Method for determining plasmid content in avian influenza tetravalent DNA vaccine and application thereof

Abstract

This invention relates to a method and its application for determining the plasmid content in a quadrivalent avian influenza DNA vaccine. Belonging to the field of molecular biology, this invention aims to provide a TaqMan quantitative real-time PCR method capable of quantitatively determining the content of each plasmid component in a quadrivalent DNA vaccine. Specifically, this invention provides a primer and probe composition for detecting the content of individual plasmids in a quadrivalent avian influenza (H5+H7) DNA vaccine based on TaqMan quantitative real-time PCR. The composition includes primers with nucleotide sequences shown in SEQ ID NO: 5-6, 8-9, 11-12, and 14-15, and TaqMan probes with nucleotide sequences shown in SEQ ID NO: 7, 10, 13, and 16. This primer and probe composition can quantitatively determine the content of each plasmid component in the quadrivalent DNA vaccine, exhibiting good specificity and reproducibility.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a combination of primers and probes and a kit for detecting the content of a single plasmid in a trivalent avian influenza DNA vaccine based on TaqMan real-time PCR. Background Technology

[0002] Avian influenza is a major and highly contagious infectious disease that affects both humans and zoonots, seriously endangering the poultry industry and public health. Some strains of highly pathogenic avian influenza viruses, specifically the H5 and H7 subtypes, can cause 100% mortality in poultry. Vaccination is a crucial strategy for controlling avian influenza, especially highly pathogenic avian influenza.

[0003] DNA vaccines have high biosafety and are simple to prepare, easy to construct into combination vaccines and multivalent vaccines. They are a promising genetically engineered vaccine for the prevention and control of highly pathogenic avian influenza (HPAI) and can respond quickly to viral mutations. To comprehensively control and effectively prevent the H5 subtype antigen variants of the 2.3.4.4h and 2.3.4.4b branches, as well as the H7 subtype antigen variants of the A and B branches currently monitored in my country, we developed a quadrivalent DNA vaccine for avian influenza (H5+H7) (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) and evaluated its immunogenicity. The results showed that the quadrivalent DNA vaccine for avian influenza (H5+H7) (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) has good immunogenicity and can provide complete immune protection against lethal challenges from the currently prevalent H5 subtype and H7 subtype avian influenza viruses. It can be used in avian influenza prevention and control practices in my country and provide strong technical support for the healthy development of my country's poultry industry. The quadrivalent vaccine is composed of a mixture of four plasmids. The quality inspection of the finished product requires the determination of the content of the four components. Conventional (ultra)micro spectrophotometer detection methods can only determine the total content and cannot distinguish and determine the content of the four components.

[0004] TaqMan probe-based quantitative PCR technology has advantages such as convenient operation, high sensitivity and good specificity, and is now widely used in the field of veterinary pathogen detection. Summary of the Invention

[0005] In view of this, in order to determine the content of each of the pH5-Re15, pH5-Re16, pH7-Re5 and pH7-Re6 strains in an equal mixture of pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain in the quadrivalent DNA vaccine for avian influenza (H5+H7) (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain), we have established a TaqMan real-time PCR method that can quantitatively determine and distinguish the content of each plasmid component in the quadrivalent DNA vaccine.

[0006] According to one aspect of the present invention, a primer and probe composition for detecting the content of a single plasmid in a quadrivalent DNA vaccine for avian influenza (H5+H7) based on TaqMan real-time PCR is provided, comprising primers with nucleotide sequences shown in SEQ ID NO: 5-6, 8-9, 11-12, 14-15, and TaqMan probes with nucleotide sequences shown in SEQ ID NO: 7, 10, 13, 16.

[0007] The avian influenza (H5+H7) quadrivalent DNA vaccine includes strains pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6.

[0008] The pH5-Re15 strain includes the nucleotide sequence SEQ ID NO: 1, the pH5-Re16 strain includes the nucleotide sequence SEQ ID NO: 2, the pH7-Re5 strain includes the nucleotide sequence SEQ ID NO: 3, and the pH7-Re6 strain includes the nucleotide sequence SEQ ID NO: 4.

[0009] In a specific embodiment of the present invention, the 5' end of the TaqMan probe is labeled with a fluorescent group such as FAM, and the 3' end is labeled with a quenching group such as BHQ1 or MGB.

[0010] In a second aspect of the invention, a detection kit is provided, comprising the aforementioned primer and probe composition.

[0011] In a third aspect of the invention, a method is provided for detecting the content of a single plasmid in a quadrivalent DNA vaccine for avian influenza (H5+H7) based on TaqMan quantitative real-time PCR, comprising using the primer and probe composition described herein, or the detection kit described herein.

[0012] In a specific embodiment of the present invention, the method includes the following steps:

[0013] (1) Using the standards of four plasmids, pH5-Re15, pH5-Re16, pH7-Re5 and pH7-Re6, as templates, a standard curve was established by performing real-time PCR with the primer and probe combination described above.

[0014] (2) Take the finished product of the avian influenza (H5+H7) quadrivalent DNA vaccine to be tested or its diluted working solution as a template, and perform real-time PCR using the primer and probe combination described above;

[0015] (3) Compare the standard curves of each plasmid and calculate the copy number of each plasmid in the avian influenza (H5+H7) quadrivalent DNA vaccine.

