WO2006050636A1 - Tube de reaction pcr nette - Google Patents

Tube de reaction pcr nette Download PDF

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Publication number
WO2006050636A1
WO2006050636A1 PCT/CN2004/001341 CN2004001341W WO2006050636A1 WO 2006050636 A1 WO2006050636 A1 WO 2006050636A1 CN 2004001341 W CN2004001341 W CN 2004001341W WO 2006050636 A1 WO2006050636 A1 WO 2006050636A1
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WIPO (PCT)
Prior art keywords
tube
pcr
reaction
round
reaction tube
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PCT/CN2004/001341
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English (en)
French (fr)
Inventor
Shengce Tao
Feijun Xian
Jing Cheng
Qiong Zhang
Min Guo
Di Jiang
Hongli Lu
Original Assignee
Capitalbio Corporation
Tsinghua University
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Publication of WO2006050636A1 publication Critical patent/WO2006050636A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5082—Test tubes per se
    • B01L3/50825—Closing or opening means, corks, bungs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/046—Function or devices integrated in the closure
    • B01L2300/047—Additional chamber, reservoir

Definitions

  • the present invention relates to a nested PCR reaction tube.
  • PCR technology has high detection sensitivity, but when the target nucleic acid in the sample to be detected is small, it is often difficult to perform effective detection.
  • the nested PCR for two rounds of amplification is much more sensitive than conventional PCR.
  • there are generally two sets of primers for a target nucleic acid one set of primers is called an external primer, which is added during the first round of amplification, and the template is the nucleic acid extracted from the sample to be detected; another set of primers It is called an internal primer and is added during the second round of amplification.
  • the template is the product of the first round of amplification.
  • the commonly used ⁇ -type PCR amplification method requires opening the cap of the first round of reaction tubes after the first round of amplification, and transferring a part of the PCR product from the first round of reaction system to the second round of the system.
  • An operational process can easily lead to cross-contamination and residual contamination, leading to false positive results and ultimately reducing the credibility of the results.
  • a common method of reducing or eliminating contamination is to integrate the nested PCR two-tube reaction into a single-tube reaction, which has been accomplished in a number of ways.
  • One way is to design the primers so that the annealing temperature of the outer primers is much higher than the annealing temperature of the inner primers.
  • the two sets of primers are in the same reaction system.
  • the external primers are amplified at a higher temperature.
  • the primers are amplified using a lower annealing temperature (LIop PA et al., 2000, Appl. Environ. Microbiol. 66: 2071-2078; Mathis AR et al" 1997, J. Clin. Microbiol.
  • the reaction system and the second round of the reaction system are physically separated in a single reaction tube.
  • the existing reports include: The second round of reaction system is placed in the Tip head to achieve isolation (Almos A. et al., 1999) , Nucleic Acids Res. 27: 1564-1565); Add trehalose in the second round of reaction system, then dry the second round of the system and adhere it to the top of the reaction tube (Wolff CD 1995, PCR methods Appl. 4:
  • the second round of the nested PCR reaction system was stored in a 0.375% agarose gel, placed on top of the reaction tube, and has a refrigerating device at the top to ensure the second round during the first step of the reaction.
  • the system is least affected (US 5,556,773).
  • single-tube nested PCR through the Tip head not only increases the complexity of the operation but may also introduce other contamination.
  • the length of the universal Tip head The degree exceeds the lumen height of the PCR reaction tube and cannot be directly placed in the lumen; and the second round reaction system is directly placed on the top of the lid with trehalose or agarose, because the top of the lid is intertwined with trehalose or agarose. The force is weak.
  • the second round reaction system is easy to partially or completely fall into the reaction tube in advance, resulting in failure of PCR amplification.
  • the nested PCR reaction tube provided by the present invention comprises a tube body and a tube cover, and the inner side surface of the tube cover is further provided with an inner tube, and the inner tube is provided with at least one sample adding hole.
  • the first reaction system of the nested PCR amplification reaction is applied to the tube, and the second reaction system is applied to the inner tube through the sample well.
  • the first round of PCR amplification is performed.
  • the second round reaction system placed in the inner tube is centrifuged, vibrated or otherwise introduced into the tube, and then a second round of PCR amplification is performed. .
  • At least one vent hole is provided in the inner tube to facilitate entry of the second round reaction system in the inner tube into the tube body.
