Primers for nucleic acid amplification in detecting housekeeping gene mrna and test method using these primers

Abstract

It is intended to provide novel primers for nucleic acid amplification to be used in detecting mRNA of a housekeeping gene, more particularly, confirming the amplification of β-actin or GAPDH. More specifically speaking, primers containing oligonucleotides having nucleotide sequences represented by any of SEQ ID NOS: 2 and 4 to 49 (in the case of β-actin) or oligonucleotides having nucleotide sequences represented by any of SEQ ID NOS: 52 to 96 (in the case of GAPDH) can be selected, combined and used.

1 . A primer for nucleic acid amplification to detect a housekeeping gene and/or housekeeping gene-related mRNA by the LAMP method.  
     
     
         2 . The primer of  claim 1 , wherein the housekeeping gene is a β-actin gene and/or a GAPDH gene.  
     
     
         3 . A primer for nucleic acid amplification to detect β-actin, which comprises an oligonucleotide having a nucleic acid sequence selected from the group consisting of: 
 1) an oligonucleotide comprising at least 5 consecutive nucleotides of the nucleotide sequence set forth as SEQ ID NO: 1 or the complementary sequence thereof which are selected from the regions of nucleotides 240-380 and 401-1060 and/or the regions complementary to these regions;    2) an oligonucleotide comprising the nucleotide sequence set forth as any one of SEQ ID NO: 2 and SEQ ID NOs: 4-34;    3) an oligonucleotide complementary to any one of the oligonucleotides defined in above 1) or 2);    4) an oligonucleotide capable of hybridizing to the oligonucleotide defined in any one of 1)-3) under stringent conditions; and    5) an oligonucleotide having the primer function, comprising the nucleotide sequence of the oligonuceotide defined in any one of above 1) to 4) in which one or more nucleotides are mutated by substitution, deletion, insertion or addition.    
     
     
         4 . A primer for nucleic acid amplification to detect β-actin, which comprises an oligonucleotide selected from the group consisting of the sequences set forth as SEQ ID NOs: 10, 14-17 and 29-50.  
     
     
         5 . A primer for nucleic acid amplification to detect GAPDH, which comprises an oligonucleotide having a nucleic acid sequence selected from the group consisting of: 
 1) an oligonucleotide comprising at least 5 consecutive nucleotides of the nucleotide sequence set forth as SEQ ID NO: 51 or the complementary sequence thereof which are selected from the region of nucleotides 110-450 and/or the region complementary to this region;    2) an oligonucleotide comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 52-79;    3) an oligonucleotide complementary to any one of the oligonucleotides defined in above 1) or 2);    4) an oligonucleotide capable of hybridizing to the oligonucleotide defined in any one of 1)-3) under stringent conditions; and    5) an oligonucleotide having the primer function, comprising the nucleotide sequence of the oligonuceotide defined in any one of above 1) to 4) in which one or more nucleotides are mutated by substitution, deletion, insertion or addition.    
     
     
         6 . A primer for nucleic acid amplification to detect GAPDH, which comprises an oligonucleotide selected from the group consisting of the sequences set forth as SEQ ID NOs: 58, 62-64 and 73-96.  
     
     
         7 . The primer for nucleic acid amplification of  claim 3 , wherein the method for nucleic acid amplification is the LAMP method.  
     
     
         8 . A primer set for nucleic acid amplification to detect β-actin, which is featured by selecting at least two primers from the primers for nucleic acid amplification that comprise oligonucleotides having nucleic acid sequences selected from the group consisting of: 
 1) an oligonucleotide comprising at least 5 consecutive nucleotides of the nucleotide sequence set forth as SEQ ID NO: 1 or the complementary sequence thereof which are selected from the region of nucleotides 240-1060 and/or the region complementary to this region;    2) an oligonucleotide comprising the nucleotide sequence set forth as any one of SEQ ID NOs: 2-34;    3) an oligonucleotide complementary to any one of the oligonucleotides defined in above 1) or 2);    4) an oligonucleotide capable of hybridizing to the oligonucleotide defined in any one of 1)-3) under stringent conditions; and    5) an oligonucleotide having the primer function, comprising the nucleotide sequence of the oligonuceotide defined in any one of above 1) to 4) in which one or more nucleotides are mutated by substitution, deletion, insertion or addition.    
     
     
         9 . A primer set for nucleic acid amplification to detect β-actin, which is featured by selecting at least four primers from the primers for nucleic acid amplification that comprise the oligonucleotides of  claim 8 .  
     
     
         10 . The primer set for nucleic acid amplification claimed in  claim 9 , which features that at least two primers contained in the primer set recognize each two gene regions in the nucleotide sequence set forth as SEQ ID NO: 1 and/or the complementary sequence thereof.  
     
