WO2017096737A1 - 一种基于毛细管微阵列的核酸高通量快速检测方法 - Google Patents

一种基于毛细管微阵列的核酸高通量快速检测方法 Download PDF

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WO2017096737A1
WO2017096737A1 PCT/CN2016/078408 CN2016078408W WO2017096737A1 WO 2017096737 A1 WO2017096737 A1 WO 2017096737A1 CN 2016078408 W CN2016078408 W CN 2016078408W WO 2017096737 A1 WO2017096737 A1 WO 2017096737A1
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nucleic acid
capillary
microarray
capillary microarray
detection method
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French (fr)
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陶生策
杨立桃
邵宁
胡佳莹
陈建伟
张大兵
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上海交通大学
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    • C12Q1/6869—Methods for sequencing
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to a detection method in the field of biomedicine, in particular to a method for rapid detection of nucleic acid high-throughput based on capillary microarray.
  • DNA/RNA nucleic acid
  • the existing commercial nucleic acid rapid detection platform is mostly based on traditional PCR (polymerase chain reaction) technology or real-time quantitative PCR technology, and is carried out in a common PCR tube or a 96-well PCR plate, which is inconvenient to use (eg, the instrument is bulky, not It is easy to carry, consumes a lot of power, complicated operation, etc., and the flux is low (each reaction can only detect one target), and it is difficult to meet the actual needs.
  • microchip platforms including microarray chips and microfluidic chips
  • microfluidic chips are increasingly being used in various biochemical reactions including nucleic acid amplification and detection.
  • Numerous nucleic acid amplification and detection methods based on microarray chips and microfluidic chips are available (Ahmad, F., et al., 2012, Analyticazia acta, 733, 1-15; Asiello, PJ, et al., 2011, Lab on a Chip, 11, 1420-1430), and some of them can achieve multiplex detection, such as Li, using microwell array chips to achieve up to 100 weight PCR amplification (Li Y., et al., 2011, Lab on a chip 11.21, 3609-3618), Guo et al.
  • Capillary is a good biochemical reaction vessel, which is low in cost and large in surface area to volume ratio. Rate (Mastichiadis C., et al., 2008, Trends in Analytical Chemistry 27.9, 771-784), siphon properties are easily loaded in the sample, and can be conveniently integrated into a capillary array for multiplex reaction and detection. Due to its excellent properties, the capillary has been in gas chromatography (Ballschmiter, K., & Zell, M. 1980, Fresenius' Journal of Analytical Chemistry, 302, 20-31), capillary electrophoresis (Ewing A., et al, 1989, Anal). .Chem., 61, 292-303), protein detection (Cao Y., 2015, Journal of fluorescence, 25, 563-568) and other fields have been widely used.
  • LAMP loop-mediated isothermal amplification
  • the object of the present invention is to provide a high-throughput rapid detection method for nucleic acid based on capillary microarray, in particular to a high-throughput detection of nucleic acid based on capillary microarray for improving detection flux and detection efficiency, reducing detection cost and sample consumption. method.
  • This method is applicable to various fields of nucleic acid detection.
  • the invention constructs a capillary microarray multiple reaction platform, which can realize the rapid parallelization of the nucleic acid amplification reagent and the rapid parallelization of the amplification reaction in a small system. Compared with the existing methods, it has its own unique advantages such as high detection flux, simple experimental operation, low sample consumption, low detection cost, and no need for expensive equipment. It can be widely used in the fields of rapid diagnosis of infectious diseases, entry-exit inspection and quarantine, on-site testing of genetically modified crop products, on-site inspection of food water source microorganisms, identification of crime scenes and bio-terrorism.
  • the invention relates to a method for rapid detection of nucleic acid high-throughput based on capillary microarray, which comprises the following steps:
  • the first step several sets of nucleic acid amplification primers are separately added to several micro-pipes on the capillary microarray, and dried.
  • the capillary microarray is then fixed in a transparent reaction tube;
  • the nucleic acid amplification reaction component other than the primer containing the sample nucleic acid is added into the micro-channel to form a nucleic acid amplification system, and the reaction nozzle is sealed;
  • the reaction tube containing the nucleic acid amplification system is subjected to an amplification reaction under temperature control conditions;
  • the real-time detection is realized by measuring the continuous fluorescence signal of the amplification reaction or the end point detection is performed by measuring the fluorescence signal after the end of the amplification reaction.
