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1. 发明授权

US5587286A Methods and kits for detection of cells in food materials 失效
标题翻译：用于检测食物中细胞的方法和试剂盒
公开(公告)号：US5587286A
公开(公告)日：1996-12-24
申请号：US3242
申请日：1993-01-11
申请人： Edward E. Pahuski , Randall L. Dimond , John H. Priest , Lisa Zandt , Kathleen K. Stebnitz , Leopoldo G. Mendoza
发明人： Edward E. Pahuski , Randall L. Dimond , John H. Priest , Lisa Zandt , Kathleen K. Stebnitz , Leopoldo G. Mendoza
IPC分类号： C12M1/34 , C07K1/36 , C12N1/02 , C12Q1/02 , C12Q1/04 , C12Q1/06 , C12Q1/10 , C12Q1/24 , C12Q1/37 , C12Q1/66 , C12Q1/68 , G01N33/48 , G01N33/50
CPC分类号： C07K1/36 , C12Q1/04 , C12Q1/10 , C12Q1/24 , G01N33/50 , C12Q2304/60 , Y10T436/25375
摘要： Processes are provided for removal of cells as cell pellets from liquid milk samples, or from cultures or extracts of other food materials or other materials of biological origin. The concentrated cells in the pellet can be analyzed by various techniques to determine the relative cell count, such as by lysing of the cells followed by measurement of ATP. Nucleic amplification, as by the polymerase chain reaction method, can be carried out using the cellular pellet directly, without need for isolation of nucleic acid from the cells. After the amplification, an assay can be carried out for amplified nucleic acid segment indicative of the presence of cells of interest in the sample. The invention thus provides methods for obtaining cellular components from samples of milk, and cultures or extracts of other materials, including food materials, and for determining relative contamination of milk and such other materials by microorganisms. The invention also provides kits for carrying out its various methods.
摘要翻译：提供了从液体乳样品或其他食品材料或其他生物来源的材料的培养物或提取物中除去作为细胞沉淀物的细胞的方法。可以通过各种技术分析沉淀中的浓缩细胞以确定相对细胞计数，例如通过裂解细胞，随后测量ATP。如通过聚合酶链反应方法的核扩增可以直接使用细胞沉淀进行，而不需要从细胞中分离核酸。在扩增后，可以对指示样品中感兴趣的细胞的存在的扩增的核酸片段进行测定。因此，本发明提供了从牛奶样品和其他材料（包括食品材料）的培养物或提取物中获得细胞成分的方法，以及用于通过微生物确定牛奶和其它物质的相对污染。本发明还提供用于实施其各种方法的试剂盒。

2. 发明授权

US5700645A Methods and kits for separation, concentration and analysis of cells 失效
标题翻译：细胞分离，浓缩和分析的方法和试剂盒
公开(公告)号：US5700645A
公开(公告)日：1997-12-23
申请号：US485428
申请日：1995-06-07
申请人： Edward E. Pahuski , Randall L. Dimond , John H. Priest , Lisa Zandt , Kathleen K. Stebnitz , Leopoldo G. Mendoza
发明人： Edward E. Pahuski , Randall L. Dimond , John H. Priest , Lisa Zandt , Kathleen K. Stebnitz , Leopoldo G. Mendoza
IPC分类号： C12M1/34 , C07K1/36 , C12N1/02 , C12Q1/02 , C12Q1/04 , C12Q1/06 , C12Q1/10 , C12Q1/24 , C12Q1/37 , C12Q1/66 , C12Q1/68 , G01N33/48 , G01N33/50 , C12N1/00 , G01N33/44
CPC分类号： C07K1/36 , C12Q1/04 , C12Q1/10 , C12Q1/24 , G01N33/50 , C12Q2304/60 , Y10T436/25375
摘要： Processes are provided for removal of cells as cell pellets from liquid milk samples, or from cultures or extracts of other food materials or other materials of biological origin. The concentrated cells in the pellet can be analyzed by various techniques to determine the relative cell count, such as by lysing of the cells followed by measurement of ATP. Nucleic amplification, as by the polymerase chain reaction method, can be carried out using the cellular pellet directly, without need for isolation of nucleic acid from the cells. After the amplification, an assay can be carried out for amplified nucleic acid segment indicative of the presence of cells of interest in the sample. The invention thus provides methods for obtaining cellular components from samples of milk, and cultures or extracts of other materials, including food materials, and for determining relative contamination of milk and such other materials by microorganisms. The invention also provides kits for carrying out its various methods.
摘要翻译：提供了从液体乳样品或其他食品材料或其他生物来源的材料的培养物或提取物中除去作为细胞沉淀物的细胞的方法。可以通过各种技术分析沉淀中的浓缩细胞以确定相对细胞计数，例如通过裂解细胞，随后测量ATP。如通过聚合酶链反应方法的核扩增可以直接使用细胞沉淀进行，而不需要从细胞中分离核酸。在扩增后，可以对指示样品中感兴趣的细胞的存在的扩增的核酸片段进行测定。因此，本发明提供了从牛奶样品和其他材料（包括食品材料）的培养物或提取物中获得细胞成分的方法，以及用于通过微生物确定牛奶和其它物质的相对污染。本发明还提供用于实施其各种方法的试剂盒。

