PCR primers and probe for identifying serum type i marek's disease vaccine virus and wild virus and application thereof

Abstract

The application discloses a PCR primer and probe for identifying serum type I Marek's vaccine virus and wild virus, wherein the primer comprises an upstream primer with a sequence as shown in SEQ ID No:1 and a downstream primer with a sequence as shown in SEQ ID No:2; and the probe comprises a wild virus-specific probe with a sequence as shown in SEQ ID No:3 and a vaccine virus-specific probe with a sequence as shown in SEQ ID No:4. Through the primer and the probe, the serum type I Marek's vaccine virus and the wild virus can be efficiently, conveniently and quickly identified, so that early warning, accurate prevention and control and purification of Marek's disease are realized.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a PCR primer and probe for differentiating serum type I Marek's vaccine virus from wild-type virus and its application. Background Technology

[0002] Marek's disease ( Marek's disease Marek's disease (MD) is caused by the Marek's disease virus (MD). Marek's disease virus Malignant tumors (MDV) are highly contagious and infectious tumor diseases in chickens, which are important immunosuppressive and malignant tumor diseases that seriously threaten the global poultry industry. They can cause mononuclear cell infiltration in various internal organs such as peripheral nerves, gonads, liver, spleen, and kidneys in affected chickens. In severe cases, solid tumors can form, leading to paralysis, emaciation, and death in the chickens. At the same time, it can cause immunosuppression and secondary infections by other pathogens, resulting in huge economic losses for large-scale chicken farms.

[0003] Marek's disease virus (MDV) belongs to the genus Marek's virus in the subfamily Alphaherpesvirinae. Based on serotype differences, it can be divided into three categories. Serotype I MDV is the only oncogenic strain, including virulent and hypervirulent wild-type strains, as well as attenuated vaccine strains used for vaccination. It is the core target for Marek's disease prevention and surveillance. Serotypes II and III are non-oncogenic strains, used only as adjunctive vaccine strains, and pose no risk of pathogenic mutation. MDV mainly replicates through feather follicle epithelial cells and spreads horizontally in chicken flocks via aerosols formed from shed dander, feathers, and dust. Intact virus particles can remain infectious for months at room temperature. Its transmission routes are insidious, its contamination range is wide, and its survival period is long. Once a flock is infected with wild-type virus, it spreads rapidly, and routine cleaning and disinfection are insufficient to completely eliminate the virus from the environment.

[0004] Currently, the core method for controlling Marek's disease globally is vaccination. The mainstream vaccines with the best control efficacy are all serum type I attenuated vaccine strains, including the internationally used CVI988 / Rispens strain and the domestically developed 814 strain. These vaccine viruses, after artificial attenuation, are non-tumorigenic and can effectively induce specific immunity in chicken flocks to resist wild-type virus invasion. However, with increasing stocking density and the continuous mutation and evolution of MDV wild-type viruses, highly virulent and extremely virulent wild-type virus strains are frequently emerging. Some wild-type viruses can break through vaccine immunity protection and cause disease. Furthermore, chicken flocks carry the vaccine virus for a long time after vaccination, and routine testing cannot distinguish between vaccine and wild-type viruses. This leads to technical blind spots in monitoring wild-type virus infection in chicken flocks, evaluating vaccine immunization effectiveness, and detecting environmental virus contamination. It is impossible to accurately determine whether the flock is in a vaccine-immunized carrier state or has been infected with wild-type viruses, making it difficult to achieve early warning, precise control, and eradication of Marek's disease. Therefore, establishing a rapid and accurate fluorescent quantitative PCR diagnostic method to distinguish between serum type I MDV vaccine virus (CVI988 / Rispens, strain 814) and wild virus is of vital importance for monitoring wild virus in chicken flocks, evaluating vaccine immunization efficacy, and purifying the breeding environment of pathogens. Summary of the Invention

[0005] The purpose of this invention is to provide PCR primers and probes, identification methods and applications for distinguishing between serum type I Marek's vaccine virus and wild-type virus, so as to quickly distinguish between serum type I Marek's vaccine virus and wild-type virus, and realize early warning, precise prevention and control and eradication of Marek's disease.

