CN106164651A - 用于热循环生物化学操作的装置和方法 - Google Patents
用于热循环生物化学操作的装置和方法 Download PDFInfo
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Abstract
一种扩增子分离装置和方法,用于能够在诸如血液的样品中识别多个目标DNA分子,所述装置包括消耗品和用于接收所述消耗品的插接设备。所述消耗品包括反应容器加热台、反应容器内容物转移台、扩增子尺寸分离台以及读取台。所述设备控制并监测所述消耗品及其操作。
Description
技术领域
本发明涉及在其中可以存在不同DNA的多样性并且希望快速检测的情况下检测特定的DNA扩增子.这种检测的一个特定目的是识别血液样品中引起败血症的细菌。
发明内容
本发明的一个目的是提供一种设备和过程,能够完成尺寸和颜色标记的扩增子的快速PCR(聚合酶链反应),并且随后以自动的方式将反应物转移至同一设备内的尺寸分离机构。该方法的优势是大大减少进行这种检验所花费的时间、简化操作和设备,并且增加单次反应中可以识别的目标数量。
根据本发明的第一方面,提供了一种消耗品,包含以下台:
加热台,反应容器被保持在其中;
反应容器内容物转移台;
扩增子尺寸分离台;以及
读取台.
典型地,加热台将反应容器保持在准确的位置中,并且消耗品被构造成允许热循环仪设备从消耗品下方被提供到第一台内,并且当PCR完成时从那里撤走热循环仪设备.
优选的反应容器是由加碳塑料材料形成的容器。利用这种反应容器,热量可以很快地被转移进出反应室。这种微量滴定容器可以具有大约2cm的总长度,并且从上到下包括帽接收边缘、过滤部以及具有底部的反应室。过滤部可以具有7-8mm的最大外直径以及大约4-5mm的深度,并且反应室从3mm逐渐缩小至2.5mm,整体上具有的壁厚为大约0.8mm.因此,反应容器可以具有基本上毛细管的尺寸,以使热传递速率最大化.
反应容器通常安装有透明盖,该透明盖可以被铰接地安装至反应容器或者安装至消耗品。
反应容器可以被设置成包含PCR过程所需的试剂.这些试剂可以是冻干的,尽管可以使用液体试剂.
所述消耗品可以包含往复设备,可操作的将所述反应容器从所述加热台转移到所述内容物转移台。
所述消耗品内容物转移台可以具有刺穿装置,用于刺穿反应容器并且允许很可能限制容器内容物通过进入尺寸分离台内。与刺穿装置相关联的可以是所述消耗品中的被设置成将所述容器向下推到那里或者反之亦然的设备.所述内容物转移台可以包括用于将反应流体吸入所述芯片上的采集井内并且因此将来自完成反应物的流体转移到所述尺寸分离芯片的装置.所述反应容器可以是加压的,可能通过被用于那里的盖子密封并且被加热,以便能够使其内容物被驱赶出来.
根据本发明的该第一方面的一个特征,所述扩增子尺寸分离台可以被设置成通过电泳操作,并且因此可以包括微流体芯片,那是具有设置成包含试剂的伸长的毛细管的芯片,以及如果必要的话能够使目标DNA样品被检测的装置。所述芯片可以包含一个或多个微制造的通道,在其中将发生尺寸分离。在优选的实施例中,为了其光学性质,该芯片具有玻璃构造,但是替代地,可以采用各种塑料和其它的材料.应当理解,在这种尺寸分离台中,扩增子将以取决于它们的尺寸的速度沿着毛细管迁移。扩增子随后沿着毛细管分别到达规定的距离,在那里它们中的每一个均能够被分析.
对于将被采用的电泳,为了实现DNA扩增子的尺寸分离,电流和筛分基质是需要的.电触点可以因此被提供到所述消耗品,连接至芯片。适当的筛分基质可以是POP4或POP6.
所述消耗品优选地包括基本上刚性的箱,并且仅具有一个移动部,该移动部包括反应容器保持器,并且被设置成将保持器从反应装置到所述内容物转移台移动一个小距离.移动部优选地包括柔性元件.所述消耗品也可以具有锁定探针,所述消耗品通过所述锁定探针能够被锁定在适当的位置中.
理想地,所述消耗品在顶表面和底表面上具有可拆卸的薄膜,所述薄膜覆盖容器和加热器入口.因此,所述消耗品能够被构造成用于被现场加载例如血液中的目标DNA.
