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

Abstract

The invention relates to a method for determining the plasmid content in an avian influenza tetravalent DNA vaccine and application. The invention belongs to the field of molecular biology, and aims to provide a TaqMan fluorescent quantitative PCR (Polymerase Chain Reaction) method capable of quantitatively determining the content of each plasmid component in a tetravalent DNA vaccine. Specifically, the invention provides a primer and probe composition for detecting the content of a single plasmid in the avian influenza (H5 + H7) tetravalent DNA vaccine based on TaqMan fluorescent quantitative PCR, and the primer and probe composition comprises primers with nucleotide sequences as shown in SEQ ID NO: 5-6, 8-9, 11-12 and 14-15, and TaqMan probes with nucleotide sequences as shown in SEQ ID NO: 7, 10, 13 and 16. The primer and probe composition can quantitatively determine the content of each plasmid component in the tetravalent DNA vaccine, and has good specificity and repeatability.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology, and particularly relates to a primer and probe combination and kit for detecting the content of a single plasmid in a trivalent DNA vaccine for avian influenza based on TaqMan fluorescent quantitative PCR. BACKGROUND

[0002] Avian influenza is a major zoonotic infectious disease that seriously endangers the poultry industry and public health safety. Some strains of H5 and H7 subtype highly pathogenic avian influenza virus can cause 100% mortality in poultry. Vaccine immunization is an important strategy for preventing and controlling avian influenza, especially highly pathogenic avian influenza.

[0003] DNA vaccines have high biological safety, and have the advantages of simple preparation, easy construction into combination vaccines and multivalent vaccines, etc. They are a kind of genetic engineering vaccine with good application prospect for preventing and controlling highly pathogenic avian influenza (HPAI) at present, and can quickly respond to virus variation. In order to comprehensively prevent and effectively prevent the 2.3.4.4h branch, 2.3.4.4b branch H5 subtype antigen variant strain and A branch and B branch H7 subtype antigen variant strain currently monitored in China, we developed an avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) and carried out immunization efficacy evaluation. The results showed that the avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) has good immunization efficacy, can provide complete immune protection against the current epidemic H5 subtype avian influenza virus and H7 subtype avian influenza virus, and can be used for avian influenza prevention and control practice in China, thereby providing strong technical support for the healthy development of the poultry industry in China. The quadrivalent vaccine is mixed by four kinds of plasmids, and the content of the four components needs to be determined for quality inspection of the finished product. The conventional (ultra) microspectrophotometer detection method can only determine the total content, and cannot distinguish and determine the content of the four components.

[0004] TaqMan probe method fluorescent quantitative PCR technology has the advantages of convenient operation, high sensitivity and good specificity, and has been widely used in the field of detection of veterinary pathogenic microorganisms. SUMMARY

[0005] Therefore, in order to determine the content of pH5-Re15 strain, pH5-Re16 strain, pH7-Re5 strain and pH7-Re6 strain after equal mixing of the avian influenza (H5+H7) tetravalent DNA vaccine (pH5-Re15 strain+pH5-Re16 strain+pH7-Re5 strain+pH7-Re6 strain), a TaqMan fluorescent quantitative PCR method capable of quantitatively determining and distinguishing the content of each plasmid component in the tetravalent DNA vaccine is established.

[0006] According to one aspect of the present application, a primer and probe composition for detecting the content of a single plasmid in an avian influenza (H5+H7) tetravalent DNA vaccine based on TaqMan fluorescent quantitative PCR is provided, which comprises a primer with the nucleotide sequence shown in SEQ ID NO: 5-6, 8-9, 11-12, 14-15, and a TaqMan probe with the nucleotide sequence shown in SEQ ID NO: 7, 10, 13, 16.

[0007] The avian influenza (H5+H7) tetravalent DNA vaccine comprises pH5-Re15 strain, pH5-Re16 strain, pH7-Re5 strain and pH7-Re6 strain.

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

[0009] In a specific embodiment of the present application, 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 present application, a detection kit is provided, which comprises the primer and probe composition.

[0011] In a third aspect of the present application, a method for detecting the content of a single plasmid in an avian influenza (H5+H7) tetravalent DNA vaccine based on TaqMan fluorescent quantitative PCR is provided, which comprises using the primer and probe composition or the detection kit.

[0012] In a specific embodiment of the present application, the method comprises the following steps:

[0013] (1) Using standard samples of the four plasmids pH5-Re15, pH5-Re16, pH7-Re5 and pH7-Re6 as templates, performing fluorescent quantitative PCR with the primer and probe composition described above to establish a standard curve;

[0014] (2) Taking the prepared avian influenza (H5+H7) tetravalent DNA vaccine or its diluted use solution as a template, and using the above-mentioned primer and probe combination to perform fluorescent quantitative PCR;

[0015] (3) Comparing the standard curves of the various plasmids, the copy number of each plasmid in the avian influenza (H5+H7) tetravalent DNA vaccine is calculated.

