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1. 发明授权

US08435742B2 Method and kit for detection/quantification of target RNA 有权
标题翻译：靶RNA的检测/定量的方法和试剂盒
公开(公告)号：US08435742B2
公开(公告)日：2013-05-07
申请号：US12739978
申请日：2008-11-04
申请人： Mutsunori Shirai , Hajime Fukunaga , Kazuhiko Fujiwara , Kanehisa Yokoyama , Kentaro Fujimoto
发明人： Mutsunori Shirai , Hajime Fukunaga , Kazuhiko Fujiwara , Kanehisa Yokoyama , Kentaro Fujimoto
IPC分类号： C12Q1/68 , C12P19/34 , C12N9/00 , C07H21/04
CPC分类号： C12Q1/6809 , C12Q2565/537 , C12Q2525/143 , C12Q2521/107
摘要： [Object] It is to provide a method and a kit capable of detecting or quantifying a target RNA simply and rapidly from trace amounts of RNA in a sample, in a case such as when one or more kinds of pathogenic microorganisms are to be detected or quantified.[Solving Means] The method comprises the steps of 1) synthesizing cDNA from a sample containing the target RNA using a liquid-phase primer having a promoter sequence and a reverse transcriptase to obtain a cDNA-RNA complex, 2) degrading the RNA of the complex, 3) synthesizing a double-stranded DNA via the cDNA obtained in the step 2) and the solid-phase primer, 4) synthesizing RNA from the double-stranded DNA, 5) synthesizing cDNA via the RNA obtained in the step 4) and the solid-phase primer to obtain a cDNA-RNA complex, 6) degrading the RNA of the complex obtained in step 5), 7) synthesizing a double-stranded DNA via the cDNA obtained in the step 6) and the liquid-phase primer, and 8) quantifying the double-stranded DNAs obtained in the steps 3) and 7). These steps can be performed in a single reaction solution.
摘要翻译：本发明提供一种方法和试剂盒，其能够在样品中的痕量RNA中简单快速地检测或定量靶RNA，例如当检测到一种或多种病原微生物或量化 [解决方法]该方法包括以下步骤：1）使用具有启动子序列和逆转录酶的液相引物合成来自含有靶RNA的样品的cDNA，得到cDNA-RNA复合物，2）降解复合物，3）通过步骤2）和固相引物获得的cDNA合成双链DNA，4）从双链DNA合成RNA，5）通过步骤4）得到的RNA合成cDNA，和固相引物以获得cDNA-RNA复合物，6）降解步骤5）中获得的复合物的RNA，7）通过步骤6）中得到的cDNA合成双链DNA和液相引物，和8）定量在步骤3）和7）中获得的双链DNA。这些步骤可以在单个反应溶液中进行。

