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1. 发明授权

US5525462A Nucleic acid sequence amplification method, detection method, and reagent kit therefor 失效
标题翻译：核酸序列扩增法，检测方法及试剂盒
公开(公告)号：US5525462A
公开(公告)日：1996-06-11
申请号：US875758
申请日：1992-04-28
申请人： Yutaka Takarada , Toshiya Aono , Shuji Shibata
发明人： Yutaka Takarada , Toshiya Aono , Shuji Shibata
IPC分类号： C12Q1/68 , C07H21/04 , C12P19/34
CPC分类号： C12Q1/6853
摘要： An oligonucleotide is provided, which has at least a base sequence A, which is 10-30 nucleotides in length and homologous to a part of a specific nucleic acid sequence to be amplified, and a base sequence B, which is 10-30 nucleotides in length and complementary to a sequence 3' to the part of the specific nucleic acid sequence to be amplified. Sequences A and B are separated by a spacer region of 0-20 nucleotides. The oligonucleotide is used in an amplification method, wherein the oligonucleotide is mixed with a sample containing a specific nucleic acid sequence to be amplified and allowed to anneal to the specific nucleic acid sequence in the sample. The annealed oligonucleotide acts as a primer in an elongation reaction, whereas any nonannealed oligonucleotide is decomposed, except for at least part of the base sequence A. The elongation product is then denatured to a single strand for use as a template to which the remaining oligonucleotide anneals to form a primer for subsequent elongation. Also provided is a detection method, which allows for detection of a specific nucleic acid sequence in a sample, as well as reagent kits for use in the amplification and detection methods.
摘要翻译：提供了寡核苷酸，其至少具有碱基序列A，其长度为10-30个核苷酸并且与待扩增的特异性核酸序列的一部分同源;以及碱基序列B，其为10-30个核苷酸长度并与要扩增的特定核酸序列部分的序列3'互补。序列A和B被0-20个核苷酸的间隔区隔开。寡核苷酸用于扩增方法，其中将寡核苷酸与含有待扩增的特异性核酸序列的样品混合并使其与样品中的特定核酸序列退火。退火的寡核苷酸在伸长反应中用作引物，而除了至少部分碱基序列A之外，任何非退火的寡核苷酸都被分解。然后将延伸产物变性为单链，用作剩余的寡核苷酸退火以形成随后伸长的底漆。还提供了一种检测方法，其允许检测样品中的特定核酸序列，以及用于扩增和检测方法的试剂盒。

2. 发明授权

US5972607A Methods for nucleic acid amplification with thermostable ribonuclease H 失效
标题翻译：用热稳定核糖核酸酶H进行核酸扩增的方法
公开(公告)号：US5972607A
公开(公告)日：1999-10-26
申请号：US893023
申请日：1997-07-15
申请人： Motohiro Kondo , Toshiya Aono , Katsuya Daimon
发明人： Motohiro Kondo , Toshiya Aono , Katsuya Daimon
IPC分类号： C12N15/09 , C07H21/04 , C12N9/22 , C12Q1/68 , C07H21/02 , C12N9/00 , C12P19/34
CPC分类号： C12Q1/6865 , C12Q1/701
摘要： Methods for nucleic acid amplification with thermostable ribonuclease H, in which single-stranded RNA (-) is prepared from RNA (+) as a target nucleic acid and the copy number of the single-stranded RNA (-) is increased through the use of thermostable ribonuclease H in combination with non-thermostable RNA-dependent DNA polymerase, non-thermostable DNA-dependent DNA polymerase and non-thermostable DNA-dependent RNA polymerase. In these methods, the number of amplification cycles is well increased and the sensitivity of detection can, therefore, be improved, as compared with the conventional methods. Also provided are methods for the detection of a target nucleic acid from RNA copies of a specific nucleic acid, obtained by any of the amplification methods, and reagent kits for use in these methods.
摘要翻译：用作为靶核酸的RNA（+）制备单链RNA（ - ）和单链RNA（ - ）拷贝数的热稳定核糖核酸酶H进行核酸扩增的方法通过使用热稳定核糖核酸酶H与非热稳定的RNA依赖性DNA聚合酶，非热稳定DNA依赖性DNA聚合酶和非热稳定DNA依赖性RNA聚合酶组合。在这些方法中，与常规方法相比，扩增循环的数量很好地增加，因此可以提高检测灵敏度。还提供了用于通过任何扩增方法获得的特异性核酸的RNA拷贝和用于这些方法的试剂盒来检测靶核酸的方法。

3. 发明授权

US5853981A Oligonucleotides, methods and kits for amplifying and detecting a nucleic acid of cytomegalovirus (CMV) using nucleic acid sequence .beta.2.7 失效
标题翻译：使用核酸序列β2.7扩增和检测巨细胞病毒（CMV）的核酸的寡核苷酸，方法和试剂盒
公开(公告)号：US5853981A
公开(公告)日：1998-12-29
申请号：US839306
申请日：1997-04-18
申请人： Motohiro Kondo , Toshiya Aono , Masaya Segawa , Koichi Yamanishi , Kazuhiro Kondo , Keiko Taya
发明人： Motohiro Kondo , Toshiya Aono , Masaya Segawa , Koichi Yamanishi , Kazuhiro Kondo , Keiko Taya
IPC分类号： C12N15/09 , C12Q1/68 , C12Q1/70
CPC分类号： C12Q1/701 , C12Q2531/143
摘要： The present invention relates to two primers for amplifying a cytomegalovirus (CMV) nucleic acid suitable for a nucleic acid sequence-based amplification (NASBA) using a DNA-dependent RNA polymerase, a first primer containing a promoter sequence and a nucleic acid sequence consisting of at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:1; a second primer containing a nucleic acid sequence consisting of at least fifteen continuous bases selected from the nucleic acid sequence of SEQ ID NO:2, NO:3 or NO:4; a detecting probe and/or a capturing probe containing a nucleic acid sequence consisting of at least continuous fifteen bases selected from the nucleic acid sequence(s) of SEQ ID NO:5, NO:6 and/or NO:7 wherein said sequence is modified if necessary; a reagent kit for detecting a CMV containing the above-mentioned primers and probes; and a nucleic acid sequence-based amplification (NASBA) using said primers. The advantages of the present invention are that CMV can be detected in an easy, rapid and specific manner with a high sensitivity.
摘要翻译：本发明涉及用于扩增适用于使用DNA依赖性RNA聚合酶的基于核酸序列的扩增（NASBA）的巨细胞病毒（CMV）核酸的两个引物，含有启动子序列的第一引物和包含启动子序列的核酸序列至少15个选自SEQ ID NO：1的核酸序列的连续碱基; 含有选自SEQ ID NO：2，NO：3或NO：4的核酸序列的至少十五个连续碱基的核酸序列的第二引物; 检测探针和/或捕获探针，其含有至少连续十五个碱基的核酸序列，所述核酸序列选自SEQ ID NO：5，NO：6和/或NO：7的核酸序列，其中所述序列是必要时修改; 用于检测含有上述引物和探针的CMV的试剂盒; 和使用所述引物的基于核酸序列的扩增（NASBA）。本发明的优点是可以以高灵敏度以容易，快速和具体的方式检测CMV。

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