[0016] In a specific embodiment of the present application, in the method for detecting the content of a single plasmid in the avian influenza (H5+H7) tetravalent DNA vaccine, the content of the pH5-Re15 recombinant plasmid is detected using primers as shown in SEQ ID NO: 5-6 and a probe 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 a probe 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 a probe 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 a probe as shown in SEQ ID NO: 16.

[0017] In a specific embodiment of the present application, the system of the fluorescent quantitative PCR comprises the following components: fluorescent quantitative PCR enzyme (2X), the amount of which can be routinely adjusted according to actual needs, for example 5-15 μl, for example 8, 10, 12 μl; RNase-free H2O, the amount of which can be routinely adjusted according to actual needs, for example 5-10 μl, for example 7 μl; upstream primer, the concentration and volume of which can be routinely adjusted according to actual needs, the concentration of which is 5-15 pmol, for example 10 pmol, and the volume of which is 0.2-0.8 μl, for example 0.4 μl; downstream primer, the concentration and volume of which can be routinely adjusted according to actual needs, the concentration of which is 5-15 pmol, for example 10 pmol, and the volume of which is 0.2-0.8 μl, for example 0.4 μl; probe, the concentration and volume of which can be routinely adjusted according to actual needs, the concentration of which is 5-15 pmol, for example 10 pmol, and the volume of which is 0.1-0.8 μl, for example 0.2 μl; template, the amount of which 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 application, the reaction procedure of the fluorescent quantitative PCR comprises the following steps: 1) digestion, time 1-5 minutes, temperature 35-40℃, for example 37℃ contamination digestion for 2 minutes; 2) pre-denaturation, temperature 90-100℃, time 3-6 minutes, for example 95℃ pre-denaturation for 5 minutes; 3) denaturation, temperature 90-100℃, time 8-12 seconds, for example 95℃ denaturation for 10 seconds, 4) annealing, temperature 50-65℃, time 25-35 seconds, for example 60℃ annealing for 30 seconds, a total of 30 cycles, and the FAM fluorescence signal is collected at the end of each annealing step.

[0019] The avian influenza (H5+H7) quadrivalent DNA vaccine is a quadrivalent DNA vaccine (pH5-Re15 strain+pH5-Re16 strain+pH7-Re5 strain+pH7-Re6 strain) against H5 and H7 subtypes, which comprises recombinant plasmid pH5-Re15 strain and recombinant plasmid pH5-Re16 strain against H5N6 subtype, and recombinant plasmid pH7-Re5 strain and recombinant plasmid pH7-Re6 strain against H7N9 subtype; the above four kinds of recombinant plasmids are mixed in equal concentration and equal volume, and the avian influenza (H5+H7) quadrivalent DNA vaccine is obtained. The avian influenza (H5+H7) quadrivalent DNA vaccine provides complete immune protection against the current epidemic different H5 subtype avian influenza virus and H7 subtype avian influenza virus lethal challenge, and has good immunopotency.

[0020] Effects

[0021] The present application provides a primer and probe composition which can be used for detecting the content of single plasmid in the avian influenza (H5+H7) quadrivalent DNA vaccine, the detection method of the present application has high accuracy and good repeatability, and is an important method for quality control of the avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain+pH5-Re16 strain+pH7-Re5 strain+pH7-Re6 strain) product. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The specific sequence region of the four sequences of the optimized HA gene of pH5-Re15 strain, pH5-Re16 strain, pH7-Re5 strain and pH7-Re6 strain is 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, probe specificity test are shown; A: pH5-Re15 strain as DNA template; B: pH5-Re16 strain as DNA template; C: pH7-Re5 strain as DNA template; D: pH7-Re6 strain as DNA template;

[0024] Figure 3 The test results of different dilution standards are shown; A1 is the amplification curve with pH5-Re15 strain as DNA template, the leftmost of the amplification curve is 10 0 fold dilution, from left to right are 10 -1 , 10 -2 , 10 -3 , 10 -4 fold dilution amplification curve; A2 is positive / negative control, the positive control takes pH5-Re15 strain as DNA template, and the negative control takes ddH2O as template; B1 is the amplification curve with pH5-Re16 strain as DNA template, the leftmost of the amplification curve is 10 0 fold dilution, from left to right are 10 -1 , 10 -2 , 10 -3 , 10 -4 fold dilution amplification curve; B2 is positive / negative control, the positive control takes pH5-Re16 strain as DNA template, and the negative control takes ddH2O as template; C1 is the amplification curve with pH7-Re5 strain as DNA template, the leftmost of the amplification curve is 10 0 fold dilution, from left to right are 10 -1 , 10 -2 , 10 -3 , 10 -4 fold dilution amplification curve; C2 is positive / negative control, the positive control takes pH7-Re5 strain as DNA template, and the negative control takes ddH2O as template; D1 is the amplification curve with pH7-Re6 strain as DNA template, the leftmost of the amplification curve is 10 0 fold dilution, from left to right are 10 -1 , 10 -2 , 10 -3 , 10 -4 fold dilution amplification curve; D2 is positive / negative control, the positive control takes pH7-Re6 strain as DNA template, and the negative control takes ddH2O as template.