2. 发明申请

US20110053150A1 Method and Kit for Detection/Quantification of Target RNA 有权
标题翻译：靶RNA的检测/定量的方法和试剂盒
公开(公告)号：US20110053150A1
公开(公告)日：2011-03-03
申请号：US12739978
申请日：2008-11-04
申请人： Mutsunori Shirai , Hajime Fukunaga , Kazuhiko Fujiwara , Kanehisa Yokoyama , Kentaro Fujimoto
发明人： Mutsunori Shirai , Hajime Fukunaga , Kazuhiko Fujiwara , Kanehisa Yokoyama , Kentaro Fujimoto
IPC分类号： C12Q1/68
CPC分类号： C12Q1/6809 , C12Q2565/537 , C12Q2525/143 , C12Q2521/107
摘要： [Object] It is to provide a method and a kit capable of detecting or quantifying a target RNA simply and rapidly from trace amounts of RNA in a sample, in a case such as when one or more kinds of pathogenic microorganisms are to be detected or quantified.[Solving Means] The method comprises the steps of 1) synthesizing cDNA from a sample containing the target RNA using a liquid-phase primer having a promoter sequence and a reverse transcriptase to obtain a cDNA-RNA complex, 2) degrading the RNA of the complex, 3) synthesizing a double-stranded DNA via the cDNA obtained in the step 2) and the solid-phase primer, 4) synthesizing RNA from the double-stranded DNA, 5) synthesizing cDNA via the RNA obtained in the step 4) and the solid-phase primer to obtain a cDNA-RNA complex, 6) degrading the RNA of the complex obtained in step 5), 7) synthesizing a double-stranded DNA via the cDNA obtained in the step 6) and the liquid-phase primer, and 8) quantifying the double-stranded DNAs obtained in the steps 3) and 7). These steps can be performed in a single reaction solution.
摘要翻译：本发明提供一种方法和试剂盒，其能够在样品中的痕量RNA中简单快速地检测或定量靶RNA，例如当检测到一种或多种病原微生物或量化 [解决方法]该方法包括以下步骤：1）使用具有启动子序列和逆转录酶的液相引物合成来自含有靶RNA的样品的cDNA，得到cDNA-RNA复合物，2）降解复合物，3）通过步骤2）和固相引物获得的cDNA合成双链DNA，4）从双链DNA合成RNA，5）通过步骤4）得到的RNA合成cDNA，和固相引物以获得cDNA-RNA复合物，6）降解步骤5）中获得的复合物的RNA，7）通过步骤6）中得到的cDNA合成双链DNA和液相引物，和8）定量在步骤3）和7）中获得的双链DNA。这些步骤可以在单个反应溶液中进行。

3. 发明申请

US20100016566A1 Method of synthesizing cdna and method of synthesizing rna chains, and nucleotide-immobilized carrier 有权
标题翻译：合成cdna的方法和合成rna链的方法以及核苷酸固定载体
公开(公告)号：US20100016566A1
公开(公告)日：2010-01-21
申请号：US11990641
申请日：2006-08-09
申请人： Kenji Kinoshita , Toru Yakabe , Kentaro Fujimoto , Kanehisa Yokoyama , Kazuhiko Fujiwara
发明人： Kenji Kinoshita , Toru Yakabe , Kentaro Fujimoto , Kanehisa Yokoyama , Kazuhiko Fujiwara
IPC分类号： C07H21/04 , C12P19/34 , C07H21/02
CPC分类号： C12P19/34 , C12N15/1096 , C12Q1/686 , C12Q2521/107 , C12Q2565/537
摘要： A method of synthesizing a cDNA chain using an insoluble carrier having on the surface thereof a polymer substance containing a first unit having a group derived from a phosphoric ester composing the hydrophilic portion of phospholipid, and a second unit having a group derived from carboxylic acid having an electron-attractive substituent bound to a carbonyl group, which includes immobilizing a polynucleotide for DNA elongation; bringing a solution containing an RNA fragment, nucleotide monomers, and a reverse transcriptase or an enzyme having polymerase activity into contact with the surface of the insoluble carrier; and allowing the polynucleotide for DNA elongation immobilized on the surface of the carrier to elongate using the RNA fragment contained in the solution as a template, to thereby form a single-strand cDNA.
摘要翻译：一种使用不溶性载体合成cDNA链的方法，所述不溶性载体在其表面上具有含有源自构成磷脂的亲水部分的磷酸酯的基团的第一单元的聚合物物质和具有衍生自羧酸的基团的第二单元，与羰基结合的电子吸引取代基，其包括固定多核苷酸用于DNA延伸; 将含有RNA片段，核苷酸单体和逆转录酶或具有聚合酶活性的酶的溶液与不溶性载体的表面接触; 并且使用溶液中包含的RNA片段作为模板，使固定在载体表面上的DNA延伸的多核苷酸伸长，从而形成单链cDNA。

