Primer probe combination for identifying peste des petits ruminants vaccine strain and wild strain and detection method of peste des petits ruminants vaccine strain and wild strain
By designing a real-time fluorescence dual identification and detection method using specific primers and probes, the problem of distinguishing between small ruminant vaccine strains and wild-type strains in existing technologies has been solved, achieving efficient and low-cost identification and detection with good sensitivity and specificity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-16
- Publication Date
- 2026-04-07
AI Technical Summary
Existing technologies are unable to effectively distinguish between small ruminant vaccine strains and wild-type strains, leading to increased detection complexity and safety risks. Furthermore, most detection technologies cannot differentiate between lineages.
Specific primers and probes were designed to distinguish PPRV vaccine strains from wild-type strains using a real-time fluorescence dual identification detection method. Amplification was performed in the same reaction tube using a pair of primers and two fluorescently labeled probes.
It enables convenient and rapid identification and detection of vaccine strains and wild-type strains, with high sensitivity, specificity and good repeatability, effectively distinguishing different lineages and reducing operational complexity and cost.
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Figure CN121802102A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology detection technology, and in particular relates to a primer-probe combination and detection method for identifying small ruminant vaccine strains and wild-type strains. Background Technology
[0002] Pestos peste des petits ruminants (PPR) is an acute, highly contagious, and deadly disease caused by the peste des petits ruminants virus (PPRV), primarily affecting small ruminants such as sheep and goats. Its clinical features include high fever, necrotizing stomatitis, and bronchopneumonia, and it is characterized by high morbidity and mortality. This disease is listed as a notifiable disease by the World Organisation for Animal Health (WOAH) and is also a Class A animal disease in my country.
[0003] Pestos petits ruminant virus (PPRV) is a member of the genus Measlesvirus in the family Paramyxoviridae. It is a single-stranded negative-sense RNA virus. Its genome encodes six structural proteins (N, P, M, F, H, L) and two non-structural proteins (C, V). Although this virus has only one serotype, based on partial sequence analysis of the F and N genes, it can be divided into four genetic lineages (I, II, III, IV). Lineages I and II are mainly distributed in West and Central Africa, lineage III in East and Middle East, and lineage IV is mainly prevalent in Asia and other regions. The PPR outbreaks in my country from 2007 to 2013 were all caused by wild-type strains of lineage IV, while the commercially available vaccine strains in China belong to lineage II.
[0004] Currently, my country's PPR prevention and control strategy primarily relies on live attenuated vaccine vaccination, supplemented by testing. However, live attenuated vaccines pose potential safety risks such as relapse and viral carriage. Most detection technologies are limited because they cannot distinguish between vaccine strains and wild-type strains. Although a few technologies can achieve broad-based differentiation, they require multiple primers and probes, increasing operational complexity and the risk of primer interference.
[0005] Therefore, establishing a method for identifying and detecting PPRV vaccine strains (lineage II) and wild-type strains (lineage IV) is crucial for the clinical diagnosis and control of this disease. Summary of the Invention
[0006] Based on the nucleotide sequences of the hypervariable region of the H gene of PPRV vaccine strain and wild-type virus strain, this study established a real-time fluorescence dual identification and detection method for PPRV vaccine strain and wild-type virus. This method can achieve convenient, rapid, and accurate identification and detection of PPRV vaccine strain and wild-type virus strain.
[0007] This invention provides a primer pair and probe for identifying small ruminant vaccine strains and wild-type strains, characterized in that it includes an upstream primer and a downstream primer;
[0008] Furthermore, the upstream primer was PPRV-qF: TCGGAAGAACATATACYGTC;
[0009] The downstream primer is PPRV-qR: CRATCTCGAAGACTCTTAA;
[0010] Furthermore, the probe used to detect the small ruminant vaccine strain has the nucleotide sequence FAM-ACCGAGCACCAGTCCAGGTAT-BHQ1;
[0011] Furthermore, the probe used to detect the wild-type peste des petits ruminants virus has the nucleotide sequence HEX-ATCCGAGCACCGATCTAGGTATC-BHQ1.
[0012] This invention provides a qPCR detection method for distinguishing between small ruminant vaccine strains and wild-type strains. The method involves adding primer pairs and probes into the same reaction tube for amplification.
[0013] Furthermore, the reaction system for the detection is: 2×PerfectStart ® II. Probe qPCR Super Mix: 10 μL; forward and reverse primers 10 uM: 1.4 μL each; probe 10 uM: 0.2 μL each; template: 2 μL; DEPC water to bring the total to 20 μL.
