RNA reaction bag and IVT reaction monitoring method

Abstract

The invention belongs to the technical field of mRNA production and manufacturing, and discloses an RNA reaction bag and an IVT reaction monitoring method. The RNA reaction bag provided by the invention comprises a bag body and a detection assembly, the bag body comprises a flexible part and a transparent hard part, the transparent hard part is structured to form a detection vessel, and in the IVT reaction process, the detection vessel is used as a cuvette to receive an IVT reaction liquid, and the detection assembly is used for detecting the IVT reaction liquid. Meanwhile, optical detectors arranged on the two opposite sides of the detection vessel are used for detection, so that detection of the IVT reaction liquid in the detection vessel can be achieved, real-time relevant information of reaction raw materials and / or products in the IVT reaction liquid is obtained, and pH value information and temperature information detected by a pH detector and a temperature detector are matched; the real-time monitoring of the IVT reaction process in the RNA reaction bag is realized, the reaction condition can be adjusted in time, and the raw materials can be supplemented in time to maintain an ideal reaction state, so that the amplification of the IVT reaction is realized, the large-scale production of RNA is realized, and the method has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of mRNA production and manufacturing, and in particular relates to an RNA reaction bag and an IVT reaction monitoring method. Background Art

[0002] Messenger RNA (mRNA) refers to a single-stranded RNA molecule transcribed from DNA, which can effectively translate the genetic information it carries into corresponding proteins on the ribosomes in cells. mRNA vaccines / drugs use a specific delivery system to carry RNA molecules encoding specific antigen targets, express corresponding proteins in the body or directly change the expression of proteins in cells, thereby stimulating the body to produce specific immune responses or change the body's metabolism, thereby achieving the effect of disease prevention and / or treatment. mRNA vaccines / drugs respond quickly to changes in disease conditions and pathogens, have a simple production process, and are easy to achieve large-scale production, and have broad application prospects.

[0003] At present, the production of mRNA mainly includes the design and production of linear plasmids, in vitro transcription (IVT) of mRNA, purification and packaging of mRNA. Among them, IVT is a key step in mRNA production, which involves transcribing and synthesizing mRNA using linear plasmids as templates in a cell-free system. Parameters such as pH value, enzyme activity, substrate concentration, product concentration and temperature in the in vitro transcription process have a significant impact on the synthesis efficiency and quality of mRNA. Therefore, during the in vitro transcription reaction, the reaction must be strictly detected and monitored to ensure that raw materials can be replenished or reaction conditions such as pH value and temperature can be adjusted in time. However, there is a gap in the prior art in real-time monitoring of IVT reaction progress without sampling. Therefore, it is of great significance to obtain a method and device that can realize real-time monitoring of IVT reaction progress without sampling. Summary of the invention

[0004] The inventor of the present invention has creatively designed the structure of the RNA reaction bag after extensive and in-depth research to address the gaps in the prior art in real-time monitoring of the progress of the IVT reaction without sampling. A transparent hard portion is introduced into the structure of the RNA reaction bag, and it is structured to form a detection dish. The detection dish is used as an optical detection container (colorimetric dish) to receive the IVT reaction liquid, and the IVT reaction liquid in the detection dish is updated in real time during the IVT reaction. At this time, the optical detectors arranged on both sides of the detection dish are used to detect the IVT reaction liquid in real time, so that the relevant information of the reaction raw materials and / or products in the IVT reaction liquid can be obtained without sampling. At the same time, the pH value information and temperature information obtained by the pH detector and temperature detector in the bag body can be used to more accurately judge the progress of the IVT reaction, which has the advantages of accuracy, convenience and high efficiency. Based on this, the technical solution of the present invention is obtained.

[0005] The first purpose of the present invention is to provide an RNA reaction bag. The RNA reaction bag is configured with a specific structure so that a detection component including multiple detectors such as an optical detector, a pH detector and a temperature detector is integrated thereon, and the detection component is used to monitor the changes of factors such as pH value, temperature, dNTP concentration, mRNA concentration, phosphate concentration, etc. during the IVT reaction in real time, so as to facilitate timely adjustment of reaction conditions and addition of raw materials to maintain a relatively ideal reaction state, which is conducive to realizing the amplification of the IVT reaction, thereby realizing large-scale production of RNA.