[0016] In a specific embodiment of the present invention, in the method for detecting the content of a single plasmid in a quadrivalent avian influenza (H5+H7) DNA vaccine, the content of the pH5-Re15 recombinant plasmid is detected using primers as shown in SEQ ID NO: 5-6 and probes as shown in SEQ ID NO: 7; the content of the pH5-Re16 recombinant plasmid is detected using primers as shown in SEQ ID NO: 8-9 and probes as shown in SEQ ID NO: 10; the content of the pH7-Re5 recombinant plasmid is detected using primers as shown in SEQ ID NO: 11-12 and probes as shown in SEQ ID NO: 13; and the content of the pH7-Re6 recombinant plasmid is detected using primers as shown in SEQ ID NO: 14-15 and probes as shown in SEQ ID NO: 16.

[0017] In a specific embodiment of the present invention, the real-time PCR system comprises the following components: real-time PCR enzyme (2X), the amount of which can be routinely adjusted according to actual needs, for example, 5-15 μl, such as 8, 10, or 12 μl; RNase-free H2O, the amount of which can be routinely adjusted according to actual needs, for example, 5-10 μl, such as 7 μl; and upstream primer, the concentration and volume of which can be routinely adjusted according to actual needs, with a concentration of 5-15 pmol, such as 10 pmol, and a volume of 0.2-0.8 μL. For example, 0.4 μl; downstream primer, the concentration and volume can be adjusted according to actual needs, the concentration is 5-15 pmol, for example 10 pmol, and the volume is 0.2-0.8 μl, for example 0.4 μl; probe, the concentration and volume can be adjusted according to actual needs, the concentration is 5-15 pmol, for example 10 pmol, and the volume is 0.1-0.8 μl, for example 0.2 μl; template, the amount can be adjusted according to actual needs, for example 1-10 μl, for example 2, 3, 4, 5 μl.

[0018] In a specific embodiment of the present invention, the reaction procedure of the real-time PCR includes the following steps: 1) digestion, time 1-5 minutes, temperature 35-40℃, for example, 37℃ for 2 minutes of contamination digestion; 2) pre-denaturation, temperature 90-100℃, time 3-6 minutes, for example, 95℃ for 5 minutes of pre-denaturation; 3) denaturation, time 90-100℃, time 8-12 seconds, for example, 95℃ for 10 seconds of denaturation; 4) annealing, temperature 50-65℃, time 25-35 seconds, for example, 60℃ for 30 seconds of annealing, for a total of 30 cycles, and collecting FAM fluorescence signal at the end of each annealing step.

[0019] The avian influenza (H5+H7) quadrivalent DNA vaccine is a quadrivalent DNA vaccine targeting both H5 and H7 subtypes (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain). It includes recombinant plasmids pH5-Re15 and pH5-Re16 targeting the H5N6 subtype, and recombinant plasmids pH7-Re5 and pH7-Re6 targeting the H7N9 subtype. Mixing these four recombinant plasmids at equal concentrations and volumes yields the avian influenza (H5+H7) quadrivalent DNA vaccine. This avian influenza (H5+H7) quadrivalent DNA vaccine provides complete immune protection against lethal challenges from currently circulating H5 and H7 subtype avian influenza viruses, exhibiting good immunogenicity.

[0020] Effective effect

[0021] This invention provides primer and probe compositions that can be used to detect the content of single plasmids in a quadrivalent avian influenza (H5+H7) DNA vaccine. The detection method of this invention has high accuracy and good repeatability, and is an important method for quality control of avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain). Attached Figure Description

[0022] Figure 1 The specific sequence regions of the four optimized HA gene sequences of strains pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6 are shown; from left to right, the first box (red) represents the upstream primer position, the third box (yellow) represents the probe position, and the second box (blue) represents the downstream primer position.

[0023] Figure 2The results of TaqMan fluorescent PCR primer and probe specificity assays are shown; where A: strain pH5-Re15 is the DNA template; B: strain pH5-Re16 is the DNA template; C: strain pH7-Re5 is the DNA template; D: strain pH7-Re6 is the DNA template.

[0024] Figure 3 The test results of standards at different dilutions are shown; among them, A1 is the amplification curve using the pH5-Re15 strain as a DNA template, and the leftmost amplification curve is 10. 0 The dilution ratios, from left to right, are 10. -1 10 -2 10 -3 10 -4 Amplification curves at multiple dilutions; A2 is the positive / negative control, where the positive control uses pH5-Re15 strain as DNA template and the negative control uses ddH2O as template; B1 is the amplification curve using pH5-Re16 strain as DNA template, where the leftmost amplification curve is 10... 0 The dilution ratios, from left to right, are 10. -1 10 -2 10 -3 10 -4 Amplification curves at multiple dilutions; B2 is the positive / negative control, where the positive control uses pH5-Re16 strain as DNA template and the negative control uses ddH2O as template; C1 is the amplification curve using pH7-Re5 strain as DNA template, where the leftmost amplification curve is 10... 0 The dilution ratios, from left to right, are 10. -1 10 -2 10 -3 10 -4 Amplification curves at multiple dilutions; C2 is the positive / negative control, where the positive control uses pH7-Re5 strain as the DNA template and the negative control uses ddH2O as the template; D1 is the amplification curve using pH7-Re6 strain as the DNA template, where the leftmost amplification curve is 10... 0 The dilution ratios, from left to right, are 10. -1 10 -2 10 -3 10 -4 Amplification curves at multiple dilutions; D2 is the positive / negative control, where the positive control uses pH7-Re6 strain as DNA template and the negative control uses ddH2O as template.

