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1. 发明授权

US5561044A Detection of mycobacteria by multiplex strand displacement nucleic acid amplification 失效
标题翻译：通过多重链置换核酸扩增检测分枝杆菌
公开(公告)号：US5561044A
公开(公告)日：1996-10-01
申请号：US398305
申请日：1995-03-03
申请人： George T. Walker , James G. Nadeau , Patricia A. Spears , Colleen M. Nycz , Daryl D. Shank , James L. Schram , Stewart R. Jurgensen
发明人： George T. Walker , James G. Nadeau , Patricia A. Spears , Colleen M. Nycz , Daryl D. Shank , James L. Schram , Stewart R. Jurgensen
IPC分类号： C12N15/09 , C12Q1/04 , C12Q1/68 , C12R1/32 , C12R1/33 , C12P19/34
CPC分类号： C12Q1/689 , C12Q1/6851 , C12Q2600/16
摘要： Primers and methods for adapter-mediated multiplex amplification of the IS6110 insertion element of Mycobacterium tuberculosis (M.tb) and the 16S ribosomal gene of Mycobacterium tuberculosis, useful for simultaneously detecting and/or identifying species of the M. tuberculosis complex and other clinically relevant Mycobacterium species. Multiplex Strand Displacement Amplification (SDA) is used in a single amplification reaction which is capable of simultaneously identifying M. tuberculosis and providing a screen for substantially all of the clinically relevant species of Mycobacteria. Also disclosed are methods for adapter-mediated multiplex amplification of multiple target sequences and a single internal control sequence for determination of sample efficacy or quantitation of the targets. In a preferred embodiment, an internal control sequence is included in the amplification reaction and coamplified with the IS6110 and 16S target sequences as an indication of sample amplification activity or to quantitate the initial amount of target sequences in the sample.
摘要翻译：针对结核分枝杆菌（M.tb）的IS6110插入元件和结核分枝杆菌的16S核糖体基因的适配器介导的多重扩增的引物和方法，可用于同时检测和/或鉴定结核分枝杆菌复合物和其他临床相关的物种分枝杆菌种。多重链位移扩增（SDA）用于能够同时鉴定结核分枝杆菌并为基本上所有临床相关分枝杆菌物种提供筛选的单一扩增反应。还公开了用于多重靶序列的衔接子介导的多重扩增的方法和用于确定靶的样品功效或定量的单个内部控制序列的方法。在优选的实施方案中，内部对照序列包括在扩增反应中并与IS6110和16S靶序列共扩增，作为样品扩增活性的指示或定量样品中靶序列的初始量。

2. 发明授权

US5470723A Detection of mycobacteria by multiplex nucleic acid amplification 失效
标题翻译：通过多重核酸扩增检测分枝杆菌
公开(公告)号：US5470723A
公开(公告)日：1995-11-28
申请号：US111076
申请日：1993-08-24
申请人： George T. Walker , James G. Nadeau , Patricia A. Spears , Colleen M. Nycz , Daryl D. Shank , James L. Schram , Stewart R. Jurgensen
发明人： George T. Walker , James G. Nadeau , Patricia A. Spears , Colleen M. Nycz , Daryl D. Shank , James L. Schram , Stewart R. Jurgensen
IPC分类号： C12N15/09 , C12Q1/04 , C12Q1/68 , C12R1/32 , C12R1/33 , C07H15/12 , C07H17/00 , C12P19/34
CPC分类号： C12Q1/689 , C12Q1/6851 , C12Q2600/16
摘要： Primers and methods for adapter-mediated multiplex amplification of the IS6110 insertion element of Mycobacterium tuberculosis (M.tb) and the 16S ribosomal gene of Mycobacterium tuberculosis, useful for simultaneously detecting and/or identifying species of the M. tuberculosis complex and other clinically relevant Mycobacterium species. Multiplex Strand Displacement Amplification (SDA) is used in a single amplification reaction which is capable of simultaneously identifying M. tuberculosis and providing a screen for substantially all of the clinically relevant species of Mycobacteria. Also disclosed are methods for adapter-mediated multiplex amplification of multiple target sequences and a single internal control sequence for determination of sample efficacy or quantitation of the targets. In a preferred embodiment, an internal control sequence is included in the amplification reaction and coamplified with the IS6110 ant 16S target sequences as an indication of sample amplification activity or to quantitate the initial amount of target sequences in the sample.
摘要翻译：针对结核分枝杆菌（M.tb）的IS6110插入元件和结核分枝杆菌的16S核糖体基因的适配器介导的多重扩增的引物和方法，可用于同时检测和/或鉴定结核分枝杆菌复合物和其他临床相关的物种分枝杆菌种。多重链位移扩增（SDA）用于能够同时鉴定结核分枝杆菌并为基本上所有临床相关分枝杆菌物种提供筛选的单一扩增反应。还公开了用于多重靶序列的衔接子介导的多重扩增的方法和用于确定靶的样品功效或定量的单个内部控制序列的方法。在优选的实施方案中，扩增反应中包含内部控制序列并与IS6110蚂蚁16S靶序列共扩增，作为样品扩增活性的指示或定量样品中靶序列的初始量。

