WO2020124510A1 - 一种极速核酸扩增的方法及其设备和应用 - Google Patents

一种极速核酸扩增的方法及其设备和应用 Download PDF

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WO2020124510A1
WO2020124510A1 PCT/CN2018/122453 CN2018122453W WO2020124510A1 WO 2020124510 A1 WO2020124510 A1 WO 2020124510A1 CN 2018122453 W CN2018122453 W CN 2018122453W WO 2020124510 A1 WO2020124510 A1 WO 2020124510A1
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temperature
reaction
low
ultra
temperature reaction
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PCT/CN2018/122453
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English (en)
French (fr)
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周荣
苏晓波
高文娟
李潇
刘文宽
周银华
周志超
许多
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广州市华南医学研究中心
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Priority to CN201880002566.4A priority Critical patent/CN110114475B/zh
Priority to PCT/CN2018/122453 priority patent/WO2020124510A1/zh
Publication of WO2020124510A1 publication Critical patent/WO2020124510A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

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  • the invention relates to a gene detection method and equipment and application thereof in the fields of biology, medicine and the like, and particularly relates to a method, equipment and application of extremely rapid nucleic acid amplification.
  • PCR Polymerase Chain Reaction
  • a typical PCR procedure is usually composed of three basic reaction steps: repeated high temperature denaturation-low temperature renaturation-temperature extension, namely, high temperature treatment at 90-95 °C for about 10-30 seconds, and then cooling to different temperatures. After maintaining at 50-65°C for about 10-30 seconds, the temperature is raised to about 72°C for 30-60 seconds, and so on for 30-40 cycles.
  • the procedure is usually: 94 °C 30 seconds, 55 °C 30 seconds, 72 °C 1 minute, 30 cycles.
  • a two-step cycle amplification method can also be performed, such as 94°C for 30 seconds and 60°C for 30 seconds.
  • the actual experimental time also includes the time required for the temperature rise and fall process of the reaction block, the current temperature change rate of the amplification instrument is mostly within 3 °C/second, and the total time required for a temperature rise and fall cycle is about 30 seconds.
  • a complete PCR process usually takes more than one hour. In fact, the most important thing for the PCR reaction itself is the temperature of the reaction solution in the PCR tube, as long as the required temperature in the PCR tube is reached, the normal reaction can be carried out.
  • PCR testing is a very effective method often used. Under special circumstances, especially in hospitals, as the timeliness of diagnosis is getting higher and higher, the more efficient and timely PCR is required.
  • the current PCR process still has a long reaction time and requires a batch reaction, that is, a long cycle, it is necessary to wait for the previous batch reaction to complete before starting the next batch of reaction, which causes the PCR instrument to be inefficient. And it also affects work efficiency. Therefore, in order to adapt to the realization of rapid amplification methods, it is also necessary to be equipped with appropriate equipment.
  • the object of the present invention is to provide a method, device and application for extremely rapid nucleic acid amplification.
  • the temperature conduction in the reaction tube is accelerated by the large temperature difference, which greatly shortens the PCR tube.
  • the temperature increase and decrease rate greatly shortens the time required for the PCR process and improves the use rate of the PCR instrument; and through the chain arrangement of the reaction tube holes of the temperature module and the design of the progressive method, it is possible to avoid only batch by batch PCR. So that you can directly add samples and reactions at any time.
  • the method for rapid nucleic acid amplification of the present invention is to place a reaction tube containing a reaction mixture in an ultra-high temperature reaction zone and an ultra-low temperature reaction zone, and perform 30-45 cycles.
  • the temperature of the ultra-high temperature reaction zone is: 100°C- A fixed temperature or temperature range in 150°C, the temperature point is selected according to the different tubes to ensure that the actual temperature of the liquid in the reaction tube in the tube can be maintained in the range of 88°C-95°C for 1-3 seconds to complete the denaturation of the double-stranded DNA fragments;
  • the temperature of the ultra-low temperature reaction zone is a fixed temperature or temperature range from 10°C to 40°C.
  • the temperature point is selected according to the different pipes to ensure that the actual temperature of the liquid in the reaction tube can be maintained in the range of 55°C-65°C for 2-4 seconds to complete the template Refolding with the primer, and at the same time complete the steps of DNA strand extension and fluorescence detection during the rapid temperature rise and fall; the cycle procedure is: react in the ultra-high temperature area for 4-10 seconds, and then react in the ultra-low temperature area 4 -10 seconds; perform 30-45 cycles to complete the nucleic acid amplification and detection process in an ultra-short time within 8-15 minutes.
  • the reaction tube is a polymerase chain reaction (PCR) tube made of polypropylene PP or a PCR reaction tube made of glass.
  • PCR polymerase chain reaction
  • the reaction mixture includes polymerase, amplification primers, dNTP, PCR buffer and sterilized double distilled water.
  • the second object of the present invention is to disclose a reactor for an extremely rapid nucleic acid amplification method, the reactor includes an ultra-high temperature reaction zone and an ultra-low temperature reaction zone; the temperature adjustment range of the ultra-high temperature reaction zone is 100°C -A fixed temperature or temperature range in -150°C; the temperature adjustment range of the ultra-low temperature reaction zone is a fixed temperature or temperature range in 10°C-40°C.
  • the ultra-low temperature reaction zone and the ultra-high temperature reaction zone are composed of two reaction tube bracket temperature-controlled metal modules, and the assembly mode of the two temperature-controlled metal modules is concentric circular rings, parallel bars or gear-like cross-embedded Way, the reaction tube bracket temperature control metal module is provided with reaction tube holes, the number of the reaction tube holes is 1-45, the reaction tube can be moved in different temperature control modules by the robot to complete automatically, when the temperature is low Complete the fluorescence detection procedure.
