DsRNA standard substance as well as preparation method and application thereof
By using specific primer compositions and multi-step purification treatment during the preparation of dsRNA standards, the problem of low purity of existing dsRNA standards was solved, and high-purity dsRNA standards were obtained, which were suitable for the quality control of mRNA vaccines, improving the safety and effectiveness of the product.
Patent Information
- Application Number
- CN202411934837.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-26
- Publication Date
- 2025-03-28
AI Technical Summary
There is a problem of low purity in the preparation of existing dsRNA standards, which contains impurities such as ssDNA, which affects the immune effect and safety of mRNA vaccines.
By a specific primer composition setting, transcription promoter and poly(T) fragments were introduced in PCR amplification of in vitro transcription templates, combined with DNA digestion and oligo(dT) enrichment treatment, followed by annealing reaction and cellulose column purification to obtain high-purity dsRNA standards.
Effective separation of impurities such as dsRNA and ssDNA was achieved, and high-purity and stable dsRNA standards were obtained, which were suitable for quality control in mRNA vaccine production, improving the safety and effectiveness of the product.
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Figure CN119709913A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology, and in particular relates to a dsRNA standard product and a preparation method and application thereof. Background Art
[0002] Messenger RNA (mRNA) is a large class of RNA molecules that is responsible for transmitting genetic information encoded in DNA and guiding cells to synthesize proteins. mRNA vaccines refer to vaccines that introduce mRNA containing encoded antigenic proteins into the human body, directly translate them, and form corresponding antigenic proteins, thereby inducing the body to produce specific immune responses and achieve preventive immune effects. It has the advantages of significant efficacy, short R&D cycle, low cost, easy large-scale production, and high safety, and has quickly become the focus of research.
[0003] The preparation of mRNA stock solution is an extremely important part of mRNA vaccine production. During the preparation of mRNA stock solution, T7 RNA polymerase may transcribe using RNA or DNA (template chain and non-template chain) as templates to form different types of dsRNA byproducts. The presence of dsRNA in the mRNA stock solution will not only affect the immune effect of the mRNA vaccine, but may also cause adverse immune reactions. Therefore, it is necessary to detect dsRNA in the mRNA stock solution.
[0004] At present, the methods for detecting dsRNA mainly include colloidal gold method, ELISA method, immunospot method (dotblot), homogeneous time-resolved fluorescence method (HTRF) and high performance liquid chromatography (HPLC), etc. Except for HPLC method, the above methods all need to use specific antibodies to specifically recognize the double-stranded structure of dsRNA, and in the above detection methods, dsRNA standards need to be introduced as controls to calculate the content of dsRNA.
[0005] However, the preparation of existing dsRNA standards contains impurities such as ssDNA, and the removal effect is not ideal. The obtained dsRNA standards have the problem of low purity and have great limitations. Summary of the invention
[0006] The first purpose of the present invention is to solve the problem of low purity of existing dsRNA standards and provide a method for preparing a dsRNA standard.
[0007] The second object of the present invention is to provide a dsRNA standard.
[0008] The third object of the present invention is to provide the use of the above-mentioned dsRNA standard in the production of mRNA vaccines.
[0009] Specifically, the preparation method of the dsRNA standard provided by the present invention specifically comprises: S1, taking a primer composition to perform PCR amplification on a DNA template to obtain an in vitro transcription template; S2, taking an RNA polymerase to perform an in vitro transcription reaction on the in vitro transcription template to obtain ssRNA; S3, taking the ssRNA to perform DNA digestion treatment and oligo (dT) enrichment treatment to obtain pure ssRNA; S4, taking the pure ssRNA to perform annealing reaction and cellulose column purification treatment to obtain the dsRNA standard; wherein the primer composition is a forward strand primer set or a reverse strand primer set, the forward strand primer set includes primer-forward strand-F and primer-forward strand-R, and the reverse strand primer set includes primer-reverse strand-F and primer-reverse strand-R; the 5' ends of the primer-forward strand-F and primer-reverse strand-F both include a transcription promoter, and the 5' ends of the primer-forward strand-R and primer-reverse strand-R both include a poly (T) fragment.
[0010] Furthermore, the transcription promoter is selected from one or more of a T7 promoter, a T3 promoter and an SP6 promoter.
[0011] Furthermore, the transcription promoter is a T7 promoter, and the nucleotide sequence of the T7 promoter is specifically shown in SEQ ID NO:1.