[0025] Figure 4 The standard curves for four standards are shown, with the five points from left to right representing 10. 0 -10 -4Dilution; A: Standard curves for primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe were validated, with strain pH5-Re15 as the DNA template; B: Standard curves for primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe were validated, with strain pH5-Re16 as the DNA template; C: Standard curves for primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe were validated, with strain pH7-Re5 as the DNA template; D: Standard curves for primers Re6 F4-Forward, Re6 R4-Reverse, and probe Re6 P4-probe were validated, with strain pH7-Re6 as the DNA template. Among them, the slope of graph A is -3.287, R2 is 0.996, and Eff% is 101.487; the slope of graph B is -3.496, R2 is 0.998, and Eff% is 93.222; the slope of graph C is -3.152, R2 is 0.999, and Eff% is 92.618; and the slope of graph D is -3.583, R2 is 0.995, and Eff% is 81.788.

[0026] Figure 5 The results of repeated assays for standards pH5-Re15 and pH5-Re16 are displayed. Specifically, A1-A4 show the repeated assay results for the pH5-Re15 standard, with the leftmost amplification curve representing 10. 0 The dilution ratios, from left to right, are 10. -1 10 -2 10 -3 10 -4 Amplification curves at multiple dilutions; A1-A3 used pH5-Re15 strain as the DNA template, A4 used pH5-Re15 strain as the positive control and ddH2O as the negative control. B1-B4 show the results of repeated assays of pH5-Re16 strain standard; the leftmost part of the amplification curve is 10. 0 The dilution ratios, from left to right, are 10. -1 10 -2 10 -3 10 -4 Amplification curves at multiple dilutions; B1-B3 used pH5-Re16 strain as DNA template, B4's positive control used pH5-Re16 strain as DNA template, and the negative control used ddH2O as template.

[0027] Figure 6The results of repeated assays for standards pH7-Re5 and pH7-Re6 are displayed. Specifically, C1-C4 show the repeated assay results for the pH7-Re5 standard; the leftmost amplification curve is 10. 0 The dilution ratios, from left to right, are 10. -1 10 -2 10 -3 10 -4 Amplification curves at multiple dilutions; C1-C3 used pH7-Re5 strain as DNA template, C4 positive control used pH7-Re5 strain as DNA template, and the negative control used ddH2O as template. D1-D4 show the results of repeated assays of pH7-Re6 strain standard; the leftmost part of the amplification curve is 10. 0 The dilution ratios, from left to right, are 10. -1 10 -2 10 -3 10 -4 Amplification curves at multiple dilutions; D1-D3 used pH7-Re6 strain as DNA template, D4 ​​used pH7-Re6 strain as DNA template for the positive control, and ddH2O as template for the negative control.

[0028] Figure 7 The standard curves for three reproducible assays of standards pH5-Re15 and pH5-Re16 are shown. Specifically, A1-A3 display the standard curves for three reproducible assays of the pH5-Re15 strain standard using primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe, with the pH5-H5-Re15 strain serving as the DNA template. The five points from left to right represent 10... 0 -10 -4 Dilution. B1-B3 show the standard curves for three reproducible assays using primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe against the pH5-Re16 strain standard, with pH5-Re16 strain as the DNA template. The five points from left to right represent 10... 0 -10 -4Dilution. The slope of A1 was -3.675, R² was 1, and Eff% was 122.272; the slope of A2 was -3.889, R² was 0.999, and Eff% was 80.763; the slope of A3 was -2.657, R² was 0.991, and Eff% was 137.884; the slope of B1 was -3.865, R² was 0.994, and Eff% was 81.434; the slope of B2 was -3.675, R² was 1, and Eff% was 87.106; the slope of B3 was -3.797, R² was 0.998, and Eff% was 83.386.

[0029] Figure 8 The standard curves for three reproducible assays of pH7-Re5 and pH7-Re6 standards are shown. Specifically, C1-C3 show the standard curves for three reproducible assays of the pH7-Re5 standard using primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe, with pH7-Re5 as the DNA template. The five points from left to right represent 10... 0 -10 -4 Dilution. D1-D3 show the standard curves for three reproducible assays using primers Re6 F4-Forward, Re6 R4-Reverse, and probe Re6 P4-probe against the pH7-Re6 strain standard, with the pH7-Re6 strain as the DNA template. The five points from left to right represent 10... 0 -10 -4 Dilution. The slope of C1 was -3.483, R² was 0.992, and Eff% was 93.698; the slope of C2 was -2.93, R² was 0.992, and Eff% was 119.413; the slope of C3 was -3.538, R² was 0.999, and Eff% was 91.696; the slope of D1 was -2.888, R² was 0.993, and Eff% was 121.971; the slope of D2 was -2.427, R² was 0.997, and Eff% was 158.255; and the slope of D3 was -2.912, R² was 0.993, and Eff% was 120.481.

[0030] Figure 9The results of Ct value test for detecting the content of avian influenza (H5+H7) quadrivalent DNA vaccine product by real-time quantitative PCR are shown. Among them, A1, B1, C1, and D1 are the results obtained by using the avian influenza (H5+H7) quadrivalent DNA vaccine product as DNA template and using its corresponding probe and primer respectively; A2, B2, C2, and D2 are the results of positive / negative controls, where A2 positive control uses pH5-Re15 strain as DNA template, B2 positive control uses pH5-Re16 strain as DNA template, C2 positive control uses pH7-Re5 strain as DNA template, and D2 positive control uses pH7-Re6 strain as DNA template. The negative controls A2, B2, C2, and D2 all use ddH2O as template.

[0031] Figure 10 The standard curve for the first repeatability test is shown, with the five points from left to right representing 10. 0 -10 -4 Dilution; A: Detection results of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe; B: Detection results of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe; C: Detection results of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe; D: Detection results of primers Re6 F4-Forward, Re6R4-Reverse, and probe Re6 P4-probe. Among them, the slope of A is -3.282, R² is 0.997, and Eff% is 101.715; the slope of B is -3.268, R² is 0.990, and Eff% is 116.203; the slope of C is -3.666, R² is 0.996, and Eff% is 88.624; and the slope of D is -3.319, R² is 0.997, and Eff% is 100.127.

