Quadruple TaqMan real-time fluorescent quantitative PCR primer group and probe group for simultaneously detecting cGPV, MDPV, MDGPV and SBDSV and kit thereof

Abstract

The invention relates to a quadruple TaqMan real-time fluorescent quantitative PCR (Polymerase Chain Reaction) primer group and a probe group for simultaneously detecting cGPV (Complementary Glutathione Virus), MDPV (Minimal Disease Papilloma Virus), MDGPV (Minimal Disease Papilloma Virus) and SBDSV (Sequence Broadcast DSV) and a kit thereof. The sequences of the primer group and the probe group are respectively shown as SEQ ID NO.1-12. The research successfully develops and verifies a multiple TaqMan-MGB real-time fluorescent PCR method capable of simultaneously detecting and distinguishing four important waterfowl parvoviruses (cGPV, MDPV, MDGPV and SBDSV). The method has good specificity, repeatability and high sensitivity. Compared with a conventional PCR method, the multiple detection method has the advantages that the clinical waterfowl parvovirus detection rate is remarkably improved, mixed infection can be effectively recognized, and the clinical diagnosis efficiency is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a quadruple TaqMan real-time fluorescent quantitative PCR primer set and probe set for simultaneously detecting cGPV, MDPV, MDGPV and SBDSV and a kit thereof. BACKGROUND

[0002] Parvoviruses are a group of small, non-enveloped DNA viruses that can infect multiple animal species. Traditionally, these viruses are named based on the host in which they were first isolated, rather than phylogenetic relationships. According to the classification of the International Committee on Taxonomy of Viruses (ICTV), waterfowl parvoviruses (WPVs) belong to the family Parvoviridae, subfamily Parvovirinae, and dependovirus genus. WPVs mainly include classical goose parvovirus (cGPV), Muscovy duck parvovirus (MDPV), Muscovy duck-origin goose parvovirus (MDGPV), and short beak and dwarfism syndrome virus (SBDSV, also known as new goose parvovirus, NGPV), which are all major pathogens in the global waterfowl farming industry.

[0003] Waterfowl parvoviruses (WPVs) have unique genomic structural features, containing inverted terminal repeats (ITRs) at both ends, which can form hairpin structures essential for viral replication. The viral genome is a linear single-stranded DNA (ssDNA) with a length of about 5.0-5.3 kb, containing two major open reading frames (ORFs). The left ORF encodes two non-structural proteins, NS1 (Rep1) and NS2 (Rep2), which not only play a key role in viral replication, transcription regulation, and particle assembly, but also have cytotoxic effects on host cells. The right ORF encodes structural proteins VP1, VP2, and VP3, which are closely related to immunogenicity, tissue tropism, pathogenicity, and host specificity. Figure 1

[0004] ​cGPV is the causative agent of Derzsy's disease, which can cause enteric exudative hepatitis and intestinal blockage in goslings and ducklings, with a mortality rate of 90-100% in the early stage of hatching. MDPV mainly infects young Muscovy ducks, causing enteritis, muscle degeneration, and growth retardation, especially with a high mortality rate in newly hatched ducks. MDGPV is a virus formed by recombination of cGPV and MDPV under natural conditions, which was first isolated in Muscovy duck farms in southern China in 1997. The virus uses the MDPV genome as a backbone, with the P9 promoter to NS (425-612 nt), NS2 (1,483-1,824 nt), and VP3 (3,124-4,248 nt) regions replaced by the corresponding regions of cGPV. The MDGPV strain is highly pathogenic to Muscovy ducks and has caused significant economic losses to the Chinese duck industry. Studies have shown that MDGPV can cause growth retardation, intestinal blockage, intestinal bleeding, and spleen atrophy in Muscovy ducklings, with similar clinical symptoms to Derzsy's disease caused by cGPV, with a mortality rate of 40-65%. Phylogenetic and epidemiological studies have shown that MDGPV has become an important recombinant strain in mixed infections and may play an important role in the continuous evolution of WPVs. Short-beak dwarf syndrome virus (SBDSV), also known as new goose parvovirus (NGPV), is a variant lineage of cGPV. Since 2015, SBDSV has gradually become an important pathogen in China, mainly causing growth retardation and skeletal abnormalities in semi-Muscovy ducks and Cherry Valley ducks, causing significant economic losses. Compared with classic cGPV, SBDSV has a wider host tropism and more complex clinical manifestations, which further increases the difficulty of its diagnosis and control.

[0005] The overall nucleotide similarity between classic GPV and MDPV is 80.6-82.6%, with NS protein homology of 80.7-83.0% and VP protein homology of 79.6-81.6%. The genomic similarity between SBDSV and cGPV strains is 91.5-98.7%, with NS protein homology of 93.4-96.9% and VP protein homology of 90.9-96.7%. These differences are mainly manifested as scattered point mutations. Although SBDSV is genetically similar to cGPV, it forms an independent phylogenetic branch, indicating that it has a unique evolutionary path and host adaptation strategy. Due to their high similarity in morphology, physicochemical properties, and genetic characteristics, it is challenging to accurately distinguish between cGPV and SBDSV.