[0006] According to a first aspect of the present invention, a PCR primer and probe for identifying Marek's disease type I vaccine virus and wild-type virus are provided. The primer comprises an upstream primer with the sequence shown in SEQ ID No:1 and a downstream primer with the sequence shown in SEQ ID No:2; the probe comprises a wild-type virus-specific probe with the sequence shown in SEQ ID No:3 and a vaccine virus-specific probe with the sequence shown in SEQ ID No:4. Thus, using this primer and probe, efficient, convenient, and accurate identification and differentiation between Marek's disease type I vaccine virus and wild-type virus can be achieved. Furthermore, it can be used for monitoring wild-type virus in chicken flocks, evaluating vaccine immunization efficacy, and purifying the breeding environment of pathogens, which has crucial significance for production and disease prevention.

[0007] In some embodiments, the 5' end of the vaccine probe is labeled with a VIC fluorescent group, the 5' end of the wild-type probe is labeled with a FAM fluorescent group, and the 3' ends of both the vaccine probe and the wild-type probe are labeled with an MGB quencher group.

[0008] According to a second aspect of the present invention, a kit for identifying Marek's disease vaccine virus type I and wild-type virus is provided, the kit comprising the aforementioned primers and probes. Thus, this kit enables efficient, convenient, and accurate identification and differentiation between Marek's disease vaccine virus type I and wild-type virus, further facilitating the monitoring of wild-type virus in chicken flocks, evaluation of vaccine immunization efficacy, and pathogen purification of the breeding environment, which has crucial significance for production and disease prevention.

[0009] In some embodiments, the kit may also include other reagents for performing quantitative real-time PCR detection.

[0010] According to a third aspect of the present invention, the PCR primers and probes described herein are provided for the preparation of a product for differentiating between serum type I Marek's vaccine virus and wild-type virus. Thus, the application of this product enables rapid identification between serum type I Marek's vaccine virus and wild-type virus, thereby achieving early warning, precise prevention and control, and eradication of Marek's disease.

[0011] According to a fourth aspect of the present invention, a non-therapeutic application is provided for the PCR primers and probes or the kit described herein in distinguishing between serum type I Marek's disease vaccine virus and wild-type virus. Thus, this application enables rapid identification between serum type I Marek's disease vaccine virus and wild-type virus, thereby achieving early warning, precise prevention and control, and eradication of Marek's disease.

[0012] According to a fifth aspect of the present invention, a real-time quantitative PCR detection method for identifying serum type I Marek's vaccine virus and wild-type virus is provided, the method comprising the following steps: S1. Synthesize the primers and probes described above, or use the kit described above; S2. Perform quantitative real-time PCR detection according to the PCR reaction system and conditions. The PCR reaction system is prepared in 20 μL volumes as follows: 10 μL Probe qPCR Mix MultiPlus, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM downstream primer, 0.2 μL of 10 μM vaccine-specific probe, 0.2 μL of 10 μM wild-type virus-specific probe, 4 μL template DNA, and 4.8 μL ddH2O. The reaction conditions are: 95℃ pre-denaturation for 20 s; 95℃ denaturation for 1 s, 62℃ annealing / extension for 20 s, for 40 cycles. S3. Result Determination: (1) Wild virus positive, vaccine virus negative: The FAM channel shows a typical S-shaped amplification curve and the Ct value is ≤35; the VIC channel shows no amplification signal; (2) Vaccine virus positive, wild virus negative: VIC channel shows a typical S-shaped amplification curve and Ct value ≤ 35, FAM channel shows no amplification signal; (3) Co-infection with wild-type virus and vaccine virus: Both FAM and VIC channels showed typical S-type amplification curves and Ct values ​​≤35; (4) Negative for both wild-type virus and vaccine virus: No amplification signal was observed in either the FAM or VIC channels. Therefore, this method establishes a rapid and accurate fluorescent quantitative PCR diagnostic method for distinguishing between serum type I MDV vaccine virus (CVI988 / Rispens, strain 814) and wild-type virus. This method is of vital importance for monitoring wild-type virus in chicken flocks, evaluating vaccine immunization efficacy, and purifying the breeding environment.

[0013] According to a sixth aspect of the present invention, a non-therapeutic application of the PCR detection method described above is provided in differentiating between serum type I Marek's vaccine virus and wild-type virus. Thus, the application of this method can rapidly differentiate between serum type I Marek's vaccine virus and wild-type virus, enabling early warning, precise prevention and control, and eradication of Marek's disease.

[0014] The beneficial effects of this invention are: 1. This invention discloses a PCR primer and probe for differentiating between serum type I Marek's vaccine virus and wild-type virus. The primer and probe are not based on conventional primers modified from existing known differential sites. Instead, they first overcome the technical difficulties of few differences in highly conserved gene sequences and difficulty in identifying single-base variations. After a large number of sequence screenings and multiple rounds of elimination verification, novel identification SNP targets were independently discovered. Then, the primers were precisely designed by combining the principle of single-base mismatch specific amplification. The overall technical solution is non-obvious and has clear inventiveness and application value.