[0032] Figure 11The results of the Ct values ​​for detecting the content of different batches of avian influenza (H5+H7) quadrivalent DNA vaccine products using the first real-time quantitative PCR method are shown. Specifically, A1-A2 represent the detection results of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe; A1 uses the finished avian influenza (H5+H7) quadrivalent DNA vaccine product as the DNA template, and A2 represents the positive / negative controls, with the positive control using the pH5-Re15 strain as the DNA template and the negative control using ddH2O as the template. Similarly, B1-B2 represent the detection results of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe; B1 uses the finished avian influenza (H5+H7) quadrivalent DNA vaccine product as the DNA template, and B2 represents the positive / negative controls, with the positive control using the pH5-Re16 strain as the DNA template and the negative control using ddH2O as the template. C1-C2: Detection results of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe; C1 uses the finished avian influenza (H5+H7) quadrivalent DNA vaccine as a DNA template, and C2 serves as a positive / negative control, with the positive control using the pH7-Re5 strain as a DNA template and the negative control using ddH2O as a template. D1-D2: Detection results of primers Re6 F4-Forward, Re6 R4-Reverse, and probe Re6 P4-probe; D1 uses the finished avian influenza (H5+H7) quadrivalent DNA vaccine as a DNA template, and D2 serves as a positive / negative control, with the positive control using the pH7-Re6 strain as a DNA template and the negative control using ddH2O as a template.

[0033] Figure 12 The standard curve for the second repeatability test is shown, with the five points from left to right representing 10... 0 -10 -4Dilution; where A: Detection results of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe; B: Detection results of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe; C: Detection results of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe; D: Detection results of primers Re6 F4-Forward, Re6R4-Reverse, and probe Re6 P4-probe. Among them, the slope of A is -3.314, R² is 1, and Eff% is 100.315; the slope of B is -3.072, R² is 0.993, and Eff% is 111.603; the slope of C is -3.666, R² is 0.999, and Eff% is 87.397; and the slope of D is -3.31, R² is 0.999, and Eff% is 100.483.

[0034] Figure 13The results of the Ct values ​​for detecting the content of different batches of avian influenza (H5+H7) quadrivalent DNA vaccine products using the second quantitative real-time PCR method are shown. Specifically, A1-A2 represent the detection results of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe; A1 uses the finished avian influenza (H5+H7) quadrivalent DNA vaccine product as the DNA template, and A2 represents the positive / negative controls, with the positive control using the pH5-Re15 strain as the DNA template and the negative control using ddH2O as the template. B1-B2 represent the detection results of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe; B1 uses the finished avian influenza (H5+H7) quadrivalent DNA vaccine product as the DNA template, and B2 represents the positive / negative controls, with the positive control using the pH5-Re16 strain as the DNA template and the negative control using ddH2O as the template. C1-C2: Detection results of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe; C1 uses the finished avian influenza (H5+H7) quadrivalent DNA vaccine as a DNA template, and C2 is a positive / negative control, where the positive control uses the pH7-Re5 strain as a DNA template, and the negative control uses ddH2O as a template. D1-D2: Detection results of primers Re6 F4-Forward, Re6 R4-Reverse, and probe Re6 P4-probe; D1 uses the finished avian influenza (H5+H7) quadrivalent DNA vaccine as a DNA template, and D2 is a positive / negative control, where the positive control uses the pH7-Re6 strain as a DNA template, and the negative control uses ddH2O as a template.

[0035] Figure 14 The standard curve for the third repeatability test is shown, with the five points from left to right representing 10... 0 -10 -4Dilution; where A: Detection results of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe; B: Detection results of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe; C: Detection results of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe; D: Detection results of primers Re6 F4-Forward, Re6R4-Reverse, and probe Re6 P4-probe. Among them, the slope of A is -3.278, R² is 0.997, and Eff% is 101.886; the slope of B is -3.231, R² is 1, and Eff% is 103.926; the slope of C is -3.503, R² is 0.999, and Eff% is 92.961; and the slope of D is -3.532, R² is 0.999, and Eff% is 91.912.

[0036] Figure 15The results of the Ct values ​​for detecting the content of different batches of avian influenza (H5+H7) quadrivalent DNA vaccine products using the third quantitative real-time PCR method were presented. A1-A2: Detection results of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15P1-Probe. A1 used the finished avian influenza (H5+H7) quadrivalent DNA vaccine product as the DNA template, and A2 served as a positive / negative control, with the positive control using the pH5-Re15 strain as the DNA template and the negative control using ddH2O as the template. B1-B2: Detection results of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe. B1 used the finished avian influenza (H5+H7) quadrivalent DNA vaccine product as the DNA template, and B2 served as a positive / negative control, with the positive control using the pH5-Re16 strain as the DNA template and the negative control using ddH2O as the template. C1-C2: Detection results of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5P3-probe; C1 uses the finished avian influenza (H5+H7) quadrivalent DNA vaccine as a DNA template, and C2 is a positive / negative control, where the positive control uses the pH7-Re5 strain as a DNA template, and the negative control uses ddH2O as a template. D1-D2: Detection results of primers Re6F4-Forward, Re6 R4-Reverse, and probe Re6 P4-probe; D1 uses the finished avian influenza (H5+H7) quadrivalent DNA vaccine as a DNA template, and D2 is a positive / negative control, where the positive control uses the pH7-Re6 strain as a DNA template, and the negative control uses ddH2O as a template. Detailed Implementation

[0037] Experimental materials

[0038] 1. Reagents

[0039] The quantitative fluorescence kit (probe method) was purchased from Vazyme, and the plasmid extraction kit was from Endofree. ® The PlasmidMaxi Kit (10) was purchased from Qiagen.