[0006] Frequent recombination and mutation are important features of WPVs evolution, which are mainly related to the compact genome structure (single-stranded DNA) and the ease of co-infection. These genetic dynamics can profoundly affect the antigenicity, virulence, host adaptability and cross-species transmission ability of the virus, producing variant strains with expanded host range and altered pathogenicity. Therefore, mixed infections between waterfowl parvoviruses and other pathogens are increasingly common in clinical practice, posing a great challenge to virus monitoring and differential diagnosis. Due to the high genomic similarity between WPVs, accurate identification usually requires whole genome sequencing, which is time-consuming and labor-intensive. Currently, there is no detection method that can accurately distinguish the four WPVs mentioned above in the clinic at the same time.

[0007] Although there are several single or duplex real-time fluorescent PCR methods for detecting cGPV, MDPV or NGPV alone, traditional single PCR detection methods are difficult to cope with the genetic complexity and co-infection of WPVs. In contrast, multiplex PCR detection technology, especially the detection method based on TaqMan real-time fluorescent quantitative PCR (qPCR) combined with secondary groove binding agent (MGB) probe, has significant advantages. MGB probe can accurately distinguish highly similar viral sequences (including single nucleotide polymorphic sites) and has excellent specificity and thermal stability, which is very suitable for multiplex reaction system.

[0008] In view of the high genetic similarity and overlapping prevalence between cGPV, MDPV, SBDSV and MDGPV, it is urgent to establish a stable, sensitive and specific multiplex detection method to realize the simultaneous identification of the four viruses. In this study, we developed and verified a new quadruple TaqMan-MGB qPCR detection method that can simultaneously detect and distinguish cGPV, MDPV, SBDSV and MDGPV. This method has good analytical performance and provides a valuable molecular diagnostic tool for the comprehensive monitoring and early detection of WPVs. SUMMARY

[0009] The purpose of the present application is to provide a quadruple TaqMan real-time fluorescent quantitative PCR primer set and probe set for simultaneously detecting and distinguishing cGPV, MDPV, SBDSV and MDGPV, and a kit thereof. The detection method established by using the primer set and probe set has good specificity and analytical performance, and provides a valuable molecular diagnostic tool for the comprehensive monitoring and early detection of waterfowl parvovirus (WPVs).

[0010] The purpose of the present application is achieved by the following technical solutions:

[0011] The present application provides a quadruple TaqMan real-time fluorescent quantitative PCR primer set and probe set for simultaneously detecting cGPV, MDPV, MDGPV and SBDSV, wherein the primer set comprises:

[0012] Primer sequences for cGPV:

[0013] cGPV-F: 5'-TCCGAATCTCGGAGGA-3',

[0014] cGPV-R: 5'-TGGTGCACGTATTCTACT-3';

[0015] Primer sequences for MDPV:

[0016] MDPV-F: 5'-AACCAGGTCCAGTAGC-3',

[0017] MDPV-R: 5'-CTGACCTGTAGGGGTC-3';

[0018] Primer sequences for MDGPV:

[0019] MDGPV-F: 5'-AACAGATTCCACTGCC-3',

[0020] MDGPV-R: 5'-GGTATCACCTTGCAGAT-3';

[0021] Primer sequences for SBDSV:

[0022] SBDSV-F: 5'-GAAGCTTACTGACCATTACCCAGTAG-3',

[0023] SBDSV-R: 5'-CCCGCACTGACTTCCTCG-3';

[0024] The probe set comprises:

[0025] Probe sequence for cGPV:

[0026] cGPV-PROBE: 5'-ACCACCGCAGGTGTTCAT-3'

[0027] Probe sequence for MDPV:

[0028] MDPV-PROBE: 5'-AGGCTTCCAGTATACCGGCT-3'

[0029] Probe sequence for MDGPV:

[0030] MDGPV-PROBE: 5'-ACTTCTCTCCAAGAGA-3'

[0031] Probe sequence for SBDSV:

[0032] SBDSV-PROBE: 5'-AAGCCTAAACTCAC-3';

[0033] wherein:

[0034] the 5'-end of cGPV-PROBE is labeled with a fluorescent group Cy5, and the 3'-end is modified with MGB;

[0035] the 5'-end of MDPV-PROBE is labeled with a fluorescent group VIC, and the 3'-end is modified with MGB;

[0036] the 5'-end of MDGPV-PROBE is labeled with a fluorescent group Texas Red, and the 3'-end is modified with MGB;

[0037] the 5'-end of SBDSV-PROBE is labeled with a fluorescent group FAM, and the 3'-end is modified with MGB.

[0038] The primer set and the probe set are used for preparing a cGPV, MDPV, MDGPV and SBDSV detection reagent.