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

[0026] Figure 5 The standard pH5-Re15 and pH5-Re16 repeated determination test results are shown. Among them: A1-A4 shows the repeated determination test results of pH5-Re15 strain standard, the leftmost of the amplification curve is 10 0 dilution, from left to right are 10 -1 , 10 -2 , 10 -3 , 10 -4 times dilution amplification curve; A1-A3 is to use pH5-Re15 strain as DNA template, A4 positive control uses pH5-Re15 strain as DNA template, and negative control uses ddH2O as template. 0 dilution, from left to right are 10 -1 , 10 -2 , 10 -3 , 10 -4 times dilution amplification curve; B1-B3 is to use pH5-Re16 strain as DNA template, B4 positive control uses pH5-Re16 strain as DNA template, and negative control uses ddH2O as template.

[0027] Figure 6Standard pH7-Re5 and pH7-Re6 repeated determination test results are shown. Among them: C1-C4 shows the repeated determination test results of pH7-Re5 strain standard, the leftmost of the amplification curve is 10 0 fold dilution, from left to right are 10 -1 , 10 -2 , 10 -3 , 10 -4 fold dilution amplification curve; C1-C3 is pH7-Re5 strain as DNA template, C4 positive control is pH7-Re5 strain as DNA template, and negative control is ddH2O as template. 0 D1-D4 shows the repeated determination test results of pH7-Re6 strain standard, the leftmost of the amplification curve is 10 -1 fold dilution, from left to right are 10 -2 , 10 -3 , 10 -4 fold dilution amplification curve; D1-D3 is pH7-Re6 strain as DNA template, D4 positive control is pH7-Re6 strain as DNA template, and negative control is ddH2O as template.

[0028] Figure 7 Standard curves of three repeated determinations of standards pH5-Re15 and pH5-Re16 are shown. Among them: A1-A3 shows the standard curves of three repeated determinations of pH5-Re15 strain standard by primer Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe, with pH5-Re15 strain as DNA template, from left to right 5 points are 10 0 -10 -4 fold dilution. B1-B3 shows the standard curves of three repeated determinations of pH5-Re16 strain standard by primer Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-Probe, with pH5-Re16 strain as DNA template, from left to right 5 points are 10 0 -10 -4Dilution. Wherein, the slope of C1 is -3.675, R2 is 1, Eff% is 122.272; the slope of A2 is -3.889, R2 is 0.999, Eff% is 80.763; the slope of A3 is -2.657, R2 is 0.991, Eff% is 137.884; the slope of B1 is -3.865, R2 is 0.994, Eff% is 81.434; the slope of B2 is -3.675, R2 is 1, Eff% is 87.106; the slope of B3 is -3.797, R2 is 0.998, Eff% is 83.386.

[0029] Figure 8 The standard curve of the standard pH7-Re5 and pH7-Re6 three repeated measurements is shown, wherein: C1-C3 shows the standard curve of the primer Re5 F3-Forward, Re5 R3-Reverse, probe Re5 P3-probe for the standard pH7-Re5 three repeated measurements, wherein the pH7-Re5 strain is used as the DNA template, and the five points from left to right are 10 0 -10 -4 Dilution. D1-D3 shows the standard curve of the primer Re6 F4-Forward, Re6 R4-Reverse, probe Re6 P4-probe for the standard pH7-Re6 three repeated measurements, wherein the pH7-Re6 strain is used as the DNA template, and the five points from left to right are 10 0 -10 -4 Dilution. Wherein, the slope of C1 is -3.483, R2 is 0.992, Eff% is 93.698; the slope of C2 is -2.93, R2 is 0.992, Eff% is 119.413; the slope of C3 is -3.538, R2 is 0.999, Eff% is 91.696; the slope of D1 is -2.888, R2 is 0.993, Eff% is 121.971; the slope of D2 is -2.427, R2 is 0.997, Eff% is 158.255; the slope of D3 is -2.912, R2 is 0.993, Eff% is 120.481.

