Method of amplifying or detecting HIV-1 RNA

Abstract

A simple, speedy and sensitive method of detecting HIV-RNA using an oligonucleotide which can bind to an intramolecularly free region of the genomic RNA of HIV-1 at relatively low and constant temperatures (at 35° C. to 50° C., preferably at 41° C.) as an oligonucleotide primer for use in amplification of a nucleic acid.

1 - 5 . (canceled)  
     
     
         6 . A method for amplifying HIV-1 RNA in a sample comprising: contacting a sample containing HIV-1 RNA with a set of primers under conditions suitable transcription and amplification of HIV-1 nucleic acid, 
 wherein said set of primers comprises at least one primer selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7 and at least one primer selected from the group consisting of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.    
     
     
         7 . The method of  claim 6 , wherein one primer in said set further comprises a promoter for RNA polymerase at the 5′ end.  
     
     
         8 . The method of  claim 6 , comprising 
 synthesizing a cDNA by the action of an RNA-dependent DNA polymerase by using a specific sequence in an RNA derived from HIV-1 anticipated in a sample as a template, and using a first primer containing a sequence complementary to the specific sequence and a second primer containing a sequence homologous to the specific sequence (either of which additionally has a promoter sequence for the RNA polymerase at the 5′ end),    denuding the cDNA to a single-stranded DNA through degradation of the RNA in the resulting RNA-DNA double strand by ribonuclease H,    forming a double-stranded DNA having a promoter sequence which can be transcribed into an RNA consisting of the specific base sequence or a sequence complementary to the specific base sequence by using the single-stranded DNA as a template by the action of a DNA-dependent DNA polymerase, and    then transcribing the double-stranded DNA into an RNA transcript, which acts as a template in the subsequent cDNA synthesis by the RNA-dependent DNA polymerase, in the presence of the RNA polymerase.    
     
     
         9 . The method of  claim 6 , wherein said first primer is SEQ ID NO: 2 and said second primer is SEQ ID NO: 13.  
     
     
         10 . The method of  claim 6 , further comprising using a third oligonucleotide which is complementary to a region of the RNA derived from HIV-1 which flanks the 5′ end of the specific sequence with an overlap (of from 1 to 10 bases) with the specific sequence to form a template used in the initial stage of the amplification by cutting the RNA derived from HIV-1 at the 5′ end of the specific sequence (by the action of the ribonuclease H).  
     
     
         11 . The method of  claim 6 , wherein transcription and amplification comprise the use of T7 phage RNA polymerase and AMV reverse transcriptase.  
     
     
         12 . A method for detecting HIV-1 RNA in a sample comprising: 
 contacting a sample suspected of containing HIV-1 RNA with a set of primers under conditions suitable transcription and amplification of HIV-1 nucleic acid, and    detecting the presence of amplified HIV-1 nucleic acids,    wherein said set of primers comprises at least one primer selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7 and at least one primer selected from the group consisting of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.    
     
     
         13 . The method of  claim 12 , wherein one primer in said set further comprises a promoter for RNA polymerase at the 5′ end.  
     
     
         14 . The method of  claim 12 , comprising: 
 synthesizing a cDNA by the action of an RNA-dependent DNA polymerase by using a specific sequence in an RNA derived from HIV-1 anticipated in a sample as a template, and using a first primer containing a sequence complementary to the specific sequence and a second primer containing a sequence homologous to the specific sequence (either of which additionally has a promoter sequence for the RNA polymerase at the 5′ end),    denuding the cDNA to a single-stranded DNA through degradation of the RNA in the resulting RNA-DNA double strand by ribonuclease H,    forming a double-stranded DNA having a promoter sequence which can be transcribed into an RNA consisting of the specific base sequence or a sequence complementary to the specific base sequence by using the single-stranded DNA as a template by the action of a DNA-dependent DNA polymerase, and    then transcribing the double-stranded DNA into an RNA transcript, which acts as a template in the subsequent cDNA synthesis by the RNA-dependent DNA polymerase, in the presence of the RNA polymerase.    
     
     
         15 . The method of  claim 12 , wherein said first primer is SEQ ID NO: 2 and said second primer is SEQ ID NO: 13.  
     
     
         16 . The method of  claim 12 , further comprising using a third oligonucleotide which is complementary to a region of the RNA derived from HIV-1 which flanks the 5′ end of the specific sequence with an overlap (of from 1 to 10 bases) with the specific sequence to form a template used in the initial stage of the amplification by cutting the RNA derived from HIV-1 at the 5′ end of the specific sequence (by the action of the ribonuclease H).  
     
     
         17 . The method of  claim 12 , wherein transcription and amplification comprise the use of T7 phage RNA polymerase and AMV reverse transcriptase.  
     
     
         18 . The method of  claim 12 , comprising detecting the presence of amplified HIV-1 nucleic acid by contacting said sample with an oligonucleotide probe and measuring the change in the fluorescence from the reaction solution, 
 wherein said probe is labeled with a fluorescent intercalative dye, has a sequence different than the first or second primer, and can bind to an HIV-1 transcript resulting from said amplification.    
     
     
         19 . The method of  claim 18 , wherein the oligonucleotide probe hybridizes with at least part of the RNA transcript and alters its fluorescence upon hybridization.  
     
     
         20 . The method of  claim 18 , wherein the oligonucleotide probe consists of at least 10 consecutive bases of SEQ ID NO: 34 or consists of at least 10 consecutive bases of the full complement of SEQ ID NO: 34.