CN106132548A - 现场病原体识别 - Google Patents
现场病原体识别 Download PDFInfo
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Abstract
一种用于生物液体样品中遗传物质的识别的过程,并且该过程包括如下步骤:将足够的纯水注入包含冻干PCR试剂和标记的引物的反应容器以溶解所述试剂和引物;将生物液体样品注入反应容器中;在所述反应容器中使反应容器内容物经受细胞破碎过程;在所述反应容器的内容物上实施病原体特异性聚合酶链反应(PCR);并且监测所述PCR并且从而确定特定遗传物质的存在。一种用于执行所述过程的设备,包括:散热模块(HRM);玻尔贴单体(TEC);容纳传热套管的反应容器;用于将反应容器保持在所述套管中的保持装置;以及用于驱动所述TEC的装置,玻尔贴单体的基板与所述HRM相邻,传热套管与所述TEC的工作面相邻。
Description
技术领域
本发明涉及对可能存在于人或者动物的血液中的病原体的识别。本发明具体地涉及对在血液样品中发现的危险的病原体的快速的现场识别。
发明内容
根据本发明的第一方面,提供了一种设备,用于实施对病原体的快速现场识别,该设备包括:
·散热模块(HRM);
·玻尔贴单体(热电单体)(TEC),玻尔贴单体的基板与HRM相邻并且优选与HRM连接;
·容纳传热套管的反应容器,该传热套管与TEC的工作面相邻并且优选与TEC的工作面连接;
·保持装置,布置成用于将反应容器保持在套管中;
·用于驱动TEC的装置;以及
·光学装置,布置成用于监测反应容器中的反应;
·以及设备被布置成用于使用微滴定反应容器和其支架进行操作的,所述支架被形成来保持反应容器以及容纳套管并且保持地装配于保持装置。
优选的微滴定反应容器是一个由碳载塑料材料形成的容器。使用这样的反应容器,热量可以迅速地传入和传出反应腔。这样的微滴定容器可以具有2cm整体长度的数量级,并且按从上往下的顺序可以包括:帽容纳边缘,填料部分,具有基部的反应腔。填料部分可具有7-8mm的最大外径和大约4-5mm的深度,并且反应腔从3mm直径向下逐渐变细为2.5mm直径,整个具有0.8mm数量级的壁厚。因此,反应容器可基本上是毛细管尺寸的,以最大化热传递的速率。反应容器将通常具有可密封反应容器的透明的盖子。
因为微滴定容器的操纵对于戴手套的手工工人来说是繁琐的,所以优选地为单独的支架装配提供以顶帽形式的东西,并且布置成在与设备的较热部分分离的平坦的表面上是可放置的,用于为过程装料或者卸料。这一单独的支架可具有形成在其上的附属物,用于承载可移除的容器盖子。因此盖子的保持和装配都是很方便的。井由塑料材料形成,而静止地保持在其中的反应容器可以在识别过程完成时易于被丢弃。
优选的热循环设备包括金属套管,该金属套管以这样的方式适于紧密地包围反应容器的反应腔,该方式在金属套管的整个长度上与反应腔相邻,并且在套管的底座处与套管形成整体的是传热脚。传热脚有利地连接至玻尔贴单体的工作面,其底面连接至布置用于在中间温度附近操作的散热模块(HRM),该温度通常在普通DNA的退火温度附近。顶帽有利地容纳套管。在套管的底座中,邻近TEC,可以是热敏电阻器,使得过程的温度能够被连续地监测和控制。套管可以由既是良好的热导体又相对耐腐蚀的金属形成,诸如黄铜。为了最小化玻尔贴单体本身的分层或者它从HRM或者传热脚上剥离的可能性,该连接可通过柔性焊料(诸如铅锡焊料183)来实现。
散热模块(HRM)可以是布置成为向所述散热模块(HRM)提供的传热液体在恒定的温度从所述散热模块(HRM)中流过的设备。在现场的上下文中,为了简化,该液体可以是水。可以有储液器以及相关联的泵,储液器包含被布置用于保持冷却剂在恒定温度的加热元件和/或风扇。
可以有固定块,该固定块被布置来包围并且保护TEC和套管,并且还充当反应容器支架的底座。此布置可以因此使得“带上顶帽”保持地装配支架到固定块,并且将反应容器带入套管中的紧密装配中。
