Primer and TaqMan-MGB probe group for fluorescent quantitative PCR (Polymerase Chain Reaction) detection of virulent and attenuated strains of classical waterfowl parvovirus

Abstract

The invention relates to a primer and a TaqMan-MGB probe group for fluorescent quantitative PCR (Polymerase Chain Reaction) detection of virulent and attenuated strains of classical waterfowl parvovirus, the primer and the TaqMan-MGB probe group comprise a fluorescent quantitative PCR detection primer and a probe group for virulent and attenuated strains of goose parvovirus respectively, and the sequences of the primer and the probe group are shown as SEQ ID NO.1-5 respectively; the primer and the TaqMan-MGB probe group further comprise a fluorescent quantitative PCR detection primer and a probe group for virulent and attenuated muscovy duck parvovirus respectively, and the sequences of the primer and the probe group are shown as SEQ ID NO.6-9 respectively. According to the invention, through systematic test verification and reaction condition optimization, an optimal primer-probe combination is screened out, a GPV TaqMan-MGB dual fluorescent quantitative PCR detection method and an MDPV TaqMan dual fluorescent quantitative PCR detection method are respectively and successfully established, and the detection of virulent and attenuated classical waterfowl parvovirus is realized.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to primers and TaqMan-MGB probe sets for the fluorescence quantitative PCR detection of strong and weak strains of classic waterfowl parvoviruses, namely goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV). Background Technology

[0002] Goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) are two classic pathogens threatening the global waterfowl farming industry, causing gosling plague and Muscovy duckling "three-week disease," respectively. Both are highly contagious and virulent, with morbidity rates reaching 50%–100% in chicks under 10 days old and mortality rates as high as 65%, causing huge economic losses to the farming industry. With the emergence of viral mutations and recombinant strains, the difficulty of disease control has further increased. Accurate identification of virulent and weak strains is crucial for epidemic early warning, vaccine selection, and the formulation of control strategies, and has become a research hotspot and technical bottleneck in the field of waterfowl disease control.

[0003] GPV and MDPV both belong to the genus *Dependoparvovirus* within the family Parvoviridae. Both are autonomous parvoviruses (replicating without helper viruses) and are classified as Anseriform dependoparvovirus 1. They exhibit clear taxonomic species specificity: GPV's primary hosts are geese and young Muscovy ducks, while MDPV is highly pathogenic only to young Muscovy ducks, with geese and other duck species showing little susceptibility. Both are single serotypes, with whole-genome sequence similarity ranging from 79.7% to 85.0%. GPV and MDPV share highly similar virological characteristics: the virus particles are non-enveloped, icosahedral in shape, and approximately 20–22 nm in diameter; both have linear single-stranded negative-sense DNA (approximately 5.8 kb long), with a conserved structure and two major open reading frames (ORFs). The left ORF encodes non-structural proteins (NS1, NS2), and the right ORF encodes structural proteins (VP1, VP2, VP3). The NS protein possesses ATPase and helicase activities, regulating viral genome replication and transcription. Its expression efficiency is directly related to viral replication capacity and virulence. The VP protein is a core component of the capsid. VP3 is the main antigen protein (a core target for vaccine development). VP2 is a virulence determinant, and its amino acid site mutations can affect host affinity, replication efficiency, and pathogenicity.

[0004] The interspecies homology between GPV and MDPV primarily reflects their evolutionary relationship, with non-structural genes showing higher conservation than structural genes. At the NS gene level, the nucleotide homology between the two is 80.0%–83.0%, and the amino acid homology is 88.9%–91.2%. At the structural gene level, the homology is significantly lower than that of non-structural genes: the nucleotide homology of VP1 gene is 79.7%–88.7%, and the amino acid homology is 85.5%–93.3%; the amino acid homology of VP2 gene is 85.5%–88.1%; and the amino acid homology of VP3 gene is 88.4%–91.0%. Unlike the high conservation of NS genes, VP genes have a higher variation rate and exhibit species-specific sequence characteristics, providing molecular targets for virus identification and differentiation between virulent and weak strains. Furthermore, the significant homology between the two genomes in terms of structural framework and core functional genes suggests a common origin, while differences in genomic details and evolutionary characteristics determine host specificity and antigenicity differences, providing key support for the establishment of precise molecular diagnostic methods (such as the design of primers specific to the VP gene variant region).

[0005] Currently, diagnostic techniques for GPV and MDPV have made some progress, mainly falling into three categories: traditional etiological detection, serological detection, and molecular biological detection. Traditional methods (virus isolation and culture, virus neutralization assays, etc.) are cumbersome and time-consuming, and cannot distinguish between virulent and weak strains, making them unsuitable for rapid clinical diagnosis. Serological methods can be used to evaluate immunization efficacy but cannot achieve early pathogen detection and are susceptible to cross-antigen interference. Among molecular biological methods, conventional PCR and dual quantitative PCR have achieved species identification of GPV and MDPV, but the former has low sensitivity, and the latter still suffers from the technical limitation of not being able to distinguish between virulent and weak strains. Existing quantitative PCR methods based on SYBR Green I can identify the two viruses through differences in melting curves, but their specificity depends on differences in GC content, is easily affected by non-specific amplification, and cannot achieve simultaneous identification of virulent and weak strains. However, there is currently no precise identification technology for virulent and weak strains of classic GPV and MDPV: existing methods can only achieve species differentiation or rely on pathogenicity tests to determine virulence, the latter being time-consuming, labor-intensive, costly, and not in line with animal welfare requirements. Developing rapid identification techniques for strong and weak strains based on molecular targets, especially highly specific and sensitive detection systems combining quantitative real-time PCR with TaqMan-MGB probes, has become a key path to overcome this technical bottleneck and is of great practical significance for improving the level of precise prevention and control of waterfowl diseases. Summary of the Invention

[0006] The purpose of this invention is to provide a primer and TaqMan-MGB probe set for the quantitative real-time PCR detection of virulent and attenuated strains of classic waterfowl parvovirus. This invention provides a primer pair and TaqMan-MGB probe for the quantitative real-time PCR identification method of virulent and attenuated GPV strains, suitable for the quantitative real-time PCR differentiation and diagnosis of virulent and attenuated GPV nucleic acids in samples; it also provides a primer pair and TaqMan-MGB probe for the quantitative real-time PCR identification method of virulent and attenuated MDPV strains, suitable for the quantitative real-time PCR differentiation and diagnosis of virulent and attenuated MDPV nucleic acids in samples. The detection system established using primers and TaqMan-MGB probes for these two virulent and attenuated strains of classic waterfowl parvovirus can amplify under the same reaction conditions, achieving simultaneous detection of two virulent and attenuated strains of classic waterfowl parvovirus (GPV, MDPV).