  • the vent hole of the vent hole is provided with a boss at the edge.
  • the inner tube is usually in the shape of a cylinder, and the sample hole and the vent hole are located on the bottom surface thereof; the inner tube and the tube cover are connected in various ways, such as a threaded connection, a ring-shaped snap connection, an elastic snap connection or a glue bond, etc. .
  • the tube body and inner tube can be made of materials such as polypropylene, polycarbonate, plexiglass, polystyrene, ABS resin, polyethylene, etc.
  • BSA and silane can be used. Pretreatment.
  • the invention sets an inner tube on the tube cover of the PCR reaction tube, and the second round PCR reaction system of the nested PCR is located therein.
  • the first round of PCR amplification is performed.
  • the second round reaction system placed in the inner tube is centrifuged, vibrated or otherwise introduced into the tube, and then a second round of PCR amplification is performed. .
  • the invention skillfully realizes the physical isolation of the nested PCR two-round amplification reaction system, avoids the pollution of the PCR system by the stepwise operation, thereby improving the reliability of the nested PCR reaction; and also can
  • the round reaction system is prepared as a dry agent and stored in the reaction tube in advance, which can facilitate the transportation and preservation of the reagent.
  • FIG. 1A is a schematic structural view of a nested PCR reaction tube of the present invention.
  • FIG. 1B is a schematic overall view of a nested PCR reaction tube of the present invention
  • Figure 2 is an inner tube diagram with a sample well and a vent;
  • Figure 3 is an inner tube diagram with a sample hole and a vent hole, wherein the vent hole has a convex structure
  • Figure 4 is an inner tube diagram with one sample well and two vents
  • Figure 5 is an inner tube diagram showing the bottom surface of the inner tube as a slope
  • Figure 6 is an inner tube diagram with a sample hole and a plurality of vent holes
  • Figure 7 is an inner tube diagram showing the bottom surface of the inner tube as a tapered surface
  • Figure 8A is a state diagram of a nested PCR reaction tube in which a liquid PCR reaction system is added to a tube body and an inner tube;
  • Figure 8B is a state diagram of the nested PCR reaction tube during the first round of PCR amplification
  • Figure 8C is a schematic view showing the reaction system in the inner tube passing through the sample hole through centrifugation, vibration or other means into the tube body;
  • Figure 8D is a state diagram of the nested PCR reaction tube for the second round of PCR amplification
  • Fig. 9 is a state diagram of a nested PCR reaction tube in which a solid PCR reaction system is added to a tube body and an inner tube;
  • Fig. 10 is an electrophoresis pattern of the RT-PCR result of Example 2.
  • the nested PCR reaction tube of the present invention comprises a tube body 1 and a tube cover 2, and an elastic snap (interference) on the inner side surface of the tube cover 2 is connected with an inner tube 7, the inner tube 7 A loading hole 8 and a venting opening 9 are provided at the bottom, and the tube cover 2 is fastened in use, and the inner tube 7 is located in the tube body 1.
  • the inner tube 7 is connected to the tube cover 2 in a variety of manners, and may be a threaded connection, a ring-shaped snap connection or a glue bond in addition to the elastic snap connection.
  • the number of the sample hole and one vent hole are also various, and can be selected according to actual conditions.
  • the inner tube 7 has a sample hole 8 and a vent hole 9; It is shown that the inner tube 7 has a sample hole 8 and a vent hole 9, and the edge of the vent hole 9 is provided with a boss;
  • FIG. 4 shows that the inner tube 7 has a sample hole 8 and The case of the two venting holes 9; Fig.
  • FIG. 5 shows the case where the bottom surface of the inner tube 7 is a slanted surface, with a sample loading hole 8 and a venting opening 9; A case where a sample hole 8 and a plurality of vent holes 9 are provided; Fig. 7 shows a case where the bottom surface of the inner tube 7 is a tapered surface (as can be seen from a perspective view of the bottom surface).
  • Example 2 PCR amplification using a nested PCR reaction tube I. Operation flow of PCR amplification of nested PCR reaction tube of the present invention
  • a first round of PCR reaction system 3 and mineral oil 4 for covering the reaction system are first added to the tube 1 of the nested PCR reaction tube, and a second round of PCR reaction system 5 is added to the inner tube 7.
  • the reaction tube is covered with a tube cover 2 and placed on a PCR machine for the first round of nested PCR amplification. If the top cover of the PCR instrument has a heating function, the heating function of the top cover is required.
