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 . A pair of oligonucleotide primers, for use as a single 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 10-50 nucleotides in length and comprising at least a fragment of 10 sequential nucleotides of a sequence selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   SEQ ID 1:  
                 
                     
                   G GGC GCC ACT GCT AGA GA; 
                 
                     
                     
                 
                     
                   SEQ ID 2:  
                 
                     
                   G TTC GGG CGC CAC TGC TAG A;  
                 
                     
                   and 
                 
                     
                     
                 
                     
                   SEQ ID 3:  
                 
                     
                   CGG GCG CCA CTG CTA; 
                 
             
                
                
                
                
                
                
                
                
                
               
            
           
         
       
       and a second oligonucleotide being 10-50 nucleotides in length and comprising at least a fragment of 10 sequential nucleotides of a sequence selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   SEQ ID 4:  
                 
                     
                   CTG CTT AAA GCC TCA ATA AA;  
                 
                     
                   and 
                 
                     
                     
                 
                     
                   SEQ ID 5:  
                 
                     
                   CTC AAT AAA GCT TGC CTT GA. 
                 
             
                
                
                
                
                
                
               
            
           
         
       
     
     
         2 . The pair of oligonucleotides of  claim 1 , consisting of a first oligonucleotide being 10-50 nucleotides in length and comprising at least a fragment of 10 sequential nucleotides of the sequence: SEQ ID 1: G GGC GCC ACT GCT AGA GA; and a second oligonucleotide being 10-50 nucleotides in length and comprising at least a fragment of 10 sequential nucleotides of the sequence SEQ ID 5: CTC AAT AAA GCT TGC CTT GA. 
     
     
         3 . The pair of oligonucleotides of  claim 1 , wherein the first oligonucleotide is operably linked to the T7 promoter sequence. 
     
     
         4 . The pair of oligonucleotides of  claim 3 , consisting of a first oligonucleotide comprising the sequence of SEQ ID 9: aat tct aat acg act cac tat agg gAG AGG GGC GCC ACT GCT AGA GA and a second oligonucleotide comprising the sequence of SEQ ID 5: CTC AAT AAA GCT TGC CTT GA. 
     
     
         5 . The pair of oligonucleotides of  claim 1 , wherein the first oligonucleotide is 10-26 nucleotides in length. 
     
     
         6 . The pair of oligonucleotides of  claim 1 , wherein the second oligonucleotide is 10-26 nucleotides in length. 
     
     
         7 . The pair of oligonucleotides of  claim 1 , wherein the first oligonucleotide is 15-50 nucleotides in length and comprises at least a fragment of 15 sequential nucleotides of a sequence selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   SEQ ID 1:  
                 
                     
                   G GGC GCC ACT GCT AGA GA; 
                 
                     
                     
                 
                     
                   SEQ ID 2:  
                 
                     
                   G TTC GGG CGC CAC TGC TAG A;  
                 
                     
                   and 
                 
                     
                     
                 
                     
                   SEQ ID 3:  
                 
                     
                   CGG GCG CCA CTG CTA. 
                 
             
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         8 . The pair of oligonucleotides of  claim 1 , wherein the second oligonucleotide is 15-50 nucleotides in length and comprises at least a fragment of 15 sequential nucleotides of a sequence selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   SEQ ID 4:  
                 
                     
                   CTG CTT AAA GCC TCA ATA AA; 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   SEQ ID 5:  
                 
                     
                   CTC AAT AAA GCT TGC CTT GA. 
                 
             
                
                
                
                
                
                
               
            
           
         
       
     
     
         9 . A method for the detection of HIV-1 nucleic acid in a sample, comprising:
 a) contacting the sample with the pair of oligonucleotides of  claim 1  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.   
     
     
         10 . The method of  claim 9 , wherein the amplified HIV-1 nucleic acid is detected by contacting products of the amplification reaction of step (a) with one or more oligonucleotide probes having a sequence selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   SEQ ID 6:  
                 
                     
                   TCT GGT AAC TAG AGA TCC CTC; 
                 
                     
                     
                 
                     
                   SEQ ID 7:  
                 
                     
                   TAG TGT GTG CCC GTC TGT;  
                 
                     
                   and 
                 
                     
                     
                 
                     
                   SEQ ID 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. 
     
     
         11 . A test kit for the detection of HIV-1 in a sample comprising:
 a) the pair of oligonucleotides of  claim 1 ; and   b) one or more detectably labeled oligonucleotides comprising a nucleic acid sequence substantially complementary to at least part of the target sequence produced in an amplification reaction comprising the pair of oligonucleotides of (a).   
     
     
         12 . The test kit of  claim 11 , wherein the one or more detectably labeled oligonucleotides is selected from the group consisting of: 
       
         
           
                 
                 
               
                     
                   SEQ ID 6:  
                 
                     
                   TCT GGT AAC TAG AGA TCC CTC; 
                 
                     
                     
                 
                     
                   SEQ ID 7:  
                 
                     
                   TAG TGT GTG CCC GTC TGT;  
                 
                     
                   and 
                 
                     
                     
                 
                     
                   SEQ ID 8:  
                 
                     
                   AGT GTG TGC CCG TCT GTT. 
                 
             
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         13 . A method for amplifying HIV-1 nucleic acid in a sample, comprising contacting the sample with the pair of oligonucleotides of  claim 1  under conditions whereby an amplification reaction can occur. 
     
     
         14 . A method for the detection of HIV-1 nucleic acid in a sample, comprising:
 a) contacting the sample with the pair of oligonucleotides of  claim 2  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.   
     
     
         15 . A method for the detection of HIV-1 nucleic acid in a sample, comprising:
 a) contacting the sample with the pair of oligonucleotides of  claim 3  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.   
     
     
         16 . A method for the detection of HIV-1 nucleic acid in a sample, comprising:
 a) contacting the sample with the pair of oligonucleotides of  claim 4  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.   
     
     
         17 . A method for the detection of HIV-1 nucleic acid in a sample, comprising:
 a) contacting the sample with the pair of oligonucleotides of  claim 5  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.   
     
     
         18 . A method for the detection of HIV-1 nucleic acid in a sample, comprising:
 a) contacting the sample with the pair of oligonucleotides of  claim 6  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.   
     
     
         19 . A method for the detection of HIV-1 nucleic acid in a sample, comprising:
 a) contacting the sample with the pair of oligonucleotides of  claim 7  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.   
     
     
         20 . A method for the detection of HIV-1 nucleic acid in a sample, comprising:
 a) contacting the sample with the pair of oligonucleotides of  claim 8  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.