设备还可以包括纯水注入站,该纯水注入站被布置用于在反应容器包含冻干材料时分配一定量的纯水进入反应容器。同样地或者可替代地,可以有被布置用于分配非冻干的这样的试剂的试剂分配器。
根据本发明的另外的方面,提供了微滴定反应容器和微滴定反应容器的支架,支架被形成来保持反应容器以及容纳套管并且保持地装配于固定块。
具体对于微滴定的内容,光学布置也尽可能地小型化。光学布置通常包括被布置用来在反应腔中激发荧光的激发光源,以及收集发出的荧光并且输送其至分光光度计的收集器。激发光源以及或许激发光收集器可以相邻或者靠近反应容器的盖子。收集器优选是光纤。在一个可替代的实施例中,光纤可以是分叉的或者具有两个或多个核,一个核被布置来用于激发,另一个被布置来用于收集。
光源可包括激光二极管或者发光二极管(LED)或者过滤为所要求的发射波长的广谱源卤素灯。优选的光学读取器是分光光度计,但是也可以使用光电二极管。
通常地,包括HRM、TEC、套管和固定块的站可以是阵列中的一个站。对于快速现场操作,该站有利地还具有包括源和收集器的光学单元。该光学单元特别优选布置成可在操作位置与存取(access)位置之间移动。可为此提供铰链装置,该铰链装置被有利地组织从而最小化光路的中断,光路的中断包括一个或多个光纤的弯折。光学单元还可具有保持装置(例如磁体)以确保在操作位置和存取位置的锁定的程度。在操作位置,保持装置优选用于保持该单元紧靠反应容器盖子,并且可包括盖子加热器。
对于现场操作,阵列可包括八个站,并且相应地被连接至病原体检测单元上的现场操作板上,该病原体检测单元还可包括电源供给和控制以及光学系统。该阵列优选组织成为对每个站进行单独控制,而HRM通过共同的加热器和泵互相联通。该单元也被优选构造,从而昂贵的电子器材和光学器材被密封在容器中,该容器的外部可被重复地、彻底地和安全地灭菌而不会有害地影响其中的内容物。
这种布置的优点是,它可以容易地携带并且使用四轮驱动车辆例如陆虎的辅助的12V电池操作。
根据本发明的第二个方面,用于生物液体样品中遗传物质的识别的过程包括如下步骤:
·将生物液体样品注入包含PCR试剂和标记的引物的反应容器中;
·如果必要,在所述反应容器中,使反应容器内容物经受细胞破碎过程;
·在反应容器的内容物上进行病原体特异性聚合酶链反应(PCR);
·监测PCR并且由此确定特定的遗传物质的存在。
优选地,反应容器包含冻干形式的PCR试剂和标记的引物,并且过程的第一步包括在容器中放置足够的纯水以溶解该试剂和引物。优选地还使用荧光实时地执行监控。
理想地,反应容器具有微滴定的比例。这具有特别的优点,例如当血液样品是使用固定容量的抽吸器/分配器例如“MICROSAFE”收集的,则恰好所需量的血液可以被输送至反应容器。不仅如此,并且随后收集和输送可由穿着防护服的卫生工作人员很容易地进行。
相应地应当理解,本发明具体旨在辅助极度危险的病原体(诸如埃博拉病毒)的快速识别。
细胞破碎过程可以包括冷冻然后解冻样品与煮沸然后冷却样品中的一者或者两者,从而其中的遗传物质细胞被破开。这种循环操作可以执行多次,例如多至五个循环。
根据本发明的一个特征,细胞破碎和PCR可使用相同的反应容器和加热/冷却装置来实施。合适的加热/冷却装置包括玻尔贴单体,下文中被称作TEC(热电单体),玻尔贴单体被布置成针对恒定的温度操作,该恒定的温度有利地是样品的冷冻温度和沸腾温度中间的一个温度。典型地,所述中间温度在普通DNA的退火温度附近。该中间的恒定的温度可通过上述的散热模块(HRM)来提供。
与TEC的工作面相邻的可以是传热套管,该传热套管被布置来紧密地容纳反应容器。
监测PCR过程优选由包括光学布置的读取器装置来执行,该光学布置优选结合依靠LED激发和CCD探测的分光光度计。该过程可随后包括将所得的光谱与目标DNA的光谱的比较。
通常地,该设备的尺寸为50mm数量级的长度和55mm的直径。这一方面符合穿防护服的操作者的方便手工处理,另一方面符合用于一次性使用的廉价的生产。用于这样的设备的合适的玻尔贴单体尺寸为9平方毫米。