[0015] 2. This invention discloses a fluorescence quantitative PCR detection method for differentiating between serum type I Marek's disease vaccine virus and wild-type virus. This method can rapidly distinguish between vaccine virus and wild-type virus in vaccines and clinical samples, with a detection time controlled within 1 hour. It has high sensitivity and strong specificity, and can accurately detect latent and early infections. It can be directly used for screening clinical samples in chicken farms, evaluating vaccine immunization efficacy, monitoring wild-type virus infection, and detecting pathogens in the breeding environment. It completely makes up for the shortcomings of existing technologies that cannot distinguish between vaccine virus and wild-type virus, and has extremely strong clinical application value and potential for large-scale promotion. Attached Figure Description

[0016] Figure 1 Figure showing the comparison results of specific single SNP sites between vaccine strain and wild-type strain; Figure 2 The image shows the results of specific detection of serum type I Malik vaccine virus and wild-type virus. Figure 3 The image shows the results of quantitative real-time PCR detection of 10-fold serial dilutions of the positive standard of serum type I Marek's vaccine. Figure 4The image shows the results of quantitative real-time PCR detection of 10-fold serial dilutions of the positive standard of wild-type Marek's vaccine type I serum. Figure 5 Standard curve of serum type I Marek's vaccine virulence; Figure 6 Standard curve of serum type I Malik wild-type toxin; Figure 7 The image shows the monitoring results of chicken feather marrow samples tested for the challenge vaccine virus and wild-type SPF virus. Detailed Implementation

[0017] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various changes and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention.

[0018] Unless otherwise specified, all chemical substances, proteins, enzymes, or reagent kits used in this invention are commercially available.

[0019] Unless otherwise stated, the methods used in this invention, such as PCR amplification and nucleic acid extraction, all employ conventional experimental procedures in the field. Related operations can be performed with reference to commonly used experimental technical manuals or the operating instructions for commercial reagent kits and instruments.

[0020] Example 1: Screening of sites used to differentiate between serum type I Marek's vaccine virus and wild-type virus.

[0021] 1.1 Design concept.

[0022] This invention breaks through the conventional design logic of SNP sites. Instead of using traditional random screening or single-strain sequence alignment, it focuses on the key functional genes of the target strain. Through comprehensive sequence alignment of multiple serotypes and strains from multiple sources, it accurately screens specific SNP sites that can achieve differential diagnosis. Then, it combines the principle of single-base mismatch specific amplification to complete the precise design and optimization of primers and probes, ensuring the stability and specificity of differential diagnosis. This solves the technical pain point that conventional probe / primer combinations cannot stably identify target strains.

[0023] 1.2 Experimental materials and reagents.

[0024] (1) Source of virus sequence samples: The serum type I Marek's vaccine strains CVI988, 814, and SC9-1 sequences were obtained by sequencing the vaccine strains. The manufacturers and catalog numbers of the vaccines are shown in Table 1. The serum type I Marek's wild-type virus sequences were obtained by downloading from the NCBI database and by sequencing the isolates. The serum type II Marek's virus strain (AF291866.1) and serum type III Marek's virus strains (GCF_008792185.1 and GCF_000838845.1) sequences were obtained by downloading from the NCBI database.

[0025] The ID numbers of Marek's disease virus serotype 1 wild strains in the NCBI database: GCA_000846265.1, GCF_000846265.1, GCA_027930745.1, GCA_027930765.1, GCA_027930775.1, GCA_027930825.1, GCA_027930895.1, GCA_027931105.1, GCA_027931135.1, GCA_027931165.1, GCA_027931185.1, GCA_027931195.1, GCA_027931205.1, GCA_027931215.1, GCA_027931225.1, GCA_027931235.1, GCA_027931255.1, GCA_027931265.1, GCA_027931275.1, GCA_027931285.1, GCA_027931335.1, GCA_027931355.1, GCA_027931365.1, GCA_027931375.1, GCA_027934365.1, GCA_027935875.1, GCA_027931415.1, GCA_027931345.1, GCA_027930755.1, GCA_027930785.1, GCA_027930795.1, GCA_027930805.1, GCA_027930815.1, GCA_027930835.1, GCA_027930845.1, GCA_027930855.1, GCA_027930865.1, GCA_027930875.1, GCA_027930885.1, GCA_027930905.1, GCA_027930915.1, GCA_027930925.1, GCA_027930935.1, GCA_027930945.1, GCA_027930955.1, GCA_027930965.1, GCA_027930975.1, GCA_027930985.1, GCA_027930995.1, GCA_027931005.1, GCA_027931025.1, GCA_027931045.1, GCA_027931055.1, GCA_027931065.1, GCA_027931075.1, GCA_027931085.1, GCA_027931095.1, GCA_027931115.1, GCA_027931125.1, GCA_027931145.1, GCA_027931155.1. GCA_027931175.1, GCA_027931245.1, GCA_027931295.1, GCA_027931305.1, GCA_027931315.1. .

