Methods and reagents for molecular detection of HIV-1 groups M, N and O

Abstract

Reagents and assays for detecting HIV-1 groups M and O and optionally HIV-1 group N and SIVcpz are provided. The reagents are nucleic acid primers for the hybridization to, amplification and subsequent detection of HIV-1 groups M, N and O and SIVcpz in a biological sample. The primers are oligonucleotides that selectively hybridize to the highly conserved regions of the env and pol regions of HIV-1. The assays employ the primers for HIV-1 nucleic acid amplification using amplification techniques such as reverse transcription and the polymerase chain reaction. The assays are useful for diagnosing an HIV-1 group M, HIV-1 group N, or an HIV-1 group O infection in a patient. Due to the high sensitivity of the assays, small concentrations of HIV in a biological sample can be detected, thereby allowing diagnosis at an early stage of infection. The assays are also useful for detecting HIV-1 contamination in a biological fluid such as blood. The assays are qualitative or quantitative and are therefore useful for viral load determinations of HIV- 1 groups M, N or O in a patient undergoing treatment for HIV-1 infection. Viral load determinations can be used to monitor the progress of the treatment regimen or the development of drug resistance and can be used to predict disease progression.

1 . A nucleic acid primer comprising an oligonucleotide that selectively hybridizes to a highly conserved region of a nucleic acid molecule of HIV-1 between nucleotide positions 4550 and 5126 or 7746 and 8459 of the HXB2 strain, wherein the oligonucleotide does not selectively hybridize to a region of the HXB2 strain between nucleotide positions 4754-4984.  
     
     
         2 . The primer of  claim 1  wherein the oligonucleotide hybridizes to a region of the env gene of HIV-1 between nucleotide positions selected from the group consisting of 7746 to 7772; 7817 to 7844; 8220 to 8258; 8432 to 8459; 7789 to 7816; 8347 to 8374; 7850 to 7879; 8265 to 8294; and 8281 to 8310.  
     
     
         3 . The primer of  claim 1  wherein the oligonucleotide hybridizes to a region of the pol gene of HIV-1 between nucleotide positions selected from the group consisting of 4550 to 4625; 4626 to 4753; 4985 to 5126; 4596 to 4625; 4724 to 4753; and 5051 to 5080.  
     
     
         4 . The primer of  claim 1  having a length of 18-40 nucleotides.  
     
     
         5 . The primer of  claim 1  wherein the oligonucleotide has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7and SEQ ID NO:8.  
     
     
         6 . A method of detecting HIV-1 groups M, N and O and SIVcpz in a biological sample comprising 
 combining the sample with a first primer under selective hybridization conditions for the selective hybridization of the first primer HIV-1 nucleic acids in the sample, wherein the first primer comprises an oligonucleotide that selectively hybridizes to a highly conserved region of the nucleic acid molecule of HIV-1 between nucleotide positions 4550 and 5126 or 7746 and 8459 of the HXB2 strain, wherein the oligonucleotide does not selectively hybridize to a region of the HXB2 strain between nucleotide positions 4754-4984; and    detecting hybridization of the primer HIV-1 nucleic acids,    wherein detection of hybridization of the primer HIV-1 nucleic acids indicates the presence of HIV-1 in the sample.    
     
     
         7 . The method of  claim 6  wherein the first primer hybridizes to a first region of the env gene of HIV-1 between nucleotide positions selected from the group consisting of HIV-1 between nucleotide positions selected from the group consisting of 7746 to 7772; 7817 to 7844; 8220 to 8258; 8432 to 8459; 7789 to 7816; 8347 to 8374; 7850 to 7879; 8265 to 8294; and 8281 to 8310.  
     
     
         8 . The method of  claim 6  wherein the first primer hybridizes to a first region of the pol gene of HIV-1 between nucleotide positions selected from the group consisting 4550 to 4625; 4626 to 4753; 4596 to 4625; 4724 to 4753; and and 8265 to 8294.  
     
     
         9 . The method of  claim 6  wherein the oligonucleotide has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7and SEQ ID NO:8.  
     
