Primer and locked nucleic acid probe set for real-time fluorescent quantitative PCR detection of two goose parvovirus strong and weak strains

Abstract

The application relates to a primer and a locked nucleic acid probe group for real-time fluorescent quantitative PCR detection of two strong and weak strains of goose parvovirus, wherein the strong and weak strains of goose parvovirus include MDGPV strong and weak viruses and SBDSV strong and weak viruses, and the sequences of the primer and the locked nucleic acid probe group are shown in SEQ ID NO. 1-8. The LNA-TaqMan real-time fluorescent quantitative PCR detection method established by using the primer and the LNA-TaqMan probe group can simultaneously detect, differentially diagnose and accurately quantify the strong and weak strains of goose parvovirus (duck small goose plague virus and / or duck short-beak dwarf syndrome virus strong and weak viruses), and is high in sensitivity, strong in specificity and good in repeatability.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to primers and locked nucleic acid (LNA)-TaqMan probe sets for the fluorescence quantitative PCR detection of two virulent strains of goose parvoviruses: Muscovy duck-origingoose parvovirus (MDGPV) and short beak and dwarfism syndrome-goose parvovirus (SBDSV). Background Technology

[0002] Goose parvovirus (GPV) infection is an acute, contagious, septicemic infectious disease that primarily affects goslings and Muscovy ducklings aged 4–20 days. It easily causes acute enteritis and inflammation of organs such as the heart, liver, and kidneys. The disease is highly contagious, with high morbidity and mortality rates, and spreads sporadically and irregularly, causing significant economic losses to the poultry industry and severely hindering its healthy development. GPV belongs to the genus Dependent Virus in the family Parvoviridae. It is a non-enveloped, single-stranded DNA virus with roughly equal numbers of positive-strand and negative-strand DNA particles. The genome size is approximately 5.0 kb and consists of two open reading frames (ORFs). The left ORF (LORF) encodes the nonstructural proteins (NS) NS1 and NS2, while the right ORF (RORF) encodes three structural proteins (viral capsid proteins, VP1, VP2, and VP3). The NS and VP proteins share a common carboxyl terminus, forming a nested structure. Like other autonomous parvoviruses, the NS protein of GPV is involved in the regulation of viral replication and its pathogenicity, while the VP protein is responsible for packaging single-stranded viral progeny DNA to form infectious progeny viral particles. Since 1997, outbreaks of Muscovy duck-origin goose parvovirus (MDGPV) have occurred in Muscovy duck-breeding areas such as Putian and Fuqing in Fujian Province. MDGPV is a new strain of Muscovy duck parvovirus that is naturally recombined from classic GPV (C-GPV) and Muscovy duck parvovirus (MDPV), which causes "three-week disease" in Muscovy ducks. It can be transmitted through direct or indirect contact and vertical transmission. In 2015, my country reported Short Beak and Dwarfism Syndrome Virus (SBDSV), a virus distantly related to MGPV. SBDSV has a wider range of natural hosts and can cause disease in commercial meat ducks such as Cherry Valley ducks, Beijing ducks, Muscovy ducks, and Taiwan White ducks. The disease occurs in ducks aged 6–40 days, with a morbidity rate of 5%–20% and a low mortality rate; the vast majority of infected ducks become stunted. Clinically, Muscovy duck gosling plague and SBDSV often circulate in farmed waterfowl in the same region, frequently exhibiting a mixed infection pattern, facilitating frequent recombination and mutation of waterfowl parvoviruses. Compared to highly virulent strains, variant strains of waterfowl parvovirus are less virulent, and the clinical symptoms in infected Muscovy duck flocks are atypical, requiring molecular biological methods for differential diagnosis.

[0003] There are many methods for GPV detection, including virus isolation and identification, ELISA, indirect immunofluorescence assay (IFA), serum neutralization assay (SN), real-time fluorescence quantitative PCR (qPCR), and loop-mediated isothermal amplification (LAMP). However, in practical production applications, most methods can only detect but not distinguish between virulent and attenuated GPV strains. Using quantitative fluorescence PCR and molecular biology techniques such as gene sequencing is currently the most effective method for identifying and detecting virulent and attenuated viral strains. Recent studies have found that modifying conventional TaqMan probes can effectively improve the specificity and sensitivity of real-time fluorescence quantitative PCR detection methods.

[0004] Locked nucleic acid (LNA) is a chemical modification that occurs through the linkage of a 2-methyl sugar between O2 and C4. LNA modification enhances the stability and affinity of DNA molecules in PCR reactions. It has been reported that the melting temperature of oligonucleotides increases by 96°C with each insertion of an additional nucleotide. Mutation modification enables significant amplification of the testing environment and the identification of single-base mismatches. Furthermore, it allows for more complex experiments in a single test tube. The unique design of LNA makes it stable in DNA or RNA environments, particularly exhibiting strong recognition and binding capabilities to DNA / RNA. Therefore, selectively modifying some bases in conventional TaqMan probes with locked nucleic acids improves sensitivity (specificity) for single-base mismatches, facilitates design, and improves the signal-to-noise ratio, partly due to reduced fluorescence from spurious bindings and the close proximity of the quencher and reporter dye. Currently, this technology is widely used in pathogen detection, pathogen identification, and gene mutation analysis. Currently, there are no research reports in China on the LNA-TaqMan probe-based quantitative PCR method for waterfowl parvovirus.