[0007] The objective of this invention is achieved through the following technical solution:

[0008] Primers and TaqMan-MGB probe set for the detection of strong and weak strains of classic waterfowl parvoviruses by real-time PCR, wherein the classic waterfowl parvoviruses include goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV).

[0009] The primer and TaqMan-MGB probe set includes real-time PCR detection primers and TaqMan-MGB probe sets targeting virulent and attenuated goose parvovirus, respectively, with the sequences as follows:

[0010] GPV-203VF: 5'-AGATAGCCTCCAAGACGACC-3' (SEQ ID NO.1),

[0011] GPV-203VR: 5'-CACCTCCCGCACTGACT-3' (SEQ ID NO.2),

[0012] GPV-203AR: 5'-ACCTCCTGCACTGACCTC-3' (SEQ ID NO.3);

[0013] NP5-Probe2: 5'-6-FAM-CCTAGTAGAAGATCCTGTC-MGB-3' (SEQ ID NO.4),

[0014] FJ-Probe12: 5'-JOE-AGACTCGATCAACACG-MGB-3' (SEQ ID NO.5);

[0015] The primer and TaqMan-MGB probe set also includes real-time PCR detection primers and TaqMan-MGB probe sets targeting virulent and attenuated Muscovy duck parvovirus, respectively, with the sequences as follows:

[0016] MDPV-134F: 5'-CCTACAGGTCAGGCAGTAGTC-3' (SEQ ID NO.6),

[0017] MDPV-134R: 5'-TGTTGTTGTGTTTTGTTCATTGGTT-3' (SEQ ID NO. 7);

[0018] P-Probe6: 5'-6-FAM-AACAGAGGAGCAAGA-MGB-3' (SEQ ID NO.8),

[0019] P1-Probe7: 5'-JOE-TGGTACCAGATGAGC-MGB-3' (SEQ ID NO.9);

[0020] This invention also provides a kit for the detection of strong and weak strains of classic waterfowl parvovirus using real-time quantitative PCR. The classic waterfowl parvovirus strains include strong and weak strains of goose parvovirus (GPV). The kit includes reaction system A, which comprises real-time quantitative PCR detection primers and a TaqMan-MGB probe set targeting strong and weak GPV strains, respectively, with the sequences as follows:

[0021] GPV-203VF: 5'-AGATAGCCTCCAAGACGACC-3',

[0022] GPV-203VR: 5'-CACCTCCCGCACTGACT-3',

[0023] GPV-203AR: 5'-ACCTCCTGCACTGACCTC-3';

[0024] NP5-Probe2: 5'-6-FAM-CCTAGTAGAAGATCCTGTC-MGB-3',

[0025] FJ-Probe12:5'-JOE-AGACTCGATCAACACG-MGB-3';

[0026] The reaction system A is 20 μL, and each 20 μL of reaction system A includes:

[0027] 2×PerfectStart ®II Probe qPCR SuperMix 10 μL, upstream universal PCR primer GPV-203VF 0.4 μL (10 μmol / L), downstream virulent PCR primer GPV-203VR 0.2 μL (10 μmol / L), downstream attenuated PCR primer GPV-203AR 0.2 μL (10 μmol / L), probe NP5-Probe2 0.2 μL (10 μmol / L), probe FJ-Probe6 0.2 μL (10 μmol / L), template DNA 2 μL, PassiveReference Dye II (50×) 0.5 μL, and Nuclease-free Water to bring the total to 20 μL.

[0028] The classic waterfowl parvovirus strains include both virulent and attenuated strains of Muscovy duck parvovirus. The kit also includes reaction system B, which comprises real-time quantitative PCR detection primers and a TaqMan-MGB probe set targeting virulent and attenuated Muscovy duck parvovirus, respectively. Their sequences are as follows:

[0029] MDPV-134F: 5'-CCTACAGGTCAGGCAGTAGTC-3',

[0030] MDPV-134R: 5'-TGTTGTTGTGTTTTGTTCATTGGTT-3';

[0031] P-Probe6: 5'-6-FAM-AACAGAGGAGCAAGA-MGB-3',

[0032] P1-Probe7: 5'-JOE-TGGTACCAGATGAGC-MGB-3'.

[0033] The reaction system B is 20 μL, and each 20 μL reaction system B contains: 2×PerfectStart ® II. ProbeqPCR SuperMix 10 μL, upstream and downstream quantitative PCR detection primers MDPV-134F and MDPV-134R (10 μmol / L each) 0.4 μL, probes P-Probe6 and P1-Probe7 (10 μmol / L each) 0.2 μL, template DNA 2 μL, Passive Reference Dye II (50×) 0.5 μL, and Nuclease-free Water to bring the total to 20 μL.

[0034] The reaction conditions for the TaqMan real-time quantitative PCR method of the kit are: 94 ℃ for 30 s; fluorescence is collected at 94 ℃ for 5 s and 60 ℃ for 30 s, for 40 cycles.

[0035] This invention also provides another kit for the detection of strong and weak strains of classic waterfowl parvovirus using real-time quantitative PCR, wherein the strong and weak strains of classic waterfowl parvovirus include strong and weak strains of Muscovy duck parvovirus. The kit includes reaction system B, which includes real-time quantitative PCR detection primers and a TaqMan-MGB probe set targeting strong and weak Muscovy duck parvovirus, respectively, with the sequences as follows:

[0036] MDPV-134F: 5'-CCTACAGGTCAGGCAGTAGTC-3',

[0037] MDPV-134R: 5'-TGTTGTTGTGTTTTGTTCATTGGTT-3';

[0038] P-Probe6: 5'-6-FAM-AACAGAGGAGCAAGA-MGB-3',

[0039] P1-Probe7: 5'-JOE-TGGTACCAGATGAGC-MGB-3'.

[0040] The reaction system B is 20 μL, and each 20 μL reaction system B contains: 2×PerfectStart ® II ProbeqPCR SuperMix 10 μL, upstream and downstream fluorescent quantitative PCR detection primers MDPV-134F and MDPV-134R (10 μmol / L each) 0.4 μL, probes P-Probe6 and P1-Probe7 (10 μmol / L each) 0.2 μL, template DNA 2 μL, Passive Reference Dye II (50×) 0.5 μL, and Nuclease-free Water to bring the total to 20 μL;

[0041] The reaction conditions for the TaqMan real-time quantitative PCR method of the kit are: 94 ℃ for 30 s; fluorescence is collected at 94 ℃ for 5 s and 60 ℃ for 30 s, for 40 cycles.