  • the effect of the drying is to attach the nucleic acid amplification primer to the inner wall of the microchannel.
  • the nucleic acid amplification primers include, but are not limited to, common PCR primers, real-time quantitative PCR primers, loop-mediated isothermal amplification primers, rolling circle amplification primers, and recombinase polymerase amplification primers;
  • the nucleic acid amplification primer is a real-time quantitative PCR primer
  • the primer is added to the microchannel together with the corresponding probe.
  • the capillary microarray further comprises a substrate
  • a plurality of micro-pipes in the capillary microarray are arranged in an array on the substrate, and a portion of the micro-pipe end exposes the surface of the substrate; the upper surface of the substrate, the outer surface of the micro-pipe exposed the base portion, and the bottom end of the micro-pipe
  • the inner surface is a hydrophobic surface
  • the hydrophobic surface can be achieved by applying a layer of hydrophobic coating to the respective surface.
  • the capillary microarray is obtained by one-time processing or assembling a capillary microarray substrate containing a micro-tube hole array with a micro-pipe; further, the processing comprises molding or Machining.
  • the material of the substrate comprises plastic, glass, metal and other polymer materials; wherein the other polymer materials such as polydimethylsiloxane, polymethyl methacrylate, polytetrafluoroethylene, Rubber, etc.; the micro-pipe is a hydrophilic capillary, the portion of the micro-pipe that exposes the substrate and the bottom end surface of the micro-pipe are hydrophobic, and the material of the micro-pipe itself is hydrophilic.
  • the other polymer materials such as polydimethylsiloxane, polymethyl methacrylate, polytetrafluoroethylene, Rubber, etc.
  • the micro-pipe is a hydrophilic capillary, the portion of the micro-pipe that exposes the substrate and the bottom end surface of the micro-pipe are hydrophobic, and the material of the micro-pipe itself is hydrophilic.
  • the adding specifically refers to dissolving the nucleic acid amplification primer in the crosslinking agent and then adding the microchannel;
  • crosslinking agent is any one of the following three mixed liquids:
  • the adding specifically refers to introducing the nucleic acid amplification reaction components into each by an inverted siphon method.
  • the adding specifically refers to adding by using a sample loading device; wherein the sample loading device comprises a sample cell and a handle, and the handle is connected to the bottom of the sample cell; the sample cell has an outer cross section smaller than the inner cross section of the reaction tube, and the sample pool The inner cross section is larger than the transverse cross section of the capillary microarray.
  • the depth of the sample cell is slightly lower than the height of the surface of the microchannel exposed to the substrate, and the inner surface of the sample cell is hydrophilic; (see Figure 3 for a schematic diagram of the sample loading process).
  • the inverted siphon mode means: inserting a sample cell filled with the nucleic acid amplification reaction component solution into the reaction tube downward, the nucleic acid amplification reaction component and the hydrophilic micro-pipe After the top end of the inner wall is in contact, it is quickly filled with a hydrophilic pipe by siphoning, and then the sample loading device is removed.
  • the measurement may be performed by means of a fluorescence detecting device or a photometric detecting device, or the color or brightness difference may be discriminated by the naked eye; specifically, a light source corresponding to the emitted wavelength is required before each measurement.
  • the reactants in the microchannels were irradiated and measured.
  • the recyclable product obtained in the fifth step can be used for subsequent detection, specifically: the product in a specific capillary can be recovered separately or all the capillary products can be recovered at one time, and further used for agarose gel electrophoresis, nucleic acid hybridization. , DNA chip, DNA sequencing and other methods of detection.
  • the temperature-controlled device in the fourth step and the measurement in the fifth step are integrated into an automation device, and the automatic operation is controlled by the software program.
  • the method can be implemented either separately in a single reaction tube or in parallel in an integrated 8-tube, 96-well or 384-well plate.
  • the present invention has the following beneficial effects:
  • the specially designed sample loading device can quickly and conveniently add the reaction solution to multiple micro-pipes at one time, thus improving the throughput and detection of one test. effectiveness;
  • micro-pipes as a reaction chamber also greatly reduces the amount of reagent samples, thereby reducing the cost of testing;
  • the invention can be applied to various high-throughput rapid detection fields of nucleic acids, such as rapid detection of infectious diseases, entry and exit inspection and quarantine, food safety and genetic testing, and criminal investigation and identification.
