Preparation method of nucleic acid fragment standard substance based on PCR (Polymerase Chain Reaction)
By using multiple constant temperature sinks, stirring mechanisms or water circulation pumps in PCR instruments, the problem of low preparation efficiency of existing PCR instruments is solved, and efficient preparation of nucleic acid fragment standards and temperature control is achieved, which is suitable for large-scale PCR preparation.
Patent Information
- Application Number
- CN202510587705.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-08
- Publication Date
- 2025-08-01
AI Technical Summary
The existing PCR instruments have low efficiency and poor temperature control effects when preparing nucleic acid fragment standards.
Multiple constant temperature water tanks are used for denaturation, annealing and extension treatment, combined with a stirring mechanism or water circulation pump, to ensure that there is no bubble attached to the surface of the reaction vessel, improve heat transfer efficiency, and quickly transfer the reaction vessel through a robot to achieve temperature changes.
It improves the preparation efficiency and yield of nucleic acid fragment standards, improves the efficiency of PCR reaction, and is suitable for large-scale PCR preparation.
Smart Images
Figure CN120400307A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of polymerase chain reaction, and particularly relates to a method for preparing a nucleic acid fragment standard based on PCR. Background Art
[0002] In criminal investigation and judicial trials, DNA analysis technology is often used to obtain evidence, which mainly includes three major steps: DNA extraction, PCR (i.e., polymerase chain reaction) amplification, and product separation and detection.
[0003] The preparation of nucleic acid fragment standards often uses the polymerase chain reaction method. The single reaction volume of existing PCR instruments is generally 20 - 50 μl, and temperature control is achieved by heating or cooling a metal well plate, resulting in low efficiency in preparing standards. Summary of the Invention
[0004] Aiming at the above deficiencies existing in the prior art, the technical problem to be solved by the present invention is to propose a method for preparing a nucleic acid fragment standard based on PCR with high preparation efficiency and good temperature control effect.
[0005] The technical solution adopted by the present invention to solve its technical problem is to propose a method for preparing a nucleic acid fragment standard based on PCR, including:
[0006] S 1. Place the reaction vessel containing the reaction materials in a first constant temperature water bath for denaturation treatment; wherein, the water temperature of the first constant temperature water bath is between 94 degrees and 100 degrees;
[0007] S 2. Place the reaction vessel after the denaturation treatment in step S 1 in a second constant temperature water bath for annealing treatment; wherein, the water temperature of the second constant temperature water bath is between 48 degrees and 68 degrees;
[0008] S 3. Place the reaction vessel after the annealing treatment in step S 2 in a third constant temperature water bath for extension treatment; wherein, the water temperature of the third constant temperature water bath is between 70 and 75 degrees;
[0009] One or more of the first constant temperature water bath, the second constant temperature water bath, and the third constant temperature water bath are provided with a stirring mechanism or a water circulation pump, and the warm water is stirred by the stirring mechanism or the water circulation pump so that air bubbles do not adhere to the surface of the reaction vessel, thereby improving the heat transfer efficiency.
[0010] Further, the stirring mechanism includes a driving motor and a stirring impeller, the stirring impeller is connected to the output rotating shaft of the driving motor, and the driving motor drives the stirring impeller to rotate to stir the warm water in the constant temperature water bath.
[0011] Further, the output rotating shaft of the driving motor is arranged along the height direction of the constant temperature water tank, and a plurality of the stirring impellers are sequentially arranged along the axial direction of the output rotating shaft, and the plurality of the stirring impellers perform layered stirring on the warm water in the constant temperature water tank.
[0012] Further, the rotational speed at which the driving motor drives the impeller to rotate is between 55 and 75 revolutions per minute.
[0013] Further, a heating module and a refrigeration module are provided in the first constant temperature water tank, the second constant temperature water tank and the third constant temperature water tank, and the heating module and the refrigeration module are respectively used to control the rise and fall of the water temperature in the constant temperature water tank.