[0040] 2. Plasmid

[0041] The recombinant plasmids pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6 were prepared, identified, and preserved by the National Avian Influenza Reference Laboratory of the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The preparation method is as follows:

[0042] Recombinant plasmid pH5-Re15 strain: The HA gene of avian influenza virus DK / HeN / SD40-1 / 24 (H5N6) was used and codons were optimized to obtain the optimized HA gene sequence SEQ ID NO: 1 (gene opti-HeNHA5), which was then ligated to the pCAGGoptiHA5 vector (see CN114524862A for the map information of plasmid pCAGGoptiHA5).

[0043] Recombinant plasmid pH5-Re16 strain: The HA gene of avian influenza virus GS / HuN / S10466 / 24 (H5N6) was used and codons were optimized to obtain the optimized HA gene sequence SEQ ID NO: 2 (gene opti-HuNHA5), which was then ligated to the pCAGGoptiHA5 vector backbone.

[0044] Recombinant plasmid pH7-Re5 strain: The HA gene of avian influenza virus CK / HeN / SD018 / 24 (H7N9) was used and codons were optimized to obtain the optimized HA gene sequence SEQ ID NO: 3 (opti-HeNHA7), which was then ligated to the pCAGGoptiHA5 vector backbone.

[0045] Recombinant plasmid pH7-Re6 strain: The HA gene of avian influenza virus CK / HeB / SD016 / 24 (H7N9) was used and codons were optimized to obtain the optimized HA gene sequence SEQ ID NO: 4 (opti-HeBHA7), which was then ligated to the pCAGGoptiHA5 vector backbone according to conventional gene editing methods.

[0046] 3. Samples: Batch 2025001, 2025002, and 2025003 of avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) were prepared by the National Avian Influenza Reference Laboratory.

[0047] The preparation method is as follows:

[0048] Recombinant plasmids pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6 were mixed in equal concentrations and volumes to prepare a quadrivalent DNA vaccine for avian influenza (H5+H7) (pH5-Re15 + pH5-Re16 + pH7-Re5 + pH7-Re6). The immunization dose of each plasmid was prepared as needed.

[0049] 4. Instrument: QuantStudio 5 real-time PCR instrument from Applied Biosystems, USA.

[0050] Example 1: Design of TaqMan Real-Time PCR Primers and Probes

[0051] The optimized HA gene sequences (i.e., SEQ ID NO: 1-4) based on strains pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6 were compared using DNAStar bioinformatics software for homology analysis. This identified unique sequence difference regions specific to the four HA gene sequences. Twenty sets of primers and TaqMan probes were designed using Primer Express 3.0 software for these differentially significant sequence fragments. (See [link to Primer description]). Figure 1 The primer and probe sequences are shown in Table 1. The probe is labeled with FAM at the 5' end and with BHQ1 or MGB quencher group at the 3' end, and was synthesized by Seven Biotech Co., Ltd.

[0052] Table 1 Primer and probe sequences

[0053]

[0054] Continued from Table 1: Primer and Probe Sequences

[0055]

[0056] Example 2: Establishment of TaqMan Real-Time PCR Method

[0057] According to the Qiagen plasmid mass extraction kit Endofree ® Plasmid Maxi Kit (10) instructions for plasmid extraction.

[0058] 1. TaqMan Real-Time PCR Reaction System and Conditions: This TaqMan Real-Time PCR method utilizes reagents from a real-time PCR kit in a 20µl reaction system. The components and their volumes are as follows:

[0059]

[0060] The reaction conditions were as follows: 37°C for 2 minutes of contamination digestion; 95°C for 5 minutes of pre-denaturation; 95°C for 10 seconds of denaturation; and 60°C for 30 seconds of annealing, for a total of 30 cycles. FAM fluorescence signals were collected at the end of each annealing step.

[0061] 2. Specificity detection of TaqMan real-time PCR primer and probe combinations

[0062] During the PCR reaction, with other reaction conditions unchanged, 20 sets of probes were used to perform TaqMan real-time PCR detection on primers and TaqMan probes for four single plasmids: pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6. Specific primers and probes were selected based on the amplification curves. The results are as follows:

[0063] Using the 20 sets of primers and probes in Table 1, and four plasmids (pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6) as DNA templates, TaqMan fluorescent PCR was performed, with a negative control included. The results showed that only samples corresponding to the appropriate primers and probes exhibited standard amplification curves. Among them, the Ct value of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe in group A1 was 13.216 when used as a DNA template in strain pH5-Re15; the Ct value of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe in group B1 was 13.053 when used as a DNA template in strain pH5-Re16; the Ct value of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe in group C3 was 14.363 when used as a DNA template in strain pH7-Re5; and the Ct value of primers Re6 F4-Forward, Re6 R4-Reverse, and probe Re6P4-probe in group D4 was 11.162 when used as a DNA template in strain pH7-Re6. All other samples showed a straight line, indicating no Ct value. The results are shown in [link to results]. Figure 2 The results indicate that primers and probes in groups A1, B1, C3, and D4 have good specificity, amplifying only their corresponding samples and showing no non-specific reactions to the other two plasmid samples. The amplification effects of the other 16 sets of primers and probes have different drawbacks compared to those described above, and they will not be used in subsequent experiments.