[0006] A second object of the present invention is to provide a method for monitoring an IVT reaction.

[0007] Specifically, the RNA reaction bag provided by the present invention includes: a bag body, the bag body includes a flexible part and a transparent hard part, the transparent hard part is structured to form a detection dish, the detection dish is connected to the inside of the bag body to receive the IVT reaction liquid; a detection component, the detection component includes an optical detector, a pH detector and a temperature detector; the pH detector and the temperature detector are arranged in the bag body; the optical detector includes a transmitter and a receiver, the transmitter and the receiver are relatively arranged on two sides of the detection dish, and the detection light emitted by the transmitter is received by the receiver through the detection dish and the IVT reaction liquid.

[0008] Furthermore, the transparent hard portion is structured to form a detection dish protruding from the bag body, a connecting piece is provided on the outer side of the detection dish, and the optical detection assembly is detachably connected to the bag body via the connecting piece.

[0009] Furthermore, the transparent hard portion is structured to form M detection dishes protruding from the bag body, where M is an integer not less than 2; the detection dishes are arranged at intervals, and a first receiving groove is formed between two adjacent detection dishes, the connecting piece is arranged in the first receiving groove, and the transmitter and / or receiver is detachably arranged in the first receiving groove.

[0010] Furthermore, the transparent hard portion is structured to form N second receiving grooves recessed in the bag body, where N is an integer not less than 2; the second receiving grooves are arranged at intervals, and a detection dish is formed between two adjacent second receiving grooves, and the transmitter and / or receiver is detachably arranged in the second receiving groove.

[0011] Furthermore, a flow guide is provided around the opening of the detection dish.

[0012] Furthermore, the material of the transparent hard part is selected from one or more of quartz, optical glass and hard optical plastic.

[0013] Furthermore, the optical detector is selected from one or more of a Fourier transform infrared spectrometer, an in-situ Raman spectrometer and a particle image analyzer.

[0014] Furthermore, the RNA reaction bag comprises an automatic sample injector, and the automatic sample injector is connected to the bag body.

[0015] In the monitoring method of the IVT reaction provided by the present invention, the IVT reaction is carried out in the above-mentioned RNA reaction bag, and the monitoring method includes: taking the detection component to detect the IVT reaction liquid in the bag body, obtaining IVT reaction parameters, and adjusting the IVT reaction conditions based on the IVT reaction parameters; wherein the IVT reaction parameters include one or more of raw material concentration, solution aging state, phosphate concentration, mRNA generation amount, pH value and temperature.

[0016] Furthermore, the method for adjusting the IVT reaction conditions based on the IVT reaction parameters includes: regulating the pH value of the IVT reaction system to be maintained at 7 to 8, regulating the deviation value of the real-time temperature in the IVT reaction from the preset reaction temperature to be 0 to 0.5°C; when the dNTP concentration in the IVT reaction system is reduced to 10 to 30% of the initial concentration, adding dNTP and Mg 2+ , cap analogs, and inorganic pyrophosphatase to 75% to 100% of the initial concentration; when precipitation occurs in the IVT reaction system, adjust the Mg 2+ When the mRNA production amount reaches 10-20 mg / mL, the IVT reaction is stopped.

[0017] Beneficial effects:

[0018] The RNA reaction bag provided by the present invention comprises a bag body and a detection component, wherein the bag body comprises a flexible part and a transparent hard part, and the detection dish is formed by structuring the transparent hard part. During the IVT reaction process, the detection dish is used as a colorimetric dish to receive the IVT reaction liquid, and the IVT reaction liquid in the detection dish can be detected by using optical detectors arranged on opposite sides of the detection dish to obtain real-time relevant information of reaction raw materials and / or products in the IVT reaction liquid, and the pH value information and temperature information detected by the pH detector and the temperature detector are used to realize real-time monitoring of the IVT reaction process in the RNA reaction bag, and then the reaction conditions can be adjusted and raw materials can be added in time to maintain a more ideal reaction state, which is conducive to realizing the amplification of the IVT reaction, thereby realizing large-scale production of RNA, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the structure of the RNA reaction bag provided in the first embodiment of the present invention;

[0020] Figure 2 for Figure 1 Sectional view of AA in the middle;

[0021] Figure 3 A schematic cross-sectional structure diagram of an RNA reaction bag provided in the second embodiment of the present invention;

[0022] Figure 4 A schematic cross-sectional structure diagram of an RNA reaction bag provided in the third embodiment of the present invention;

[0023] Figure 5 This is a schematic cross-sectional structure diagram of an RNA reaction bag provided in the fourth embodiment of the present invention.