[0012] Furthermore, the length of the poly(T) fragment is 40 to 75 nt.
[0013] Furthermore, in step S2, the RNA polymerase is selected from one or more of T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase.
[0014] Furthermore, in step S2, in the in vitro transcription reaction, the added amount of the RNA polymerase is 1-5 U / μL, and the added amount of the in vitro transcription template is 10-20 ng / μL.
[0015] Furthermore, in step S2, the temperature of the in vitro transcription is 35-42° C., and the time is 1-12 h.
[0016] Furthermore, in step S3, the DNA digestion treatment includes: using DNase I to treat ssRNA to achieve DNA degradation.
[0017] Furthermore, in step S3, based on the mass of the ssRNA, the amount of DNase I added is 1-2 U / μg.
[0018] Furthermore, in step S3, the temperature of the DNA digestion treatment is 35-42° C. and the time is 15-30 min.
[0019] Furthermore, in step S3, the oligo(dT) enrichment treatment is selected from one or more of oligo(dT) chromatography enrichment treatment, oligo(dT) magnetic bead enrichment treatment and oligo(dT) column high performance liquid chromatography enrichment treatment.
[0020] Furthermore, in step S4, the annealing reaction includes: taking pure ssRNA and subjecting it to a heat preservation treatment at 90-98° C. for 3-10 minutes, followed by a slow cooling treatment.
[0021] Furthermore, in step S4, the cooling rate of the slow cooling treatment is 0.1-2°C / min, and the final temperature is 20-30°C.
[0022] Furthermore, in step S4, the cellulose column purification treatment is wood cellulose column chromatography and / or microcrystalline cellulose column chromatography.
[0023] The dsRNA standard provided by the present invention is prepared by the above-mentioned method for preparing the dsRNA standard.
[0024] The present invention also provides the use of the above-mentioned dsRNA standard in the production of mRNA vaccines.
[0025] Beneficial effects:
[0026] In the preparation method of the dsRNA standard provided by the present invention, a primer composition used in the preparation process of an in vitro transcription template is specially set, a transcription promoter is introduced into the 5' end of the forward primer in the forward strand primer set and the reverse strand primer set, and a poly(T) fragment is also introduced into the 5' end of the reverse primer, so that the ssRNA includes a poly(A) fragment, and the ssRNA before annealing is subjected to oligo(dT) enrichment treatment, so that the dsRNA and impurities such as ssDNA are fully separated effectively, the purity of the dsRNA obtained after annealing is high, and the separation between dsRNA and ssRNA can be quickly and effectively achieved by using a cellulose column purification treatment, so as to obtain a high-quality dsRNA standard. The dsRNA standard has high purity and is stable, can be well applied to the production of mRNA vaccines, and has excellent application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 The gel electrophoresis diagram of the in vitro transcription template-positive strand and the in vitro transcription template-reverse strand provided in the embodiments of the present invention;
[0028] Figure 2 This is a gel electrophoresis diagram of the dsRNA standards provided in the embodiments of the present invention. DETAILED DESCRIPTION
[0029] The nucleotide sequences involved in the present invention are specifically shown in Table 1.
[0030] Table 1.
[0031]
[0032] Based on a deep understanding of the impurities in existing dsRNA standards, the inventors of the present invention have found through extensive and in-depth research and a large number of experiments that by specially setting the primer composition used in the preparation of the in vitro transcription template, the 5' end of the forward primer in the forward primer set and the reverse primer set are both introduced with a transcription promoter and the 5' end of the reverse primer is both introduced with a poly(T) fragment, and the 3' end of the prepared forward ssRNA and reverse ssRNA includes a poly(A) fragment, and oligo(dT) enrichment treatment can be used to achieve effective separation of ssRNA and in vitro transcription template, thereby obtaining high-purity ssRNA, and the ssRNA is subjected to annealing reaction and cellulose column purification treatment to obtain a high-purity and stable dsRNA standard. Based on this, the technical solution of the present invention is obtained.
[0033] In the present invention, the preparation method of the dsRNA standard specifically comprises: S1, taking a primer composition to perform PCR amplification on a DNA template to obtain an in vitro transcription template; S2, taking an RNA polymerase to perform an in vitro transcription reaction on the in vitro transcription template to obtain ssRNA; S3, taking the ssRNA to perform DNA digestion treatment and oligo (dT) enrichment treatment to obtain pure ssRNA; S4, taking the pure ssRNA to perform annealing reaction and cellulose column purification treatment to obtain the dsRNA standard.