[0026] Table 1. List of manufacturers and product codes of commercially available vaccines

[0027] (2) Target genes: Meq gene, PP38 gene, PP24 gene, ICP4 gene, gB gene, gI gene, gH gene, gE gene (all of which are key functional genes specifically expressed by Marek's virus strains and are the core targets for identifying different serotypes of the virus strain).

[0028] (3) Main reagents: nucleic acid extraction reagents, PCR amplification reagents, primers / probes, etc. All reagents meet the experimental standards to ensure experimental repeatability.

[0029] 1.3 Experimental steps and results.

[0030] (1) Gene sequence sequencing and collection.

[0031] Full-length sequencing of the Meq, PP38, PP24, ICP4, gB, gI, gH, and gE genes was performed on serotype I Marek's vaccine strain and the isolated serotype I Marek's wild-type virus strain. Simultaneously, the target gene sequences corresponding to the above serotype strains were downloaded from the NCBI database, and all sequences were integrated to form a sequence library to ensure that the sequences cover strains from different sources and serotypes, thereby reducing the impact of sequence bias on the screening results.

[0032] (2) SNP site screening.

[0033] Bioinformatics analysis tools were used to perform multiple sequence alignment analysis on all integrated target gene sequences, focusing on comparing sequence differences between different serotypes of the virus strain, and between the same serotype vaccine strain and wild-type virus strain, to screen out SNP sites with potential for differential diagnosis: the site must be unique in the target differential strain and highly conserved in the same type of virus from different sources, excluding identification failure caused by sequence variation.

[0034] Comprehensive sequencing and alignment analysis of the Meq, PP38, PP24, ICP4, gB, gI, gH, and gE gene sequences of serotype I Marek's vaccine strain, serotype I Marek's wild-type virus strain, serotype II Marek's virus strain, and serotype III Marek's virus strain revealed only one SNP locus that met the requirements for differential diagnosis. This locus is located in the Meq gene and exhibits high specificity and conservation in the target differential strain. It can serve as the core target for different serotype strains and for differentiating serotype I vaccine strains from wild-type strains. The screening results are shown in Tables 2 and 3 below.

[0035] Table 2 Site screening results target gene Coverage of strain types SNP site screening results Remark Meq gene Serum type I One identifying SNP site that meets the requirements was selected. The gene is missing in serum type II and III, and the SC9-1 vaccine strain for serum type I is also missing. A qualified SNP site was screened in this gene, and this SNP site showed regular differences in the CVI988 / 814 wild-type vaccine strain for serum type I (Table 3). PP38 gene Serum type I, serum type II No matching identifying SNP sites were found. However, stable differentially expressed sites could not be screened between the serum type I vaccine and wild-type virus. PP24 gene Serum type I No matching identifying SNP sites were found. The base differences between serum type I vaccines and wild-type viruses are unstable and cannot be used as stable identification targets. ICP4 gene Serum type I, serum type II, serum type III No matching identifying SNP sites were found. The base differences between serum type I vaccines and wild-type viruses are unstable and cannot be used as stable identification targets. gB gene Serum type I, serum type II, serum type III No matching identifying SNP sites were found. Base differences are unstable and cannot be used as stable identification targets. gI gene Serum type I, serum type II, serum type III No matching identifying SNP sites were found. The base differences between serum type I vaccines and wild-type viruses are unstable and cannot be used as stable identification targets. gH gene Serum type I, serum type II, serum type III One SNP locus that meets the requirements for differential diagnosis was selected. The base differences between serum type I vaccines and wild-type viruses are unstable and cannot be used as stable identification targets. gE gene Serum type I, serum type II, serum type III No matching identifying SNP sites were found. The base differences between serum type I vaccines and wild-type viruses are unstable and cannot be used as stable identification targets. As shown in Table 2, only one suitable SNP site for identification was found in the Meq gene; none were found in other genes. Further analysis of this suitable SNP site yielded the results shown in Table 3.

