, T, U, A or I;
X.sub.2 =T, U, C or I;
N is any nucleotide;
X.sub.3 =G, C or I;
X.sub.4 =T or U;
and X.sub.5 =C, A or I;
an inverted sequence of SEQ ID NO:5 and SEQ ID NO:7 and,
5'-GGX.sub.1 GTX.sub.2 GGNCX.sub.3 TCCTX.sub.2 ATX.sub.4 TT-3'
wherein X.sub.1 =C, T, U, A or I;
X.sub.2 =A, G or I;
N is any nucleotide;
X.sub.3 =G, C or I;
and X.sub.4 =G, T, U or I;
an inverted complementary sequence of SEQ ID NO:5 and SEQ ID NO:7;
and the second oligonucleotide has a nucleotide sequence selected from the group consisting of:
5'-X.sub.1 X.sub.2 NAX.sub.2 X.sub.3 X.sub.3 X.sub.4 AX.sub.3 AACCCAX.sub.5 CCA-3'
wherein X.sub.1 =T, U, C or I;
X.sub.2 =A, G or I;
N is any nucleotide;
X.sub.3 =T or U;
X.sub.4 =C, A, T, U or I;
and X.sub.5 =G, T, U or I;
designated SEQ ID NO:6 and SEQ ID NO:8 in the Sequence Listing;
3'-X.sub.1 X.sub.2 NX.sub.3 X.sub.2 AAX.sub.4 X.sub.3 AX.sub.3 X.sub.3 GGGX.sub.3 X.sub.5 GGX.sub.3 -5'
wherein X.sub.1 =A, G or I;
X.sub.2 =T, U, C or I;
N is any nucleotide;
X.sub.3 =T or U;
X.sub.4 =G, A, T, U or I;
and X.sub.5 =C, A or I;
a complementary sequence of SEQ ID NO:6 and SEQ ID NO:8;
and
3'-X.sub.1 X.sub.2 NAX.sub.2 X.sub.3 X.sub.3 X.sub.4 AX.sub.3 AACCCAX.sub.5 CCA-5'
wherein X.sub.1 =T, U, C or I;
X.sub.2 =A, G or I;
N is any nucleotide;
X.sub.3 =T or U;
X.sub.4 =C, A, T, U or I;
and X.sub.5 =G, T, U or I;
an inverted sequence of SEQ ID NO:6 and SEQ ID NO:8;
5'-X.sub.1 X.sub.2 NX.sub.3 X.sub.2 AAX.sub.4 X.sub.3 AX.sub.3 X.sub.3 GGGX.sub.3 X.sub.5 GGX.sub.3 -3'
wherein X.sub.1 =A, G or I;
X.sub.2 =T, U, C or I;
N is any nucleotide;
X.sub.3 =T or U;
X.sub.4 =G, A, T, U or I;
and X.sub.5 =C, A or I an inverted complementary sequence of SEQ ID NO:6 and SEQ ID NO:8, and degenerate sequences thereof wherein the conserved nucleotide sequence LV I encodes the amino acid sequence:
PHPX.sub.1 GLX.sub.2 KX.sub.3 X.sub.3 X.sub.4 X.sub.5 T
wherein X.sub.1 =A, P or G, X.sub.2 =K or A, X.sub.3 =K or R, X.sub.4 =S or R, and X.sub.5 =V or I, designated SEQ ID NO:1 in the Sequence Listing, and the conserved nucleotide sequence LV II encodes the amino acid sequence:
WX.sub.1 GX.sub.2 X.sub.3 LX.sub.4 PX.sub.5 KWX.sub.6
wherein X.sub.1 =M, L or I;
X.sub.2 =Y, F or T;
X.sub.3 =E or Q;
X.sub.4 =H or W;
X.sub.5 =D or T;
and X.sub.6 =T or K, designated SEQ ID NO:2 in the Sequence Listing, in combination with reagents for use in an amplification system.

69 more elements

A method of detecting lentiviral nucleic acid in a biological sample comprising:

combining the sample with a first oligonucleotide probe, wherein the first oligonucleotide has a nucleotide sequence selected from the group consisting of:
5'-CCX.sub.1 CAX.sub.2 CCNGX.sub.3 AGGAX.sub.2 X.sub.4 AX.sub.5 AA-3'
wherein X.sub.1 =G, T, U, A or I;
X.sub.2 =T, U, C or I;
N is any nucleotide;
X.sub.3 =G, C or I;
X.sub.4 =T or U;
and X.sub.5 =C, A or I;
designated SEQ ID NO:5 and SEQ ID NO:7 in the Sequence Listing;
3'-GGX.sub.1 GTX.sub.2 GGNCX.sub.3 TCCTX.sub.2 ATX.sub.4 TT-5'
wherein X.sub.1 =C, T, U, A or I;
X.sub.2 =A, G or I;
N is any nucleotide;
X.sub.3 =G, C or I;
and X.sub.4 =G, T, U or I;
a complementary sequence of SEQ ID NO:5 and SEQ ID NO:7;
3'-CCX.sub.1 CAX.sub.2 CCNGX.sub.3 AGGAX.sub.2 X.sub.4 AX.sub.5 AA-5'
wherein X.sub.1 =G, T, U, A or I;
X.sub.2 =T, U, C or I;
N is any nucleotide;
X.sub.3 =G, C or I;
X.sub.4 =T or U;
and X.sub.5 =C, A or I;
an inverted sequence of SEQ ID NO:5 and SEQ ID NO:7 and,
5'-GGX.sub.1 GTX.sub.2 GGNCX.sub.3 TCCTX.sub.2 ATX.sub.4 TT-3'
wherein X.sub.1 =C, T, U, A or I;
X.sub.2 =A, G or I;
N is any nucleotide;
X.sub.3 =G, C or I;
and X.sub.4 =G, T, U or I;
an inverted complementary sequence of SEQ ID NO:5 and SEQ ID NO:7;
and
a second oligonucleotide has a nucleotide sequence selected from the group consisting of:
5'- X.sub.1 X.sub.2 NAX.sub.2 X.sub.3 X.sub.3 X.sub.4 AX.sub.3 AACCCAX.sub.5 CCA-3'
wherein X.sub.1 =T, U, C or I;
X.sub.2 =A, G or I;
N is any nucleotide;
X.sub.3 =T or U;
X.sub.4 =C, A, T, U or I;
and X.sub.5 =G, T, U or I;
designated SEQ ID NO:6 and SEQ ID NO:8 in the Sequence Listing;
3'-X.sub.1 X.sub.2 NX.sub.3 X.sub.2 AAX.sub.4 X.sub.3 AX.sub.3 X.sub.3 GGGX.sub.3 X.sub.5 GGX.sub.3 -5'
wherein X.sub.1 =A, G or I;
X.sub.2 =T, U, C or I;
N is any nucleotide;
X.sub.3 =T or U;
X.sub.4 =G, A, T, U or I;
and X.sub.5 =C, A or I;
a complementary sequence of SEQ ID NO:6 and SEQ ID NO:8;
and
3'-X.sub.1 X.sub.2 NAX.sub.2 X.sub.3 X.sub.3 X.sub.4 AX.sub.3 AACCCAX.sub.5 CCA-5'
wherein X.sub.1 =T, U, C or I;
X.sub.2 =A, G or I;
N is any nucleotide;
X.sub.3 =T or U;
X.sub.4 =C, A, T, U or I;
and X.sub.5 =G, T, U or I;
an inverted sequence of SEQ ID NO:6 and SEQ ID NO:8;
5'- X.sub.1 X.sub.2 NX.sub.3 X.sub.2 AAX.sub.4 X.sub.3 AX.sub.3 X.sub.3 GGGX.sub.3 X.sub.5 GGX.sub.3 -3'
wherein X.sub.1 =A, G or I;
X.sub.2 =T, U, C or I;
N is any nucleotide;
X.sub.3 =T or U;
X.sub.4 =G, A, T, U or I;
and X.sub.5 =C, A or I an inverted complementary sequence of SEQ ID NO:6 and SEQ ID NO:8, and
degenerate sequences thereof wherein the conserved nucleotide sequence LV I encodes the amino acid sequence:
PHPX.sub.1 GLX.sub.2 KX.sub.3 X.sub.3 X.sub.4 X.sub.5 T
wherein X.sub.1 =A, P or G, X.sub.2 =K or A, X.sub.3 =K or R, X.sub.4 =S or R, and X.sub.5 =V or I, designated SEQ ID NO:1 in the Sequence Listing, and the conserved nucleotide sequence LV II encodes the amino acid sequence:
WX.sub.1 GX.sub.2 X.sub.3 LX.sub.4 PX.sub.5 KWX.sub.6
wherein X.sub.1 =M, L or I;
X.sub.2 =Y, F or T;
X.sub.3 =E or Q;
X.sub.4 =H or W;
X.sub.5 =D or T;
and X.sub.6 =T or K, designated SEQ ID NO:2in the Sequence Listing;
incubating the combination for a sufficient period of time at a temperature which promotes hybridization of the oligonucleotide probe to the lentiviral-specific nucleic acid sequence, not to a nucleic acid sequence of retroviruses outside of the lentivirus family;
and
detecting hybridization.

75 more elements

Dependencies are inferred from the claim text.