[0034] In the present invention, in step S1, the DNA template is used as a template for chain extension in PCR amplification in order to obtain an in vitro transcription template of the expected length. Those skilled in the art can make conventional selections from the prior art, and the present invention does not impose any particular limitation thereto. Specific examples thereof include but are not limited to: one or more of a plasmid vector, a bacterial genomic DNA, and an artificial sequence.
[0035] In the present invention, in step S1, the primer composition is specifically a forward-strand primer set or a reverse-strand primer set. The forward-strand primer set includes primer-forward-F and primer-forward-R, and the reverse-strand primer set includes primer-reverse-F and primer-reverse-R; and the 5' ends of the primer-forward-F and primer-reverse-F both include a transcription promoter, and the 5' ends of the primer-forward-R and primer-reverse-R both include a poly(T) fragment.
[0036] In the present invention, the transcription promoter is a sequence used to guide the start of RNA transcription. Those skilled in the art can make adaptive selections from existing known transcription promoters, so as to achieve in vitro transcription of RNA. The present invention does not impose any particular limitation on it. Specific examples thereof include but are not limited to: one or more of T7 promoter, T3 promoter and SP6 promoter.
[0037] In some specific embodiments, the transcription promoter is preferably a T7 promoter, and the nucleotide sequence of the T7 promoter is specifically shown in SEQ ID NO:1.
[0038] In the present invention, the length of the poly (T) fragment is preferably 40 to 75 nt, such as 40 nt, 43 nt, 45 nt, 49 nt, 50 nt, 55 nt, 58 nt, 60 nt, 65 nt, 70 nt or any integer value therebetween.
[0039] In the present invention, the primer-positive strand-F specifically includes a transcription promoter and a complementary sequence 1 along the 5' to 3' direction, and the complementary sequence 1 is complementary to the 3' end base of the obtained ssRNA-reverse strand. Those skilled in the art can design it according to the sequence of the DNA template used, and the present invention does not impose any particular limitation on it.
[0040] In the present invention, the primer-positive strand-R specifically includes a poly(T) fragment and a complementary sequence 2 along the 5' to 3' direction, and the complementary sequence 2 is complementary to the 3' end base of the obtained ssRNA-positive strand. Those skilled in the art can design it according to the sequence of the DNA template used, and the present invention does not impose any particular limitation on it.
[0041] In the present invention, the primer-reverse strand-F specifically includes a transcription promoter and a complementary sequence 3 along the 5' to 3' direction, and the complementary sequence 3 is complementary to the 3' end base of the obtained ssRNA-positive strand. Those skilled in the art can design it according to the sequence of the DNA template used, and the present invention does not impose any particular limitation on it.
[0042] In the present invention, the primer-reverse strand-R specifically includes a poly(T) fragment and a complementary sequence 4 along the 5' to 3' direction, and the complementary sequence 4 is complementary to the 3' end base of the obtained ssRNA-reverse strand. Those skilled in the art can design it according to the sequence of the DNA template used, and the present invention does not impose any particular limitation on it.
[0043] In the present invention, in step S1, the PCR amplification is a technique routinely used in the field of genetic engineering. Those skilled in the art can make adaptive choices for the reaction system and reaction conditions of PCR amplification according to the primer composition, DNA template and in vitro transcription template to be obtained, and the present invention does not impose any particular limitation thereto.
[0044] In the present invention, in step S1, the in vitro transcription template can be more specifically divided into an in vitro transcription template-positive strand or an in vitro transcription template-negative strand, depending on whether the primer composition used is a positive strand primer set or a reverse strand primer set. It should be noted that the in vitro transcription template-positive strand or the in vitro transcription template-negative strand is prepared by independent PCR amplification.
[0045] In the present invention, in step S2, the RNA polymerase is adaptively selected according to the transcription promoter included in the in vitro transcription template, and specific examples thereof include but are not limited to: one or more of T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase.
[0046] In some specific embodiments, in step S2, the amount of RNA polymerase added in the in vitro transcription reaction is preferably 1-5U / μL, 1U / μL, 1.3U / μL, 1.6U / μL, 1.95U / μL, 2U / μL, 2.5U / μL, 3U / μL, 4U / μL, 5U / μL or any value therebetween; the amount of the in vitro transcription template added is preferably 10-20ng / μL, such as 10ng / μL, 11.3ng / μL, 13ng / μL, 15ng / μL, 20ng / μL or any value therebetween.