应当理解,建议所述消耗品被构造成确保反应容器内容物在任何时间都不会泄漏.同样应当理解,根据本发明的消耗品可以是一个简单的设备,具有合理可能的最小尺寸,并且被构造成用于最少量的人工干预,并且成本最小以符合一次性使用.所述消耗品因此可以是一个长方形盒,具有大约12cm的总长度,23mm宽度和28mm深度。
根据本发明的第二方面,提供了一种以插接台形式的设备,被构造成接收并且保持本发明第一方面的消耗品,所述插接台包含热循环仪设备。
一种优选的热循环仪设备包括金属套管以及在套管的底部与套管一体的热传递模块,金属套管适合于以这样一种方式紧紧地围绕所述反应容器反应室,该方式使得贯穿其长度与其连续。热传递模决可以是贴到减热模块(HRM)的珀尔帖单元(peltier cell),减热模块被设置成用于在中值温度附近操作,该温度典型地在平均DNA的退火温度附近.作为对珀尔帖单元的一种替换,可以具有围绕套管的加热线绕丝,覆盖着所述容器反应室.
所述设备优选地包括以下中的一个、多个或所有:
消耗品接收舱;
可操作的消耗品位置锁;
提供装置,被设置成将热循环仪插入到所述消耗品内,从而套管围绕所述反应容器;
·装置,典型地光学地装置,用于监测反应容器中的PCR过程;
·推进器,用于将反应容器从加热台移动到内容物转移台;
·装置,或许是盖子上的加热器,用于为盖子除雾并且对反应容器的内容物加压;
·设备,用于使反应容器被推到所述容器内容物转移台上;
·加热器装置,可操作的将所述扩增子尺寸分离台维持在恒定的温度;
·照相机装置,优选光学的照相机装置,被设置成询问所述消耗品读取台;
·设置成用于为所述芯片触点供电的装置;
·控制装置;以及
·结果读取装置.
插接台可以被构造成接收并且处理多个消耗品,优选地具有能力在第一个仍在进行中的同时去开始第二个运转.
优选地,读取器装置包括光学装置,典型地包含依赖于LED激发和CCD检测的分光光度计。读取器装置也可以包含维持时间基线的装置,以便使荧光能够相对于时间绘图并且因此可以外推出产品尺寸.
应当理解,所述插接台可以包含控制软件和用户界面和/或与计算机相连,用于控制、监测和记录目的.软件可以包含自动操作装置的能力.因此,用户可能仅需要将芯片载入所述插接台内,启动,并且设备将做剩下的事情.软件可以因此提供反馈装置,用于监测过程、时间、温度等。软件也可以通过将观察的尺寸和光谱与已知数据库进行比较从而将结果提供给终端用户.
在消耗品和插接台之间,温度控制装置可以被包含在所述尺寸分离台处,这是由于电泳产热,同时温度变化能够改变样品在尺寸分离台中迁移的速率。因此,在插接台中可以具有热交换器,诸如减热模块(HRM),被设置成用于芯片温度控制。为了再现性的原因,这能够用于维持特定的恒定温度,或者为了迎合不同的测试而维持不同的恒定温度.
根据本发明的第三方面,提供了一种利用消耗品和设备的过程,所述过程用于快速的检测大量的DNA目标,例如存在于血液样品中的病原体。
所述过程可以包括以下中的至少大多数:
·将提取的DNA载入消耗品的反应容器内,消耗品已经包含优选荧光标记的、检测目标DNA样品所必需的试剂和引物;
·将加载的消耗品载入插接台内;
·使容器进行热循环;
·当完成热循环时,从消耗品中撤出加热装置;
·将容器从加热装置转移到容器内容物转移台;
·将容器推动到容器底部刺穿器上,并且因此刺穿容器底部并允许容器内容物流入尺寸分离台中的尺寸分离通道内;
·同时控制尺寸分离通道的温度,引起标记的扩增子沿着通道前进;
·光学地监测扩增子到达特定台;
·产生标记的扩增子的光谱图;
·绘制相对于时间基线观察到的光谱;
·通过与具有已知尺寸的等位基因分型标准物的对比来确定尺寸和颜色,并且因此识别任何检测的扩增子的身份。
在允许容器内容物流入尺寸分离通道内的步骤之后,容器内容物进入筛分基质.优选地,电泳沿着分离通道驱动扩增子.