[0014] Furthermore, the reaction procedure for the detection is as follows: pre-denaturation at 94℃ for 30 s; denaturation at 94℃ for 5 s; annealing and extension at 58℃ for 30 s, and fluorescence collection for a total of 40 cycles.
[0015] This invention provides a detection reagent for rapid detection of peste des petits ruminants virus, comprising primer pairs and probes.
[0016] This invention provides the use of a reagent in the preparation of a kit for detecting small ruminant vaccine strains and wild-type strains.
[0017] Compared with the prior art, the present invention has the following beneficial effects:
[0018] This study established a real-time fluorescence dual-identification method for the detection of peste des petits ruminants (PPR) vaccine strains and wild-type strains. This method requires only one pair of primers and two probes labeled with different fluorescent groups to achieve the identification and detection of PPR vaccine strains and wild-type strains. Standard curve R 2 All values are greater than 0.998, indicating a good linear relationship; the sensitivity of all values can reach 10. 2 It exhibits high copy number and sensitivity; shows no cross-reactivity with other pathogens, demonstrating strong specificity; and exhibits low coefficients of variation within and between groups, resulting in good reproducibility. The detection rate of artificially simulated samples is significantly higher than that of the nationally recommended PCR detection method, and is completely consistent with commercially available detection kits.
[0019] Through optimization of the reaction system and procedure, and evaluation of sensitivity, specificity, and repeatability, a real-time fluorescence dual identification method for small ruminant vaccine strains and wild-type strains was successfully established. This method exhibits high sensitivity, good specificity and repeatability, and is convenient to operate, low in cost, and can effectively achieve cross-strain identification and detection. Attached Figure Description
[0020] Figure 1 This figure shows the preliminary results of establishing a dual identification and detection method for small ruminant vaccine strains and wild-type strains in Example 1.
[0021] Figure 2 Figure 1 shows the results of primer concentration optimization for the dual identification and detection method of peste des petits ruminants (PPR) vaccine strain and wild-type strain in Example 1. Figure A shows the final primer concentration of 200 nmol / L; Figure B shows 400 nmol / L; Figure C shows 600 nmol / L; Figure D shows 800 nmol / L; Figure E shows 1000 nmol / L; Figure F shows 1200 nmol / L; Figure G shows 1400 nmol / L; Figure H shows 1600 nmol / L; and Figure I shows 1800 nmol / L.
[0022] Figure 3Figure 1 shows the optimized probe concentration results for the dual identification and detection method of peste des petits ruminants (PPR) vaccine strain and wild-type strain in Example 1. Figure A shows the final probe concentrations of the PPR vaccine strain (100 nmol / L) and wild-type PPR strain (100 nmol / L); Figure B shows the final probe concentrations of the PPR vaccine strain (100 nmol / L) and wild-type PPR strain (150 nmol / L); Figure C shows the final probe concentrations of the PPR vaccine strain (100 nmol / L) and wild-type PPR strain (200 nmol / L); Figure D shows the final probe concentrations of the PPR vaccine strain (150 nmol / L) and wild-type PPR strain (100 nmol / L); Figure E shows the final probe concentrations of the PPR vaccine strain (150 nmol / L) and wild-type PPR strain (150 nmol / L); Figure F shows the final probe concentrations of the PPR vaccine strain (150 nmol / L) and wild-type PPR strain (150 nmol / L). Figure G shows the final concentration of the peste des petits ruminants (PPR) vaccine strain probe at 200 nmol / L and the final concentration of the PPR wild-type strain probe at 100 nmol / L. Figure H shows the final concentration of the PPR vaccine strain probe at 200 nmol / L and the final concentration of the PPR wild-type strain probe at 150 nmol / L. Figure I shows the final concentration of the PPR vaccine strain probe at 200 nmol / L and the final concentration of the PPR wild-type strain probe at 200 nmol / L.
[0023] Figure 4 The graph shows the optimized reaction procedure results of the dual identification and detection method for peste des petits ruminants (PPR) vaccine strain and wild-type strain in Example 1. In Figure A, the temperature is 58℃, Figure B is 59℃, Figure C is 60℃, Figure D is 61℃, and Figure E is 62℃.
[0024] Figure 5 Figure A shows the standard curves for the dual identification and detection method of peste des petits ruminants (PPR) vaccine strain and wild-type strain in Example 1. Figure B shows the standard curves for the PPR vaccine strain and wild-type PPR strain.