[0039] The application provides a quadruple TaqMan real-time fluorescent quantitative PCR detection method for detecting cGPV, MDPV, MDGPV and SBDSV by using the primer set and the probe set, and the method is not for diagnosis purposes,

[0040] The total volume of the quadruple TaqMan-MGB real-time fluorescent PCR reaction system is 20 muL, and contains: 10 muL of 2xAll-Powerful qPCRPreMix, 0.4 muL of cGPV, MDPV, MDGPV and SBDSV corresponding forward and reverse primers with a final concentration of 0.2 muM, 0.2 muL of probes cGPV-PROBE, MDPV-PROBE, MDGPV-PROBE and SBDSV-PROBE with a final concentration of 0.1 muM, 2 muL of mixed template DNA, and the volume is made up with nuclease-free water.

[0041] The PCR reaction condition is: contamination digestion at 53 DEG C for 10 min; pre-denaturation at 95 DEG C for 30 s; 95 DEG C denaturation for 10 s, 60 DEG C annealing / extension for 20 s, a total of 45 cycles.

[0042] The application also provides a quadruple TaqMan real-time fluorescent quantitative PCR kit for simultaneously detecting four kinds of waterfowl parvovirus (cGPV, MDPV, MDGPV and SBDSV), and the kit comprises primers and probes for detecting cGPV, MDPV, MDGPV and SBDSV, respectively.

[0043] The primer and probe sequences for detecting cGPV are as follows:

[0044] cGPV-F: 5'-TCCGAATCTCGGAGGA-3' (SEQ ID NO. 1),

[0045] cGPV-R: 5'-TGGTGCACGTATTCTACT-3' (SEQ ID NO. 2);

[0046] cGPV-PROBE: 5'-ACCACCGCAGGTGTTCAT-3' (SEQ ID NO. 3);

[0047] The primer and probe sequences for detecting MDPV are as follows:

[0048] MDPV-F: 5'-AACCAGGTCCAGTAGC-3' (SEQ ID NO. 4),

[0049] MDPV-R: 5'-CTGACCTGTAGGGGTC-3' (SEQ ID NO. 5);

[0050] MDPV-PROBE: 5'-AGGCTTCCAGTATACCGGCT-3' (SEQ ID NO. 6);

[0051] The primer and probe sequences for detecting MDGPV are as follows:

[0052] MDGPV-F: 5'-AACAGATTCCACTGCC-3' (SEQ ID NO. 7),

[0053] MDGPV-R: 5'-GGTATCACCTTGCAGAT-3' (SEQ ID NO. 8);

[0054] MDGPV-PROBE: 5'-ACTTCTCTCCAAGAGA-3' (SEQ ID NO. 9);

[0055] The primer and probe sequences for detecting SBDSV are as follows:

[0056] SBDSV-F: 5'-GAAGCTTACTGACCATTACCCAGTAG-3' (SEQ ID NO. 10), SBDSV-R: 5'-CCCGCACTGACTTCCTCG-3' (SEQ ID NO. 11);

[0057] SBDSV-PROBE: 5'-AAGCCTAAACTCAC-3' (SEQ ID NO. 12);

[0058] The 5'-end of the cGPV-PROBE is labeled with a fluorescent group Cy5, and the 3'-end is modified with MGB;

[0059] The 5'-end of the MDPV-PROBE is labeled with a fluorescent group VIC, and the 3'-end is modified with MGB;

[0060] The 5'-end of the MDGPV-PROBE is labeled with a fluorescent group Texas Red, and the 3'-end is modified with MGB;

[0061] The 5'-end of the SBDSV-PROBE is labeled with a fluorescent group FAM, and the 3'-end is modified with MGB.

[0062] Further, the total volume of the quadruple TaqMan-MGB real-time fluorescence PCR reaction system established therefrom is 20 μL, containing: 10 μL 2×All-Powerful qPCR PreMix, 0.4 μL of each of the forward and reverse primers corresponding to cGPV, MDPV, MDGPV, and SBDSV at a final concentration of 0.2 μM, 0.2 μL of each of the probes cGPV-PROBE, MDPV-PROBE, MDGPV-PROBE, and SBDSV-PROBE at a final concentration of 0.1 μM, 2 μL of mixed template DNA, and the volume is made up with nuclease-free water;

[0063] The PCR reaction conditions are as follows: contamination digestion at 53℃ for 10 min; pre-denaturation at 95℃ for 30 s; 95℃ denaturation for 10 s, 60℃ annealing / extension for 20 s, for a total of 45 cycles.

[0064] Compared with the prior art, the present application has the following advantages:

[0065] 1. The present application develops a new type of multiplex TaqMan-MGB qPCR method, which adopts highly specific primers and MGB modified probes for GPV, MDPV, MDGPV, and SBDSV VP1 genes. Given the high sequence similarity (>90%) between SBDSV and cGPV, designing specific primers that can distinguish between the two viruses is a great challenge. The application of MGB probe technology effectively solves this problem, and this technology can accurately distinguish single nucleotide polymorphisms (SNPs). It is worth noting that the SBDSV and GPV specific probes only differ by one nucleotide. MGB modification significantly improves the thermal stability and mismatch recognition ability of the probe, making it possible to accurately and simultaneously detect highly homologous virus species.

