US2015292043A1PendingUtilityA1
Nucleic acid sequences that can be used as primers and probes in the amplification and detection of all subtypes of hiv-1
Est. expiryAug 8, 2017(expired)· nominal 20-yr term from priority
C12Q 1/703C12Q 1/70C12Q 1/6848Y10S435/81
62
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Claims
Abstract
The present invention is related to nucleic acid sequences that can be used in the field of virus diagnostics, more specifically the diagnosis of infections with the AIDS causing Human Immuno-deficiency Virus (HIV). With the present invention nucleotide sequences are provided that can be used as primers and probes in the amplification and detection of HIV-1 nucleic acid. The oligonucleotide sequences provided with the present invention are located in the LTR part of the HIV viral genome. It has been found that, by using the sequences of the present invention in methods for the amplification and detection of nucleic acid a sensitive and specific detection of HIV-1 can be obtained. The benefit of the sequences of the present invention primarily resides in the fact that, with the aid of primers and probes comprising the sequences according to the invention the nucleic acid of all presently known subtypes of HIV-1 can be detected with high accuracy and sensitivity. So far no primer pairs or hybridization probes have been developed that would allow the detection of such a broad range of HIV-1 variants. The oligonucleotide sequences according to the present invention are especially useful in methods for the amplification of nucleic acid.
Claims
exact text as granted — not AI-modified1 - 20 . (canceled)
21 . A pair of oligonucleotide primers for the amplification of a target sequence between two primer binding sites located within the LTR region of the HIV-1 genome, said pair consisting of a first primer and a second primer,
wherein said first primer consists essentially of a first oligonucleotide that is fully complementary to a sequence of the LTR region at a first primer binding site and binds thereto under conditions whereby nucleic acid amplification can occur, said oligonucleotide being 15-26 nucleotides in length and comprising at least 15 sequential nucleotides of the nucleotide sequence of:
SEQ ID NO: 1:
G GGC GCC ACT GCT AGA GA;
said first oligonucleotide being operably linked to a T3, T7, or SP6 promoter; and
wherein said second primer consists essentially of a second oligonucleotide that is fully complementary to a sequence which is the reverse complement of a sequence of the LTR region at a second primer binding site and binds thereto under conditions whereby nucleic acid amplification can occur, said oligonucleotide being 10-26 nucleotides in length and comprising at least 10 sequential nucleotides of the nucleotide sequence of:
SEQ ID NO: 4:
CTG CTT AAA GCC TCA ATA AA
wherein said primers amplify the target sequence between the two primer binding sites located within the LTR region of the HIV-1 genome when contacted with HIV-1 nucleic acid under conditions whereby nucleic acid amplification can occur.
22 . The pair of oligonucleotide primers of claim 21 , wherein said first oligonucleotide comprises the nucleotide sequence of:
SEQ ID NO: 1:
G GGC GCC ACT GCT AGA GA.
23 . A primer set for the amplification of a target sequence between two primer binding sites located within the LTR region of the HIV-1 genome, said set consisting of a first primer and a second primer,
wherein said first primer consists essentially of a first hybridizing oligonucleotide that is fully complementary to a sequence of the LTR region at a first primer binding site and binds thereto under conditions whereby nucleic acid amplification can occur, said oligonucleotide being 15-26 nucleotides in length and comprising at least 15 sequential nucleotides of the nucleotide sequence of:
SEQ ID NO: 1:
G GGC GCC ACT GCT AGA GA;
said first hybridizing oligonucleotide being operably linked to a T3, T7, or SP6 promoter; and
wherein said second primer consists essentially of a second hybridizing oligonucleotide that is fully complementary to a sequence which is the reverse complement of a sequence of the LTR region at a second primer binding site and binds thereto under conditions whereby nucleic acid amplification can occur, said oligonucleotide being 10-26 nucleotides in length and comprising at least 10 sequential nucleotides of the nucleotide sequence of:
SEQ ID NO: 4:
CTG CTT AAA GCC TCA ATA AA
wherein said primers amplify the target sequence between the two primer binding sites located within the LTR region of the HIV-1 genome when contacted with HIV-1 nucleic acid under conditions whereby nucleic acid amplification can occur.