  • Figure 2 is a capillary microarray
  • 21 is a microchannel
  • 22 is a hydrophobic surface at the top of the substrate
  • 23 is a substrate.
  • Figure 3 is a schematic diagram of the principle of loading
  • 31 is a hydrophilic loading tank in the loading device
  • 32 is a handle in the loading device
  • 33 is a reaction tube
  • 34 is a capillary microarray.
  • Figure 4 is a schematic view showing the arrangement of primers in Example 1 and Example 2;
  • 1-10 are: P-CaMV35S, bar, CP4epsps, P-FMV35S, pat, T-nos, nptII, ADH1, blank control, blank control;
  • Figure 5 is a schematic diagram showing the results of Example 1.
  • Figure 6 is a schematic diagram showing the results of Example 2.
  • Example 1 Multiple detection of known transgenic materials using loop-mediated isothermal amplification (LAMP)
  • the seed powder of the existing transgenic corn event MON863 in the laboratory was extracted and purified by a commercial DNA extraction kit, and the concentration of the DNA was verified by NanoDrop 1000 as a sample to be tested.
  • the multiple LAMP reaction is carried out using a capillary microarray (see Fig. 2, specifically including the microchannel 21 and the substrate 23, the top surface of the substrate 23 and the portion of the microchannel that exposes the portion 22 of the top surface of the substrate is hydrophobic).
  • LAMP primers 8 groups were previously pipetted (microchannels 1-10 in microarray were: P-CaMV35S, bar, CP4epsps, P-FMV35S, pat, T-nos, nptII, ADH1, blank control, blank control) ) separately added to each of the arrays
  • the capillary was fixed in a dry state, and a LAMP reaction system containing a DNA template was added by an inverted siphon method to carry out a multiple LAMP reaction. There is only 1.6 ⁇ L per reaction system. The specific operation process is shown in Figure 1.
  • the specific sample loading process is shown in FIG. 3, and the sample loading device is used for loading.
  • the sample loading device includes a sample cell 31 and a handle 32.
  • the handle 32 is connected to the bottom of the sample cell 31; the sample cell 31 has an outer cross section smaller than the inner cross section of the reaction tube.
  • the inner cross section of the sample cell 31 is larger than the cross section of the capillary microarray, the depth of the sample cell 31 is slightly smaller than the height of the portion of the microchannel exposed substrate surface, and the inner surface of the sample cell 31 is hydrophilic.
  • the sample cell 31 filled with the DNA template containing the LAMP reaction system (except the primer) is inserted downward into the reaction tube, and the nucleic acid amplification reaction component is in contact with the top end of the inner wall of the hydrophilic micro-tube, and is quickly filled with hydrophilic by siphon action. Slight micro-pipes, then remove the sample loading device.
  • reaction conditions are conventional nucleic acid amplification conditions.
  • Results detection and analysis After the reaction is completed, a hand-held UV lamp with an emission wavelength of 365 nm is irradiated from the side of the reaction tube to excite the reactants in the microchannel to fluoresce, and then a digital camera is used to photograph the fluorescent signal in each capillary of the microarray from the top of the reaction tube. .
  • the photo format was converted to the 16Bit TIFF format using Photoshop 7.0 (Adobe Systems Inc., USA), and the specific fluorescence intensity in each capillary was read using GenePix Pro 6.1 (Molecular Devices, USA).
  • the positive signals in the results are the sequence numbers: 1, 6, 7, and 8, respectively.
  • the corresponding target names are: P-CaMV35s, T-nos, nptII, and maize endogenous genes. ADH1. Looking up the relevant databases and literature, it is theoretically found that the transgenic elements contained in the transgenic maize and the maize endogenous genes were all detected at one time in the capillary microarray multiple LAMP reaction. For the two replicates of the same sample, the results were identical and consistent with expectations.
  • the corn sample M2 collected from Shanghai Port and having unknown composition was tested by Shanghai Entry-Exit Inspection and Quarantine Bureau.
  • the detection result is shown in Fig. 6; from Fig. 6 and in combination with the mode Fig. 4, the positive signals in the result are the serial numbers: 1, 4, 5, 6, and 8, respectively, and the corresponding target names are: P-CaMV35s, FMV- 35S, pat, T-nos and maize endogenous gene ADH1.