[0066] 2.The multiplex detection method developed by the application can reliably distinguish GPV, MDPV, MDGPV and SBDSV, and no cross-reaction occurs even in samples containing mixed virus nucleic acids, while the conventional PCR method often leads to false positive results. In addition, the application of TaqMan-MGB chemistry technology significantly improves the specificity and sensitivity of detection by increasing the probe melting temperature (Tm) and enhancing the recognition ability of minor sequence differences. The detection limit of the method is 10 1 to 10 3 copies / μL, which is more sensitive than conventional PCR and SYBR Green qPCR methods based on gel electrophoresis. The coefficient of variation is less than 2% both within and between batches, which ensures the consistency of detection results even in low viral load cases, which is particularly important for early diagnosis and subclinical monitoring.

[0067] 3.The application applies the method to 235 clinical samples collected from waterfowl farms in southern China in the past five years, and the overall detection rate (54.9%) is significantly higher than that of the conventional PCR method (51.9%). The detection rate of MDPV is 11%, while the detection rate of cGPV is relatively low (5.1%), and is only found in goose samples, indicating that the virus still maintains host specificity and may be replaced by MDGPV in duck flocks. The detection rate of SBDSV is 7.2%, which confirms that the virus is still prevalent and has the potential risk of causing biological safety incidents. The high detection rate of MDGPV (42.6%) may be related to its recombinant characteristics and field prevalence trend, which is consistent with the results of previous epidemiological studies. The method also detects 26 cases (11%) of MDGPV-MDPV mixed infection, suggesting that the two viruses exist in co-circulation and potential synergistic effect, which is likely related to their overlapping host range and genomic recombination events.

[0068] The multiplex qPCR method has high sensitivity and specificity in detecting single and mixed infection pathogens, highlighting its practical value in clinical diagnosis. Overall, compared with conventional PCR, the method exhibits superior sensitivity, specificity and diagnostic efficiency in field sample detection, providing a powerful tool for monitoring and control of waterfowl parvovirus infection.

[0069] 4.The application provides a quadruple TaqMan real-time fluorescent quantitative PCR primer set and probe set for simultaneously detecting cGPV, MDPV, MDGPV and SBDSV, and a kit thereof, and establishes a detection method capable of simultaneously detecting cGPV, MDPV, MDGPV and SBDSV, which has the following advantages and effects:

[0070] 4-1. Simultaneous detection, rapid detection, high efficiency: the four TaqMan real-time fluorescent quantitative PCR detection method established by the primer set and the probe set of the application can simultaneously detect, differentially diagnose and accurately quantify cGPV, MDPV, MDGPV and SBDSV, simplify the operation procedure, save the cost, and simultaneously, the detection method does not need to be detected by conventional agarose gel electrophoresis, and the result can be determined through the program of the real-time fluorescent quantitative PCR machine after the reaction is completed.

[0071] 4-2. Accurate quantification: by preparing the standard product and drawing the standard curve, the cGPV, MDPV, MDGPV and SBDSV in the detected sample can be directly accurately quantified according to the Ct value of the detected sample.

[0072] 4-3. High sensitivity: the minimum detection limit of cGPV is 10 2 copies / μL; the minimum detection limit of MDPV is 10 1 copies / μL; the minimum detection limit of MDGPV is 10 2 copies / μL; the minimum detection limit of cGPV is 10 3 copies / μL.

[0073] 4-4. Strong specificity: no positive amplification fluorescence signal is observed for eight common pathogens (such as DAdV-B2, DTMUV, DEV, NDRV, GoAstV, DPMV, MDRV and DHAV-1), and only amplification signals are observed for cGPV (Cy5 channel), MDPV (VIC channel), MDGPV (Texas Red channel) and SBDSV (FAM channel), which indicates that the method has high specificity for the four waterfowl parvoviruses. In addition, when cGPV, MDPV, SBDSV and MDGPV nucleic acids are mixed for detection, only specific amplification signals are observed in the respective channels, and no cross reaction occurs, which further confirms that the multiplex detection method has excellent specificity and reliability.

[0074] 4-5. Good repeatability: the batch variation coefficient of the real-time fluorescent quantitative PCR detection method established for cGPV detection is 0.78-1.90%, the batch variation coefficient is 0.75-0.89%; the batch variation coefficient of MDPV detection is 0.59-1.28%, the batch variation coefficient is 0.66-1.26%; the batch variation coefficient of MDGPV detection is 0.76-1.18%, the batch variation coefficient is 1.21-1.43%; the batch variation coefficient of SBDSV detection is 0.53-0.83%, the batch variation coefficient is 0.82-1.69%. All the CV values are lower than 2%, which indicates that the multiplex qPCR method established has excellent repeatability and stability under different template concentrations.

