Method for detecting mRNA copy number of novel crown RSV combined vaccine

Abstract

The invention discloses a method for detecting the copy number of mRNA (messenger ribonucleic acid) of a novel crown RSV (respiratory syncytial virus) combined vaccine. A primer probe combination adopted by the detection method consists of primer pairs as shown in SEQ ID NO.2 and SEQ ID NO.3 and a probe as shown in SEQ ID NO.4, the 5'end of the probe is marked with a fluorescence reporter group, and the 3 'end of the probe is marked with a fluorescence quenching group. According to the detection method, correlation coefficients of equations between the Cq value and the template copy number are both larger than 0.99, amplification efficiency is both 90%-110%, and specificity and accuracy are good; the method can be used for quality control processes such as release detection in the combined vaccine development process and toxicological experiments, and has important significance and value for research and development and quality control of combined vaccines.

Description

[0001] The present invention claims priority to Chinese patent application No. 202410307151.5 filed with the Patent Office of China on March 18, 2024, with application number 202410307151.5 and invention name “Mutants, nucleic acids and mRNA vaccines of RSV pre-fusion F protein”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present application belongs to the field of vaccine detection technology, and specifically relates to a method for detecting the mRNA copy number of a new coronavirus RSV combined vaccine. Background Art

[0003] As public health restrictions are lifted, RSV and SARS-CoV-2 co-infections are becoming more common. Recent studies have shown that the proportion of RSV co-infection in people infected with SARS-CoV-2 in the UK and the US is about 3%. In Brazil, the co-infection rate in children under 2 years old is as high as 18.7%, and in Iran, 9.7% of children who died from SARS-CoV-2 were co-infected with RSV. It is worth noting that co-infection may lead to more severe illness compared with single infection.

[0004] In order to respond to the health threats of RSV virus and SARS-CoV-2 virus to human beings, RSV & SARS-CoV-2 mRNA combination vaccine came into being. In the process of research and development and production release of mRNA combination vaccine, the content of mRNA vaccine is one of the key indicators that determine its effectiveness and safety, and it 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 vaccine, but RiboGreen and ultraviolet spectroscopy can only detect the total content of mRNA in mRNA vaccine, and cannot distinguish and accurately quantify the mRNA of different components in mRNA combination vaccine, and are easily affected by impurities in the sample; although dPCR (digital PCR) technology can accurately quantify the components of bivalent mRNA vaccine, it is limited by its high system cost, limited throughput, cumbersome operation and high system complexity; although the method of nucleic acid chip has also been reported, in addition to designing specific probes, it is also necessary to design nucleic acids or antibodies that bind to fluorescence, and specific instrument consumables are required, and the operation is complicated and cumbersome. Summary of the invention

[0005] The purpose of this application is to provide a method for detecting the copy number of mRNA of the new crown RSV combined vaccine, so as to solve the technical problems in the detection methods in the prior art that RiboGreen and ultraviolet spectroscopy are unable to distinguish and accurately quantify the mRNA of different components in the new crown RSV mRNA combined vaccine, and that dPCR and nucleic acid chips are high in cost and cumbersome in operation.

[0006] In order to achieve the above-mentioned objectives, the first aspect of the present application provides a primer-probe combination for detecting the copy number of mRNA, wherein the mRNA is used to encode RSV pre-fusion F protein, and the sequence is shown in SEQ ID NO.1. The primer-probe combination consists of a primer pair shown in SEQ ID NO.2 and 3 and a probe shown in SEQ ID NO.4, and the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

[0007] In one or more embodiments, the fluorescent reporter group is selected from FAM, HEX, VIC, ROX or Cy5, and the fluorescent quencher group is selected from TAMRA, MGB, BHQ1, JOE, BHQ2 or BHQ3.

[0008] In one or more embodiments, the 5' end of the probe is labeled with FAM, and the 3' end is labeled with TAMRA.

[0009] In order to achieve the above-mentioned purpose, the second aspect of the present application provides an application of a primer-probe combination as described in any of the above embodiments in detecting the number of mRNA copies encoding RSV pre-fusion F protein in the new crown RSV combined vaccine.