3. 发明授权

US5736365A Multiplex nucleic acid amplification 失效
标题翻译：多重核酸扩增
公开(公告)号：US5736365A
公开(公告)日：1998-04-07
申请号：US705225
申请日：1996-08-29
申请人： George Terrance Walker , James G. Nadeau , Patricia Anne Spears , Colleen M. Nycz , Daryl Dee Shank , James L. Schram , Stewart Russel Jurgensen
发明人： George Terrance Walker , James G. Nadeau , Patricia Anne Spears , Colleen M. Nycz , Daryl Dee Shank , James L. Schram , Stewart Russel Jurgensen
IPC分类号： C12N15/09 , C12Q1/04 , C12Q1/68 , C12R1/32 , C12R1/33 , C12P19/34 , C07H21/04 , C12Q1/70
CPC分类号： C12Q1/689 , C12Q1/6851 , C12Q2600/16
摘要： Primers and methods for adapter-mediated multiplex amplification of the IS6110 insertion element of Mycobacterium tuberculosis (M.tb) and the 16S ribosomal gene of Mycobacterium tuberculosis, useful for simultaneously detecting and/or identifying species of the M. tuberculosis complex and other clinically relevant Mycobacterium species. Multiplex Strand Displacement Amplification (SDA) is used in a single amplification reaction which is capable of simultaneously identifying M. tuberculosis and providing a screen for substantially all of the clinically relevant species of Mycobacteria. Also disclosed are methods for adapter-mediated multiplex amplification of multiple target sequences and a single internal control sequence for determination of sample efficacy or quantitation of the targets. In a preferred embodiment, an internal control sequence is included in the amplification reaction and coamplified with the IS6110 and 16S target sequences as an indication of sample amplification activity or to quantitate the initial amount of target sequences in the sample.
摘要翻译：针对结核分枝杆菌（M.tb）的IS6110插入元件和结核分枝杆菌的16S核糖体基因的适配器介导的多重扩增的引物和方法，可用于同时检测和/或鉴定结核分枝杆菌复合物和其他临床相关的物种分枝杆菌种。多重链位移扩增（SDA）用于能够同时鉴定结核分枝杆菌并为基本上所有临床相关分枝杆菌物种提供筛选的单一扩增反应。还公开了用于多重靶序列的衔接子介导的多重扩增的方法和用于确定靶的样品功效或定量的单个内部控制序列的方法。在优选的实施方案中，内部对照序列包括在扩增反应中并与IS6110和16S靶序列共扩增，作为样品扩增活性的指示或定量样品中靶序列的初始量。

4. 发明授权

US6066458A Methods, apparatus and computer program products for determining quantities of nucleic acid sequences in samples using standard curves and amplification ratio estimates 失效
标题翻译：用于使用标准曲线和扩增率估计来确定样品中核酸序列数量的方法，装置和计算机程序产品
公开(公告)号：US6066458A
公开(公告)日：2000-05-23
申请号：US80589
申请日：1998-05-18
申请人： Perry D. Haaland , James G. Nadeau , Colleen M. Nycz , Cheryl H. Dean , Catherine A. Spargo
发明人： Perry D. Haaland , James G. Nadeau , Colleen M. Nycz , Cheryl H. Dean , Catherine A. Spargo
IPC分类号： C12N15/00 , C12Q1/68
CPC分类号： C12Q1/6851
摘要： Methods for determining quantities of nucleic acid sequences in samples undergoing amplification utilize amplification ratio estimates (R*) in operations to accurately perform absolute quantitation even when amplification factors for the target and control sequences undergoing amplification are different, time dependent or vary as a function of starting concentrations of nucleic acid sequences. These operations also take into account conversion efficiencies associated with the conversion of probes upon generation of target or control amplicons, but do not require the explicit calculation of such efficiencies. The operations also recognize that a preferred R* should be determined based on a preferred statistical criterion to improve quantitation. In addition, the use of standard samples having known starting concentrations of target and control sequences therein may enable accurate absolute quantitation without the explicit calculation of amplification ratio estimates.
摘要翻译：用于确定正在进行扩增的样品中核酸序列数量的方法利用操作中的扩增比率估计（R *）来准确地执行绝对定量，即使目标和经历放大的控制序列的放大因子不同，时间依赖或随着核酸序列的起始浓度。这些操作还考虑了在靶或扩增子产生时与探针转化相关的转化效率，但不需要明确计算这种效率。操作还认识到，优选的R *应基于优选的统计标准来确定，以改进定量。此外，使用具有已知起始浓度的目标和控制序列的标准样品可以使得能够进行精确的绝对定量，而无需显式计算扩增率估计值。

