Method for determining the content of each component of multivalent COVID-19 mRNA vaccine

Abstract

The present disclosure relates to a method for determining the content of each component of a multivalent COVID-19 mRNA vaccine, wherein the mRNA vaccine comprises an mRNA having a nucleotide sequence number of SEQ ID NO: 1. In a bivalent mRNA vaccine comprising YKYY021P04 and YKYY021P12, P04‑2 is used as a primer to detect the content of YKYY021P04; P12‑2 is used as a primer to detect the content of YKYY021P12. The method has good specificity and specificity, high sensitivity and accuracy, and can accurately distinguish and quantify the content of each component in a bivalent mRNA vaccine, which is of great significance and value to the quality control of bivalent mRNA vaccines.

Description

Field of the Invention

[0001] The present invention belongs to the field of drug detection technology, and specifically relates to a method for detecting the content of each component of a multivalent new coronavirus mRNA vaccine. Background of the Invention

[0002] The development of mRNA vaccine technology is limited by its molecular instability and its low delivery efficiency. The first study on this aspect was reported in 1990. Wolf et al. injected mRNA into human muscle cells and found that it could be expressed in muscle cells for a long time. Later, in 2012, German researchers developed and modified an mRNA vaccine with long-term efficacy. With the continuous modification of its structure and the development of efficient delivery technology, mRNA vaccine technology has gradually matured. Compared with traditional peptide and protein vaccines, this vaccine can induce the body to produce humoral immunity and cellular immunity. The vaccine production process is simple, the research and development cycle is short, the cost is low, and it is easy to standardize production. It is suitable for vaccine development and production during pandemics and infectious diseases. It is a vaccine with broad application prospects. Due to the continuous mutation and evolution of the virus during the transmission process, in order to prevent the occurrence of immune escape, the bivalent mRNA vaccine came into being. Compared with the monovalent mRNA vaccine, the bivalent mRNA vaccine can induce a wider range of immune antibodies and may also neutralize new variants.

[0003] During the production and release of bivalent mRNA vaccines, the content of mRNA vaccines is one of the key indicators that determine its effectiveness and safety, and is an important quality parameter that must be controlled. At present, RiboGreen, dPCR and ultraviolet spectroscopy are commonly used to detect the content of mRNA vaccines, but RiboGreen and ultraviolet spectroscopy can only detect the total content of mRNA in mRNA vaccines, and cannot distinguish and accurately quantify the mRNA of different components in bivalent mRNA vaccines, and are easily affected by impurities in the sample; although dPCR (digital PCR) technology can accurately quantify the components of bivalent mRNA vaccines, it is limited by its high system cost, limited throughput, cumbersome operation and high system complexity; although the nucleic acid chip method has also been reported, in addition to designing specific probes, it also requires the design of nucleic acids or antibodies that bind to fluorescence, and requires specific instrument consumables, and the operation is complicated and cumbersome.

[0004] Therefore, it is urgent to establish a method with good specificity, high accuracy and sensitivity, good amplification efficiency, simple operation and accurate quantification of the content of each component mRNA vaccine in a bivalent mRNA vaccine. Summary of the invention

[0005] The sequences of the mRNA vaccine (YKYY021P04) designed for the S protein of the wild strain of the new coronavirus and the mRNA vaccine (YKYY021P12) designed for the S protein of the Omicron variant are more than 95% similar. Their structures both include a 5' cap structure, a 5' untranslated region (UTR), an open reading frame (ORF) encoding an antigen (i.e., S protein), a 3' untranslated region (UTR) and a 3' poly (A) tail structure, and most of the sequence differences are located in the ORF region.

[0006] RT-PCR is a method for quantitative determination of nucleic acid vaccines, and there are limitations in the detection process of multivalent nucleic acid vaccines. For the bivalent nucleic acid vaccine disclosed in the present invention, since the mRNA vaccine sequences designed based on the S proteins of wild strains and variants are very different, it is very difficult to find primers that can specifically identify and distinguish the wild strains and variants. Even if complementary primers with large sequence differences are found, the size of the amplified product band (the size of the qPCR amplified product is preferably around 70bp to 150bp) will also affect the qPCR quantitative method. If the primer amplification span is too small, the specificity is not good, non-specific amplification products are prone to appear, and it is difficult to distinguish the target product from primer dimers by agarose gel electrophoresis. There will also be a situation where the amplification efficiency is low or there is no amplification product; if the amplification span is too large, that is, the amplification product fragment is too long, the molecular structure is unstable, and it is easy to break, resulting in a decrease in amplification efficiency. At the same time, if the sequence is too long, repeated sequences are prone to appear, and primer dimers are prone to appear.

[0007] The present invention discloses a bivalent mRNA vaccine designed for the S protein of the wild strain and the variant strain of the novel coronavirus, and designs specific primers for each component in the vaccine, and detects the content of each component by the RT-qPCR method. The method has good specificity, high quantitative accuracy, and a recovery rate deviation as low as 7%, which is much less than the pharmacopoeia requirement. The primers used have a single peak type of expansion and melting curve with the bivalent mRNA vaccine template, a single Tm value, and no amplification product with the non-detection component template; the annealing temperature is optimal, the electrophoresis band of the amplified product is single, and the brightness is high; the primer concentration and reaction system are optimal, the linear correlation coefficient of the amplification reaction is the highest, and the amplification efficiency is closest to 100%; the content of each component in the bivalent mRNA vaccine can be accurately distinguished and quantified.

[0008] The present disclosure provides the following technical solution, an mRNA vaccine, which comprises an mRNA with a nucleotide sequence number of SEQ ID NO: 1.

[0009] The present disclosure provides the following technical solution, an mRNA vaccine, which comprises an mRNA with a nucleotide sequence number of SEQ ID NO: 2.

[0010] The present disclosure also provides the following technical solution: a bivalent mRNA vaccine comprising mRNA with nucleotide sequences numbered SEQ ID NO: 1 and SEQ ID NO: 2.

[0011] In some preferred embodiments, the mRNA is encapsulated by LNP to obtain mRNA-LNP.

[0012] The present disclosure also provides a primer designed based on the bivalent mRNA vaccine, including primers P04-2 and / or P12-2; wherein:

[0013] The upstream primer sequence number of the primer P04-2 is SEQ ID NO: 3 (AACACAGCTGCCTCCAGC), and the downstream primer sequence number is SEQ ID NO: 4 (ATTGGTGCCGGACACGTG);

[0014] The upstream primer sequence number of the primer P12-2 is SEQ ID NO: 5 (CACGCCATCAGCGGCACC), and the downstream primer sequence number is SEQ ID NO: 6 (TTCTTGTGGTCCAGGAAT).