[0010] In order to achieve the above-mentioned purpose, the third aspect of the present application provides a primer-probe combination as described in any of the above embodiments for detecting the intracellular half-life of mRNA of RSV pre-fusion F protein in the new crown RSV combined vaccine.

[0011] In one or more embodiments, the COVID-19 RSV combined vaccine comprises:

[0012] mRNA encoding RSV prefusion F protein as shown in SEQ ID NO.1;

[0013] A nucleotide sequence encoding a recombinant spike protein, wherein the recombinant spike protein is derived from a novel coronavirus spike protein and has an S-2P site mutation relative to a wild-type novel coronavirus spike protein;

[0014] Vaccine carrier and excipients, wherein the vaccine carrier is lipid nanoparticles.

[0015] In order to achieve the above objectives, the fourth aspect of the present application provides a method for detecting the copy number of mRNA encoding RSV pre-fusion F protein in a new crown RSV combined vaccine, wherein the new crown RSV combined vaccine comprises:

[0016] mRNA encoding RSV prefusion F protein as shown in SEQ ID NO.1;

[0017] A nucleotide sequence encoding a recombinant spike protein, wherein the recombinant spike protein is derived from a novel coronavirus spike protein and has an S-2P site mutation relative to a wild-type novel coronavirus spike protein;

[0018] Vaccine carriers and excipients, wherein the vaccine carrier is lipid nanoparticles;

[0019] The detection method comprises:

[0020] After diluting the RSV plasmid standard to different standard concentrations, the plasmids of different standard concentrations are used as templates, and a PCR reaction is performed in combination with the primer-probe combination described in any of the above embodiments to detect the sample threshold Cq, and a standard curve of the sample threshold and the mRNA concentration is prepared;

[0021] The membrane of the COVID-19 RSV combined vaccine is broken and reverse transcribed into cDNA. Using cDNA as a template, a PCR reaction is carried out in combination with the primer-probe combination described in any one of claims 1 to 3. The sample threshold Cq is detected and substituted into the standard curve to obtain the copy number of preF protein mRNA in the COVID-19 RSV combined vaccine.

[0022] In order to achieve the above objectives, the fifth aspect of the present application provides a method for detecting the half-life of mRNA encoding RSV pre-fusion F protein in cells in a new crown RSV combined vaccine, wherein the new crown RSV combined vaccine comprises:

[0023] mRNA encoding RSV prefusion F protein as shown in SEQ ID NO.1;

[0024] A nucleotide sequence encoding a recombinant spike protein, wherein the recombinant spike protein is derived from a novel coronavirus spike protein and has an S-2P site mutation relative to a wild-type novel coronavirus spike protein;

[0025] Vaccine carriers and excipients, wherein the vaccine carrier is lipid nanoparticles;

[0026] The detection method comprises:

[0027] After diluting the RSV plasmid standard to different standard concentrations, the plasmids of different standard concentrations are used as templates, and a PCR reaction is performed in combination with the primer-probe combination described in any of the above embodiments to detect the sample threshold Cq, and a standard curve of the sample threshold and the initial mRNA concentration is prepared;

[0028] The COVID-19 RSV combination vaccine is incubated with the target cells, the cells are collected at different times after incubation and total RNA is extracted, the total RNA is reverse transcribed into cDNA, and a PCR reaction is performed using the cDNA as a template in combination with the primer-probe combination described in any one of claims 1 to 3, the sample threshold Cq is detected, and the standard curve is substituted to obtain the copy number of the preF protein mRNA of the COVID-19 RSV combination vaccine after incubation of the target cells for different times, and the half-life of the preF protein mRNA in the COVID-19 RSV combination vaccine in the target cells is obtained.