  • the reactor includes manual, semi-automatic and fully automatic.
  • the reactor includes a low-temperature reaction ring, a high-temperature reaction disk, a heat insulation belt, a cover plate, a rotating shaft, and a stepper motor; the low-temperature reaction ring and the high-temperature reaction disk have the same center; The diameter is larger than the diameter of the high-temperature reaction plate; the outer edge of the high-temperature reaction plate is gear-like; the inner edge of the low-temperature reaction ring is gear-like; The heat insulation bands are separated from each other and bite each other to form a complete circular disk, and the low-temperature reaction ring and the high-temperature reaction disk bite form a gear-like bite line; samples are distributed along the same circumferential line of the disk surface circumference on the bite line Tube holes; the total number of sample tube holes can be 60-90.
  • the positions of the low-temperature reaction ring and the high-temperature reaction disk are fixed; the cover plate is connected to the rotating shaft, and the axis of the rotating shaft is concentric with the center of the low-temperature reaction ring or the high-temperature reaction disk; The cover plate is connected to the stepping motor through the rotating shaft; the low-temperature reaction ring and the high-temperature reaction plate are located below the cover plate; the temperature of the low-temperature reaction ring is set at a fixed one of 10°C-40°C Temperature or temperature range; the temperature of the high-temperature reaction plate is set at a fixed temperature or temperature range from 100°C to 150°C.
  • a T-slot opening card is provided on the side periphery of the cover plate, and the T-slot opening card corresponds to the sample tube hole.
  • the bottom of the sample tube hole is provided with a hole for installing a laser diode; the sample tube hole on the low-temperature reaction ring is provided with a hole for installing an optical fiber.
  • the third object of the present invention is to disclose a nucleic acid detection device, which comprises the above-mentioned reactor.
  • the fourth object of the present invention is to disclose the use of the aforementioned nucleic acid amplification method in a PCR-based nucleic acid amplification and analysis method.
  • the above-mentioned applications include real-time fluorescence quantitative PCR, multiplex PCR, RT-PCR, nested PCR and sequencing.
  • the fifth object of the present invention is to disclose an operating system for the above-mentioned extremely rapid nucleic acid amplification method, which includes a high temperature control component, a low temperature control component, a fluorescence reading control component, and a computer control system.
  • the high temperature control component, low temperature control component, and fluorescence reading control component are connected.
  • the nucleic acid amplification method of the present invention revolutionizes traditional nucleic acid amplification, changes the three-stage reaction mode of traditional PCR, and utilizes the method of fixing the required temperature in advance and allowing batch reaction Mode, puts forward a pipelined PCR working mode, improves the utilization rate of PCR equipment, saves the user's time, realizes follow-up and follow-up testing, and makes the system more flexible and applicable.
  • the use of extreme temperature difference steps to increase the rate of temperature change greatly reduces the time required for the reaction liquid in the tube to reach the optimal temperature, achieves the purpose of rapid nucleic acid detection, and enables nucleic acid detection technology to be more widely used.
  • Figure 1 shows the results of conventional PCR of amplified fragments with lengths of 70bp, 100bp, 150bp, and 180bp, and electrophoresis results of PCR amplification products mediated by extreme ambient temperatures of 100°C and 10°C, where M lane is a molecular weight standard reference.
  • Lane 1 is the result of conventional PCR product electrophoresis
  • Lane 2 is the result of extreme environmental temperature-mediated PCR product electrophoresis
  • Figure 2 shows the results of conventional PCR of amplified fragments with lengths of 70bp, 100bp, 150bp, and 180bp, and electrophoresis results of PCR amplification products mediated by extreme ambient temperatures of 130°C and 20°C, in which lane M is a molecular weight standard reference.
  • Lane 1 is the result of conventional PCR product electrophoresis
  • Lane 2 is the result of extreme environmental temperature-mediated PCR product electrophoresis
  • Figure 3 shows the results of conventional PCR of amplified fragments with lengths of 70bp, 100bp, 150bp, and 180bp, and electrophoresis results of PCR amplification products mediated by extreme ambient temperatures of 150°C and 40°C, in which lane M is a molecular weight standard reference.
  • Lane 1 is the result of conventional PCR product electrophoresis
  • lane 2 is the result of extreme environmental temperature-mediated PCR product electrophoresis
  • FIG. 4 is a schematic plan view of a preferred embodiment of the ultrafast nucleic acid amplification reactor of the present invention.
  • FIG. 5 is a schematic side view of a preferred embodiment of the ultrafast nucleic acid amplification reactor of the present invention.
  • Example 2 The preliminary experiment of the effect of low temperature ambient temperature on the temperature change rate of cooling
  • Example 3 A preferred embodiment of the comparative experiment between the extremely fast PCR method and the conventional PCR method
  • the four sets of target amplification sequences and the corresponding forward primer and reverse primer sequences are as follows:
  • the first set of target amplification sequences SEQ ID NO: 1:
  • the second set of target amplification sequences SEQ ID NO: 4:
  • the reaction solution of the PCR reaction system is prepared as follows.
  • the PCR reaction buffer is selected from the products of TAKARA:
  • Independent high-temperature modules and low-temperature modules are used as reaction environment temperature brackets for PCR reactions, wherein the temperature of the high-temperature module is set to 100°C and the temperature of the low-temperature module is set to 10°C.
  • thermometer Place a thermometer in the PCR reaction tube to measure the temperature of the reaction solution in the PCR tube.