  • Cancel as shown in FIG.
  • the reaction tube is taken out from the PCR machine, and the second round reaction system in the inner tube 7 is introduced into the reaction tube by centrifugation, vibration, or the like.
  • amplification can be performed by agarose gel electrophoresis or DNA chip hybridization.
  • the first round of PCR reaction system 3 and the second may be pre-loaded in the tube body 1 and the inner tube 7, respectively.
  • a spherical solid solution such as a spherical reaction system 5, It was dissolved at the time of use, and then subjected to PCR amplification as described above.
  • the nested PCR reaction tube of the present invention performs SARS-Cov multiplex nested RT-PCR
  • RNA PCR Buffer MgCl 2 (25 mM), dNTP Mixture (10 mM), RNase Inhibitor (40 U/ ⁇ ), AMV RTase XL (5U ⁇ 1), AMV-Optimized Taq (5 ⁇ / ⁇ 1)) (Dalian TaKaRa); Taq PCR Master Mixture (Beijing Tianwei Times); dUTP (100 mM) (Shanghai Shenggong); Uracil Glycosidase UNG (Invitrogen, USA); DNA molecular weight reference DL 2 000 (Dalian TaKaRa); mineral oil (Sigma); sterilized water; sterilized DEPC-water.
  • Instrument consumables MJ Research PCR thermal cycler PTC200 (MJ Research Inc. Miami, FL); UVP Bioimaging System and analyzed with Labworks 4.0 (UVP, Inc., Upland, CA); nested PCR reaction tubes installed as shown in Figure 1, And sterilized.
  • PCR amplification primers used were as shown in Table 1, and were synthesized by Shanghai Shenggong Bioengineering Co., Ltd., and were subjected to DHPLC purity determination and UV quantification.
  • Nucleic acid template The SARS-Cov genome RA obtained from the SARS-Cov VERO cell culture supernatant provided by the Chinese Center for Disease Control and Prevention (CDC) has a concentration of 10 8 copies/L.
  • the first and second rounds of PCR reaction systems were prepared according to Tables 2 and 3, respectively.
  • the final volume of the first round reaction of nested PCR is 10 ⁇ 1, taking 7 ⁇ 1 of the system prepared according to Table 2, adding 3 ⁇ 1 10'
  • the diluted SARS-Cov genomic RNA was overlaid with 20 ⁇ of mineral oil; 40 ⁇ l of the system prepared according to Table 3 was added through the wells in the inner tube 2 mounted in the cap.
  • a 1.2% agarose gel was prepared using 0.5XTBE, and the concentration of EB in the gel was 0.5 g ⁇ L.
  • the amount of PCR product loaded was 2 L per well, and the molecular weight of the electrophoresis was referred to as DL2000.
  • the electrophoresis conditions were 100 V for 30 minutes.
  • Electrophoresis results were recorded using a UVP bioimaging system and analyzed using Labworks 4.0 (UVP, Inc., Upland, CA). The electrophoresis results are shown in Figure 10.
  • the M channel is the DNA molecular weight reference DL2000, and the 1-3 channels are three replicate samples of single-tube multiplex nested PCR.