[0005] The quantitative real-time PCR detection method based on the gene sequence of functional protein coding regions is currently the main method used to distinguish different strains of waterfowl parvovirus. Therefore, this invention focuses on MGPV and SBDSV strains, establishing quantitative real-time PCR detection methods using LNA-TaqMan qPCR of the VP1 gene to distinguish between virulent and attenuated MGPV and SBDSV strains, aiming to provide a new methodological option for the early diagnosis and quantitative detection of MGPV and SBDSV. Summary of the Invention

[0006] To achieve the above objectives, this invention has developed LNA-TaqMan real-time quantitative PCR primers and LNA-TaqMan probe sets for detecting two virulent and attenuated strains of goose parvovirus, including virulent and attenuated MDDPV and SBDSV. The primers and LNA-TaqMan probe sets are suitable for the differential diagnosis of virulent and attenuated MDDPV and SBDSV strains in samples using real-time quantitative PCR.

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

[0008] To achieve the above objectives, the present invention provides primers and locked nucleic acid probe sets for LNA-TaqMan real-time fluorescence quantitative PCR detection of two virulent and weak strains of goose parvovirus, the two goose parvoviruses being Muscovy goose plague virus (MDGPV) and duck short-beaked dwarf syndrome virus (SBDSV).

[0009] The primer and locked nucleic acid probe set includes LNA-TaqMan real-time quantitative PCR detection primers and LNA-TaqMan probe sets targeting virulent and attenuated Muscovy duck goose plague virus, respectively, and their specific sequences are as follows (Note: lowercase letters represent LNA modified bases):

[0010] MDGPV-121F: 5'-CCCCCAAGCCAAAATCAAACC-3' (SEQ ID NO. 1), MDGPV-121R: 5'-CCGTTACCAGGCCCAAGAT-3' (SEQ ID NO. 2);

[0011] PT-Probe: 6-FAM-ACCCCAACgAAAAG-MGB (SEQ ID NO.3),

[0012] D-Probe: VIC-ACCCCGACgAAAA-MGB (SEQ ID NO.4);

[0013] The primer and locked nucleic acid probe set also includes LNA-TaqMan real-time quantitative PCR detection primers and LNA-TaqMan probe sets targeting virulent and attenuated Duck Short-beaked Dwarf Syndrome Virus (DSDS) viruses, respectively, with the specific sequences as follows (Note: lowercase letters represent LNA-modified bases):

[0014] SBDSV-124F: 5'-GCAAACTGGAACATCTGGA-3' (SEQ ID NO.5),

[0015] SBDSV-150R: 5'-TGAGCTGGGATGCTGG-3' (SEQ ID NO. 6);

[0016] M15-Probe: 6-FAM-CACACAgAAGGGGA-MGB (SEQ ID NO.7),

[0017] M15F92-Probe: JOE-ACAGAAGAGgAGGC-MGB (SEQ ID NO. 8).

[0018] The present invention also provides a kit for LNA-TaqMan real-time fluorescence quantitative PCR detection of strong and weak strains of goose parvovirus, wherein the strong and weak strains of goose parvovirus include strong and weak strains of Muscovy goose plague virus, and the kit includes reaction system A.

[0019] The reaction system A includes LNA-TaqMan real-time quantitative PCR detection primers and LNA-TaqMan probe sets targeting virulent and attenuated Muscovy duck goose plague virus, respectively, with the specific sequences as follows (Note: lowercase letters represent LNA-modified bases):

[0020] MDGPV-121F: 5'-CCCCCAAGCCAAAATCAAACC-3',

[0021] MDGPV-121R: 5'-CCGTTACCAGGCCCAAGAT-3';

[0022] PT-Probe: 6-FAM-ACCCCAACgAAAAG-MGB,

[0023] D-Probe: VIC-ACCCCGACgAAAA-MGB.

[0024] The reaction system A is 20 μL, and each 20 μL of reaction system A includes:

[0025] 10 μL of IIProbe qPCR SuperMix, 0.8 μL each of primers MDDPV-121F and MDDPV-121R (both at 0.4 μmol / L), 0.4 μL of probe PT-Probe (at 0.2 μmol / L), 1.0 μL of probe D-Probe (at 0.5 μmol / L), 2.0 μL of template DNA, 0.5 μL of Passive Reference Dye II (50×), and Nuclease-free Water to bring the total volume to 20 μL.

[0026] The goose parvovirus strains of varying strengths and weaknesses also include duck short-beaked dwarf syndrome virus strains of varying strengths and weaknesses, and the kit also includes reaction system B;

[0027] The reaction system B includes LNA-TaqMan real-time quantitative PCR detection primers and LNA-TaqMan probe sets targeting virulent and attenuated Duck Short-beaked Dwarf Syndrome Virus (DSDS) viruses, respectively, with the specific sequences as follows (Note: lowercase letters represent LNA-modified bases):

[0028] SBDSV-124F: 5'-GCAAACTGGAACATCTGGA-3',

[0029] SBDSV-150R: 5'-TGAGCTGGGATGCTGG-3';

[0030] M15-Probe: 6-FAM-CACACAgAAGGGGA-MGB,

[0031] M15F92-Probe: JOE-ACAGAAGAGgAGGC-MGB.

[0032] The reaction system B is 20 μL, and each 20 μL of reaction system B includes:

[0033] The kit contains 10 μL of IIProbe qPCR SuperMix, 0.8 μL each of primers SBDSV-124F and SBDSV-150R (both at 0.4 μmol / L), 0.8 μL of probe M15-Probe (at 0.4 μmol / L), 0.6 μL of probe M15F92-Probe (at 0.3 μmol / L), 2.0 μL of template DNA, 0.5 μL of Passive Reference Dye II (50×), and Nuclease-free Water to a final volume of 20 μL. The reaction conditions for LNA-TaqMan real-time quantitative PCR using this kit are: 94℃ for 30 s; fluorescence is collected at 94℃ for 5 s and 60℃ for 30 s, for 40 cycles.

