DsRNA standard substance concentration detection method and application thereof

Abstract

The invention belongs to the technical field of molecular biology, and particularly provides a dsRNA standard substance concentration detection method which comprises the following steps: S1, designing a specific reverse transcription primer according to a dsRNA sequence to be detected; s2, mixing a dsRNA standard substance to be detected with the specific reverse transcription primer to obtain a mixture; s3, adding a reverse transcription system into the mixture for reverse transcription; s4, carrying out gradient dilution on reverse transcription products, and detecting reverse transcription products with different gradients by adopting qPCR (Quantitative Polymerase Chain Reaction); s5, according to a qPCR result, selecting a reverse transcription product with a proper concentration gradient to carry out ddPCR; s6, calculating the concentration of the dsRNA standard substance according to the CT value obtained through qPCR detection and the copy number obtained through ddPCR. The method provided by the invention is not influenced by modification types and is only related to dsRNA sequences, so that the difference influence of different modifications is avoided in principle, and the method can be used for strictly controlling the content of dsRNA in the production process of mRNA vaccine drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for detecting the concentration of a dsRNA standard product and an application thereof. Background Art

[0002] dsRNA, the full name is double-stranded RNA, translated as double-stranded ribonucleic acid. During the IVT (in vitro synthesis of mRNA), T7 RNA polymerase may transcribe using RNA or DNA (template strand and non-template strand) as templates to form different types of dsRNA byproducts. The immunogenicity exhibited by dsRNA, on the one hand, will reduce the translation efficiency of mRNA due to the feedback regulation between the autoimmune system and the translation system; on the other hand, dsRNA enables the body to form memory immunity, and when mRNA drugs need to be repeatedly administered, the drug efficacy will be reduced. Therefore, it is necessary to strictly control the content of dsRNA during the production process of mRNA vaccine drugs, which requires manufacturers to be able to accurately and quantitatively detect dsRNA. The commercial kits currently used by manufacturers are mainly based on the ELISA method. ELISA is based on in vitro antigen-antibody reaction, a detection method that combines antigen-antibody specific reaction with the efficient catalytic action of enzymes, and the sensitivity can reach (pg / mL) level. It is one of the most mature immunoassay methods currently used in commercial applications, and is also widely used in medical experiments, clinical diagnosis, and biopharmaceuticals. This kit needs to establish a standard curve through dsRNA standards for quantification, and the assignment of dsRNA standards becomes the key to the accuracy of the kit. Currently, the main quantitative methods for dsRNA standards are ultraviolet spectrophotometry, fluorescent dye method, and capillary electrophoresis.

[0003] The principle of ultraviolet spectrophotometry is to use the bases that make up the nucleic acid molecules. Due to the aromatic ring structure, they have the characteristics of ultraviolet absorption. The absorption value is between 250-270nm, and the maximum absorption wavelength is 260nm. By measuring the absorbance value of nucleic acid under the condition of 260nm wavelength, the quantitative detection of nucleic acid can be achieved. Since free nucleotides, salts and organic compounds also have certain ultraviolet absorption, therefore, when the sample purity is poor or other types of samples are mixed, the spectrophotometry method may not accurately reflect the total amount of nucleic acid.

[0004] The fluorescent dye method uses dyes that specifically bind to RNA. These fluorescent dyes can only emit fluorescent signals when they bind to RNA. The fluorescent signal is proportional to the RNA concentration. The standard curve established based on RNA standards can accurately quantify RNA. The detection process requires the use of an enzyme reader or a fluorometer. However, since there is no dye that can specifically bind to double-stranded RNA, if RNA quantitative reagents are used directly to quantify dsRNA, there will be inaccurate quantification. The special structure of double-stranded RNA will also affect the specific binding of the dye.

[0005] Capillary electrophoresis is based on the fluorescent dye method to quantify nucleic acids, which can provide both band and concentration information. Using a ladder with known fragment size, a standard curve of migration time and fragment size is generated, and the fragment size of the sample can be determined by different migration times. The sample is quantified by the ratio of the sample peak area to the area of ​​the marker or ladder of known concentration. Similarly, due to the particularity of the dsRNA structure, the dye specifically binds to single-stranded RNA, and double-stranded RNA will also affect the specific binding of the dye.