[0029] In order to achieve the above-mentioned purpose, the sixth aspect of the present application provides a kit for detecting the copy number of mRNA encoding RSV pre-fusion F protein in the new crown RSV combined vaccine, wherein the new crown RSV combined vaccine comprises:

[0030] mRNA encoding RSVpreF protein as shown in SEQ ID NO.1;

[0031] A nucleotide sequence mRNA encoding a recombinant spike protein, wherein the recombinant spike protein is derived from a novel coronavirus spike protein and has an S-2P site mutation relative to a wild-type novel coronavirus spike protein;

[0032] Vaccine carriers and excipients, wherein the vaccine carrier is lipid nanoparticles;

[0033] The kit comprises:

[0034] The primer-probe combination described in any one of the above embodiments;

[0035] Reverse transcription reagents;

[0036] Fluorescence quantitative PCR reagents.

[0037] In one or more embodiments, it further includes:

[0038] RSV plasmid standards;

[0039] RNA extraction reagents.

[0040] Different from the prior art, the beneficial effects of this application are:

[0041] This application successfully established a method for detecting the copy number of RSV preF mRNA in the RSV & SARS-CoV-2 mRNA combined vaccine. The correlation coefficient (R2) of the equation between the Cq value and the template copy number of this method was greater than 0.99, the amplification efficiency was between 90% and 110%, and the specificity and accuracy were good;

[0042] The detection method of the present application designs 3 sets of primers through sequence optimization, which can be used for release testing and other quality control processes and toxicology experiments during the development of RSV & SARS-CoV-2 mRNA combined vaccines, such as the tissue distribution evaluation of mRNA vaccines in small animals, etc., which is of great significance and value to the research and development and quality control of RSV & SARS-CoV-2 mRNA combined vaccines; and experiments have shown that compared with the other two groups, the primer-probe combination RSV1 has better detection effect, high amplification efficiency, high detection sensitivity, and is more conducive to the detection of the copy number of RSV & SARS-CoV-2 mRNA combined vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 It is the fluorescence quantitative PCR amplification curve of the plasmid standard combined with the primer-probe combination RSV1 in Example 2 of the present application;

[0045] Figure 2 It is the standard curve for RSV Pre F gene detection using the primer-probe combination RSV1 in Example 2 of the present application;

[0046] Figure 3 It is the fluorescence quantitative PCR amplification curve of the plasmid standard combined with the primer-probe combination RSV3 in Example 2 of the present application;

[0047] Figure 4 It is the standard curve for RSV Pre F gene detection using primer-probe combination RSV3 in Example 2 of the present application;

[0048] Figure 5 This is the cellular half-life curve of RSV preF mRNA in the combined vaccine of Example 3 of the present application. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work should fall within the scope of protection of the present application.

[0050] In order to detect the effectiveness of the COVID-19 RSV combined vaccine developed by the applicant, it is necessary to accurately measure the content of each mRNA in the vaccine. Due to the diversity of mRNA in the combined vaccine, it is urgent to establish a detection method with good specificity, accuracy and sensitivity.

[0051] To this end, the applicant designed specific primers and probes for the PreF mRNA component in the mRNA combination vaccine of the S protein of the Omicron variant EG5.1 of the new coronavirus and the pre-fusion F protein (hereinafter referred to as preF protein) of the RSV virus, and detected the content of the Pre F protein mNA component by the RT-qPCR method.

[0052] The sources of the following experimental materials and reagents are shown in the following table:

[0053] Table 1 Sources of experimental materials and reagents

[0054]

[0055]

[0056] Example 1: Primer and probe design

[0057] This application is for a SARS-CoV-2 & RSV mRNA combined vaccine (for specific vaccine information, please refer to the prior application with application number 2024103071515). The combined vaccine includes mRNA for encoding the preF protein of the RSV virus, and its sequence is shown in SEQ ID NO.1.

[0058] The combination vaccine also includes mRNA for encoding a recombinant spike protein, whose sequence is shown in SEQ ID NO.5. The recombinant spike protein is derived from the new coronavirus spike protein and has an S-2P site mutation relative to the wild-type new coronavirus spike protein.