4. 发明授权

US07968290B2 Method of detecting gene 失效
标题翻译：检测基因的方法
公开(公告)号：US07968290B2
公开(公告)日：2011-06-28
申请号：US11920559
申请日：2006-05-16
申请人： Kenji Kinoshita , Kazuhiko Fujiwara , Kanehisa Yokoyama , Kentaro Fujimoto , Toru Yakabe
发明人： Kenji Kinoshita , Kazuhiko Fujiwara , Kanehisa Yokoyama , Kentaro Fujimoto , Toru Yakabe
IPC分类号： C12Q1/68 , C12Q1/26 , C12Q1/28 , C12P19/34 , C07H21/02 , C07H21/04
CPC分类号： C12N15/1096 , C12Q1/6834 , C12Q2565/537 , C12Q2527/107
摘要： A method of detecting a gene including immobilizing a primer for DNA elongation onto an insoluble carrier having on the surface thereof a polymer substance containing a first unit having a phosphorylcholine group and a second unit having a carboxylic acid-derived group having an electron-attractive substituent bound to a carbonyl group; annealing the template DNA fragments or RNA fragments with the primer for DNA elongation, so as to elongate the DNA primer while incorporating therein an enzyme, thereby allowing coloration of a chromogenic reagent by its enzymatic action; and judging whether the DNA fragments or RNA fragments of the gene presents or not, based on the degree of coloration.
摘要翻译：一种检测基因的方法，包括将DNA延伸引物固定在具有表面的不溶性载体上，所述不溶性载体具有含有具有磷酸胆碱基团的第一单元的聚合物物质和具有吸电子取代基的羧酸衍生基团的第二单元与羰基结合; 用引物退火模板DNA片段或RNA片段以进行DNA延伸，以便在引入酶的同时延长DNA引物，从而通过其酶作用使着色试剂着色; 并基于着色程度来判断该基因的DNA片段或RNA片段是否存在。

5. 发明申请

US20090137406A1 RNA Detection Method 有权
标题翻译： RNA检测方法
公开(公告)号：US20090137406A1
公开(公告)日：2009-05-28
申请号：US12227904
申请日：2007-06-01
申请人： Kenji Kinoshita , Kanehisa Yokoyama , Kentaro Fujimoto , Toru Yakabe , Shin Saito , Kazuhiko Fujiwara
发明人： Kenji Kinoshita , Kanehisa Yokoyama , Kentaro Fujimoto , Toru Yakabe , Shin Saito , Kazuhiko Fujiwara
IPC分类号： C40B30/00 , C12Q1/68
CPC分类号： C12Q1/6837
摘要： The present invention provides an RNA detection method detecting, from a reaction system containing a target sample, a target RNA chain originated from the target sample, using a surface having on the surface thereof a polymer substance which contains a first unit having a group derived from a phosphate ester composing the hydrophilic portion of a phospholipid and a second unit having a carboxylic acid derivative group composed of an electron-attractive substitutional group bound to a carbonyl group, while being provided with at least one reaction space, the reaction space having an immobilized nucleic acid primer immobilized therein.
摘要翻译：本发明提供一种RNA检测方法，其从含有目标样品的反应体系中检测源自目标样品的靶RNA链，其表面上具有含有第一单元的表面，该第一单元含有来自构成磷脂的亲水部分的磷酸酯和具有由与羰基结合的吸电子取代基组成的羧酸衍生物基团的第二单元，同时具有至少一个反应空间，所述反应空间具有固定化的固定在其中的核酸引物。