[0034] The present invention also provides a kit for LNA-TaqMan real-time fluorescence quantitative PCR detection of strong and weak strains of goose parvovirus, wherein the strong and weak strains of goose parvovirus include strong and weak strains of duck short-beaked dwarf syndrome virus; the kit includes reaction system B;

[0035] The reaction system B includes LNA-TaqMan real-time quantitative PCR detection primers and LNA-TaqMan probe sets targeting virulent and attenuated Duck Short-beaked Dwarf Syndrome Virus (DSDS) viruses, respectively, with the specific sequences as follows (Note: lowercase letters represent LNA-modified bases):

[0036] SBDSV-124F: 5'-GCAAACTGGAACATCTGGA-3',

[0037] SBDSV-150R: 5'-TGAGCTGGGATGCTGG-3';

[0038] M15-Probe: 6-FAM-CACACAgAAGGGGA-MGB,

[0039] M15F92-Probe: JOE-ACAGAAGAGgAGGC-MGB.

[0040] The reaction system B is 20 μL, and each 20 μL of reaction system B includes:

[0041] 10 μL of IIProbe qPCR SuperMix, 0.8 μL each of primers SBDSV-124F and SBDSV-150R (both at a concentration of 0.4 μmol / L), 0.8 μL of probe M15-Probe (at a concentration of 0.4 μmol / L), 0.6 μL of probe M15F92-Probe (at a concentration of 0.3 μmol / L), 2.0 μL of template DNA, 0.5 μL of Passive Reference Dye II (50×), and Nuclease-free Water to bring the total volume to 20 μL.

[0042] The reaction conditions for the LNA-TaqMan real-time quantitative PCR method of the kit are: 94℃ for 30s; fluorescence is collected at 94℃ for 5s and 60℃ for 30s, for 40 cycles.

[0043] Compared with the prior art, the advantages of the present invention are as follows:

[0044] 1. Simultaneous detection, rapid and efficient detection: The LNA-TaqMan real-time quantitative PCR detection method established by the primers and LNA-TaqMan probe set of this invention can simultaneously detect, differentiate and diagnose strong and weak strains of goose parvovirus (Muscovy duck goose plague virus and / or duck short-beak dwarf syndrome virus), and accurately quantify them. It simplifies the operation procedure and saves costs. At the same time, this detection method does not require conventional agarose gel electrophoresis detection. After the reaction, the results can be judged by the program built into the real-time quantitative PCR machine.

[0045] 2. Accurate quantification: By preparing standards and plotting standard curves, the virulence of Muscovy duck goose plague virus and duck short-beak dwarf syndrome virus infecting the sample can be accurately quantified directly based on the Ct values ​​of the strong and weak virulence of Muscovy duck goose plague virus and duck short-beak dwarf syndrome virus.

[0046] 3. High sensitivity: The method established in this invention has a minimum detection limit of 2.0 × 10⁻⁶ for the template of the recombinant standard plasmid pUC57-PTVP1 of the virulent MDGPV VP1 gene. 0 The method established, using copies / μL, has a minimum detection limit of 2.0 × 10⁻⁶ for the recombinant standard plasmid pUC57-DVP1 of the attenuated VP1 gene of MDDPV. 1 The copy number / μL indicates that the dual-fluorescence quantitative LNA-TaqMan PCR method established using primers MDDPV-121F, MDDPV-121R, probe PT-Probe, and probe D-Probe has high sensitivity. The minimum detection limit of the method established in this invention for the recombinant standard plasmid pUC57-M15VP1 of the highly virulent SBDSV VP1 gene is 1.0 × 10⁻⁶ copies / μL. 0 The method established, using copies / μL, had a minimum detection limit of 1.0 × 10⁻⁶ for the recombinant standard plasmid pUC57-M15F92VP1 of the attenuated SBDSV VP1 gene. 1 Copy / μL. This indicates that the dual-fluorescence quantitative LNA-TaqMan PCR method established in this invention using primers SBDSV-124F, primers SBDSV-150R, probe M15-Probe, and probe M15F92-Probe has high sensitivity.

[0047] 4. High Specificity: A dual real-time quantitative PCR method based on reaction system A was established and optimized to detect the nucleic acids of pUC57-PTVP1, pUC57-DVP1 positive plasmids, and MDDPV PT, MDDPV D, MDPV, C-GPV, SBDSV, DEV, DPMV, DHV-I, and DTMUV. Results showed that the nucleic acids of virulent MDDPV PT and attenuated MDDPV D, as well as a mixture of their plasmid standards, all exhibited amplification curves. No amplification curves were observed for other pathogen nucleic acids and the negative control. This indicates that the method has high specificity and enables the differential diagnosis of virulent and attenuated MDDPV strains.

[0048] A dual LNA-TaqMan real-time quantitative PCR method, established and optimized using reaction system B, was used to detect the nucleic acids of positive plasmids pUC57-M15VP1 and pUC57-M15F92VP1, as well as SBDSV-M15, SBDSV-M15F92, MDPV, C-GPV, MDGPV, DEV, DPMV, DHV-I, and DTMUV. The results showed that the nucleic acids of virulent SBDSV-M15 and attenuated SBDSV-M15F92, as well as a mixture of their plasmid standards, all exhibited amplification curves. No amplification curves were observed for other pathogen nucleic acids and the negative control. This indicates that the method has high specificity and enables the differential diagnosis of virulent and attenuated SBDSV strains.

[0049] 5. Good reproducibility: The quantitative real-time LNA-TaqMan PCR method established in this invention has good reproducibility and stability. Attached Figure Description

[0050] Figure 1 These are the standard curves for LNA-TaqMan dual real-time quantitative PCR in Example 1, where A is the standard curve for LNA-TaqMan PCR of the MDGPV-PTVP1 gene dual real-time quantitative PCR, and B is the standard curve for LNA-TaqMan PCR of the MDGPV-DVP1 gene dual real-time quantitative PCR.