[0059] In this application, three pairs of specific primer probe combinations were designed for the above-mentioned mRNA sequence encoding preF protein using the design website https: / / www.genscript.com.cn / tools / real-time-pcr-taqman-primer-design-tool. The specific primer probe sequences are shown in Table 2 below, and the primer probe combinations RSV-F1, RSV-R1, RSV-P1 and RSV-F3, RSV-R3, RSV-P3 in Table 2 were selected to further verify their effects. They were synthesized by GenScript Biotech Co., Ltd. and the primer probe combination RSV-F1, RSV-R1, RSV-P1 was named RSV1, and the primer probe combination RSV-F3, RSV-R3, RSV-P3 was named RSV3.

[0060] It should be noted that in the primer-probe combination of Example 1, the 5' end of the probe is labeled with FAM, and the 3' end is labeled with TAMRA; in other embodiments, the 5' end of the probe may also be labeled with other fluorescent reporter groups, such as HEX, VIC, ROX, Cy5, etc., and the 3' end of the probe may also be labeled with other fluorescent quenching groups, such as MGB, BHQ1, JOE, BHQ2, BHQ3, etc.

[0061] Use TE solution to prepare the above primers and probes into 10 μM working solutions for use. The preparation process is as follows: (1) Preparation of 1×TE diluent: take 500 μL 20×TE buffer, add 9.5 mL sterile enzyme-free water and mix well, and use it immediately; (2) Centrifuge the primer and probe dry powder at 12000 rpm for 1 min, then add 250 μL 1×TE solution to the tube to dissolve it into 10 μM storage solution, package it at 25 μL / tube, and store it at -20°C. The shelf life is 6 months.

[0062] Table 2 Primer probe combinations

[0063]

[0064]

[0065] Example 2: Standard curve establishment

[0066] (1) Preparation of plasmid standards

[0067] The RSV plasmid glycerol bacteria stored in the laboratory was taken out from -80°C, the RSV plasmid included mRNA (SEQ ID NO.1) for encoding the preF protein of the RSV virus, which was made in the laboratory; after thawing at room temperature, 50 μL was inoculated into 5 mL of LB medium containing 50 mg / L kanamycin, and cultured at 30°C and 220 rpm for 7 h to prepare an activated seed solution;

[0068] Transfer 1 mL of seed solution to 1 L of LB medium containing 50 mg / L kanamycin, and culture at 30°C and 220 rpm for 16 h. Then, collect E. coli cells by centrifugation at 4°C and 5000 rpm for 10 min. Extract plasmid DNA with NucleoBond Xtra Maxi Plus kit, and detect its concentration and purity with NanoDrop ONE spectrophotometer. The A260 / A280 ratio of plasmid DNA is in the range of 1.8 to 2.0. Divide the plasmid DNA into small portions and store them at -80°C for use as plasmid standards.

[0069] The copy number of the proposed plasmid standard was calculated according to the following formula: Plasmid copy number (copies / μL) = C × 10-9 ×6.02×10 23 / (M×660), where C is the plasmid concentration (ng / μL) and M is the number of bases in the plasmid. Based on the calculated results, the plasmid standard was diluted 10-fold to a copy number concentration of 1×10 8 , 3×10 7 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 The obtained standards of different concentrations were used to prepare the standard curve.

[0070] (2) Establishment of RSV1 standard curve

[0071] The plasmid standards of different concentrations obtained in step (1) were used as templates, and fluorescence quantitative PCR was performed in combination with the primer-probe combination RSV1 of Example 1. Three parallels were set for each PCR sample, and the experiment was repeated 3 times to obtain Figure 1 , the specific experimental data are shown in Table 3 below, Figure 1 This is the fluorescence quantitative PCR amplification curve of the plasmid standard combined with the primer-probe combination RSV1 in Example 2 of the present application.

[0072] Table 3 Amplification data of different concentrations of plasmid combined with RSV1

[0073]

[0074]

[0075] The standard curve of RSV Pre F gene detection was drawn based on the Cq value of the standard DNA solution PCR reaction and the logarithm of the initial template copy number. Figure 2 , Figure 2 This is the standard curve for RSV Pre F gene detection using the primer-probe combination RSV1 in Example 2 of the present application.