6. 发明授权

US08822670B2 Method of synthesizing cDNA and method of synthesizing RNA chains, and nucleotide-immobilized carrier 有权
标题翻译：合成cDNA的方法和合成RNA链的方法以及核苷酸固定载体
公开(公告)号：US08822670B2
公开(公告)日：2014-09-02
申请号：US11990641
申请日：2006-08-09
申请人： Kenji Kinoshita , Toru Yakabe , Kentaro Fujimoto , Kanehisa Yokoyama , Kazuhiko Fujiwara
发明人： Kenji Kinoshita , Toru Yakabe , Kentaro Fujimoto , Kanehisa Yokoyama , Kazuhiko Fujiwara
IPC分类号： C07H21/00
CPC分类号： C12P19/34 , C12N15/1096 , C12Q1/686 , C12Q2521/107 , C12Q2565/537
摘要： A method of synthesizing a cDNA chain using an insoluble carrier having on the surface thereof a polymer substance containing a first unit having a group derived from a phosphoric ester composing the hydrophilic portion of phospholipid, and a second unit having a group derived from carboxylic acid having an electron-attractive substituent bound to a carbonyl group, which includes immobilizing a polynucleotide for DNA elongation; bringing a solution containing an RNA fragment, nucleotide monomers, and a reverse transcriptase or an enzyme having polymerase activity into contact with the surface of the insoluble carrier; and allowing the polynucleotide for DNA elongation immobilized on the surface of the carrier to elongate using the RNA fragment contained in the solution as a template, to thereby form a single-strand cDNA.
摘要翻译：一种使用不溶性载体合成cDNA链的方法，所述不溶性载体在其表面上具有含有源自构成磷脂的亲水部分的磷酸酯的基团的第一单元的聚合物物质和具有衍生自羧酸的基团的第二单元，与羰基结合的电子吸引取代基，其包括固定多核苷酸用于DNA延伸; 将含有RNA片段，核苷酸单体和逆转录酶或具有聚合酶活性的酶的溶液与不溶性载体的表面接触; 并且使用溶液中包含的RNA片段作为模板，使固定在载体表面上的DNA延伸的多核苷酸伸长，从而形成单链cDNA。

7. 发明授权

US08088580B2 RNA detection method 有权
标题翻译： RNA检测方法
公开(公告)号：US08088580B2
公开(公告)日：2012-01-03
申请号：US12227904
申请日：2007-06-01
申请人： Kenji Kinoshita , Kanehisa Yokoyama , Kentaro Fujimoto , Toru Yakabe , Shin Saito , Kazuhiko Fujiwara
发明人： Kenji Kinoshita , Kanehisa Yokoyama , Kentaro Fujimoto , Toru Yakabe , Shin Saito , Kazuhiko Fujiwara
IPC分类号： C12Q1/68 , C12P19/34 , C07H21/02 , C07H21/04
CPC分类号： C12Q1/6837
摘要： The present invention provides an RNA detection method detecting, from a reaction system containing a target sample, a target RNA chain originated from the target sample, using a surface having on the surface thereof a polymer substance which contains a first unit having a group derived from a phosphate ester composing the hydrophilic portion of a phospholipid and a second unit having a carboxylic acid derivative group composed of an electron-attractive substitutional group bound to a carbonyl group, while being provided with at least one reaction space, the reaction space having an immobilized nucleic acid primer immobilized therein.
摘要翻译：本发明提供一种RNA检测方法，其从含有目标样品的反应体系中检测源自目标样品的靶RNA链，其表面上具有含有第一单元的表面，该第一单元含有来自构成磷脂的亲水部分的磷酸酯和具有由与羰基结合的吸电子取代基组成的羧酸衍生物基团的第二单元，同时具有至少一个反应空间，所述反应空间具有固定化的固定在其中的核酸引物。

8. 发明申请

US20090042190A1 Method of detecting gene 失效
标题翻译：检测基因的方法
公开(公告)号：US20090042190A1
公开(公告)日：2009-02-12
申请号：US11920559
申请日：2006-05-16
申请人： Kenji Kinoshita , Kazuhiko Fujiwara , Kanehisa Yokoyama , Kentaro Fujimoto , Toru Yakabe
发明人： Kenji Kinoshita , Kazuhiko Fujiwara , Kanehisa Yokoyama , Kentaro Fujimoto , Toru Yakabe
IPC分类号： C12Q1/68
CPC分类号： C12N15/1096 , C12Q1/6834 , C12Q2565/537 , C12Q2527/107
摘要： A method of detecting a gene including immobilizing a primer for DNA elongation onto an insoluble carrier having on the surface thereof a polymer substance containing a first unit having a phosphorylcholine group and a second unit having a carboxylic acid-derived group having an electron-attractive substituent bound to a carbonyl group; annealing the template DNA fragments or RNA fragments with the primer for DNA elongation, so as to elongate the DNA primer while incorporating therein an enzyme, thereby allowing coloration of a chromogenic reagent by its enzymatic action; and judging whether the DNA fragments or RNA fragments of the gene presents or not, based on the degree of coloration.
摘要翻译：一种检测基因的方法，包括将DNA延伸引物固定在具有表面的不溶性载体上，所述不溶性载体具有含有具有磷酸胆碱基团的第一单元的聚合物物质和具有吸电子取代基的羧酸衍生基团的第二单元与羰基结合; 用引物退火模板DNA片段或RNA片段以进行DNA延伸，以便在引入酶的同时延长DNA引物，从而通过其酶作用使着色试剂着色; 并基于着色程度来判断该基因的DNA片段或RNA片段是否存在。