  • the detected targets are completely identical, so the detection method can be considered to have high specificity and accuracy.
  • the results of the real-time PCR experiment are as follows:

Abstract

提供了一种基于毛细管微阵列的核酸高通量快速检测方法。

Description

一种基于毛细管微阵列的核酸高通量快速检测方法 技术领域
本发明涉及生物医学领域的检测方法，具体涉及一种基于毛细管微阵列的核酸高通量快速检测方法。
背景技术
基于核酸(DNA/RNA)的便携式快速检测在传染病快速诊断、出入境检验检疫、转基因作物产品现场检测、食品水源微生物现场检测、犯罪现场证物鉴定及生物反恐等领域有着广泛而迫切的需求(Yager P.，et al，2008，Annu.Rev.Biomed.Eng.，10，107-144；Niemz A.，et al，2010，Trends in Biotechnology,29,240-250)。现有的商业化核酸快速检测平台大多基于传统PCR(聚合酶链式反应)技术或者实时定量PCR技术，在普通PCR管或96孔PCR板中进行，存在使用不便(如仪器体积较大，不便于携带、耗电量大、操作复杂等)、通量较低(每个反应基本只能检测一种靶标)等问题，难以满足实际需求。
随着微加工技术的发展，微芯片平台，包括微阵列芯片和微流控芯片，开始越来越多地应用于包括核酸扩增及检测在内的各种生化反应中。现已有很多基于微阵列芯片和微流控芯片的核酸扩增及检测方法(Ahmad,F.,et al.，2012，Analytica chimica acta,733,1-15；Asiello,P.J.,et al.，2011，Lab on a Chip,11,1420-1430)，并且其中有一些能实现多重检测，如Li等利用微孔阵列芯片实现了多达100重的PCR扩增(Li Y.,et al.，2011，Lab on a chip 11.21,3609-3618)，Guo等利用微流控液滴芯片结合毛细管电泳实现了超过20重的PCR扩增(Guo J.,et al.,2011,Analytical chemistry,83.5,1579-1586)，Fang等利用PDMS微流控芯片和环介导等温扩增实现了对10种核酸靶标的并行检测(Fang,X.,et al.,2010，Analytical chemistry,83,690-695)。虽然上述基于微阵列芯片和微流控芯片的核酸扩增及检测平台大大缩小了反应的体积进而减少了试剂样本耗费，同时增加了便携性，并且通量也得到了提高，但依然存在一些问题，如制作工艺复杂难以标准化、成本较高、操作较为复杂、依赖专门的仪器设备等，限制了其实用性。
毛细管是一种良好的生化反应容器，其成本低廉、表面积体积比大提高生化反应效 率(MastichiadisC.,et al.，2008，Trends in Analytical Chemistry 27.9，771-784)、虹吸特性易于反应中样品自动载入、而且可方便地集成为毛细管阵列而用于多重反应和检测。由于其种种优良特性，毛细管已在气相色谱(Ballschmiter,K.,&Zell,M.1980，Fresenius'Journal of Analytical Chemistry,302,20-31)、毛细管电泳(Ewing A.，et al，1989,Anal.Chem.，61,292-303)、蛋白质检测(Cao Y.，2015，Journal of fluorescence,25，563-568)等领域得到了广泛应用。