3. 发明授权

US6090589A Nucleic acid amplification with DNA-dependent RNA polymerase activity of RNA replicases 失效
标题翻译：核酸扩增与RNA复制酶的DNA依赖性RNA聚合酶活性
公开(公告)号：US6090589A
公开(公告)日：2000-07-18
申请号：US480041
申请日：1995-06-06
申请人： Randall L. Dimond , Steven J. Ekenberg , James R. Hartnett , Geoffrey R. Hudson , Leopoldo G. Mendoza , Katharine M. Miller , John E. Monahan , Christopher L. Jones , Mark A. Maffitt , Richard A. Martinelli , Edward E. Pahuski , James W. Schumm
发明人： Randall L. Dimond , Steven J. Ekenberg , James R. Hartnett , Geoffrey R. Hudson , Leopoldo G. Mendoza , Katharine M. Miller , John E. Monahan , Christopher L. Jones , Mark A. Maffitt , Richard A. Martinelli , Edward E. Pahuski , James W. Schumm
IPC分类号： C12N15/09 , C12Q1/68 , C07H19/00 , C07H21/04 , C12P19/34
CPC分类号： C12Q1/6867 , C12Q1/6806 , C12Q1/6853
摘要： The present invention entails methods, and kits for carrying them out, based on the discovery that an RNA replicase, such as Q.beta. replicase, has DNA-dependent RNA polymerase ("DDRP") activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2'-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase. The discovery of this DDRP activity provides methods of the invention for nucleic acid amplification wherein a nucleic acid, with a DNA segment with the sequence of an RNA that is autocatalytically replicatable by an RNA replicase, is provided as a substrate for the replicase. Assays of the invention include those wherein a nucleic acid analyte is hybridized with one or more nucleic acid probes, which include or are processed to generate a DNA segment which is amplifiable through production from the segment, catalyzed by the DDRP activity of an RNA replicase, of an autocatalytically replicatable RNA, which is autocatalytically replicated to provide an abundance of readily detectable reporter molecules.
摘要翻译：基于发现RNA复制酶，例如Qβ复制酶，具有核酸片段的DNA依赖性RNA聚合酶（“DDRP”）活性（包括DNA片段和DNA）的发现，本发明需要用于进行它们的方法和试剂盒：RNA嵌合片段，其包含2'-脱氧核糖核苷酸或其类似物，并且具有由复制酶自动催化复制的RNA序列。该DDRP活性的发现提供了用于核酸扩增的本发明的方法，其中提供具有可由RNA复制酶自动催化复制的RNA序列的DNA区段的核酸作为复制酶的底物。本发明的测定包括其中核酸分析物与一种或多种核酸探针杂交的核酸探针，其包括或被处理以产生可通过RNA复制酶的DDRP活性催化的片段产生可扩增的DNA区段，的自催化复制RNA，其自动催化复制以提供丰富的容易检测的报告分子。

4. 发明授权

US06677140B2 Nucleic acid amplification with DNA-dependent RNA polymerase activity of RNA replicases 失效
标题翻译：核酸扩增与RNA复制酶的DNA依赖性RNA聚合酶活性
公开(公告)号：US06677140B2
公开(公告)日：2004-01-13
申请号：US10071057
申请日：2002-02-07
申请人： Randall L. Dimond , Steven J. Ekenberg , James R. Hartnett , Geoffrey R. Hudson , Leopoldo G. Mendoza , Katharine M. Miller , John E. Monahan , Christopher L. Jones , Mark A. Maffitt , Richard A. Martinelli , Edward E. Pahuski , James W. Schumm
发明人： Randall L. Dimond , Steven J. Ekenberg , James R. Hartnett , Geoffrey R. Hudson , Leopoldo G. Mendoza , Katharine M. Miller , John E. Monahan , Christopher L. Jones , Mark A. Maffitt , Richard A. Martinelli , Edward E. Pahuski , James W. Schumm
IPC分类号： C12P1934
CPC分类号： C12Q1/6867 , C12Q1/6806 , C12Q1/6853 , C12Q2521/501 , C12Q2521/325 , C12Q2521/119 , C12Q2521/107 , C12Q2563/137 , C12Q2531/149 , C12Q2525/121
摘要： The present invention entails methods and kits for carrying them out based on the discovery that an RNA replicase, such as Q&bgr; replicase, has DNA-dependent RNA polymerase (“DDRP”) activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2′-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase.
摘要翻译：本发明涉及用于携带RNA的复制酶如Qbeta复制酶具有核酸片段的DNA依赖性RNA聚合酶（“DDRP”）活性的发现的方法和试剂盒，包括DNA片段和DNA：RNA嵌合其包含2'-脱氧核糖核苷酸或其类似物，并且具有由复制酶自动催化复制的RNA序列。