[0051] Figure 2 This is the specificity of LNA-TaqMan dual real-time quantitative PCR in Example 1; wherein, 1: pUC57-PTVP1 plasmid standard; 2: MDGPV PT virulent strain; 3: pUC57-DVP1 plasmid standard; 4: MDGPV D attenuated strain; 5-11: MDPV, C-GPV, SBDSV, DEV, DPMV, DHV-I, DTMUV; 12: negative control.

[0052] Figure 3 This is a graph showing the sensitivity results of LNA-TaqMan dual real-time quantitative PCR in Example 1, where A represents the sensitivity test results of LNA-TaqMan PCR for the VP1 gene of the MDGPV PT strain (1-7: the concentrations of the pUC57-PTVP1 recombinant plasmid were 2.0 × 10⁻⁶). 6 Copies / μL ~ 2.0 × 10⁻⁶ 0 Copy / μL; 8: negative control); B is the result of the dual real-time quantitative PCR sensitivity test of the VP1 gene of MDDPV D strain (1~7: the concentration of pUC57-DVP1 recombinant plasmid is 2.0×10⁻⁶). 6 Copies / μL ~ 2.0 × 10⁻⁶ 0Copy / μL; 8: negative control).

[0053] Figure 4 These are the standard curves for dual real-time quantitative LNA-TaqManPCR in Example 2, where A is the standard curve for dual real-time quantitative LNA-TaqManPCR of the SBDSV-M15 VP1 gene, and B is the standard curve for dual real-time quantitative LNA-TaqManPCR of the SBDSV-M15F92 VP1 gene.

[0054] Figure 5 This is a diagram showing the specific results of LNA-TaqMan dual real-time quantitative PCR in Example 2; where 1: pUC57-M15VP1 plasmid standard; 2: SBDSV-M15 virulent strain; 3: pUC57-M15F92VP1 plasmid standard; 4: SBDSV-M15F92 attenuated strain; 5-11: MDPV, MDGPV, C-GPV, DEV, DPMV, DHV-I, DTMUV; 12: negative control.

[0055] Figure 6 This is a graph showing the sensitivity results of LNA-TaqMan dual real-time quantitative PCR in Example 2; where A represents the sensitivity test results of LNA-TaqMan dual real-time quantitative PCR for the VP1 gene of SBDSV-M15 strain (1-7: the concentrations of pUC57-M15VP1 recombinant plasmids were 1.0×10⁻⁶). 6 Copies / μL ~ 1.0 × 10⁻⁶ 0 Copy / μL; 8: negative control); B is the result of LNA-TaqMan dual real-time quantitative PCR sensitivity test of VP1 gene in SBDSV-M15F92 strain (1~7: the concentration of pUC57-M15F92VP1 recombinant plasmid is 1.0×10 6 Copies / μL ~ 1.0 × 10⁻⁶ 0 Copy / μL; 8: negative control). Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the embodiments of this invention will be further described in detail below with reference to the accompanying drawings. Materials and instruments not described in this invention are conventional materials and instruments in the art, and operational details not described in this invention are conventional operations in the art. The nucleic acid sequences shown in this invention are all written from left to right in a 5' to 3' direction.

[0057] In this invention, "upstream" refers to the 5' end or the 5' direction of the nucleic acid, and "downstream" refers to the 3' end or the 3' direction of the nucleic acid.

[0058] Example 1: Establishment of a quantitative real-time LNA-TaqMan PCR method for differential diagnosis of virulent and attenuated Muscovy duck and gosling plague virus 1. Materials and Methods

[0059] 1.1 Viruses and Pathogens

[0060] The virulent MDGPV-PT strain, the attenuated MDGPV-D strain, MDPV, C-GPV, SBDSV, duck plague virus, duck paramyxovirus, duck hepatitis virus type I, and duck Tembusu virus were all preserved in the laboratory of the Fujian Academy of Agricultural Sciences. Pathological samples were collected since 2023 from a Muscovy duck farm in Fujian Province suspected of having gosling plague, including liver, spleen, pancreas, and kidney tissue samples submitted by the ducks. All samples were preserved in the laboratory of the Fujian Academy of Agricultural Sciences.

[0061] 1.2 Main Reagents and Instruments

[0062] IIProbe 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).

[0063] 1.3 Primer Design and Synthesis

[0064] The VP1 sequences of the genomes of eight MGPV lineage virus strains included in GenBank were analyzed using DNAStar software. A pair of universal primers for MGPV and two specific LNA-TaqMan probes targeting virulent and attenuated MGPV strains were designed using the online primer design tool Primer-BLAST. The probes were labeled with different luminescent groups to distinguish between virulent and attenuated strains. The primer and LNA-TaqMan probe sequences are shown in Table 1 (Note: lowercase letters represent LNA modified bases).

[0065] Table 1 Primer and probe sequences for quantitative real-time LNA-TaqMan PCR

[0066]

[0067] 1.4 Viral Nucleic Acid Extraction

[0068] Nucleic acid extraction was performed on MDDPV-PT, MDDPV-D, C-GPV, MDPV, 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.

[0069] 1.5 Construction of recombinant plasmid standards

[0070] Variable region gene fragments of the VP1 gene from virulent PT and attenuated D strains of MDDPV were synthesized and cloned into the pUC57 vector to construct recombinant plasmid standards pUC57-PTVP1 and pUC57-DVP1. 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-PTVP1 and pUC57-DVP1 had a concentration of 6.0 × 10⁻⁶. 9 Copy / μL, 8.0×10 9 Copy / μL. Dilute both to a uniform concentration of 2.0 × 10⁻⁶. 9 Copies / μL, stored at -20℃ as a recombinant plasmid standard for future use.