9. 发明授权

US08088340B2 Polymer compound for biomedical use and biochip substrate using such a polymer compound 有权
标题翻译：用于生物医学用途的高分子化合物和使用这种高分子化合物的生物芯片底物
公开(公告)号：US08088340B2
公开(公告)日：2012-01-03
申请号：US11908170
申请日：2006-03-14
申请人： Mitsutaka Matsumoto , Sumio Shibahara , Takayuki Matsumoto , Kanehisa Yokoyama , Sohei Funaoka , Daisuke Masuda , Michael Patrick Coleman , Dominic Zichi
发明人： Mitsutaka Matsumoto , Sumio Shibahara , Takayuki Matsumoto , Kanehisa Yokoyama , Sohei Funaoka , Daisuke Masuda , Michael Patrick Coleman , Dominic Zichi
IPC分类号： G01N33/52
CPC分类号： C08J7/047 , C08F220/28 , C08F220/30 , C08F220/36 , C08F220/38 , C08F2220/286 , C08F2220/306 , C08J2365/00 , C08J2443/00 , G01N33/54393 , Y10T428/31504 , Y10T428/31938
摘要： A biochip substrate capable of realizing the high detection accuracy by restricting a nonspecific adsorption or bonding of a substance to be detected, when used for a detection or analysis of protein, nucleic acids and the like. The biochip substrate is for fixing a biologically active substance on a surface of a solid substrate, and characterized in that it has a layer comprising a polymer compound obtained by copolymerizing an ethylenically unsaturated polymerizable monomer having an alkylene glycol residue, an ethylenically unsaturated polymerizable monomer having a functional group for fixing a biologically active substance and an ethylenically unsaturated polymerizable monomer having a cross-linkable functional group, on the surface of the substrate.
摘要翻译：一种生物芯片基板，当用于检测或分析蛋白质，核酸等时，能够通过限制待检测物质的非特异性吸附或结合来实现高检测精度。生物芯片基板用于将生物活性物质固定在固体基材的表面上，其特征在于，其具有包含通过使具有亚烷基二醇残基的烯属不饱和可聚合单体和具有亚烷基二醇残基的烯键式不饱和可聚合单体共聚得到的聚合物，用于固定生物活性物质的官能团和具有可交联官能团的烯属不饱和可聚合单体。

10. 发明申请

US20150005180A9 Biochip 审中-公开
标题翻译：生物芯片
公开(公告)号：US20150005180A9
公开(公告)日：2015-01-01
申请号：US10572332
申请日：2004-09-17
申请人： Kazuhiko Ishihara , Sohei Funaoka , Kanehisa Yokoyama
发明人： Kazuhiko Ishihara , Sohei Funaoka , Kanehisa Yokoyama
IPC分类号： C40B30/04 , C40B60/12 , C40B40/08 , C40B40/12 , C40B50/18 , C40B99/00 , C40B40/10
CPC分类号： C12Q1/003
摘要： A biochip is provided that suppresses nonspecific adsorption or bonding of a detection target substance without coating with an adsorption inhibitor and that has excellent detection sensitivity. The constitution is such that it has a macromolecular substance containing a phosphorylcholine group and an active ester group on a substrate surface of a biochip substrate.
摘要翻译：提供一种生物芯片，其抑制检测对象物质的非特异性吸附或结合而不用吸附抑制剂涂覆并且具有优异的检测灵敏度。其结构是在生物芯片基板的基板表面上具有含有磷酰胆碱基和活性酯基的大分子物质。

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