WO2011079746A1

WO2011079746A1 - Tube comprising fusible layer

Info

Publication number: WO2011079746A1
Application number: PCT/CN2010/080195
Authority: WO
Inventors: 周国华; 梁超; 成思佳
Original assignee: 华东医学生物技术研究所
Priority date: 2009-12-30
Filing date: 2010-12-23
Publication date: 2011-07-07
Also published as: CN101787347A; CN101787347B

Abstract

The present invention provides a tube, which comprises at least one layer that seals reagent for a chemical reaction. The layer is made up of fusible material. When temperature reaches the melting point of the fusible material, the layer melts down and releases the reagent so as to control the process of the chemical reaction by the change of temperature. The tube is portable, and can avoid contamination due to exposure of the reagent.

Description

Specification tube containing fusible layer

Technical field

The invention belongs to the field of genetic engineering and relates to a reaction tube which controls the progress of a reaction by temperature.

Polymerase Chain Reaction (PCR) is an in vitro nucleic acid amplification technique developed in the mid-1980s. It has the outstanding advantages of being specific, sensitive, high yield, fast, simple, reproducible and easy to automate; it can amplify the target gene or a DNA fragment to be studied in a test tube to 100,000 or even in a few hours. Millions times, allowing the naked eye to directly observe and judge; a sufficient amount of DNA can be amplified from a hair, a drop of blood, or even a cell for analysis and identification. PCR technology is a revolutionary initiative and milestone in the biomedical field.

Rolling Circle Amplification (RCA) is an isothermal signal amplification method. DNA can be exponentially amplified in a short time, so it can be used for the detection of trace molecules. At present, this technology can amplify circular DNA, RA, linear DNA, and even whole genome DNA, mainly for whole genome amplification, nucleic acid sequencing, single nucleotide polymorphism, and DNA chips and protein chips. Analysis and other broad areas.

Loop-mediated isothermal amplification (LAMP) technology is a sensitive, specific and convenient nucleic acid amplification technology that has been developed in recent years. The reaction is carried out in less than an hour to amplify the nucleic acid, and its efficiency can reach the order of 10 ⁹ -10 ^1Q copies. The method is simple, fast, accurate, inexpensive, and easy to detect.

With the development of biotechnology, nucleic acid amplification techniques such as polymerase chain reaction (PCR), rolling circle amplification (RCA) and loop-mediated isothermal nucleic acid amplification (LAMP) are used in biological analysis, pathogenic microbial detection and disease diagnosis. It is widely used in clinical and basic research, but as the nucleic acid amplification technology, the biggest shortcoming is that it is easy to produce hysteresis pollution of the product, especially when the expansion product is detected, it is easy to cause the product. Pollution. The reaction process of these nucleic acid amplification techniques, including the initiation, termination and detection of the reaction, can be carried out by adding or removing reagents by opening the reaction tube, especially if some reagents are added in advance in the course of the reaction. It will cause interference and even affect the continuation of the reaction. These deficiencies make the corresponding technology have high requirements for operators and experimental environments, which limits its application range, especially on-site rapid detection. As a nucleic acid amplification technique, since the polymerase is still active at low temperatures, mis-annealing of the primer may result in non-specific expansion. At present, this non-specific amplification is overcome mainly by hot-starting polymerase, and its cost is much higher than that of ordinary polymerases, which increases the cost of experiments and the promotion of unfavorable techniques. There is currently no temperature-controlled reaction tube that does not require the addition of reagents during the reaction, reduces operational contamination, and can control the progress of the reaction by simply adjusting the temperature.

Summary of the invention

It is an object of the present invention to provide a reaction tube that utilizes temperature to control the progress of the reaction.

The object of the invention is achieved by the following technical solutions:

A reaction tube for controlling the progress of a reaction by temperature, wherein the reaction tube is designed with a sealing layer in the tube, and the reaction reagent required for the reaction process to be controlled is sealed in the sealing layer, and the sealing material is melted and released by controlling the temperature change. The reagents are sealed to control the progress of the reaction.