[0076] Quantitative PCR reaction system and reaction conditions:

[0077] The reaction system is 25 μL: 0.5 μL each of forward and reverse primers, 0.25 μL of fluorescent probe, 12.5 μL of Premix Ex TaqTM, 1 μL of reverse transcription product diluted 10 to 10,000 times, and 10.25 μL of water;

[0078] The reaction conditions were: 95°C for 5 s, 60°C for 30 s, for a total of 40 cycles.

[0079] Please refer to Table 3. Figure 1 and Figure 2 The results showed that the standard deviation of the Cq value in the three repeated experiments of the primer-probe combination RSV1 was less than 0.5, indicating that the detection data was reliable. The correlation coefficient (R2) of the equation between the detection system Cq value and the template copy number was greater than 0.99, indicating a good linear relationship. The amplification efficiency calculated based on the slope was between 90% and 110%. The correlation coefficient and slope results met the recommended standards of the GCC industry white paper. Therefore, it was shown that the primer-probe combination RSV1 can be used to detect the copy number of mRNA encoding RSV preF protein.

[0080] (3) Establishment of RSV3 standard curve

[0081] The same method as step (2) was used to perform fluorescence quantitative PCR using plasmid standards of different concentrations as templates in combination with the primer-probe combination RSV3 of Example 1 to obtain Figure 3 , the specific experimental data are shown in Table 4 below, Figure 3 This is the fluorescence quantitative PCR amplification curve of the plasmid standard combined with the primer-probe combination RSV3 in Example 2 of the present application.

[0082] Table 4 Amplification data of different concentrations of plasmid combined with RSV3

[0083]

[0084] The standard curve of RSV Pre F gene detection was drawn based on the Cq value of the standard DNA solution PCR reaction and the logarithm of the initial template copy number. Figure 4 , Figure 4 This is the standard curve for RSV Pre F gene detection using the primer-probe combination RSV3 in Example 2 of the present application.

[0085] Please refer to Table 4. Figure 3 and Figure 4 The results showed that the standard deviation of the Cq value in the experiment of the primer-probe combination RSV3 varied greatly, and the standard deviation at some standard concentrations was greater than 0.6, indicating poor data reliability. The correlation coefficient (R2) of the equation between the Cq value of the detection system and the template copy number was greater than 0.99, indicating a good linear relationship; however, the amplification efficiency calculated based on the slope was only about 87.3%, significantly weaker than RSV1.

[0086] Therefore, the primer-probe combination RSV1 was selected for further vaccine copy number detection experiments and half-life detection experiments.

[0087] Example 3: Verification of copy number detection effect

[0088] (1) SARS-CoV-2 & RSV mRNA combined vaccine membrane rupture treatment

[0089] Take 250 μL of mRNA combined vaccine and add 27 μL of 10% Triton-100. After mixing evenly, heat at 40°C for 10 min to obtain the combined vaccine after membrane rupture.

[0090] (2) Copy number detection experiment

[0091] The combined vaccine after membrane rupture was reverse transcribed to obtain cDNA, and then fluorescent quantitative PCR was performed in the same manner as in Example 2 using cDNA as a template in combination with the primer probe combination RSV1. A negative control and a blank control were set up to obtain the data in the following Table 5.

[0092] mRNA reverse transcription system and reaction conditions:

[0093] Prepare the reaction solution on ice. The total reaction system is 20 μL: Random 6mers S 20 μM, PrimeScript RT Enzyme Mix I 1 μL, 5× PrimeScript Buffer (for Real Time) 4 μL, mRNA 5 pg~10 ng, RNase Free dH 2 O to 20μL; reaction conditions: 37℃, 15min; 85℃, 5sec.

[0094] Table 5 Results of RSV preF mRNA copy number detection after combined vaccine membrane rupture

[0095]

[0096] As shown in the above table, the negative control and blank control were not detected, and the standard deviation of the Cq value of the sample test was less than 0.5, indicating that the test data was reliable. According to the standard curve equation, the RSV PreF copy number in the SARS-CoV-2 & RSV mRNA combined vaccine was 2.6×10 6 Copies / μL.