根据本发明的设备和过程的特别重要的特征是该过程可在10分钟内完成,换言之,患者立等可取。
附图说明
现在将参考附图通过示例来描述过程和设备的实施例,该附图中:
图1是设备的分解图;
图2是微滴定反应容器和其支架的截面;
图3是组装的设备的截面;
图4是八站阵列单元的等距视图;
图5和图6是八站阵列单元的分解图;
图7、图8和图9示出了光学单元;以及
图10示出了阵列单元控制面板和屏幕。
具体实施方式
如图所示,用于病原体的识别的设备包括散热模块HRM、玻尔贴单体TEC、传热套管30、保持装置40、微滴定反应容器50和容器支架60。
散热模块HRM包括容器10和至容器10的可密封的盖子11。容器10具有液体入口12和出口13,盖子具有用于准确和经济地容纳TEC的凹陷14。
TEC具有在凹陷14内连接至盖子11的基面20,以及工作面21。
套管30由黄铜形成并且包括容纳部31和热传输脚32。容纳部31适于紧密地容纳反应容器50的反应腔部分51。用与脚32连接的TEC工作面21的宽度相同的底直径来形成脚32。连接至套管30的是热敏电阻器33。
安装至盖子11的PCB 15将TEC和热敏电阻器33连接,以提供电路和控制电路。
保持装置40包括基板41和连接至基板41的盖42。基板41连接至HRM盖子11。保持装置40的功能是收容和保护套管30以及容纳反应容器50及反应容器50的支架60。
反应容器50是由碳载塑料材料形成的微滴定容器,具有2cm的整体长度。它包括,从上往下的顺序,帽容纳边缘51,填料部分52,以及具有基部54的反应腔53。填料部分52具有7mm的最大外径和5mm的深度。反应腔53从3mm向下逐渐变细为2.5mm,整体具有0.8mm的壁厚。因此反应腔50大致具有毛细管尺寸。
支架60容纳和保持反应腔50。它的形状为顶帽,并且因此容纳套管30。形成在支架60上的是柔性附属物61,在柔性附属物61的远端承载透明的盖子62,盖子62可密封反应容器50的边缘51。
如图2所示,容器50适于从固定容量的毛细管抽吸器/分配器(MICROSAFE)70接收样品。这继而可以从标准的血液提取设备接收它的样品。
在图4至图10中示意性地示出了图1至图3中所描绘的识别设备的阵列的一个实施例。装配于阵列板80的是八个这样的设备。在装配时,保持装置的盖42或者支架60可在板80以上被看到。在板80下是HRM。HRM的入口12和出口13与其它设备的入口和出口联通,也与加热器兼泵单元81联通,从而液体可以以恒定的温度通过设备的HRM连续地循环。板80安装至壳体90。
光学布置包括在位置83处铰接到安装在阵列板80上的支柱84的光学单元82,以及安装在壳体90中的分光光度计。光学单元82在图7至图9中具体地示出,并且包括分叉光纤电缆85,其中一个芯体是激发光纤而另一个是收集器。铰接部的位置83在最小化线缆85的干扰的位置。
光学单元82能够在操作位置和存取位置之间通过切换件86移动。光学单元82具有保持磁体87和88,保持磁体87和88布置为分别在存取位置和操作位置保持单元82。支柱84上的对应的磁体87a和88a用于留住磁体87和88。单元82还结合一个盖子加热器89和相关联的传感器,从而在操作位置,保持装置用于保持盖加热器89紧靠盖子62,以防止盖子62起雾。
在壳体90内是密封的盒子91,密封的盒子91包含设备电子器材、光源和收集的光的读取器。因此这些被保护防止溢出的病原体的进入,而盒子91的外部可被消毒。柔性管92在密封的盒子91和光学单元83之间连通,柔性管92对二者中每一者密封,并且包含用于加热器89和光缆85的电输入。
在一个可替代的实施例中,光源和/或光传感器安装在单元82中。在另一个可替代的实施例中,该设备结合了布置为用于输送一定量的纯水至反应容器中的站,从而液化冻干试剂。在另一个实施例中,该设备结合了布置成将试剂输送至反应容器中的站。