[0025] Figure 6 Figure A shows the sensitivity evaluation results of the dual identification and detection method for peste des petits ruminants (PPR) vaccine strain and wild-type strain in Example 1. Figure B shows the sensitivity of the PPR vaccine strain and the sensitivity of the PPR wild-type strain.
[0026] Figure 7 This is a graph showing the specificity evaluation results of the dual identification and detection method for small ruminant vaccine strains and wild-type strains in Example 1. Detailed Implementation
[0027] Material
[0028] 2×PerfectStart®ⅡProbe qPCR Super Mix and 2K Marker were purchased from Beijing TransGen Biotech Co., Ltd.; 2×HiAmp PCR Master Mix (Dye) was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd.; RNA Reverse Transcription Kit (RT Easy™ Ⅱ) was purchased from Chengdu Fuji Biotechnology Co., Ltd.; Agarose and SuperRed nucleic acid dye were purchased from Lanjieke Technology Co., Ltd.; FastPure Viral DNA / RNA Mini Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; Small Ruminant Disease Live Vaccine (Clone) was purchased. Nine strains of foot-and-mouth disease (FMD) were purchased from Xinjiang Tiankang Biopharmaceutical Co., Ltd.; the real-time fluorescence quantitative PCR kit for FMD types A and O was purchased from Lanzhou Veterinary Research Biotechnology Co., Ltd.; the PPRV fluorescence quantitative RT-PCR detection kit was purchased from Lanzhou Veterinary Research Biotechnology Co., Ltd.; the goat pox live vaccine was purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd.; the live vaccine for sheep infectious pustular dermatitis (HCE strain) was purchased from Shandong Huahong Bioengineering Co., Ltd.; the nucleic acids of Mycoplasma sheepii, Pasteurella multocida, Proteus mirabilis, Streptococcus, and Mansonia hemolyticus were preserved in our laboratory; and the nucleic acid material of the wild-type strain of peste des petits ruminants was provided by Alashankou Customs Technology Center.
[0029] Example 1
[0030] 1. Construction of recombinant plasmids
[0031] Based on the whole genome sequence analysis of the peste des petits ruminants (PPRV) vaccine strain (HQ197753.1) and wild-type strains (FJ905304.1, KM091959.1, KP260624.1), specific PCR primers were designed with the PPRV H gene as the target gene to amplify the target gene of the PPRV vaccine strain. Primer information is shown in Table 1. The PCR reaction system contained: 7.5 μL of 2×HiAmp PCR MasterMix (Dye), 0.5 μL each of forward and reverse primers, 2 μL of PPRV vaccine strain nucleic acid, and DEPC water to a total volume of 15 μL. The reaction program was: 95℃ pre-denaturation for 5 min; 35 cycles (95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s); and 72℃ final extension for 10 min. The PCR amplification products were analyzed by agarose gel electrophoresis and purified before being ligated into the pMD19-T vector to construct a recombinant plasmid of the peste des petits ruminants (PPR) vaccine strain. The recombinant plasmid of the wild-type PPR strain was constructed by artificially synthesizing the corresponding fragment of the H gene (KM091959.1) by Xinjiang Youkang Biotechnology Co., Ltd., and cloning it into the pUC57 vector.
[0032] Table 1. Primer sequences for Small Ruminant Disease PCR
[0033]
[0034] 2. Using the H gene and recombinant plasmid sequences of the peste des petits ruminants (PPR) vaccine strain and wild-type strain as references, qPCR primers and identification probes were designed. Both primers and probes were synthesized by Xinjiang Youkang Biotechnology Co., Ltd. The primer and probe sequences are shown in Table 2.
[0035] Table 2. Primer and probe sequences for qPCR of small ruminant disease vaccine strain and wild-type strain.
[0036]
[0037] 3. Establishment of a dual identification and detection method for peste des petits ruminants (PPR) vaccine strains and wild-type strains.
[0038] Using recombinant plasmids of the peste des petits ruminants (PPR) vaccine strain and wild-type virus strain as positive controls, and DEPC water as a negative control, PPR qPCR primers and two differential probes were placed in the same reaction system to preliminarily establish a dual differential detection method for PPR vaccine strain and wild-type virus strain. The reaction system consisted of 2×PerfectStart... ® II. Probe qPCR Super Mix: 10 μL, forward and reverse primers (10 μM): 0.4 μL each, probe (10 μm): 0.4 μL each, template: 2 μL, DEPC water to 20 μL. The reaction program was set as follows: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 5 s, 60℃ annealing and extension for 30 s, for a total of 40 cycles, and fluorescence was collected. The preliminary results of the dual identification and detection method for peste des petits ruminants vaccine strain and wild-type strain are as follows. Figure 1 As shown in the diagram, in the dual-reaction system, when the recombinant plasmid of the peste des petits ruminants (PPR) vaccine strain was used as a template, only the FAM channel produced fluorescence, and the negative control showed no fluorescence signal; when the recombinant plasmid of the PPR wild-type strain was used as a template, only the HEX channel produced fluorescence, and the negative control showed no fluorescence. The results indicate that this method is feasible and can effectively distinguish between the vaccine strain and the wild-type strain using two identification probes in the same reaction system.