[0042] Compared with the prior art, the advantages of the present invention are as follows:

[0043] To achieve accurate identification and quantitative detection of virulent and attenuated strains of goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV), this invention focuses on the conserved regions of the VP1 gene of these two classic waterfowl parvoviruses and the virulent and attenuated strain-specific sites. Based on the full genome sequences published in GenBank and related literature, a specific real-time PCR detection system was designed and constructed: For GPV, one universal upstream primer GPV-203VF, two virulent and attenuated strain-specific downstream primers GPV-203VR and GPV-203AR, and two TaqMan-MGB probes NP5-Probe2 and FJ-Probe12 (labeled with differentially luminescent groups) targeting different strains were synthesized; for MDPV, one pair of universal primers MDPV-134F and MDPV-134R, and two specific TaqMan probes P-Probe6 and P1-Probe7 (labeled with differentially luminescent groups) targeting virulent and attenuated strains were synthesized. Through systematic experimental verification and reaction condition optimization, the optimal primer-probe combination was screened, and detection methods for GPV (Gross Virus) and MDPV (MDPV) using TaqMan-MGB dual-fluorescence quantitative PCR (for rapid detection and differentiation of virulent and aberrant strains) were successfully established. The detection systems established using primers and TaqMan-MGB probes targeting these two classic waterfowl parvovirus strains (Gross Virus and MDPV) can amplify under the same reaction conditions, enabling simultaneous detection of both strains.

[0044] Performance validation results show that the detection methods established using primers and TaqMan-MGB probes targeting virulent and attenuated strains of these two classic waterfowl parvoviruses are highly specific. The GPV detection method established using primers and TaqMan-MGB probes targeting virulent and attenuated goose parvoviruses can accurately identify virulent and attenuated strains and shows no cross-reactivity with related viruses such as MDPV, MGPPV, SBDSV, DEV, DPMV, DHV-I, and DTMUV. Similarly, the MDPV detection method established using primers and TaqMan-MGB probes targeting virulent and attenuated Muscovy duck parvoviruses can specifically distinguish between virulent and attenuated strains and shows no cross-reactivity with C-GPV, MGPV, SBDSV, DEV, DPMV, DHV-I, and DTMUV. In terms of sensitivity, the detection limits of the established GPV detection methods for virulent and attenuated VP1 genes are 1.5 × 10⁻⁶. 0The limits of detection for the virulent and attenuated VP1 gene of the MDPV detection methods established above were both 1.25 × 10¹ copies / μL and 1.5 × 10¹ copies / μL. Both methods demonstrated excellent repeatability, with coefficients of variation less than 2%, and the results from clinical samples were completely consistent with sequencing analysis. The TaqMan-MGB PCR detection method established in this study provides an efficient and reliable technical means for the accurate identification and diagnosis of virulent and attenuated GPV and MDPV strains in clinical samples, and also provides crucial laboratory technical support for the prevention and control of goose parvovirus and Muscovy duck parvovirus diseases.

[0045] This invention establishes a TaqMan-MGB dual-fluorescence quantitative PCR method for detecting GPV and a dual-fluorescence quantitative PCR method for detecting MDPV. Using two different fluorophores, FAM and JOE, with high fluorescence signal intensity and low interference, probes for detecting the virulent and attenuated VP1 genes of GPV and MDPV are respectively labeled. This method can easily distinguish between virulent and attenuated GPV strains and virulent and attenuated MDPV strains, exhibiting high specificity, high sensitivity, and good repeatability. It provides a specific, efficient, and convenient technical means for rapid clinical detection of related pathogens and has broad market application value. Attached Figure Description

[0046] Figure 1 This is the standard curve for TaqMan dual real-time quantitative PCR in Example 1 of this invention, wherein... Figure 1 A represents the standard curve for TaqMan dual real-time quantitative PCR of the GPVNP5 VP1 gene. Figure 1 B is the standard curve for TaqMan dual real-time quantitative PCR of the GPV FJ VP1 gene.

[0047] Figure 2 This is the specificity of TaqMan dual real-time quantitative PCR in Example 1; wherein, 1: pUC57-NP5VP1 plasmid standard; 2: GPV NP5 virulent strain; 3: pUC57-FJVP1 plasmid standard; 4: GPV FJ attenuated strain; 5~11: MDPV, MDGPV, SBDSV, DEV, DPMV, DHV-I, DTMUV; 12: negative control.

[0048] Figure 3 This refers to the sensitivity of the TaqMan-MGB dual real-time quantitative PCR in Example 1; where, Figure 3 A: Results of TaqMan-MGB dual real-time quantitative PCR sensitivity test of VP1 gene in GPVNP5 strain (1-7): The concentration of pUC57-NP5VP1 recombinant plasmid was 1.5×10⁻⁶.6 Copy / μL ~ 1.5 × 10 0 copies / μL; 8: negative control), Figure 3 B: Results of TaqMan-MGB dual real-time quantitative PCR sensitivity test of VP1 gene in GPV FJ strain (1-7: the concentration of pUC57-NP5VP1 recombinant plasmid was 1.5×10⁻⁶). 6 Copy / μL ~ 1.5 × 10 1 copies / μL; 8: negative control)

[0049] Figure 4 This is the standard curve for TaqMan dual real-time quantitative PCR in Example 2, where... Figure 4 A. Standard curve for TaqMan dual real-time quantitative PCR of the MDPV-PVP1 gene. Figure 4 B. Standard curve for TaqMan dual real-time quantitative PCR of MDPV-P1 VP1 gene.

[0050] Figure 5 This refers to the specificity of TaqMan dual real-time quantitative PCR in Example 2; wherein, 1: pUC57-PVP1 plasmid standard; 2: MDPV-P virulent strain; 3: pUC57-P1VP1 plasmid standard; 4: MDPV-P1 attenuated strain; 5~11: C-GPV, MDGPV, SBDSV, DEV, DPMV, DHV-I, DTMUV; 12: negative control.