[0006] Since mRNA drug manufacturers use different modified UTPs to synthesize mRNA, the use of different chemically modified nucleotides to synthesize mRNA can effectively reduce the autoimmunogenicity of mRNA. Because TLR7 and TLR8 bind to the GU-rich region of mRNA, modified uracil such as pUTP(ψ) (pseudouridine), N1-Me-pUTP (N1-methylpseudouridine) and 5-OMe-UTP (5-methyluridine) can inhibit the recognition of mRNA by PRRs (pattern recognition receptors). Therefore, dsRNA standards with different modifications are generally used in dsRNA detection kits (ELISA method). These differently modified dsRNA standards also have different absorbances in ultraviolet spectrophotometry, may also affect the specific binding of the dye in the fluorescent dye method, and will also affect the migration time in capillary electrophoresis due to differences in molecular weight. Therefore, a quantitative method that is not affected by the type of modification is needed. Summary of the invention

[0007] The purpose of the present invention is to overcome the problems in the prior art of poor detection accuracy and inability to avoid the influence of different modification types.

[0008] To this end, the present invention provides a method for detecting the concentration of a dsRNA standard, comprising the following steps:

[0009] S1. Design specific reverse transcription primers according to the dsRNA sequence to be tested;

[0010] S2, mixing the dsRNA standard to be tested with a specific reverse transcription primer to obtain a mixture;

[0011] S3, adding a reverse transcription system to the mixture for reverse transcription;

[0012] S4, performing gradient dilution on the reverse transcription products, and using qPCR to detect the reverse transcription products of different gradients;

[0013] S5. According to the qPCR results, select the reverse transcription products with appropriate concentration gradients for ddPCR;

[0014] S6. Calculate the concentration of dsRNA standard using the CT value obtained by qPCR and the copy number obtained by ddPCR.

[0015] Specifically, the above step S2 specifically includes: performing quantitative detection on the dsRNA standard to be detected, diluting it to a certain concentration according to the quantitative result, and mixing it with a specific reverse transcription primer to obtain a mixture.

[0016] Specifically, in the above step S2, ultraviolet spectrophotometry, fluorescent dye method or capillary electrophoresis is used to quantitatively detect the dsRNA standard to be tested.

[0017] Specifically, in the above step S2, the dilution concentration of the dsRNA standard is 0.02-2 ng / μL.

[0018] Specifically, before adding the reverse transcription system in the above step S3, the mixture is further subjected to heat treatment.

[0019] Specifically, in the above step S3, the heat treatment temperature is 65-95° C., and the heat treatment time is 5 minutes.

[0020] Specifically, the appropriate concentration gradient in the above step S5 is a gradient with a CT value in the range of 23-28 detected by qPCR.

[0021] Specifically, the above step S6 is specifically as follows: calculating the difference between the CT value of the reverse transcription product after 100-fold dilution in the qPCR test result and the CT value corresponding to the dilution tested by ddPCR, calculating the conversion multiple between gradients according to the CT difference, and calculating the concentration of the dsRNA standard by the conversion multiple and the copy number obtained by ddPCR.

[0022] Specifically, the mass concentration of the above dsRNA standard product = dsRNA molecular weight * copy number * conversion multiple.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] The dsRNA standard concentration detection method provided by the present invention utilizes the property that dsRNA can be denatured into a single strand under high temperature. The dsRNA and specific primers are mixed and heated, and then quickly cooled to allow RNA to bind to the primers. A high-efficiency reverse transcriptase is used for reverse transcription. The obtained reverse transcription product is gradient diluted and qPCR and ddPCR are detected. The original sample concentration is calculated by the CT difference and the copy number concentration of the absolute quantification of ddPCR, so as to achieve absolute quantification of the dsRNA standard. This method is not affected by the modification type, but only by the dsRNA sequence. In principle, it avoids the difference in the influence of different modifications and can be used to strictly control the content of dsRNA in the production process of mRNA vaccine drugs.