[0036] Table 3. Comparison of screened SNP sites in vaccine virus and wild-type virus. Strain classification Sample size Representative strains Target SNP site base Sequence Consistency Verification results Serum type I vaccine strain 6 strains CVI988 / Rispens (supplied by Boehringer Ingelheim, Rispens, and Lingyu), 814 plants (supplied by Shihua and Dahua Agricultural), SC9-1 (supplied by Lingyu). T 100% All vaccine strains showed T at this site, with no variation. Serotype I wild strain 100 strains Highly virulent strains RB1B, Md5, GA, GX0101, and highly virulent strain JM (domestic clinical isolates from 2022-2026) (Guangdong, Guangxi Zhuang Autonomous Region, Yunnan, Jiangsu, Zhejiang, etc.) G 100% All wild-type strains showed G at this site, with no variation. The results in Table 3 show that the SNP site identified in the analysis of the sequences of 6 vaccine strains and 100 wild-type virus strains exhibited stable and regular variations. Specifically, the base at this site was T in the vaccine strains, while it was G in the wild-type virus strains. Therefore, this site can be used as an SNP site to identify and differentiate between serum type I Marek's vaccine virus and wild-type virus.

[0037] (3) Primer / probe design and optimization.

[0038] For the unique differential diagnostic serum type I Marek's virus strain identified through screening, located in the Meq gene, universal upstream and downstream primers were designed to simultaneously detect serum type I Marek's virus vaccine strain and wild-type virus, taking into account the region where this site is located. Then, based on the specific single SNP sites of CVI988 strain, 814 strain vaccine strain, and wild-type virus (comparison results are shown in...),... Figure 1 As shown in the image, the red box indicates the SNP site, and probes were designed accordingly.

[0039] Based on gene sequence characteristics, multiple different primer / probe combinations were designed (including primers of different lengths and annealing temperatures, as well as probes with different labels), and each combination was specifically optimized and screened.

[0040] First, universal primers capable of detecting serum type I Marek's vaccine virus and wild-type virus were designed, and their sequences are as follows: Upstream primer: 5-AGGAGAAACAGAAGCTGGAAAG-3 (SEQ ID No:1); Downstream primer: 5-TCCTTACGTAGGTGTTCATTGG-3 (SEQ ID No:2).

[0041] Then, multiple probe sequences were designed based on specific single SNP sites. The probe sequences and detection results are shown in Table 4.

[0042] Table 4. Multiple probe sequences and detection results Group Vaccine virus probe (5'-3') Wild-type venom probe (5'-3') result 1 5'-VIC-CCTCTCGGAGAAG-MGB-3' 5'-FAM-CCGCTCGGAGAAG-MGB-3' × 2 5'-VIC-GACCTCTCGGAGAAG-MGB-3' 5'-FAM-GACCGCTCGGAGAAG-MGB-3' × 3 5'-VIC-CGTGACGCCTCTCGGAGAA-MGB-3' 5'-FAM-CGTGACGCCGCTCGGAGAA-MGB-3' × 4 5'-VIC-GTGACGCCTCTCGGAGAAG-MGB-3' 5'-FAM-GTGACGCCGCTCGGAGAAG-MGB-3' × 5 5'-VIC-TCGTGACGCCTCTCGGAGA-MGB-3' 5'-FAM-TCGTGACGCCGCTCGGAGA-MGB-3' × 6 5'-VIC-CGTGACGCCTCTCGGAGAAGACGCA-BHQ1-3' 5'-FAM-CGTGACGCCGCTCGGAGAAGACGCA-TAMRA-3' × 7 5'-VIC-TGACGCCTCTCGGA-MGB-3' 5'-FAM-TGACGCCGCTCGGA-MGB-3' √ As shown in Table 4, only the 7th group of probes could distinguish between serum type I Marek's vaccine virus and wild-type virus. Therefore, the best set of primers and probes was finally selected, and the specific sequences are as follows: Upstream primer: 5-AGGAGAAACAGAAGCTGGAAAG-3 (SEQ ID No:1); Downstream primer: 5-TCCTTACGTAGGTGTTCATTGG-3 (SEQ ID No:2); Wild-type toxicant specific probe: 5'-FAM-TGACGCCGCTCGGA-MGB-3' (SEQ ID No:3); Vaccine-specific probe: 5'-VIC-TGACGCCTCTCGGA-MGB-3' (SEQ ID No:4); The vaccine virus probe is labeled with the VIC fluorescent group at its 5' end, the wild virus probe is labeled with the FAM fluorescent group at its 5' end, and both are labeled with the MGB quencher group at their 3' ends.