In the reaction tube, when the sealing layer is a plurality of layers, the sealing layer is disposed on the sealing layer to be melted according to the sequence of different reaction processes (actually, the sealing material constituting the sealing layer is melted, and the sealing is adopted for convenience of expression) After the layer melting "described, the same below), the reaction reagent in the sealing layer can be in contact with the reaction system of the previous reaction process (usually the reaction liquid), and the melting temperature of each sealing layer is less than or equal to the reaction of the corresponding reaction process. The temperature is higher than the reaction temperature of the previous reaction process and lower than the melting temperature of the sealing layer corresponding to the post-reaction process (the first reaction process if the sealing layer is used, the sealing layer is not considered due to the absence of the prior reaction process) Whether the melting temperature is higher than the reaction temperature of the previous reaction process. Similarly, the final molten sealing layer is not present in the sealing layer corresponding to the post-reaction process without considering whether the melting temperature below the sealing layer is lower than the subsequent reaction. The melting temperature of the sealing layer corresponding to the process). The order of melting of each sealing layer is set in the order of the reaction temperature from low to high.

The reaction tube is provided with a sealing layer in sequence according to the sequence of different reaction processes controlled by the sealing layer, and the outermost sealing layer (ie, the sealing layer closest to the reaction tube nozzle) corresponds to the previous reaction process.

In the reaction tube, the temperature regulation of the reaction tube is adjusted in stages according to a reaction course to be controlled, which is higher than or equal to the reaction temperature of the corresponding reaction process and lower than the melting temperature of the sealing layer corresponding to the subsequent reaction process. Preferably, the temperature of the reaction tube is adjusted to a reaction temperature required for the corresponding reaction process, and the sealing layer corresponding to the corresponding reaction progress is melted because the melting point is less than or equal to the reaction temperature, and the reagent previously sealed in the sealing layer is released to cause a corresponding reaction. The process can proceed.

The reaction tube, wherein the temperature adjustment of the reaction tube should be adjusted from a low temperature to a high temperature according to the sequence of the reaction process. The reaction tube, wherein different sealing layers use substances having different melting points as sealing materials. The reaction tube, wherein the substances having different melting points are paraffin waxes having different melting points or low melting point polytetrafluoroethylene having different melting points.

In the reaction tube, the number of sealing layers is designed according to the number of reaction stages required; the reaction processes at different temperatures have different melting points of the corresponding sealing layers.

The reaction tube in which the reactants in the sealing layer are mixed in or separated by a sealing material. When preparing the temperature-controlled reaction tube, first add the reagents required for the corresponding reaction stage in the tube, and then add the appropriate paraffin wax to the surface of the desired reagent (for example, paraffin wax, other sealing layer materials may also be used), heat until the paraffin is melted, and then cooled to It is actually sealed with paraffin after room temperature. According to the number of different processes controlled by the sealing layer and the reaction temperature in the whole reaction, the number and order (or position) of the sealing layers in the reaction tube are designed, and the wax having the appropriate melting point is selected as the sealing layer material according to the temperature of the different reaction processes.

The temperature-controlled reaction tube of the present invention is not limited to application in the field of genetic engineering.

Advantageous Effects of Invention The present invention provides a reaction tube capable of controlling the progress of a reaction by temperature change, and by releasing a reagent which is previously stored in a reaction tube by changing the temperature, thereby controlling the initiation, termination, and detection of the reaction. The reaction tube effectively avoids the pollution caused by the frequent opening of the reaction tube in the reaction process and the hysteresis pollution after the end of the reaction, reduces the interference of the repeated addition of the reagent to the reaction process, is easy to be automatically controlled, and can control the reaction to some extent. The start time of the process increases the specificity of the reaction. Such a reaction tube can be widely used in basic research in the field of biomedicine and in the fields of biological analysis, pathogenic microorganism detection, and disease diagnosis. In addition, the temperature-controlled reaction tube of the invention seals the required reaction reagents in advance according to the progress of the reaction, and is convenient for carrying and transportation, and does not need to be temporarily prepared, thereby saving operation time.