[0025] The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a flow chart of the method for detecting the concentration of dsRNA standards provided by the present invention. DETAILED DESCRIPTION

[0027] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be limited by the attached claims and their equivalents.

[0028] To this end, the present invention provides a method for detecting the concentration of a dsRNA standard, comprising the following steps:

[0029] S1. Design corresponding reverse transcription primers, qPCR and ddPCR primers, and qPCR and ddPCR probes according to the sequence specificity of the dsRNA standard to be tested.

[0030] S2. Perform preliminary quantitative detection on the dsRNA standard to be tested by ultraviolet spectrophotometry, fluorescent dye method, capillary electrophoresis or other feasible methods, dilute the dsRNA standard to be tested to a certain concentration (concentration that meets the requirements of the reverse transcription experiment) according to the quantitative results, and mix the diluted dsRNA standard to be tested with a specific reverse transcription primer to obtain a mixture.

[0031] The sample diluent is preferably TE buffer, and more preferably, the TE buffer component is a solution containing 10mM Tris-HCl and 1mM EDTA at a pH of 8.0. Nuclease-free water can also be used as a diluent.

[0032] The dilution concentration of the dsRNA standard to be tested can be determined by referring to the input amount recommended in the instruction manual of the reverse transcription kit. The dilution concentration of the dsRNA standard is preferably 0.02-2 ng / μL.

[0033] The amount of primers in the mixture is preferably 10-100 times the amount of dsRNA template to ensure sufficient amount.

[0034] S3. Use a PCR instrument or other feasible equipment to heat-treat the mixture to allow the RNA to bind to the reverse transcription primer.

[0035] The heat treatment temperature is 65-95°C and the heat treatment time is 5 minutes. After the heat treatment, the sample is quickly placed on ice to cool down for later use.

[0036] A reverse transcription system is prepared and added to the heat-treated mixture for reverse transcription.

[0037] The reverse transcription reagent can be a conventional commercial kit. The reverse transcription system preferably contains a certain amount of RNase inhibitor to ensure that RNA is not degraded.

[0038] S4. Perform ten-fold or five-fold gradient dilution on the reverse transcription product, preferably using TE buffer at pH 8.0 as the diluent. Use qPCR to detect the reverse transcription products of different gradients, perform multiple repeated tests on each gradient sample, and take the average value.

[0039] The qPCR detection reagents may be conventional commercial kits, including hot-start Taq DNA polymerase, dNTPs, buffer, primers, probes and other substances necessary for qPCR detection.

[0040] The qPCR detection program was as follows: hot start condition was 95°C for 30 sec; 35-45 cycles were 95°C for 10 sec, 60°C for 30 sec (fluorescence collection).

[0041] S5. Select the reverse transcription products corresponding to the gradient with CT values ​​in the range of 23-28 detected by qPCR for ddPCR.

[0042] ddPCR detection reagents contain hot-start Taq DNA polymerase, dNTPs, buffer, primers, probes and other substances necessary for ddPCR detection.

[0043] The ddPCR detection program was as follows: hot start condition was 95°C for 10 min; 40-45 cycles were 94°C for 30 sec, 60°C for 45 sec; and cold storage condition was 4°C∞.

[0044] S6. Calculate the difference △CT between the CT value of the reverse transcription product after 100-fold dilution in the qPCR test result and the CT value corresponding to the dilution tested by ddPCR, calculate the conversion factor between the gradients according to 2^△CT, and calculate the concentration of the dsRNA standard by the conversion factor and the copy number obtained by ddPCR.

[0045] Specifically, the mass concentration of the dsRNA standard = dsRNA molecular weight * copy number * conversion multiple.

[0046] The following specific examples are used to study the dsRNA standard concentration detection method of the present invention and the effects of its application.

[0047] Embodiment 1:

[0048] Reference Figure 1 This embodiment provides a method for detecting the concentration of a dsRNA standard, comprising the following steps:

[0049] S1. Prepare four differently modified dsRNA standards (unmodified, pUTP modified, N1-Me-pUTP modified, and 5-OMe-UTP modified).