[0064] Example 3: Detection of plasmid content in a quadrivalent avian influenza (H5+H7) DNA vaccine using TaqMan real-time PCR.

[0065] 1. Preparation of standards: Extract pH5-Re15, pH5-Re16, pH7-Re5 and pH7-Re6 plasmids using an endotoxin-free plasmid extraction kit, aliquot 1µg into each tube, vacuum dry to prepare standards, and dissolve in 100µl ddH2O (10ng / µl) before use.

[0066] 2. Establishment of the standard curve: Four plasmid standards were serially diluted 10-fold.0 -10 -4 After serial dilution, the samples were used as test samples and amplified using the selected primers and probes. Standard curves were plotted for each plasmid. The results are as follows:

[0067] The copy number is 1.4 × 10⁻⁶. 9 After serially diluting the plasmid at 10-fold concentrations of 1 copy / µl, take 10... 0 -10 -4 Dilution analysis revealed a strong linear correlation. The slopes of the four plasmids were -3.287, -3.496, -3.512, and -3.853, respectively, with correlation coefficients R0. 2 The values ​​were 0.996, 0.998, 0.999, and 0.995, respectively. The results are shown in Table 2. Figure 3 and Figure 4 .

[0068] Table 2. Ct value test results of standards at different dilutions

[0069]

[0070] 3. Standard Curve Repeatability Experiment: Following the method described in "2. Establishment of Standard Curve," the amplification was repeated three times under the same conditions. The reproducibility of the standard curve and Ct values ​​was observed. The results are as follows:

[0071] The copy number is 1.4 × 10⁻⁶. 9 After performing a 10-fold serial dilution of the standard (copy / µl), take 10... 0 -10 -4 Dilution was measured, and three PCR amplifications were performed under the same conditions. The Ct values, average Ct values, and coefficients of variation are shown in Table 3. The coefficient of variation within each group was less than 10%. Good linear correlation was observed between plasmids at different dilutions in the three assays. 2 All values ​​are greater than 0.99, as shown in the results below. Figures 5-8 This indicates that the amplification stability of the standard curve is relatively high.

[0072] Table 3. Ct values ​​and coefficients of variation for repeatability testing of standards at different dilutions

[0073]

[0074] Among them, A1-A3 show the results of primer Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe; B1-B3 show the results of primer Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe; C1-C3 show the results of primer Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe; D1-D3 show the results of primer Re6 F4-Forward, Re6 R4-Reverse, and probe Re6 P4-probe.

[0075] 4. TaqMan real-time PCR method for detecting the copy number of each plasmid in avian influenza (H5+H7) quadrivalent DNA vaccine: The vaccine sample from batch 2025001 and its diluted working solution were serially diluted 10-fold, and 10 μL of each was taken... -2 The dilution was determined, and the Ct value was detected by real-time PCR. The standard curve established in "2. Establishment of Standard Curve" was compared with the standard curve established. The copy number of each plasmid was calculated. The copy number calculation formula is: (6.02 * 10(23)) * (ng / μl * 10(-9) ) / (DNA length * 660) = copies / μl, and its content was calculated.

[0076] The results of TaqMan quantitative PCR testing on avian influenza (H5+H7) quadrivalent DNA vaccine samples with different concentrations are as follows: The vaccine product from batch 2025001 was diluted to 10... -2 The Ct values ​​were measured, and the standard curves of each plasmid were compared to calculate the copy number of each plasmid in the finished avian influenza (H5+H7) quadrivalent DNA vaccine and its application solution. The content of each plasmid was then calculated based on its copy number. The results are shown in Tables 4, 5, and 6. Figure 9 .

[0077] Table 4. Ct values ​​for detecting the content of avian influenza (H5+H7) quadrivalent DNA vaccine using quantitative real-time PCR.

[0078]

[0079] In this table, A represents the detection results of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe; B represents the detection results of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe; C represents the detection results of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe; and D represents the detection results of primers Re6 F4-Forward, Re6 R4-Reverse, and probe Re6 P4-probe.

[0080] Table 5. Copy number calculation results for detecting the content of avian influenza (H5+H7) quadrivalent DNA vaccine by real-time PCR.

[0081]

[0082] Unit: copies / µl

[0083] A: Detection results of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe; B: Detection results of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe; C: Detection results of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe; D: Detection results of primers Re6 F4-Forward, Re6R4-Reverse, and probe Re6 P4-probe.

[0084] Table 6. Results of plasmid content in quadrivalent DNA vaccine samples of avian influenza (H5+H7)

[0085]

[0086] Unit: ng / µl

[0087] A: Detection results of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe; B: Detection results of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe; C: Detection results of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe; D: Detection results of primers Re6 F4-Forward, Re6R4-Reverse, and probe Re6 P4-probe.

[0088] The above results demonstrate that the TaqMan real-time quantitative PCR method for detecting single plasmids in the quadrivalent avian influenza (H5+H7) DNA vaccine established in this invention has high accuracy. The detected contents of the four plasmids (pH5-Re15, pH5-Re16, pH7-Re5, pH7-Re6) in the quadrivalent avian influenza (H5+H7) DNA vaccine are consistent with the actual contents of each plasmid in the quadrivalent avian influenza (H5+H7) DNA vaccine. It also exhibits good repeatability, with the coefficient of variation (CV) of the standard repeatability detection being less than 5%, indicating method stability. Furthermore, it demonstrates high sensitivity, with the quantitative method detecting concentrations as low as 0.001 ng / μl, fully meeting the content detection requirements of the quadrivalent avian influenza (H5+H7) DNA vaccine. Finally, it exhibits strong specificity, unaffected by homologous sequence interference, and can effectively distinguish the four plasmids (pH5-Re15, pH5-Re16, pH7-Re5, pH7-Re6).