[0014] Further, in step S1, the reaction vessel is immersed in the first constant temperature water tank and does not contact the tank wall of the first constant temperature water tank, and the warm water in the first constant temperature water tank contacts all the outer walls of the reaction vessel for heat transfer;
[0015] In step S2, the reaction vessel is immersed in the second constant temperature water tank and does not contact the tank wall of the second constant temperature water tank, and the warm water in the second constant temperature water tank contacts all the outer walls of the reaction vessel for heat transfer;
[0016] In step S3, the reaction vessel is immersed in the third constant temperature water tank and does not contact the tank wall of the third constant temperature water tank, and the warm water in the third constant temperature water tank contacts all the outer walls of the reaction vessel for heat transfer.
[0017] Further, a plurality of the reaction vessels each containing reaction materials are arranged in multiple rows and multiple columns, and adjacent two reaction vessels are arranged at intervals, and are together placed into the first constant temperature water tank or the second constant temperature water tank or the third constant temperature water tank.
[0018] Further, the water temperature of the first constant temperature water tank is 95 degrees, the water temperature of the second constant temperature water tank is between 55 degrees and 65 degrees, and the water temperature of the third constant temperature water tank is between 70 degrees and 75 degrees.
[0019] Further, an antifoaming agent is added to the warm water in the first constant temperature water tank, the second constant temperature water tank and the third constant temperature water tank.
[0020] Further, steps S1, S2 and S3 are cycled multiple times. In the first cycle, the water bath time of the reaction vessel in the first constant temperature water tank is 15 seconds to 30 seconds, the water bath time of the reaction vessel in the second constant temperature water tank is 15 seconds to 45 seconds, and the water bath time of the reaction vessel in the third constant temperature water tank is 30 seconds to 2 minutes, and the reaction time can be adjusted according to the volume of the reaction materials contained in the reaction vessel.
[0021] Further, the reaction vessel is in the shape of a flat bag, and the volume of the reaction material contained in each reaction vessel can reach 10 - 50 mL, so as to increase the volume of a single PCR reaction and improve the preparation efficiency of the nucleic acid fragment standard.
[0022] Compared with the prior art, the present invention has at least the following beneficial effects:
[0023] In the present invention, denaturation treatment is carried out in the first constant temperature water bath, annealing treatment is carried out in the second constant temperature water bath, and extension treatment is carried out in the third constant temperature water bath. The water temperatures in the first constant temperature water bath, the second constant temperature water bath, and the third constant temperature water bath are respectively set to the water temperatures required for denaturation treatment, annealing treatment, and extension treatment, ensuring a high temperature change efficiency of the reaction vessel, improving the efficiency of the polymerase chain reaction, and further improving the preparation efficiency and yield of the standard. Moreover, a stirring mechanism or a water circulation pump is provided in the constant temperature water bath to ensure that no bubbles adhere to the outer surface of the reaction vessel, thereby ensuring the heat transfer efficiency of the reaction vessel and improving the reaction efficiency.
[0024] In the present invention, a plurality of stirring impellers are sequentially arranged on the output rotating shaft of the driving motor, and the water in the constant temperature water bath is stirred in layers by the plurality of stirring impellers to improve the efficiency of the bubbles rising upward, thereby ensuring that no bubbles adhere to the outer surface of the reaction vessel and improving the heat transfer efficiency.
[0025] In the present invention, a plurality of reaction vessels are arranged in multiple rows and columns and are spaced apart, and are simultaneously placed in the constant temperature water bath, so that reactions can be carried out simultaneously in the plurality of reaction vessels, improving the preparation efficiency and being applicable to the situation of large-scale PCR preparation of standards. Among them, the reaction vessel is in the shape of a flat bag, and the volume of the reaction material contained in each reaction vessel can reach 10 - 50 mL, which can greatly increase the volume of a single PCR reaction and improve the preparation efficiency of the nucleic acid fragment standard. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 is a schematic diagram of the method for preparing a nucleic acid fragment standard based on PCR according to the present invention;
[0027] Figure 2 is a flow chart for the preparation of the standard. DETAILED DESCRIPTION OF THE INVENTION
[0028] The following are specific embodiments of the present invention in combination with the accompanying drawings to further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.
[0029] It should be noted that all directional indications (such as up, down, left, right, front, back...) in the embodiments of the present invention are only used to explain the relative positional relationship, movement conditions, etc. between components in a specific posture (as shown in the attached drawings). If the specific posture changes, the directional indications will also change accordingly.