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

[0031] Figure 10 The standard curve of the first repeatability detection is shown, and from left to right, the 5 points are 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, Re6 R4-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 Ct value test results of the first fluorescent quantitative PCR method for detecting the content of different batches of avian influenza (H5+H7) tetravalent DNA vaccine finished products are shown. Among them, A1-A2: the detection results of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe; A1 takes the avian influenza (H5+H7) tetravalent DNA vaccine finished product as the DNA template, and A2 is the positive / negative control, wherein the positive control takes pH5-Re15 strain as the DNA template, and the negative control takes ddH2O as the template. B1-B2: the detection results of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe; B1 takes the avian influenza (H5+H7) tetravalent DNA vaccine finished product as the DNA template, and B2 is the positive / negative control, wherein the positive control takes pH5-Re16 strain as the DNA template, and the negative control takes ddH2O as the template. C1-C2: the detection results of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe; C1 takes the avian influenza (H5+H7) tetravalent DNA vaccine finished product as the DNA template, and C2 is the positive / negative control, wherein the positive control takes pH7-Re5 strain as the DNA template, and the negative control takes ddH2O as the template. D1-D2: the detection results of primers Re6 F4-Forward, Re6 R4-Reverse, and probe Re6 P4-probe; D1 takes the avian influenza (H5+H7) tetravalent DNA vaccine finished product as the DNA template, and D2 is the positive / negative control, wherein the positive control takes pH7-Re6 strain as the DNA template, and the negative control takes ddH2O as the template.

[0033] Figure 12 The standard curve of the second repeatability detection is shown, and the 5 points from left to right are 10 0 -10 -4Dilution; wherein, A: detection results of primer Re15 F1-Forward, Re15 R1-Reverse, probe Re15 P1-Probe; B: detection results of primer Re16 F1-Forward, Re16 R1-Reverse, probe Re16 P1-probe; C: detection results of primer Re5 F3-Forward, Re5 R3-Reverse, probe Re5 P3-probe; D: detection results of primer Re6 F4-Forward, Re6 R4-Reverse, probe Re6 P4-probe. Wherein, the slope of A is -3.314, R2 is 1, Eff% is 100.315; the slope of B is -3.072, R2 is 0.993, Eff% is 111.603; the slope of C is -3.666, R2 is 0.999, Eff% is 87.397; the slope of D is -3.31, R2 is 0.999, Eff% is 100.483.

[0034] Figure 13The Ct value test results of the second fluorescent quantitative PCR method for detecting the content of different batches of avian influenza (H5+H7) quadrivalent DNA vaccine finished products are shown; among them, A1-A2: detection results of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15 P1-Probe; A1 takes the avian influenza (H5+H7) quadrivalent DNA vaccine finished product as the DNA template, and A2 is the positive / negative control, wherein the positive control takes pH5-Re15 strain as the DNA template, and the negative control takes ddH2O as the template. B1-B2: detection results of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe; B1 takes the avian influenza (H5+H7) quadrivalent DNA vaccine finished product as the DNA template, and B2 is the positive / negative control, wherein the positive control takes pH5-Re16 strain as the DNA template, and the negative control takes ddH2O as the template. C1-C2: detection results of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5 P3-probe; C1 takes the avian influenza (H5+H7) quadrivalent DNA vaccine finished product as the DNA template, and C2 is the positive / negative control, wherein the positive control takes pH7-Re5 strain as the DNA template, and the negative control takes ddH2O as the template. D1-D2: detection results of primers Re6 F4-Forward, Re6 R4-Reverse, and probe Re6 P4-probe; D1 takes the avian influenza (H5+H7) quadrivalent DNA vaccine finished product as the DNA template, and D2 is the positive / negative control, wherein the positive control takes pH7-Re6 strain as the DNA template, and the negative control takes ddH2O as the template.

[0035] Figure 14 The standard curve of the third repeatability detection is shown, and the five points from left to right are 10 0 -10 -4Dilution; wherein, A: detection results of primer Re15 F1-Forward, Re15 R1-Reverse, probe Re15 P1-Probe; B: detection results of primer Re16 F1-Forward, Re16 R1-Reverse, probe Re16 P1-probe; C: detection results of primer Re5 F3-Forward, Re5 R3-Reverse, probe Re5 P3-probe; D: detection results of primer Re6 F4-Forward, Re6 R4-Reverse, probe Re6 P4-probe. The slope of A is -3.278, R2 is 0.997, Eff% is 101.886; the slope of B is -3.231, R2 is 1, Eff% is 103.926; the slope of C is -3.503, R2 is 0.999, Eff% is 92.961; the slope of D is -3.532, R2 is 0.999, Eff% is 91.912.

[0036] Figure 15The Ct value test results of different batches of avian influenza (H5+H7) quadrivalent DNA vaccine finished product content by the third fluorescence quantitative PCR method; among them, A1-A2: the detection results of primers Re15 F1-Forward, Re15 R1-Reverse, and probe Re15P1-Probe; A1 takes the avian influenza (H5+H7) quadrivalent DNA vaccine finished product as the DNA template, and A2 is the positive / negative control, wherein the positive control takes pH5-Re15 strain as the DNA template, and the negative control takes ddH2O as the template. B1-B2: the detection results of primers Re16 F1-Forward, Re16 R1-Reverse, and probe Re16 P1-probe; B1 takes the avian influenza (H5+H7) quadrivalent DNA vaccine finished product as the DNA template, and B2 is the positive / negative control, wherein the positive control takes pH5-Re16 strain as the DNA template, and the negative control takes ddH2O as the template. C1-C2: the detection results of primers Re5 F3-Forward, Re5 R3-Reverse, and probe Re5P3-probe; C1 takes the avian influenza (H5+H7) quadrivalent DNA vaccine finished product as the DNA template, and C2 is the positive / negative control, wherein the positive control takes pH7-Re5 strain as the DNA template, and the negative control takes ddH2O as the template. D1-D2: the detection results of primers Re6 F4-Forward, Re6 R4-Reverse, and probe Re6 P4-probe; D1 takes the avian influenza (H5+H7) quadrivalent DNA vaccine finished product as the DNA template, and D2 is the positive / negative control, wherein the positive control takes pH7-Re6 strain as the DNA template, and the negative control takes ddH2O as the template. DETAILED DESCRIPTION