[0039] (1) To improve amplification efficiency, the reaction system of the initially established dual identification detection method was optimized. Nine gradients were set in the range of 200-1800 nmol / L for the final concentrations of upstream and downstream primers (A = 200 nmol / L, B = 400 nmol / L, C = 600 nmol / L, D = 800 nmol / L, E = 1000 nmol / L, F = 1200 nmol / L, G = 1400 nmol / L, H = 1600 nmol / L, I = 1800 nmol / L), with a gradient interval of 200 nmol / L, to optimize primer concentration. Three gradients were set in the range of 100-200 nmol / L for the final concentrations of vaccine strain and wild-type strain probes, with a gradient interval of 50 nmol / L, and the probe combination was optimized using a matrix method. The specific optimization scheme is shown in Table 3. The primer and probe concentration combination with high amplification efficiency was the optimal reaction system. The optimization results of the reaction system for the dual identification and detection method of small ruminant disease vaccine strain and wild-type strain are shown in […]. Figure 2 and Figure 3 . Figure 2 The primer concentration optimization results showed that the amplification efficiency was high when the final concentration of upstream and downstream primers was 1400 nmol / L. Figure 3 The results of probe concentration optimization showed that a high amplification efficiency could be obtained when the final probe concentration of both the vaccine strain and the wild-type strain was 100 nmol / L.
[0040] Table 3. Optimization scheme for probe concentration in the dual identification and detection method for peste des petits ruminants vaccine strain and wild-type strain.
[0041]
[0042] (2) To further improve amplification efficiency, the annealing temperature in the reaction program was optimized based on the established optimal reaction system. Five temperature gradients (A = 58℃, B = 59℃, C = 60℃, D = 61℃, E = 62℃) were set within the annealing temperature range of 58℃ to 62℃, with each gradient spaced 1℃ apart. The reaction program for the dual identification detection method was optimized, and the annealing temperature with higher amplification efficiency was selected as the optimal reaction program. The optimization results of the reaction program for the dual identification detection method of the peste des petits ruminants vaccine strain and the wild-type strain are shown below. Figure 4 As shown, annealing temperature has a significant impact on amplification efficiency. As the temperature increases, the amplification efficiency gradually decreases, with the highest amplification efficiency observed at an annealing temperature of 58℃.
[0043] (3) The concentration is 7.13×10 10 The recombinant plasmid of the small ruminant vaccine strain was copied and contained 6.33 × 10⁻⁶. 10 The recombinant plasmid of the small ruminant plague wild-type virus was serially diluted 10-fold to 10^6 times. 7 ~103 Using the recombinant plasmid as a template, a standard curve was established using the optimized reaction system and procedure. The linearity and amplification efficiency of this method were then evaluated. The results of the standard curve establishment for the dual identification and detection method of the peste des petits ruminants (PPR) vaccine strain and the wild-type strain are shown below. Figure 5 As shown, the R² values for both the vaccine strain and the wild-type strain are greater than 0.998, and the amplification efficiencies are both greater than 98%, indicating that this method has good linearity and amplification efficiency.
[0044] (4) To evaluate the limit of detection of the established dual identification method for peste des petits ruminants (PPR) vaccine strains and wild-type strains, a 10-1T04 Sensitivity tests were conducted using ~10¹ copies of the vaccine strain and wild-type virus recombinant plasmid as templates. The sensitivity test results of the dual identification detection method for the peste des petits ruminants (PPR) vaccine strain and wild-type virus strain are as follows: Figure 6 As shown in the figure, the method has a detection limit of 10² copies for both vaccine strains and wild-type strains, indicating its high sensitivity.