  • the PCR tube was placed in an independent high-temperature tank of 100°C, and the temperature of the reaction solution in the PCR tube was measured to rise from 60°C ( ⁇ 3°C) to 92°C ( ⁇ 3°C). Time 14s; place the PCR tube in a separate 10°C low temperature bath, and measure the temperature of the reaction solution in the PCR tube from 92°C ( ⁇ 3°C) to 60°C ( ⁇ 3°C), which takes 4s. Therefore, we designed the reaction procedure of the extreme ambient temperature-mediated PCR experiment as follows:
  • the temperature of the reaction solution in the PCR tube is 92°C ( ⁇ 3°C) when the PCR tube is placed in an independent high-temperature bath, and the lowest temperature is 60°C ( ⁇ 3°C) during the period when it is placed in an independent high-temperature bath at 10°C .
  • the actual temperature of the reaction solution in the PCR reaction tube is repeatedly circulated at 60-92°C ( ⁇ 3°C) to achieve the amplification of the target gene fragment.
  • FIG. 1 shows the conventional PCR of amplified fragments with lengths of 70bp, 100bp, 150bp, and 180bp.
  • °C Extreme ambient temperature mediated PCR amplification product electrophoresis results diagram where M lane is a molecular weight standard reference, lane 1 is the conventional PCR product electrophoresis results, lane 2 is the extreme environment temperature mediated PCR product electrophoresis results.
  • Embodiment 4 The second preferred embodiment of the comparison experiment between the extremely fast PCR method and the conventional PCR method
  • the temperature of the reaction solution in the PCR tube is 92°C ( ⁇ 3°C) when the PCR tube is placed in an independent high-temperature bath at 130°C, and 60°C ( ⁇ 3°C) when it is placed in an independent low-temperature bath at 20°C .
  • the actual temperature of the reaction solution in the PCR reaction tube is repeatedly circulated in 60-92°C ( ⁇ 3°C) to achieve the amplification of the target gene fragment.
  • Example 5 The third preferred embodiment of the comparison experiment between the extremely fast PCR method and the conventional PCR method
  • the temperature of the reaction solution in the PCR tube is 92°C ( ⁇ 3°C) when the PCR tube is placed in a 150°C independent high-temperature bath, and 60°C ( ⁇ 3°C) when it is placed in a 20°C independent low-temperature bath .
  • the actual temperature of the reaction solution in the PCR reaction tube is repeatedly circulated at 60-92°C ( ⁇ 3°C) to achieve the amplification of the target gene fragment.
  • FIG. 3 shows the conventional PCR of the amplified fragments with lengths of 70bp, 100bp, 150bp, and 180bp.
  • lane M is a molecular weight standard reference
  • lane 1 is the conventional PCR product electrophoresis result
  • lane 2 is the extreme environment temperature-mediated PCR product electrophoresis result.
  • the conventional PCR method and the extreme ambient temperature-mediated PCR method can effectively achieve PCR amplification for short gene fragments of different sizes; the extreme ambient temperature-mediated PCR method can be performed within 8-12 minutes
  • the time can be shortened to about one-tenth of conventional PCR.
  • the combination of temperature and time in the high and low temperature regions and the length of placement can be used to select a combination of temperature and time to achieve control of the temperature of the reaction liquid in the reaction tube at 60-92°C ( ⁇ 3°C) and achieve the target fragment The purpose of amplification.
  • Embodiment 6 A preferred embodiment of the ultrafast nucleic acid amplification reactor of the present invention
  • FIGS. 4 and 5 is a schematic top view of a preferred embodiment of the ultrafast nucleic acid amplification reactor of the present invention.
  • FIG. 5 is a schematic side view of a preferred embodiment of the ultrafast nucleic acid amplification reactor of the present invention. It can be seen from FIG.
  • the reactor for nucleic acid amplification of the present invention includes a low-temperature reaction ring 1, a high-temperature reaction disk 2, an insulating tape 3, a cover plate 4, a rotating shaft 5, and a stepping motor 6; the low-temperature reaction ring 1 and The high temperature reaction disk 2 has the same center; the diameter of the low temperature reaction ring 1 is larger than the diameter of the high temperature reaction disk 2; the outer edge of the high temperature reaction disk 2 is gear-like; the inner edge of the low temperature reaction ring 1 is Gear-like; the low-temperature reaction ring 1 and the high-temperature reaction disk 2 are isolated from each other by the heat insulation belt 3 and interlock with each other to form a complete circular disk, the low-temperature reaction ring 1 and the high-temperature reaction disk 2 A gear-like bite line is formed at the bite; sample tube holes 7 are distributed along the same circumferential line of the disc surface on the bite line; the positions of the low-temperature reaction ring 1 and the high-temperature reaction plate 2 are fixed; the
  • a T-slot opening card 41 is provided on the side periphery of the cover plate 4, and the T-slot opening card 41 corresponds to the sample tube hole 7.
  • the bottom of the sample tube hole 7 is provided with a hole position 8 for installing a laser diode; the sample tube hole 7 is provided with a hole position 9 for installing an optical fiber.
  • sample tube holes 7 there can be 60-90 sample tube holes 7 so that the adjacent sample cup holes are actually located in different temperature zones. If a sample is moved from one hole to another adjacent hole, it will go from one temperature zone to another temperature zone. If this sample moves 60 holes along the circumference, then this sample has undergone 30 cycles of high and low temperature changes, which means that the entire process of a PCR reaction environment is completed.
  • the sample cup moves from one hole to the adjacent hole in sequence along the circumference through the cover plate that can be rotated and moved up and down.
  • 5 is a schematic side view of a preferred embodiment of the ultrafast nucleic acid amplification reactor of the present invention.
  • the reactor includes a cover plate 4 in addition to the low-temperature reaction ring 1, the high-temperature reaction plate 2 and the heat insulation band 3 It is connected to the rotating shaft 5 and the cover plate 4 connected to the rotating shaft 5.
