Method of nucleic acid amplification and measuring reagent and reagent kit therefor

Abstract

The object of the present invention is to provide a method that allows stable amplification of an internal standard material while maintaining an accurate assay value for a target nucleic acid in a nucleic acid detection system involving the use of an internal standard material and a reagent kit used therefor. The present invention relates to a method for nucleic acid amplification comprising preventing an internal standard amplification product from affecting amplification reaction of a target nucleic acid by performing amplification of an internal standard material prior to amplification of the target nucleic acid in the method for amplifying a target nucleic acid in a sample using an internal standard material and a reagent and reagent kit used therefor.

1 . An assay reagent or reagent kit for nucleic acid amplification wherein amplification of an internal standard material is performed prior to amplification of a target nucleic acid, comprising at least the following reagents:
 (a) an internal standard material with a known concentration or copy number having a nucleotide sequence different from that of a target nucleic acid at least in the primer design region, wherein the concentration of the internal standard material is set to be higher than that of an amplification reaction solution used for an assay involving simultaneous amplification of the internal standard material and the target nucleic acid;   (b) an oligonucleotide primer used for amplification of an internal standard material that allows performance of amplification of an internal standard material prior to amplification of the target nucleic acid, wherein the concentration of the oligonucleotide primer used for amplification of the internal standard material is set to be lower than that of an amplification reaction solution used for an assay involving simultaneous amplification of the internal standard material and the target nucleic acid, and the oligonucleotide primer for amplification of the internal standard material is primer (b1) and/or (b2):   (b1) wherein when a first arbitrary sequence F1c and a second arbitrary sequence F2c are successively selected from the 3′ side of the target sequence of a template nucleic acid in the internal standard material toward the 3′ terminal on the template nucleic acid, the primer (b1) comprises a sequence identical to F1c and a sequence F2 complementary to F2c in that order from the 5′ side toward the 3′ side;   (b2) wherein when a third arbitrary sequence R1 and a fourth arbitrary sequence R2 are successively selected from the 5′ side of the target sequence of a template nucleic acid in the internal standard material toward the 5′ terminal on the template nucleic acid, the primer (b2) comprises a sequence R1c complementary to R1 and a sequence R2 identical to R2 in that order form the 5′ side toward the 3′ side; and   (c) an oligonucleotide primer used for amplification of a target nucleic acid, wherein the oligonucleotide primer for amplification of a target nucleic acid is primer (c1) and/or (c2):   (c1) wherein when a first arbitrary sequence F1c and a second arbitrary sequence F2c are successively selected from the 3′ side of the target sequence of a template nucleic acid in the target nucleic acid toward the 3′ terminal on the template nucleic acid, the primer (c1) comprises a sequence identical to F1c and a sequence F2 complementary to F2c in that order form the 5′ side toward the 3′ side;   (c2) wherein when a third arbitrary sequence R1 and a forth arbitrary sequence R2 are successively selected from the 5′ side of the target sequence of a template nucleic acid in the target nucleic acid toward the 5′ terminal on the template nucleic acid, the primer (c2) comprises a sequence R1c complementary to R1 and a sequence R2 identical to R2 in that order from the 5′ side toward the 3′ side.   
     
     
         2 . The assay reagent or reagent kit according to  claim 1 , which further comprises the following reagents:
 (a) an oligonucleotide probe used for detecting an internal standard material, and   (b) an oligonucleotide probe used for detecting a target nucleic acid.