[0097] Example 4: Verification of half-life detection effect

[0098] The RSV & SARS-CoV-2 mRNA combined vaccine was directly incubated with HEK293 cells, and the cells were collected after 6h, 12h, 24h, 48h, and 72h to extract total RNA. The total RNA was reverse transcribed to obtain cDNA using the same experimental method in Example 3. Then, the same experimental method as in Example 2 was used to perform fluorescent quantitative PCR with cDNA as a template and in combination with the primer probe combination RSV1. Three parallels were set for each sample, and the experiment was repeated three times. At the same time, negative controls and blank controls were set to obtain the data in the following Table 6.

[0099] Table 6 Results of half-life detection of RSV preF mRNA in cells in combined vaccines

[0100]

[0101] As shown in the above table, the negative control and blank control were not detected, and the standard deviation of the Cq values ​​of the standard curve and sample detection was less than 0.5, indicating that the detection data was reliable. According to the standard curve equation, the RSV preF mRNA copy number of HEK293 cells transfected with RSV&SARS-CoV-2 mRNA combined vaccine at different times is shown in the following Table 7.

[0102] Table 7 Calculation results of RSV preF mRNA copy number in cells in combined vaccines

[0103]

[0104]

[0105] Based on the data obtained from the above calculations, the half-life curve of RSV preF mRNA in cells in the SARS-CoV-2 & RSV mRNA combined vaccine was calculated using GraphPad Prism 10 software to fit the curve. Figure 5 , Figure 5 This is the cellular half-life curve of RSV preF mRNA in the combined vaccine of Example 3 of the present application.

[0106] like Figure 5 As shown, the half-life of RSV preF mRNA in the SARS-CoV-2 & RSV mRNA combination vaccine in cells is 28.79h.

[0107] Based on the above experiments, this application successfully established a method for detecting the copy number of RSV preFmRNA in the RSV & SARS-CoV-2mRNA combined vaccine. The correlation coefficient (R2) of the equation between the Cq value and the template copy number of this method was greater than 0.99, and the amplification efficiency was between 90% and 110%, indicating that the method had good specificity and accuracy.

[0108] The method of obtaining the RSV preF mRNA copy number after membrane disruption, reverse transcription and RT-PCR analysis of the combined vaccine can be used in quality control processes such as release testing during the development of RSV & SARS-CoV-2 mRNA combined vaccines.

[0109] After transfection, total RNA extraction, reverse transcription and RT-PCR analysis of the RSV & SARS-CoV-2 mRNA combined vaccine, the method for obtaining the RSV preF mRNA copy number can be used for toxicological experiments in the development process of the RSV & SARS-CoV-2 mRNA combined vaccine, such as tissue distribution evaluation of mRNA vaccines in small animals, etc. Therefore, the present invention has important significance and value for the research and development and quality control of the RSV & SARS-CoV-2 mRNA combined vaccine.

[0110] It is obvious to those skilled in the art that the present application is not limited to the details of the exemplary embodiments described above, and that the present application can be implemented in other specific forms without departing from the spirit or essential features of the present application. Therefore, the embodiments should be regarded as exemplary and non-limiting in all respects, and the scope of the present application is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present application. Any reference numeral in a claim should not be regarded as limiting the claim to which it relates.

[0111] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A primer-probe combination for detecting the copy number of mRNA, characterized in that: The mRNA is used to encode RSV pre-fusion F protein, and its sequence is shown in SEQ ID NO.

1. The primer-probe combination consists of a primer pair shown in SEQ ID NO.2 and 3 and a probe shown in SEQ ID NO.

4. The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group.

2. The primer-probe combination according to claim 1, characterized in that: The fluorescent reporter group is selected from FAM, HEX, VIC, ROX or Cy5, and the fluorescent quencher group is selected from TAMRA, MGB, BHQ1, JOE, BHQ2 or BHQ3.

3. The primer-probe combination according to claim 2, characterized in that: The 5' end of the probe was labeled with FAM, and the 3' end was labeled with TAMRA.

4. An application of a primer-probe combination as described in any one of claims 1 to 3 in detecting the number of copies of mRNA encoding RSV pre-fusion F protein in a new coronavirus RSV combined vaccine.