[0030] In addition, in the present invention, descriptions such as "first", "second", "one", etc. are only for descriptive purposes and should not be construed as indicating or implying their relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "a plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
[0031] In the present invention, unless otherwise clearly specified and limited, terms such as "connection" and "fixation" should be understood in a broad sense. For example, "fixation" can be a fixed connection, a detachable connection, or integrated; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and can be the communication inside two elements or the interaction relationship between two elements, unless otherwise clearly limited. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.
[0032] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the ability of those of ordinary skill in the art to implement. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope required by the present invention.
[0033] In PCR amplification, it includes designing primers, designing specific primers to amplify a DNA fragment of the required length. It includes PCR amplification, performing a PCR reaction to generate the required DNA fragment. It includes purification. Similarly, the PCR product is purified using gel electrophoresis or a column purification kit.
[0034] As Figure 1 - Figure 2 shown, a method for preparing a nucleic acid fragment standard based on PCR in this embodiment mainly includes the following steps:
[0035] S1. Place the reaction container containing the reaction materials in a first constant temperature water bath for denaturation treatment; wherein, the water temperature of the first constant temperature water bath is between 94 degrees and 100 degrees;
[0036] S2. Place the reaction container after the denaturation treatment in step S1 in a second constant temperature water bath for annealing treatment; wherein, the water temperature of the second constant temperature water bath is between 48 degrees and 68 degrees;
[0037] S3. Place the reaction vessel after the annealing treatment in step S2 in a third constant temperature water bath for extension treatment; wherein, the water temperature of the third constant temperature water bath is between 70 and 75 degrees Celsius.
[0038] One or more of the first constant temperature water bath, the second constant temperature water bath, and the third constant temperature water bath are provided with a stirring mechanism or a water circulation pump. The warm water is stirred by the stirring mechanism or the water circulation pump so that air bubbles do not adhere to the surface of the reaction vessel, thereby improving the heat transfer efficiency. The temperature changes quickly, which can effectively improve the reaction efficiency, and then improve the preparation efficiency of the standard product and increase the production capacity. It is applicable to large-scale PCR instruments.
[0039] During actual use, in this embodiment, denaturation treatment is carried out in the first constant temperature water bath, annealing treatment is carried out in the second constant temperature water bath, and extension treatment is carried out in the third constant temperature water bath. The water temperatures in the first constant temperature water bath, the second constant temperature water bath, and the third constant temperature water bath are respectively set to the water temperatures required for denaturation treatment, annealing treatment, and extension treatment, ensuring a high temperature change efficiency of the reaction vessel, improving the efficiency of the polymerase chain reaction, and then improving the preparation efficiency and yield of the standard product. Moreover, a stirring mechanism or a water circulation pump is provided in the constant temperature water bath to ensure that air bubbles do not adhere to the outer surface of the reaction vessel, thereby ensuring the heat transfer efficiency of the reaction vessel and improving the reaction efficiency. In the prior art, semiconductor heating / cooling technology (such as Peltier effect elements) or metal heating blocks are usually used to achieve heating and cooling.
[0040] Preferably, as a preferred implementation manner of this embodiment, the water temperature of the first constant temperature water bath is 95 degrees Celsius. At this temperature, double-stranded DNA can be unwound into single-stranded DNA. The water temperature of the second constant temperature water bath is between 55 and 65 degrees Celsius. By directly transferring the reaction vessel from the first constant temperature water bath to the second constant temperature water bath, it is not necessary to reduce the water temperature of the first constant temperature water bath from 95 degrees Celsius to between 55 and 65 degrees Celsius, ensuring a faster temperature change of the reaction vessel, effectively improving the reaction efficiency, and then improving the preparation efficiency of the standard product. Among them, the transfer of the reaction vessel from the first constant temperature water bath to the second constant temperature water bath can be carried out by grasping with a manipulator. Controlling the water temperature of the second constant temperature water bath within this temperature range, the specific value depends on the Tm value of the primer, and the purpose is to enable the specific primer to bind to the target DNA sequence. The water temperature of the third constant temperature water bath is 70 degrees Celsius. Similarly, by transferring the reaction vessel from the second constant temperature water bath to the third constant temperature water bath, it is not necessary to heat the water temperature of the second constant temperature water bath to 70 degrees Celsius, and the reaction efficiency can also be effectively improved, and then the preparation efficiency of the standard product can be improved. The transfer of the reaction vessel from the second constant temperature water bath to the third constant temperature water bath can also be carried out by grasping with a manipulator. During the extension treatment, Taq DNA polymerase synthesizes a new DNA strand using dNTPs as raw materials.