[0075] From the above, the detection method established by the application has good specificity, repeatability and high sensitivity. Compared with the conventional PCR method, the multiplex detection method not only significantly improves the detection rate of clinical waterfowl parvovirus, but also effectively identifies mixed infection, and greatly improves the clinical diagnosis efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0076] Figure 1 is a schematic diagram of the genome structure of waterfowl parvovirus (WPVs).

[0077] Figure 2 is the amplification curve and standard curve of the TaqMan-MGB qPCR detection method of the application. The concentration gradient of the standard plasmid is 10 8 to 10 3 copies / μL. (A1-A2), (B1-B2), (C1-C2) and (D1-D2) are the amplification curves and standard curves of p-cGPV, p-MDPV, p-MDGPV and p-SBDSV standard plasmids, respectively.

[0078] Figure 3 is the specificity analysis of the multiplex TaqMan-MGB qPCR detection method of the application. N represents the control samples used in this study, including DAdV-B2, DTMUV, DEV, NDRV, DPMV, MDRV, DHAV-1 and ddH2O. No positive signal was detected in all control samples.

[0079] Figure 4 is the amplification curve of the 10-fold gradient dilution of the standard plasmid for the sensitivity evaluation of the multiplex TaqMan-MGB qPCR method of the application. The dilution gradient is 10 9 to 10 0 copies / μL. (A) Cy5 channel detects cGPV; (B) VIC channel detects MDPV; (C) Texas Red channel detects MDGPV; (D) FAM channel detects SBDSV. The minimum detection limit of the method for cGPV, MDPV, MDGPV and SBDSV is 10 2 , 10 1 , 10 2 and 10 3 copies / μL. DETAILED DESCRIPTION

[0080] The content of the application will be described in detail below in combination with the drawings and examples of the specification:

[0081] 1. Materials and methods

[0082] 1.1 Virus strains and clinical samples

[0083] The virus strains used in the present application include: goose parvovirus (cGPV) NP5 strain (GenBank accession number: PQ272760), muscovy duck parvovirus (MDPV) P strain (GenBank accession number: KU844281), semi-muscovy duck parvovirus (MDGPV) D strain (GenBank accession number: OQ301813), and small bantam duck star virus (SBDSV) M15 strain (GenBank accession number: OR777281), which are isolated and preserved by the laboratory. Other virus strains used as negative controls include: duck adenovirus type B2 (DAdV-B2), duck tembusu virus (DTMUV), duck plague virus (DEV), new duck reovirus (NDRV), duck paramyxovirus (DPMV), muscovy duck reovirus (MDRV), and duck hepatitis A virus type 1 (DHAV-1).

[0084] From 2021 to 2024, 235 clinical samples suspected of waterfowl parvovirus (WPV) infection were collected from waterfowl farms in southern China. The samples were from the liver, spleen, kidney and intestinal tissue of sick or dead birds.

[0085] 1.2 Nucleic acid extraction

[0086] The total DNA and RNA of the clinical samples and virus isolates were extracted using FastPure Viral DNA / RNA Mini Kit (Novogene Bioinformatics Technology, Nanjing, China) according to the manufacturer's instructions. The extracted nucleic acids were immediately stored at -20°C for later use.

[0087] 1.3 Sequence alignment and primer design

[0088] Waterfowl parvovirus full-genome sequences were obtained from the GenBank database, including classic goose parvovirus (cGPV) strains (B, Y, DY16, YZ99-6, LH, and NP5); muscovy duck parvovirus (MDPV) strains (P, YY, FZ91, FJM2, FJV1, and FJM5); semi-muscovy duck parvovirus (MDGPV) strains (D, PT, ZW, 2022JS, SAAS-SHNH, and GD201911); and duck short-beak dwarf syndrome virus (SBDSV) strains (sdlco1, SC16, AA, SD, AH, and M15). DNASTAR software was used for multiple sequence alignment of 18 waterfowl parvovirus strains to identify their conserved regions and variable regions.

[0089] Based on the alignment results, Primer Premier 5.0 software was used to design specific primers and TaqMan-MGB probes for the highly conserved region of the VP gene. The specificity of the primers and probes was evaluated using the BLAST tool on NCBI to ensure no cross-reactivity with non-target sequences. The PrimerSelect module of the DNASTAR software was used to analyze and minimize potential secondary structures such as primer dimer formation and hairpin structures.

[0090] A number of primer-probe combinations were initially designed, and the best-performing optimized combination was obtained through experimental screening. In the final protocol, the TaqMan-MGB probes were labeled with different fluorescent groups at their 5' ends: cGPV probe labeled with Cy5, MDPV probe labeled with VIC, MDGPV probe labeled with Texas Red, and SBDSV probe labeled with FAM. All probes were modified with a minor groove binder (MGB) group at their 3' ends to enhance binding specificity and increase the melting temperature. The final sequences of the primers and probes are shown in Table 1, and all were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0091] Table 1 - qPCR primers and probes for simultaneous detection of GPV, MDPV, MDGPV, and SBDSV

[0092]

[0093] 1.4 Standard plasmid construction

[0094] Specific primer pairs (primer sequences in Table 2) were designed for the VP gene regions of cGPV, MDPV, MDGPV, and SBDSV for amplifying fragments containing the TaqMan-qPCR target sequences. The PCR reaction system was 20 μL, containing: 10 μL 2x TaqMasterMix (Dye Plus) (Qiagen, Nanjing, China), 2 μL cDNA template, 1 μL each primer (10 μM), and 6 μL nuclease-free water (ddH2O). The reaction program was set as: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 30 s, for a total of 35 cycles; and finally 72°C terminal extension for 5 min.