[0015] The present disclosure also provides a method for detecting components in a bivalent mRNA vaccine using the primers, comprising the following steps:

[0016] 1) preparing the test solution of the bivalent mRNA vaccine;

[0017] The bivalent mRNA vaccine undergoes the following pre-treatment process: demulsification and / or purification of the bivalent mRNA vaccine;

[0018] 2) Preparation of standard solutions: using the standard products of each component mRNA in the bivalent mRNA vaccine to prepare a series of standard solutions with different gradient concentrations;

[0019] 3) Reverse Transcription-PCR: Reverse transcribe the mRNA in the test solution in step 1) and the standard solution in step 2) to obtain cDNA samples;

[0020] 4) qPCR detection: adding primers to a series of cDNA samples obtained from a series of standard solutions and a cDNA sample obtained from a test solution, respectively, and performing qPCR detection after amplification;

[0021] 5) Using the series of cDNA samples obtained from the series of standard solutions, a CT value-concentration standard curve is obtained; using the CT value of the cDNA sample obtained from the test solution, the mRNA concentration in the test solution is quantitatively detected.

[0022] In some preferred embodiments, the qPCR detection method in step 4) is:

[0023] i) preparing a solution for a qPCR reaction system;

[0024] ii) The solution is subjected to pre-denaturation, denaturation, annealing and melting.

[0025] In some preferred embodiments, the detection method, wherein:

[0026] The annealing temperature of the reaction system of the primer P04-2 is 34-39°C, preferably 38°C;

[0027] The annealing temperature of the reaction system of the primer P12-2 is 54-60°C, preferably 58°C.

[0028] In some preferred embodiments, the annealing time is 10 to 40 seconds, more preferably 30 seconds.

[0029] In some preferred embodiments, the annealing cycle number is 30 to 50 times, more preferably 40 times.

[0030] In some more preferred embodiments, the pre-denaturation condition is 95° C. for 30 s; and the number of cycles is 1.

[0031] In some more preferred embodiments, the denaturation condition is 95° C. for 10 s and the number of cycles is 40 times.

[0032] In some preferred embodiments, the melting process is melting at a high temperature of 90-100° C. for 10-20 seconds, then melting at an annealing temperature for 30-60 seconds, and finally melting at a high temperature of 90-100° C. for a second time for 10-20 seconds.

[0033] In some preferred embodiments, the primer concentration in the qPCR reaction system solution in step i) is 0.1-0.3 μM, more preferably 0.2 μM.

[0034] In some preferred embodiments, the reaction system in the qPCR reaction system solution in step i) is 10 to 50 μL, more preferably 20 μL.

[0035] The present disclosure also provides a kit for detecting a bivalent mRNA vaccine, the kit comprising primers P04-2 and / or P12-2; wherein:

[0036] The upstream primer sequence number of the primer P04-2 is SEQ ID NO: 3, and the downstream primer sequence number is SEQ ID NO: 4;

[0037] The upstream primer sequence number of the primer P12-2 is SEQ ID NO: 5, and the downstream primer sequence number is SEQ ID NO: 6.

[0038] The beneficial effects of the present invention are as follows:

[0039] The method for detecting the content of each component of the bivalent mRNA vaccine described in the present invention, in the bivalent mRNA vaccine containing YKYY021P04 and YKYY021P12, uses P04-2 as a primer to detect the content of YKYY021P04; uses P12-2 as a primer to detect the content of YKYY021P12. The method of the present invention has good specificity, high quantitative accuracy, and a recovery rate deviation as low as 7%, which is much less than the pharmacopoeia requirement. The peak type of the expansion and melting curve of the primers used and the bivalent mRNA vaccine template is single, the Tm value is single, and there is no amplification product with the non-detection component template; the annealing temperature is optimal, the electrophoresis band of the amplification product is single, and the brightness is high; the primer concentration and the reaction system are optimal, the linear correlation coefficient of the amplification reaction is the highest, and the amplification efficiency is closest to 100%; the content of each component in the bivalent mRNA vaccine can be accurately distinguished and quantified. Specifically manifested as:

[0040] 1. Primer:

[0041] 1-1. Specificity

[0042] Using P04-2 as primer, ① when detecting YKYY021P04, the Tm of its melting curve was 86.772, and the peak shape and Tm value were single, and the electrophoresis band of the amplified product was single, and it was the target band, indicating that the qPCR amplification system was specific, and the amplified product was single and accurate; ② when detecting non-YKYY021P04, the electrophoresis of the amplified product had no band, indicating that P04-2 was a specific primer for YKYY021P04, and the detection method had good specificity.

[0043] Using P12-2 as primer, ① when detecting YKYY021P12, the melting curve Tm was 88.073, and the peak shape and Tm value were single, and the electrophoresis band of the amplified product was single and the target band, indicating that the qPCR amplification system was specific, and the amplified product was single and accurate; ② when detecting non-YKYY021P12, the amplified product had no band in the electrophoresis, indicating that P12-2 was the specific primer of YKYY021P12, and the detection method had good specificity.

[0044] When using primers with other different sequences (for example, primers P04-1, P04-3, P12-1, P12-3), when detecting YKYY021P04 and YKYY021P12, bands appeared in the electrophoresis of the amplified products, which were not specific and could not detect YKYY021P04 and / or YKYY021P12.

[0045] 1-2. Good accuracy

[0046] The recovery rate deviation of the determination result (ie, the deviation between the measured concentration and the actual concentration) is controlled at about 10%, as low as about 7%, which is significantly better than the pharmacopoeia PCR quantitative recovery limit standard of 50% to 150%.

[0047] 1-3. Good amplification efficiency

[0048] The amplification efficiency of primers P04-2 and P12-2 was close to 100%, and the deviation was small and controlled within 5%, which proved that the linear correlation coefficient was high and the quantitative effect was the best.

[0049] The nucleotide sequences of primers P04-2 and P12-2 were shifted forward and backward to be used as new primers (for example, primers P04-2-02, P04-2-03, P12-2-02, P12-2-03). However, their amplification efficiency deviation was large (more than 10%), which did not meet the quantitative requirements of the standard curve and could not be used for the quantification of bivalent mRNA vaccines.

[0050] 2. Optimal annealing temperature:

[0051] The optimum annealing temperature of each primer is 38° C. for P04-2 and 58° C. for P12-2. Under the annealing temperature conditions, the amplified product band is single, there is no non-specific amplified product or primer dimer, the band brightness is the highest, and the size is consistent with the target band, and the electrophoresis amplification effect is the best.

[0052] 3. Optimal primer concentration:

[0053] When detecting the content of bivalent mRNA vaccine components, the primer concentration in the qPCR reaction was controlled to be 0.1-0.3 μM, and the linear correlation coefficient (around 0.99) and amplification efficiency (close to 100%, with small deviation, controlled at 10%) were good, with high quantitative accuracy.

[0054] When the primer concentration is 0.2 μM, the correlation coefficient (above 0.996) and amplification efficiency (close to 100%, with small deviation, controlled within 3%) are optimal, and the quantitative accuracy is high.

[0055] 4. Optimal reaction system:

[0056] When testing the content of the components of the bivalent mRNA vaccine, the reaction system in the controlled qPCR reaction was 10 to 50 μL, and the linear correlation coefficient (above 0.99) and amplification efficiency (within 10% deviation) were good, and the quantification was accurate.