[0089] Example 4: Repeatability test of TaqMan real-time PCR method

[0090] Two hundred microliters of each of the three batches of the quadrivalent avian influenza (H5+H7) DNA vaccine were taken as test samples and diluted to 10 μL. -2 Each batch was replicated in three places, and each batch was subjected to quantitative real-time PCR by three different operators on different instruments. The Ct values ​​were measured, and the copy number of each plasmid in the finished vaccine was calculated by comparing the standard curve of each plasmid with the Ct values. The content of each plasmid was calculated based on the copy number of each plasmid. Based on this, the coefficients of variation between batches and within batches were calculated.

[0091] Three batches of avian influenza (H5+H7) quadrivalent DNA vaccine were subjected to three separate TaqMan quantitative PCR tests, performed by three different operators on different instruments. The results are as follows:

[0092] 1. Results of the first repeatability test: The first repeatability test was conducted by operator 1. The test results showed that the inter-batch coefficient of variation was within 15%, and the intra-batch coefficient of variation was also within 15%. The results are shown in Tables 7, 8, and 9. Figure 10 , Figure 11 .

[0093] Table 7. Ct values ​​for detecting plasmid content in finished vaccine product using quantitative real-time PCR.

[0094]

[0095] Table 8. Copy number calculation results for detecting the content of each plasmid in the finished vaccine product by real-time PCR (10⁻¹⁰) 8 Copy / µl)

[0096]

[0097] Table 9. Content (ng / µl) and coefficient of variation of each plasmid in different batches of finished vaccine products.

[0098]

[0099] 2. Results of the second repeatability test: The second repeatability test was conducted by operator 2. The test results showed that the inter-batch coefficient of variation was within 10%, and the intra-batch coefficient of variation was within 10%. The results are shown in Tables 10, 11, and 12. Figure 12 , Figure 13 .

[0100] Table 10. Ct values ​​for detecting plasmid content in finished vaccine product using quantitative real-time PCR.

[0101]

[0102] Table 11. Copy number calculation results for detecting the content of each plasmid in the finished vaccine product by real-time PCR (10⁻¹⁰) 8 Copy / µl)

[0103]

[0104] Table 12. Content (ng / µl) and coefficient of variation of each plasmid in different batches of finished vaccine products.

[0105]

[0106] 3. Results of the third repeatability test: The third repeatability test was conducted by operator 3. The test results showed that the inter-batch coefficient of variation was within 15%, and the intra-batch coefficient of variation was within 20%. The results are shown in Tables 13, 14, and 15. Figure 14 , Figure 15 .

[0107] Table 13. Ct values ​​for detecting plasmid content in finished vaccine product using quantitative real-time PCR.

[0108]

[0109] Table 14. Copy number calculation results for detecting the content of each plasmid in the finished vaccine product by real-time PCR (10⁻¹⁰) 8 Copy / µl)

[0110]

[0111] Table 15. Content (ng / µl) and coefficient of variation of each plasmid in different batches of finished vaccine products.

[0112]

[0113] Based on the results of the three repeatability tests, the inter-batch coefficient of variation was within 15%, and the intra-batch coefficient of variation was within 20%. The results show that the TaqMan real-time PCR method we established has a coefficient of variation within 20%, indicating good repeatability.

[0114] Example 5: Determination of the range of identification method for each component in a quadrivalent DNA vaccine for avian influenza (H5+H7) by real-time quantitative PCR.

[0115] The total standard deviation was calculated based on the results of three repeatability tests (Table 16). According to the principle of normal distribution in statistics, 2 times the standard deviation covers 95% of the detected values. Therefore, the determination range for the content of each plasmid component in the quadrivalent DNA vaccine for avian influenza (H5+H7) is determined to be 2 times the total standard deviation. That is, when performing quantitative real-time PCR identification, in the quadrivalent DNA vaccine for avian influenza (H5+H7) (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain), the content of pH5-Re15 strain per milliliter should be within the range of 0.249±0.026 mg, the content of pH5-Re16 strain per milliliter should be within the range of 0.244±0.027 mg, the content of pH7-Re5 strain per milliliter should be within the range of 0.249±0.023 mg, and the content of pH7-Re6 strain per milliliter should be within the range of 0.214±0.012 mg.

[0116] Table 16 Judgment range for detecting the content of each plasmid in the quadrivalent DNA vaccine of avian influenza (H5+H7) using the real-time quantitative PCR method.

[0117]

[0118] discuss

[0119] To accurately detect the content of four plasmids in a quadrivalent DNA vaccine for avian influenza (H5+H7) (pH5-Re15 + pH5-Re16 + pH7-Re5 + pH7-Re6) prepared by mixing equal volumes of four plasmids of the same concentration (pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6), this experiment established a targeted TaqMan real-time PCR detection method.

[0120] Based on the laboratory sequencing results, homology analysis was performed using DNAStar software to identify specific sequence regions of the HA gene in strains pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6. Twenty sets of primers and TaqMan probes targeting the insertion site were designed using PrimerExpress 3.0 software. Using these 20 sets of primers and probes, and with strains pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6 as templates, TaqMan fluorescent PCR reactions were performed. It was found that four sets of primers all produced amplification curves, and only samples corresponding to each primer and probe set showed standard amplification curves; other samples did not show amplification curves, indicating that each primer and probe set had good specificity.