[0095] After purification, the PCR products were cloned into the pMD18-T vector (TAKARA, Shiga, Japan) to construct recombinant plasmids p-cGPV, p-MDPV, p-MDGPV, and p-SBDSV, respectively. The correctness of the recombinant plasmids was verified by Sanger sequencing, and the plasmid concentration and purity were determined using a DS-11 spectrophotometer (DeNovix, Wilmington, DE, USA). The copy number of each recombinant plasmid was calculated using the following formula:

[0096] Copy number / μL = (6.02 x 1023 ) x (X ng / μL x 10 -9 ) / (plasmid length (bp) x 660) where X represents plasmid concentration (ng / μL). The prepared standard plasmids were stored at -20 °C for later use.

[0097] Table 2 Primers used for construction of recombinant plasmid standards

[0098]

[0099] 1.5 Preparation of mixed plasmid standards

[0100] According to the calculated copy numbers, four positive control plasmids (1 x 101 0 copies / μL) of cGPV, MDPV, MDGPV and SBDSV were mixed with equal volume of 1 x TE buffer to prepare mixed standard solution with final concentration of 10 9 copies / μL. By 10-fold gradient dilution, standard series from 10 9 to 10 0 copies / μL were obtained for detection sensitivity evaluation.

[0101] 1.6 Establishment and optimization of multiplex real-time fluorescent PCR reaction system

[0102] Based on the established single-target real-time fluorescent PCR reaction conditions, multiplex detection system was developed and optimized. Matrix method was used to optimize primer and probe concentrations. Main optimization parameters included reaction temperature, primer concentration and probe concentration.

[0103] Firstly, primers and probes of cGPV, MDPV, MDGPV and SBDSV were diluted to a final concentration range of 0.1-0.6 μM, and gradient PCR was performed at annealing temperature range of 58-65 °C to determine the optimal conditions. By testing real-time fluorescent PCR reactions of different primer / probe concentration combinations and annealing temperatures, the optimal primer / probe concentration and optimal annealing temperature of the multiplex detection system were finally determined according to amplification efficiency, specificity and fluorescence signal intensity, etc.

[0104] 1.7 Establishment of standard curve

[0105] Recombinant plasmids p-GPV, p-MDPV, p-MDGPV and p-SBDSV were 10-fold gradient diluted to prepare concentration range of 10 8 -10 3Standard samples were prepared by 10-fold serial dilution of the recombinant plasmids p-GPV, p-MDPV, p-MDGPV and p-SBDSV, respectively, to a final concentration of 10 copies / μL. Three technical replicates were set for each dilution, and the established multiplex real-time PCR method was used for detection. The standard curve was plotted with the logarithmic values of the initial copy number of the plasmid (x-axis) and the corresponding cycle threshold (Ct value, y-axis). The quantitative standard curves of GPV, MDPV, MDGPV and SBDSV were established by regression analysis, respectively, to provide the basis for detection sensitivity evaluation and absolute quantification.

[0106] 1.8 Sensitivity, specificity and repeatability evaluation

[0107] Specificity evaluation:

[0108] The nucleic acid extracted from the control strains DAdV-B2, DTMUV, DEV, NDRV, GPMV, MDRV and DHAV-1 was used as a template, and normal muscovy duck fibroblasts were set as negative controls. The specificity of the method was evaluated by multiplex TaqMan-MGB qPCR detection.

[0109] Sensitivity evaluation:

[0110] The recombinant plasmids p-GPV, p-MDPV, p-MDGPV and p-SBDSV were serially diluted by 10 times, and the multiplex qPCR detection was performed until the template could not be detected. The lowest detection copy number of each target was recorded as the limit of detection (LOD) of the method. At the same time, the detection performance was compared with that of the conventional PCR method, the sensitivity improvement effect was evaluated, and the correlation between the dilution concentration and the positive detection rate of qPCR was analyzed.

[0111] Repeatability evaluation:

[0112] The plasmid standard (p-GPV, p-MDPV, p-MDGPV and p-SBDSV) was detected at different time points by three independent experiments, and the Ct value of each target was recorded. The coefficient of variation (CV) was calculated by the formula: CV (%) = (standard deviation (SD) / average value (X)) x 100

[0113] The repeatability and stability of the evaluation method were evaluated.

[0114] 1.9 Clinical sample detection

[0115] The method was applied to the detection of 235 clinical samples collected from waterfowl farms in southern China in the past five years. The nucleic acid DNA of the samples was extracted using the viral nucleic acid extraction kit FastPure Viral DNA / RNA Mini Kit (Novozyme Biological Technology, Nanjing, China), and the optimized multiplex TaqMan-MGB real-time fluorescent PCR was used for detection.