[0043] Example 2: Preparation of vaccine virus and wild-type virus standards.

[0044] Genomic DNA extraction: Genomic DNA was extracted from CVI988 / Rispens vaccine strain and MD5 wild-type standard strain (China Institute of Veterinary Drug Control) according to the instructions of the commercial viral DNA extraction kit. After the concentration was found to be qualified, the DNA was stored at -20℃ for later use.

[0045] Target fragment PCR amplification: Universal primers were used to amplify the target fragments of the vaccine strain and the wild-type strain, respectively. The PCR reaction system (25 μL) consisted of: 12.5 μL of 2×Premix Taq (TaKaRa), 0.5 μL each of the upstream and downstream primers, 3 μL of template DNA, and 8.5 μL of ddH2O. Reaction conditions: 95℃ pre-denaturation for 4 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, for 30 cycles; final extension at 72℃ for 10 min. The PCR products were detected by 2% agarose gel electrophoresis; a single band matching the size of the target fragment was considered acceptable.

[0046] Vector ligation and transformation: After purification of qualified PCR products, they were ligated with pMD19-T vector (pMD™ 19-T VectorCloning Kit, Takara, catalog number: 6013) at 12°C overnight. The ligation products were transformed into DH5α competent cells, plated on LB solid medium containing Amp resistance, and cultured overnight at 37°C inverted.

[0047] Recombinant bacterial culture and plasmid extraction: Single colonies were picked and inoculated into LB liquid medium containing Amp resistance. The culture was carried out at 37°C and 200 r / min for 12 h with shaking. Recombinant plasmids were extracted using a commercial plasmid mini-prep kit and named pMD19-CVI988-Meq and pMD19-MD5-Meq, respectively.

[0048]

[0049]

[0050] Standard identification and storage: The recombinant plasmid is sequenced. Plasmids with sequencing results that match the target sequence and have no base mutations are considered qualified standards. After nucleic acid copy number determination, standard curve templates are prepared by serial dilution, aliquoted and stored at -80℃, avoiding repeated freeze-thaw cycles, for subsequent quantitative PCR detection.

[0051] Example 3: Establishment of a real-time quantitative PCR reaction for differentiating serum type I Marek's vaccine virus and wild-type virus.

[0052] The reaction system (20 μL) was prepared using TaKaRa Probe qPCR Mix MultiPlus probe-based real-time PCR enzyme as an example (as shown in Table 5): Table 5. Real-time PCR reaction system reagents Usage Probe qPCR Mix MultiPlus 10μL Upstream primer (10 μM) 0.4μL Downstream primer (10 μM) 0.4μL Vaccine-specific probe (10 μM) 0.2μL Wild-type venom-specific probe (10 μM) 0.2μL stencil 4μL <![CDATA[ddH2O]]> 4.8μL Mix the reaction mixture thoroughly, and then amplify it on a PCR instrument. The specific amplification program is as follows: 95℃ pre-denaturation for 20 s; 95℃ denaturation for 1 s, 62℃ annealing / extension for 20 s, for 40 cycles; fluorescence signal acquisition is performed during the annealing / extension stage at 62℃.

[0053] Result interpretation criteria: (1) Wild-type virus positive: The FAM channel shows a typical S-shaped amplification curve and the Ct value is ≤35, while the VIC channel shows no amplification signal; (2) Positive for vaccine virus: The VIC channel shows a typical S-shaped amplification curve and the Ct value is ≤35, while the FAM channel shows no amplification signal; (3) Co-infection with wild-type virus and vaccine virus: Both FAM and VIC channels showed typical S-type amplification curves, and the Ct value was ≤35; (4) Wild-type virus negative: No amplification signal in the FAM channel (Ct value shows Undetermined); (5) Vaccine virus negative: No amplification signal in the VIC channel (Ct value shows Undetermined).

[0054] Possible outcomes: (1) Wild virus positive, vaccine virus negative: The FAM channel shows a typical S-shaped amplification curve and the Ct value is ≤35; the VIC channel shows no amplification signal; (2) Vaccine virus positive, wild virus negative: VIC channel shows a typical S-shaped amplification curve and Ct value ≤ 35, FAM channel shows no amplification signal; (3) Co-infection with wild-type virus and vaccine virus: Both FAM and VIC channels showed typical S-type amplification curves and Ct values ​​≤35; (4) Wild virus negative, vaccine virus negative: No amplification signal in FAM and VIC channels.