DRAWINGS

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic view of a temperature control reaction tube of the present invention. Among them: 1, sealing material one; 2, sealing material 2; 3, reagent one; 4, reagent two; 5, the body; 6, tube cover. The sealing material 1 and the reagent 1 form a sealing layer 1, and the sealing material 2 and the reagent 2 constitute a sealing layer 2.

2 is a schematic view of a temperature sealing layer of a temperature-controlled reaction tube of the present invention.

detailed description

The invention is further illustrated by the following examples.

Example 1: Detector construction and working process 1, construction:

1 to 5, the detector includes a reaction tube oscillation device, a temperature adjustment device, a time adjustment device 2 (timing module 2), and a fluorescence color observation device connected to the central control circuit 1, respectively; and may also include a central control circuit 1 connected reaction tube lifting device; (may also include temperature-controlled reaction tube 9, temperature control reaction tube details see Example 2). Its towel:

The reaction tube oscillating device comprises a reaction tube holder 6 and a bracket oscillating motor (such as a cam motor) 5, and the bracket oscillating motor is connected with the reaction tube bracket; the reaction tube lifting device comprises a reaction tube holder lifting drive motor 17, a reaction tube holder lifting rod 18, and a reaction The tube bracket lifting drive motor is connected to the reaction tube bracket lifting rod, and the reaction tube bracket lifting rod is connected with the reaction tube bracket. The reaction tube oscillating device can vibrate the reaction tube during the reaction process, mix the reaction system and accelerate the completion of the reaction process; the reaction tube support lifting device can conveniently take the reaction tube, or the reaction tube can be placed close to (positioned) or away from the heating device .

The temperature adjusting device comprises a temperature control module 3, a heating device 4, and a temperature measuring device 20; the heating device and the temperature measuring device are respectively connected with the temperature control module, and the temperature control module is connected with the central control circuit or directly forms part of the central control circuit, and the temperature is measured. The temperature sensor of the device is located near the lower part of the reaction tube or near the heating device (when the reaction tube is close to or placed on the heating device, the temperature of the heating device is substantially the same as the temperature of the portion of the reaction tube containing the reaction liquid); the heating device can be located Below the reaction tube holder, or placed in other parts of the instrument, the heated hot air is sent to the area where the reaction tube is located by means of air supply or the like (when the amount of liquid in the reaction tube is large, the local temperature can be avoided by directly heating the bottom of the reaction tube) Too high to keep the temperature of the reaction solution uniform; the temperature adjustment device may further include a heat sink 19 connected to the temperature control module.

The fluorescence color observation device comprises a fluorescence excitation light source 10, a fluorescence color observation or acquisition device; a fluorescence color observation device is an observation window 7 with or without a filter; the fluorescence color collection device is an image acquisition device or photoelectric conversion data acquisition. The analysis device; the fluorescence excitation light source 10 emits light to illuminate the reaction liquid in the reaction tube 9, and the fluorescence color observation or collection device can observe or collect the fluorescent signal emitted by the reaction liquid in the reaction tube. In order to save instrument cost, it is preferable to adopt an observation window method.