[0057] When the reaction system was 20 μL, the linear correlation coefficient (above 0.996) and amplification efficiency (within 3% deviation) were optimal, and the quantitative accuracy was the highest. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Screening electrophoresis results for the optimal annealing temperature of primer P04-1;

[0059] Figure 2 Screening electrophoresis results for the optimal annealing temperature of primer P04-2;

[0060] Figure 3 Screening electrophoresis results for the optimal annealing temperature of primer P04-3;

[0061] Figure 4 Screening electrophoresis results for the optimal annealing temperature of primer P12-1;

[0062] Figure 5 Screening electrophoresis results for the optimal annealing temperature of primer P12-2;

[0063] Figure 6 Screening electrophoresis results for the optimal annealing temperature of primer P12-3;

[0064] Figure 7 The electrophoresis results of primer P04-1 and different template amplifications;

[0065] Figure 8 The electrophoresis results of primer P04-2 and different template amplifications;

[0066] Fig. 9 The electrophoresis results of primer P04-3 and different template amplifications;

[0067] Fig.10 The electrophoresis results of primer P12-1 and different template amplifications;

[0068] Fig.11 The electrophoresis results of primer P12-2 and different template amplifications;

[0069] Fig.12 The electrophoresis results of primer P12-3 and different template amplifications;

[0070] Fig.13 is the melting curve of primer P04 and template YKYY021P04;

[0071] Fig.14 is the melting curve of the mixed template of primer P04 and YKYY021P04 and YKYY021P04;

[0072] Fig.15 is the melting curve of primer P12 and template YKYY021P12;

[0073] Fig.16 is the melting curve of the mixed template of primer P12 and YKYY021P04 and YKYY021P04;

[0074] Fig.17 The amplification electrophoresis results of primer P04 and template YKYY021P04, template YKYY021P12 and mixed template of YKYY021P04 and YKYY021P12;

[0075] Fig.18 The amplification electrophoresis results of primer P12 and template YKYY021P04, template YKYY021P12 and mixed template of YKYY021P04 and YKYY021P12;

[0076] Fig.19 It is the standard curve of Example 9;

[0077] Fig. 20 is the standard curve of primer P04-2;

[0078] Fig.21 is the standard curve of primer P12-2. DETAILED DESCRIPTION

[0079] Table 1 Experimental reagents

[0080]

[0081]

[0082] Table 2 Experimental instruments

[0083]

[0084] Example 1 Reverse transcription

[0085] 1-1. Preparation of mRNA stock solution and bivalent mRNA vaccine solution

[0086] i) Preparation of mRNA stock solution:

[0087] Linearized plasmid templates (purchased from GenScript Biotech Co., Ltd.) were designed using the S protein sequences of the wild-type SARS-CoV-2 strain and the S protein sequence of the Omicron variant SARS-CoV-2 strain, respectively. In the presence of T7 RNA polymerase, mRNA complementary to one chain in the linearized plasmid template was synthesized using nucleoside triphosphates (NTPs) as substrates. The stability of the mRNA was enhanced by adding a cap structure to the 5' end, and two high-purity mRNA stock solutions, YKYY021P04 and YKYY021P12, were prepared through a series of separation and purification processes; that is, YKYY021P04 stock solution and YKYY021P12 stock solution.

[0088] Table 3

[0089] mRNA name Sequence number YKYY021P04 SEQ ID NO:1 YKYY021P12 SEQ ID NO:2

[0090] The specific synthesis process of mRNA stock solution is as follows:

[0091] 1) Thawing of reagents

[0092] a) Thaw 10× transcription buffer and four ribonucleotides (ATP, CTP, GTP, UTP) at room temperature, mix thoroughly, and centrifuge briefly; keep 10× transcription buffer at room temperature and keep four ribonucleotides on ice for later use.

[0093] b) T7 RNA polymerase, inorganic pyrophosphatase and mouse RNase inhibitor were mixed separately, centrifuged briefly, and placed on ice for later use.

[0094] 2) Transcription

[0095] If you choose the co-transcriptional capping method, prepare the co-transcriptional capping system according to the following table:

[0096] Table 4 Co-transcriptional capping system

[0097]

[0098] Note: Template DNA needs to be added later. Since 10× transcription buffer contains spermidine, too high spermidine concentration will cause template DNA precipitation.

[0099] 3) Incubation

[0100] The above reaction solutions were mixed evenly, centrifuged briefly, and incubated at 37°C for 2 h.

[0101] Note: ① If the transcript length is less than 100 nt, increase the reaction time to 4-8h. ② It is recommended to perform the reaction in a PCR instrument with the hot lid open to prevent the reaction solution from evaporating for a long time. ③ The reaction product may have a white precipitate. This is the free pyrophosphate that forms magnesium pyrophosphate with the magnesium ions in the reaction solution during the reaction, which does not affect subsequent experiments. If you want to remove it, add EDTA and it will disappear. If adding EDTA affects subsequent experiments, you can also centrifuge to recover the supernatant.

[0102] 4) DNase I treatment

[0103] After the transcription reaction was completed, 1 μL DNase I (nuclease-free) was added to each tube, mixed, centrifuged briefly, and incubated at 37°C for 30 min to remove the template DNA.

[0104] 5) mRNA purification

[0105] When using the lithium chloride precipitation method, the RNA length must be greater than 300 nt and the concentration must not be less than 100 ng / μL.

[0106] i) To 20 μL of the reaction mixture, add 30 μL of nuclease-free water and 30 μL of 7.5 M lithium chloride.

[0107] ii) After mixing well, place at -20°C for at least 30 min, centrifuge at 12000 rpm and 4°C for 15 min for purification, and collect the precipitate.

[0108] iii) Add 500 μL of ice-cold 70% ethanol to wash the RNA pellet.

[0109] iv) Dissolve the mRNA precipitate in 20 μL of nuclease-free water to obtain an mRNA stock solution, which is stored at -80°C.

[0110] ii) Preparation of bivalent mRNA vaccine solution

[0111] Using nanolipid particles (LNP) as the delivery system, the above two mRNA stock solutions (YKYY021P04 stock solution and YKYY021P12 stock solution) were encapsulated separately, and then mixed in a 1:1 ratio to obtain a mixed bivalent mRNA vaccine solution.

[0112] 1-2. Solution to be tested:

[0113] The encapsulated bivalent mRNA vaccine solution was demulsified and purified using isopropanol to obtain a test solution.

[0114] 1-3. Standard solution:

[0115] The YKYY021P04 stock solution and the YKYY021P12 stock solution were gradiently diluted to form eight gradient standard solutions of different concentrations: 10, 50, 100, 200, 300, 500, 1000, 1500 ng / μL; that is, YKYY021P04 gradient standard solution and YKYY021P12 gradient standard solution.