[0051] Figure 6 This refers to the sensitivity of the TaqMan-MGB dual real-time quantitative PCR in Example 2; where... Figure 6 A: Results of TaqMan-MGB dual real-time quantitative PCR sensitivity assay for the VP1 gene of MDPV strain P (1-7): The concentrations of the pUC57-PVP1 recombinant plasmid were 1.25 × 10⁻⁶. 6 Copy / μL ~1.25×10 0 copies / μL; 8: negative control); Figure 6 B: Results of TaqMan-MGB dual real-time quantitative PCR sensitivity test of VP1 gene in MDPV P1 strain (1-7): The concentrations of pUC57-P1VP1 recombinant plasmids were 1.25 × 10⁻⁶. 6 Copy / μL ~1.25×10 0 copies / μL; 8: negative control). Detailed Implementation

[0052] The present invention will now be described in detail with reference to the accompanying drawings and embodiments:

[0053] Example 1: Establishment of a quantitative real-time TaqMan-MGB PCR method for differentiating between strong and weak virulent goose parvoviruses

[0054] 1. Materials and Methods

[0055] 1.1 Viruses and Pathogens

[0056] The virulent GPV strain NP5 (GPV NP5), the attenuated GPV strain FJ (GPV FJ), Muscovy duck parvovirus (MDPV), Muscovy duck goose plague virus (MDGPV), duck short-beaked dwarf syndrome virus (SBDSV), duck plague virus (DEV), duck paramyxovirus (DPMV), duck hepatitis virus type I (DHV-I), and duck Tembusu virus (DTMUV) were all preserved in the laboratory of the Fujian Academy of Agricultural Sciences. Pathological samples were collected since 2021 from a suspected goose plague outbreak farm in Fujian Province, including liver, spleen, pancreas, and kidney tissue samples submitted by geese. All samples were preserved in the laboratory of the Fujian Academy of Agricultural Sciences.

[0057] 1.2 Main Reagents and Instruments

[0058] PerfectStart ® II. Probe qPCR SuperMix was purchased from Beijing TransGen Biotech Co., Ltd. FastPure Viral DNA / RNA Mini kit (RC311-01), FastPure Gel DNA Extraction Mini Kit, and FastPure Plasmid Mini Kit were all purchased from Nanjing Novizan Biotechnology Co., Ltd. A real-time PCR instrument (ABI 7500) was purchased from Roche, Inc. (USA).

[0059] 1.3 Primer and Probe Design

[0060] The VP1 sequences of 20 GPV lineage virus strains included in GenBank were analyzed using DNAStar software. Multiple sets of GPV identification primers and specific probes for virulent and attenuated GPV strains were designed using Oligo 6.0 software. The probes were labeled with different luminescent groups to distinguish between virulent and attenuated strains. The primer and probe sequences are shown in Table 1.

[0061] Table 1 Primer and probe sequences for quantitative real-time PCR of GPV virulence and attenuation using TaqMan-MGB.

[0062]

[0063]

[0064] 1.4 Viral Nucleic Acid Extraction

[0065] Nucleic acid extraction was performed on GPV NP5, GPV FJ, MDGPV, MDPV, SBDSV, DEV, DPMV, DHV-I, and DTMUV according to the FastPure Viral DNA / RNA Extraction Kit instructions. Viral RNA was reverse transcribed into cDNA and stored at -20°C for later use, along with the viral DNA.

[0066] 1.5 Construction of recombinant plasmid standards

[0067] Variable region gene fragments of the VP1 gene from virulent GPV strain NP5 and attenuated strain FJ were synthesized and cloned into the pUC57 vector to construct recombinant plasmid standards pUC57-NP5VP1 and pUC57-FJVP1. Gene synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd. The concentration of the recombinant plasmids was determined using a full-wavelength microplate reader, and the copy number (copy / μL) was calculated. The results showed that pUC57-NP5VP1 and pUC57-FJVP1 had a concentration of 7.5 × 10⁻⁶. 9 Copies / μL, 9.0×10 9 Copy / μL. Dilute both to a uniform concentration of 1.5 × 10⁻⁶. 9 The sample was collected at 1 copy / μL and stored at -20 °C as a recombinant plasmid standard.

[0068] 1.7 Optimization of TaqMan-MGB Real-Time Quantitative PCR Method

[0069] Two recombinant plasmid standards, pUC57-NP5VP1 and pUC57-FJVP1, were mixed in equal volumes and used as templates. Two pairs of specific primers for strong and weak GPV, as shown in Table 1, and the TaqMan-MGB probe were used for real-time quantitative PCR amplification in the same system. The TaqMan-MGB real-time quantitative PCR reaction system was set to 20 μL. The annealing temperature (56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃), final primer concentration (0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L), and final probe concentration (0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L) were optimized using a matrix method to obtain the optimal reaction conditions for TaqMan real-time quantitative PCR.

[0070] 1.8 Establishment of the standard curve for the TaqMan-MGB real-time quantitative PCR method

[0071] The pUC57-NP5VP1 and pUC57-FJVP1 plasmid standards were serially diluted 10-fold, respectively, and the final concentrations were taken as 1.5 × 10⁻⁶. 6 Copy / μL - 1.5 × 10 0 A plasmid standard mixture of copies / μL was used as a template, and Nuclease-free Water was set up as a negative control. Amplification was performed using optimized TaqMan-MGB real-time quantitative PCR. A standard curve was plotted with the logarithm of the plasmid copy number at different concentrations as the X-axis and the corresponding Ct value as the Y-axis.

[0072] 1.9 Specificity test of TaqMan-MGB real-time quantitative PCR method

[0073] DNA from GPV NP5, GPV FJ, MDPV, MDGPV, SBDSV, and DEV, and cDNA from DPMV, DHV-I, and DTMUV stored in our laboratory were used. A mixture of pUC57-NP5VP1 and pUC57-FJVP1 plasmid standards was used as a positive control, and Nuclease-free Water was used as a negative control. The optimized TaqMan-MGB real-time quantitative PCR method for identifying the virulence of GPV was used for detection.

[0074] 1.10 Sensitivity test of TaqMan-MGB real-time quantitative PCR method

[0075] Two recombinant plasmid standards, pUC57-NP5VP1 and pUC57-FJVP1, were serially diluted 10-fold and mixed in equal volumes. The final concentrations were then determined to be 1.5 × 10⁻⁶. 6 Copy / μL - 1.5 × 10 0 Using a plasmid standard mixture of copies / μL as a template, the method was amplified using optimized TaqMan-MGB real-time PCR to evaluate its sensitivity.

[0076] 1.11 Repeatability test of TaqMan-MGB real-time quantitative PCR method

[0077] Three concentrations of pUC57-NP5VP1 (1.5 × 10⁻⁶) were selected. 6 Copy / μL, 1.5×10 5 Copy / μL, 1.5×10 4 pUC57-FJVP1 (1.5×10) 6 Copy / μL, 1.5×10 5Copy / μL, 1.5×10 4 Using a template, the optimized TaqMan-MGB real-time PCR method was used to perform intra-assay and inter-assay repeatability tests. Intra-assay repeatability tests were performed with three replicates per sample, and inter-assay repeatability tests were performed on samples at three different time periods. The results were statistically analyzed to verify the repeatability of the method.

[0078] 1.12 Detection of clinical samples

[0079] Twelve tissue samples (liver, spleen, pancreas, kidneys, etc.) from geese collected since 2021 from a suspected goose farm in Fujian Province experiencing goose plague were ground and processed. Viral nucleic acid was extracted from the supernatant using a nucleic acid extraction kit, and GPV was detected using an optimized dual TaqMan-MGB real-time quantitative PCR method. Positive samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the concordance rate between the PCR method and the sequencing results was calculated.