[0121] The copy number is 1.4 × 10⁻⁶. 9 After serially diluting the plasmid at 10-fold concentrations of 1 copy / µl, take 10... 0 -10 -4 Dilution analysis revealed a strong linear correlation. The slopes of the four plasmids were -3.287, -3.496, -3.512, and -3.853, respectively, with correlation coefficients R0. 2 The values ​​were 0.996, 0.998, 0.999, and 0.995, respectively. The standard curve repeatability test demonstrated that the prepared standard was relatively stable; the Ct values ​​showed good reproducibility in three amplifications under the same conditions, and the coefficient of variation was less than 10%. This indicates that the standard curve preparation of this detection method has good repeatability and high stability.

[0122] Take batch 2025001 of avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) and its diluted working solution as samples, and dilute them to 10 μL. -2 The Ct value was detected, and the standard curves of each plasmid were compared to calculate the copy number of each plasmid in different contents of the avian influenza (H5+H7) quadrivalent DNA vaccine. The results showed that the plasmid content calculated based on the detected copy number was similar to the content of each plasmid when the avian influenza (H5+H7) quadrivalent DNA vaccine was prepared, indicating that the detection method of the present invention has high accuracy.

[0123] TaqMan quantitative PCR was used to detect three batches of avian influenza (H5+H7) quadrivalent DNA vaccine products. The vaccine samples were diluted to 10... -2Each batch was performed in triplicate, with three different operators using different instruments. The Ct values ​​were detected by quantitative real-time PCR, and the copy number and content of each plasmid in the finished vaccine were calculated by comparing with the standard curves of each plasmid. The inter-batch and intra-batch coefficients of variation were also calculated. The results showed that the inter-batch and intra-batch coefficients of variation were all within 20% in the three repeatability tests, indicating that the TaqMan quantitative real-time PCR detection method we established has good repeatability.

[0124] Based on repeatability testing, the concentrations of the following avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) should be within the range of 0.249±0.026 mg / mL, pH5-Re16 strain within the range of 0.244±0.027 mg / mL, pH7-Re5 strain within the range of 0.249±0.023 mg / mL, and pH7-Re6 strain within the range of 0.214±0.012 mg / mL.

[0125] in conclusion

[0126] This study successfully established a TaqMan quantitative real-time PCR method for detecting the content of four plasmids in a quadrivalent avian influenza (H5+H7) DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain). This method is an important identification test in the quality standards of the quadrivalent avian influenza (H5+H7) DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain), and is a crucial method for quality control of this product. The detection method exhibits good specificity and repeatability. When detecting the content of each component in the quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) of avian influenza (H5+H7) using the real-time quantitative PCR method, the content of pH5-Re15 strain per milliliter should be within the range of 0.249±0.026 mg, the content of pH5-Re16 strain per milliliter should be within the range of 0.244±0.027 mg, the content of pH7-Re5 strain per milliliter should be within the range of 0.249±0.023 mg, and the content of pH7-Re6 strain per milliliter should be within the range of 0.214±0.012 mg.

Claims

1. A primer and probe composition for determining the plasmid content in a quadrivalent avian influenza DNA vaccine, comprising primers with nucleotide sequences shown in SEQ ID NO:5-6, 8-9, 11-12, 14-15, and TaqMan probes with nucleotide sequences shown in SEQ ID NO:7, 10, 13, 16; wherein The quadrivalent DNA vaccine for avian influenza includes recombinant plasmids pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6. The recombinant plasmid pH5-Re15 includes the nucleotide sequence SEQ ID NO: 1, the recombinant plasmid pH5-Re16 includes the nucleotide sequence SEQ ID NO: 2, the recombinant plasmid pH7-Re5 includes the nucleotide sequence SEQ ID NO: 3, and the recombinant plasmid pH7-Re6 includes the nucleotide sequence SEQ ID NO:

4.

2. The primer and probe composition according to claim 1, wherein, The TaqMan probe has a fluorescent group labeled at its 5' end and a quenching group labeled at its 3' end.

3. A detection kit comprising the primer and probe composition as described in claim 1 or 2.

4. A method for determining the plasmid content in a quadrivalent avian influenza DNA vaccine, comprising using the primer and probe composition as described in claim 1 or 2, or the detection kit as described in claim 3; in, The quadrivalent DNA vaccine for avian influenza includes recombinant plasmids pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6. The recombinant plasmid pH5-Re15 includes the nucleotide sequence SEQ ID NO: 1, the recombinant plasmid pH5-Re16 includes the nucleotide sequence SEQ ID NO: 2, the recombinant plasmid pH7-Re5 includes the nucleotide sequence SEQ ID NO: 3, and the recombinant plasmid pH7-Re6 includes the nucleotide sequence SEQ ID NO:

4.

5. The method according to claim 4, comprising the following steps: (1) Using the standards of four recombinant plasmids, pH5-Re15, pH5-Re16, pH7-Re5 and pH7-Re6, as templates, a standard curve was established by performing real-time PCR with the primer and probe combination described above. (2) Take the finished product of the quadrivalent DNA vaccine of avian influenza to be tested or its diluted working solution as a template, and perform real-time PCR using the primer and probe combination described above; (3) Compare the standard curves of each plasmid and calculate the copy number of each plasmid in the quadrivalent DNA vaccine for avian influenza.

6. The method according to claim 5, wherein, The system of the real-time PCR includes the following components: real-time PCR enzyme, RNase-free H2O, primers, probes, and template.

7. The method according to claim 5, wherein, The reaction procedure for quantitative real-time PCR includes the following steps: digestion; pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 10 seconds, annealing at 60°C for 30 seconds, for a total of 30 cycles, with fluorescence signals collected at the end of each annealing step.

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