[0055] Example 4: Specificity test of the detection method.

[0056] CVI988 live vaccine, 814 live vaccine, SC9-1 live vaccine, HVT live vaccine, MDV wild virus, and various common DNA viruses were collected, as detailed in Table 6 below. Viral nucleic acid was extracted using the Axygen Virus Total RNA / DNA Extraction Kit, and the method established in Example 3 was used for testing.

[0057] Table 6 List of different viruses Sample number Virus type source 1 Marek's serum type I vaccine strain 814 Guangdong Wenshi Dahua Agricultural Biotechnology Co., Ltd. - Likewei 2 Marek's serum type I vaccine strain 814 Shihua Animal Health Technology (Beijing) Co., Ltd. - Weikebao 3 Marek's serum type I vaccine strain CVI988 Nanchang Boehringer Ingelheim Animal Health Co., Ltd. - Dongkeling 4 Marek's serum type I vaccine strain CVI988 + serum type III vaccine strain HVT strain Ruipu (Baoding) Biopharmaceutical Co., Ltd. - Shuangxinlike 5 Marek's serum type III vaccine strain HVT Nanchang Boehringer Ingelheim Animal Health Co., Ltd. - Willick 6 Marek's wild-type poison - MD5 strain Guangdong Wenshi Dahua Agricultural Biotechnology Co., Ltd. 7 Marek's serum type I wild-type strain 1 Wens Foodstuff Group Co., Ltd. since separation 8 Marek's serum type I wild-type strain 2 Wens Foodstuff Group Co., Ltd. since separation 9 Marek's serum type I wild-type strain 3 Wens Foodstuff Group Co., Ltd. since separation 10 Marek's serum type I wild-type strain 4 Wens Foodstuff Group Co., Ltd. since separation 11 Throatpox vaccine Zhaoqing Dahua Agricultural Biopharmaceutical Co., Ltd. - Throat Pox Control Agent 12 Avian adenovirus wild-type virus Wens Foodstuff Group Co., Ltd. since separation 13 Wild-type chicken infectious anemia virus Wens Foodstuff Group Co., Ltd. since separation 14 fowlpox virus live vaccine Guangdong Wenshi Dahua Agricultural Biotechnology Co., Ltd. Test results as follows Figure 2 As shown in the results, this detection method can distinguish between Marek's serum type I CVI988 and 814 vaccine viruses and wild viruses, and it does not cross-react with common avian DNA viruses, indicating that the detection method has high specificity.

[0058] Example 5: Methodological Validation.

[0059] 5.1 Vaccine virulence sensitivity test.

[0060] The vaccine positive standard (pMD19-CVI988-Meq plasmid) was serially diluted 10-fold with ddH2O to a concentration of 3.31 × 10⁻⁶. 8 ~3.31×10 1 Copies / μL were used as nucleic acid templates and detected according to the method established in Example 3, with each concentration measured three times. The detection sensitivity reached 3.31 × 10⁻⁶. 2 Copy number, with the common logarithm of plasmid copy number LgC (copy number) on the x-axis and Ct value on the y-axis, was used to plot the standard curve of the established Marek's vaccine qPCR detection method. The fitted curve was y = -3.434x + 49.581 (R² = 0.999). (The results of the vaccine positive standard test are shown below.) Figure 3 As shown, the standard curve is as follows Figure 5 (As shown).

[0061] 5.2 Wild Poison Sensitivity Test.

[0062] The wild-type virus positive standard (pMD19-MD5-Meq plasmid) was diluted 10-fold with ddH2O to a concentration of 2.35 × 10⁻⁶. 8 ~2.35×10 1 Copies / μL were used as nucleic acid templates and detected according to the method established in Example 3, with each concentration measured three times. The detection sensitivity reached 2.35 × 10⁻⁶. 1The standard curve of the established RT-qPCR detection method was plotted with the common logarithm of plasmid copy number (LgC) on the x-axis and Ct value on the y-axis. The fitted curve was y = -3.729x + 46.269 (R² = 0.998). (The results of the wild-type virus standard test are shown below.) Figure 4 As shown, the standard curve is as follows Figure 6 (As shown).

[0063] 5.3 Sample testing and verification.