[0050] Corresponding reverse transcription primers, qPCR and ddPCR primers, and qPCR and ddPCR probes were designed according to the sequence specificity of the dsRNA standard to be tested.

[0051] The synthesized primers and probes were centrifuged at 4000 rpm for 1 min, carefully removed after centrifugation, and diluted with TE buffer to prepare a 10 μM working solution. The TE buffer component is a solution with a pH value of 8.0 containing 10 mM Tris-HCl and 1 mM EDTA.

[0052] S2. Use UV spectrophotometry to perform preliminary quantitative detection on four differently modified dsRNA standards, and use TE buffer to dilute the four differently modified dsRNA standards according to the quantitative results.

[0053] Take 10 μL of each of the four diluted dsRNA standards to be tested and add them to the eight-tube concatenation tubes. Then add 1 μL of dsR-RT-Primer-R primers to each concatenation tube and cover the tube caps. Vortex to mix and centrifuge briefly for later use.

[0054] S3. Place the eight-tube strip on a common PCR instrument and heat treat it at 95℃ for 5 minutes. Before the end of the program, quickly take out the eight-tube strip and insert it into ice to allow the eight-tube strip to cool quickly so that the RNA and primers can fully combine.

[0055] Prepare the reverse transcription system according to Table 1, take out the eight-tube strip from ice, open the tube cap, add 9 μL / well reverse transcription mix, cover the tube cap, vortex and centrifuge briefly.

[0056] Place the eight-tube strip on a common PCR instrument and perform reverse transcription according to the reverse transcription program in Table 2.

[0057] Table 1 Reverse transcription system

[0058] Components Single serving μL 10×HH RTL Buffer 2 dNTP Mix (10 mM each) 1 RNase Inhibitor (40U / μL) 0.5 RTL reverse transcriptase 2.0 (15U / μL) 0.5 Nuclease-free Water 5 Previous step product 11 Total volume 20

[0059] Table 2 Reverse transcription procedures

[0060] Temperature ℃ Time (min:s) 25 05:00 55 10:00 80 10:00 8 ∞

[0061] S4. After reverse transcription, the reverse transcription product was diluted 10 times step by step. Use a dispenser to take 5 μL + 45 μL TE buffer and dilute it downward five times. The four dsRNA reverse transcription products were diluted to obtain five gradient samples of 1E-02 / 1E-03 / 1E-04 / 1E-05 / 1E-06. Each gradient sample was repeated twice.

[0062] Prepare qPCR mix according to Table 3, dispense qPCR mix into new eight-tube strips at 15 μL / well, add diluted reverse transcription product at 15 μL / well, vortex mix the eight-tube strips, centrifuge briefly to avoid bubbles in the solution, and perform qPCR detection according to the procedure in Table 4.

[0063] Table 3 qPCR reaction system

[0064]

[0065] Table 4 qPCR reaction procedure

[0066]

[0067] S5. Prepare ddPCR Mix according to the system in Table 5, select the reverse transcription product with a gradient of 1E-06 for ddPCR, and set up 2 duplicate wells, and a total of 8 wells for ddPCR detection of the four modified samples.

[0068] Table 5 ddPCR reaction system

[0069]

[0070] Aliquot ddPCR Miix into a new column of a new 96-well plate at 15 μL / well, add four 1E-06 gradient samples at 5 μL / well, and repeat twice for a total of 8 wells.

[0071] Attach the membrane, vortex carefully to mix, and centrifuge using a plate shaker to keep the liquid at the bottom of the tube.

[0072] The Automated Droplet Generator was used to generate droplets. After the instrument generated the droplets, the 96-well plate was sealed with aluminum foil using the PX1 PCR Plate Sealer. The 96-well plate was placed on the PCR instrument for amplification. The amplification program is shown in Table 6.

[0073] Table 6 ddPCR reaction program

[0074]

[0075] After amplification, the plate was read using a QX200 Droplet Reader. After reading, the channel was streaked for analysis, and the Threshold was set to 3400 for analysis to obtain the positive copy number. The results are shown in Tables 7-10.

