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    Simultaneous amplification of multiple targets
    1.
    发明授权
    Simultaneous amplification of multiple targets 失效
    同时放大多个目标

    公开(公告)号：US5422252A

    公开(公告)日：1995-06-06

    申请号：US73197

    申请日：1993-06-04

    申请人： George T. Walker ,  James G. Nadeau ,  Michael C. Little

    发明人： George T. Walker ,  James G. Nadeau ,  Michael C. Little

    IPC分类号： G01N33/566 ,  C07H21/04 ,  C12N15/09 ,  C12P19/34 ,  C12Q1/68 ,  G11B5/00 ,  G11B5/09 ,  G11B5/187 ,  G11B5/31 ,  G11B5/455 ,  G11B5/465 ,  G11B19/04 ,  C12Q1/70

    CPC分类号： C12Q1/6853

    摘要： Methods for multiplex amplification of target nucleic acid sequences using a single pair of primers. Defined sequences are appended to the ends of multiple target sequences as part of the amplification reaction so that no steps in addition to amplification are required. The target sequences with the appended defined sequences need not be isolated prior to amplification. In one embodiment for coamplification of two target sequences, a sequence corresponding to a terminal segment of the first target sequence is appended to one end of the second target sequence and a sequence corresponding to a terminal segment of the second target sequence is appended to one end of the first target sequence. Amplification of the two targets then requires only a single pair of primers. Alternatively, a single defined sequence may be appended to the 5' and 3' ends of any number of selected targets. All such modified target sequences may then be amplified using a single pair of primers which hybridize to the defined end-sequences.

    摘要翻译： 使用单对引物多重扩增靶核酸序列的方法。 定义的序列作为扩增反应的一部分附加到多个靶序列的末端，因此不需要扩增步骤。 具有附加定义序列的靶序列不需要在扩增前分离。 在两个目标序列的共放大的一个实施例中，对应于第一目标序列的末端片段的序列被附加到第二目标序列的一端，并且与第二目标序列的末端片段相对应的序列被附加到一端 的第一个靶序列。 两个靶标的扩增只需要一对引物即可。 或者，单个定义的序列可以附加到任意数目的所选目标的5'和3'端。 然后可以使用与定义的末端序列杂交的单对引物扩增所有这些修饰的靶序列。

    Simultaneous amplification of multiple targets
    2.
    发明授权
    Simultaneous amplification of multiple targets 失效
    同时放大多个目标

    公开(公告)号：US5624825A

    公开(公告)日：1997-04-29

    申请号：US451313

    申请日：1995-05-26

    申请人： George T. Walker ,  James G. Nadeau ,  Michael C. Little

    发明人： George T. Walker ,  James G. Nadeau ,  Michael C. Little

    IPC分类号： G01N33/566 ,  C07H21/04 ,  C12N15/09 ,  C12P19/34 ,  C12Q1/68 ,  G11B5/00 ,  G11B5/09 ,  G11B5/187 ,  G11B5/31 ,  G11B5/455 ,  G11B5/465 ,  G11B19/04

    CPC分类号： C12Q1/6853

    摘要： Methods for multiplex amplification of target nucleic acid sequences using a single pair of primers. Defined sequences are appended to the ends of multiple target sequences as part of the amplification reaction so that no steps in addition to amplification are required. The target sequences with the appended defined sequences need not be isolated prior to amplification. In one embodiment for coamplification of two target sequences, a sequence corresponding to a terminal segment of the first target sequence is appended to one end of the second target sequence and a sequence corresponding to a terminal segment of the second target sequence is appended to one end of the first target sequence. Amplification of the two targets then requires only a single pair of primers. Alternatively, a single defined sequence may be appended to the 5' and 3' ends of any number of selected targets. All such modified target sequences may then be amplified using a single pair of primers which hybridize to the defined end-sequences.