The central control circuit can also include a control information input device. The time setting and temperature setting device can also be integrated into the control information input device. Others such as whether the reaction tube needs vibration reaction tube, the reaction tube vibration time and frequency, whether the reaction tube holder needs to be lifted or the like can be input from the control information. A central control circuit is input to the device, or directly written into the program of the central control circuit. Information such as time adjustment, temperature adjustment, vibration adjustment, etc., which control the progress of the reaction, can be written into the program of the central control circuit in advance, and the type of the corresponding program-controlled reaction can be selected on the control information input device. After receiving the control information and feedback information, the central control circuit adjusts the operation of the corresponding device (heating device, heat sink, reaction tube holder lifting or shaking device, excitation light source, etc.). 2. Working process:

1), (power on) The temperature-controlled reaction tube 9 containing the reaction system is placed in the instrument through the sample inlet 8 and placed on the reaction tube holder 6 (if the reaction tube holder lifting device is provided, it can be conveniently taken and placed) Reaction tube).

2), setting the reaction time of the corresponding reaction process through the timing module 2, setting the reaction temperature of the corresponding reaction process through the temperature control module 3, and the relevant control information is regulated by the central control circuit 1 (or selecting a program written in advance to the central control circuit) Reaction type). After the setting is completed, the heating device 4 starts to work, is heated to the set temperature, and is heated by the temperature sensor feedback of the temperature measuring device 20 to stop the heating (if the temperature is too high, the heat sink device 19 can be activated), and the nucleic acid amplification reaction starts.

(According to the reaction needs to determine whether to oscillate the reaction tube).

3) After the nucleic acid amplification reaction is completed, the temperature is set to the temperature required for the sealing layer to be melted (or the type of the program-controlled reaction preset by the central control circuit) by setting the temperature control module 3, and the heating device is operated and raised. To the set temperature, the sealing layer in which the fluorescent dye is sealed in the reaction tube is melted. The fluorescent dye and the reaction product are uniformly mixed by the oscillating device 5.

4) Start the excitation light source 10 to illuminate the reaction tube, observe the fluorescence generation through the observation window 7 or collect and analyze the fluorescence information in the reaction tube by using a photographic or photoelectric sensor. Example 2: Temperature-controlled reaction tube

Paraffin supplier: Nanyang paraffin fine chemical plant.

Paraffin specifications and characteristics

Paraffin model melting point (°c)

52# 52-54

54# 54-56

56# 56-58

58# 58-60

60# 60-62

62# 62-64

64# 64-66

70# 67-72

75# 72-77 80# 77-82

85# 82-87

90# 87-92

95# 92-97

Referring to FIG. 7, the sealing layer is disposed in the order of different reaction processes after the sealing layer is melted, and the reaction reagent in the sealing layer can react with the previous reaction process (usually the reaction liquid, for the first reaction, since there is no a reaction process, so the reaction system is usually in contact with the analyte and other reaction reagents that do not contain the sealed reagent required for the first reaction, and the melting temperature of each sealing layer is less than or equal to the reaction temperature of the corresponding reaction process. And higher than the reaction temperature of the previous reaction process (corresponding to the first reaction sealing layer does not consider this condition, such as the 30 ° C sealing layer in this example) and lower than the melting temperature of the sealing layer corresponding to the post-reaction process (corresponding to the final The molten sealing layer does not take into account this condition, such as the 80 ° C sealing layer in this example). The reaction reagents in the respective sealing layers are respectively mixed in or separated from the sealing material constituting the sealing layer corresponding to each reaction course. When the reaction is carried out, the temperature regulation of the reaction tube is adjusted in stages according to the reaction process to be controlled, which is higher than or equal to the reaction temperature of the corresponding reaction process and lower than the melting temperature of the sealing layer corresponding to the subsequent reaction process (to avoid the reaction tube temperature being higher than The reaction temperature of the corresponding reaction process may adversely affect some reaction processes. In this example, the temperature of the reaction tube in each stage is adjusted to the reaction temperature required for the corresponding reaction process. It is assumed that the reaction temperature of each reaction process in Fig. 7 is 30 ° C, 40 ° C, 50 ° C, 60 ° C, 70 ° C, 80 ° C, and the sealing material (such as paraffin wax) constituting the sealing layer corresponding to each reaction process. The melting temperature is 28~30°C, 38~40°C, 48~50°C, 58-60°C, 68~70°C, 78~80°C, and the initial reaction temperature is 30°C. . Adding the test substance, when the temperature of the reaction tube rises to 30 ° C, the sealing layer melts at 30 ° C, releasing the reagent required for the first stage, the first stage reaction proceeds, and the molten paraffin floats on the upper part of the reaction liquid; After that, the temperature of the reaction tube is raised to 40 ° C, the sealing layer is melted at 40 ° C, the reagent required for the second stage is released, the second stage reaction is carried out, and the molten paraffin floats on the upper portion of the reaction liquid; after the appropriate time, Then, the temperature of the reaction tube is raised to 50 ° C, the sealing layer is melted at 50 ° C, and the reagent required for the third stage is released, the third stage reaction proceeds, and the molten paraffin floats on the upper portion of the reaction liquid; and so on, at 60 The fourth-stage reaction was carried out at °C, and the fifth-stage reaction was carried out at 80 °C. After the reaction is completed, the temperature (or cooling) is lowered, and all or part of the paraffin floating on the upper portion of the reaction liquid is solidified to seal the reaction liquid to avoid hysteresis contamination. Example 3