[0076] Table 7 ddPCR results of unmodified dsRNA

[0077]

[0078] Table 8 pUTP modified dsRNA ddPCR results

[0079]

[0080]

[0081] Table 9 N1-Me-pUTP modified dsRNA ddPCR results

[0082]

[0083] Table 10 5-OMe-UTP modified dsRNA ddPCR results

[0084]

[0085] S6. Calculate the difference between the CT value of the reverse transcription product after 100-fold dilution in the qPCR test results and the CT value corresponding to the dilution tested by ddPCR. Calculate the conversion multiple between gradients based on the CT difference. Calculate the concentration of the dsRNA standard by the conversion multiple and the copy number obtained by ddPCR. The results are shown in Table 11.

[0086] Mass concentration of dsRNA standard = dsRNA molecular weight * copy number * conversion factor / 6.02 * 10 23 .

[0087] Table 11 dsRNA standard mass concentration test results

[0088]

[0089] It should be noted that, for the aforementioned method embodiments, for the sake of simplicity, they are all expressed as a series of action combinations, but those skilled in the art should be aware that the present application is not limited by the described order of actions, because according to the present application, certain steps can be performed in other orders or simultaneously. Secondly, those skilled in the art should also be aware that the embodiments described in the specification are all preferred embodiments, and the actions and modules involved are not necessarily required by the present application.

[0090] The embodiments of the present application are introduced in detail above. Specific examples are used in this article to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method and core idea of ​​the present application. At the same time, for general technical personnel in this field, according to the idea of ​​the present application, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A method for detecting the concentration of a dsRNA standard, characterized in that: The following steps are involved: S1. Design specific reverse transcription primers according to the dsRNA sequence to be tested; S2, mixing the dsRNA standard to be tested with a specific reverse transcription primer to obtain a mixture; S3, adding a reverse transcription system to the mixture for reverse transcription; S4, performing gradient dilution on the reverse transcription products, and using qPCR to detect the reverse transcription products of different gradients; S5. According to the qPCR results, select the reverse transcription products with appropriate concentration gradients for ddPCR; S6. Calculate the concentration of dsRNA standard using the CT value obtained by qPCR and the copy number obtained by ddPCR.

2. The method for detecting the concentration of a dsRNA standard according to claim 1, wherein: The step S2 specifically includes: performing quantitative detection on the dsRNA standard to be detected, diluting it to a certain concentration according to the quantitative result, and mixing it with a specific reverse transcription primer to obtain a mixture.

3. The method for detecting the concentration of a dsRNA standard according to claim 2, wherein: In the step S2, ultraviolet spectrophotometry, fluorescent dye method or capillary electrophoresis is used to quantitatively detect the dsRNA standard to be tested.

4. The method for detecting the concentration of a dsRNA standard according to claim 2, wherein: The dilution concentration of the dsRNA standard in step S2 is 0.02-2 ng / μL.

5. The method for detecting the concentration of a dsRNA standard according to claim 1, wherein: Before adding the reverse transcription system in step S3, the mixture is further subjected to heat treatment.

6. The method for detecting the concentration of a dsRNA standard according to claim 1, wherein: In step S3, the heat treatment temperature is 65-95° C. and the heat treatment time is 5 min.

7. The method for detecting the concentration of a dsRNA standard according to claim 1, wherein: The appropriate concentration gradient in step S5 is a gradient in which the CT value detected by qPCR is in the range of 23-28.

8. The method for detecting the concentration of a dsRNA standard according to claim 1, wherein: The step S6 is specifically as follows: calculating the difference between the CT value of the reverse transcription product after 100-fold dilution in the qPCR test result and the CT value corresponding to the dilution tested by ddPCR, calculating the conversion multiple between gradients according to the CT difference, and calculating the concentration of the dsRNA standard by the conversion multiple and the copy number obtained by ddPCR.

9. The method for detecting the concentration of a dsRNA standard according to claim 8, wherein: The mass concentration of the dsRNA standard product = dsRNA molecular weight * copy number * conversion multiple.

10. Use of the dsRNA standard concentration detection method according to any one of claims 1 to 9 in the production of mRNA vaccine drugs.

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