    摘要翻译： 使用单对引物多重扩增靶核酸序列的方法。 定义的序列作为扩增反应的一部分附加到多个靶序列的末端，因此不需要扩增步骤。 具有附加定义序列的靶序列不需要在扩增前分离。 在两个目标序列的共放大的一个实施例中，对应于第一目标序列的末端片段的序列被附加到第二目标序列的一端，并且与第二目标序列的末端片段相对应的序列被附加到一端 的第一个靶序列。 两个靶标的扩增只需要一对引物即可。 或者，单个定义的序列可以附加到任意数目的所选目标的5'和3'端。 然后可以使用与定义的末端序列杂交的单对引物扩增所有这些修饰的靶序列。

    Detection of nucleic acid amplification
    4.
    再颁专利
    Detection of nucleic acid amplification 失效
    检测核酸扩增

    公开(公告)号：USRE39885E1

    公开(公告)日：2007-10-16

    申请号：US09082247

    申请日：1998-05-20

    申请人： James G. Nadeau ,  George T. Walker

    发明人： James G. Nadeau ,  George T. Walker

    IPC分类号： C12Q1/68 ,  C12P19/34

    CPC分类号： C12Q1/6844 ,  C12Q2565/525 ,  C12Q2561/113 ,  C12Q2531/119

    摘要： Methods for detecting, immobilizing or localizing primer extension products of a Strand Displacement Amplification reaction which are coupled to, and an indication of, amplification of the target sequence. The primer extension products are secondary, target-specific DNA products generated concurrently with SDA of the target sequence and can therefore be used to detect and/or measure target sequence amplification in real-time. In general, the secondary amplification products are not amplifiable and remain inert in the SDA reaction after they are formed without interfering with amplification of the target sequence. The secondary amplification products may be designed or modified to contain special features to facilitate their detection, immobilization or localization.

    摘要翻译： 用于检测，固定或定位连接于链序列扩增反应的引物延伸产物的方法，以及扩增靶序列的指示。 引物延伸产物是与靶序列的SDA同时产生的次要靶特异性DNA产物，因此可用于实时检测和/或测量靶序列扩增。 通常，二次扩增产物在其形成后不能扩增并在SDA反应中保持惰性，而不干扰靶序列的扩增。 二次扩增产物可以被设计或修改以包含特殊特征以促进其检测，固定或定位。

    Detection of mycobacteria by multiplex strand displacement nucleic acid amplification
    5.
    发明授权
    Detection of mycobacteria by multiplex strand displacement nucleic acid amplification 失效
    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。

    Detection of mycobacteria by multiplex nucleic acid amplification
    6.
    发明授权
    Detection of mycobacteria by multiplex nucleic acid amplification 失效
    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。

    Detection of nucleic acid amplification
    7.
    发明授权
    Detection of nucleic acid amplification 失效
    检测核酸扩增

    公开(公告)号：US5547861A

    公开(公告)日：1996-08-20

    申请号：US229281

    申请日：1994-04-18

    申请人： James G. Nadeau ,  George T. Walker

    发明人： James G. Nadeau ,  George T. Walker

    IPC分类号： C12N15/09 ,  C12Q1/68 ,  C12R1/32 ,  C12P19/34 ,  C07H21/04 ,  C12Q1/70

    CPC分类号： C12Q1/6844

    摘要： Methods for detecting, immobilizing or localizing primer extension products of a Strand Displacement Amplification reaction which are coupled to, and an indication of, amplification of the target sequence. The primer extension products are secondary, target-specific DNA products generated concurrently with SDA of the target sequence and can therefore be used to detect and/or measure target sequence amplification in real-time. In general, the secondary amplification products are not amplifiable and remain inert in the SDA reaction after they are formed without interfering with amplification of the target sequence. The secondary amplification products may be designed or modified to contain special features to facilitate their detection, immobilization or localization.

    摘要翻译： 用于检测，固定或定位连接于链序列扩增反应的引物延伸产物的方法，以及扩增靶序列的指示。 引物延伸产物是与靶序列的SDA同时产生的次要靶特异性DNA产物，因此可用于实时检测和/或测量靶序列扩增。 通常，二次扩增产物在其形成后不能扩增并在SDA反应中保持惰性，而不干扰靶序列的扩增。 二次扩增产物可以被设计或修改以包含特殊特征以促进其检测，固定或定位。

    Sequence-specific methods for homogeneous, real-time detection of lamp products
    8.
    发明授权
    Sequence-specific methods for homogeneous, real-time detection of lamp products 有权
    灯具产品均匀，实时检测的序列特异性方法

    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。