所述过程也可以在必要时包括细胞破碎步骤,其中在PCR之前,反应容器的内容物可以是冷冻和冻融以及沸腾和冷却中的一种或两种,这使细胞破裂以获取特定的DNA。
本发明的装置和方法能够检测一个或多个目标样品,但是数量上限高于现有的方法。
在某些情况下,可能希望实时观察反应尤其是扩增子在分离通道中的进展,并且这可以在根据本发明的装置中容易实现.
附图说明
现在将参照附图通过举例来描述本发明的实施例,其中:
图1是消耗品在第一种构造中的截面侧视图;
图2是接合微量滴定反应容器的PCR加热器的截面侧视图;
图3是消耗品在第二种构造中的截面侧视图;
图4是消耗品在第一种构造中的截面俯视图;
图5是消耗品在第二种构造中的截面俯视图;
图6例示了反应容器内容物转移台的细节;
图7是消耗品的插入端视图;
图8是芯片的等距视图;
图9是芯片的示意性平面图;
图10例示了插接台的外部;
图11描述了插接台的内容物;
图12是PCR加热器的截面图;
图13是插接台工作台区域的细节图;
图14例示了插接台屏幕上的显示器。
具体实施方式
在图1至图9中描述了一种扩增子分离消耗品,包括箱10并且包含以下台:
保持反应容器30的加热台20;
反应容器内容物转移台40;
扩增子尺寸分离台50;以及
读取台60。
在加热台20处是微量滴定反应容器30。反应容器30由加碳塑料材料形成,并且总长度为2cm。它以从上到下包括帽接收边缘31,具有柔软地连接至此的盖子32,填料部33,具有底座35的反应室34.填料部33具有7-8mm的最大外直径以及5mm的深度.反应室34的直径从3mm逐渐减小至2.5mm.反应室34具有大约0.8mm的壁厚。因此,反应容器基本上具有毛细管的尺寸,以使往返于容器内容物的热传递速率最大.盖子32密封地安装到容器边缘31。
反应容器30被保持在加热台20中,配置成使容器30易接近盖子加热器以及上面的光学读取器和下面的加热器.反应容器30实际上被保持在消耗品10中的可经由孔10a接近的往复装置11内,以便从消耗品的外面驱动,并且从而可从加热台20移动到内容物转移台40.在消费品箱10上也已经形成了探针10b,消耗品通过所述探针10b能够被可移除地锁定到插接台内。消费品箱10同样被构造成使它能够以唯一的一种定向被提供到插接台.
在反应容器内容物转移台40处,在消耗品的地板上的是孔41,其尺寸适合于密封地接收反应容器30的底座35.孔41的中央是针42,可操作的刺穿底座35并允许容器内容物流入孔41内.在台40处,消耗品10的顶部允许接近螺线管设备,可操作的推动反应容器30向下进入孔41内并且到针42上.针42是当刺穿底座35时允许液体从其流出的针.针42是背对背的“C”.
孔41被形成在玻璃电泳芯片12中并且与在那里形成的第一毛细管13连通.毛细管13终止于芯片12中的孔41a内.沿着第一毛细管13的中途是与第二毛细管15的接合点14.第二毛细管15在电源孔16和终端孔17之间延伸.消耗品被设置成使读取台刚好位于终端孔17之前.电极18与四个孔41、41a、16和17中的每一个相关联,用于驱动电泳.毛细管13、15内是筛分基质.
在图10至图14中示出的插接台具有带对接锁101的消耗品接收工作台100.在工作台100的上方与消耗品加热台20相对的是光学读取器102和相关连的容器盖子加热器102a,而与流体转移台40相对的是以螺钉操作的推进器103形式的设备,用于将容器向下推动到针42上。在对接锁101的上方并且在消耗品的孔10a之前的是螺线管操作的活塞104。
工作台100包括PCR热循环设备105。该设备包括HRM 106,具有底面108和工作面109的珀尔帖单元107,包括热传递底座110和套筒111的热交换器,以及驱动器112。HRM106,珀尔帖单元107和热传递底座110通过柔性焊料而彼此相连。套筒111被形成为紧紧地但是可移除地附接到消耗品10中的反应容器30。HRM处于包括散热器113和风扇114以及泵115的热传递液体回路中。
在工作台100上被适当放置的是用于电泳电极18的触点116.工作台100的外面并且低于其水平的是相机117.工作台也包括位于下面的并且贯穿分离台50的加热器118.光学读取器102和相机117供给光谱仪119.