[0080] 2 Results

[0081] 2.1 Screening and comparison of TaqMan-MGB real-time quantitative PCR primer and probe combinations for GPV NP5 and FJ VP1 genes, and establishment of standard curve equations.

[0082] The recombinant plasmid standards of pUC57-NP5VP1 and pUC57-FJVP1 were serially diluted 10-fold (1.5 × 10⁻⁶). 6 Copy / μL ~ 1.5 × 10 0 Using a mixture of copies / μL as a template, primer and probe combinations for GPV NP5 and FJ VP1 gene amplification as shown in Table 1 were selected one by one, referring to PerfectStart. ®Amplification was performed using the reaction system and conditions recommended in the Probe qPCR SuperMix instruction manual. A standard curve was plotted with the logarithm of the initial template number as the X-axis and the cycle threshold (Ct value) as the Y-axis. The results (Table 2) show that, through analysis of the amplification efficiency and correlation coefficient in the standard curve equation, primer and probe combination 2 for GPV NP5 VP1 gene amplification and primer and probe combination 12 for GPV FJ VP1 gene amplification meet the requirements for establishing a dual TaqMan-MGB real-time quantitative PCR method for GPV NP5 and GPV FJVP1 genes. The classic goose parvovirus virulent and attenuated viral fluorescence quantitative TaqMan-MGB PCR differential diagnostic method established by this invention can simultaneously detect virulent and attenuated GPV viruses using only one universal upstream primer GPV-203VF, two virulent and attenuated virus-specific primers, and corresponding probes. This saves detection costs compared to the traditional method which requires designing two pairs of primers (4 strips) for simultaneous detection of two viruses.

[0083] Table 2 Standard curve equations for primer and probe combinations used in GPV NP5 and FJ VP1 gene amplification

[0084]

[0085] 2.2 Optimization of reaction conditions for TaqMan-MGB real-time quantitative PCR method

[0086] After optimization experiments, the optimal reaction system for dual TaqMan-MGB real-time quantitative PCR targeting the GPV NP5 and GPV FJ VP1 genes was determined: 2×PerfectStart ®II Probe qPCR SuperMix 10 μL, upstream universal PCR primer GPV-203VF (10 μmol / L) 0.4 μL, virulent downstream PCR primer GPV-203VR (10 μmol / L) 0.2 μL, attenuated downstream PCR primer GPV-203AR (10 μmol / L) 0.2 μL, probe NP5-Probe2 (10 μmol / L) 0.2 μL, probe FJ-Probe6 (10 μmol / L) 0.2 μL, recombinant plasmid standard mixture template DNA 2 μL, Passive Reference Dye II (50×) 0.5 μL, Nuclease-free Water to bring the total to 20 μL. The optimal reaction conditions for TaqMan-MGB real-time quantitative PCR targeting GPV NP5 and GPV FJ VP1 genes are: 94 ℃ for 30 s; fluorescence is collected at 94 ℃ for 5 s and 60 ℃ for 30 s, for 40 cycles.

[0087] 2.3 Establishment of dual TaqMan-MGB real-time quantitative PCR standard curves for GPV NP5 and GPV FJ VP1 genes

[0088] The recombinant plasmid standards of pUC57-NP5VP1 and pUC57-FJVP1 were serially diluted 10-fold (1.5 × 10⁻⁶). 6 Copy / μL ~ 1.5 × 10 0 The plasmid was mixed with a sample of 1 copy / μL as a template and amplified using the optimized TaqMan-MGB real-time quantitative PCR method. A standard curve was plotted with the logarithm of the initial template number as the X-axis and the cycle threshold (Ct value) as the Y-axis. The results showed that the recombinant plasmid standard pUC57-NP5VP1 was 1.5 × 10⁻⁶ μL. 6 Copy / μL ~ 1.5 × 10 0 Copy / μL, pUC57-FJVP1 at 1.5×10 6 Copy / μL ~ 1.5 × 10 0 Both copy number and μL showed good linearity at their respective Ct values. The standard curve for pUC57-NP5VP1 was Y = -3.282X + 15.744, with a correlation coefficient R0. 2 =0.998, amplification efficiency of 101.694; the standard curve of pUC57-FJVP1 is Y=-3.194X+14.381, correlation coefficient R 2 =0.993, amplification efficiency of 105.607.

[0089] 2.4 Specificity test results

[0090] An optimized dual TaqMan real-time quantitative PCR method was used to detect the nucleic acids of pUC57-NP5VP1, pUC57-FJVP1 positive plasmids, and GPV NP5, GPV FJ, MDPV, MDGPV, SBDSV, DEV, DPMV, DHV-I, and DTMUV. The results (Figure 1) showed that the nucleic acids of virulent GPV NP5 and attenuated GPV FJ, as well as a mixture of their plasmid standards, all exhibited amplification curves. No amplification curves were observed for the nucleic acids of other pathogens and the negative control. Figure 2 This indicates that the method has high specificity and can achieve differential diagnosis of strong and weak GPV strains.

[0091] 2.5 Sensitivity Test Results

[0092] The established TaqMan dual real-time quantitative PCR method for the virulent and attenuated GPV VP1 gene was used to detect the sensitivity of a mixture of two recombinant standard plasmids, pUC57-NP5VP1 and pUC57-FJVP1, corresponding to the virulent and attenuated GPV VP1 gene. The results showed that the lowest detectable amount of the established method for the recombinant standard plasmid of the virulent GPV VP1 gene was 1.5 × 10⁻⁶. 0 copies / μL ( Figure 3 A) The method established has a minimum template detection limit of 1.5 × 10⁻⁶ for the GPV attenuated VP1 gene recombinant standard plasmid. 1 copies / μL ( Figure 3 B). This indicates that the dual-fluorescence quantitative PCR method established in this experiment has high sensitivity.

[0093] 2.6 Results of Repeatability Tests

[0094] Following the optimized reaction conditions, three dilutions of pUC57-NP5VP1 and pUC57-FJVP1 plasmid standards were selected as templates for testing. Three intra-batch and inter-batch replicates were performed for each dilution of the template. The results showed that the intra-batch and inter-batch coefficients of variation for the same template at different concentrations were all within 2% (Table 3), indicating that the established quantitative real-time PCR method has good reproducibility and high stability.