[0037] EXPERIMENTAL MATERIALS

[0038] 1. Reagents

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

[0040] 2. Plasmid

[0041] The recombinant plasmids pH5-Re15 strain, pH5-Re16 strain, pH7-Re5 strain, and pH7-Re6 strain were prepared, identified, and preserved by the National Avian Influenza Reference Laboratory of 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 codon optimized to obtain the optimized HA gene sequence SEQ ID NO: 1 (gene opti-HeNHA5), which was then connected with the pCAGGoptiHA5 vector (see CN114524862A for the map information of plasmid pCAGGoptiHA5) backbone to obtain the recombinant plasmid.

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

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

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

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

[0047] The preparation method is as follows:

[0048] The recombinant plasmids pH5-Re15 strain, pH5-Re16 strain, and pH7-Re5 strain and pH7-Re6 strain were mixed at the same concentration and volume to prepare the avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain), wherein the immunization dose of each plasmid was prepared as needed.

[0049] 4. Instrument: QuantStudio5 fluorescence quantitative PCR instrument of American Applied Biosystems Company.

[0050] Design of TaqMan fluorescent quantitative PCR primers and probes

[0051] The optimized HA gene sequences (i.e., SEQ ID NO: 1-4) based on the pH5-Re15 strain, pH5-Re16 strain, pH7-Re5 strain and pH7-Re6 strain were subjected to homology analysis comparison using DNAStar biological software, and the sequence difference regions specific to the four HA gene sequences were found. Primer Express 3.0 software was used to design 20 groups of primers and TaqMan probes for the differentially significant difference sequence fragments, and the positions are shown in Table 1. Figure 1 The primer and probe sequences are shown in Table 1. The probe is labeled with FAM at the 5' end and BHQ1 or MGB quenching group at the 3' end, and is synthesized by Saivon Biotechnology Co., Ltd.

[0052] Table 1 Primer and probe sequences

[0053]

[0054] Table 1 Primer and probe sequences (continued)

[0055]

[0056] Establishment of TaqMan fluorescent quantitative PCR method

[0057] According to the Qiagen plasmid large extraction kit Endofree ® Plasmid Maxi Kit (10) instruction manual to extract plasmid.

[0058] 1. Reaction system and conditions of TaqMan fluorescent quantitative PCR: The TaqMan fluorescent quantitative PCR method uses fluorescent quantitative kit reagents to complete in a 20 μl reaction system, and the components and their volumes are as follows:

[0059]

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

[0061] 2. Specific detection of TaqMan fluorescent quantitative PCR primer and probe combination

[0062] The other reaction conditions were unchanged when performing PCR reaction. The primers and TaqMan probes of 20 groups were used respectively to detect the primers and TaqMan probes of four single plasmids pH5-Re15, pH5-Re16, pH7-Re5 and pH7-Re6 by TaqMan fluorescent quantitative PCR. The primers and probes with specificity were screened according to the amplification curve. The results were as follows:

[0063] The 20 groups of primers and probes in Table 1 were used to perform TaqMan fluorescent PCR detection with pH5-Re15 strain, pH5-Re16 strain, pH7-Re5 strain and pH7-Re6 strain as DNA templates, and a negative control was set up. The results showed that only the samples corresponding to the appropriate primers and probes of each group appeared standard amplification curve. Among them, the Ct value of A1 group primer Re15 F1-Forward, Re15 R1-Reverse and probe Re15 P1-Probe was 13.216 when pH5-Re15 strain was used as DNA template; the Ct value of B1 group primer Re16 F1-Forward, Re16 R1-Reverse and probe Re16 P1-Probe was 13.053 when pH5-Re16 strain was used as DNA template; the Ct value of C3 group primer Re5 F3-Forward, Re5 R3-Reverse and probe Re5 P3-Probe was 14.363 when pH7-Re5 strain was used as DNA template; the Ct value of D4 group primer Re6 F4-Forward, Re6 R4-Reverse and probe Re6 P4-Probe was 11.162 when pH7-Re6 strain was used as DNA template; the other samples showed straight line as No Ct value, and the results were shown in Figure 2 , which indicated that the primers and probes of A1, B1, C3 and D4 groups had good specificity and could only amplify the corresponding samples without non-specific reaction to the other two plasmid samples. The amplification effect of the other 16 sets of primers and probes had different disadvantages compared with the above described primers and probes, and was not used in subsequent experiments.