[0116] 1-4. Reverse transcription:

[0117] The standard solution, the test solution, the YKYY021P04 stock solution and the YKYY021P12 stock solution were reverse transcribed to obtain cDNA. The specific process is as follows:

[0118] Use 5 μL of 5×gDNA wiper mix and 1 μL of the following solutions:

[0119] ①YKYY021P04 gradient standard solution, the concentrations are: 10, 50, 100, 200, 300, 500, 1000, 1500 ng / μL;

[0120] ②YKYY021P12 gradient standard solution, concentrations are: 10, 50, 100, 200, 300, 500, 1000, 1500 ng / μL;

[0121] ③Solution to be tested;

[0122] ④YKYY021P04 original solution;

[0123] ⑤YKYY021P12 original solution;

[0124] Mix, dilute the samples to 10 μL with sterile enzyme-free water, and place at 42°C for 2 min. After the mixture is placed, prepare the following reverse transcription reaction systems (Table 5).

[0125] Table 5: Reverse transcription reaction system

[0126] Component name Addition volume (μL) Mixed liquid 10 10×RT mix 2 HiScriptⅢEnzyme Mix 2 Oligo(dT)20VN 1 Random hexamers 1 Sterile enzyme-free water 4

[0127] Reverse transcription reaction: Place the prepared system reaction solution in a PCR instrument and perform reverse transcription at 37°C for 15 min; or at 85°C for 5 s to obtain cDNA.

[0128] The cDNA products were diluted 1000-fold for later use.

[0129] Example 2 Primer design and annealing temperature determination

[0130] When the annealing temperatures of the primers are 68°C for P04-1, 38°C for P04-2, 58°C for P04-3, 68°C for P12-1, 58°C for P12-2 and 54°C for P12-3, the electrophoresis band of the amplified product with the test solution is single, which is the optimal annealing temperature.

[0131] 2-1. Primer design:

[0132] The mRNA vaccines (YKYY021P04) and (YKYY021P12) designed for the wild-type and Omicron variant S proteins of the novel coronavirus mentioned above are more than 95% similar in sequence, including at least the following difference sequences:

[0133] Table 6 Differential sequence information

[0134]

[0135] For the above difference sequence:

[0136] ① Primers P04-1, P04-2 and P04-3 were designed based on template YKYY021P04, all of which were primer pairs including an upstream primer and a downstream primer;

[0137] ② Primers P12-1, P12-2 and P12-3 were designed based on template YKYY021P12, all of which were primer pairs including an upstream primer and a downstream primer.

[0138] The above primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The primers were dissolved in sterile enzyme-free water and diluted to a concentration of 10 μM and divided for use.

[0139] According to the Tm value of the primer (i.e., melting temperature), a suitable gradient temperature range (gradient of 2°C) was designed to screen the best annealing temperature of different primers, as shown in the following table:

[0140] Table 7: Primer sequences and annealing temperatures

[0141]

[0142]

[0143] 2-2.qPCR amplification (qualitative analysis):

[0144] The amplification reaction was carried out using different primers and annealing temperatures from 2 to 1, with the cDNA product obtained from the test solution in "1-4 Reverse Transcription" in Example 1 as a template.

[0145] 2-2-1. qPCR reaction system

[0146] The primers in 2-1 were mixed with the cDNA products of the test solution (Example 1) respectively, as shown in Table 8 for details.

[0147] Table 8: qPCR reaction system (20 μL)

[0148] Reagent name Addition volume (μL) cDNA 1 2×Taq Pro Universal SYBR qPCR Master Mix 10 Upstream primer 0.4 Downstream primer 0.4 Sterile enzyme-free water 8.2

[0149] 2-2-2. qPCR reaction conditions

[0150] qPCR amplification was performed under the gradient annealing temperature conditions of each primer, as shown in Table 9:

[0151] Table 9: qPCR reaction conditions

[0152]

[0153] 2-3. Electrophoresis:

[0154] The qPCR amplification product obtained in 2-2 was subjected to agarose gel electrophoresis. The specific operation is as follows:

[0155] 1) Preparation of agarose gel: Take 1g of agarose, add 100mL of 1×TBE buffer, heat and dissolve in a microwave oven, take out after the agarose in the solution is completely dissolved, dry it at room temperature to 50-60°C, add 10μL of nucleic acid dye to the solution, mix well, pour the gel into a gel plate, and dry it until the gel is completely solidified;

[0156] 2) Place the prepared gel into an electrophoresis tank filled with 1×TBE buffer, correctly connect the electrophoresis tank and the electrophoresis instrument circuit, and place the sample well of the gel close to the cathode of the electrophoresis tank;

[0157] 3) Take 10 μL of the 2-2 amplified product, mix 10 μL of the sample and 2 μL of 6× loading buffer thoroughly and add them to the spotting wells of the agarose gel (sample and DNA molecular weight standard). After spotting the sample, turn on the electrophoresis instrument and perform electrophoresis at a constant voltage of 120 V for 30 minutes;

[0158] 4) After the electrophoresis is completed, the gel is placed on the stage of the gel imager and an image of the electrophoresis result is obtained under UV mode;

[0159] Analyze the electrophoresis bands, and the results are as follows Figures 1 to 6 As shown, the amplification effect is as follows: the band in the electrophoresis result of the amplified product obtained is a single band with bright brightness, and the band position and band size are confirmed to be consistent with the target band according to the DNA molecular weight standard, which proves that the temperature is the appropriate annealing temperature for the corresponding primer.

[0160] If there are multiple bands in the electrophoresis results, it proves that non-specific amplification products or primer dimers exist in the amplification products, and this temperature cannot be used as a suitable annealing temperature for the corresponding primers.

[0161] Table 10: Amplification effects at different annealing temperatures

[0162]

[0163]

[0164] Result analysis:

[0165] From the experimental results (Table 10), it can be seen that the annealing temperature of primer P04-1 is between 44 and 64°C, and the electrophoresis results of the amplified product have multiple bands, indicating the presence of non-specific amplified products or primer dimers, so the optimal annealing temperature of the primer is not within this temperature range; between 34 to 42°C and 66 to 76°C, the amplified product band is single and consistent with the target band size. Under the same other conditions, the brightness of the electrophoresis band of the amplified product with an annealing temperature of 68°C is the highest, and the amplification effect is the best.

[0166] The annealing temperature of primer P04-2 is between 40 and 66°C, and the electrophoresis results of the amplified product show multiple bands, indicating that nonspecific amplified products or primer dimers are produced. Therefore, the optimal annealing temperature of the primer is not within this temperature range; but between 34 and 38°C, the amplified product band is single and consistent with the size of the target band. Under the same other conditions, the electrophoresis band of the amplified product with an annealing temperature of 38°C has the highest brightness and the best amplification effect.

[0167] The product band amplified by primer P04-3 between 56 and 66°C is single and consistent with the designed target band size. Under the same other conditions, the electrophoresis band of the amplified product with 58°C as the annealing temperature has the highest brightness and the best amplification effect.

[0168] When the annealing temperature of primer P12-1 is between 56 and 66°C, the electrophoresis results of the amplified product show multiple bands, indicating the presence of nonspecific amplified products or primer dimers. When the annealing temperature is higher than 74°C, the electrophoresis results of the amplified product show no bands, so the optimal annealing temperature of the primer is not within this temperature range. When other conditions are the same, the electrophoresis band of the amplified product with an annealing temperature of 68°C has the highest brightness and the best amplification effect.