In this example, six primers were designed for the HA gene of swine influenza H1N1. The LAMP amplification system is as follows: Table 1. H1N1 LAMP primers SW H1-F3: 5 '-GGTGCTATAAACACCAGCC-3 '

SW H1-B3: 5 '-TGATGGTGATAACCGTACC-3 '

SW H1-LF: 5 '-GGACATTYTCCAATTGTG-3 '

SW H1-LB 5 ' -TTGCCGGTTTCATTGAAGG-3 '

SW Hl-FIP(Flc+F2): 5'-CTGTRGCCAGTCTCAATTTTGTGttttCTGAAGTY

CCATTTCAGAATATACATCCR-3 '

SW Hl-BIP(Blc+B2): 5 ' -ATCCCGTCTATTCAATCTAGAGGCttttCTGAAGAT

CCATCTACCATCCCTGTC-3,

Y: t/u or c; R: g or a.

25 μL LAMP reaction system:

Buffer 1.875 L

BIP, FIP 2.31

B3, F3 each 0.19 μ

LB, LF each 1

Bst DNA polymerase 0.5 L

Fluorescent dye 0.5 L

H ₂ 0 1.625 L

Mineral oil 8 L

H1N1 template 1 L

LAMP reaction procedure: 63 ° C / 1.5 h 80 ° C / 5 min

The temperature-controlled reaction tube is provided with a sealing layer which is sealed with a fluorescent dye (such as SYBR Green l) in advance by using paraffin wax (such as 90#), and after adding the nucleic acid amplification system and the sample to be tested in the reaction tube, the reaction tube is placed in the present invention. In the designed detector, the reaction system is heated to the temperature required for the reaction according to the LAMP reaction procedure. After the reaction is completed, the tube is not required to be opened, the temperature is raised to melt the sealing layer, the fluorescent dye is released, and the nucleic acid is amplified. The product is thoroughly mixed with the fluorescent dye, and the excitation light source is irradiated to the reaction tube at an appropriate time. The fluorescence of the reaction tube can be observed from the observation window, and the sample to be tested contains the pathogenic microorganism. If the fluorescence is generated, the sample to be tested contains the detection pathogen. microorganism. The method can perform nucleic acid amplification reaction and fluorescence detection directly in the instrument without removing the reaction tube. Example 4: Using a temperature-controlled reaction tube to terminate the reaction

The solution realizes the temperature control reaction termination by temperature control of the release of the reaction terminator in the sealing layer, and mainly targets the termination of the constant temperature reaction such as RCA and LAMP. This solution can be used to control the reaction using a simple thermostat. It is suitable for use under rapid test conditions such as pathogenic microorganisms and disease diagnosis.