     
         11 . The primer set for nucleic acid amplification claimed in  claim 9 , which features that the primers contained in the primer set recognize at least six gene regions in the nucleotide sequence set forth as SEQ ID NO: 1 and/or the complementary sequence thereof.  
     
     
         12 . A primer set comprising a combination of each one primer selected from each of (a) FIP: SEQ ID NOs: 35-42; (b) RIP: SEQ ID NOs: 43-50; (c) F3: SEQ ID NOs: 10 and 14-17 and (d) R3: SEQ ID NOs: 29-32, which categories consist of the primers for nucleic acid amplification to detect β-actin that comprise the oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 10, 14-17, 29-32, 35-42 and 43-50.  
     
     
         13 . The primer set of  claim 12 , which further comprises the oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 33 and 34.  
     
     
         14 . A primer set for nucleic acid amplification to detect GAPDH, which is featured by selecting at least two primers from the primers for nucleic acid amplification that comprise the oligonucleotides of  claim 5 .  
     
     
         15 . A primer set for nucleic acid amplification to detect GAPDH, which is featured by selecting at least four primers from the primers for nucleic acid amplification that comprise the oligonucleotides of  claim 5 .  
     
     
         16 . The primer set for nucleic acid amplification claimed in  claim 14 , which features that at least two primers contained in the primer set recognize each two gene regions in the nucleotide sequence set forth as SEQ ID NO: 51 and/or the complementary sequence thereof.  
     
     
         17 . The primer set for nucleic acid amplification claimed in cliam  14 , which features that the primers contained in the primer set recognize at least six gene regions in the nucleotide sequence set forth as SEQ ID NO: 51 and/or the complementary sequence thereof.  
     
     
         18 . A primer set comprising a combination of each one primer selected from each of (a) FIP: SEQ ID NOs: 80-87 and (b) RIP: SEQ ID NOs: 88-96, which categories consist of the primers for nucleic acid amplification to detect GAPDH that comprise the oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 80-96.  
     
     
         19 . The primer set of  claim 18 , which further comprises an oligonucleotide having either one of the nucleotide sequences set forth as SEQ ID NO: 58 or SEQ ID NOs: 62-64 and an oligonucleotide having any one of the nucleotide sequences set forth as SEQ ID NOs: 73-77.  
     
     
         20 . The primer set of  claim 18 , further comprising, as primers, oligonucleotides having the nucleotide sequences forth as SEQ ID NOs: 78 and 79.  
     
     
         21 . The primer set of  claim 8 , wherein the method for nucleic acid amplification is the LAMP method.  
     
     
         22 . A primer set for nucleic acid amplification to detect β-actin, which comprises any one of the following primer sets: 
 1) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 35, 43, 14 and 29 as primers;    2) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 36, 44, 15 and 30 as primers;    3) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 37, 45, 10 and 31 as primers;    4) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 41, 50, 17 and 32 as primers;    5) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 38, 45, 10 and 31 as primers;    6) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 39, 45, 10 and 31 as primers;    7) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 40, 45, 16 and 31 as primers;    8) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 41, 45, 16 and 31 as primers;    9) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 37, 46, 16 and 31 as primers;    10) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 37, 47, 16 and 31 as primers;    11) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 37, 48, 16 and 31 as primers; and    12) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 37, 49, 16 and 31 as primers;    
     
     
         23 . A primer set for nucleic acid amplification to detect GAPDH, which comprises any one of the following primer sets: 
 1) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 80, 88, 62 and 73 as primers;    2) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 81, 89, 63 and 75 as primers;    3) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 86, 95, 58 and 76 as primers;    4) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 87, 96, 64 and 77 as primers;    5) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 81, 89, 62 and 75 as primers;    6) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 82, 89, 62 and 75 as primers;    7) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 83, 89, 63 and 75 as primers;    8) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 83, 89, 62 and 75 as primers;    9) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 84, 89, 62 and 75 as primers;    10) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 85, 89, 62 and 75 as primers;    12) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 81, 90, 63 and 75 as primers;    13) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 81, 91, 63 and 75 as primers;    14) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 81, 92, 63 and 75 as primers;    15) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 81, 93, 63 and 75 as primers;    16) a primer set comprising oligonucleotides having the nucleotide sequences set forth as SEQ ID NOs: 81, 94, 63 and 75 as primers.    
     
     
         24 . A method for detecting nucleic acid, wherein the primer of  claim 1  is used.  
     
     
         25 . A method for detecting nucleic acid, wherein the primer of  claim 8  is used.  
     
     
         26 . A reagent and/or reagent kit used in the method for detecting nucleic acid defined in  claim 24  or  25 .  
     
     
         27 . A system for detecting nucleic acid, which uses the nucleic acid detection method of  claim 24.