[0064] Example 3 Detection of the content of each plasmid in avian influenza (H5+H7) tetravalent DNA vaccine by TaqMan fluorescent quantitative PCR method

[0065] 1. Preparation of standard: The plasmids pH5-Re15, pH5-Re16, pH7-Re5 and pH7-Re6 were extracted by endotoxin-free plasmid extraction kit, 1 µg of each was divided into each tube, vacuum dried, and prepared into standard, which was dissolved in 100 µl ddH2O (10 ng / µl) when used.

[0066] 2. Establishment of standard curve: The four plasmid standards were diluted according to 10-fold gradient in turn, 100 -10 -4 After gradient dilution, the selected primer probe was used for amplification as the sample to be tested, and the standard curve of each plasmid was drawn. The results are as follows:

[0067] The copy number of the standard sample was 1.4 x 10 9 After 10-fold gradient dilution of the plasmid with a copy number of 1.4 x 10 0 -10 -4 dilution for detection, and the results showed that there was a good linear correlation, and the slopes of the four plasmids were -3.287, -3.496, -3.512, and -3.853, respectively, and the correlation coefficients R 2 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 standard samples at different dilutions

[0069]

[0070] 3. Standard curve repeatability experiment According to the method of "2. Establishment of standard curve", amplification was repeated 3 times under the same conditions, and the reproducibility of the standard curve graph and the Ct value was observed. The results are as follows:

[0071] The copy number of the standard sample was 1.4 x 10 9 After 10-fold gradient dilution of the plasmid with a copy number of 1.4 x 10 0 -10 -4 dilution for detection, and the results showed that there was a good linear correlation, and the slopes of the four plasmids were -3.287, -3.496, -3.512, and -3.853, respectively, and the correlation coefficients R 2 were 0.996, 0.998, 0.999, and 0.995, respectively. The results are shown in Table 2, Figures 5-8 , indicating that the amplification stability of the standard curve was high.

[0072] Table 3 Ct value and coefficient of variation of repeatability detection of standard samples at different dilutions

[0073]

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

[0075] 4. TaqMan fluorescent quantitative PCR method for detecting the copy number of each plasmid in avian influenza (H5+H7) tetravalent DNA vaccine: the 2025001 batch vaccine sample and its diluted use solution were respectively diluted by 10 times gradient, and 10 -2 copies / μl, and the content was calculated.

[0076] The detection results of TaqMan fluorescent quantitative PCR method for different content of avian influenza (H5+H7) tetravalent DNA vaccine sample are as follows: the 2025001 batch of vaccine finished product was diluted to 10 -2 copies / μl, and the content was calculated. Figure 9 .

[0077] Table 4 Ct value results of fluorescent quantitative PCR method for detecting the content of avian influenza (H5+H7) tetravalent DNA vaccine

[0078]

[0079] A: detection result of primer Re15 F1-Forward, Re15 R1-Reverse, probe Re15 P1-Probe; B: detection result of primer Re16 F1-Forward, Re16 R1-Reverse, probe Re16 P1-probe; C: detection result of primer Re5 F3-Forward, Re5 R3-Reverse, probe Re5 P3-probe; D: detection result of primer Re6 F4-Forward, Re6 R4-Reverse, probe Re6 P4-probe.

[0080] Table 5 Copy number calculation results of the content of avian influenza (H5+H7) quadrivalent DNA vaccine detected by the fluorescent quantitative PCR method

[0081]

[0082] Unit: copy / μl

[0083] A: detection result of primer Re15 F1-Forward, Re15 R1-Reverse, probe Re15 P1-Probe; B: detection result of primer Re16 F1-Forward, Re16 R1-Reverse, probe Re16 P1-probe; C: detection result of primer Re5 F3-Forward, Re5 R3-Reverse, probe Re5 P3-probe; D: detection result of primer Re6 F4-Forward, Re6 R4-Reverse, probe Re6 P4-probe.

[0084] Table 6 Content results of each plasmid in the avian influenza (H5+H7) quadrivalent DNA vaccine sample

[0085]

[0086] Unit: ng / μl

[0087] A: detection result of primer Re15 F1-Forward, Re15 R1-Reverse, probe Re15 P1-Probe; B: detection result of primer Re16 F1-Forward, Re16 R1-Reverse, probe Re16 P1-probe; C: detection result of primer Re5 F3-Forward, Re5 R3-Reverse, probe Re5 P3-probe; D: detection result of primer Re6 F4-Forward, Re6 R4-Reverse, probe Re6 P4-probe.