[0071] Optimization of the 1.6LNA-TaqMan real-time quantitative PCR method

[0072] Two recombinant plasmid standards, pUC57-PTVP1 and pUC57-DVP1, were mixed in equal volumes and used as templates. Quantitative real-time PCR amplification was then performed in the same system (reaction system A) using two specific primers (MDGPV-121F and MDGPV-121R) and LNA-TaqMan probes (PT-Probe and D-Probe). The TaqMan real-time quantitative PCR reaction system was set to 20 μL. The annealing temperature (56℃, 57℃, 58℃, 59℃, 60℃), final primer concentration (0.1μmol / L, 0.2μmol / L, 0.3μmol / L, 0.4μmol / L, 0.5μmol / L, 0.6μmol / L, 0.7μmol / L, 0.8μmol / L, 0.9μmol / L, 1.0μmol / L), and final probe concentration (0.1μmol / L, 0.2μmol / L, 0.3μmol / L, 0.4μmol / L, 0.5μmol / L) were optimized using a matrix method to obtain the optimal reaction conditions for TaqMan real-time quantitative PCR.

[0073] 1.7 Establishment of the standard curve for the LNA-TaqMan real-time quantitative PCR method

[0074] The pUC57-PTVP1 and pUC57-DVP1 plasmid standards were serially diluted 10-fold, and the final concentrations were taken as 2.0 × 10⁻⁶. 6 Copy / μL - 2.0 × 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. Optimized TaqMan real-time quantitative PCR was used for amplification. 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.

[0075] Specificity test of the 1.8LNA-TaqMan real-time quantitative PCR method

[0076] DNA from MDGPV-PT, MDGPV-D, MDPV, C-GPV, SBDSV, and DEV, and cDNA from DPMV, DHV-I, and DTMUV stored in our laboratory were used. A mixture of pUC57-PTVP1 and pUC57-DVP1 plasmid standards was used as a positive control, and nuclease-free water was used as a negative control. The optimized dual TaqMan real-time quantitative PCR method with varying viral loads was employed for detection. 1.9 Sensitivity test of the LNA-TaqMan real-time quantitative PCR method.

[0077] Two recombinant plasmid standards, pUC57-PTVP1 and pUC57-DVP1, were serially diluted 10-fold and mixed in equal volumes. The final concentrations were then determined to be 2.0 × 10⁻⁶. 6 Copy / μL - 2.0 × 10 0 Using a plasmid standard mixture of copies / μL as a template, the method was amplified using optimized dual TaqMan real-time PCR to evaluate its sensitivity.

[0078] 1.10 Repeatability test of LNA-TaqMan real-time quantitative PCR method

[0079] Three concentrations of pUC57-PTVP1 (2.0 × 10⁻⁶) were selected. 6 Copies / μL, 2.0 × 10 5 Copies / μL, 2.0 × 10 4 (copy / μL), pUC57-DVP1 (2.0×10) 6 Copies / μL, 2.0 × 10 5 Copies / μL, 2.0 × 10 4Using copies / μL as templates, intra-assay and inter-assay repeatability tests were performed using the optimized dual TaqMan real-time PCR method. Intra-assay repeatability tests were performed with 3 replicates per sample, and inter-assay repeatability tests were performed on samples at 3 different time periods. The results were statistically analyzed to verify the repeatability of the method.

[0080] 1.11 Detection of clinical samples

[0081] Twenty-five tissue samples (liver, spleen, pancreas, kidneys, etc.) from Muscovy ducklings collected since 2023 from a suspected Muscovy duck farm in Fujian Province experiencing gosling plague were ground and processed. Viral nucleic acid was extracted from the supernatant using a nucleic acid extraction kit, and MDDPV was detected using an optimized dual LNA-TaqMan 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.

[0082] 2 Results

[0083] 2.1 Optimization of reaction conditions for LNA-TaqMan real-time quantitative PCR method

[0084] After optimization experiments, the optimal reaction system (reaction system A) for dual LNA-TaqMan real-time quantitative PCR targeting the MGPV-PT and MGPV-D VP1 genes was determined: The following reagents were used: 10 μL of IIProbe qPCR SuperMix, 0.8 μL each of upstream specific PCR primer MDDPV-121F (0.4 μmol / L) and downstream specific PCR primer MDDPV-121R (0.4 μmol / L), 0.4 μL of probe PT-Probe (0.2 μmol / L), 1.0 μL of probe D-Probe (0.5 μmol / L), 2.0 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 LNA-TaqMan real-time quantitative PCR targeting the MDDPV PT and MDDPV DVP1 genes were: 94℃ for 30 s; fluorescence was collected at 94℃ for 5 s and 60℃ for 30 s, for 40 cycles.

[0085] 2.2 Establishment of the Standard Curve

[0086] The recombinant plasmid standards of pUC57-PTVP1 and pUC57-DVP1 were serially diluted 10-fold (2.0 × 10⁻⁶). 6 Copies / μL ~ 2.0 × 10⁻⁶ 0The sample was mixed with plasmids (copies / μL) as a template and amplified using an optimized dual LNA-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 showed ( Figure 1 The recombinant plasmid standard pUC57-PTVP1 was measured at 2.0 × 10⁻⁶. 6 Copies / μL ~ 2.0 × 10⁻⁶ 0 Copy / μL, pUC57-DVP1 at 2.0×10 6 Copies / μL ~ 2.0 × 10⁻⁶ 0 Both copy number and μL showed good linearity at their respective Ct values. The standard curve for pUC57-PTVP1 was Y = -3.4X + 17.045, with a correlation coefficient R0. 2 =0.995, amplification efficiency of 96.858; the standard curve of pUC57-DVP1 is Y = -3.303X + 17.545, correlation coefficient R 2 =0.994, amplification efficiency of 100.779.