[0045] (5) To evaluate the specificity of the established dual identification and detection method for PPR vaccine strain and wild-type strain, nucleic acids of foot-and-mouth disease virus (types A and O), goatpox virus, Mycoplasma sheep, Pasteurella multocida, Proteus mirabilis, Streptococcus, Mansonia hemolyticus, and sheep pox virus were used as detection templates. Recombinant plasmids of PPR vaccine strain and wild-type strain were used as positive controls, and DEPC water was used as a negative control. The specificity of this method was evaluated. The specificity test results of the dual identification and detection method for PPR vaccine strain and wild-type strain are as follows: Figure 7 As shown in the figure. This method can specifically detect peste des petits ruminants vaccine strains and wild-type strains, and shows no cross-reactivity with other pathogens such as foot-and-mouth disease virus and goat pox virus, indicating that it has good specificity.
[0046] (6) To evaluate the repeatability stability of the established dual identification and detection method for peste des petits ruminants (PPR) vaccine strains and wild-type strains, a 10-year test was conducted. 6 ~10 4 The recombinant plasmids of the vaccine strain and wild-type virus strain were used as templates for the experiment. Three replicate wells were used for each plasmid concentration, and two replicate experiments were performed. The method was evaluated by calculating intra- and inter-group differences. The repeatability results of the dual identification and detection method for peste des petits ruminants (PPR) vaccine strain and wild-type virus strain are shown in Table 4. The coefficients of variation for both intra- and inter-group tests were less than 2%, indicating good repeatability.
[0047] Table 4. Repeatability evaluation results of the dual identification detection method for peste des petits ruminants (PPR) vaccine strain and wild-type virus strain.
[0048]
[0049] (7) Using the established method for identifying and detecting the PPR vaccine strain and wild-type strain, along with a commercially available PPR virus fluorescence quantitative RT-PCR detection kit and the PCR detection method recommended in "Diagnostic Techniques for PPR" (GB / T27982-2011), eight PPR vaccine strains and eight PPR wild-type cDNA artificial simulation samples were tested. The results are shown in Table 5. The detection rate of the PPR vaccine strain and wild-type strain identified in this invention is completely consistent with that of the commercially available PPR virus detection kit, and significantly higher than the PCR detection method recommended by the national standard. Furthermore, both the commercially available PPR virus detection kit and the PCR detection method recommended by the national standard are general nucleic acid detection methods and cannot achieve inter-lineage differentiation. The results indicate that the PPR vaccine strain and wild-type strain identification and detection method established in this study can be used for inter-lineage differentiation in clinical samples.
[0050] Table 5. Test results of artificially simulated samples
[0051]
[0052] This invention, based on the whole-genome sequence analysis of multiple peste des petits ruminants (PPR) viruses, selected the H gene as the target gene and designed qPCR primers and specific identification probes. By optimizing the reaction system and procedure, and systematically evaluating its sensitivity, specificity, and repeatability, a dual identification method for PPR vaccine strains and wild-type strains was successfully established. This method requires only one pair of universal primers and two identification probes to effectively distinguish between vaccine and wild-type strains, and has advantages such as high sensitivity, strong specificity, and good repeatability. The detection rate of artificially simulated samples is significantly higher than that of the PCR detection method recommended by national standards, and is completely consistent with commercially available detection kits.
[0053] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A primer pair and probe for identifying small ruminant vaccine strains and wild-type strains, characterized in that, Including upstream and downstream primers; The upstream primer is PPRV-qF: TCGGAAGAACATATACYGTC; The downstream primer is PPRV-qR: CRATCTCGAAGACTCTTAA; The probe used to detect the small ruminant vaccine strain has the nucleotide sequence FAM-ACCGAGCACCAGTCCAGGTAT-BHQ1. The probe used to detect the wild-type peste des petits ruminants virus has the nucleotide sequence HEX-ATCCGAGCACCGATCTAGGTATC-BHQ1.
2. A method for distinguishing between peste des petits ruminants (PPR) vaccine strains and wild-type strains, characterized in that, The method involves adding the primer pair and probe described in claim 1 into the same reaction tube for amplification.
3. The detection method according to claim 2, characterized in that, The reaction system for the detection is: 2×PerfectStart ® II. Probe qPCR Super Mix: 10 μL; forward and reverse primers 10 uM: 1.4 μL each; probe 10 uM: 0.2 μL each; Template: 2 μL; DEPC water to make up to 20 μL.
4. The detection method according to claim 3, characterized in that, The reaction procedure for the detection was as follows: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 5 s; 58℃ annealing extension for 30 s and fluorescence collection for a total of 40 cycles.
5. A diagnostic reagent for rapid detection of peste des petits ruminants virus, characterized in that, Includes the primer pair and probe as described in claim 1.
6. Use of the reagent as described in claim 5 in the preparation of a kit for detecting small ruminant vaccine strains and wild-type strains.