A reaction terminator such as sodium edetate (EDTA) solution or other metal complexing agent and a protein denaturant are added to the bottom of the reaction tube. Add ΙΟμΙ paraffin (Li) to the upper surface. The reaction tube is heated to the melting point of the paraffin to melt the paraffin, and the reaction tube is taken out and cooled to re-solidify the paraffin to form a sealing layer. In the RCA or LAMP reaction, a reaction system is added to the paraffin seal layer. After the constant temperature reaction (60 ° C), the reaction tube is heated to the melting point of the paraffin. Since the paraffin wax density is less than water, the paraffin wax will float on the upper surface of the liquid surface after melting, and the reaction system will be mixed with the reaction terminator under the paraffin seal layer. The reaction was terminated.

After the reaction tube is taken out and cooled, the paraffin will solidify again, so that the surface of the liquid is isolated from the air, avoiding the hysteresis contamination that may be caused by the reaction product. Example 5: Product detection using a temperature-controlled reaction tube

The solution controls the release of the product indicator in the sealing layer by temperature control, and the amplification reaction and the product indication are carried out in the same tube. This program is mainly aimed at RCA, LAMP and other temperature amplification reactions, which can realize rapid, simple and visual detection of pathogenic microorganisms.

A product indicator such as SYB Green, Gold View or the like is added to the bottom of the reaction tube. ΙΟμΙ paraffin (64#) was added to the upper surface. The reaction tube is heated to the melting point of the paraffin to melt the paraffin. The reaction tube was taken out and cooled to room temperature to re-solidify the paraffin to form a sealing layer.

In the RCA or LAMP reaction, a reaction system is added to the paraffin seal layer. After the constant temperature reaction (60 ° C), the reaction tube is heated to the melting point of the paraffin. Since the paraffin wax density is less than water, the paraffin wax will float on the upper surface of the liquid surface after melting, and the reaction system will be mixed with the product indicator to be visually observable. The change enables visualization of the amplified product.

The detection method designed by the scheme avoids the inhibition of the amplification reaction caused by the addition of the product indicator in the reaction system, and realizes the reaction and monitoring in the same reaction tube on the other hand, without opening the reaction tube and avoiding the product. Hysteresis pollution. Example 6: Using a temperature-controlled reaction tube to achieve ordinary heat-resistant polymerase thermal initiation and color reaction

Referring to FIG. 6, the solution controls the release of key components in the reaction system of the sealing layer by temperature control, so that the non-specific amplification reaction is suppressed at a low temperature, and the release of the coloring reagent in the sealing layer is controlled by a higher temperature. That is, the hot start and color reaction of the ordinary heat-resistant polymerase are realized in a single tube. This protocol is mainly for the PCR reaction, and can perform hot-start PCR using a low-cost ordinary heat-resistant polymerase and perform color detection on the PCR product.

A product indicator such as SYB Green, Gold View or the like (i.e., reagent one) is added to the bottom of the reaction tube 15. On the upper surface, ΙΟμΙ paraffin (95#) (ie, sealing material 1) was added. The reaction tube is heated to a melting point of paraffin to melt the paraffin. The reaction tube was taken out and cooled to room temperature to re-solidify the paraffin to form a sealing layer 1.

Adding a key component of the PCR reaction to a surface of the sealing layer, such as a thermostable polymerase, magnesium ion, dNTP, etc. Species or several (ie reagent II). On the upper surface thereof, ΙΟμΙ paraffin di(85#) (i.e., sealing material 2) was added, and the temperature was lower than the PCR denaturation temperature and lower than the melting point of paraffin one of the sealing layer 1. The reaction tube is heated to the melting point of paraffin two to melt the paraffin. The reaction tube was taken out and cooled to room temperature, and the paraffin wax was re-solidified to form a sealing layer 2.