壳体90上安装有具有触摸屏94的显示面板93。触摸屏94包括操作者用于启动和控制任何一个设备中的过程并且观察过程的结果的装置。显示面板包括每个设备的一个LED单元,并且提供简单的指示,该指示是特定的设备在使用中以及其中的过程的进展。
用于血液样品中遗传物质的识别并且使用该设备的过程包括如下步骤:
·向上述的包含冻干的PCR试剂和标记的引物的微滴定反应容器中注入足够量的纯水;
·向反应容器中注入生物液体样品;
·如果必要,在所述反应容器中使反应容器内容物经受细胞破碎过程;
·在反应容器中,在反应容器的内容物上实施病原体特异性聚合酶链反应(PCR);以及
·监控PCR并且由此确定特定的遗传物质的存在。
血液样品将采用固定容量的毛细管抽吸器/分配器(如“MICROSAFE”)来收集,从而恰好所需量的血液可被输送至反应容器。采用本文所述的设备,该收集和输送可方便地由穿着防护服的卫生工作人员来执行。
对于血液,细胞破碎过程包括煮沸然后冷却样品,从而其中的遗传物质细胞被破开。此循环操作可执行多次,例如多至五次。
由于TEC 14的基面20保持在稍微高于普通DNA/RNA的退火温度的恒定的温度,至工作面21的正电流引起该面21加热至高于基面20的温度的一个温度。提供给工作面21的负电流引起其温度下降至低于基面20的温度。
PCR过程被读取器装置监控,该读取器装置包括光学布置,该光学布置典型地结合依靠LED激发和CCD探测的分光光度计。得到的光谱然后与目标DNA/RNA的光谱比较,并且因此确定样品是否包含目标DNA。
一旦任何特定设备中的过程完成,承载密封的反应容器50的支架60可从板80/设备上被移除并且被丢弃,优选被焚化。
设备的典型尺寸是40mm的长度和55mm的直径。这一方面符合穿防护服的操作者方便手工处理,另一方面符合用于一次性使用的廉价的生产。这意味着支架60可以是大约55mm的直径和2cm的深度。用于这样的设备的合适的玻尔贴单体尺寸为9平方毫米。壳体90的整体尺寸(包括光学单元)为55cm长×45cm宽×35cm深。
尽管该设备和过程被描述为适合于检测血液中的目标DNA/RNA,它们也可方便地用于检测尿液或唾液中目标DNA/RNA。
Claims (53)
1.一种用于实施病原体的快速现场识别的设备,所述设备包括:
·散热模块(HRM);
·热电单体(TEC),热电单体(TEC)的基板与HRM相邻;
·容纳传热套管的反应容器,所述传热套管与TEC的工作面相邻;
·保持装置,布置成用于将反应容器保持在套管中;
·用于驱动TEC的装置;以及
·光学装置,布置成用于监测所述反应腔中的过程的进展;
·以及布置成用于使用微滴定反应容器和微滴定反应容器的支架进行操作的设备,所述支架被形成来保持所述反应容器以及容纳套管并且保持地装配于所述保持装置。
2.如权利要求1所述的设备,并且其中TEC基板连接至HRM并且套管连接至TEC的工作面。
3.如权利要求2所述的设备,其中所述连接使用焊料实现。
4.如权利要求1-3中任一权利要求所述的设备,并且布置成用于使用具有反应腔的微滴定反应容器的操作。
5.如前述任一权利要求所述的设备,并且其中所述反应容器是碳载塑料材料。
6.如前述任一权利要求所述的设备,并且其中所述反应容器具有2cm数量级的整体长度并且按照下降顺序包括:帽容纳边缘,填料部分,具有基部的反应腔,所述填料部分具有7-8mm的最大外径和大约4-5mm的深度,并且所述反应腔从3mm向下逐渐变细为2.5mm,整个具有0.8mm数量级的壁厚。
7.如前述任一权利要求所述的设备,并且其中所述反应容器具有能够密封至所述反应容器的透明的盖子。
8.如前述任一权利要求所述的设备,并且其中所述传热套管适于紧密地包围所述反应容器反应腔。
9.如前述任一权利要求所述的设备,并且其中所述套管由黄铜形成。
10.如前述任一权利要求所述的设备,并且布置用于在普通DNA的退火温度附近的操作。
11.如前述任一权利要求所述的设备,并且结合使所述过程的温度能够被连续地监测和控制的热敏电阻器。