[0095] Table 3. Repeatability results of TaqMan-MGB real-time PCR detection

[0096]

[0097] 2.7 Test results of clinical samples

[0098] This study collected 12 clinical samples suspected of having goose plague and used the established dual real-time fluorescence TaqMan-MGB PCR technology for detection. Three samples tested positive for virulent GPV (25%), while no virulent GPV was detected. Positive samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and sequence alignment was performed using online BLAST software. The results showed that the dual real-time fluorescence PCR method established in this study had a 100% concordance rate with the sequencing results, indicating that the established dual real-time fluorescence TaqMan-MGB PCR technology has high sensitivity and strong stability, and is suitable for the detection of clinical samples.

[0099] Example 2: Establishment of a quantitative real-time TaqMan-MGB PCR method for differentiating between strong and weak Muscovy duck parvoviruses

[0100] 1. Materials and Methods

[0101] 1.1 Viruses and Pathogens

[0102] The virulent MDPV strain P (MDPV-P), the attenuated MDPV strain P1 (MDPV-P1), classic goose parvovirus (C-GPV), Muscovy goose plague virus (MDGPV), duck short-beaked dwarf syndrome virus (SBDSV), duck plague virus (DEV), duck paramyxovirus (DPMV), duck hepatitis virus type I (DHV-I), and duck Tembusu virus (DTMUV) were all preserved in the laboratory of the Fujian Academy of Agricultural Sciences. Pathological samples were collected since 2023 from suspected Muscovy ducklings with viral enteritis in Fujian Province, including liver, spleen, pancreas, and kidney tissue samples, all preserved in the laboratory of the Fujian Academy of Agricultural Sciences.

[0103] 1.2 Main Reagents and Instruments

[0104] PerfectStart ® II. Probe qPCR SuperMix was purchased from Beijing TransGen Biotech Co., Ltd. FastPure Viral DNA / RNA Mini kit (RC311-01), FastPure Gel DNA Extraction Mini Kit, and FastPure Plasmid Mini Kit were all purchased from Nanjing Novizan Biotechnology Co., Ltd. A real-time PCR instrument (ABI 7500) was purchased from Roche, Inc. (USA).

[0105] 1.3 Primer and Probe Design

[0106] The VP1 sequences of the genomes of eight MDPV lineage virus strains included in GenBank were analyzed using DNAStar software. Multiple sets of TaqMan-MGB PCR primers and specific probes targeting virulent and attenuated MDPV strains were designed using Oligo 6.0 software. The probes were labeled with different luminescent groups to distinguish between virulent and attenuated strains. The primer and probe sequences are shown in Table 4.

[0107] Table 4 Primer and probe sequences for quantitative real-time TaqMan-MGB PCR

[0108]

[0109]

[0110] 1.4 Viral Nucleic Acid Extraction

[0111] Nucleic acid extraction was performed on MDPV-P, MDPV-P1, MDGPV, C-GPV, SBDSV, DEV, DPMV, DHV-I, and DTMUV according to the FastPure Viral DNA / RNA Extraction Kit instructions. RNA was reverse transcribed into cDNA and stored at -20 °C for later use, along with the viral DNA.

[0112] 1.5 Construction of recombinant plasmid standards

[0113] The variable region gene fragments of the VP1 gene from virulent MDPV strain P and attenuated MDPV strain P1 were synthesized separately and cloned into the vector pUC57 to construct recombinant plasmid standards pUC57-PVP1 and pUC57-P1VP1. Gene synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd. The concentration of the recombinant plasmids was determined using a full-wavelength microplate reader, and the copy number (copy / μL) was calculated. The results showed that pUC57-PVP1 and pUC57-P1VP1 had a concentration of 8.0 × 10⁻⁶. 9 Copy / μL, 6.25×10 9 Copy / μL. Dilute both to a uniform concentration of 1.25 × 10⁻⁶. 9 The sample was collected at 1 copy / μL and stored at -20 °C as a recombinant plasmid standard.

[0114] 1.6 Optimization of TaqMan-MGB Real-time Quantitative PCR Method

[0115] Two recombinant plasmid standards, pUC57-PVP1 and pUC57-P1VP1, were mixed in equal volumes and used as templates. Two pairs of specific primers for strong and weak MDPV and the TaqMan-MGB probe, as shown in Table 4, were used to perform real-time quantitative PCR amplification in the same system. The TaqMan real-time quantitative PCR reaction system was set to 20 μL. The annealing temperature (56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃), final primer concentration (0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L, 0.6 μmol / L, 0.7 μmol / L, 0.8 μmol / L, 0.9 μmol / L), and final probe concentration (0.1 μmol / L, 0.2 μmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.5 μmol / L) were optimized using a matrix method to obtain the optimal reaction conditions for TaqMan real-time quantitative PCR.

[0116] 1.7 Establishment of the standard curve for the TaqMan-MGB real-time quantitative PCR method

[0117] The pUC57-PVP1 and pUC57-P1VP1 plasmid standards were serially diluted 10-fold, and the final concentrations were 1.25 × 10⁻⁶. 6 Copy / μL - 1.25 × 10 0 A plasmid standard mixture of copies / μL was used as a template, and Nuclease-free Water was set up as a negative control. Amplification was performed using optimized TaqMan-MGB real-time quantitative PCR. A standard curve was plotted with the logarithm of the plasmid copy number at different concentrations as the X-axis and the corresponding Ct value as the Y-axis.

[0118] 1.8 Specificity test of TaqMan-MGB real-time quantitative PCR method

[0119] DNA from MDPV-P, MDPV-P1, C-GPV, MDGPV, SBDSV, and DEV, and cDNA from DPMV, DHV-I, and DTMUV stored in our laboratory were used as positive controls, with a mixture of pUC57-PVP1 and pUC57-P1VP1 plasmid standards as a positive control and Nuclease-free Water as a negative control. The optimized dual TaqMan-MGB real-time quantitative PCR method with strong and weak GPVs was used for detection.

[0120] 1.9 Sensitivity test of TaqMan-MGB real-time quantitative PCR method

[0121] Two recombinant plasmid standards, pUC57-PVP1 and pUC57-P1VP1, were serially diluted 10-fold and mixed in equal volumes. The final concentration of each standard was 1.25 × 10⁻⁶. 6 Copy / μL - 1.25 × 10 0 Using a plasmid standard mixture of copies / μL as a template, the method was amplified by optimized dual TaqMan-MGB real-time PCR to evaluate its sensitivity.

[0122] 1.10 Repeatability test of TaqMan-MGB real-time quantitative PCR method

[0123] Three concentrations of pUC57-PVP1 (1.25 × 10⁻⁶) were selected. 6 Copy / μL, 1.25×10 5 Copy / μL, 1.25×10 4 ), pUC57-P1VP1 (1.25×10 6 Copy / μL, 1.25×10 5 Copy / μL, 1.25×10 4 Using a template, the optimized dual TaqMan-MGB real-time PCR method was used to perform intra-assay and inter-assay repeatability tests. Intra-assay repeatability tests were performed with three replicates per sample, and inter-assay repeatability tests were performed on samples at three different time periods. The results were statistically analyzed to verify the repeatability of the method.