[0024] Figure numerals: 1. bag body; 101. flexible part; 102. transparent hard part; 2. detection component; 201. optical detector; 202. pH detector; 203. temperature detector; 3. detection dish; 4. transmitter; 5. receiver; 6. flow guide part; 7. automatic sample injector. DETAILED DESCRIPTION

[0025] Although the present invention can be easily embodied in different forms of embodiments, only some of the specific embodiments are shown in the drawings and will be described in detail in this specification. It should be understood that this description should be regarded as an exemplary illustration of the principles of the invention and is not intended to limit the invention to that described herein.

[0026] Thus, a feature indicated in this specification will be used to illustrate one of the features of an embodiment of the present invention, rather than implying that each embodiment of the present invention must have the described feature. In addition, it should be noted that this specification describes many features. Although some features can be combined together to illustrate possible system designs, these features can also be used in other combinations that are not explicitly described. Thus, unless otherwise stated, the described combinations are not intended to be limiting.

[0027] In the embodiments shown in the drawings, the indications of directions (such as up, down, left, right, front and back) used to explain the structure and movement of various elements of the present invention are not absolute but relative. These descriptions are appropriate when these elements are in the positions shown in the drawings. If the description of the positions of these elements changes, the indications of these directions also change accordingly.

[0028] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0029] See also Figure 1 to Figure 2 , Figure 1 Schematic diagram of the structure of the RNA reaction bag according to the first embodiment of the present invention. Figure 2 yes Figure 1 The RNA reaction bag provided by the present invention comprises a bag body 1 and a detection component 2.

[0030] The bag body 1 can be divided into a flexible part 101 and a transparent hard part 102 according to hardness. The transparent hard part 102 is structured to form a detection dish 3 protruding from the bag body, and the opening of the detection dish 3 faces the inside of the bag body 1 to achieve communication with the inside of the bag body 1. During the IVT reaction process, the IVT reaction liquid in the bag body 1 can enter the detection dish 3, and with the shaking of the bag body 1 and / or the stirring of the IVT reaction liquid in the bag body 1, the IVT reaction liquid in the detection dish 3 can be updated in real time, so that the IVT reaction liquid in the detection dish 3 maintains a high consistency with the overall IVT reaction liquid in the bag body 1.

[0031] The material of the flexible portion 101 is a material commonly used in the prior art for preparing bioreactor bags. Those skilled in the art may make an adaptive choice according to the actual needs of use, and the present invention does not impose any particular limitation thereon. In some specific embodiments, specific examples of the material of the flexible portion 101 include, but are not limited to, one or more of polyethylene terephthalate, low-density polyethylene, polyethylene, polypropylene, polyvinyl alcohol, and polyvinyl chloride.

[0032] The material of the transparent hard part 102 is a material conventionally used in the prior art for preparing a cuvette. A person skilled in the art may make an adaptive choice according to the actual needs of use, and the present invention does not impose any particular limitation thereto. In some specific embodiments, the material of the transparent hard part 102 is preferably selected from one or more of quartz, optical glass, and hard optical plastic. Specific examples of the hard optical plastic include, but are not limited to, one or more of polymethyl methacrylate, polystyrene, polycarbonate, styrene acrylonitrile, and transparent polyamide.

[0033] The detection assembly 2 includes an optical detector 201, a pH detector 202 and a temperature detector 203. The pH detector 202 and the temperature detector 203 are detachably mounted in the bag body 1; the optical detector 201 includes a transmitter 4 and a receiver 5, and a protruding snap-in block is structured on the transparent hard part 102 on opposite sides of the detection dish 3, and the transmitter 4 and the receiver 5 are respectively provided with snap-in grooves, and the transmitter 4 and the receiver 5 are detachably mounted on opposite sides of the detection dish 3 through the snap-in grooves and the snap-in blocks.

