Oligonucleotide for detection of HIV-1 and detection method

Abstract

An oligonucleotide useful for detection of an RNA derived from HIV-1 consisting of at least 10 consecutive bases in any of SEQ ID NOS:1, 2, 4 to 10 and 13 to 17, which can bind to a specific site of the RNA.

1 . An oligonucleotide useful for detection of an RNA derived from HIV-1 consisting of at least 10 consecutive bases in any of SEQ ID NOS:1, 2, 4 to 10 and 13 to 17, which can bind to a specific site of the RNA.  
     
     
         2 . The oligonucleotide according to  claim 1 , which is an oligonucleotide primer for DNA elongation.  
     
     
         3 . The oligonucleotide according to  claim 1 , which is an oligonucleotide probe which is partly modified or labeled with a detectable label.  
     
     
         4 . A step of amplifying an RNA derived from HIV-1, which comprises 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, 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 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 an RNA polymerase, wherein the first primer is an oligonucleotide consisting of at least 10 consecutive bases in any of SEQ ID NOS:1, 2, 4 to 10 and 13 to 17, and the second primer is an oligonucleotide consisting of at least 10 consecutive bases in any of SEQ ID NOS: 19 to 27.  
     
     
         5 . The step according to  claim 4 , wherein the combination of the first primer and the second primer is any of the following combinations: 
 (1) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 5 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 19 as the second primer,    (2) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 5 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 20 as the second primer,    (3) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 6 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 20 as the second primer,    (4) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 5 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 21 as the second primer,    (5) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 6 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 21 as the second primer,    (6) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 8 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 22 as the second primer,    (7) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 9 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 22 as the second primer,    (8) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 10 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 22 as the second primer,    (9) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 8 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 23 as the second primer,    (10) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 9 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 23 as the second primer,    (11) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 10 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 23 as the second primer,    (12) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 9 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 24 as the second primer,    (13) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 10 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 24 as the second primer,    (14) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 11 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 24 as the second primer,    (15) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 13 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 25 as the second primer,    (16) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 17 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 26 as the second primer,    (17) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 18 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 26 as the second primer,    (18) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 16 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 27 as the second primer,    (19) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 17 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 27 as the second primer, and    (20) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 18 as the fist primer and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 27 as the second primer.    
     
     
         6 . The step according to  claim 4 , which further comprises adding 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 rebonuclease H), wherein the combination of the first primer, the second primer and the third primer is any of the following combinations: 
 (1) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 5 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 19 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 2 as the third primer,    (2) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 5 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 20 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 3 as the third primer,    (3) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 6 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 20 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 3 as the third primer,    (4) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 5 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 21 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 4 as the third primer,    (5) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 6 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 21 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 4 as the third primer,    (6) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 8 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 22 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 5 as the third primer,    (7) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 9 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 22 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 5 as the third primer,    (8) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 10 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 22 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 5 as the third primer,    (9) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 8 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 23 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 6 as the third primer,    (10) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 9 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 23 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 6 as the third primer,    (11) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 10 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 23 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 6 as the third primer,    (12) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 9 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 24 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 7 as the third primer,    (13) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 10 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 24 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 7 as the third primer,    (14) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 11 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 24 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 7 as the third primer,    (15) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 13 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 25 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 11 as the third primer,    (16) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 17 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 26 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 13 as the third primer,    (17) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 18 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 26 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 13 as the third primer,    (18) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 16 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 27 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 14 as the third primer,    (19) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 17 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 27 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 14 as the third primer, and    (19) the combination of an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 18 as the fist primer, an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 27 as the second primer, and an oligonucleotide consisting of at least 10 consecutive bases in SEQ ID NO: 14 as the third primer.    
     
     
         7 . A step of detecting HIV-1, which comprises conducting a step as defined in any of  claims 4  to  6  in the presence of an oligonucleotide probe (having a sequence different from those of the first primer and the second primer) which can specifically bind to the RNA transcript resulting from the amplification and is labeled with an fluorescent intercalative dye, and measuring the change in the fluorescence from the reaction solution.  
     
     
         8 . The step according to  claim 7 , wherein the oligonucleotide probe is designed to hybridize with at least part of the RNA transcript and alters its fluorescence upon hybridization.