插接台也包含适当的软件.
插接台具有包含消耗品接收舱121和触摸控制显示屏122的箱120.
为了使用装置,容器反应室34被充满适当的试剂以及荧光标记的引物.如果这些是冻干的,则在加入待研究的样品之前添加适当量的纯水,并且将透明盖子32安置就位.
启动插接台,当HRM回路中的液体将受热升温至选定的温度时,刚好高于目标DNA的退火温度,电路将为触摸屏提供信号,以指示液体温度.
消耗品随后经由舱121被推入对接设备120中,直到探针10b被对接锁101接合并且在工作台100上被锁定就位,此时电触点116接合电泳电极18.舱121也用于将消耗品保持到工作台100。
开启光学读取器102和盖子加热器,并且热循环仪105被驱动器112提升,从而套筒111紧紧地接合反应容器30。如果需要的话,热循环仪105首先被设置成在PCR循环之前进行细胞破碎循环,后者被光学读取器102观察.来自读取器102的报告能够使屏幕122指示PCR已完成,并且关闭且撤回热循环仪105.热循环仪105在稍低于其最高温度时被关闭,以确保反应容器30内的压力,同时避免进一步的DNA分离.
活塞104将消耗品往复装置11从加热台20推动到内容物转移台40.推进器103向往复装置11施加向下的压力,并且推动反应容器向下进入孔41内并且到针42上,其穿透反应容器的底座35.由于保留在反应容器30中的压力,其内容物很容易被驱逐出来并进入孔41内,在那里它们消散进入毛细管13中的筛分基质内,并且沿着那里迁移.在接合点14处,扩增子被捕获并且通过电泳沿着第二毛细管15驱动.较小的扩增子行进最快,并且因此所有的扩增子以不用的时间到达读取台。每个扩增子的尺寸和颜色被相机检测,并且产生每个扩增子的光谱图。插接台中的相关软件将根据该信息识别扩增子的DNA,并且在屏幕上显示身份.
该实施例的消耗品是一个长方形盒,总长度为12cm,宽度为23mm并且深度为28mm.玻璃芯片12为7.5cm长,5mm宽和1.5mm深.毛细管15的总长度是7.0cm。消耗品箱10在其底座中具有1mm凹槽,以接收并保护芯片.结果,芯片具有大约最小的、与相对确保的完整性和有效操作一致的可能尺寸,因此允许消耗品的结构具有对于一次性制品所期望的经济性。
插接台120的长度和宽度的总尺寸是20cm并且高度是15cm。
部件列表
消耗品台:
加热台20;反应容器30;容器内容物转移台40;
扩增子尺寸分离台50;读取台60。
部件:
消费品箱10;往复装置11;孔10a探针10b;容器帽接收边缘31;
盖子32;填料部33;反应室34;底座35;电泳芯片12;第一毛细管13;接合点14;第二毛细管15;电源孔16;阅读台孔17;电极18;接收器孔41;针42;沉井41a。
插接台
接收工作台100;对接锁101;光学读取器102;螺线管操作的活塞103;螺线管操作的活塞104;热循环仪105;HRM 106;珀尔帖单元107;底面108;工作面109;热交换器热传递底座110;套筒111;驱动器112;散热器113;风扇114;泵115;触点116;相机117;箱118;舱119;屏幕120。
Claims (62)
1.一种扩增子分离消耗品,包括以下台:
加热台,反应容器被保持于加热台中;
反应容器内容物转移台;
扩增子尺寸分离台;以及
读取台。
2.根据权利要求1所述的消耗品,其中所述加热台被构造为可撤回地接收热循环仪设备。
3.根据权利要求2所述的消耗品,被设置成从所述消耗品的下面将所述热循环仪设备提供到所述第一台内。
4.根据权利要求4所述的消耗品,被设置成将所述反应容器的反应室部分紧密安装在所述热循环仪设备内。
5.根据权利要求1至4中任一项所述的消耗品,其中所述反应容器包含PCR过程所需的试剂和引物。
6.根据权利要求7所述的消耗品,其中所述试剂和引物是冻干的。
7.根据权利要求5或6所述的消耗品,其中所述引物是荧光标记的。
8.根据前述权利要求中任一项所述的消耗品,其中所述反应容器从上到下包括帽接收边缘、填料部、带有底座的反应室。
9.根据前述权利要求中任一项所述的消耗品,其中为所述反应容器设置帽。
10.根据权利要求9所述的消耗品,其中所述帽是透明的。
11.根据权利要求9或10所述的消耗品,其中所述帽被可移动地连接至此。