  • the rotating shaft 5 is located at the center of the low-temperature reaction ring 1 or the high-temperature reaction plate 2; the low-temperature reaction ring 1 and the high-temperature reaction plate 2 are located under the cover plate 4; 40 T-slot opening cards 41 are provided on the side periphery, and the sample tube 71 is caught on the cover plate 4 through the T-slot opening cards 41.
  • the T-groove opening card 41 corresponds to the sample tube hole 7; the sample tube 71 is installed in the T-groove opening card 41 on the periphery of the side of the cover plate 4, following the cover plate 4 in a circular stepping motion and up and down motion, 1-6 seconds After the clock, the cover plate 4 moves upward, pulling all the sample tubes 71 out of the sample tube holes 7.
  • the cover plate 4 rotates one hole position, and then moves downward. In this way, all sample tubes 71 are replaced with the temperature environment together.
  • the up and down movement and stepping rotation movement of the cover plate 4 can be driven by the stepping motor 6 according to a predetermined program.
  • the cover plate 4 carries the sample tube 71 for up, down, and circular motions, and moves once every 5 seconds, and 40 motions make one rotation, which is equivalent to 20 temperature cycles. After the sample tube 71 has completed 30 temperature cycles, it will be removed from the cover.
  • the bottom of the sample tube hole 7 is provided with thirty hole positions 8 for installing laser diodes; the side of the sample tube hole 7 on the low temperature reaction ring 1 is provided with thirty hole positions 9 for installing optical fibers for reading Get signal.
  • the laser tube emits excitation light, stimulating the sample in the sample tube 71 to emit possible fluorescence, which is captured by the optical fiber and transmitted to the photoelectric detection section.

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Abstract

本发明公开了一种极速核酸扩增检测的方法，将核酸扩增反应管在超高温区和超低温区重复短时放置，超高温区为100℃-150℃中的一个温度点；超低温区为10℃-40℃中的一个温度点；如此30-45个循环、在8-15分钟内完成核酸扩增检测过程。本发明还公开了一种用于极速核酸扩增的反应器，包括调节范围为100℃-150℃中的一个固定温度的超高温反应区和调节范围为10℃-40℃中的一个固定温度超低温反应区。本发明还公开了一种用于上述极速核酸扩增的操作软件系统和设备。

Description

一种极速核酸扩增的方法及其设备和应用 技术领域
本发明涉及用于生物学、医学等领域的基因检测方法及其设备和应用，具体涉及一种极速核酸扩增的方法及其设备和应用。
背景技术
聚合酶链式反应(Polymerase Chain Reaction)简称PCR，已经成为核酸检测的常规技术。一个典型的PCR程序通常是将反应样品经过反复的高温变性—低温复性—适温延伸三个基本反应步骤构成，即在90-95℃高温处理10-30秒左右，然后以不同方式降温至50-65℃、维持约10-30秒后升温到72℃左右维持30-60秒，如此往复30-40个周期。按照《分子克隆实验指南》一书中记载的典型的PCR三步循环扩增过程程序通常为：94℃30秒，55℃30秒，72℃1分钟，循环30次。针对短片段也可以进行两步循环的扩增方法，如94℃30秒，60℃30秒。整个反应过程中，实际实验时间还包括反应块升降温过程所需的时间、目前扩增仪的变温速率多在3℃/秒之内，一个升降温循环共需时间共约30秒之上，通常一个完整的PCR过程一般需要一个小时以上。而实际上，其中对PCR反应本身最重要的是PCR管内反应液温度，只要达到要求的PCR管内温度即可进行正常反应。