24 . The primer set of claim 23 , wherein said first oligonucleotide comprises the nucleotide sequence of:
SEQ ID NO: 1:
G GGC GCC ACT GCT AGA GA.
25 . A pair of oligonucleotide primers for the amplification of a target sequence between two primer binding sites located within the LTR region of the HIV-1 genome, said pair consisting of a first primer and a second primer,
wherein said first primer consists essentially of a first hybridizing oligonucleotide that is fully complementary to a sequence of the LTR region at a first primer binding site and binds thereto under conditions whereby nucleic acid amplification can occur, said oligonucleotide being 15-26 nucleotides in length, wherein said first hybridizing oligonucleotide comprises at least 15 sequential nucleotides of the nucleotide sequence of:
SEQ ID NO: 1:
G GGC GCC ACT GCT AGA GA;
said hybridizing oligonucleotide being operably linked to a T3, T7, or SP6 promoter; and
wherein said second primer consists essentially of a second hybridizing oligonucleotide that is fully complementary to a sequence which is the reverse complement of a sequence of the LTR region at a second primer binding site and binds thereto under conditions whereby nucleic acid amplification can occur, said oligonucleotide being 10-26 nucleotides in length, wherein said second hybridizing oligonucleotide comprises at least 10 sequential nucleotides of the nucleotide sequence of:
SEQ ID NO: 4:
CTG CTT AAA GCC TCA ATA AA
wherein said primers amplify the target sequence between the two primer binding sites located within the LTR region of the HIV-1 genome when contacted with HIV-1 nucleic acid under conditions whereby nucleic acid amplification can occur.
26 . The pair of oligonucleotide primers of claim 25 , wherein said first oligonucleotide comprises the nucleotide sequence of:
SEQ ID NO: 1:
G GGC GCC ACT GCT AGA GA.
27 . A method for amplifying HIV-1 nucleic acid in a sample, comprising:
a) contacting the sample with a pair of oligonucleotide primers that bind to a first primer binding site and a second primer binding site located within the LTR region of the HIV-1 genome; and b) performing a nucleic acid amplification under conditions wherein said oligonucleotide primers bind only to said first and second primer binding sites, thereby amplifying HIV-1 nucleic acid in the sample; wherein said pair of oligonucleotide primers consists of a first primer and a second primer, wherein said first primer consists essentially of a first oligonucleotide that is fully complementary to a sequence of the LTR region at the first primer binding site, said oligonucleotide being 15-26 nucleotides in length and comprising at least 15 sequential nucleotides of the nucleotide sequence of:
SEQ ID NO: 1:
G GGC GCC ACT GCT AGA GA;
said first oligonucleotide being operably linked to a promoter; and
wherein said second primer consists essentially of a second oligonucleotide that is fully complementary to a sequence which is the reverse complement of a sequence of the LTR region at the second primer binding site, said oligonucleotide being 10-26 nucleotides in length and comprising at least 10 sequential nucleotides of the nucleotide sequence of:
SEQ ID NO: 4:
CTG CTT AAA GCC TCA ATA AA.
28 . The method of claim 27 , wherein said first oligonucleotide comprises the nucleotide sequence of:
SEQ ID NO: 1:
G GGC GCC ACT GCT AGA GA.
29 . The method of claim 27 , wherein performing a nucleic acid amplification comprises contacting the sample with one or more probe oligonucleotides selected from:
SEQ ID NO: 6:
TCT GGT AAC TAG AGA TCC CTC
SEQ ID NO: 7:
TAG TGT GTG CCC GTC TGT
SEQ ID NO: 8:
AGT GTG TGC CCG TCT GTT
one or more of which are provided with a detectable label, under conditions where hybridization can occur; and
detecting the presence of the label in any hybrids formed between the amplified sequence and the one or more probe oligonucleotides.
30 . The method of claim 27 , wherein said promoter is a T3, T7, or SP6 promoter.