[0034] During the IVT reaction process, the transmitter 4 in the optical detection 201 emits detection light that passes through the side wall of the detection dish 4 and the IVT reaction liquid therein and is received by the receiver 5. Analysis and calculation are performed based on the optical signal received by the receiver 5 to obtain relevant information about the raw materials and / or products in the IVT reaction liquid in a sampling-free and contactless manner. At the same time, the pH detector 202 and the temperature detector 203 in the bag body 1 that are in direct contact with the IVT reaction liquid are used to measure the pH value and temperature of the IVT reaction liquid in real time. The relevant information about the raw materials and / or products in the IVT reaction liquid and the physical and chemical information such as the pH and temperature of the IVT reaction liquid are combined to achieve real-time monitoring of the IVT reaction process in the RNA reaction bag, and based on the real-time monitoring results, the reaction conditions are adjusted in time and raw materials are added to maintain a more ideal reaction state, which is conducive to the amplification of the IVT reaction and thus the large-scale production of RNA, and has good application prospects.

[0035] The optical detector 201 is a type of instrument commonly used in the prior art that uses the absorption and / or reflection of specific light by a substance to determine the chemical composition, concentration, purity and other information of a solution. In the RNA reaction bag, based on the inventor's deep understanding of the relationship between the substances present in the IVT reaction system and their changes and the IVT reaction process, one or more instruments selected from Fourier transform infrared spectrometer, in-situ Raman spectrometer and particle image analyzer are preferably used as optical detectors to more accurately obtain relevant information about the raw materials and / or products in the IVT reaction solution.

[0036] The pH detector 202 is a type of instrument commonly used in the prior art for measuring the pH value of a solution. Those skilled in the art may make an adaptive choice according to actual needs, and the present invention does not impose any particular limitation thereto.

[0037] The temperature detector 203 is a type of instrument commonly used in the prior art for measuring the temperature value of a solution. Those skilled in the art may make an adaptive choice according to actual needs, and the present invention does not impose any particular limitation thereto.

[0038] It should be noted that Figure 1 and Figure 2 The sizes of the detection dish 3 and the bag body 1 shown in are only for the convenience of display and do not represent the actual size relationship between the two. In some actual products, the volume of the detection dish 3 is preferably set to be much smaller than the volume of the bag body 1. More specifically, the volume of the detection dish 3 is preferably set to 0.7-7mL, such as 0.7mL, 0.8mL, 1mL, 1.5mL, 2mL, 3mL, 5mL, 7mL or any value therebetween. The optical path of the detection dish 3 is preferably set to 0.5-2mm, such as 0.5mm, 0.8mm, 1mm, 1.2mm, 1.5mm, 2mm or any value therebetween. In the first embodiment, the volume of the detection dish 3 is 0.7mL and the optical path length is 2mm.

[0039] During the IVT reaction process, compared with directly optically detecting the bag body 1, detecting the IVT reaction liquid in the detection dish 3 has the characteristics of small detection solution volume and short detection light path, so that the detection is less disturbed by the shaking and flow of the solution. Therefore, the optical signal received by the receiver 5 can better reflect the relevant information of the reaction raw materials and / or products in the IVT reaction liquid, and the detection result has higher accuracy and reliability.

[0040] In the first embodiment, a guide portion 6 is preferably provided on the opening circumference of the detection dish 3 to guide the IVT reaction liquid in the bag 1 to flow toward the detection dish 3, which is conducive to the real-time update of the IVT reaction liquid in the detection dish 3 during the IVT reaction.

[0041] In the first embodiment, the RNA reaction bag preferably also includes an automatic sampler 7, which is connected to the bag body 1 through a pipeline. During the IVT reaction process, the automatic sampler 7 can realize the accurate addition of the reaction raw materials, and can also add the corresponding reaction raw materials according to the progress of the IVT reaction measured by the detection component 2, which is conducive to the realization of RNA automated large-scale production.