[0116] 2 Results

[0117] 2.1 Optimization of multiplex real-time fluorescent PCR reaction system and reaction conditions

[0118] After systematic optimization, the final multiplex TaqMan-MGB real-time fluorescent PCR reaction system was established with a total volume of 20 μL, containing: 10 μL 2x All-Powerful qPCR PreMix (Novozyme Biotech, Nanjing), 0.4 μL of cGPV / MDPV / MDGPV / SBDSV forward and reverse primers (final concentration 0.2 μM / primer), 0.2 μL of corresponding TaqMan-MGB probes (final concentration 0.1 μM / probe), 2 μL mixed template DNA, and the volume was made up with nuclease-free water. The optimized thermal cycling conditions were: contamination digestion at 53°C for 10 min; pre-denaturation at 95°C for 30 s; 95°C denaturation for 10 s, 60°C annealing / extension for 20 s, for a total of 45 cycles. The optimized conditions ensured efficient amplification and high specificity of simultaneous detection of cGPV, MDPV, MDGPV and SBDSV.

[0119] 2.3 Preparation and evaluation of standard curve

[0120] The standard plasmid solution was diluted 10-fold gradient with DNase / RNase-free water (10 8 -10 3 copies / μL) as the template for multiplex TaqMan-MGB real-time fluorescent PCR, and the optimized multiplex real-time fluorescent PCR reaction system and reaction conditions were used for amplification to obtain the corresponding amplification kinetics curves. The standard curve was plotted with the logarithmic value of the initial copy number of the plasmid (x-axis) and the cycle threshold value (Ct value, y-axis). Figure 2

[0121] Linear regression equation and detection performance parameters showed:

[0122] • cGPV: regression equation y = -3.866x + 39.52, amplification efficiency 97%, R 2 0.995

[0123] • MDPV: regression equation y = -3.59x + 40.97, amplification efficiency 100%, R 2 0.997

[0124] • MDGPV: regression equation y = -3.32x + 40.20, amplification efficiency 100%, R 2 0.998

[0125] • SBDSV: regression equation y = -3.98x + 40.23 (the highest correlation among the four targets), amplification efficiency 100%, R 2 0.999 ​

[0126] Results showed that the established multiplex qPCR method presented excellent linear relationship and high amplification efficiency in a wide range of concentrations. The accurate quantification of viral load could be achieved by substituting the Ct value of unknown samples into the corresponding equation.

[0127] 2.4 Specificity evaluation:

[0128] To evaluate the specificity of the established multiplex TaqMan-MGB qPCR method, DNA / cDNA samples extracted from eight major waterfowl viruses (DAdV-B2, DTMUV, DEV, NDRV, GoAstV, DPMV, MDRV and DHAV-1) were detected. The optimized multiplex real-time fluorescence PCR reaction system and reaction conditions were used for detection, with the nucleic acids of cGPV, MDPV, MDGPV and SBDSV as positive controls, and normal Muscovy duck fibroblast DNA as negative control, to evaluate the specificity of the established multiplex TaqMan-MGB qPCR method.

[0129] Results showed that fluorescence signals only appeared specifically in the corresponding detection channels: cGPV in Cy5 channel, MDPV in VIC channel, MDGPV in Texas Red channel, and SBDSV in FAM channel. No amplification signals were observed for the other eight viruses and negative control ( Figure 3 A-D), indicating that the method had high specificity for the four waterfowl parvoviruses. In addition, when cGPV, MDPV, SBDSV and MDGPV nucleic acids were mixed for detection, specific amplification signals were only observed in the respective corresponding channels, without cross-reaction ( Figure 3 E), further confirming that the multiplex detection method had excellent specificity and reliability.

[0130] Repeatability evaluation

[0131] To evaluate the repeatability of the multiplex TaqMan-MGB real-time fluorescence PCR method, the optimized multiplex TaqMan real-time fluorescence quantitative PCR method was used to detect three dilution gradients (10 7 -10 5 copies / μL) of p-cGPV, p-MDPV, p-MDGPV and p-SBDSV standard plasmids for intra-batch and inter-batch variation detection. The reproducibility of the method was evaluated by calculating the coefficient of variation (CV) of each repeated Ct value.

[0132] As shown in Table 3: Intra-batch CV: 0.53%-1.90% (p-cGPV, p-MDPV, p-MDGPV and p-SBDSV); Inter-batch CV: 0.75%-1.69%. All CV values were lower than 2%, indicating that the established multiplex qPCR method had excellent repeatability and stability under different template concentrations.