近来也有人在毛细管内成功实现了核酸扩增反应，如Manage等人在毛细管凝胶内实现了PCR扩增反应(Manage D.，et al，2013,Lab Chip,13,2576-2584)，McCarthy等人利用padlock探针和RCA(滚环扩增)在毛细管中成功扩增并检测了两种病毒的DNA(McCarthy,E.,et al.，2006，Analytical and bioanalytical chemistry，386，1975-1984)，Zhang等在毛细管中用LAMP(环介导等温扩增)技术实现了对血液中一种SNP的快速检测(Zhang L.，et al，2014,Anal.Chem.，86,10461-10466)，Liu等利用LAMP技术在毛细管阵列中实现了对多份样本中结核分枝杆菌的并行检测。这些方法都利用了毛细管的优良特性，降低了试剂和样品的耗费，同时大大降低了能耗而增加了便携性，但均难以在一次反应中实现对较多靶标的并行检测。如将微型毛细管微阵列多重反应平台与现有核酸检测技术相结合，将是一个很好的高通量核酸快检解决方案。
发明内容
本发明的目的在于提供一种基于毛细管微阵列的核酸高通量快速检测方法，具体是一种提高检测通量和检测效率、降低检测成本和样品耗费的基于毛细管微阵列的核酸高通量检测方法。该方法适用于各种核酸检测领域。
本发明构建了一套毛细管微阵列多重反应平台，可非常简单地在微小体系内实现核酸扩增试剂的快速并行进样及扩增反应的快速并行进行。相比较现有方法，有其特有的优点如，检测通量高、实验操作简单、样品耗费少、检测费用低、无需昂贵设备等。可以在传染病快速诊断、出入境检验检疫、转基因作物产品现场检测、食品水源微生物现场检测、犯罪现场证物鉴定及生物反恐等领域得到广泛的应用。
本发明的目的是通过以下技术方案来实现的：
本发明涉及一种基于毛细管微阵列的核酸高通量快速检测方法，其特征在于，包含以下步骤：
第一步、将若干组核酸扩增引物分别加入毛细管微阵列上的若干微管道中，干燥， 再将毛细管微阵列固定于透明反应管中；
第二步、将含有样品核酸的除引物外的其他核酸扩增反应组分加入微管道中，形成核酸扩增体系，封住反应管口；
第三步、将装有核酸扩增体系的反应管置于控温条件下进行扩增反应；
第四步、通过对扩增反应连续荧光信号测量实现实时检测或对扩增反应结束后一次荧光信号测量实现终点检测。
优选地，第一步中，所述干燥的作用是为了将核酸扩增引物附于微管道内壁。
优选地，第一步中，所述核酸扩增引物包括但不限于：普通PCR引物、实时定量PCR引物、环介导等温扩增引物、滚环扩增引物、重组酶聚合酶扩增引物；
其中，当所述核酸扩增引物为实时定量PCR引物时，引物是同相应探针一并加入微管道中的。
优选地，第一步中，所述毛细管微阵列还包括基底；
其中，毛细管微阵列中若干个微管道以阵列方式贯穿排布于基底上，并且微管道端部有少部分露出基底表面；基底的上表面、微管道露出基底部分的外表面以及微管道底端内表面均为疏水性表面；
所述疏水性表面可通过在相应表面涂覆一层疏水性涂料来实现。
优选地，第一步中，所述毛细管微阵列的获得方式包括一次性加工成型或将含微管道孔阵列的毛细管微阵列基底与微管道组装；进一步地，所述加工成型包括制模浇铸或机械加工。
进一步优选地，所述基底的材质包括塑料、玻璃、金属及其它高分子材料；其中，所述其它高分子材料如聚二甲基硅氧烷、聚甲基丙烯酸甲酯、聚四氟乙烯、橡胶等；微管道为亲水性毛细管，微管道露出基底的部分及微管道的底端表面为疏水的，而微管道本身的材质为亲水的。
优选地，第一步中，所述加入具体指将核酸扩增引物溶于交联剂中后再加入微管道中；
其中，所述交联剂为以下三种混合液中的任意一种：
a、质量百分比为0.1-1％的壳聚糖的乙酸水溶液，pH为4.5-6.0；
b、质量百分比为0.1-1％的明胶水溶液；
c、质量百分比为0.05-5％的聚乙二醇水溶液。
优选地，第二步中，所述加入具体指通过倒置虹吸方式将核酸扩增反应组分引入各 微管道中；
进一步地，所述加入具体指采用加样装置加入；其中，所述加样装置包括样品池和手柄，手柄连接于样品池底部；样品池为外横截面小于反应管的内横截面，样品池的内横截面大于毛细管微阵列横的截面，样品池的深度略小于微管道露出基底表面部分的高，样品池的内表面为亲水性；(加样过程示意图见附图3)。
优选地，第二步中，所述倒置虹吸方式是指：将充满核酸扩增反应组分溶液的加样装置样品池朝下插入反应管中，核酸扩增反应组分与亲水性微管道内壁顶端接触后靠虹吸作用迅速充满亲水性管道，之后再移除加样装置。
优选地，第五步中，所述测量可以是借助荧光检测设备或光度检测设备进行检测，也可以是通过肉眼辨别颜色或亮度差异；具体是指：每次测量前需用相应发射波长的光源照射微管道内反应物，再进行测量。