现在升降温方式有多种，比如气体加温、水加温及电热块、半导体块升降温等，而且主流PCR仪通过半导体反复升降温实现自动扩增和检测，但是最快的升降温速率也不超过3℃/秒，而管内反应液的温度变化速率则更低，故在PCR全程时间上受到限制。
随着PCR技术不断得到广泛的应用，利用PCR技术对不同类型的样本检测及样本检测量的增多，使得对分析效率和成本提出了更高的要求。因此，进一步缩短PCR扩增时间，发展快速PCR技术，对于大样本量的快速检测及特殊样 品如临床样品的时效性具有十分重要的意义。
在医院或实验室，PCR检测法是经常需要用到的非常有效的手段。在特殊情况下，特别是医院中，由于对诊断的时效性要求越来越高，就越要求PCR的高效及时。但是由于目前PCR过程仍存在反应时间较长，并需要批次反应即周期较长的问题，要等待上一个批次反应完成才能开始下一个批次的反应，这样造成PCR仪器使用效率不高，而且也很影响工作效率。因此，为适应快速扩增的方法的实现，也需要配备合适的设备。
发明内容
为克服上述技术缺陷，本发明的目的是提供一种极速核酸扩增的方法及其设备和应用。通过设置PCR管外反应块温度与PCR管内目标反应温度的较大温差，并直接通过机械运动将PCR管转移到相应反应块上，如此，通过大温差促使反应管内温度传导加速，大大缩短PCR管内温度升降温速率，使PCR过程所需的时间大大缩短，提高PCR仪器的使用率；而且通过温度模块反应管孔的链式排列及递进方式的设计，避免只能一批一批进行PCR，从而实现直接可随时添加样本及反应。
为实现上述目的，采用如下的技术方案：
本发明的极速核酸扩增的方法为将装有反应混合液的反应管置于超高温反应区和超低温反应区，进行30-45个循环，所述超高温反应区的温度为：100℃-150℃中的一个固定温度或温度区间，温度点选择根据管材的不同、以确保管内反应管内液体实际温度在88℃-95℃区间能维持1-3秒使得DNA片段双链完成变性；所述超低温反应区的温度为10℃-40℃中的一个固定温度或温度区间，温度点选择根据管材的不同、以确保反应管内液体实际温度在55℃-65℃区间能维持2-4秒完成模板与引物的复性，同时在快速升降温过程中完成DNA链延伸及荧光检测等步骤；所述循环的程序为：在所述超高温区反应4-10秒，再在所述超低温区反应4-10秒；进行30-45个循环、在8-15分钟内超短时间内完成核酸 扩增检测过程。
优选的，所述反应管为聚丙烯PP材质的多聚酶链式反应(PCR)管或玻璃材质的PCR反应管。
优选的，所述反应混合液包括聚合酶、扩增引物、dNTP、PCR缓冲液和灭菌双蒸水。
本发明的第二个目的是公开了一种用于极速核酸扩增方法的反应器，所述反应器包括超高温反应区和超低温反应区；所述超高温反应区的温度调节范围为100℃-150℃中的一个固定温度或温度区间；所述超低温反应区的温度调节范围为10℃-40℃中的一个固定温度或温度区间。
优选的，所述超低温反应区和超高温反应区由两块反应管托槽温控金属模块构成，所述两温控金属模块的组装模式为同心圆环、平行条块或齿轮样交叉互嵌方式，所述反应管托槽温控金属模块上设置有反应管孔，所述反应管孔的数量为1-45个，反应管在不同温控模块中可移动以机械手自动完成，在低温时完成荧光检测程序。反应器包括手动、半自动、全自动。
优选的，所述反应器包括低温反应环、高温反应盘、隔热带、盖板、旋转轴和步进电机；所述低温反应环和所述高温反应盘同一圆心；所述低温反应环的直径大于所述高温反应盘的直径；所述高温反应盘的外边沿为齿轮样；所述低温反应环的内边沿为齿轮样；所述低温反应环与所述高温反应盘之间用所述隔热带相互分隔并相互咬合形成完整的圆形盘，所述低温反应环与所述高温反应盘咬合处形成齿轮样咬合线；在所述咬合线上沿盘面圆周的同一圆周线上分布样品管孔；样品管孔总数可以为60-90个。所述低温反应环和所述高温反应盘的位置固定；所述盖板与所述旋转轴相连，所述旋转轴的轴心与所述低温反应环或高温反应盘的圆心同圆心；所述盖板通过所述旋转轴与所述步进电机连接；所述低温反应环和高温反应盘位于所述盖板的下方；所述低温反应环的温度设置在10℃-40℃中的一个固定温度或温度区间；所述高温反应盘的温度设置在100℃-150℃中的一个固定温度或温度区间。
优选的，所述盖板的侧面周边设置了T型槽开口卡，所述T型槽开口卡与所述样品管孔相对应。
优选的，所述样品管孔的底部设置了用于安装激光二极管的孔位；所述低温反应环上的样品管孔设置了用于安装光导纤维的孔位。
本发明的第三个目的是公开了一种核酸检测设备，所述设备包含上述的反应器。
本发明的第四个目的是公开了将上述核酸扩增方法用在以PCR为基础的核酸扩增和分析方法中。优选的，上述应用包括实时荧光定量PCR、多重PCR、RT-PCR、巢式PCR和测序等。
本发明的第五个目的是公开了一种用于上述极速核酸扩增方法的操作系统，包括高温控制部件、低温控制部件和荧光读数控制部件和电脑控制系统，所述电脑控制系统分别与所述高温控制部件、低温控制部件、荧光读数控制部件连接。
与现有技术相比，本发明的核酸扩增方法对传统核酸扩增进行了革新性的改进，改变了传统PCR三阶段的反应模式，利用了提前固定所需温度、并可批次反应的模式，提出了流水线式的PCR工作模式，提高PCR设备的使用率，节省使用者的时间，实现随来随测，使得系统有更大的灵活性和适用性。采用极端温度差阶以提高温度变化速率，就大大减少了管内反应液达到最适温度所需要的时间，实现了快速核酸检测的目的，使得核酸检测技术能够获得更广泛的应用。
附图说明