[0087] 2.3 Specificity test results

[0088] An optimized dual real-time quantitative PCR method (LNA-TaqMan) was used to detect the nucleic acids of pUC57-PTVP1, pUC57-DVP1 positive plasmids, and MDGPV PT, MDGPV D, MDPV, C-GPV, SBDSV, DEV, DPMV, DHV-I, and DTMUV. The results showed that the nucleic acids of virulent MDGPVPT and attenuated MDGPV D, as well as a mixture of their plasmid standards, all exhibited amplification curves. No amplification curves were observed for other pathogen nucleic acids or the negative control. Figure 2 This indicates that the method has high specificity and can achieve differential diagnosis of strong and weak MDGPV strains.

[0089] 2.4 Sensitivity Test Results

[0090] The established dual real-time quantitative PCR method for the virulent and attenuated VP1 gene of MDDPV was used to detect the sensitivity of a mixed sample of recombinant standard plasmids pUC57-PTVP1 and pUC57-DVP1. The results showed that the lowest detectable amount of the template for the recombinant standard plasmid pUC57-PTVP1 of the virulent VP1 gene of MDDPV was 2.0 × 10⁻⁶. 0 copy / μL ( Figure 3 A) The method established has a minimum detection limit of 2.0 × 10⁻⁶ templates for the MDDPV attenuated VP1 gene recombinant standard plasmid pUC57-DVP1. 1 copy / μL ( Figure 3B). This indicates that the dual-fluorescence quantitative LNA-TaqMan PCR method established in this experiment has high sensitivity.

[0091] 2.6 Repeatability Test Results

[0092] Following the optimized reaction conditions, three dilutions of pUC57-PTVP1 and pUC57-DVP1 plasmid standards were selected as templates for testing. Three intra-batch and inter-batch replicates were performed for each template dilution. The results showed that the intra-batch and inter-batch coefficients of variation for the same template at different concentrations were all within 2.0% (Table 2), indicating that the established quantitative LNA-TaqMan PCR method has good repeatability and stability.

[0093] Table 2. Repeatability results of LNA-TaqMan real-time PCR detection

[0094]

[0095] 2.7 Test results of clinical samples

[0096] This study collected 25 clinical samples suspected of having Muscovy duck gosling plague and used the established dual real-time fluorescence PCR technology for detection. Two samples tested positive for virulent MGPV (8.0%), while no virulent MGPV samples were 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 real-time fluorescence LNA-TaqMan PCR method established in this study had a 100% concordance rate with the sequencing results, indicating that the established MGPV fluorescence quantitative LNA-TaqMan PCR method has high sensitivity and strong stability, and is suitable for the detection of clinical samples.

[0097] Example 2: Establishment of a strong and weak virulence quantitative LNA-TaqMan PCR differential diagnostic method for duck short-beak dwarf syndrome

[0098] 1. Materials and Methods

[0099] 1.1 Virus and Clinical Samples

[0100] The virulent SBDSV strain M15, the attenuated SBDSV strain M15F92, Muscovy duck parvovirus, goose parvovirus, Muscovy duck gosling plague virus, duck plague virus, duck paramyxovirus, duck hepatitis type I virus, and duck Tembusu virus were all preserved in the laboratory of the Fujian Academy of Agricultural Sciences. Pathological samples were collected from ducklings in Fujian Province between 2023 and 2024 from a suspected case of duck short-beaked dwarf syndrome, including liver, spleen, pancreas, and kidney tissues, and were all preserved in the laboratory of the Fujian Academy of Agricultural Sciences.

[0101] 1.2 Main Reagents and Instruments

[0102] IIProbe 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).

[0103] 1.3 Primer Design and Synthesis

[0104] The VP1 sequences of 15 SBDSV lineage virus strains included in GenBank were analyzed using DNAStar software. A pair of universal primers and two specific probes targeting virulent and attenuated SBDSV strains were designed using the online primer design tool Primer-BLAST. The probes were labeled with different luminescent groups to distinguish between virulent and attenuated strains. The primer and probe sequences are shown in Table 3 (Note: lowercase letters represent LNA-modified bases).

[0105] Table 3 Primer and probe sequences for quantitative real-time LNA-TaqMan PCR

[0106]

[0107] 1.4 Viral Nucleic Acid Extraction

[0108] Nucleic acid extraction was performed on SBDSV-M15, SBDSV-M15F92, MDPV, C-GPV, MDGPV, 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.

[0109] 1.5 Construction of recombinant plasmid standards

[0110] The variable region gene fragments of the VP1 gene from the virulent and attenuated SBDSV strains M15 and M15F92 were synthesized and cloned into the pUC57 vector to construct recombinant plasmid standards pUC57-M15VP1 and pUC57-M15F92VP1. Gene synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd. The copy number (copy / μL) was calculated after determining the concentration of the recombinant plasmids using a full-wavelength microplate reader. The results showed that pUC57-M15VP1 and pUC57-M15F92VP1 had a concentration of 2.25 × 10⁻⁶. 9Copy / μL, 1.75×10 9 Copy / μL. Dilute both to 1.0×10⁻⁶. 9 Copies / μL, stored at -20℃ as a recombinant plasmid standard for future use.

[0111] Optimization of the 1.6LNA-TaqMan real-time quantitative PCR method

[0112] Two recombinant plasmid standards, pUC57-M15VP1 and pUC57-M15F92VP1, were mixed in equal volumes and used as templates. Quantitative real-time PCR amplification was performed in the same system (reaction system B) using two specific primers (SBDSV-124F and SBDSV-150R) and LNA-TaqMan probes (M15-Probe and M15F92-Probe). The TaqMan real-time quantitative PCR reaction system was set to 20 μL. The annealing temperature (56℃, 57℃, 58℃, 59℃, 60℃), final primer concentration (0.1μmol / L, 0.2μmol / L, 0.3μmol / L, 0.4μmol / L, 0.5μmol / L, 0.6μmol / L, 0.7μmol / L, 0.8μmol / L, 0.9μmol / L, 1.0μmol / L), and final probe concentration (0.1μmol / L, 0.2μmol / L, 0.3μmol / L, 0.4μmol / L, 0.5μmol / L) were optimized using a matrix method to obtain the optimal reaction conditions for LNA-TaqMan real-time quantitative PCR.