优选地，第五步得到的可回收产物可用于后续其它检测，具体是指：可单独回收特定毛细管内的产物或一次性回收所有毛细管内产物，进一步用于包括琼脂糖凝胶电泳、核酸杂交、DNA芯片、DNA测序等方法的检测。
优选地，所述方法中，第四中的温控的装置及第五步的所述测量可集成于一个自动化装置，由软件程序控制其自动运行。
优选地，所述方法既可以在单个反应管中单独实现，也可以在集成的8连管、96孔板或384孔板中并行实现。
与现有技术相比，本发明具有如下有益效果：
1、利用毛细管微阵列和亲疏水特性，采用特殊设计的加样装置(见附图3)可快速便捷地将反应液一次性加入多个微管道中，从而提高了一次检测的通量和检测效率；
2、采用微管道作为反应腔也大大减小了试剂样品用量，从而降低了检测成本；
3、本发明可适用于各种核酸高通量快速检测领域，如传染病快速检测、出入境检验检疫、食品安全和转基因检测、刑侦鉴定等。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为本发明以实施例1为例的工作流程图；
图2为毛细管微阵列；
其中，21是微管道，22是基底顶部的疏水表面，23是基底。
图3为加样原理示意图；
其中31是加样装置中的亲水性加样池，32是加样装置中的手柄，33是反应管，34是毛细管微阵列。
图4为实施例1和实施例2中引物排列模式图；
其中，1-10分别为：P-CaMV35S、bar、CP4epsps、P-FMV35S、pat、T-nos、nptII、ADH1、空白对照、空白对照；
图5为实施例1结果示意图；
图6为实施例2结果示意图。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
实施例1：利用环介导等温扩增(LAMP)对成分已知转基因材料的多重检测
针对目前转基因产品中常检出的7种转基因元件和玉米的内源参照基因，查询相关文献，找到针对这些基因的LAMP引物组。所有引物由英潍捷基公司(上海)合成。用含钙黄绿素染料的常规LAMP反应对所有引物进行验证筛选，每种基因筛选出一套可以成功检出的LAMP引物组。引物具体信息如下表1所示：
表1
Figure PCTCN2016078408-appb-000001
Figure PCTCN2016078408-appb-000002
将实验室已有的转基因玉米事件MON863的种子粉末，利用商业化的DNA提取试剂盒进行基因组DNA的提取和纯化，再利用NanoDrop 1000验证DNA的浓度，作为待测样品。
利用毛细管微阵列(见图2，具体包括微管道21和基底23，基底23的顶部表面和微管道露出基底顶部表面的部分22是疏水性的)平台进行多重LAMP反应。即事先用移液器将8组LAMP引物(微阵列中微管道1-10分别为：P-CaMV35S、bar、CP4epsps、P-FMV35S、pat、T-nos、nptII、ADH1、空白对照、空白对照)分别加入阵列中的各 毛细管中并干燥固定，采用倒置虹吸法加入含DNA模板的LAMP反应体系，进行多重LAMP反应。每个反应体系仅有1.6μL。具体操作流程见图1。
具体加样过程见图3，加样采用加样装置，加样装置包括样品池31和手柄32，手柄32连接于样品池31底部；样品池31为外横截面小于反应管的内横截面，样品池31的内横截面大于毛细管微阵列横的截面，样品池31的深度略小于微管道露出基底表面部分的高，样品池31的内表面为亲水性。加样时，将充满含DNA模板的LAMP反应体系(引物除外)样品池31朝下插入反应管中，核酸扩增反应组分与亲水性微管道内壁顶端接触后靠虹吸作用迅速充满亲水性微管道，之后再移除加样装置即可。
其他未明确给出的反应条件均为常规核酸扩增条件。
结果检测和分析。待反应结束后用发射波长为365nm的手持式紫外灯从反应管侧面照射，激发微管道内反应物发出荧光，再用数码相机从反应管顶部朝下拍照记录微阵列中各个毛细管内的荧光信号。用Photoshop7.0(Adobe Systems Inc.,USA)将照片格式转换为16Bit TIFF格式，再使用GenePix Pro 6.1(Molecular Devices,USA)读取每个毛细管内的具体荧光强度。
