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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WO
WIPO (PCT)
Prior art keywords
tube
pcr
reaction
round
reaction tube
Prior art date
Application number
PCT/CN2004/001341
Other languages
English (en)
Chinese (zh)
Inventor
Shengce Tao
Feijun Xian
Jing Cheng
Qiong Zhang
Min Guo
Di Jiang
Hongli Lu
Original Assignee
Capitalbio Corporation
Tsinghua University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Capitalbio Corporation, Tsinghua University filed Critical Capitalbio Corporation
Publication of WO2006050636A1 publication Critical patent/WO2006050636A1/fr

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Classifications

    • 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)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un tube de réaction PCR nette qui comprend une substance de tube et un bouchon de tube, il y a un tube intérieur sur la surface médicale du bouchon et au moins un orifice d’alimentation sur le tube intérieur. Le tube de réaction possède un tube intérieur sur le bouchon, dans lequel est fourni le système de réaction PCR de seconde phase. Le système de seconde réaction entrera dans la substance de tube pour réaliser l’amplification PCR de seconde phase par centrifugation, vibrations ou autres modes après la fin de l’amplification de première phase. Ce type de tube de réaction PCR réalise l’isolation physique entre les deux phases d’amplification PCR nette et évite la pollution du système de PCR par l'opération par étapes, et de ce fait augmente la fiabilité de la PCR nette. En outre, il peut pré-réserver les réactifs secs des systèmes de réaction PCR nette des deux phases dans le tube de réaction, et il est pratique à transporter ou à conserver. La structure de ce type de tube de réaction PCR est simple et le tube peut être couramment utilisé pour toutes sortes de réactions PCR nette.
PCT/CN2004/001341 2004-11-10 2004-11-23 Tube de reaction pcr nette WO2006050636A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNU2004201125256U CN2784420Y (zh) 2004-11-10 2004-11-10 一种巢式pcr反应管
CN200420112525.6 2004-11-10

Publications (1)

Publication Number Publication Date
WO2006050636A1 true WO2006050636A1 (fr) 2006-05-18

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WO (1) WO2006050636A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008047272A3 (fr) * 2006-10-17 2008-06-12 Koninkl Philips Electronics Nv Dispositif d'amplification et de détection d'acides nucléiques
DE102012222351A1 (de) 2012-12-05 2014-06-05 Gna Biosolutions Gmbh Reaktionsgefäß mit magnetischem Verschluss

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CN103103118B (zh) * 2011-11-15 2015-04-08 厦门万泰沧海生物技术有限公司 一种核酸扩增与检测反应管
JP6359754B1 (ja) * 2017-12-29 2018-07-18 ヤマトエスロン株式会社 Ｐｃｒ用容器及び試薬入りｐｃｒ用容器
JP6371892B1 (ja) * 2017-08-24 2018-08-08 ヤマトエスロン株式会社 試薬カセット
CN108641899A (zh) * 2018-05-24 2018-10-12 金陵科技学院 一种离心管的预分盖
CN109337899A (zh) * 2018-10-25 2019-02-15 宁波艾捷康宁生物科技有限公司 一种核酸提取与pcr检测试剂盒
CN111534641B (zh) * 2020-04-01 2021-06-04 上海科技大学 一种核酸检测试剂盒、检测方法及应用
CN112760417B (zh) * 2021-01-20 2022-05-17 中国疾病预防控制中心病毒病预防控制所 一种rap基因检测试剂盒、检测方法和应用及rap病毒检测试剂盒
CN112877191A (zh) * 2021-02-22 2021-06-01 西安交通大学 一种防污染耗材以及应用该防污染耗材进行crispr分子诊断的方法
CN113265457A (zh) * 2021-05-25 2021-08-17 上海真测生物科技有限公司 一种遗传性耳聋多重检测的crRNA组合、试剂盒及方法
CN116445261B (zh) * 2023-05-31 2023-09-08 山东东大检测科技有限公司 一种pcr反应管以及移液器

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5604101A (en) * 1993-10-22 1997-02-18 Abbott Laboratories Method of minimizing contamination in amplification reactions using a reaction tube with a penetrable membrane
CN1324397A (zh) * 1998-08-27 2001-11-28 埃克斯特兰那公司 综合核酸提取、扩增及检测的自携式装置
CN1448500A (zh) * 2003-05-01 2003-10-15 东南大学 可直接检测基因的免冲洗pcr扩增管

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5604101A (en) * 1993-10-22 1997-02-18 Abbott Laboratories Method of minimizing contamination in amplification reactions using a reaction tube with a penetrable membrane
CN1324397A (zh) * 1998-08-27 2001-11-28 埃克斯特兰那公司 综合核酸提取、扩增及检测的自携式装置
CN1448500A (zh) * 2003-05-01 2003-10-15 东南大学 可直接检测基因的免冲洗pcr扩增管

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008047272A3 (fr) * 2006-10-17 2008-06-12 Koninkl Philips Electronics Nv Dispositif d'amplification et de détection d'acides nucléiques
DE102012222351A1 (de) 2012-12-05 2014-06-05 Gna Biosolutions Gmbh Reaktionsgefäß mit magnetischem Verschluss
EP2928605B1 (fr) * 2012-12-05 2019-09-04 GNA Biosolutions GmbH Récipient de réaction à fermeture magnétique

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