  • Reagent name 10 ⁇ system should be added to the final concentration
  • the invention has simple structure, convenient and practical, and can be widely applied to various nested PCR reactions.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Clinical Laboratory Science (AREA)
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Description

一种巢式 PCR反应管 技术领域
本发明涉及一种巢式 PCR反应管。
背景技术
常规 PCR技术具有较高的检测灵敏度，但是当待检测样品中的目标核酸 较少时， 往往难以进行有效的检测， 进行两轮扩增的巢式 PCR由于检测灵敏 度远高于常规 PCR， 应用更为广泛。 对于巢式 PCR， 针对一种目标核酸一般 有两套引物， 其中一套引物称为外引物， 在第一轮扩增时加入， 模板为待检 测样品中所提取出的核酸； 另一套引物称为内引物， 在第二轮扩增时加入， 模板为第一轮扩增的产物。 目前， 通常使用的臬式 PCR扩增方法需要在第一 轮扩增结束后，开启第一轮反应管的管盖，从第一轮反应体系转移一部分 PCR 产物到第二轮的体系中， 这一操作过程极易导致交叉污染和残留污染， 导致 假阳性结果并最终降低结果的可信度。
降低或者消除污染的常用方法是将巢式 PCR两管反应整合成单管反应， 已有多种方式可以实现这一整合。 一种方式是在引物设计时使得外引物的退 火温度远高于内引物的退火温度， 两套引物同处于一种反应体系中， 在 PCR 反应时先采用较高的温度进行外引物的扩增， 然后采用较低的退火温度进行 内引物的扩增 (LIop P.A. et al.， 2000, Appl. Environ. Microbiol. 66:2071-2078; Mathis A.R. et al" 1997, J. Clin. Microbiol. 35: 1691-1695, EP0519338A1)。 但 是外引物在进行第二轮扩增反应时有可能发生作用， 进而导致非特异性扩增 产生， 降低 PCR结果的可信度。 另一种方式是将第一轮的反应体系与第二轮 的反应体系在单个反应管中通过物理隔离来实现， 已有的报道包括： 将第二 轮反应的体系置于 Tip头中来实现隔离 （Almos A. et al., 1999, Nucleic Acids Res. 27: 1564-1565) ；在第二轮反应体系中添加海藻糖，然后干燥第二轮体系 并将其黏附于反应管的盖顶 （Wolff C.D. 1995, PCR methods Appl. 4:
376-379)； 将巢式 PCR第二轮反应体系存于 0.375%的琼脂糖凝胶中， 置于反 应管的顶部， 并在顶部有一制冷装置以保证在第一步反应过程中第二轮体系 所受的影响最小（US5，556，773)。但是， 通过 Tip头来实现的单管巢式 PCR 不但会增加操作的复杂度而且还可能引入其它污染， 另外， 通用 Tip头的长 度均超过 PCR反应管的内腔高度而不能直接置于内腔； 而用海藻糖或琼脂糖 将第二轮反应体系直接置于盖顶上的方式， 由于盖顶与海藻糖或琼脂糖相互 作用力较弱， 在进行 PCR操作时第二轮反应体系易于提前部分或全部掉入反 应管， 从而导致 PCR扩增失败。
发明公开
本发明的目的是提供一种结构简单、 使用方便的巢式 PCR反应管。 本发明所提供的巢式 PCR反应管， 包括管体和管盖， 所述管盖的内侧面 上还设有内管， 所述内管上设有至少一个加样孔。
巢式 PCR扩增反应的第一轮反应体系加于管体， 第二轮反应体系通过加 样孔加于内管中。 进行 PCR扩增时， 先进行第一轮 PCR扩增， 结束后将置于 内管中的第二轮反应体系通过离心、 振动或其它方式进入到管体中， 再进行 第二轮 PCR扩增。
作为本发明的一种改进， 在其内管上还设有至少一个排气孔， 以方便内 管中的第二轮反应体系进入到管体中。 为了防止在加样过程中排气孔被液膜 堵塞， 所述排气孔的排气孔的边缘设有凸台。
内管通常呈筒状， 加样孔和排气孔位于其底面； 内管与管盖的连接方式 有多种选择， 如螺紋连接、 环状卡扣连接、 弹性卡扣连接或胶粘结等。
管体、 内管可以选择聚丙烯、 聚碳酸酯、 有机玻璃、 聚苯乙烯、 ABS树 脂、 聚乙烯等材料制作， 在进行 PCR扩增时为消除材料对 PCR可能的抑制， 可以采用 BSA和硅烷化进行预处理。
本发明在 PCR反应管的管盖上套设一个内管， 而巢式 PCR的第二轮 PCR 反应体系位于其中。 进行 PCR扩增时， 先进行第一轮 PCR扩增， 结束后将置 于内管中的第二轮反应体系通过离心、 振动或其它方式进入到管体中， 再进 行第二轮 PCR扩增。 本发明巧妙地实现了巢式 PCR两轮扩增反应体系的物理 隔离， 避免了分步操作对 PCR体系的污染， 从而提高了巢式 PCR反应的可靠 性； 而且还可以将巢式 PCR的两轮反应体系分别制备成干剂预先存放于反应 管中， 可以方便试剂的运输、 保存。
附图说明
图 1A为本发明巢式 PCR反应管的结构示意图；
图 1B为本发明巢式 PCR反应管的整体示意图； 图 2为带有一个加样孔和一个排气孔的内管图；
图 3为带有一个加样孔和一个排气孔的内管图， 其中排气孔有一个凸起 的结构；
图 4为带有一个加样孔和两个排气孔的内管图；
图 5为内管底面为斜面的内管图；
图 6为带有一个加样孔和多个排气孔的内管图；
图 7为内管底面为锥面的内管图；
图 8A为在管体和内管中加有液体 PCR反应体系的巢式 PCR反应管状态 图；
图 8B为巢式 PCR反应管进行第一轮 PCR扩增时状态图；
图 8C为将内管中反应体系通过加样孔通过离心、振动或其它方式进入到 管体中的示意图；
图 8D为巢式 PCR反应管进行第二轮 PCR扩增时状态图； .