[0041] The temperatures of multiple constant-temperature water baths are the temperatures required for each stage of the polymerase chain reaction. According to the progress of each stage of the reaction, the reaction vessel is placed in the corresponding constant-temperature water bath, which is simple to operate and has a fast heating and cooling speed.
[0042] Among them, the first constant-temperature water bath, the second constant-temperature water bath, and the third constant-temperature water bath can be arranged in sequence on the workbench surface, because according to the preparation process of this embodiment, the switching order of the reaction vessel among the first constant-temperature water bath, the second constant-temperature water bath, and the third constant-temperature water bath is like this, ensuring that the time for the manipulator to grasp and move the reaction vessel is short, saving time and improving efficiency.
[0043] In the actual preparation process, steps S1, S2, and S3 are cycled multiple times to achieve exponential amplification of the target DNA. By repeating the above steps, usually 25 to 35 cycles, the target DNA fragment grows exponentially. Theoretically, the amplification quantity doubles in each round. Among them, in the first cycle, the water bath time of the reaction vessel in the first constant-temperature water bath is 15 seconds to 30 seconds, the water bath time of the reaction vessel in the second constant-temperature water bath is 15 seconds to 45 seconds, and the water bath time of the reaction vessel in the third constant-temperature water bath is 30 seconds to 2 minutes.
[0044] Specifically, in this embodiment, a stirring mechanism or a water circulation pump is provided in each of the first constant-temperature water bath, the second constant-temperature water bath, and the third constant-temperature water bath. The warm water in the constant-temperature water bath is stirred by the stirring mechanism or the water circulation pump, so that air bubbles will not adhere to the surface of the reaction vessel, thereby improving the heat transfer efficiency and ensuring that the reaction vessel can be heated or cooled rapidly.
[0045] When a water circulation pump is provided in each of the first constant-temperature water bath, the second constant-temperature water bath, and the third constant-temperature water bath, water is pumped by the water circulation pump to realize the internal circulation of the warm water in each constant-temperature water bath. The warm water in the first constant-temperature water bath, the second constant-temperature water bath, and the third constant-temperature water bath is circulated, so that the heat transfer efficiency of the reaction vessel placed in the corresponding constant-temperature water bath is higher.
[0046] When a stirring mechanism is provided in each of the first constant-temperature water bath, the second constant-temperature water bath, and the third constant-temperature water bath, the stirring mechanism includes a driving motor and a stirring impeller. The stirring impeller is connected to the output rotating shaft of the driving motor. The driving motor drives the stirring impeller to rotate, stirring the warm water in the constant-temperature water bath, so that the air bubbles in the warm water in each of the first constant-temperature water bath, the second constant-temperature water bath, and the third constant-temperature water bath escape and will not adhere to the outer surface of the reaction vessel, so as not to affect the heat transfer efficiency between the warm water and the reaction vessel.
[0047] Further, the output rotating shaft of the driving motor is arranged along the height direction of the constant temperature water tank, and a plurality of the stirring impellers are sequentially arranged along the axial direction of the output rotating shaft. The plurality of stirring impellers perform stratified stirring on the warm water in the constant temperature water tank. Compared with the single-layer stirring method, multi-layer stirring can make the bubbles in the warm water escape from different dimensions.
[0048] Among them, the rotational speed at which the driving motor drives the impeller to rotate is between 55 and 75 revolutions per minute, that is, the stirring mechanism in this embodiment belongs to the category of low-speed stirring. Low-speed stirring can avoid the splashing of warm water and the mixing of air. High-speed stirring is likely to introduce more bubbles because the high-speed rotation of the stirring impeller will break the liquid surface, destroy the liquid surface tension, and entrain air, resulting in the generation of bubbles. The stirring duration can last for three or four minutes, and long-term stirring should be avoided to prevent the re-mixing of bubbles.