[0113] 1.7 Establishment of the standard curve for the LNA-TaqMan real-time quantitative PCR method

[0114] pUC57-M15VP1, pUC57-M15F92VP1, and plasmid standards were serially diluted 10-fold to obtain a final concentration of 1.0 × 10⁻⁶. 6 Copy / μL - 1.0 × 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. Optimized LNA-TaqMan real-time quantitative PCR was used for amplification. 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.

[0115] Specificity test of the 1.8LNA-TaqMan real-time quantitative PCR method

[0116] DNA from SBDSV-M15, SBDSV-M15F92, MDPV, C-GPV, MDGPV, and DEV, and cDNA from DPMV, DHV-I, and DTMUV stored in our laboratory were used. A mixture of pUC57-M15VP1 and pUC57-M15F92VP1 plasmid standards was used as a positive control, and Nuclease-free Water was used as a negative control. The optimized dual real-time quantitative PCR method for MDDSV with strong and weak virulence was used for detection.

[0117] Sensitivity test of 1.9LNA-TaqMan real-time quantitative PCR method

[0118] Two recombinant plasmid standards, pUC57-M15VP1 and pUC57-M15F92VP1, were serially diluted 10-fold and mixed in equal volumes. The final concentrations were then determined to be 1.0 × 10⁻⁶. 6 Copy / μL - 1.0 × 10 0 Using a plasmid standard mixture of copies / μL as a template, the method was amplified using an optimized dual-fluorescence quantitative LNA-TaqMan PCR method to evaluate its sensitivity.

[0119] 1.10 Repeatability test of LNA-TaqMan real-time quantitative PCR method

[0120] Three concentrations of pUC57-M15VP1 (1.0×10⁻⁶) were selected respectively. 6 Copy / μL, 1.0×10 5 Copy / μL, 1.0×10 4 (copy / μL), pUC57-M15F92VP1 (1.0×10⁻⁶) 6 Copy / μL, 1.0×10 5 Copy / μL, 1.0×10 4 Using copies / μL as templates, intra-assay and inter-assay repeatability tests were performed using the optimized dual-quantitative real-time LNA-TaqManPCR method. Intra-assay repeatability tests were performed with 3 replicates per sample, and inter-assay repeatability tests were performed on samples at 3 different time periods. The results were statistically analyzed to verify the repeatability of the method.

[0121] 1.11 Detection of clinical samples

[0122] Twenty clinical tissue samples from a duck farm were ground and processed. Viral nucleic acid was extracted from the supernatant according to the nucleic acid extraction kit instructions, and SBDSV was detected using an optimized dual real-time quantitative PCR method (LNA-TaqMan PCR). 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.

[0123] 2 Results

[0124] 2.1 Optimization of reaction conditions for real-time quantitative LNA-TaqMan PCR method

[0125] After optimization experiments, the optimal reaction system (reaction system B) for dual TaqMan real-time quantitative PCR targeting the SBDSV-M15 and SBDSV-M15F92 VP1 genes was determined: The following reagents were used: 10 μL of Probe qPCR SuperMix, 0.8 μL each of upstream specific PCR primer SBDSV-124F (0.4 μmol / L) and downstream specific PCR primer SBDSV-150R (0.4 μmol / L), 0.8 μL of probe M15-Probe (0.4 μmol / L), 0.6 μL of probe M15F92-Probe (0.3 μmol / L), 2.0 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 SBDSV-M15 and SBDSV-M15F92 VP1 genes were: 94℃ for 30 s; fluorescence was collected at 94℃ for 5 s and 60℃ for 30 s, for 40 cycles.

[0126] 2.2 Establishment of the Standard Curve

[0127] The recombinant plasmid standards of pUC57-M15VP1 and pUC57-M15F92VP1 were serially diluted 10-fold (1.0 × 10⁻⁶). 6 Copies / μL ~ 1.0 × 10⁻⁶ 0 A mixture of plasmid copies / μL was used as a template, and amplification was performed using an optimized dual real-time quantitative LNA-TaqMan 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 ( Figure 4 The recombinant plasmid standard pUC57-M15VP1 was tested at 1.0 × 10⁻⁶. 6 Copies / μL ~ 1.0 × 10⁻⁶ 0 Copy / μL, pUC57-M15F92VP1 at 1.0×106 Copies / μL ~ 1.0 × 10⁻⁶ 0 Both copies / μL showed good linearity at their respective Ct values. The standard curve for pUC57-M15VP1 was Y = -3.584X + 16.46, with a correlation coefficient R0. 2 =0.996, amplification efficiency of 99.112; the standard curve of pUC57-M15F92VP1 is Y = -3.289X + 13.974, correlation coefficient R 2 =0.998, amplification efficiency of 101.393.

[0128] 2.3 Specificity test results

[0129] An optimized dual LNA-TaqMan real-time quantitative PCR method was used to detect the nucleic acids of pUC57-M15VP1, pUC57-M15F92VP1 positive plasmids, as well as SBDSV-M15, SBDSV-M15F92, MDPV, C-GPV, MDGPV, DEV, DPMV, DHV-I, and DTMUV. The results showed ( Figure 5 The nucleic acids of virulent and attenuated SBDSV-M15 and the plasmid mixture of the two viruses showed amplification curves, while nucleic acids of other pathogens and the negative control did not. This indicates that the method has high specificity and can achieve the differential diagnosis of virulent and attenuated SBDSV strains.

