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 hybridizing primers, for use as a hybridizing primer set in the amplification of a target sequence located within the LTR region of the genome of HIV-1, said hybridizing primer pair consisting of a first hybridizing oligonucleotide being 10-50 nucleotides in length and comprising 10 sequential nucleotides of a hybridizing 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 a second hybridizing oligonucleotide being 10-50 nucleotides in length and comprising 10 sequential nucleotides of a hybridizing nucleotide sequence selected from the group consisting of:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   SEQ ID NO:4: 
                     
                 
                     
                   CTG CTT AAA GCC TCA ATA AA; 
                 
                     
                     
                 
                     
                   SEQ ID NO:5: 
                 
                     
                   CTC AAT AAA GCT TGC CTT GA; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   SEQ ID NO:12: 
                 
                     
                   GAT GCA TGC TCA ATA AAG CTT GCC TTG AGT. 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         15 . A pair of hybridizing primers, for use as a hybridizing primer set in the amplification of a target sequence located within the LTR region of the genome of HIV-1, said hybridizing primer pair consisting of a first hybridizing oligonucleotide being 10-26 nucleotides in length and comprising 10 sequential nucleotides of a hybridizing 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 a second hybridizing oligonucleotide being 10-26 nucleotides in length and comprising 10 sequential nucleotides of a hybridizing nucleotide sequence selected from the group consisting of:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   SEQ ID NO:4: 
                     
                 
                     
                   CTG CTT AAA GCC TCA ATA AA; 
                 
                     
                     
                 
                     
                   SEQ ID NO:5: 
                 
                     
                   CTC AAT AAA GCT TGC CTT GA; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   SEQ ID NO:12: 
                 
                     
                   GAT GCA TGC TCA ATA AAG CTT GCC TTG AGT. 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         16 . The pair of hybridizing primers of  claim 14 , wherein the first hybridizing oligonucleotide comprises at least 10 sequential nucleotides of the hybridizing nucleotide sequence of SEQ ID NO: 1: G GGC GCC ACT GCT AGA GA; and wherein the second hybridizing oligonucleotide comprises at least 10 sequential nucleotides of the hybridizing nucleotide sequence of SEQ ID NO:5: CTC AAT AAA GCT TGC CTT GA.  
     
     
         17 . The pair of hybridizing primers of  claim 14 , wherein the first hybridizing oligonucleotide comprises at least 10 sequential nucleotides of the hybridizing nucleotide sequence of SEQ ID NO: 1: G GGC GCC ACT GCT AGA GA; and wherein the second hybridizing oligonucleotide comprises at least 10 sequential nucleotides of the hybridizing nucleotide sequence of SEQ ID NO:5: CTC AAT AAA GCT TGC CTT GA.  
     
     
         18 . The pair of hybridizing primers of  claim 14 ,  15 ,  16  or  17 , in combination with a promoter sequence recognized by a DNA dependent RNA polymerase, wherein the first hybridizing oligonucleotide is operably associated with the promoter sequence recognized by a DNA dependent RNA polymerase.  
     
     
         19 . The pair of hybridizing primers of  claim 18 , wherein the promoter sequence is a T7 promoter sequence.  
     
     
         20 . The pair of hybridizing primers of  claim 19 , wherein the first hybridizing oligonucleotide is operably associated with the promoter sequence recognized by a DNA dependent RNA polymerase and consists essentially of the nucleotide sequence of SEQ ID NO:9: aat tct aat acg act cac tat agg gAG AGG GGC GCC ACT GCT AGA GA and wherein the second hybridizing oligonucleotide consists essentially of the hybridizing nucleotide sequence of SEQ ID NO:5: CTC AAT AAA GCT TGC CTT GA.  
     
     
         21 . A method for the detection of HIV-1 nucleic acid in a sample, comprising: 
 a) contacting the sample with the pair of hybridizing primers of any of claims  14 - 20  under conditions whereby an amplification reaction can occur; and    b) detecting the presence of amplified HIV-1 nucleic acid, thereby detecting HIV-1 nucleic acid in the sample.    
     
     
         22 . The method of  claim 21 , wherein the detection of amplified nucleic acid is carried out by reacting the sample with one or more oligonucleotides selected from the group consisting of:  
       
         
           
                 
                 
                 
                 
               
                     
                     
                 
                     
                   SEQ ID NO:6: 
                   TCT GGT AAC TAG AGA TCC CTC; 
                     
                 
                     
                     
                 
                     
                   SEQ ID NO:7: 
                   TAG TGT GTG CCC GTC TGT; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   SEQ ID NO:8: 
                   AGT GTG TGC CCG TCT GTT, 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
               
            
           
         
       
       under conditions whereby a hybridization complex can form, and detecting the presence of the hybridization complex, thereby detecting HIV-1 nucleic acid in the sample.  
     
     
         23 . A method for amplifying a target sequence located within the LTR region of the genome of HIV-1, comprising contacting the sample with the pair of oligonucleotide primers of any of claims  14 - 20  under conditions whereby an amplification reaction can occur.  
     
     
         24 . The method of  claim 20  or  22 , wherein the amplification reaction is a transcription based amplification system reaction.  
     
     
         25 . The method of  claim 24 , wherein the transcription based amplification system reaction is a nucleic acid based amplification reaction.  
     
     
         26 . A test kit for the detection of HIV-1 in a sample, comprising: 
 a) the pair of hybridizing primers of any of claims  14 - 20 ;    b) one or more detectably labeled oligonucleotides comprising a nucleic acid sequence substantially complementary to at least part of a target sequence produced in an amplification reaction comprising the pair of oligonucleotides of (a); and    c) suitable amplification reagents.