[0049] During actual use, a plurality of stirring impellers are sequentially arranged on the output rotating shaft of the driving motor, and the water in the constant temperature water tank is subjected to stratified stirring by the plurality of stirring impellers, so as to improve the efficiency of the upward escape of bubbles, and further ensure that no bubbles adhere to the outer surface of the reaction vessel, thereby improving the heat transfer efficiency. That is, by arranging the stirring impellers in layers, the bubbles at different depths can be processed directionally, accelerating the upward escape and rupture of bubbles, and improving the bubble elimination efficiency.
[0050] Furthermore, in order to reduce or even eliminate the bubbles in the warm water in the constant temperature water tank, an antifoaming agent can also be added to the warm water in the first constant temperature water tank, the second constant temperature water tank, and the third constant temperature water tank.
[0051] In this embodiment, in order to realize the temperature control of the first constant temperature water tank, the second constant temperature water tank, and the third constant temperature water tank, a heating module and a refrigeration module are provided in the first constant temperature water tank, the second constant temperature water tank, and the third constant temperature water tank. The heating module and the refrigeration module are respectively used to control the rise and fall of the water temperature in the constant temperature water tank. Moreover, the temperature control accuracy of each constant temperature water tank can reach ±0.1°C to ±1.0°C. By accurately controlling the water temperature of the warm water in the constant temperature water tank, the reaction efficiency is ensured.
[0052] Among them, in step S1, the reaction vessel is immersed in the first constant temperature water tank and does not contact the tank wall of the first constant temperature water tank. The warm water in the first constant temperature water tank contacts all the outer walls of the reaction vessel for heat transfer.
[0053] In step S2, the reaction vessel is immersed in the second constant temperature water tank and does not contact the tank wall of the second constant temperature water tank. The warm water in the second constant temperature water tank contacts all the outer walls of the reaction vessel for heat transfer.
[0054] In step S3, the reaction vessel is immersed in the third constant temperature water bath without contacting the wall of the third constant temperature water bath, and the warm water in the third constant temperature water bath contacts all the outer walls of the reaction vessel for heat transfer.
[0055] When the reaction vessel is placed in the corresponding constant temperature water bath, ensure that the reaction vessel can be in full contact with the warm water to ensure the efficiency of heat transfer. For example, the reaction vessel can be supported or suspended by a support wire to achieve full contact between the reaction vessel and the hot water. Increase the heat conduction contact area, improve the heat conduction efficiency, and further improve the production efficiency of the standard product.
[0056] Furthermore, multiple reaction vessels filled with reaction materials can be arranged in multiple rows and columns, and adjacent two reaction vessels are arranged at intervals and placed together in the first constant temperature water bath or the second constant temperature water bath or the third constant temperature water bath. The temperature range of the constant temperature water bath in this embodiment can reach -5 to 100 degrees Celsius, or even a larger range, the internal circulation flow rate can reach 5 to 10 L / min, and the production efficiency can reach 400 mL / round or more.
[0057] In the actual operation process, multiple reaction vessels are arranged in multiple rows and columns, and are arranged at intervals, and are placed in the constant temperature water bath at the same time. Reactions can be carried out simultaneously in multiple reaction vessels to improve the preparation efficiency, which is applicable to the case of preparing standard products for large-scale PCR. The reaction vessel is in the shape of a flat bag, and the volume of the reaction material contained in each reaction vessel can reach 10 - 50 mL, which can greatly increase the volume of a single PCR reaction and improve the preparation efficiency of nucleic acid fragment standard products.
[0058] In this solution, the efficiency of preparing nucleic acid fragment standard products by the preparation method of this embodiment is high, and the temperature control effect is good.