[0169] The product band amplified by primer P12-2 between 54 and 60°C is single and consistent with the designed target band size. When other conditions are the same, the brightness of the electrophoresis band of the amplified product with 58°C as the annealing temperature is the darkest.

[0170] The primer P12-3 produced a single amplification product at 54°C, while under other conditions, non-specific amplification products or primer dimers were produced.

[0171] in conclusion:

[0172] The optimal annealing temperature of each primer was 68°C for P04-1, 38°C for P04-2, 58°C for P04-3, 68°C for P12-1, 58°C for P12-2, and 54°C for P12-3.

[0173] Example 3 Specific primer screening

[0174] The electrophoresis band of the amplification product mixed with primer P04-2 and template YKYY021P04 is single and is the target band, while the electrophoresis band of the amplification product mixed with template YKYY021P12 is bandless, which is specific and can be used to detect the content of YKYY021P04 in the bivalent mRNA vaccine;

[0175] The electrophoresis band of the amplification product mixed with primer P12-2 and template YKYY021P12 is single and is the target band, while the electrophoresis band of the amplification product mixed with template YKYY021P04 is bandless, which is specific and can be used to detect the content of YKYY021P12 in bivalent mRNA vaccines;

[0176] The mixed amplification products of other primers and two templates showed bands in electrophoresis, which are not specific and cannot be used to detect the content of each component of the bivalent mRNA vaccine.

[0177] In order to accurately determine the content of each component in the bivalent mRNA vaccine, specific primers for each component are required, that is, the primers are mixed with the cDNA products of the corresponding components for qPCR amplification to obtain the target product, and there is no amplification product when mixed with the cDNA products of other components. Therefore, the YKYY021P04 and YKYY021P12 stock solution cDNA products (Example 1) were used as templates for amplification at the optimal annealing temperature of each primer (Example 2);

[0178] Primer P04-1 was mixed with the cDNA products of YKYY021P04 stock solution and YKYY021P12 stock solution (Example 1) respectively;

[0179] Primer P04-2 was mixed with the cDNA products of YKYY021P04 stock solution and YKYY021P12 stock solution (Example 1) respectively;

[0180] Primer P04-3 was mixed with the cDNA products of YKYY021P04 stock solution and YKYY021P12 stock solution (Example 1) respectively;

[0181] Primer P12-1 was mixed with the cDNA products of YKYY021P04 stock solution and YKYY021P12 stock solution (Example 1) respectively;

[0182] Primer P12-2 was mixed with the cDNA products of YKYY021P04 stock solution and YKYY021P12 stock solution (Example 1) respectively;

[0183] Primer P12-3 was mixed with the cDNA products of YKYY021P04 stock solution and YKYY021P12 stock solution (Example 1) respectively;

[0184] The amplified product was subjected to agarose gel electrophoresis. Other reagents and steps were consistent with 2-2 and 2-3 in Example 2. The bands after electrophoresis of the product were analyzed ( Figures 7 to 12 ), the judgment criteria and results are as follows (Table 11):

[0185] Table 11 Banding of cDNA products after electrophoresis --- Judgment criteria

[0186]

[0187] When the primer is directed to one of the mRNAs in the mRNA component (e.g., mRNA 1 ) is shown as a band in the electrophoresis results, and the amplified band is single and consistent with the size of the target band (i.e., the amplified product is single and is the target product); for another mRNA (e.g., mRNA 2 ) electrophoresis results show no band (i.e., no amplification product); then it proves that the primer (e.g., primer 1) is a specific primer.

[0188] Conversely, when the primers are specific for two or more mRNAs in the mRNA component (e.g., mRNA 1 and mRNA 2 ) electrophoresis results all show bands; this proves that the primer (for example, primer 2) is not a specific primer.

[0189] Table 12 Bands of cDNA products after electrophoresis --- Results

[0190]

[0191] Result analysis:

[0192] From the results in Table 12, it can be seen that primer P04-1 was used to amplify the cDNA products of YKYY021P04 and YKYY021P12 as templates under the annealing condition of 68°C, and the amplified products showed bands in the electrophoresis, indicating that primer P04-1 is not specific and is not suitable for quantitative detection of mixed templates YKYY021P04 and YKYY021P12.

[0193] Primer P04-3 used an annealing temperature of 58°C, and the electrophoresis results of the amplified product were similar to those of P04-1, and it was also not suitable for quantitative detection of mixed templates.

[0194] Primer P04-2 was amplified under annealing conditions at 38°C. The amplified band with template YKYY021P04 was single and consistent with the target band size. No band was produced by electrophoresis of the amplified product with template YKYY021P12, indicating that primer P04-2 is suitable as a primer pair for quantitative detection of mixed templates YKYY021P04 and YKYY021P12.

[0195] The annealing temperature of primer P12-1 was 68°C, and the amplified products showed bands in electrophoresis, indicating that primer P12-1 was not suitable for quantitative detection of mixed templates YKYY021P04 and YKYY021P12.

[0196] Primer P12-3 used an annealing temperature of 54°C, and the electrophoresis results of the amplified product were similar to those of P12-1, so it is also not suitable for quantitative detection of mixed templates.

[0197] Primer P12-2 was amplified under annealing conditions at 58°C. The electrophoresis band of the amplified product with template YKYY021P12 was single and the target band; the electrophoresis band of the amplified product with template YKYY021P04 was no band, indicating that primer P12-2 is suitable as a primer pair for quantitative detection of mixed templates YKYY021P04 and YKYY021P12.

[0198] in conclusion:

[0199] Primer P04-2 was mixed with the cDNA product of YKYY021P04 for qPCR amplification, and the amplified product had a single electrophoresis band, which was the target band; when mixed with the cDNA product of YKYY021P12, no amplified product was found. Therefore, P04-2 is a specific primer for YKYY021P04 and can be used to detect the content of YKYY021P04 in a bivalent mRNA vaccine composed of a mixture of YKYY021P04 and YKYY021P12 mRNA;

[0200] When primer P12-2 was mixed with the cDNA product of YKYY021P04, no amplification product was produced; when it was mixed with the cDNA product of YKYY021P12, the amplification product had a single electrophoresis band, which was the target band. Therefore, P12-2 is a specific primer for YKYY021P12 and can be used to detect the content of YKYY021P12.

[0201] When other primers were mixed with the cDNA products of the two original solutions, the amplified products showed amplified product bands when subjected to electrophoresis. These bands are not specific and cannot be used to detect the content of the components in the bivalent mRNA vaccine.

[0202] Example 4 qPCR (quantitative analysis)

[0203] qPCR quantitative detection was performed using primers P04-2 and P12-2, and the contents of YKYY021P04 and YKYY021P12 in the bivalent mRNA vaccine were measured to be 47.302 ng / μL and 52.902 ng / μL, respectively.

[0204] 4-1.qPCR amplification:

[0205] According to Example 3, primers suitable for detecting the content of the solution to be tested were screened out, as shown in the following table:

[0206] Table 13 Primer information

[0207]

[0208] The obtained cDNA products were diluted respectively;

[0209] The cDNA products of the YKYY021P04 gradient standard solution and the test solution (Example 1) were diluted and mixed with primer P04-2 respectively;

[0210] The cDNA products of the YKYY021P12 gradient standard solution and the test solution (Example 1) were diluted and mixed with primer P12-2 respectively;

[0211] The specific qPCR reaction system is shown in Table 14, and qPCR amplification is performed under the conditions in Tables 15 and 16.