图1显示了长度分别为70bp、100bp、150bp、180bp的扩增片段常规PCR和在100℃和10℃极端环境温度介导的PCR扩增产物电泳结果图，其中，M泳道为分子量标准参照物，泳道1为常规PCR产物电泳结果，泳道2为极端环境温度介导的PCR产物电泳结果；
图2显示了长度分别为70bp、100bp、150bp、180bp的扩增片段常规PCR和在130℃和20℃极端环境温度介导的PCR扩增产物电泳结果图，其中，M泳道为分子量标准参照物，泳道1为常规PCR产物电泳结果，泳道2为极端环境温度介导的PCR产物电泳结果；
图3显示了长度分别为70bp、100bp、150bp、180bp的扩增片段的常规PCR和在150℃和40℃极端环境温度介导的PCR扩增产物电泳结果图，其中，M泳道为分子量标准参照物，泳道1为常规PCR产物电泳结果，泳道2为极端环境温度介导的PCR产物电泳结果；
图4是本发明的极速核酸扩增反应器的一个优选实施例的俯视示意图；
图5是本发明的极速核酸扩增反应器的一个优选实施例的侧视示意图。
具体实施方式
为使本发明更加容易理解，下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围，下列实施例中未提及的具体实验方法，通常按照常规实验方法进行。
实施例1高温环境温度对升温的温变速率的影响的初步试验
进行环境温度对温变效率影响的初步试验，具体操作如下：
设置PCR仪反应孔槽的温度为100℃，用点温计测试PCR反应管内液体的起始温度为60℃，将此反应管放置于PCR仪反应孔槽内，当PCR反应管内液体的温度达到92℃时，耗时14秒。
设置PCR仪反应孔槽的温度为95℃，用点温计测试PCR反应管内液体的起始温度为60℃，将此反应管放置于PCR仪反应孔槽内，当PCR反应管内液体的温度达到92℃时，耗时22秒。
将特制加温仪模块反应孔槽的温度设为130℃，用点温计测试PCR反应管内液体的起始温度为60℃，将此反应管放置于PCR仪反应孔槽内，当PCR反 应管内液体的温度达到92℃时，耗时6秒。
将特制加温仪模块反应孔槽的温度设为150℃，用点温计测试PCR反应管内液体的起始温度为60℃，将此反应管放置于PCR仪反应孔槽内，当PCR反应管内液体的温度达到92℃时，耗时5秒。
通过上述试验，得到以下结论：在150℃环境温度的情况下，由60℃升至到92℃时，温变速率为6.4℃/秒；在130℃环境温度的情况下，由60℃升至到92℃时，温变速率为5.3℃/秒；在100℃环境温度的情况下，由60℃升至到92℃时，温变速率为2.13℃/秒；在95℃环境温度的情况下，由60℃升至到92℃时，温变速率为1.45℃/秒。
以上结论说明在对相同起始温度的反应液进行同样梯度的升温时，环境温度与反应液初始温度相差越大，则反应液中实际升温的温变速率越快。
实施例2低温环境温度对降温的温变速率的影响的初步试验
进行环境温度对温变效率影响的初步试验，具体操作如下：
设置PCR仪反应孔槽的温度为10℃，用点温计测试PCR反应管内液体的起始温度为92℃，将此反应管放置于PCR仪反应孔槽内，当PCR反应管内液体的温度降到60℃时，耗时4秒。
设置PCR仪反应孔槽的温度为20℃，用点温计测试PCR反应管内液体的起始温度为92℃，将此反应管放置于PCR仪反应孔槽内，当PCR反应管内液体的温度降到60℃时，耗时5秒。
设置PCR仪反应孔槽的温度为40℃，用点温计测试PCR反应管内液体的起始温度为92℃，将此反应管放置于PCR仪反应孔槽内，当PCR反应管内液体的温度降到60℃时，耗时8秒。
设置PCR仪反应孔槽的温度为45℃，用点温计测试PCR反应管内液体的起始温度为92℃，将此反应管放置于PCR仪反应孔槽内，当PCR反应管内液体的温度降到60℃时，耗时11秒。
设置PCR仪反应孔槽的温度为60℃，用点温计测试PCR反应管内液体的起始温度为92℃，将此反应管放置于PCR仪反应孔槽内，当PCR反应管内液体的温度降到60℃时，耗时45秒。
通过上述试验，得到以下结论：在10℃环境温度的情况下，由92℃降至到60℃时，实际温变速率为8℃/秒；在20℃环境温度的情况下，由92℃降至到60℃时，实际温变速率为6.4℃/秒；在40℃环境温度的情况下，由92℃降至到60℃时，实际温变速率为4℃/秒；在45℃环境温度的情况下，由92℃降至到60℃时，实际温变速率为2.9℃/秒；在60℃环境温度的情况下，由92℃降至到60℃时，实际温变速率为0.7℃/秒。
以上结论说明在对相同起始温度的反应液进行同样梯度的降温时，当环境温度与反应液初始温度相差越大，则降温的温变速率越快。
实施例3极速PCR方法与常规PCR方法的比较实验的一个优选实施例
以乙肝病毒(HBV)基因的四组不同短片段作为目标片段进行扩增，采用极速PCR方法与常规PCR方法进行对照。
1、四组目标扩增序列和对应的正向引物和反向引物序列如下：
第一组目标扩增序列SEQ ID NO:1：
Figure PCTCN2018122453-appb-000001
第一组正向引物SEQ ID NO:2：5’-CCCCAACCTCCAATCACTCA-3’
第一组反向引物SEQ ID NO:3：5’-CGCAGACACATCCAGCGATAAC-3’
第二组目标扩增序列SEQ ID NO:4：
Figure PCTCN2018122453-appb-000002
第二组正向引物SEQ ID NO:5：5’-CCCCAACCTCCAATCACTCA-3’
第二组反向引物SEQ ID NO:6：
5’-AGCAGGATGAAGAGGAAGATGATAAA-3’
第三组目标扩增序列SEQ ID NO:7：
Figure PCTCN2018122453-appb-000003
第三组正向引物SEQ ID NO:8：5’-CCCCAACCTCCAATCACTCA-3’
第三组反向引物SEQ ID NO:9：
Figure PCTCN2018122453-appb-000004
第四组目标扩增序列SEQ ID NO:10：
Figure PCTCN2018122453-appb-000005