[0042] Among them, the automatic sampler 7 is a type of instrument commonly used in the prior art for quantitatively delivering raw materials into a reaction container. Those skilled in the art can make an adaptive choice according to actual needs, and the present invention does not impose any special limitation on it.

[0043] The pipeline on the bag body 1 communicating with the automatic sample injector 7 is preferably arranged at a position of the bag body 1 away from the transparent hard portion 102 , so as to reduce the interference of raw material sampling on the optical detection result of the IVT reaction solution in the detection dish 3 .

[0044] It should be noted that the test dish 3 formed by the structured transparent hard part 102 provided in the above embodiment is only one example of the present invention. The transparent hard part 102 described in the present invention can form M test dishes 3 protruding from the bag body 1 in the same structural manner, where M is an integer not less than 2. That is, the technical solution of forming one or more test dishes 3 protruding from the bag body 1 by structuring the transparent hard part of the bag body 1 itself on the RNA reaction bag is included in the protection scope of the claims of the present invention.

[0045] See also Figure 3 . Figure 3 It is a schematic diagram of the cross-sectional structure of the RNA reaction bag of the second embodiment of the present invention. Compared with the first embodiment, in this embodiment, the transparent hard part 102 is structured to form two detection dishes 3 protruding from the bag body 1, and the two detection dishes 3 are arranged at intervals to form a first accommodating groove. The side wall of the first accommodating groove is structured to form a protruding snap-in block, and a protruding snap-in block is also structured to form a protruding snap-in block on the transparent hard part 102 of the two detection dishes 3 that are separated from each other; the transmitter 4 is correspondingly provided with a snap-in groove, and the transmitter 4 is detachably installed in the first accommodating groove through the snap-in groove and the snap-in block; the number of receivers 5 is set to two, and the two receivers 5 are correspondingly provided with a snap-in groove, and the two receivers 5 are detachably installed on the side of the two detection dishes 3 that are separated from each other through the snap-in groove and the snap-in block.

[0046] Among them, the transmitter 4 preferably has a plurality of windows capable of emitting detection light, so as to emit detection light to a plurality of detection dishes 3 at the same time. In some actual products, the transmitter 4 preferably emits the same detection light to different detection dishes 3 at the same time, so as to realize parallel multiple repeated tests on the IVT reaction liquid, reduce the interference of errors on the detection results, and more accurately obtain the relevant information of the raw materials and / or products in the IVT reaction liquid. In other actual products, the transmitter 4 preferably emits different detection lights to different detection dishes 3, and the receiver 5 is configured as an instrument capable of receiving corresponding detection light, so as to realize the detection of multiple detection targets, so that the relevant information of the raw materials and / or products in the IVT reaction liquid obtained is more comprehensive, which is conducive to realizing more accurate monitoring of the IVT reaction process.

[0047] It should be noted that the test dish 3 formed by the structured transparent hard part 102 provided in the above embodiment is only one example of the present invention, and the transparent hard part 102 described in the present invention can form a test dish 3 having different structural relationships with the bag body 1 in different structural ways. That is, the technical solution of forming a structure having the function of a cuvette by structuring the transparent hard part of the bag body 1 itself on the RNA reaction bag is included in the protection scope of the claims of the present invention.

[0048] See also Figure 4 . Figure 4 : is a schematic diagram of the cross-sectional structure of the RNA reaction bag of the third embodiment of the present invention. Compared with the first embodiment, in this embodiment, the transparent hard part 102 near the edge of the bag body 1 is structured to form a second receiving groove recessed in the bag body 1, and the second receiving groove and the bag body 1 are enclosed to form the above-mentioned detection dish 3. That is, the detection dish 3 in this embodiment is indirectly formed by the spatial structural relationship between the second receiving groove formed by the structured transparent hard part 102 and the bag body 1.

[0049] The side wall of the second accommodating groove is structured to form a protruding snap-in block, and the outer surface of the detection dish 3 on the side away from the second accommodating groove is structured to form a protruding snap-in block; the transmitter 4 and the receiver 5 are respectively provided with snap-in grooves, and the transmitter 4 can be removably installed in the second accommodating groove through the snap-in groove and the snap-in block, and the receiver 5 can be removably installed on the outer surface of the detection dish 3 through the snap-in groove and the snap-in block.