由图5，并结合模式图4可知，结果图中阳性信号分别是序号：1、6、7、8，其对应的靶标名称分别是：P-CaMV35s、T-nos、nptII以及玉米内源基因ADH1。查找相关数据库和文献得出，理论上该转基因玉米所含转基因元件和玉米內源基因在毛细管微阵列多重LAMP反应中一次性全部检出。对于同一样品的两次重复，检测结果完全一致，且都与预期相符。
实施例2：对成分未知转基因材料的多重检测
利用上述实验过程对由上海市出入境检验检疫局收集自上海港口、成分未知的玉米样本M2进行检测。检测结果如附图6；由图6并结合模式图4可知，结果图中阳性信号分别是序号：1、4、5、6、8，其对应的靶标名称分别是：P-CaMV35s、FMV-35S、pat、T-nos以及玉米内源基因ADH1。将此结果与独立进行的real-time PCR实验结果进行比较，所检出靶标完全一致，故可以认为该检测方法具有很高的特异性和准确性。real-time PCR实验结果如下：
表2
Figure PCTCN2016078408-appb-000003
Figure PCTCN2016078408-appb-000004
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。

Claims (10)

  1. 一种基于毛细管微阵列的核酸高通量快速检测方法，其特征在于，包含以下步骤：
    第一步、将若干组核酸扩增引物分别加入毛细管微阵列上的若干微管道中，干燥，再将毛细管微阵列固定于反应管中；
    第二步、将含有样品核酸的除引物外的其他核酸扩增反应组分加入微管道中，形成核酸扩增体系；
    第三步、将装有核酸扩增体系的反应管置于控温条件下进行扩增反应；
    第四步、通过对扩增反应连续荧光信号测量实现实时检测或对扩增反应结束后一次荧光信号测量实现终点检测。
  2. 根据权利要求1所述的基于毛细管微阵列的核酸高通量快速检测方法，其特征在于，第一步中，所述核酸扩增引物包括但不限于：普通PCR引物、实时定量PCR引物、环介导等温扩增引物、滚环扩增引物、重组酶聚合酶扩增引物；
    其中，当所述核酸扩增引物为实时定量PCR引物时，引物是同相应探针一并加入微管道中的。
  3. 根据权利要求1所述的基于毛细管微阵列的核酸高通量快速检测方法，其特征在于，第一步中，所述毛细管微阵列还包括基底；
    其中，毛细管微阵列中若干个微管道以阵列方式贯穿排布于基底上，并且微管道顶端有少部分露出基底表面。
  4. 根据权利要求3所述的基于毛细管微阵列的核酸高通量快速检测方法，其特征在于，所述基底的上表面、微管道露出基底部分的外表面以及微管道底端内表面均为疏水性表面，微管道的其余面为亲水性面。
  5. 根据权利要求1所述的基于毛细管微阵列的核酸高通量快速检测方法，其特征在于，第一步中，所述加入具体指将核酸扩增引物溶于交联剂中后再加入微管道中；
    其中，所述交联剂为以下三种混合液中的任意一种：
    a、质量百分比为0.1-1％的壳聚糖的乙酸水溶液，pH为4.5-6.0；
    b、质量百分比为0.1-1％的明胶水溶液；
    c、质量百分比为0.05-5％的聚乙二醇水溶液。
  6. 根据权利要求1所述的基于毛细管微阵列的核酸高通量快速检测方法，其特征 在于，第二步中，所述加入具体指通过倒置虹吸方式将其他核酸扩增反应组分引入各微管道中。
  7. 根据权利要求1或6所述的基于毛细管微阵列的核酸高通量快速检测方法，其特征在于，所述加入具体指采用加样装置加入；
    其中，所述加样装置包括样品池和手柄，手柄连接于样品池底部；
    所述样品池为外横截面小于反应管的内横截面，样品池的内横截面大于毛细管微阵列横的截面，样品池的深度略小于微管道露出基底表面部分的高度。
  8. 根据权利要求7所述的基于毛细管微阵列的核酸高通量快速检测方法，其特征在于，所述样品池的内表面为亲水性。
  9. 根据权利要求1所述的基于毛细管微阵列的核酸高通量快速检测方法，其特征在于，第五步中，所述测量可以是借助荧光检测设备或光度检测设备进行检测，也可以是通过肉眼辨别颜色或亮度差异。
  10. 根据权利要求1至6或8至9中任一项所述的基于毛细管微阵列的核酸高通量快速检测方法，其特征在于，所述方法既可以在单个反应管中单独实现，也可以在集成的8连管、96孔板或384孔板中并行实现。
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