第四组正向引物SEQ ID NO:11：5’-CCCCAACCTCCAATCACTCA-3’
第四组反向引物SEQ ID NO:12：
Figure PCTCN2018122453-appb-000006
2、PCR反应体系反应液配制如下，其中，PCR反应缓冲液选自TAKARA公司的产品：
Figure PCTCN2018122453-appb-000007
3、常规PCR反应程序如下：
Figure PCTCN2018122453-appb-000008
4、极端环境温度介导的PCR实验反应操作如下：
使用独立的高温模块、低温模块作为PCR反应的反应环境温度托槽，其中，高温模块的温度设置为100℃，低温模块的温度设置为10℃。
用点温计放置于PCR反应管内以测定PCR管内反应液的温度。
参考实施例1和2得到的实验结果，将PCR管放置于独立的100℃高温槽中，测定PCR管内反应液温度从60℃(±3℃)升至92℃(±3℃)，需要耗时14s；将PCR管放置于独立的10℃低温槽中，测定PCR管内反应液温度从92℃(±3℃)降至60℃(±3℃)，需要耗时4s。因此，我们设计的极端环境温度介导的PCR实验的反应程序如下：
Figure PCTCN2018122453-appb-000009
PCR管内的反应液的温度在PCR管放置于100℃独立高温槽期间，最高温为92℃(±3℃)，放置于10℃独立高温槽期间内，最低温为60℃(±3℃)。
通过人工手动方式使PCR反应管在高温槽和低温槽之间运动，使得PCR反应管内反应液实际温度在60-92℃(±3℃)中反复循环、以实现目标基因片段的扩增。
实验结果显示，用常规PCR方法，用时约1小时25分钟，用极端环境温度介导的PCR反应，用时约10分钟。两种方法的扩增效果用2％琼脂糖凝胶电泳进行检测，电泳结果见图1，图1显示了长度分别为70bp、100bp、150bp、180bp 的扩增片段常规PCR和在100℃和10℃极端环境温度介导的PCR扩增产物电泳结果图，其中，M泳道为分子量标准参照物，泳道1为常规PCR产物电泳结果，泳道2为极端环境温度介导的PCR产物电泳结果。
由图1结果可见，极端环境温度介导的PCR扩增检测过程可以在11分钟左右即完成，而且其扩增效果可以达到常规PCR扩增检测类似的效果。
实施例4极速PCR方法与常规PCR方法的比较实验的第二个优选实施例
试剂体系、模板、引物、常规PCR对照实验同实施例3。
实验的反应程序稍作改变如下：
Figure PCTCN2018122453-appb-000010
PCR管内的反应液的温度在PCR管放置于130℃独立高温槽期间，最高温为92℃(±3℃)，放置于20℃独立低温槽期间时，最低温为60℃(±3℃)。
通过人工手动方式使PCR反应管在高温槽和低温槽之间运动，使得PCR反应管内反应液实际温度在60-92℃(±3℃)中反复循环、以实现目标基因片段的扩增。
实验结果显示，用常规PCR方法，用时约1小时25分钟，用此条件极端环境温度介导的PCR反应，用时约8分钟。两种方法的扩增效果用2％琼脂糖凝胶电泳进行检测，电泳结果见图2，图2显示了长度分别为70bp、100bp、150bp、180bp的扩增片段常规PCR和在120℃和25℃极端环境温度介导的PCR扩增产物电泳结果图，其中，M泳道为分子量标准参照物，泳道1为常规PCR产物电泳结果，泳道2为极端环境温度介导的PCR产物电泳结果。
由图2结果可见，极端环境温度介导的PCR扩增检测过程可以在8分钟内即完成，而且其扩增效果可以达到常规PCR扩增检测近似的效果。
实施例5极速PCR方法与常规PCR方法的比较实验的第三个优选实施例
试剂体系、模板、引物、常规PCR对照实验同实施例3。
实验的反应程序稍作改变如下：
Figure PCTCN2018122453-appb-000011
PCR管内的反应液的温度在PCR管放置于150℃独立高温槽期间，最高温为92℃(±3℃)，放置于20℃独立低温槽期间时，最低温为60℃(±3℃)。
通过人工手动方式使PCR反应管在高温槽和低温槽之间运动，使得PCR反应管内反应液实际温度在60-92℃(±3℃)中反复循环、以实现目标基因片段的扩增。
实验结果显示，用常规PCR方法，用时约1小时25分钟，用极端环境温度介导的PCR反应，用时约11.5分钟。两种方法的扩增效果用2％琼脂糖凝胶电泳进行检测，电泳结果见图3，图3显示了长度分别为70bp、100bp、150bp、180bp的扩增片段的常规PCR和在100℃和30℃极端环境温度介导的PCR扩增产物电泳结果图，其中，M泳道为分子量标准参照物，泳道1为常规PCR产物电泳结果，泳道2为极端环境温度介导的PCR产物电泳结果。
由图3结果可见，极端环境温度介导的PCR扩增检测过程可以在11.5分钟即完成，而且其扩增效果可以达到常规PCR扩增检测近似的效果。
由实施例3-5的结果可见，常规PCR方法和极端环境温度介导的PCR方法对不同大小短基因片段均可有效实现PCR扩增；极端环境温度介导PCR法在8-12min内即能够实现高效、超快速扩增，用时可缩短到常规PCR的约十分之一之内。可以据此发明，在具体的实验中根据高、低温区温度点与放置时长相结合去选择温度和时间组合，达到控制反应管内反应液温度在60-92℃(±3℃) 并实现目标片段扩增的目的。
实施例6本发明的极速核酸扩增反应器的一个优选实施例