[0064] The results of testing and verification on SPF chicken immunized and challenged samples are as follows: Figure 7 As shown: all 10 SPF samples immunized with CVI988 vaccine showed vaccine-specific signals, with no positive results for wild-type virus; all 10 SPF blank chicken samples challenged with MDV wild-type virus showed wild-type virus-specific signals, with no positive results for vaccine-type virus; and all 10 negative control samples showed no specific amplification, with no false positives or false negatives. This indicates that the accuracy of the method of this invention is 100%.

[0065] In summary, this invention discloses PCR primers and probes for differentiating between serum type I Marek's disease vaccine virus and wild-type virus. These primers and probes are not based on conventional primer modifications of known differentially expressed sites. Instead, they overcome the technical challenges of low sequence differences in highly conserved genes and difficulty in identifying single-base variations. Through extensive sequence screening and multiple rounds of elimination verification, novel identifying SNP targets were independently discovered. The primers were then precisely designed using the principle of single-base mismatch specific amplification. Furthermore, based on these primers and probes, a real-time quantitative PCR detection method for differentiating between serum type I Marek's disease vaccine virus and wild-type virus was established. This method can rapidly distinguish between vaccine virus and wild-type virus in vaccines and clinical samples, with a detection time controlled within 1 hour. It exhibits high sensitivity and specificity, enabling accurate detection of latent and early infections. It can be directly used for screening clinical samples in chicken farms, evaluating vaccine immunization efficacy, monitoring wild-type virus infection, and detecting pathogens in the breeding environment. This completely overcomes the shortcomings of existing technologies in differentiating between vaccine virus and wild-type virus, possessing strong clinical application value and potential for large-scale promotion.

Claims

1. PCR primers and probes for differentiating serum type I Marek's vaccine virus from wild-type virus, among which, The primers include an upstream primer with the sequence shown in SEQ ID No:1 and a downstream primer with the sequence shown in SEQ ID No:2; the probes include a wild-type virus-specific probe with the sequence shown in SEQ ID No:3 and a vaccine-specific probe with the sequence shown in SEQ ID No:

4.

2. The PCR primers and probes according to claim 1, wherein, The 5' end of the vaccine probe is labeled with a VIC fluorescent group, the 5' end of the wild-type probe is labeled with a FAM fluorescent group, and the 3' ends of both the vaccine probe and the wild-type probe are labeled with an MGB quenching group.

3. A kit for differentiating serum type I Marek's vaccine virus from wild-type virus, wherein, The kit contains the primers and probes as described in claim 1 or 2.

4. The kit according to claim 2, wherein, The kit also includes other reagents for achieving real-time quantitative PCR detection.

5. The use of the PCR primers and probes according to claim 1 or 2 in the preparation of products for differentiating serum type I Marek's vaccine virus from wild-type virus.

6. The non-therapeutic use of the PCR primers and probes of claim 1 or 2 or the kit of claim 3 or 4 in the identification of serum type I Marek's vaccine virus and wild-type virus.

7. A real-time quantitative PCR method for differentiating serum type I Marek's vaccine virus from wild-type virus, wherein, The method includes the following steps: S1. Synthesize the primers and probes as described in claim 1 or 2, or use the kit as described in claim 3 or 4; S2. Perform quantitative real-time PCR detection according to the PCR reaction system and conditions. The PCR reaction system is prepared in 20 μL as follows: 10 μL Probe qPCR Mix MultiPlus, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM downstream primer, 0.2 μL of 10 μM vaccine-specific probe, 0.2 μL of 10 μM wild-type virus-specific probe, 4 μL template DNA, and 4.8 μL ddH2O. The reaction conditions are: 95℃ pre-denaturation for 20 s; 95℃ denaturation for 1 s, 62℃ annealing / extension for 20 s, for 40 cycles. S3. Result Determination: (1) Wild virus positive, vaccine virus negative: The FAM channel shows a typical S-shaped amplification curve and the Ct value is ≤35; the VIC channel shows no amplification signal; (2) Vaccine virus positive, wild virus negative: VIC channel shows a typical S-shaped amplification curve and Ct value ≤ 35, FAM channel shows no amplification signal; (3) Co-infection with wild-type virus and vaccine virus: Both FAM and VIC channels showed typical S-type amplification curves and Ct values ​​≤35; (4) Wild virus negative, vaccine virus negative: No amplification signal in FAM and VIC channels.

8. The non-therapeutic application of the PCR detection method according to claim 7 in differentiating serum type I Marek's vaccine virus from wild-type virus.

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