图 9为在管体和内管中加有固体 PCR反应体系的巢式 PCR反应管状态图； ' 图 10为实施例 2RT-PCR结果电泳图。
实施发明的最佳方式
实施例 1、 巢式 PCR反应管
如图 1A和 1B所示， 本发明的巢式 PCR反应管， 包括管体 1和管盖 2， 管盖 2的内侧面上弹性卡扣（过盈）连接有内管 7， 内管 7的底部设有一个加 样孔 8和一个排气孔 9， 使用时扣上管盖 2， 内管 7即位于管体 1中。
在上述实施例中， 内管 7与管盖 2的连接方式是多样的， 除了弹性卡扣 连接外， 还可以是螺紋连接、 环状卡扣连接或胶粘结。 加样孔和一个排气孔 的数量也是多样的， 可以根据实际情况进行选择， 如图 2所示的是内管 7带 有一个加样孔 8和一个排气孔 9的情况； 图 3所示的是内管 7带有一个加样 孔 8和一个排气孔 9， 排气孔 9的边缘设有凸台的情况； 图 4所示的是内管 7 带有一个加样孔 8和两个排气孔 9的情况； 图 5所示的是内管 7的底面为斜 面， 带有一个加样孔 8和一个排气孔 9的情况； 图 6所示的是内管 7带有一 个加样孔 8和多个排气孔 9的情况； 图 7所示的是内管 7的底面为锥面的情 况 （由底面的透视图可以看出） 。
实施例 2、 应用巢式 PCR反应管进行 PCR扩增 一、 本发明巢式 PCR反应管进行 PCR扩增的操作流程
如图 8A所示，先在巢式 PCR反应管的管体 1中加入第一轮 PCR反应体系 3和用于覆盖反应体系的矿物油 4, 在内管 7中加入第二轮 PCR反应体系 5; 如图 8B所示， 然后将反应管盖上管盖 2， 置于 PCR仪上进行巢式 PCR第一 轮扩增， 若 PCR仪的顶盖具有加热功能， 则需要将顶盖的加热功能取消； 如 图 8C所示， 巢式 PCR的第一轮扩增反应结束后， 从 PCR仪中取出反应管， 通过离心、 振动等方式使在内管 7中的第二轮反应体系进入反应管的管体 1 ; 如图 8D所示， 将反应管重新置于 PCR仪中， 进行第二轮 PCR扩增， 扩增结 束后通过琼脂糖凝胶电泳、 DNA芯片杂交等方式即可以对扩增产物进行检 另外为了利于运输和保存， 以及减少操作中的误差和污染， 还可以如图 9所示， 在管体 1和内管 7中分别预先装入第一轮 PCR反应体系 3、 第二轮 PCR反应体系 5的球形等固体干剂， 使用时溶解， 然后按照如上所述的方法 进行 PCR扩增。
二、 本发明巢式 PCR反应管进行 SARS-Cov多重巢式 RT-PCR
1、 实验材料
化学试剂： 一步法 RT-PCR试剂盒 （组份有： lOxOne Step RNA PCR Buffer, MgCl2 (25 mM), dNTP Mixture (10 mM), RNase Inhibitor (40 U/μΙ), AMV RTase XL(5U^1), AMV-Optimized Taq (5υ/μ1)) (大连 TaKaRa)； Taq PCR Master Mixture (北京天为时代） ； dUTP (lOO mM) (上海生工） ； 尿嘧 啶糖苷酶 UNG (美国 Invitrogen); DNA分子量参照 DL2000 (大连 TaKaRa); 矿物油 （Sigma) ； 灭菌水； 灭菌 DEPC—水。
仪器耗材： MJ Research PCR thermal cycler PTC200 (MJ Research Inc. Miami, FL); UVP Bioimaging System and analyzed with Labworks 4.0 (UVP, Inc., Upland, CA); 按图 1安装好的巢式 PCR反应管， 并进行灭菌处理。
引物：所用 PCR扩增引物如表 1所示，均由上海生工生物工程公司合成， 且经过 DHPLC纯度测定以及紫外定量。
表 1. 所用 PCR扩增引物
引物性
引物对 统一名称 引物序列 目标区域 质
SARS-CoV 外引物 PMSU— 00002 GCATCGTTGACTATGGTGTCCGATTCT ORFlab Setl PMSL— 00001 ACATCACAGCTTCTACACCCGTTAAGGT ORFlab