12.如前述任一权利要求所述的设备,并且其中所述散热模块(HRM)是布置成为向所述散热模块(HRM)提供的传热液体在恒定的温度从所述散热模块(HRM)中流过的设备。
13.如权利要求12所述的设备,并且其中所述散热模块包括包含加热元件的储液器以及相关联的泵,所述加热元件被布置用来保持所述传热液体在恒定的温度。
14.如权利要求12或13所述的设备,并且其中所述传热液体是水。
15.如前述任一权利要求所述的设备,并且其中所述保持装置包括固定块,所述固定块被布置来包围并且保护TEC和套管,并且还充当所述反应容器的底座。
16.如前述任一权利要求所述的设备,并且其中用于驱动TEC的装置包括与套管相邻的热敏电阻器。
17.如前述任一权利要求所述的设备,并且布置成由12伏电源驱动。
18.如前述任一权利要求所述的设备,并且具有包括源和收集器的光学单元。
19.如权利要求18所述的设备,并且其中所述光学单元被布置成能够在操作位置和存取位置之间移动。
20.如权利要求19所述的设备,并且具有铰链装置,所述铰链装置布置成用于在操作位置和存取位置之间移动所述光学单元。
21.如权利要求18至20中任一所述的设备,并且其中所述光学单元具有保持装置,以保持所述单元在所述操作位置和存取位置。
22.如权利要求21所述的设备,并且其中所述保持装置包括磁体。
23.如权利要求18至22中任一所述的设备,并且其中所述光学单元结合反应容器盖子加热器。
24.如前述任一权利要求所述的设备,并且其中HRM、TEC、套管和保持装置形成站的阵列中的站。
25.如权利要求24所述的设备,并且其中所述阵列包括八个站。
26.如权利要求24或权利要求25所述的设备,并且其中所述阵列安装在板中。
27.如权利要求24至26中任一权利要求所述的设备,并且布置成用于单独的站的单独的操作和控制。
28.如前述任一权利要求所述的设备,并且结合纯水分配器。
29.如前述任一权利要求所述的设备,并且结合试剂分配器。
30.一种微滴定反应容器和滴定反应容器的支架,所述支架被形成来保持所述反应容器以及容纳套管并且保持地装配于权利要求1至29任一所述的设备的保持装置。
31.如权利要求30所述的微滴定反应容器,并且由碳载塑料材料形成。
32.如权利要求30或31所述的容器,并且具有2cm量级的整体长度,并且以下降顺序包括,帽容纳边缘,填料部分,具有基部的反应腔,所述填料部分具有7-8mm的最大外径和大约4-5mm的深度,并且所述反应腔从3mm向下逐渐变细为2.5mm,整个具有0.8mm数量级的壁厚。
33.如权利要求30至32中任一权利要求所述的容器,并且具有能够密封至所述容器的透明的盖子。
34.如前述权利要求中任一权利要求所述的容器,并且其中所述支架具有形成在支架上的承载可移除的容器盖子的附属物。
35.如权利要求30至34中任一权利要求所述的容器,并且其中所述支架具有顶帽的形式。
36.一种用于生物液体样品中遗传物质的识别的过程,并且包括如下步骤:
·将所述生物液体样品注入包含PCR试剂和标记的引物的反应容器中;
·在所述反应容器的内容物上进行病原体特异性聚合酶链反应(PCR);以及
·监测PCR并且由此确定特定的遗传物质的存在;
·并且使用如权利要求1至29中任一所述的设备。
37.如权利要求36所述的过程,并且进一步包括细胞破碎步骤。
38.如权利要求36或权利要求37所述的过程,并且其中PCR试剂和标记的引物是冻干的,并且第一步骤包括将足够的纯水放入容器中以溶解试剂和引物。
39.如权利要求36至38中任一权利要求所述的过程,并且其中所述反应容器具有微滴定的比例。
40.如权利要求36至39中任一权利要求所述的过程,并且其中固定容量的毛细管抽吸器/分配器例如“MICROSAFE”能够被用于输送样品至所述反应容器。
41.如权利要求36至40中任一权利要求所述的过程,并且其中所述细胞破碎过程包括冷冻然后解冻所述样品与煮沸然后冷却所述样品中的一者或两者,从而其中的遗传物质细胞被破开。