12.根据前述权利要求中任一项所述的消耗品,其中所述反应容器由加碳塑料材料形成。
13.根据前述权利要求中任一项所述的消耗品,其中所述反应容器具有微量滴定部分、填料部和反应室,所述微量滴定部分长度为2cm,填料部的最大外直径为7-8mm,并且深度为大约4-5mm,所述反应室从3mm逐渐减小到2.5mm,整体上具有的壁厚为大约0.8mm,所述反应容器因此具有基本上毛细管的尺寸。
14.根据前述权利要求中任一项所述的消耗品,还包括往复设备,所述往复设备可操作的将所述反应容器从所述加热台转移到所述内容物转移台。
15.根据前述权利要求中任一项所述的消耗品,其中所述消耗品内容物转移台具有刺穿装置,所述刺穿装置被设置成用于刺穿所述反应容器底座并允许所述容器内容物进入所述尺寸分离台内。
16.根据权利要求15所述的消耗品,其中与所述刺穿装置相关联的是所述消耗品中可操作的将所述容器向下推到所述刺穿装置上的装置。
17.根据前述权利要求中任一项所述的消耗品,其中所述内容物转移台包括用于将反应流体吸入所述芯片上的采集井内并且因此将完成反应的流体转移到所述尺寸分离芯片的装置。
18.根据前述权利要求中任一项所述的消耗品,其中所述扩增子尺寸分离台被设置成通过电泳操作。
19.根据前述权利要求中任一项所述的消耗品,其中所述扩增子尺寸分离台包括微流体芯片。
20.根据权利要求20所述的消耗品,其中所述芯片具有玻璃结构。
21.根据前述权利要求中任一项所述的消耗品,其中所述扩增子尺寸分离台包含筛分基质。
22.根据权利要求21所述的消耗品,其中所述筛分基质包括POP4或POP6。
23.根据前述权利要求中任一项所述的消耗品,还包括连接到所述尺寸分离台的电触点,电流通过所述电触点被提供,以便驱动扩增子尺寸分离。
24.根据前述权利要求中任一项所述的消耗品,还包括基本上刚性的箱。
25.根据权利要求24所述的消耗品,在顶表面和底表面上具有可拆卸的薄膜,所述薄膜覆盖反应容器和加热器的入口。
26.根据前述权利要求中任一项所述的消耗品,并且具有长度为120mm,宽度为20mm,深度为30mm的大致尺寸。
27.一种插接台形式的扩增子分离仪器,被构造成接收并保持如前述权利要求中任一项所述的消耗品,所述插接台包含热循环仪设备。
28.根据权利要求27所述的仪器,其中所述热循环仪设备包括金属套管,所述金属套管适合于紧紧地围绕所述反应容器反应室,使得随后贯穿反应容器反应室的长度上与反应容器反应室是连续的,以及在反应容器反应室底座处与所述套管一体的热传递模块。
29.根据权利要求28所述的仪器,其中所述热传递模块是附接到减热模块(HRM)并且设置成在中值温度附近操作的珀尔帖单元,该温度在平均DNA的退火温度附近。
30.根据权利要求27至29中任一项所述的仪器,还包括提供装置,被设置成将所述热循环仪插入所述消耗品内从而使所述套管围绕所述反应容器。
31.根据权利要求27至30中任一项所述的仪器,还包括监测装置,可操作地监测所述反应容器内的PCR进展。
32.根据权利要求31所述的仪器,其中所述监测装置可操作地确定PCR完成了。
33.根据权利要求27至32中任一项所述的仪器,还包括用于确定所述反应容器能够被移动并且引起所述反应容器在所述消耗品中从所述加热台移动到所述内容物转移台的设备。
34.根据权利要求27至33中任一项所述的仪器,还包括可操作地使所述反应容器被推到所述容器内容物转移台上的设备。
35.根据权利要求33或34所述的仪器,其中所述设备包括螺线管驱动的活塞。
36.根据权利要求27至35中任一项所述的仪器,还包括被设置成询问所述消耗品读取台的读取装置。
37.根据权利要求36所述的仪器,其中所述读取装置包括光学设备。
38.根据权利要求37所述的仪器,其中所述光学设备包含依赖LED激发和CCD检测的分光光度计。
39.根据权利要求36至38中任一项所述的仪器,其中所述读取装置包含维持时间基线以便荧光能够相对于时间绘图的装置。
40.根据权利要求27至39中任一项所述的仪器,还被设置成用于为所述消耗品内容物转移台供电。
41.根据权利要求27至40中任一项所述的仪器,还包括可操作地控制所述消耗品尺寸分离台的温度的温度控制装置。