Claims
1. A method for preparing a nucleic acid fragment standard based on PCR, characterized in that, Including: S1. Place the reaction vessel containing the reaction materials in the first constant temperature water bath for denaturation treatment; wherein, the water temperature of the first constant temperature water bath is between 94 degrees and 100 degrees; S2. Place the reaction vessel after the denaturation treatment in step S1 in the second constant temperature water bath for annealing treatment; wherein, the water temperature of the second constant temperature water bath is between 48 degrees and 68 degrees; S3. Place the reaction vessel after the annealing treatment in step S2 in the third constant temperature water bath for extension treatment; wherein, the water temperature of the third constant temperature water bath is between 70 and 75 degrees; One or more of the first constant temperature water bath, the second constant temperature water bath, and the third constant temperature water bath are provided with a stirring mechanism or a water circulation pump, and the warm water is stirred by the stirring mechanism or the water circulation pump so that air bubbles do not adhere to the surface of the reaction vessel, thereby improving the heat transfer efficiency.
2. The method for preparing a nucleic acid fragment standard based on PCR according to claim 1, wherein The stirring mechanism includes a driving motor and a stirring impeller. The stirring impeller is connected to the output rotating shaft of the driving motor, and the driving motor drives the stirring impeller to rotate to stir the warm water in the constant temperature water bath.
3. The method for preparing a nucleic acid fragment standard based on PCR according to claim 2, wherein The output rotating shaft of the driving motor is arranged along the height direction of the constant temperature water bath, and a plurality of the stirring impellers are sequentially arranged along the axial direction of the output rotating shaft, and the plurality of stirring impellers perform layered stirring on the warm water in the constant temperature water bath.
4. The method for preparing a nucleic acid fragment standard product based on PCR according to claim 2, wherein The rotational speed at which the driving motor drives the impeller to rotate is between 55 and 75 revolutions per minute.
5. The method for preparing a nucleic acid fragment standard based on PCR according to claim 1, wherein The first constant temperature water bath, the second constant temperature water bath, and the third constant temperature water bath are provided with a heating module and a refrigeration module, and the heating module and the refrigeration module are respectively used to control the rise and fall of the water temperature in the constant temperature water bath.
6. The method for preparing a nucleic acid fragment standard based on PCR according to claim 1, wherein In step S1, the reaction vessel is immersed in the first constant temperature water bath and does not contact the wall of the first constant temperature water bath, and the warm water in the first constant temperature water bath contacts all the outer walls of the reaction vessel for heat transfer; In step S2, the reaction vessel is immersed in the second constant temperature water bath and does not contact the wall of the second constant temperature water bath, and the warm water in the second constant temperature water bath contacts all the outer walls of the reaction vessel for heat transfer; In step S3, the reaction vessel is immersed in the third constant temperature water bath and does not contact the wall of the third constant temperature water bath, and the warm water in the third constant temperature water bath contacts all the outer walls of the reaction vessel for heat transfer.
7. The method for preparing a nucleic acid fragment standard product based on PCR according to claim 1, wherein Arrange a plurality of the reaction vessels all containing reaction materials in multiple rows and multiple columns, and adjacent two reaction vessels are arranged at intervals, and place them together in the first constant temperature water bath or the second constant temperature water bath or the third constant temperature water bath.
8. The method for preparing a nucleic acid fragment standard product based on PCR according to claim 1, wherein The water temperature of the first constant temperature water bath is 95 degrees, the water temperature of the second constant temperature water bath is between 55 degrees and 65 degrees, and the water temperature of the third constant temperature water bath is between 70 degrees and 75 degrees.
9. The method for preparing a nucleic acid fragment standard based on PCR according to claim 1, wherein An antifoaming agent is added to the warm water in one or more of the first constant temperature water bath, the second constant temperature water bath, and the third constant temperature water bath.
10. The method for preparing a nucleic acid fragment standard based on PCR according to claim 1, wherein Steps S1, S2, and S3 are cycled multiple times. In the first cycle, the water bath time of the reaction vessel in the first constant temperature water bath is 15 seconds to 30 seconds, the water bath time of the reaction vessel in the second constant temperature water bath is 15 seconds to 45 seconds, the water bath time of the reaction vessel in the third constant temperature water bath is 30 seconds to 2 minutes, and the reaction time can be adjusted according to the volume of the reaction materials contained in the reaction vessel.