5. 发明授权

US5919630A Detection of nucleic acids by fluorescence quenching 失效
公开(公告)号：US5919630A
公开(公告)日：1999-07-06
申请号：US186030
申请日：1998-11-04
申请人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
发明人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
IPC分类号： G01N21/64 , C07H21/00 , C12N15/09 , C12Q1/68 , G01N33/58 , C07H21/04 , C12P19/34
CPC分类号： C12Q1/6818 , C12Q1/6825
摘要： Single-stranded signal primers are modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned in sufficiently close spatial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition site (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not cleavable or nickable by the restriction endonuclease. In the presence of target, however, signal primer and the restriction endonuclease recognition site are rendered double-stranded and cleavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indication of the presence of the target sequence or of target sequence amplification.

6. 发明授权

US5928869A Detection of nucleic acids by fluorescence quenching 失效
公开(公告)号：US5928869A
公开(公告)日：1999-07-27
申请号：US865675
申请日：1997-05-30
申请人： James G. Nadeau , J. Bruce Pitner , C. Preston Linn , James L. Schram
发明人： James G. Nadeau , J. Bruce Pitner , C. Preston Linn , James L. Schram
IPC分类号： G01N33/50 , C12N15/09 , C12Q1/68 , G01N33/566 , C07H21/00 , C07H21/04 , C12P19/34
CPC分类号： C12Q1/6827 , C12Q1/6818 , C12Q1/6858
摘要： A detector oligonucleotide having a sequence which forms an intramolecularly base-paired secondary structure is described for use in detecting nucleic acid target sequences and target sequence amplification. The detector oligonucleotide is further modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned on the detector oligonucleotide such that they are in close spatial proximity in the base-paired, folded secondary structure, thereby causing quenching of donor fluorescence. The detector oligonucleotide may optionally further comprise a restriction endonuclease recognition site (RERS) which remains partially or entirely single-stranded in the base-paired secondary structure. The RERS is flanked by the two dyes. In the presence of target, the base-paired secondary structure is unfolded or linearized, increasing the distance between the donor and acceptor dyes and causing a change in fluorescence of the donor and/or the acceptor. If an RERS is present, it is rendered double-stranded in the presence of target, allowing cleavage or nicking by a restriction endonuclease and separation of the two dyes onto separate nucleic acid fragments. This may further contribute to the magnitude of the change in fluorescence.

7. 发明授权

US5846726A Detection of nucleic acids by fluorescence quenching 失效
公开(公告)号：US5846726A
公开(公告)日：1998-12-08
申请号：US855085
申请日：1997-05-13
申请人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
发明人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
IPC分类号： G01N21/64 , C07H21/00 , C12N15/09 , C12Q1/68 , G01N33/58 , C07H21/04 , C12P19/34
CPC分类号： C12Q1/6818 , C12Q1/6825
摘要： Single-stranded signal primers are modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned in sufficiently close spatial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition site (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not cleavable or nickable by the restriction endonuclease. In the presence of target, however, signal primer and the restriction endonuclease recognition site are rendered double-stranded and cleavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indication of the presence of the target sequence or of target sequence amplification.