[0047] In some specific embodiments, in step S2, the conditions of the in vitro transcription include a temperature of preferably 35-42°C, such as 35°C, 35.5°C, 37°C, 39°C, 40°C, 41°C, 42°C or any value therebetween; and a time of preferably 1-12h, such as 1h, 1.3h, 1.8h, 2h, 2.5h, 4h, 8h, 10h, 12h or any value therebetween.
[0048] In the present invention, in step S3, the DNA digestion treatment specifically includes: using DNase I to treat ssRNA to achieve DNA degradation.
[0049] In some specific embodiments, in step S3, based on the mass of the ssRNA, the added amount of the DNase I is preferably 1-2 U / μg, such as 1 U / μg, 1.3 U / μg, 1.5 U / μg, 1.8 U / μg, 2 U / μg or any value therebetween.
[0050] In some specific embodiments, the conditions for the DNA digestion treatment include a temperature of preferably 35 to 42°C, such as 35°C, 36°C, 37°C, 39°C, 40°C, 42°C or any value therebetween; and a time of preferably 15 to 30 min, such as 15 min, 17.5 min, 20 min, 25 min, 30 min or any value therebetween.
[0051] In the present invention, in step S3, specific examples of the oligo(dT) enrichment treatment include but are not limited to: one or more of oligo(dT) chromatography enrichment treatment, oligo(dT) magnetic bead enrichment treatment and oligo(dT) column high performance liquid chromatography enrichment treatment.
[0052] In the present invention, in step S4, the annealing reaction specifically includes: taking pure ssRNA and subjecting it to a heat preservation treatment at 90-98° C. for 3-10 minutes, followed by a slow cooling treatment to obtain the dsRNA standard.
[0053] In some specific embodiments, in step S4, the cooling rate of the slow cooling treatment is preferably 0.1-2°C / min, such as 0.1°C / min, 0.5°C / min, 1°C / min, 1.5°C / min, 2°C / min or any value therebetween; the final temperature is preferably 20-30°C, such as 20°C, 20.5°C, 23°C, 25°C, 28°C, 30°C or any value therebetween.
[0054] In the present invention, in step S4, the cellulose column purification process refers to utilizing the selective adsorption capacity of cellulose for ssRNA and dsRNA, using it as a separation medium to achieve effective separation of dsRNA and ssRNA, thereby obtaining high-purity dsRNA. In some specific embodiments, specific examples of cellulose column purification processes include but are not limited to: wood cellulose column chromatography and / or microcrystalline cellulose column chromatography. Among them, the wood cellulose column chromatography refers to a technology that uses wood fiber as a filler to separate ssRNA and dsRNA by means of column chromatography, and the wood cellulose is a substance obtained by using wood as a raw material and removing lignin, hemicellulose and other components through simple treatment. The microcrystalline cellulose column chromatography refers to a technology that uses microcrystalline cellulose as a filler to separate ssRNA and dsRNA by means of column chromatography, and the microcrystalline cellulose refers to a natural polysaccharide composed of glucose molecules connected by β-1,4-glucosidic bonds.
[0055] Based on the technical effects of the above-mentioned preparation method of dsRNA standard, the present invention further provides a dsRNA standard. The dsRNA standard is prepared by the above-mentioned preparation method, has high purity, and can be well applied to the quality control of mRNA vaccine production.
[0056] Based on the application potential of the above-mentioned dsRNA in the production of mRNA vaccines, the present invention further provides the application of the dsRNA in the production of mRNA vaccines.
[0057] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.
[0058] The reagents and sources involved in the following examples specifically include:
[0059] Takara Premixed High-Fidelity Enzyme HS (Takara, item number R040A);
[0060] DNA purification and recovery kit (Novozymes, catalog number DC301);
[0061] 10X Transcription Buffer (Nearshore Protein, Catalog No. EB231)
[0062] T7 RNA polymerase (Nearshore Protein, Catalog No. E121-H200);
[0063] Inorganic pyrophosphatase (Hanhai Biological, catalog number HBP000501);
[0064] RNase inhibitor (Kaixia Biotechnology, product number RNI-ME101);
[0065] DNase I (Novozymes, catalog number GMP4104);
[0066] Oligo dT18 chromatographic column (Sartorius, product number 311.1219-2);
[0067] DNA marker (Takara, catalog number 3428Q).