31 . A method for amplifying HIV-1 nucleic acid in a sample, comprising:
a) contacting the sample with a primer set that bind to a first primer binding site and a second primer binding site located within the LTR region of the HIV-1 genome; and b) performing a nucleic acid amplification under conditions wherein said oligonucleotide primers bind only to said first and second primer binding sites, thereby amplifying HIV-1 nucleic acid in the sample; wherein said primer set consists of a first primer and a second primer, wherein said first primer consists essentially of a first hybridizing oligonucleotide that is fully complementary to a sequence of the LTR region at the first primer binding site, said oligonucleotide being 15-26 nucleotides in length and comprising at least 15 sequential nucleotides of the nucleotide sequence of:
SEQ ID NO: 1:
G GGC GCC ACT GCT AGA GA;
said first hybridizing oligonucleotide being operably linked to a promoter; and
wherein said second primer consists essentially of a second hybridizing oligonucleotide that is fully complementary to a sequence which is the reverse complement of a sequence of the LTR region at the second primer binding site, said oligonucleotide being 10-26 nucleotides in length and comprising at least 10 sequential nucleotides of the nucleotide sequence of:
SEQ ID NO: 4:
CTG CTT AAA GCC TCA ATA AA.
32 . The method of claim 31 , wherein said first oligonucleotide comprises the nucleotide sequence of:
SEQ ID NO: 1:
G GGC GCC ACT GCT AGA GA.
33 . The method of claim 31 , wherein performing a nucleic acid amplification comprises contacting the sample with one or more probe oligonucleotides selected from:
SEQ ID NO: 6:
TCT GGT AAC TAG AGA TCC CTC
SEQ ID NO: 7:
TAG TGT GTG CCC GTC TGT
SEQ ID NO: 8:
AGT GTG TGC CCG TCT GTT
one or more of which are provided with a detectable label, under conditions where hybridization can occur; and
detecting the presence of the label in any hybrids formed between the amplified sequence and the one or more probe oligonucleotides.
34 . The method of claim 31 , wherein said promoter is a T3, T7, or SP6 promoter.
35 . A method for amplifying HIV-1 nucleic acid in a sample, comprising:
a) contacting the sample with a pair of oligonucleotide primers that bind to a first primer binding site and a second primer binding site located within the LTR region of the HIV-1 genome; and b) performing a nucleic acid amplification under conditions wherein said oligonucleotide primers bind only to said first and second primer binding sites, thereby amplifying HIV-1 nucleic acid in the sample; wherein said pair of oligonucleotide primers consists of a first primer and a second primer, wherein said first primer consists essentially of a first hybridizing oligonucleotide that is fully complementary to a sequence of the LTR region at the first primer binding site, said oligonucleotide being 15-26 nucleotides in length, wherein said first hybridizing oligonucleotide comprises at least 15 sequential nucleotides of the nucleotide sequence of:
SEQ ID NO: 1:
G GGC GCC ACT GCT AGA GA;
said hybridizing oligonucleotide being operably linked to a promoter; and
wherein said second primer consists essentially of a second hybridizing oligonucleotide that is fully complementary to a sequence which is the reverse complement of a sequence of the LTR region at the second primer binding site, said oligonucleotide being 10-26 nucleotides in length, wherein said second hybridizing oligonucleotide comprises at least 10 sequential nucleotides of the nucleotide sequence of:
SEQ ID NO: 4:
CTG CTT AAA GCC TCA ATA AA.
36 . The method of claim 35 , wherein said first oligonucleotide comprises the nucleotide sequence of:
SEQ ID NO: 1:
G GGC GCC ACT GCT AGA GA.
37 . The method of claim 35 , wherein performing a nucleic acid amplification comprises contacting the sample with one or more probe oligonucleotides selected from:
SEQ ID NO: 6:
TCT GGT AAC TAG AGA TCC CTC
SEQ ID NO: 7:
TAG TGT GTG CCC GTC TGT
SEQ ID NO: 8:
AGT GTG TGC CCG TCT GTT
one or more of which are provided with a detectable label, under conditions where hybridization can occur; and
detecting the presence of the label in any hybrids formed between the amplified sequence and the one or more probe oligonucleotides.
38 . The method of claim 35 , wherein said promoter is a T3, T7, or SP6 promoter.Join the waitlist — get patent alerts
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