[0212] Table 14: qPCR reaction system (20 μL)

[0213]

[0214] Table 15: qPCR reaction conditions: Primer P04-2

[0215]

[0216] Table 16: qPCR reaction conditions: Primer P12-2

[0217]

[0218] 4-2. Standard curve:

[0219] The CT detection value of the obtained standard solution is used as the ordinate and the logarithm of the theoretical concentration is used as the abscissa to draw a standard curve. The result is as follows: Fig. 20 and Fig.21 The amplification efficiency was calculated according to the formula E% (amplification efficiency) = 10^(-1 / k), where k is the slope of the standard curve.

[0220] Table 17 Standard curves of each component

[0221] Components Standard curve R2 Amplification efficiency YKYY021P04 y=-3.2468x+26.802 0.9975 103.23％ YKYY021P12 y=-3.2846x+28.659 0.9961 101.58％

[0222] 4-3. Determination of content:

[0223] The CT detection values ​​of the test solutions were respectively substituted into the above standard curves to calculate the contents of the components YKYY021P04 and YKYY021P12 in the test solutions. The results are shown in Table 17.

[0224] Table 18 Results of determination of the content of each component in the bivalent mRNA vaccine

[0225] Components CT value Standard curve Content (ng / μL) YKYY021P04 21.364 y=-3.2468x+26.802 47.302 YKYY021P12 22.872 y=-3.2951x+28.551 52.902

[0226] in conclusion:

[0227] In the bivalent mRNA vaccine, the contents of YKYY021P04 and YKYY021P12 were 47.302 ng / μL and 52.902 ng / μL, respectively.

[0228] Example 5 Specificity Test

[0229] Primer P04-2 was used to perform qPCR amplification with cDNA products of YKYY021P04 and the test solution (mixed YKYY021P04 and YKYY021P12). The peak shape and Tm value of the melting curve were single, and the electrophoresis band of the amplified product was single and was the target band; and there was no band in the electrophoresis with the amplified product of YKYY021P12; indicating that the qPCR amplification system was specific, the amplified product was single and accurate, P04-2 was a specific primer for YKYY021P04, and the detection method had good specificity;

[0230] Primer P12-2 was used for qPCR amplification with YKYY021P12 and the cDNA product of the test solution. The peak shape and Tm value of the melting curve were single, the electrophoresis band of the amplified product was single, and it was the target band; and there was no band when electrophoresed with the amplified product of YKYY021P04; this showed that the qPCR amplification system was specific, the amplified product was single and accurate, P12-2 was a specific primer for YKYY021P12, and the detection method had good specificity.

[0231] Primer P04-2 of Example 4 was used to perform qPCR amplification using the cDNA products of the YKYY021P04 stock solution, the YKYY021P12 stock solution and the solution to be tested after reverse transcription (Example 1) as templates;

[0232] Primer P12-2 of Example 4 was used to perform qPCR amplification using the cDNA products of the YKYY021P04 stock solution, the YKYY021P12 stock solution and the solution to be tested after reverse transcription (Example 1) as templates;

[0233] Other reagents and methods are consistent with those in 4-1 and 4-2 of Example 4. The melting curves were analyzed ( Figures 13-16 ); and the amplified product was subjected to agarose gel electrophoresis, and other reagents and methods were consistent with 2-3 in Example 2, and the electrophoresis bands (such as Fig.17 and 18 shown).

[0234] Table 19cDNA product melting curve and bands after electrophoresis

[0235]

[0236] in conclusion:

[0237] From the results in Table 19, it can be seen that P04-2 was used as a primer, and ①YKYY021P04, ②YKYY021P12 and ③ the cDNA products of the test solution (mixed with YKYY021P04 and YKYY021P12) were used as templates for qPCR amplification, wherein the Tm of the melting curves of ① and ③ were both 86.772, and the peak shape and Tm value were single, and the electrophoresis band of the amplified product was single, and it was the target band; indicating that the qPCR amplification system was specific, and the amplified product was single and accurate; and the electrophoresis of ② amplified product had no band; indicating that P04-2 was a specific primer for YKYY021P04, and the detection method had good specificity.

[0238] Primer P12-2 performed qPCR amplification with cDNA products of ①YKYY021P04, ②YKYY021P12 and ③ test solution (mixture of YKYY021P04 and YKYY021P12) as templates, respectively. The Tm of melting curves of ② and ③ were both 88.073, and the peak shape and Tm value were single. The electrophoresis band of the amplified product was single and the target band, indicating that the qPCR amplification system was specific and the amplified product was single and accurate. At the same time, the amplified product of ① showed no band in the electrophoresis, indicating that P12-2 was a specific primer for YKYY021P12 and the detection method had good specificity.

[0239] Example 6 Accuracy Test

[0240] The recoveries of YKYY021P04 and YKYY021P12 in the bivalent vaccine were 110.26% and 107.34% respectively (between 50% and 150% of the pharmacopoeia PCR quantitative recovery limit standard), and the recovery rate and accuracy of the detection method were good.

[0241] Accuracy test sample: YKYY021P04 and YKYY021P12 stock solutions were mixed to prepare accuracy test samples; the actual concentrations were 150ng / μL and 150ng / μL respectively;

[0242] The test solution was reverse transcribed using the method of Example 1 to obtain cDNA products, and qPCR quantitative detection was performed using the method of Example 4. The measured concentrations were 165.39 ng / μL and 161.01 ng / μL, respectively.

[0243] Recovery rate (%) = (measured concentration ÷ actual concentration) × 100%

[0244] The calculated recoveries were 110.26% and 107.34%, respectively.

[0245] in conclusion:

[0246] The components in the bivalent vaccine were quantitatively analyzed by fitting the standard curve. The recoveries of YKYY021P04 and YKYY021P12 template detection were 110.26% and 107.34% respectively (between 50% and 150% of the pharmacopoeia PCR quantitative recovery limit standard), which were close to 100%, indicating that the recovery rate and accuracy of the detection method were good.

[0247] Example 7 Primer sequence optimization

[0248] The linear correlation coefficient R of primer P04-2 2 The slope was 0.996, the slope was -3.407, and the amplification efficiency was 96.56%, which met the quantitative requirements of the standard curve (R 2 ≥0.98, slope -3.1 to -3.8, i.e., amplification efficiency is 83.30 to 110.17%), which can accurately quantify YKYY021P04;

[0249] The linear correlation coefficient of primer P12-2 was 0.998, the slope was -3.289, and the amplification efficiency was 101.44%, which met the quantitative requirements of the standard curve and could accurately quantify YKYY021P12;

[0250] The slopes and amplification efficiencies of the forward primer P04-2-02 and the backward primer P04-2-03 of primer P04-2, and the forward primer P12-2-02 and the backward primer P12-2-03 of primer P12-2 all exceed the quantitative requirements of the standard curve, do not meet the requirements, and cannot be used for quantitative detection.