8. 发明授权

US5811269A Detection of mycobacteria by multiplex nucleic acid amplification 失效
标题翻译：通过多重核酸扩增检测分枝杆菌
公开(公告)号：US5811269A
公开(公告)日：1998-09-22
申请号：US640378
申请日：1996-04-30
申请人： James G. Nadeau , Cheryl H. Dean , James L. Schram , Deborah R. Howard , Margaret S. Dey , David J. Wright
发明人： James G. Nadeau , Cheryl H. Dean , James L. Schram , Deborah R. Howard , Margaret S. Dey , David J. Wright
IPC分类号： C12N15/09 , C07H21/04 , C12Q1/04 , C12Q1/68 , C12R1/32 , C12R1/325 , C12R1/33 , C12P19/34
CPC分类号： C12Q1/689 , C12Q2600/16
摘要： Primers and methods for adapter-mediated multiplex amplification of the IS6110 insertion element of the Mycobacterium tuberculosis (M.tb) complex and a 16S rDNA target common to essentially all mycobacteria are described. In certain embodiments, the primers are optimized for efficient multiplex amplification in thermophilic SDA. The multiplex Strand Displacement Amplification methods of the invention are capable, in a single amplification reaction, of simultaneously identifying M. tuberculosis and providing a screen for substantially all of the clinically relevant species of mycobacteria. Also disclosed are internal control sequences designed for coamplification with the two targets, allowing assessment of amplification efficiency and/or quantitation of the targets.
摘要翻译：描述了针对结核分枝杆菌（M.tb）复合物的IS6110插入元件和基本上所有分枝杆菌共同的16S rDNA靶的衔接子介导的多重扩增的引物和方法。在某些实施方案中，针对嗜热SDA中的有效多重扩增优化引物。本发明的多重链位移扩增方法能够在单个扩增反应中同时鉴定结核分枝杆菌并提供基本上所有临床相关分枝杆菌物种的筛选。还公开了设计用于与两个靶标共扩增的内部控制序列，允许评估靶标的扩增效率和/或定量。

9. 发明授权

US5681705A Amplification and detection of mycobacterium avium complex species 失效
标题翻译：鸟分枝杆菌复合物的扩增和检测
公开(公告)号：US5681705A
公开(公告)日：1997-10-28
申请号：US520194
申请日：1995-08-28
申请人： James L. Schram , James G. Nadeau , Cheryl H. Dean
发明人： James L. Schram , James G. Nadeau , Cheryl H. Dean
IPC分类号： C12N15/09 , C07H21/04 , C12Q1/04 , C12Q1/68 , G01N33/53 , G01N33/566 , G01N33/569 , G01N33/58
CPC分类号： C12Q1/689
摘要： Amplification primers and methods are disclosed for complex-specific amplification of target sequences in the dnaJ genes of the Mycobacterium Avium Complex species. Also provided are assay probes for detection of the amplification products and/or identification of the MAC species which is present.
摘要翻译：公开了扩增引物和方法用于复合物特异性扩增分枝杆菌复合物种的dnaJ基因中的靶序列。还提供了用于检测扩增产物的检测探针和/或存在的MAC物种的鉴定。

10. 发明授权

US6054279A Detection of nucleic acids by fluorescence quenching 失效
标题翻译：通过荧光猝灭检测核酸
公开(公告)号：US6054279A
公开(公告)日：2000-04-25
申请号：US120916
申请日：1998-07-20
申请人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
发明人： James G. Nadeau , J. Bruce Pitner , James L. Schram , C. Preston Linn , Glenn P. Vonk , G. Terrance Walker
IPC分类号： G01N21/64 , C07H21/00 , C12N15/09 , C12Q1/68 , G01N33/58 , C21Q1/68
CPC分类号： C12Q1/6818 , C12Q1/6825
摘要： Single-stranded signal primers are modified by linkage to two dyes which form a donor/acceptor dye pair. The two dyes are positioned in sufficiently close spatial proximity on the signal primer that the fluorescence of the first dye is quenched by the second dye. The signal primer may further comprise a restriction endonuclease recognition site (RERS) between the two dyes. As the signal primer is initially single-stranded and remains single-stranded in the absence of target, the restriction endonuclease recognition site is not cleavable or nickable by the restriction endonuclease. In the presence of target, however, signal primer and the restriction endonuclease recognition site are rendered double-stranded and cleavable or nickable by the restriction endonuclease. Cleavage or nicking separates the two dyes and a change in fluorescence due to decreased quenching is detected as an indication of the presence of the target sequence or of target sequence amplification.
摘要翻译：通过连接到形成供体/受体染料对的两种染料来修饰单链信号引物。这两种染料位于信号引物足够接近的空间上，第一种染料的荧光被第二种染料淬灭。信号引物还可以包含两种染料之间的限制性内切核酸酶识别位点（RERS）。由于信号引物最初是单链的，并且在不存在靶标的情况下保持单链，限制性内切核酸酶识别位点不受限制性内切核酸酶的切割或切割。然而，在靶的存在下，信号引物和限制性内切核酸酶识别位点被限制性内切核酸酶双链和切割或切割。切割或切口分离两种染料，并且由于猝灭降低引起的荧光变化被检测为目标序列或靶序列扩增的存在的指示。

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