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。

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。

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。

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。

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。

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。

公开(公告)号：US06379888B1

公开(公告)日：2002-04-30

申请号：US09406074

申请日：1999-09-27

申请人： James G. Nadeau ,  C. Preston Linn ,  J. Bruce Pitner ,  Cheryl H. Dean ,  G. Terrance Walker

发明人： James G. Nadeau ,  C. Preston Linn ,  J. Bruce Pitner ,  Cheryl H. Dean ,  G. Terrance Walker

IPC分类号： C12Q168

CPC分类号： C12Q1/6818 ,  Y10T436/143333 ,  C12Q2563/137 ,  C12Q2537/1373

摘要： Signal primers are employed for detection of nucleic acid target sequences by fluorescence quenching mechanisms. The signal primer comprises a first and a second oligonucleotide and is partially single-stranded and partially double-stranded. In the presence of target, the second oligonucleotide of the signal primer is displaced from the first and a conformational change in a reporter probe occurs which changes the distance between the members of a donor/quencher dye pair linked to the reporter probe. The change in proximity between the dyes causes an increase or a decrease in fluorescence quenching, which is detected as an indication of the presence of the target sequence.

摘要翻译： 信号引物用于通过荧光猝灭机制检测核酸靶序列。 信号引物包含第一和第二寡核苷酸并且是部分单链和部分双链的。 在靶的存在下，信号引物的第二寡核苷酸从第一个位点移位，并且发生报告物探针中的构象变化，其改变与报道探针连接的供体/猝灭剂染料对的成员之间的距离。 染料之间接近度的变化导致荧光淬灭的增加或减少，其被检测为靶序列存在的指示。

公开(公告)号：US09315863B2

公开(公告)日：2016-04-19

申请号：US13505598

申请日：2010-11-04

申请人： James G. Nadeau

发明人： James G. Nadeau

IPC分类号： C12Q1/68

CPC分类号： C12Q1/6865 ,  C12Q2525/307

摘要： Presented herein are methods and compositions for generating sequence-specific, secondary amplification products during Loop-mediated Isothermal Amplification (LAMP). Conventional LAMP produces a preponderance of high molecular weight DNA structures concatenated into self-complementary hairpins, which are not amenable to detection by routine probe-based hybridization methods, making multiplex detection of two or more targets or sequence variants in closed-tube formats extremely difficult. Provided herein, for example, are methods for generating secondary LAMP products bearing a fragment of the original target sequence embedded within low-molecular weight products that are devoid of competitive hairpin structures, the lack of which enhances probe-based detection of target sequences. These secondary products can, for example, be produced in real-time, during the LAMP process, and can provide the option of detecting multiple target sequences within a single tube using, e.g., a homogenous, real-time fluorescence format.

摘要翻译： 本文提出了在环介导的等温扩增（LAMP）期间产生序列特异性二级扩增产物的方法和组合物。 常规的LAMP产生连接到自身互补发夹中的高分子量DNA结构的优势，其不适于通过常规基于探针的杂交方法的检测，使得封闭管形式中的两个或更多个目标或序列变体的多重检测非常困难 。 例如，本文提供的是产生具有嵌入低分子量产物中的原始靶序列的片段的次级LAMP产物的方法，所述片段不含有竞争力的发夹结构，其缺乏可增强靶序列的基于探针的检测。 这些次要产物可以例如在LAMP过程期间实时产生，并且可以提供使用例如均匀的实时荧光格式在单个管内检测多个靶序列的选项。

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.

公开(公告)号：US5840487A

公开(公告)日：1998-11-24

申请号：US801542

申请日：1997-02-18

申请人： James G. Nadeau ,  Michael C. Little

发明人： James G. Nadeau ,  Michael C. Little

IPC分类号： C12Q1/68 ,  C12P19/34

CPC分类号： C12Q1/6865 ,  C12Q1/6851 ,  C12Q1/689

摘要： Methods employing internal oligonucleotide standards in isothermal nucleic acid amplification reactions to determine the efficacy of the amplification reaction and to quantify pre-amplification target levels.