[0088] The above results show that the established TaqMan fluorescent quantitative PCR method for detecting single plasmid in avian influenza (H5+H7) tetravalent DNA vaccine has high accuracy, the content of the four plasmids (pH5-Re15, pH5-Re16, pH7-Re5, pH7-Re6) detected in the avian influenza (H5+H7) tetravalent DNA vaccine is consistent with the actual content of each plasmid in the avian influenza (H5+H7) tetravalent DNA vaccine; good repeatability, the coefficient of variation (CV) of the repeated detection of the standard is less than 5%, the method is stable; high sensitivity, the quantitative method can detect as low as 0.001 ng / μl, which fully meets the content detection requirements of the avian influenza (H5+H7) tetravalent DNA vaccine; strong specificity, not interfered by homologous sequences, and can effectively distinguish the four plasmids (pH5-Re15, pH5-Re16, pH7-Re5, pH7-Re6).

[0089] Example 4 Repeatability test of TaqMan fluorescent quantitative PCR method

[0090] Take 200 μl of each of three batches of avian influenza (H5+H7) tetravalent DNA vaccine finished products as detection samples, dilute the samples to 10 -2 ng / μl, and perform fluorescent quantitative PCR on each batch of 3 repeats by 3 different operators on different instruments, detect the Ct value, compare the standard curve of each plasmid, calculate the copy number of each plasmid in the finished vaccine, and calculate the content of each plasmid according to the copy number of each plasmid. On this basis, the inter-batch and intra-batch coefficients of variation are calculated.

[0091] The three batches of avian influenza (H5+H7) tetravalent DNA vaccine finished products were repeatedly detected by TaqMan fluorescent quantitative PCR for three times, respectively by 3 different operators on different instruments. The results are as follows:

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

[0093] Table 7 Ct value results of detecting the content of each plasmid in the vaccine finished product by fluorescent quantitative PCR method

[0094]

[0095] Table 8 Calculation results of copy number of each plasmid in the vaccine finished product when detecting the content of each plasmid by fluorescent quantitative PCR method (10 8 copies / µl)

[0096]

[0097] Table 9 The content of each plasmid (ng / μΐ) and the coefficient of variation in different batches of vaccine products

[0098]

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

[0100] Table 10 Ct value results when detecting the content of each plasmid in vaccine products by fluorescence quantitative PCR method

[0101]

[0102] Table 11 Calculation results of copy number when detecting the content of each plasmid in vaccine products by fluorescence quantitative PCR method (10 8 copies / μΐ)

[0103]

[0104] Table 12 The content of each plasmid (ng / μΐ) and the coefficient of variation in different batches of vaccine products

[0105]

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

[0107] Table 13 Ct value results when detecting the content of each plasmid in vaccine products by fluorescence quantitative PCR method

[0108]

[0109] Table 14 Calculation results of copy number when detecting the content of each plasmid in vaccine products by fluorescence quantitative PCR method (10 8 copies / μΐ)

[0110]

[0111] Table 15 The content of each plasmid (ng / μΐ) and the coefficient of variation in different batches of vaccine products

[0112]

[0113] The batch variation coefficient of the three repeated tests is within 15%, and the within-batch variation coefficient is within 20%. The results show that the variation coefficient of the TaqMan fluorescent quantitative PCR method established by us is within 20%, and the method has good repeatability.

[0114] Example 5 Determination of the determination range of the fluorescent quantitative PCR identification method for each component in the avian influenza (H5+H7) quadrivalent DNA vaccine

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

[0116] Table 16 Determination range of the fluorescent quantitative PCR method for detecting the content of each plasmid in the avian influenza (H5+H7) quadrivalent DNA vaccine

[0117]

[0118] Discussion

[0119] In order to accurately detect the content of the four plasmids pH5-Re15 strain, pH5-Re16 strain, and pH7-Re5 strain, and pH7-Re6 strain in the avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) mixed in equal volumes, a specific TaqMan fluorescent quantitative PCR detection method was established.

[0120] According to the laboratory sequencing results, homology analysis and comparison are carried out by using DNAStar biological software, specific sequence regions of HA gene sequences of the pH5-Re15 strain, the pH5-Re16 strain, the pH7-Re5 strain and the pH7-Re6 strain are found, 20 groups of primers and TaqMan probes for the insertion site are designed by using PrimerExpress 3.0 software. The TaqMan fluorescent PCR reaction is carried out by using the 20 groups of primers and probes and taking the pH5-Re15 strain, the pH5-Re16 strain, the pH7-Re5 strain and the pH7-Re6 strain as templates, it is found that the amplification curves can appear for 4 groups of primers, and only the samples corresponding to each group of primers and probes appear standard amplification curves, and no amplification curve appears for other samples, which indicates that each group of primers and probes has good specificity.

[0121] The copy number is 1.4 x 10 9 After 10-fold gradient dilution of 1.4 x 10 0 -10 -4 copies / ul of the plasmid, 10 2 copies / ul of the plasmid are taken for detection, and the results show that there is a good linear correlation, the slopes of the four plasmids are -3.287, -3.496, -3.512 and -3.853 respectively, and the correlation coefficients R -2 are 0.996, 0.998, 0.999 and 0.995 respectively. The repeatability test of the standard curve proves that the prepared standard product is relatively stable, the Ct values are good in reproducibility when amplified under the same condition for 3 times, and the coefficient of variation is less than 10%, so it can be seen that the repeatability of the standard curve preparation of the detection method is good, and the stability is high.