[0251] 7-1. Primer sequence:

[0252] In this example, the primer sequences in the qPCR reaction system are optimized, and the primers (P04-2 and P12-2) in Example 4 are moved forward or backward by 2 nucleotides, as follows:

[0253] Table 20: Primer sequences at different sites

[0254]

[0255] 7-2. Experimental steps and results:

[0256] Primers P04-2, P04-2-02 and P04-2-03 were used as templates for the cDNA product (Example 1) reverse transcribed from the YKYY021P04 gradient standard solution; primers P12-2, P12-2-02 and P12-2-03 were used as templates for amplification to establish a standard curve. Other reagents and methods were consistent with 4-1 to 4-2 in Example 4.

[0257] The amplification efficiency and linear correlation coefficient of the standard curves fitted by different primers were analyzed, and the results are shown in Table 21:

[0258] Table 21: Amplification curves of primers at different positions

[0259] Primer name <![CDATA[R 2 ]]> Slope Amplification efficiency % P04-2 0.996 -3.407 96.56％ P04-2-02 0.992 -3.025 114.08％ P04-2-03 0.989 -3.4002 115.33％ P12-2 0.998 -3.289 101.44％ P12-2-02 0.990 -2.9812 116.50％ P12-2-03 0.986 -3.089 110.71％

[0260] in conclusion:

[0261] From the results, it can be seen that the linear correlation coefficient R of the primer P04-2 selected in Example 4 is compared with the primers with the corresponding base sequences shifted forward or backward. 2 The slope was 0.996, the slope was -3.407, and the amplification efficiency was 96.56%, which met the quantitative requirements of the standard curve (R 2 ≥0.98, slope -3.1 to -3.8, i.e. amplification efficiency is 83.30 to 110.17%); the forward primer P04-2-02 and the backward primer P04-2-03, the slopes are -3.025 and -3.4002, respectively, and the amplification efficiencies are 114.08% and 115.33%, respectively, which exceed the quantitative requirements of the standard curve, do not meet the requirements, and cannot be used for quantitative detection;

[0262] The linear correlation coefficient of the primer P12-2 selected in Example 4 is 0.998, the slope is -3.289, and the amplification efficiency is 101.44%, which meets the quantitative requirements of the standard curve; the forward primers P12-2-02 and P12-2-03 have slopes of -2.9812 and -3.089, respectively, and amplification efficiencies of 116.50% and 110.71%, respectively, which exceed the quantitative requirements of the standard curve, do not meet the requirements, and cannot be used for quantitative detection.

[0263] Since the higher the linear correlation coefficient, the closer the amplification efficiency is to 100%, the better the quantitative effect is. Therefore, the primer pair P04-2 and P12-2 in Example 4 is indeed the best primer pair.

[0264] Example 8 Primer concentration in qPCR reaction

[0265] When the primer concentration was 0.2 μM, the correlation coefficient and amplification efficiency were optimal, and the quantitative accuracy was high.

[0266] In the qPCR reaction system, three different primer concentrations of 0.1 μM, 0.2 μM, and 0.3 μM were selected for screening.

[0267] Primer P04-2 uses the cDNA product (Example 1) reverse transcribed from the YKYY021P04 gradient standard solution as a template;

[0268] Primer P12-2 uses the cDNA product (Example 1) reverse transcribed from the YKYY021P12 gradient standard solution as a template;

[0269] Amplification was performed in reaction systems with primer concentrations of 0.1 μM, 0.2 μM, and 0.3 μM, respectively, to establish a standard curve;

[0270] Other reagents and methods are the same as those in 4-1 to 4-2 of Example 4.

[0271] The reaction system is as follows:

[0272] Table 22 Amount of each reagent in different reaction systems (μL)

[0273]

[0274] Obtain the linear correlation coefficient R of the standard curve 2 And the amplification efficiency is as follows:

[0275] Table 23 Amplification under different primer concentrations

[0276]

[0277] in conclusion:

[0278] The logarithmic value of the theoretical concentration after amplification is the horizontal coordinate of the standard curve, and the average CT value detected at each point of the standard curve is used as the vertical coordinate for linear fitting. When the primer concentration is 0.2 μM, the linear correlation coefficient is the highest, which is 0.9975 and 0.9961 respectively; the amplification efficiency is 97.74% and 101.57% respectively, 0.1 μM is 95.25% and 105.77%, 0.3 μM is 105.11% and 110.51%, and 0.2 μM is closer to 100%. The higher the linear correlation coefficient, the closer the amplification efficiency is to 100%, and the best quantitative effect. Therefore, in the qPCR reaction, when the primer concentration is 0.2 μM (Example 4), the results of the correlation coefficient and amplification efficiency are optimal, and the quantitative accuracy is high.

[0279] Example 9 qPCR reaction system

[0280] When the qPCR system was 20 μL, the amplification efficiency after analysis was optimal.

[0281] Three different reaction systems (three commonly used systems) of 10 μL, 20 μL and 50 μL were selected for screening.

[0282] Primer P04-2 uses the cDNA product (Example 1) reverse transcribed from the YKYY021P04 gradient standard solution as a template;

[0283] Primer P12-2 uses the cDNA product (Example 1) reverse transcribed from the YKYY021P12 gradient standard solution as a template;

[0284] Amplification was performed in 10 μL, 20 μL and 50 μL reaction systems respectively to establish a standard curve;

[0285] Other reagents and methods are the same as those in 4-1 to 4-2 of Example 4.

[0286] The reaction system is as follows:

[0287] Table 24 Amount of each reagent in different reaction systems (μL)

[0288]

[0289]

[0290] Obtain the linear correlation coefficient R of the standard curve 2 And the amplification efficiency is as follows:

[0291] Table 25 Amplification under different systems

[0292]

[0293] in conclusion:

[0294] When the qPCR system is 20 μL, the linear correlation coefficients are 0.9975 and 0.9961, respectively, which are higher than the reaction system of 10 μL (0.9951 and 0.9925) and 50 μL (0.9947 and 0.9905), and the amplification efficiency is 97.74% and 101.57%, respectively, which is closer to 100% than the reaction system of 10 μL (91.89% and 109.49%) and 50 μL (102.67% and 107.14%). Therefore, when the qPCR system is 20 μL (Example 4), the result of the amplification efficiency after analysis is optimal.

[0295] Example 10 Standard Curve

[0296] The concentration of the standard solution was in the range of 10 to 1500 ng / μL, and the linear relationship and amplification efficiency of the standard curve were good with high accuracy.

[0297] 10-1. Standard curve range

[0298] According to the concentration of the vaccine stock solution and the requirements of the reverse transcription kit for samples, the range of the standard curve was preliminarily determined to be 0-2000 ng / μL. The YKYY021P04 mRNA stock solution with known concentration was diluted to eight concentrations of 1, 2, 10, 20, 100, 200, 1000, and 2000 ng / μL to explore the range of the standard curve.