PMV— 00023 TCACTTGCTTCCGTTGAGGAGCCGCTTGTCACAATGCCAATT ORFlab 内引物
PMV— 00024 GGTTTCGGATGTTACAGCGTCATCACCAAGCTCGCCAACAGTT ORFlab
PMSU— 00003 GCTGCATTGGTTTGTTATATCGTTATGC ORFlab 外引物
SARS-CoV PMSL— 00002 ATACAGAATACATAGATTGCTGTTATCC ORFlab Set2 PMV_00031 TCACTTGCTTCCGTTGAGGTAGCCAGCGTGGTGGTTCATACAA ORFlab 内引物
PMV_00032 GGTTTCGGATGTTACAGCGTCTCCCGGCAGAAAGCTGTAAGCT ORFlab
PMSU— 00006 ATACAGAATACATAGATTGCTGTTATCC N 外引物
SARS-CoV PMSL_00005 CACGTCTCCCAAATGCTTGAGTGACG N
Set3 PMV— 31007 TCACTTGCTTCCGTTGAGGTCCTCATCACGTAGTCGCGGTAATTC N
内引物
PMV— 31012 GGTTTCGGATGTTACAGCGTGGCTTTTTAGATGCCTCAGCAGCA N
PMSU_00005 TTAAATGCACCGGCCACGGTTTG S 外引物
SARS-CoV PMSL— 00003 CCAGCTCCAATAGGAATGTCGCACTC S
Set4 PMV— 00045 TCACTTGCTTCCGTTGAGGATGCACCGGCCACGGTTTGTG S
内引物
PMV 00046 GGTTTCGGATGTTACAGCGTATGCGCCAAGCTGGTGTGAGTTGA S
PMV— 00072 ATGGGGAAGGTGAAGGTCGG Human G3PDH 外引物
PMV— 00073 TGGTGAAGACGCCAGTGGAC Human G3PDH
IC
PMV— 40013 TCACTTGCTTCCGTTGAGGCGTATTGGGCGCCTGGTCAC Human G3PDH 内引物
PMV— 40014 GGTTTCGGATGTTACAGCGTCCAGCATCGCCCCACTTGAT Human G3PDH
PMV— 10001 TCACTTGCTTCCGTTGAGG
通用引物
PMV_10002 GGTTTCGGATGTTACAGCGT
*IC: internal control
核酸模板： 中国疾病预防控制中心 （中国 CDC) 提供的从 SARS-Cov VERO细胞培养上清中所获得的 SARS-Cov基因组 R A， 浓度为 108 copies/ L。
1、 实验方法
1 ) PCR反应体系配制
分别按表 2和表 3配制第一轮和第二轮 PCR反应体系。 巢式 PCR第一 轮反应体系的最终体积为 10 μ 1， 取 7 μ 1按表 2配制的体系， 加入 3 μ 1 10' 稀释的 SARS-Cov基因组 RNA， 并在其上覆盖以 20 μΐ的矿物油； 在安装 于顶盖的内管 2中通过加样孔加入 40μ 1按表 3配制的体系。
2) PCR扩增
盖上反应管的管盖 2，按照表 4的热循环程序进行进行第一轮 PCR反应， 设置 PCR仪顶盖为不加热。第一轮热循环程序结束后，从 PCR仪中取出 PCR 反应管， 置于 Eppendorf的台式离心机中 6000 rpm离心 1分钟使得顶盖中的 第二轮反应体系完全进入反应管的底部， 将 PCR反应管重新置于 PCR仪中， 按照表 5的热循环程序进行第二轮 PCR反应。
3) PCR结果电泳检测