[0122] The 2025001 batch of avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15 strain + pH5-Re16 strain + pH7-Re5 strain + pH7-Re6 strain) finished product and the use liquid diluted therefrom are taken as samples, and are diluted to 10 -2 copies / ul respectively, and the Ct values are detected, the standard curves of the plasmids are compared, the copy numbers of the plasmids in the avian influenza (H5+H7) quadrivalent DNA vaccine with different contents are calculated, and the results show that the plasmid content calculated according to the detected copy number of the plasmid is similar to the content of each plasmid when the avian influenza (H5+H7) quadrivalent DNA vaccine is prepared, which indicates that the detection method has high accuracy.

[0123] The TaqMan fluorescent quantitative PCR detection is carried out on three batches of avian influenza (H5+H7) quadrivalent DNA vaccine finished products, the vaccine samples are diluted to 10 -2Each batch was repeated 3 times, respectively by 3 different operators on different instruments. The Ct value was detected by fluorescence quantitative PCR, and the standard curve of each plasmid was compared to calculate the copy number and content of each plasmid in the finished vaccine, and the coefficient of variation between batches and within batches was calculated. Results: In the three repeated tests, the coefficient of variation between batches and within batches was less than 20%, indicating that the established TaqMan fluorescence quantitative PCR detection method had good repeatability.

[0124] On the basis of repeated detection, in the avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15+pH5-Re16+pH7-Re5+pH7-Re6), the content of pH5-Re15 should be in the range of 0.249±0.026 mg per milliliter, the content of pH5-Re16 should be in the range of 0.244±0.027 mg per milliliter, the content of pH7-Re5 should be in the range of 0.249±0.023 mg per milliliter, and the content of pH7-Re6 should be in the range of 0.214±0.012 mg per milliliter.

[0125] Conclusion

[0126] This experiment successfully established a TaqMan fluorescence quantitative PCR detection method for the content of four plasmids in the avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15+pH5-Re16+pH7-Re5+pH7-Re6). This method is an important identification method in the quality standard of the avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15+pH5-Re16+pH7-Re5+pH7-Re6), and is an important method for quality control of the avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15+pH5-Re16+pH7-Re5+pH7-Re6) product. This detection method has good specificity and repeatability. When detecting the content of each component in the avian influenza (H5+H7) quadrivalent DNA vaccine (pH5-Re15+pH5-Re16+pH7-Re5+pH7-Re6) by fluorescence quantitative PCR, the content of pH5-Re15 should be in the range of 0.249±0.026 mg per milliliter, the content of pH5-Re16 should be in the range of 0.244±0.027 mg per milliliter, the content of pH7-Re5 should be in the range of 0.249±0.023 mg per milliliter, and the content of pH7-Re6 should be in the range of 0.214±0.012 mg per milliliter.

Claims

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

4.

2. The primer and probe composition of claim 1, wherein, The 5' end of the TaqMan probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group. 3.A detection kit comprising the primer and probe composition of claim 1 or 2. 4.A method for determining the plasmid content of a quadrivalent avian influenza DNA vaccine, comprising using the primer and probe composition of claim 1 or 2, or the detection kit of claim 3. wherein The quadrivalent avian influenza DNA vaccine comprises pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6. The pH5-Re15 comprises the nucleotide sequence SEQ ID NO: 1, the pH5-Re16 comprises the nucleotide sequence SEQ ID NO: 2, the pH7-Re5 comprises the nucleotide sequence SEQ ID NO: 3, and the pH7-Re6 comprises the nucleotide sequence SEQ ID NO:

4. 5.The method of claim 4, comprising the following steps: (1) using standard samples of the four plasmids pH5-Re15, pH5-Re16, pH7-Re5, and pH7-Re6 as templates, and performing fluorescent quantitative PCR with the primer and probe composition to establish a standard curve; (2) using the primer and probe composition, performing fluorescent quantitative PCR with the detected quadrivalent avian influenza DNA vaccine product or its diluted use solution as a template; (3) comparing the standard curves of each plasmid to calculate the copy number of each plasmid in the quadrivalent avian influenza DNA vaccine.

6. The method of claim 5, wherein, The system of the fluorescent quantitative PCR comprises the following components: fluorescent quantitative PCR enzyme, RNase-free H2O, upstream primer, downstream primer, probe, and template.

7. The method of claim 5, wherein, The reaction program of the fluorescent quantitative PCR comprises the following steps: digestion; 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 10 seconds, 60℃ annealing for 30 seconds, a total of 30 cycles, and collecting the fluorescent signal at the end of each annealing step.

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