Before the PCR reaction, a reaction system other than the key components in the sealing layer is added to the paraffin seal layer 2, and the tube cover 16 is covered. When the reaction is carried out, it is first heated to the melting point of paraffin two and kept at a constant temperature for 5 minutes to completely melt the paraffin 2. Since the paraffin wax density is less than water, the paraffin wax will float on the upper surface of the liquid surface after melting, and the reaction system will be mixed with the key component of the reaction under paraffin 2 (ie, reagent 2), and the temperature of the reaction tube meets the temperature required for the amplification reaction, thereby The amplification reaction is initiated.

After the end of the amplification reaction, the reaction tube is heated to the melting point of paraffin wax. Since the paraffin wax density is less than water, the paraffin wax will float on the upper surface of the liquid surface after melting, and the reaction system will be mixed with the product indicator (ie, reagent one). The tube temperature is in accordance with the desired temperature of the product indicator, producing visually observable changes that enable visual inspection of the amplified product.

The present scheme is designed to perform a hot start reaction using an ordinary thermostable polymerase, suppresses a non-specific amplification reaction, and enhances the specificity of the PCR reaction while performing a color reaction of the amplified product in the same tube. (The reaction tubes of the two sealing layers can also be subjected to temperature amplification reaction for RCA, LAMP, etc., and the reagents required for the nucleic acid amplification reaction are all or partially sealed in the sealing layer 2 (lower temperature melting), and the fluorescent dye is sealed on the sealing layer. In one (higher temperature melting), two sealing layers are sequentially dissolved by controlling the temperature change, the reaction progress is controlled, and nucleic acid amplification reaction and fluorescence visual inspection are performed.

Claims

Claim

1. A reaction tube for controlling the progress of a reaction by temperature, the main feature of which is to design a sealing layer in the tube, sealing the reaction reagent required for the reaction process to be controlled in the sealing layer, and melting the sealing material by controlling the temperature change. , releasing the sealed reagent to achieve control of the reaction process.

2. The reaction tube according to claim 1, wherein when the sealing layer is a plurality of layers, the sealing layer is disposed in the order of different reaction processes, and the reaction reagent in the sealing layer can be melted after the sealing layer is melted. Where the reaction process is in contact with the reaction system, the melting temperature of each sealing layer is less than or equal to the reaction temperature of the corresponding reaction progress and higher than the reaction temperature of the previous reaction process and lower than the melting temperature of the sealing layer corresponding to the subsequent reaction course.

The reaction tube according to claim 2, wherein the sealing layer is sequentially disposed in a sequential order of different reaction processes controlled by the sealing layer, and the outermost sealing layer corresponds to the progress of the previous reaction.

4. The reaction tube according to claim 2, wherein the temperature regulation of the reaction tube is adjusted in stages according to a reaction course to be controlled, which is higher than or equal to the reaction temperature of the corresponding reaction process and lower than the sealing layer corresponding to the subsequent reaction process. The melting temperature.

The reaction tube according to claim 4, wherein the temperature of the reaction tube is adjusted to a reaction temperature required for the corresponding reaction process, and the sealing layer corresponding to the corresponding reaction progress is melted because the melting point is less than or equal to the reaction temperature, and will be The release of the reagent enclosed in the sealing layer enables the corresponding reaction process to proceed.

6. A reaction tube according to claim 4 or 5, characterized in that the temperature regulation of the reaction tube is regulated from a low temperature to a high temperature in the order of the progress of the reaction.

A reaction tube according to claim 2, wherein the different sealing layers are made of a material having a different melting point as a sealing material.

The reaction tube according to claim 2, wherein the substances having different melting points are paraffin waxes having different melting points or low melting point polytetrafluoroethylene having different melting points.

9. A reaction tube according to claim 2, characterized in that the number of sealing layers is designed according to the number of reaction stages required; the reaction processes at different temperatures differ in the melting point of their respective sealing layers.

10. A reaction tube according to claim 1, wherein the reactants in the sealing layer are mixed in or separated by a sealing material.