Nucleic acid sequences that can be used as primers and probes in the amplification and detection of all subtypes of HIV-1

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.

1 - 13 . (canceled)  
     
     
         14 . A pair of oligonucleotide primers, for use as a primer set in the amplification of a target sequence located within the LTR region of the genome of HIV-1, said primer pair consisting of: 
 a first oligonucleotide being 15-50 nucleotides in length and comprising:    a) at least 15 sequential nucleotides of a nucleotide sequence selected from the group consisting of: 
 SEQ ID NO: 1: G GGC GCC ACT GCT AGA GA;  
 SEQ ID NO: 2: G TTC GGG CGC CAC TGC TAG A; and  
 SEQ ID NO: 3: CGG GCG CCA CTG CTA; and  
   b) a T7 promoter sequence operably associated with the nucleotide sequence of (a), and    a second oligonucleotide being 15-50 nucleotides in length and comprising at least 15 sequential nucleotides of the nucleotide sequence of SEQ ID NO:5: CTC AAT AAA GCT TGC CTT GA.    
     
     
         15 . A method for the amplification of HIV-1 nucleic acid in a sample in an isothermal continuous transcription based nucleic acid amplification reaction, comprising contacting the sample with: 
 a) the primer pair of  claim 14;  and    b) a T7 RNA polymerase.    
     
     
         16 . A method for detection of HIV-1 nucleic acid in a sample in an isothermal continuous transcription based nucleic acid amplification reaction, comprising: 
 a) contacting the sample with: 
 i) the primer pair of  claim 14;  and  
 ii) a T7 RNA polymerase,  
   under conditions whereby the isothermal continuous transcription based nucleic acid amplification reaction can occur to amplify HIV-1 nucleic acid; and    b) detecting the presence of amplified HIV-1 nucleic acid, thereby detecting HIV-1 nucleic acid in the sample.    
     
     
         17 . A method for the detection of HIV-1 nucleic acid in a sample in a transcription based amplification system reaction, comprising: 
 a) contacting the sample with a pair of oligonucleotide primers, said primer pair consisting of:    a first oligonucleotide being 15-50 nucleotides in length and comprising: 
 i) at least 15 sequence nucleotides of a nucleotide sequence selected from the group consisting of: 
 SEQ ID NO: 1: G GGC GCC ACT GCT AGA GA;  
 SEQ ID NO:2: G TTC GGG CGC CAC TGC TAG A; and  
 SEQ ID NO:3: CGG GCG CCA CTG CTA; and  
 
 ii) a DNA dependent RNA promoter operably associated with the nucleotide sequence of (i), and  
   a second oligonucleotide being 15-50 nucleotides in length and comprising at least 15 sequential nucleotides of the nucleotide sequence of SEQ ID NO:5: CTC AAT AAA GCT TGC CTT GA,    under conditions whereby the transcription based amplification system reaction can occur to amplify the HIV-1 nucleic acid; and 
 b) detecting the presence of amplified HIV-1 nucleic acid, thereby detecting HIV-1 nucleic acid in the sample.