5. An application of a primer-probe combination as described in any one of claims 1 to 3 in detecting the intracellular half-life of mRNA encoding RSV prefusion F protein in a new coronavirus RSV combined vaccine.

6. The use according to claim 4 or 5, characterized in that: The novel coronavirus RSV combined vaccine comprises: mRNA encoding RSV prefusion F protein as shown in SEQ ID NO.1; A nucleotide sequence encoding a recombinant spike protein, wherein the recombinant spike protein is derived from a novel coronavirus spike protein and has an S-2P site mutation relative to a wild-type novel coronavirus spike protein; Vaccine carrier and excipients, wherein the vaccine carrier is lipid nanoparticles.

7. A method for detecting the copy number of mRNA encoding RSV prefusion F protein in a COVID-19 RSV combined vaccine, characterized in that: The novel coronavirus RSV combined vaccine comprises: mRNA encoding RSV prefusion F protein as shown in SEQ ID NO.1; A nucleotide sequence encoding a recombinant spike protein, wherein the recombinant spike protein is derived from a novel coronavirus spike protein and has an S-2P site mutation relative to a wild-type novel coronavirus spike protein; Vaccine carriers and excipients, wherein the vaccine carrier is lipid nanoparticles; The detection method comprises: After diluting the RSV plasmid standard to different standard concentrations, using the plasmids of different standard concentrations as templates, performing PCR reaction in combination with the primer-probe combination described in any one of claims 1 to 3, detecting the sample threshold Cq, and preparing a standard curve of the sample threshold and mRNA concentration; The membrane of the COVID-19 RSV combined vaccine is broken and reverse transcribed into cDNA. Using cDNA as a template, a PCR reaction is carried out in combination with the primer-probe combination described in any one of claims 1 to 3. The sample threshold Cq is detected and substituted into the standard curve to obtain the copy number of preF protein mRNA in the COVID-19 RSV combined vaccine.

8. A method for detecting the half-life of mRNA encoding RSV pre-fusion F protein in a new coronavirus RSV combined vaccine in cells, characterized in that: The novel coronavirus RSV combined vaccine comprises: mRNA encoding RSV prefusion F protein as shown in SEQ ID NO.1; A nucleotide sequence encoding a recombinant spike protein, wherein the recombinant spike protein is derived from a novel coronavirus spike protein and has an S-2P site mutation relative to a wild-type novel coronavirus spike protein; Vaccine carriers and excipients, wherein the vaccine carrier is lipid nanoparticles; The detection method comprises: After diluting the RSV plasmid standard to different standard concentrations, using the plasmids of different standard concentrations as templates, performing PCR reaction in combination with the primer-probe combination described in any one of claims 1 to 3, detecting the sample threshold Cq, and preparing a standard curve of the sample threshold and the initial mRNA concentration; The COVID-19 RSV combination vaccine is incubated with the target cells, the cells are collected at different times after incubation and total RNA is extracted, the total RNA is reverse transcribed into cDNA, and a PCR reaction is performed using the cDNA as a template in combination with the primer-probe combination described in any one of claims 1 to 3, the sample threshold Cq is detected, and the standard curve is substituted to obtain the copy number of the preF protein mRNA of the COVID-19 RSV combination vaccine after incubation of the target cells for different times, and the half-life of the preF protein mRNA in the COVID-19 RSV combination vaccine in the target cells is obtained.

9. A kit, characterized in that: Used to detect the copy number of mRNA encoding RSV pre-fusion F protein in the new crown RSV combined vaccine, the new crown RSV combined vaccine comprising: mRNA encoding RSVpreF protein as shown in SEQ ID NO.1; A nucleotide sequence mRNA encoding a recombinant spike protein, wherein the recombinant spike protein is derived from a novel coronavirus spike protein and has an S-2P site mutation relative to a wild-type novel coronavirus spike protein; Vaccine carriers and excipients, wherein the vaccine carrier is lipid nanoparticles; The kit comprises: The primer-probe combination according to any one of claims 1 to 3; Reverse transcription reagents; Fluorescence quantitative PCR reagents.

10. The kit according to claim 9, characterized in that Also includes: RSV plasmid standards; RNA extraction reagents.

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