[0124] 1.11 Testing of Clinical Samples

[0125] Twenty-seven tissue samples (liver, spleen, pancreas, kidneys, etc.) from Muscovy ducks collected since 2021 from a suspected Muscovy duck viral enteritis farm in Fujian Province were ground and processed. Viral nucleic acid was extracted from the supernatant according to the nucleic acid extraction kit instructions, and MDPV was detected using an optimized dual TaqMan-MGB real-time quantitative PCR method. Positive samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the concordance rate between the PCR method and the sequencing results was calculated.

[0126] 2 Results

[0127] 2.1 Screening and comparison of TaqMan-MGB real-time quantitative PCR primer and probe combinations for MDPV-P and P1VP1 genes, and establishment of standard curve equations.

[0128] The recombinant plasmid standards of pUC57-PVP1 and pUC57-P1VP1 were serially diluted 10-fold (1.25 × 10⁻⁶). 6 Copy / μL ~1.25×10 0The plasmid was mixed with a sample of 1 copy / μL as a template and amplified using an optimized dual TaqMan real-time quantitative PCR method. A standard curve was plotted with the logarithm of the initial template number as the X-axis and the cycle threshold (Ct value) as the Y-axis. The results (Table 5) show that, through the analysis of amplification efficiency and correlation coefficients in the standard curve equation, primer and probe combinations 6 and 7 for MDPV-P1 VP1 gene amplification meet the requirements for establishing a dual TaqMan-MGB real-time quantitative PCR method for MDPV-P and MDPV-P1 VP1 genes. The classic Muscovy duck parvovirus virulent and attenuated virus quantitative PCR differential diagnostic method established in this invention only requires one pair of universal primers, MDPV-134F and MDPV-134R, combined with the corresponding probe to achieve simultaneous detection of MDPV virulent and MDPV attenuated viruses. Compared with the traditional method, which requires the design of two pairs of primers (4 strips) for simultaneous detection of two viruses, this method greatly saves detection costs.

[0129] Table 5. Standard curve equations corresponding to primer and probe combinations for MDPV-P and P1 VP1 gene amplification.

[0130]

[0131] 2.2 Optimization of reaction conditions for TaqMan-MGB real-time quantitative PCR method

[0132] After optimization experiments, the optimal reaction system for dual TaqMan real-time quantitative PCR targeting the MDPV-P and MDPV-P1 VP1 genes was determined: 2×PerfectStart ® The following reagents were used: 10 μL of Probe qPCR SuperMix, 0.4 μL each of upstream and downstream specific PCR primers (10 μmol / L), 0.2 μL each of probe (10 μmol / L), 2 μL of template DNA in the recombinant plasmid standard mixture, 0.5 μL of Passive Reference Dye II (50×), and Nuclease-free Water to a final volume of 20 μL. The optimal reaction conditions for TaqMan real-time quantitative PCR targeting the MDPV-P and MDPV-P1 VP1 genes were: 94 ℃ for 30 s; fluorescence was collected at 94 ℃ for 5 s and 60 ℃ for 30 s, for 40 cycles.

[0133] 2.3 Establishment of the Standard Curve

[0134] The recombinant plasmid standards of pUC57-PVP1 and pUC57-P1VP1 were serially diluted 10-fold (1.25 × 10⁻⁶). 6Copy / μL ~1.25×10 0 The plasmid was mixed with a sample of 1 copy / μL as a template and amplified using an optimized dual TaqMan real-time quantitative PCR method. A standard curve was plotted with the logarithm of the initial template number as the X-axis and the cycle threshold (Ct value) as the Y-axis. Figure 4 (As shown). The results showed that the recombinant plasmid standard pUC57-PVP1 was at 1.25 × 10⁻⁶. 6 Copy / μL ~1.25×10 0 Copy / μL, pUC57-P1VP1 at 1.25×10 6 Copy / μL ~1.25×10 0 Both copy number and μL showed good linearity at their respective Ct values. The standard curve for pUC57-PVP1 was Y = -3.268X + 13.542, with a correlation coefficient R0. 2 =0.999, amplification efficiency of 102.301; the standard curve of pUC57-P1VP1 is Y=-3.163X+14.590, and the correlation coefficient R is 0.999. 2 =0.996, amplification efficiency of 107.074.

[0135] 2.4 Specificity test results

[0136] An optimized dual TaqMan-MGB real-time quantitative PCR method was used to detect the nucleic acids of pUC57-PVP1, pUC57-P1VP1 positive plasmids, and MDPV-P, MDPV-P1, C-GPV, MDGPV, SBDSV, DEV, DPMV, DHV-I, and DTMUV. The results (Figure 5) showed that the nucleic acids of virulent MDPV-P and attenuated MDPV-P1, as well as a mixture of their plasmid standards, all exhibited amplification curves. Other pathogen nucleic acids and the negative control did not show amplification curves. Figure 5 This indicates that the method has high specificity and can achieve differential diagnosis of strong and weak MDPV strains.

[0137] 2.5 Sensitivity Test Results

[0138] The established TaqMan-MGB dual real-time quantitative PCR method for the MDPV virulent and attenuated VP1 gene was used to detect the sensitivity of a mixture of two recombinant standard plasmids pUC57-PVP1 and pUC57-P1VP1 corresponding to the MDPV virulent and attenuated VP1 gene. The results showed that the lowest detection limit of the established method for the template of the MDPV virulent VP1 gene recombinant standard plasmid pUC57-PVP1 was 1.25 × 10⁻⁶. 1 copies / μL ( Figure 6A) The method established has a minimum detection limit of 1.25 × 10⁻⁶ template for the MDPV attenuated VP1 gene recombinant standard plasmid pUC57-P1VP1. 1 copies / μL ( Figure 6 B). This indicates that the dual-fluorescence quantitative PCR method established in this experiment has high sensitivity.

[0139] 2.6 Results of Repeatability Tests

[0140] Following the optimized reaction conditions, three dilutions of pUC57-PVP1 and pUC57-P1VP1 plasmid standards were selected as templates for testing. Three intra-batch and inter-batch replicates were performed for each dilution of the template. The results showed that the intra-batch and inter-batch coefficients of variation for the same template at different concentrations were all within 2% (Table 6), indicating that the established quantitative real-time PCR method has good reproducibility and high stability.