采用 0.5XTBE来配制 1.2%的琼脂糖凝胶， 胶中的 EB的浓度为 0.5 g^L。 PCR产物的上样量为每孔 2 L， 电泳的分子量参照为 DL2000。 电泳 条件为 100 V， 30分钟。 电泳结果采用 UVP生物成像系统来记录并且采用 Labworks 4.0 (UVP, Inc., Upland, CA)来进行结果分析。电泳结果如图 10所示， M道为 DNA分子量参照 DL2000, 1— 3道为单管多重巢式 PCR的三个重复 样品。 实验中所进行的为 5重 PCR，'由于最短的两个 PCR产物长度接近， 而 最长的两个 PCR产物长度亦接近， 在常规的琼脂糖凝胶电泳上不容易区分， 电泳条带应为 3条。 结果表明， 本发明巢式 PCR反应管可以有效地实现单管 多重一步法 RT-PCR, 且具有高的可靠性。
表 2. 巢式 PCR第一轮反应体系
反应物名称 10 μΐ体系中应加入体积 终浓度
10 X One Step RNA PCR Buffer 1 1 IX
MgCl2(25 raM) 2 U 1 ' 5 mM
dNTP Mixture (10 mM) 1 μ 1 1 mM
RNase Inhibitor (40 U/μ 1) 0.2 μΐ 0.8 U/μ 1
AMV RTase XL (5 U/u 1) 0.2 μΐ 0.1 U/μ 1
AMV- Optimized Taq(5 U/μ 1) 0.2 μΐ 0.1 U/μ 1
UNG(1 U/μ 1) 0.1 μΐ 0.01 U/μ 1
PMSU— 00002 (10 UM) 0.1 μΐ 0.1 μΜ
PMSL— 00001 (10 μΜ) 0.1 μΐ 0.1 μΜ
PMSU_00003 (10 Μ) 0.1 μΐ 0.1 μΜ
PMSL_00002 (10 uM) 0.1 μΐ 0.1 μΜ
PMSU— 00006 (10 ) 0.1 U l 0.1 μΜ
PMSL_00005 (10 M) 0.1 μΐ 0.1 μΜ PMSU_00005 (10 μΜ) 0.1 μΐ 0.1 μΜ
PMSL— 00003 (10 uM) 0.1 μΐ 0.1 μΜ
PMV_00072 (10 μΜ) 0.05 u 1 0.05 UM
PMV_00073 (10 μΜ) 0.05 μΐ 0.05 μΜ 灭菌 DEPC—水 1.4 ul
Total 7 μ 1 表 3. 巢式 PCR第二轮反应体系
Figure imgf000009_0001
表 4. 第一轮反应的热循环程序
50 °C 30min RT反应
37 'C 10 miti UNG处理
94 'C 10 min 灭活 RTase以及 UNG,
94 °C 30sec \
55 °C 30sec 1 30循环
72 'C 1 min /
72 "C lOmin 表 5.第二轮反应的热循环程序
94'C 3min 预变性
94 °C 30sec \
60°C 30sec | 30循环
72 °C 1 min Z
72V lOmin 工业应用
本发明结构简单， 方便实用， 可以广泛应用于各种巢式 PCR反应。

Claims

权 利 要 求 书
1、一种巢式 PCR反应管， 包括管体和管盖， 其特征在于： 所述管盖的内 侧面上还设有内管， 所述内管上设有至少一个加样孔。
2、根据权利要求 1所述的反应管， 其特征在于： 所述内管上还设有至少 一个排气孔。
3、根据权利要求 2所述的反应管， 其特征在于： 所述排气孔的排气孔的 边缘设有凸台。
4、 根据权利要求 2所述的反应管， 其特征在于： 所述内管呈筒状， 所述 加样孔和排气孔位于所述内管底部。
5、 根据权利要求 1或 2或 3或 4所述的反应管， 其特征在于： 所述内管 与所述管盖的连接方式为螺紋连接。 '
6、 根据权利要求 1或 2或 3或 4所述的反应管， 其特征在于： 所述内管 与所述管盖的连接方式为环状卡扣连接。
7、根据权利要求 1或 2或 3或 4所述的反应管， 其特征在于： 所述内管 与所述管盖的连接方式为弹性卡扣连接。
8、根据权利要求 1或 2或 3或 4所述的反应管， 其特征在于： 所述内管 与所述管盖的连接方式为胶粘结。
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