[0068] Example
[0069] This example is used to illustrate a method for preparing a dsRNA standard, which specifically includes:
[0070] 1. Preparation of in vitro transcription template: (1) Using the dsRNA template sequence shown in SEQ ID NO: 6 as a template, primer set 1 and primer set 2 shown in Table 2 were used respectively, and the 5' ends of primer-positive strand-F and primer-reverse strand-F both included a T7 promoter, and the 5' ends of primer-positive strand-R and primer-reverse strand-R both included a poly(T) fragment with a length of 50 nt, and Takara premixed high-fidelity enzyme was used. HS was amplified by PCR according to the instructions to obtain PCR products.
[0071] Table 2.
[0072]
[0073] The PCR amplification reaction system includes: 25 μL (2×) HS (Premix), 1 μL of primer-positive strand-F (10 mM), 1 μL of primer-positive strand-R (10 mM), 1 ng of plasmid vector and 22 μL of ddHO 2 O. The reaction procedure included: 95°C for 5 min; 95°C for 10 s, 64°C for 5 s, 72°C for 30 s, 30 cycles; 72°C for 5 min.
[0074] (2) Take the PCR product and perform agarose gel electrophoresis. The results are as follows: Figure 1 As shown. Figure 1 It can be seen that the molecular weight of the PCR product is about 550bp; then the DNA purification recovery kit is used and the DNA is recovered according to the instructions to obtain the in vitro transcription template-positive chain and the in vitro transcription template-reverse chain. Among them, the concentration of the in vitro transcription template-positive chain is 269ng / μL, and the concentration of the in vitro transcription template-positive chain is 283ng / μL.
[0075] 2. Preparation of ssRNA: (1) Take 1 μL of in vitro transcription template-positive strand or in vitro transcription template-reverse strand, 1.5 μL of ATP (100 mM), 1.5 μL of CTP (100 mM), 1.5 μL of GTP (100 mM), 1.5 μL of UTP (100 mM), 2 μL of 10X Transscription Buffer, 2 μL of T7 RNA polymerase (0.1 U / μL), 1 μL of inorganic pyrophosphatase and 1 μL of RNase inhibitor (40 U / μL), mix, and use nuclease-free water to make up the volume to 20 μL to obtain an in vitro transcription reaction solution; take the in vitro transcription reaction solution and react at 37° C. for 4 hours to obtain a product solution I including the ssRNA-positive strand and a product solution II including the ssRNA-negative strand. The nucleotide sequence of the ssRNA-positive strand is specifically as shown in SEQ ID NO: 7.
[0076] (2) Based on the mass of ssRNA in the solution, DNase I was added to product solution I and product solution II at an addition amount of 2 U / μg, and the mixture was incubated at 37°C for 20 min to obtain a crude ssRNA-positive strand sample solution and a crude ssRNA-negative strand sample solution.
[0077] 3. Purification of ssRNA: (1) Use eluent I to make up the volume of the crude ssRNA-positive strand solution and the crude ssRNA-negative strand solution to 1 mL, respectively, and perform FPLC purification using an oligo dT column and the procedure shown in Table 3 to obtain a pure ssRNA-positive strand solution and a pure ssRNA-negative strand solution.
[0078] Table 3.
[0079]
[0080] Among them, the balancing solution used in the balancing is flushing solution I; the flushing solution I used in flushing I includes 50mM Tris-HCl buffer (pH=7.0), 5mM EDTA and 500mM NaCl; the flushing solution II used in flushing II includes 50mM Tris-HCl buffer (pH=7.0) and 5mM EDTA; the elution solution used in elution is 10mM Tris-HCl buffer (pH=7.5).
[0081] (2) Using a PALL 30Kd ultrafiltration tube, the pure ssRNA-positive strand solution and the pure ssRNA-negative strand solution were desalted and concentrated at 4°C and 4500g to obtain ssRNA-positive strand and ssRNA-negative strand, respectively.
[0082] 4. Preparation of dsRNA: According to the reaction system shown in Table 4, ssRNA-positive strand, ssRNA-negative strand, Tris-HCl buffer (pH=7.0) and NaCl were mixed to prepare a denaturation annealing reaction solution; the denaturation annealing reaction solution was subjected to annealing reaction to obtain dsRNA.
[0083] Table 4.
[0084] substance Final concentration ssRNA-positive strand 0.1ug / uL ssRNA-minus strand 0.1ug / uL Tris-HCl buffer (pH = 7.0) 10mM NaCl 50mM
[0085] The annealing reaction conditions include: keeping at 95°C for 5 minutes; cooling from 95°C to 25°C at a cooling rate of 1°C / min; and storing at 4°C.