[0050] See also Figure 5 . Figure 5 : is a schematic diagram of the cross-sectional structure of the RNA reaction bag of the fourth embodiment of the present invention. Compared with the first embodiment, in this embodiment, the transparent hard part 10 is structured to form two second receiving grooves recessed in the bag body 1, and the two second receiving grooves are arranged at intervals to enclose the above-mentioned detection dish 3. That is, the detection dish 3 in this embodiment is indirectly formed by the spatial structural relationship between the multiple second receiving grooves formed by the structured transparent hard part 102.

[0051] The side wall of the second receiving groove is structured to form a protruding threaded column, and the transmitter 4 and the receiver 5 are respectively provided with threaded holes. The transmitter 4 and the receiver 5 are respectively detachably installed in the two second receiving grooves through the screw connection of the threaded column and the threaded hole.

[0052] In this embodiment, the transmitter 4 and the receiver 5 in the optical detector 201 have a closer and more stable connection with the bag body 1, which can effectively reduce the position deviation between the optical detector 201 and the detection dish 3 caused by mechanical forces such as shaking during the detection process, and further improve the accuracy and reliability of the detection results.

[0053] It should be noted that the “first” and “second” in the “first containing groove” and the “second containing groove” described in the above embodiments only serve to distinguish and facilitate description, and do not limit the protection scope of the present invention.

[0054] In the RNA reaction bags provided in the first to fourth embodiments above, the optical detector is set as a Fourier transform infrared spectrometer, and the same volume of IVT reaction solution is contained in the RNA reaction bags respectively, and placed on a shaker at 100 rpm for IVT reaction, and the Fourier transform infrared spectrometer is used to monitor the absorbance changes corresponding to the phosphate and mRNA in the IVT reaction solution in real time, and the absorbance trend is converted into the absolute concentration curve of phosphate and mRNA. At the same time, during the IVT reaction process, three samples are taken at different time points, and the following tests are performed:

[0055] (1) Detection of phosphate concentration: The phosphate concentration of the sample was detected using a phosphate fluorescence detection kit (Biyuntian, catalog number S0192S) and referring to the instructions.

[0056] (2) Detection of mRNA concentration: Take 2 μL of IVT reaction solution and dilute it 20 times, 50 times and 100 times with DEPC water, then take 4 μL of each dilution and analyze it by electrophoresis with 1% non-denaturing agarose gel; use RiboRuler high range RNA molecular weight standard (Thermo Fisher Scientific, SM1821) as Yangshen, 1×TAE as electrophoresis buffer, and perform electrophoresis at 150V for 30mi. Use the Yangshen band of RNA molecular weight similar to the mRNA band to be tested as a reference to convert the gray value and concentration of the mRNA product to be tested. The test results are shown in Table 1.

[0057] Table 1.

[0058]

[0059]

[0060] From the test results shown in Table 1, it can be seen that by using the test dish 3 in the RNA reaction bag provided in the first to fourth embodiments of the present invention as the test container, the measured phosphate and mRNA concentrations are highly consistent with the results obtained by the traditional method, and the test results are highly reliable.

[0061] The embodiment of the present invention also provides a method for monitoring an IVT reaction. In this embodiment, the IVT reaction is carried out in the RNA reaction bag provided in the above embodiment, and the detection method uses the detection component 2 in the RNA reaction bag to detect the IVT reaction solution, specifically comprising the following steps:

[0062] The detection component is used to detect the IVT reaction liquid in the bag to obtain one or more IVT reaction parameters including raw material concentration, solution aging state, phosphate concentration, mRNA production, pH value and temperature; and the reaction conditions of the IVT reaction are adjusted based on the IVT reaction parameters.

[0063] The method for adjusting the IVT reaction conditions based on the IVT reaction parameters includes: adjusting the pH value of the IVT reaction system to maintain at 7 to 8, adjusting the deviation value of the real-time temperature in the IVT reaction from the preset reaction temperature to 0 to 0.5°C; when the dNTP concentration in the IVT reaction system is reduced to 10 to 30% of the initial concentration, adding dNTP and Mg 2+ , cap analogs, and inorganic pyrophosphatase to 75% to 100% of the initial concentration; when precipitation occurs in the IVT reaction system, adjust the Mg 2+ When the mRNA production amount reaches 10-20 mg / mL, the IVT reaction is stopped.