[0299] The mRNA solution was reverse transcribed to obtain reverse transcribed cDNA, and an amplification reaction was performed to establish a standard curve. Other reagents and steps were consistent with 1-5 to 1-7 in Example 1.

[0300] The values ​​obtained for each point of the standard curve are as follows:

[0301] Table 26 CT value and theoretical concentration at each point

[0302]

[0303]

[0304] The CT values ​​of STD2-8 and the logarithm of the theoretical concentration were fitted linearly to establish a standard curve ( Fig.19 ), the result is as follows:

[0305] Table 27 Standard curve

[0306] Components Standard curve equation <![CDATA[R 2 ]]> STD2~8 y=-3.3966x+23.845 0.9941

[0307] in conclusion:

[0308] The point detection CT value of STD1 (theoretical concentration 2000ng / μL) (mean 14.559) is larger than the CT value of STD2 (1000ng / μL) (mean 14.191), indicating that the corresponding concentration of STD1 has a greater impact on the experimental detection results, and the concentration of 2000ng / μL cannot be used to establish a standard curve.

[0309] STD2-8 (concentration of 1-1000 ng / μL) was fitted, and the correlation coefficient of the fitting curve was 0.9941, which was significantly greater than 0.98, and the slope was -3.3966, between -3.8 and -3.1, indicating that the standard curve of the mRNA stock solution was good within the concentration range of 1-1000 ng / μL (according to the requirements of the pharmacopoeia for the standard curve), and the sample could be accurately quantified.

[0310] 10-2. Establishment of standard curve

[0311] Taking into account the needs of sample testing and the results of the above tests, the standard curve was set to eight points: 10, 50, 100, 200, 300, 500, 1000, and 1500 ng / μL. The 7-1 experiment was repeated with YKYY021P04 and YKYY021P12 stock solutions. The standard curve ( Fig. 20 and 21 )The fitting results are as follows:

[0312] Table 28 Standard curve

[0313] Components Standard curve equation <![CDATA[R 2 ]]> YKYY021P04 y=-3.3773x+29.67 0.9975 YKYY021P12 y=-3.2849x+28.649 0.9961

[0314] in conclusion:

[0315] From the fitting results, it can be seen that P04-2 and P12-2 were used as primers, and the YKYY021P04 and YKYY021P12 stock solutions were diluted to eight concentrations of 10, 50, 100, 200, 300, 500, 1000, and 1500 ng / μL to establish a standard curve, and the linear correlation coefficients were 0.9975 and 0.9961, respectively, which were significantly greater than 0.98, and the slopes of the curves were -3.3773 and -3.2849, respectively, which were between -3.8 and -3.1, indicating that the concentration of the standard solution was within the above range (Example 1), and the linear relationship and amplification efficiency of the standard curve were good with high accuracy.

Claims

1. A method for detecting components in a bivalent mRNA vaccine using primers, comprising the following steps: 1) preparing a test solution of a bivalent mRNA vaccine; the bivalent mRNA vaccine comprises mRNA with nucleotide sequences numbered as SEQ ID NO: 1 and SEQ ID NO: 2; 2) Preparation of standard solutions: using the standard products of each component mRNA in the bivalent mRNA vaccine to prepare a series of standard solutions with different gradient concentrations; 3) Reverse Transcription-PCR: Reverse transcribe the mRNA in the test solution in step 1) and the standard solution in step 2) to obtain cDNA samples; 4) qPCR detection: primers are added to a series of cDNA samples obtained from a series of standard solutions and a cDNA sample obtained from a test solution, and qPCR detection is performed after amplification; wherein: The primers include primer P04-2 and primer P12-2; The upstream primer sequence number of the primer P04-2 is SEQ ID NO: 3, and the downstream primer sequence number is SEQ ID NO: 4; The upstream primer sequence number of the primer P12-2 is SEQ ID NO: 5, and the downstream primer sequence number is SEQ ID NO: 6; The qPCR detection method is: i) preparing a solution for a qPCR reaction system; The primer concentration in the qPCR reaction system solution in step i) is 0.1-0.3 μM; The reaction system in the qPCR reaction system solution in step i) is 10 to 50 μL; ii) pre-denaturing, denaturing, annealing and melting the solution; The annealing temperature of the reaction system of the primer P04-2 is 34-39°C; The annealing temperature of the reaction system of the primer P12-2 is 54-60°C; 5) Using the series of cDNA samples obtained from the series of standard solutions, a CT value-concentration standard curve is obtained; using the CT value of the cDNA sample obtained from the test solution, the mRNA concentration in the test solution is quantitatively detected.

2. The method according to claim 1, wherein: The annealing temperature of the reaction system of the primer P04-2 is 38°C; The annealing temperature of the reaction system of the primer P12-2 is 58°C.

3. The method according to claim 1 or 2, wherein: The annealing time is 10 to 40 seconds.

4. The method according to claim 3, wherein: The annealing time is 30s.

5. The method according to claim 1 or 2, wherein: The annealing cycle number is 30 to 50 times.

6. The method according to claim 5, wherein: The annealing cycle number is 40 times.

7. The method according to claim 1 or 2, wherein: The condition of the pre-denaturation in step ii) of the step 4) qPCR detection is 95° C. for 30 s; and the number of cycles is 1.

8. The method according to claim 1 or 2, wherein: The denaturation condition in step ii) of step 4) qPCR detection is 95° C. for 10 s; and the number of cycles is 40 times.

9. The method according to claim 1 or 2, wherein: The melting process in step ii) of step 4) qPCR detection is high temperature melting at 90-100° C. for 10-20 seconds, then at annealing temperature for 30-60 seconds, and finally a second high temperature melting at 90-100° C. for 10-20 seconds.

10. The method according to claim 1 or 2, wherein: The primer concentration in the qPCR reaction system solution in step i) of the step 4) qPCR detection is 0.2 μM.

11. The method according to claim 1 or 2, wherein: The reaction system in the qPCR reaction system solution in step i) of the step 4) qPCR detection is 20 μL.

12. The method according to claim 1 or 2, wherein: The mRNA is encapsulated by LNP to obtain mRNA-LNP.

13. A kit for detecting a bivalent mRNA vaccine, the kit comprising primers P04-2 and P12-2, and a solution of a qPCR reaction system; the bivalent mRNA vaccine comprises mRNA having nucleotide sequences numbered SEQ ID NO: 1 and SEQ ID NO: 2; wherein: The upstream primer sequence number of the primer P04-2 is SEQ ID NO: 3, and the downstream primer sequence number is SEQ ID NO: 4; The upstream primer sequence number of the primer P12-2 is SEQ ID NO: 5, and the downstream primer sequence number is SEQ ID NO: 6; The primer concentration in the qPCR reaction system solution is 0.1-0.3 μM; The reaction system in the qPCR reaction system solution is 10 to 50 μL.

14. The kit according to claim 13, wherein The primer concentration in the qPCR reaction system solution was 0.2 μM.

15. The kit according to claim 13 or 14, wherein The reaction system in the qPCR reaction system solution is 20 μL.

16. The kit according to claim 13 or 14, wherein The mRNA is encapsulated by LNP to obtain mRNA-LNP.

Images