[0086] 5. Purification of dsRNA: (1) Use microcrystalline cellulose (manufacturer: Sigma-Aldrich, product number: C6288) as the filler in the chromatography column shell, then add the annealed dsRNA and the balance solution in a 1:1 (V:V) ratio and mix them on the machine, collect the eluate, and obtain a pure dsRNA solution.
[0087] The balancing solution includes 10 mM HEPES, 125 mM NaCl, 0.1 mM EDTA and 16% (V:V) ethanol; and the eluting solution is sterile water treated with DEPC.
[0088] (2) Using a PALL 30Kd ultrafiltration tube, the pure dsRNA solution was desalted and concentrated at 4°C and 4500g to obtain a dsRNA standard. The obtained dsRNA standard was subjected to agarose gel electrophoresis. The results are as follows: Figure 2 shown.
[0089] Depend on Figure 2 The test results shown show that the dsRNA standard has clear bands, no tailing or other unexpected bands, has high purity, and can be well applied to dsRNA detection of mRNA stock solution.
[0090] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.
Claims
1. A method for preparing a dsRNA standard, characterized in that: The preparation method comprises: S1, taking a primer composition to perform PCR amplification on a DNA template to obtain an in vitro transcription template; S2, taking an RNA polymerase to perform an in vitro transcription reaction on the in vitro transcription template to obtain ssRNA; S3, taking the ssRNA to perform DNA digestion treatment and oligo (dT) enrichment treatment to obtain pure ssRNA; S4, taking the pure ssRNA to perform annealing reaction and cellulose filling column purification treatment to obtain the dsRNA standard; Wherein, the primer composition is a forward-strand primer set or a reverse-strand primer set, the forward-strand primer set includes primer-forward-F and primer-forward-R, and the reverse-strand primer set includes primer-reverse-F and primer-reverse-R; the 5' ends of the primer-forward-F and primer-reverse-F both include a transcription promoter, and the 5' ends of the primer-forward-R and primer-reverse-R both include a poly(T) fragment.
2. The method for preparing a dsRNA standard according to claim 1, characterized in that: The transcription promoter is selected from one or more of a T7 promoter, a T3 promoter and an SP6 promoter; Optionally, the transcription promoter is a T7 promoter, and the nucleotide sequence of the T7 promoter is specifically shown in SEQ ID NO:
1.
3. The method for preparing a dsRNA standard according to claim 1, characterized in that: The length of the poly(T) fragment is 40 to 75 nt.
4. The method for preparing a dsRNA standard according to claim 1, characterized in that: In step S2, the RNA polymerase is selected from one or more of T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase; Optionally, in the in vitro transcription reaction, the amount of RNA polymerase added is 1-5 U / μL, and the amount of in vitro transcription template added is 10-20 ng / μL; Optionally, the temperature of the in vitro transcription is 35-42° C., and the time is 1-12 h.
5. The method for preparing a dsRNA standard according to claim 1, characterized in that: In step S3, the DNA digestion treatment includes: treating ssRNA with DNase I to achieve DNA degradation; Optionally, based on the mass of the ssRNA, the amount of DNase I added is 1 to 2 U / μg; Optionally, the temperature of the DNA digestion treatment is 35-42° C. and the time is 15-30 min.
6. The method for preparing a dsRNA standard according to claim 1, characterized in that: In step S3, the oligo(dT) enrichment treatment is selected from one or more of oligo(dT) chromatography enrichment treatment, oligo(dT) magnetic bead enrichment treatment and oligo(dT) column high performance liquid chromatography enrichment treatment.
7. The method for preparing a dsRNA standard according to claim 1, characterized in that: In step S4, the annealing reaction includes: taking pure ssRNA and subjecting it to a heat preservation treatment at 90 to 98° C. for 3 to 10 minutes, followed by a slow cooling treatment to obtain the dsRNA standard; Optionally, the slow cooling treatment has a cooling rate of 0.1 to 2°C / min, and a final temperature of 20 to 30°C.
8. The method for preparing a dsRNA standard according to claim 1, characterized in that: In step S4, the cellulose column purification treatment is lignocellulose column chromatography and / or microcrystalline cellulose column chromatography.
9. A dsRNA standard, characterized in that: The dsRNA standard is prepared by the preparation method of any one of claims 1 to 8.
10. Use of the dsRNA standard product according to claim 9 in the production of mRNA vaccines.