[0064] Although the present invention has been described with reference to several typical embodiments, it should be understood that the terms used are illustrative and exemplary, rather than restrictive. Since the present invention can be embodied in a variety of forms without departing from the spirit or essence of the invention, it should be understood that the above embodiments are not limited to any of the foregoing details, but should be interpreted broadly within the spirit and scope defined by the appended claims, so all changes and modifications falling within the scope of the claims or their equivalents should be covered by the appended claims.

Claims

1. An RNA reaction bag, characterized in that: The RNA reaction bag includes: A bag body, the bag body comprising a flexible portion and a transparent hard portion, the transparent hard portion being structured to form a test dish, the test dish being connected to the interior of the bag body to receive the IVT reaction liquid; The detection component includes an optical detector, a pH detector and a temperature detector; the pH detector and the temperature detector are arranged in a bag body; the optical detector includes a transmitter and a receiver, the transmitter and the receiver are arranged on two sides of the detection dish relative to each other, and the detection light emitted by the transmitter is received by the receiver through the detection dish and the IVT reaction liquid.

2. The RNA reaction bag according to claim 1, characterized in that: The transparent hard part is structured to form a detection dish protruding from the bag body. A connecting piece is arranged on the outer side of the detection dish. The optical detection assembly is detachably connected to the bag body through the connecting piece.

3. The RNA reaction bag according to claim 2, characterized in that: The transparent hard portion is structured to form M detection dishes protruding from the bag body, where M is an integer not less than 2; the detection dishes are arranged at intervals, and a first receiving groove is formed between two adjacent detection dishes, the connecting member is arranged in the first receiving groove, and the transmitter and / or receiver is detachably arranged in the first receiving groove.

4. The RNA reaction bag according to claim 1, characterized in that The transparent hard part is structured to form N second containing grooves recessed in the bag body, where N is an integer not less than 2; the second containing grooves are arranged at intervals, and a detection dish is formed between two adjacent second containing grooves, and the transmitter and / or receiver is detachably arranged in the second containing groove.

5. The RNA reaction bag according to claim 4, characterized in that: A flow guide is arranged around the opening of the detection dish.

6. The RNA reaction bag according to claim 1, characterized in that: The material of the transparent hard part is selected from one or more of quartz, optical glass and hard optical plastic.

7. The RNA reaction bag according to claim 1, characterized in that: The optical detector is selected from one or more of a Fourier transform infrared spectrometer, an in-situ Raman spectrometer and a particle image analyzer.

8. The RNA reaction bag according to claim 1, characterized in that: The RNA reaction bag comprises an automatic sample injector, and the automatic sample injector is communicated with the bag body.

9. A method for monitoring an IVT reaction, characterized in that: The IVT reaction is carried out in the RNA reaction bag described in any one of claims 1 to 8, and the monitoring method comprises: taking the detection component to detect the IVT reaction liquid in the bag body, obtaining IVT reaction parameters, and adjusting the IVT reaction conditions based on the IVT reaction parameters; wherein the IVT reaction parameters include one or more of raw material concentration, solution aging state, phosphate concentration, mRNA production, pH value and temperature.

10. The method for monitoring an IVT reaction according to claim 9, characterized in that: The method for adjusting the IVT reaction conditions based on the IVT reaction parameters includes: The pH value of the IVT reaction system is regulated to be maintained at 7-8, and the deviation value of the real-time temperature in the IVT reaction compared with the preset reaction temperature is regulated to be 0-0.5°C; When the dNTP concentration in the IVT reaction system decreases to 10-30% of the initial concentration, add dNTP and Mg 2+ , cap analogs, inorganic pyrophosphatase to 75% to 100% of the initial concentration; When precipitation occurs in the IVT reaction system, adjust the Mg 2+ To 75% to 100% of the initial concentration; When the amount of mRNA produced reached 10-20 mg / mL, the IVT reaction was stopped.

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