42.根据权利要求27至41中任一项所述的仪器,还具有自动打开的抽屉,被设置成接收和弹出消耗品。
43.根据权利要求27至42中任一项所述的仪器,还具有抓持部,所述抓持部被设置成接收并且保持所述消耗品,并且当相关的过程结束时,释放并且弹出所述消耗品。
44.根据权利要求27至43中任一项所述的仪器,被构造成接收并处理多个消耗品。
45.根据权利要求44所述的仪器,可操作地彼此独立地接收并处理多个装填好的消耗品。
46.根据权利要求27至45中任一项所述的仪器,还包括控制软件和用户界面。
47.根据权利要求27至46中任一项所述的仪器,所述仪器与计算机关联,用于控制、监测和备案目的。
48.根据权利要求46或47所述的仪器,被设置成提供将观察到的扩增子尺寸和光谱与已知数据库进行比较的结果。
49.一种DNA分离方法,用于采用前述权利要求中任一项所述的消耗品和仪器快速检测大量的DNA目标。
50.根据权利要求49所述的方法,还被设置成用于检测血液样品中存在的病原体。
51.根据权利要求49或50所述的方法,还包括:
将提取的DNA载入所述消耗品的所述反应容器内;
将加载的消耗品安装到所述仪器内;
使所述反应容器经受热循环,以诱导聚合酶链反应;
将所述反应容器从所述加热台转移到所述容器内容物转移台;
转移所述容器内容物,以便流入所述尺寸分离台中的尺寸分离通道内;
使扩增子沿着所述尺寸分离台前进;
监测扩增子到达特定台;以及
确定到达的扩增子的身份。
52.根据权利要求51所述的方法,还包括细胞破碎阶段。
53.根据权利要求51或52所述的方法,还包括控制所述尺寸分离通道的温度。
54.根据权利要求49至53中任一项所述的方法,其中采用电泳沿着所述分离通道驱动所述扩增子。
55.根据权利要求54所述的方法,其中电泳筛分凝胶是POP4或POP6。
56.根据权利要求49至55中任一项所述的方法,其中光学地监测扩增子的到达。
57.根据权利要求56所述的方法,还包括产生到达的扩增子的光谱图。
58.根据权利要求49至57中任一项所述的过程,还包括识别所述扩增子。
59.根据权利要求49至58中任一项所述的方法,还包括在进行PCR之前的细胞破碎步骤。
60.一种扩增子分离消耗品,基本上如以上参照附图所描述的。
61.一种扩增子分离仪器,基本上如以上参照附图所描述的。
62.一种扩增子分离和识别方法,基本上如以上参照附图所描述的。
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CN106457251A (zh) | 2017-02-22 |
JP2017505617A (ja) | 2017-02-23 |
US20170225171A1 (en) | 2017-08-10 |
CN106132548A (zh) | 2016-11-16 |
US20170051335A1 (en) | 2017-02-23 |
EP3100029A1 (en) | 2016-12-07 |
GB201401584D0 (en) | 2014-03-19 |
JP2017505616A (ja) | 2017-02-23 |
CN106461554A (zh) | 2017-02-22 |
WO2015114297A1 (en) | 2015-08-06 |
JP2017504340A (ja) | 2017-02-09 |
US20170056879A1 (en) | 2017-03-02 |
JP2017510796A (ja) | 2017-04-13 |
WO2015114296A1 (en) | 2015-08-06 |
EP3100027A1 (en) | 2016-12-07 |
WO2015114294A1 (en) | 2015-08-06 |
EP3100028A1 (en) | 2016-12-07 |
WO2015114295A1 (en) | 2015-08-06 |
US20170232441A1 (en) | 2017-08-17 |
EP3099412A1 (en) | 2016-12-07 |
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