     
         10 . The method of  claim 6  further comprising the step of combining the sample with a second primer under selective hybridization conditions for the selective hybridization of the second primer HIV-1 nucleic acids in the sample, wherein the second primer comprises an oligonucleotide that selectively hybridizes to a second highly conserved region of the nucleic acid molecule of HIV-1 between nucleotide positions 4550 and 5126 or 7746 and 8459 of the HXB2 strain wherein the oligonucleotide does not selectively hybridize to a region of the HXB2 strain between nucleotide positions 4754-4984, wherein the first and second primers are a first primer pair, and wherein the primer pair and the sample are incubated under nucleic acid amplification conditions to amplify HIV-1 nucleic acids in the sample.  
     
     
         11 . The method of  claim 10  wherein the second primer hybridizes to a second region of the env gene of HIV-1 between nucleotide positions selected from the group consisting of 7746 to 7772; 7817 to 7844; 8220 to 8258; 8432 to 8459; 7789 to 7816; 8347 to 8374; 7850 to 7879; 8265 to 8294; and 8281 to 8310.  
     
     
         12 . The method of  claim 10  wherein the second primer hybridizes to a second region of the pol gene of HIV-1 between nucleotide positions selected from the group consisting of 4550 to 4625; 4626 to 4753; 4985-5126, 4596 to 4625; 4724 to 4753; and 5051 to 5080.  
     
     
         13 . The method of  claim 10  wherein the second primer has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.  
     
     
         14 . The method of  claim 10  wherein the first primer pair comprises a forward primer and a reverse primer, wherein the forward primer comprises the nucleotide sequence of SEQ ID NO: 1 and the reverse primer comprises the nucleotide sequence of SEQ ID NO:2, or wherein the forward primer comprises the nucleotide sequence of SEQ ID NO:6 and the reverse primer comprises the nucleotide sequence of SEQ ID NO:7.  
     
     
         15 . The method of  claim 10  further comprising a second primer pair, wherein the first and second primer pairs are nested.  
     
     
         16 . The method of  claim 15  wherein the second primer pair comprises a forward primer and a reverse primer, wherein the forward primer comprises the nucleotide sequence of SEQ ID NO:3 and the reverse primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:5, or wherein the forward primer comprises the nucleotide sequence of SEQ ID NO:8 and the reverse primer comprises the nucleotide sequence of SEQ ID NO:9.  
     
     
         17 . A method for detecting HIV-1 group N or chimpanzee SIV in a biological sample comprising 
 combining the sample with a first primer under selective hybridization conditions for the selective hybridization of the first primer HIV-1 or chimpanzee SIV nucleic acids in the sample, wherein the first primer comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, and    detecting hybridization of the primer HIV-1 or chimpanzee SIV nucleic acids,    wherein detection of hybridization of the primer HIV-1 or chimpanzee SIV nucleic acids indicates the presence of HIV-1 or chimpanzee SIV in the sample.    
     
     
         18 . The method of  claim 17  further comprising a second primer that hybridizes to a second highly conserved region of the nucleic acid molecule between nucleotide positions 4450 and 5126 or 7746 and 8459 of the HXB2 strain of HIV-1, wherein the oligonucleotide does not selectively hybridize to a region of the HXB2 strain between nucleotide positions 4754-4984, wherein the first and second primers are a primer pair, and wherein the primer pair and the sample are incubated under nucleic acid amplification conditions to amplify HIV-1 nucleic acids in the sample.  
     
     
         19 . The method of  claim 18  wherein the second primer has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.  
     
     
         20 . The primer of  claim 1 , wherein amplification occurs under amplification conditions comprising 30-40 cycles of heating at 93-97° C. for 30-90 seconds, at 45-57° C. for 30-90 seconds, and at 70-74° C. for 30-90 seconds.  
     
     
         21 . The method of  claim 6 , wherein amplification occurs under amplification conditions comprising 30-40 cycles of heating at 93-97° C. for 30-90 seconds, at 45-57° C. for 30-90 seconds, and at 70-74° C. for 30-90 seconds.