5. 发明授权

US06369207B1 Nucleic acid amplification with DNA-dependent RNA polymerase activity of RNA replicases 失效
公开(公告)号：US06369207B1
公开(公告)日：2002-04-09
申请号：US09396001
申请日：1999-09-14
申请人： Randall L. Dimond , Steven J. Ekenberg , James R. Hartnett , Geoffrey R. Hudson , Leopoldo G. Mendoza , Katharine M. Miller , John E. Monahan , Christopher L. Jones , Mark A. Maffitt , Richard A. Martinelli , Edward E. Pahuski , James W. Schumm
发明人： Randall L. Dimond , Steven J. Ekenberg , James R. Hartnett , Geoffrey R. Hudson , Leopoldo G. Mendoza , Katharine M. Miller , John E. Monahan , Christopher L. Jones , Mark A. Maffitt , Richard A. Martinelli , Edward E. Pahuski , James W. Schumm
IPC分类号： C07H1900
CPC分类号： C12Q1/6867 , C12Q1/6806 , C12Q1/6853 , C12Q2521/501 , C12Q2521/325 , C12Q2521/119 , C12Q2521/107 , C12Q2563/137 , C12Q2531/149 , C12Q2525/121
摘要： The present invention entails methods, and kits for carrying them out, based on the discovery that an RNA replicase, such as Q&bgr; replicase, has DNA-dependent RNA polymerase (“DDRP”) activity with nucleic acid segments, including DNA segments and DNA:RNA chimeric segments, which comprise a 2′-deoxyribonucleotide or an analog thereof and which have sequences of RNAs that are autocatalytically replicatable by the replicase. The discovery of this DDRP activity provides methods of the invention for nucleic acid amplification wherein a nucleic acid, with a DNA segment with the sequence of an RNA that is autocatalytically replicatable by an RNA replicase, is provided as a substrate for the replicase. The replicase catalyzes synthesis, from the DNA segment, of the RNA, which the replicase then autocatalytically replicates. The invention entails use of the amplification methods in detecting nucleic acid analytes, as in nucleic acid probe hybridization assays. Such assays of the invention include those wherein a nucleic acid analyte is hybridized with one or more nucleic acid probes, which include or are processed to generate a DNA segment which is amplifiable through production from the segment, catalyzed by the DDRP activity of an RNA replicase, of an autocatalytically replicatable RNA, which is autocatalytically replicated to provide an abundance of readily detectable reporter molecules. The invention permits replacement of an RNA, that is autocatalytically replicatable with an RNA replicase and employed as a reporter or label in prior art assays, such as nucleic acid probe hybridization assays or immunoassays, with a nucleic acid comprising a DNA segment with the same base sequence as the RNA. The invention also includes the methods of the invention with Mn+2, Co+2, or Zn+2 in the solutions in which the DDRP activity occurs.

6. 发明授权

US6100024A Methods and compositions for nucleic acid detection by target extension and probe amplification 失效
标题翻译：通过靶扩增和探针扩增进行核酸检测的方法和组合物
公开(公告)号：US6100024A
公开(公告)日：2000-08-08
申请号：US652888
申请日：1991-02-08
申请人： Geoffrey R. Hudson , James W. Schumm , Randall L. Dimond
发明人： Geoffrey R. Hudson , James W. Schumm , Randall L. Dimond
IPC分类号： C12Q1/68 , C07H21/04
CPC分类号： C12Q1/6865
摘要： The present invention provides a novel, single-stranded DNA probe which comprises an anti-target segment, a strand of a promoter, and a reporter segment, arranged so that a target segment, which has a 3'-hydroxyl at its terminus, can prime DNA polymerase-catalyzed extension of the target segment along the probe as template, when the target segment is hybridized to the anti-target segment of the probe, to provide an extension product from which transcripts, with the sequence complementary to that of the reporter segment of the probe, can be made by transcription from the promoter corresponding to the promoter segment of the probe. The transcripts, optionally after further amplification or other processing, can be detected. In one embodiment of the invention, the transcripts will be autocatalytically replicatable by an RNA replicase such as Q.beta. replicase. The invention also provides methods of using a probe of the invention in testing a sample of nucleic acid for the presence of a nucleic acid which comprises target segment corresponding to the anti-target segment of the probe and test kits for carrying out such methods with a probe of the invention.
摘要翻译：本发明提供一种新颖的单链DNA探针，其包含抗靶区段，启动子链和报道区段，其排列使得其末端具有3'-羟基的靶区段可以当靶区段与探针的抗靶区段杂交时，提供靶向片段沿着探针的初步DNA聚合酶催化延伸作为模板，以提供延伸产物，其中转录本与序列的序列互补可以通过从对应于探针的启动子区段的启动子的转录来制备探针的片段。可以检测转录本，任选地在进一步扩增或其他处理之后。在本发明的一个实施方案中，转录本将通过RNA复制酶例如Qβ复制酶自动催化复制。本发明还提供了使用本发明的探针来测试核酸样品中存在核酸的方法，所述核酸包含对应于探针的抗靶节段的靶区段，以及用于用本发明的探针。

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