[0141] Table 6. Repeatability results of TaqMan-MGB real-time PCR detection

[0142]

[0143] 2.7 Test results of clinical samples

[0144] This study collected 27 clinical samples suspected of having Muscovy duck viral enteritis (MDPV) and detected them using the established dual real-time fluorescence TaqMan-MGB PCR technique. Two samples showed virulent MDPV, with a positive rate of 7.4%, while no attenuated MDPV was detected. Positive samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and sequence alignment was performed using online BLAST software. The results showed that the established dual real-time fluorescence TaqMan-MGB PCR method had a 100% concordance rate with the sequencing results, indicating that the established dual real-time fluorescence TaqMan-MGB PCR technique has high sensitivity and strong stability, making it suitable for the detection of clinical samples.

Claims

1. A primer and TaqMan-MGB probe set for the real-time quantitative PCR detection of strong and weak strains of classic waterfowl parvovirus, characterized in that: The classic waterfowl parvoviruses include goose parvovirus and Muscovy duck parvovirus; The primer and TaqMan-MGB probe set includes real-time PCR detection primers and a TaqMan-MGB probe set targeting virulent and attenuated goose parvoviruses, respectively, with the sequences as follows: GPV-203VF: 5'-AGATAGCCTCCAAGACGACC-3', GPV-203VR: 5'-CACCTCCCGCACTGACT-3', GPV-203AR: 5'-ACCTCCTGCACTGACCTC-3'; NP5-Probe2: 5'-6-FAM-CCTAGTAGAAGATCCTGTC-MGB-3', FJ-Probe12:5'-JOE-AGACTCGATCAACACG-MGB-3'; The primer and TaqMan-MGB probe set also includes real-time PCR detection primers and TaqMan-MGB probe sets targeting virulent and attenuated Muscovy duck parvovirus, respectively, with the following sequences: MDPV-134F: 5'-CCTACAGGTCAGGCAGTAGTC-3', MDPV-134R: 5'-TGTTGTTGTGTTTTGTTCATTGGTT-3'; P-Probe6: 5'-6-FAM-AACAGAGGAGCAAGA-MGB-3', P1-Probe7: 5'-JOE-TGGTACCAGATGAGC-MGB-3'.

2. A kit for the detection of strong and weak strains of classic waterfowl parvovirus using real-time quantitative PCR, characterized in that: The classic waterfowl parvovirus strains include both virulent and attenuated goose parvovirus strains. The kit includes reaction system A, which comprises real-time quantitative PCR detection primers and a TaqMan-MGB probe set targeting virulent and attenuated goose parvovirus, respectively. Their sequences are as follows: GPV-203VF: 5'-AGATAGCCTCCAAGACGACC-3', GPV-203VR: 5'-CACCTCCCGCACTGACT-3', GPV-203AR: 5'-ACCTCCTGCACTGACCTC-3'; NP5-Probe2: 5'-6-FAM-CCTAGTAGAAGATCCTGTC-MGB-3', FJ-Probe12:5'-JOE-AGACTCGATCAACACG-MGB-3'.

3. The reagent kit according to claim 2, characterized in that: The reaction system A is 20 μL, and each 20 μL of reaction system A includes: 2×PerfectStart ® II Probe qPCR SuperMix 10 μL, upstream universal PCR primer GPV-203VF 0.4 μL (10 μmol / L), downstream virulent PCR primer GPV-203VR 0.2 μL (10 μmol / L), downstream attenuated PCR primer GPV-203AR 0.2 μL (10 μmol / L), probe NP5-Probe2 0.2 μL (10 μmol / L), probe FJ-Probe6 0.2 μL (10 μmol / L), template DNA 2 μL, Passive Reference Dye II (50×) 0.5 μL, and Nuclease-free Water to bring the total to 20 μL.

4. The reagent kit according to claim 2, characterized in that: The classic waterfowl parvovirus strains also include Muscovy duck parvovirus (MDPV) strains. The kit also includes reaction system B, which includes real-time quantitative PCR detection primers and TaqMan-MGB probe sets targeting virulent and attenuated Muscovy duck parvovirus, respectively. Their sequences are as follows: MDPV-134F: 5'-CCTACAGGTCAGGCAGTAGTC-3', MDPV-134R: 5'-TGTTGTTGTGTTTTGTTCATTGGTT-3'; P-Probe6: 5'-6-FAM-AACAGAGGAGCAAGA-MGB-3', P1-Probe7: 5'-JOE-TGGTACCAGATGAGC-MGB-3'.

5. The reagent kit according to claim 4, characterized in that: The reaction system B is 20 μL, and each 20 μL reaction system B contains: 2×PerfectStart ® II Probe qPCR SuperMix 10 μL, upstream and downstream fluorescent quantitative PCR detection primers MDPV-134F and MDPV-134R (10 μmol / L each) 0.4 μL, probes P-Probe6 and P1-Probe7 (10 μmol / L each) 0.2 μL, template DNA 2 μL, Passive Reference Dye II (50×) 0.5 μL, and Nuclease-free Water to bring the total to 20 μL.

6. The kit according to any one of claims 2-5, characterized in that: The reaction conditions for the TaqMan real-time quantitative PCR method of the kit are: 94 ℃ for 30 s; fluorescence is collected at 94 ℃ for 5 s and 60 ℃ for 30 s, for 40 cycles.

7. A kit for the detection of strong and weak strains of classic waterfowl parvovirus using real-time quantitative PCR, characterized in that: The classic waterfowl parvovirus strains include Muscovy duck parvovirus strains. The kit includes reaction system B, which includes real-time quantitative PCR detection primers and a TaqMan-MGB probe set targeting both virulent and attenuated Muscovy duck parvovirus strains. Their sequences are as follows: MDPV-134F: 5'-CCTACAGGTCAGGCAGTAGTC-3', MDPV-134R: 5'-TGTTGTTGTGTTTTGTTCATTGGTT-3'; P-Probe6: 5'-6-FAM-AACAGAGGAGCAAGA-MGB-3', P1-Probe7: 5'-JOE-TGGTACCAGATGAGC-MGB-3'.

8. The reagent kit according to claim 7, characterized in that: The reaction system B is 20 μL, and each 20 μL reaction system B contains: 2×PerfectStart ® II Probe qPCR SuperMix 10 μL, upstream and downstream fluorescent quantitative PCR detection primers MDPV-134F and MDPV-134R (10 μmol / L each) 0.4 μL, probes P-Probe6 and P1-Probe7 (10 μmol / L each) 0.2 μL, template DNA 2 μL, Passive Reference Dye II (50×) 0.5 μL, and Nuclease-free Water to bring the total to 20 μL; The reaction conditions for the TaqMan real-time quantitative PCR method in the kit are: 94 ℃ for 30 s; fluorescence is collected at 94 ℃ for 5 s and 60 ℃ for 30 s, for 40 cycles.

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