[0130] 2.4 Sensitivity Test Results

[0131] The established dual real-time quantitative PCR method for the SBDSV virulent VP1 gene was used to detect the sensitivity of a mixed sample containing recombinant standard plasmids pUC57-M15VP1 and pUC57-M15F92VP1. The results showed that the lowest detectable amount of the template for the SBDSV virulent VP1 gene recombinant standard plasmid pUC57-M15VP1 was 1.0 × 10⁻⁶. 0 copy / μL ( Figure 6 A) The method established has a minimum detection limit of [missing information] for the template of the SBDSV attenuated VP1 gene recombinant standard plasmid pUC57-M15F92VP1.

[0132] 1.0×10 1 copy / μL ( Figure 6 B). This indicates that the dual-fluorescence quantitative LNA-TaqMan PCR method established in this experiment has high sensitivity.

[0133] 2.6 Repeatability Test Results

[0134] Following the optimized reaction conditions, three dilutions of pUC57-M15VP1 and pUC57-M15F92VP1 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 2.0% (Table 4), indicating that the established quantitative real-time PCR method has good repeatability and stability.

[0135] Table 4. Repeatability results of LNA-TaqMan real-time PCR detection

[0136]

[0137] 2.7 Test results of clinical samples

[0138] This study collected 20 clinical samples suspected of having duck short-beaked dwarf syndrome and used a newly established dual real-time fluorescence PCR technique for detection. Two samples tested positive for highly virulent SBDSV (10%), while no samples tested positive for attenuated SBDSV. 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 real-time fluorescence LNA-TaqMan PCR method established in this study had a 100% concordance rate with the sequencing results, indicating that the established SBDSV fluorescence quantitative LNA-TaqMan PCR method has high sensitivity and strong stability, and is suitable for the detection of clinical samples.

Claims

1. A primer and locked nucleic acid probe set for LNA-TaqMan real-time fluorescence quantitative PCR detection of two virulent and attenuated strains of goose parvovirus, characterized in that: The two goose parvoviruses mentioned include Muscovy goose plague virus and duck short-beak dwarf syndrome virus; The primer and locked nucleic acid probe set includes LNA-TaqMan real-time quantitative PCR detection primers and LNA-TaqMan probe sets targeting virulent and attenuated Muscovy duck goose plague virus, respectively, and their specific sequences are as follows: MDGPV-121F: 5'-CCCCCAAGCCAAAATCAAACC-3', MDGPV-121R: 5'-CCGTTACCAGGCCCAAGAT-3'; PT-Probe: 6-FAM-ACCCCAACgAAAAG-MGB, D-Probe: VIC-ACCCCGACgAAAA-MGB; The primer and locked nucleic acid probe set also includes LNA-TaqMan real-time quantitative PCR detection primers and LNA-TaqMan probe sets targeting virulent and attenuated Duck Short-beaked Dwarf Syndrome Virus (DSDS) viruses, respectively, with the specific sequences as follows: SBDSV-124F: 5'-GCAAACTGGAACATCTGGA-3', SBDSV-150R: 5'-TGAGCTGGGATGCTGG-3'; M15-Probe: 6-FAM-CACACAgAAGGGGA-MGB, M15F92-Probe: JOE-ACAGAAGAGgAGGC-MGB.

2. A kit for detecting virulent and attenuated strains of goose parvovirus using LNA-TaqMan real-time fluorescence quantitative PCR, characterized in that: The goose parvovirus strains include Muscovy duck goose plague virus strains, and the kit includes reaction system A. The reaction system A includes LNA-TaqMan real-time quantitative PCR detection primers and LNA-TaqMan probe sets targeting virulent and attenuated Muscovy duck goose plague virus, respectively, with the specific sequences as follows: MDGPV-121F: 5'-CCCCCAAGCCAAAATCAAACC-3', MDGPV-121R: 5'-CCGTTACCAGGCCCAAGAT-3'; PT-Probe: 6-FAM-ACCCCAACgAAAAG-MGB, D-Probe: VIC-ACCCCGACgAAAA-MGB; The goose parvovirus strains of varying strengths and weaknesses also include duck short-beaked dwarf syndrome virus strains of varying strengths and weaknesses, and the kit also includes reaction system B; The reaction system B includes LNA-TaqMan real-time quantitative PCR detection primers and LNA-TaqMan probe sets targeting virulent and attenuated Duck Short-beaked Dwarf Syndrome Virus (DSDS) viruses, respectively, with the specific sequences as follows: SBDSV-124F: 5'-GCAAACTGGAACATCTGGA-3', SBDSV-150R: 5'-TGAGCTGGGATGCTGG-3'; M15-Probe: 6-FAM-CACACAgAAGGGGA-MGB, M15F92-Probe: JOE-ACAGAAGAGgAGGC-MGB.

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: 10 μL of 2.0×PerfectStart II Probe qPCR SuperMix, 0.8 μL each of primers MDDPV-121F and MDDPV-121R (both at 0.4 μmol / L), 0.4 μL of probe PT-Probe (at 0.2 μmol / L), 1.0 μL of probe D-Probe (at 0.5 μmol / L), 2.0 μL of template DNA, 0.5 μL of Passive Reference Dye II (50×), and Nuclease-free Water to bring the total volume to 20 μL.

4. The kit according to claim 2, characterized in that: The reaction system B is 20 μL, and each 20 μL of reaction system B includes: 10 μL of 2.0×PerfectStart II Probe qPCR SuperMix, 0.8 μL each of primers SBDSV-124F and SBDSV-150R (both at a concentration of 0.4 μmol / L), 0.8 μL of probe M15-Probe (at a concentration of 0.4 μmol / L), 0.6 μL of probe M15F92-Probe (at a concentration of 0.3 μmol / L), 2.0 μL of template DNA, 0.5 μL of Passive Reference Dye II 50×, and Nuclease-free Water to bring the total volume to 20 μL.

5. The reagent kit according to claim 2, characterized in that: The reaction conditions for the LNA-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.

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