本实施例为本发明的极速核酸扩增反应器的一个优选实施例，其结构如图4和图5所示。图4是本发明的极速核酸扩增反应器的一个优选实施例的俯视示意图；图5是本发明的极速核酸扩增反应器的一个优选实施例的侧视示意图。由图4可见，本发明的核酸扩增的反应器包括低温反应环1、高温反应盘2、隔热带3、盖板4、旋转轴5和步进电机6；所述低温反应环1和所述高温反应盘2同一圆心；所述低温反应环1的直径大于所述高温反应盘2的直径；所述高温反应盘2的外边沿为齿轮样；所述低温反应环1的内边沿为齿轮样；所述低温反应环1与所述高温反应盘2之间用所述隔热带3相互隔离并相互咬合形成完整的圆形盘，所述低温反应环1与所述高温反应盘2咬合处形成齿轮样咬合线；在所述咬合线上沿盘面圆周的同一圆周线上分布样品管孔7；所述低温反应环1和高温反应盘2的位置固定；所述盖板4与所述旋转轴5相连，所述旋转轴5的轴心位于所述低温反应环1或高温反应盘2的圆心；所述盖板4通过所述旋转轴5与所述步进电机6连接；所述低温反应环1和高温反应盘2位于所述盖板4的下方；所述低温反应环1的温度设置在10℃-40℃中的一个固定温度或温度区间；所述高温反应盘2的温度设置在100℃-150℃中的一个固定温度或温度区间。所述盖板4的侧面周边设置了T型槽开口卡41，所述T型槽开口卡41与所述样品管孔7相对应。所述样品管孔7的底部设置了用于安装激光二极管的孔位8；所述样品管孔7设置了用于安装光导纤维的孔位9。
样品管孔7可以为60-90个，这样，相邻的试样杯孔实际上位于不同的温度区上。如果一个试样从一个孔被移入相邻的另一个孔中，就从一个温度区到了另一个温度区。如果这个试样沿着圆周移动了60个孔位，那么这个试样就经历了30个高、低温变化的周期，也就是说完成了一个PCR反应环境的全过程。
试样杯沿着圆周依次从一个孔向相邻的孔移动是通过可转动并可上下移动 的盖板来实现的。图5是本发明的极速核酸扩增反应器的一个优选实施例的侧视示意图，结合图5，该反应器除了低温反应环1、高温反应盘2和隔热带3，还包括盖板4和旋转轴5，盖板4与旋转轴5相连，旋转轴5位于低温反应环1或高温反应盘2的圆心；低温反应环1和高温反应盘2位于盖板4的下方；盖板4的侧面周边设置了40个T型槽开口卡41，样品管71通过T型槽开口卡41卡在盖板4上。T型槽开口卡41与样品管孔7相对应；样品管71安装在盖板4侧面周边的T型槽开口卡41内，跟随盖板4做圆周步进动作和上下动作，1-6秒钟之后，盖板4向上移动，将所有样品管71拔出样品管孔7。盖板4转动一个孔位，再向下移动。这样，所有样品管71就一起更换了温度环境。盖板4的上下移动和步进旋转移动可以由步进电机6按预定程序驱动。盖板4携带样品管71作上下和圆周运动，每5秒动作一次，40次动作旋转一周，相当于是20个温度循环。当这个样品管71已经完成了30个温度周期后，就会被从盖板上移出。
样品管孔7底部设置了用于安装激光二极管的三十个孔位8；低温反应环1上的样品管孔7的侧面设置了用于安装光导纤维的三十个孔位9，用于读取信号。在样品管71停留于低温试样孔中时，激光管发出激发光，刺激样品管71中的试样发出可能的荧光，可能的荧光被光纤捕捉并传送到光电检测部分。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (10)

  1. 一种极速核酸扩增方法，所述方法将装有反应混合液的反应管置于超高温反应区和超低温反应区，进行30-45个循环；
    所述超高温反应区的温度为：100℃-150℃中的一个固定温度；
    所述超低温反应区的温度为：10℃-40℃中的一个固定温度；
    所述循环的程序为：在所述超高温反应区和所述超低温反应区依次放置4-10秒，作为一个循环。
  2. 根据权利要求1所述的方法，其中，
    所述反应管为聚丙烯材质的多聚酶链式反应管或玻璃材质的多聚酶链式反应管；
    所述反应混合液包括聚合酶、扩增引物、dNTP、PCR缓冲液和灭菌双蒸水。
  3. 一种用于权利要求1所述极速核酸扩增方法的反应器，其中，所述反应器包括超高温反应区和超低温反应区；
    所述超高温反应区的温度调节范围为100℃-150℃中的一个固定温度或温度区间；
    所述超低温反应区的温度调节范围为10℃-40℃中的一个固定温度或温度区间。
  4. 根据权利要求3所述的反应器，其中，所述超高温反应区和所述超低温反应区分别由两块反应管托槽温控金属模块构成；
    所述两块反应管托槽温控金属模块的组装方式为同心圆环、平行条块或齿轮样交叉互嵌方式；
    所述反应管托槽温控金属模块上有反应管孔，所述反应管孔的数量为1-45个；
    所述反应管孔内用于放置反应管，所述反应管在所述两块反应管托槽温控金属模块间通过机械手自动完成位置移动；
    所述反应器设置荧光检测系统，所述荧光检测系统在低温时完成荧光检测程序。
  5. 根据权利要求3所述的反应器，其中，所述超高温反应区为高温反应盘，所述超低温反应区为低温反应环，所述反应器还包括隔热带、盖板、旋转轴和步进电机；
    所述低温反应环和所述高温反应盘为同一圆心；
    所述低温反应环的直径大于所述高温反应盘的直径；
    所述高温反应盘的外边沿为齿轮样；
    所述低温反应环的内边沿为齿轮样；
    所述低温反应环与所述高温反应盘之间用所述隔热带分隔并相互咬合形成完整的圆形盘，所述低温反应环与所述高温反应盘咬合处形成齿轮样咬合线；
    在所述咬合线上沿盘面圆周的同一圆周线上分布样品管孔；
    所述低温反应环和高温反应盘的位置固定；
    所述盖板与所述旋转轴相连，所述旋转轴的轴心与所述低温反应环或高温反应盘的圆心相同；
    所述盖板通过所述旋转轴与所述步进电机连接；
    所述低温反应环和所述高温反应盘位于所述盖板的下方。
  6. 根据权利要求5所述的反应器，其中，所述盖板的侧面周边设置了T型槽开口卡，所述T型槽开口卡与所述样品管孔相对应。
  7. 根据权利要求6所述的反应器，其中，所述样品管孔的底部设置了用于安装激光二极管的孔位；所述低温反应环上的样品管孔设置了用于安装光导纤 维的孔位。
  8. 一种核酸检测设备，所述设备包含权利要求4-7之一所述的反应器。
  9. 权利要求8所述核酸扩增设备在以PCR为基础的核酸扩增和分析方法中的应用。
  10. 一种用于权利要求1所述极速核酸扩增方法的操作系统，包括高温控制部件、低温控制部件、荧光读数控制部件和电脑控制系统，所述电脑控制系统分别与所述高温控制部件、低温控制部件、荧光读数控制部件连接。
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