42.如权利要求38所述的过程,并且其中随后使用相同的反应容器和加热/冷却装置实施PCR。
43.如权利要求36至42中任一权利要求所述的过程,并且其中所述加热/冷却装置包括玻尔贴单体,所述玻尔贴单体布置为针对恒定的温度操作。
44.如权利要求43所述的过程,并且其中所述恒定的温度介于所述样品的冷冻温度和沸腾温度的中间。
45.如权利要求44所述的过程,并且其中所述中间温度在普通DNA的退火温度附近。
46.如权利要求36至45中任一权利要求所述的过程,并且其中与TEC的工作面相邻的是传热套管,所述传热套管布置为紧密地容纳反应容器。
47.如权利要求36至46中任一权利要求所述的过程,并且其中监测PCR过程由包括光学布置的读取器装置来执行。
48.如权利要求47所述的过程,并且其中所述光学布置结合依靠LED激发和CCD探测的分光光度计。
49.如权利要求48所述的过程,并且包括对得到的光谱与目标DNA的光谱的比较。
50.一种基本上如上文参考所述附图所述的用于生物液体样品中遗传物质的识别的过程。
51.一种基本上如上文参考所述附图所述的用于生物液体样品中遗传物质的识别的设备。
52.一种基本上如上文参考所述附图所述的用于生物液体样品中遗传物质的识别的微滴定反应容器及微滴定反应容器的支架。
53.一种基本上如上文结合所述附图所述的一种阵列单元,用于安装设备的阵列以及控制设备中的热循环过程。
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CN201580014422.7A Pending CN106164651A (zh) | 2014-01-29 | 2015-01-28 | 用于热循环生物化学操作的装置和方法 |
CN201580014424.6A Withdrawn CN106132548A (zh) | 2014-01-29 | 2015-01-28 | 现场病原体识别 |
CN201580014421.2A Pending CN106457251A (zh) | 2014-01-29 | 2015-01-28 | 用于反应的过程和设备 |
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JP2017505617A (ja) | 2017-02-23 |
US20170051335A1 (en) | 2017-02-23 |
EP3099412A1 (en) | 2016-12-07 |
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EP3100028A1 (en) | 2016-12-07 |
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JP2017504340A (ja) | 2017-02-09 |
JP2017510796A (ja) | 2017-04-13 |
WO2015114294A1 (en) | 2015-08-06 |
GB201401584D0 (en) | 2014-03-19 |
CN106164651A (zh) | 2016-11-23 |
CN106457251A (zh) | 2017-02-22 |
WO2015114295A1 (en) | 2015-08-06 |
US20170232441A1 (en) | 2017-08-17 |
EP3100029A1 (en) | 2016-12-07 |
JP2017505616A (ja) | 2017-02-23 |
EP3100027A1 (en) | 2016-12-07 |
US20170056879A1 (en) | 2017-03-02 |
CN106461554A (zh) | 2017-02-22 |
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