[0133]

[0134] 2.5 Clinical detection results

[0135] Using the multiplex TaqMan-MGB real-time fluorescence PCR method established in the present application, 235 clinical samples were detected. The overall detection rate (54.9%) was significantly higher than that of the conventional PCR method (51.9%). The detection rate of MDPV was 11%, while the detection rate of cGPV was relatively low (5.1%) and was only found in goose samples, indicating that the virus still maintains host specificity and may be replaced by MDGPV in duck flocks. The detection rate of SBDSV was 7.2%, confirming that the virus is still prevalent and has the potential risk of causing biological safety incidents. The high detection rate of MDGPV (42.6%) may be related to its recombinant characteristics and field prevalence trend, which is consistent with the results of previous epidemiological studies. The method also detected 26 cases (11%) of MDGPV-MDPV mixed infection, suggesting that there is co-circulation and potential synergistic effect between the two viruses, which is likely related to their overlapping host range and genomic recombination events.

[0136] The present application successfully develops and verifies a multiplex TaqMan-MGB real-time fluorescence PCR method that can simultaneously detect and distinguish four important waterfowl parvoviruses (cGPV, MDPV, MDGPV and SBDSV). The method has excellent specificity, high sensitivity, good linearity and reliable repeatability. Compared with conventional PCR, the multiplex detection method not only significantly improves the detection rate, but also exhibits excellent mixed infection recognition ability.

[0137] The present application establishes a quadruple TaqMan real-time fluorescence quantitative PCR method that can simultaneously detect and distinguish cGPV, MDPV, MDGPV and SBDSV. The specific primers and taqman probes are designed based on the VP gene sequence alignment. The method has strong specificity and no cross-reaction with other major waterfowl viruses. The detection sensitivity is: GPV 10 2 copies / μL, MDPV 10 1 copies / μL, MDGPV 10 2 copies / μL, SBDSV 10 3 copies / μL. The standard curve shows excellent linearity (R 2The results showed that the method had high specificity (≥0.995) and high amplification efficiency (97%-100%). The intra- and inter-batch coefficient of variation were both less than 2.0%, which confirmed the good repeatability and stability of the method. Clinical detection showed that the multiplex detection system had higher total detection rate (54.9% vs 51.9%) than routine PCR and could better identify mixed infections. A total of 26 samples were found to be co-infected with MDGPV and MDPV. The multiplex TaqMan-MGB qPCR method established in this study provided a rapid, sensitive, and reliable simultaneous detection tool for the four waterfowl parvoviruses, and its application would help to strengthen disease monitoring, optimize epidemic control, and provide more effective means for the prevention and control of waterfowl parvovirus disease.

[0138] The application of minor groove binder (MGB) probes enabled accurate identification of highly homologous virus strains, even strains with single nucleotide differences, effectively solving the diagnostic problems caused by the high genetic similarity and recombination events between GPV, MDPV, MDGPV, and SBDSV.

[0139] The successful application of this method in clinical samples from different waterfowl farms in southern China confirmed its practical value in large-scale monitoring and epidemic investigation. This diagnostic platform provides a powerful tool for the early detection, epidemiological monitoring, and effective prevention and control of waterfowl parvovirus-related diseases, and is expected to be widely used in veterinary diagnostic laboratories.

Claims

1. A quadruple TaqMan real-time fluorescence quantitative PCR primer set and probe set for simultaneous detection of cGPV, MDPV, MDGPV, and SBDSV, characterized by: The primer set comprises: The primer sequence for cGPV: cGPV-F: 5'-TCCGAATCTCGGAGGA-3', cGPV-R: 5'-TGGTGCACGTATTCTACT-3'; The primer sequence for MDPV: MDPV-F: 5'-AACCAGGTCCAGTAGC-3', MDPV-R: 5'-CTGACCTGTAGGGGTC-3'; The primer sequence for MDGPV: MDGPV-F: 5'-AACAGATTCCACTGCC-3', MDGPV-R: 5'-GGTATCACCTTGCAGAT-3'; The primer sequence for SBDSV: SBDSV-F: 5'-GAAGCTTACTGACCATTACCCAGTAG-3', SBDSV-R: 5'-CCCGCACTGACTTCCTCG-3'; The probe set comprises: The probe sequence for cGPV: cGPV-PROBE: 5'-ACCACCGCAGGTGTTCAT-3' The probe sequence for MDPV: MDPV-PROBE: 5'-AGGCTTCCAGTATACCGGCT-3' The probe sequence for MDGPV: MDGPV-PROBE: 5'-ACTTCTCTCCAAGAGA-3' The probe sequence for SBDSV: SBDSV-PROBE: 5'-AAGCCTAAACTCAC-3'; Wherein: The 5'-end of cGPV-PROBE is labeled with a fluorescent group Cy5, and the 3'-end is modified with MGB; The 5'-end of MDPV-PROBE is labeled with a fluorescent group VIC, and the 3'-end is modified with MGB; The 5'-end of MDGPV-PROBE is labeled with a fluorescent group Texas Red, and the 3'-end is modified with MGB; The 5'-end of SBDSV-PROBE is labeled with a fluorescent group FAM, and the 3'-end is modified with MGB.

2. Use of the primer set and probe set of claim 1 in the preparation of a reagent for detecting cGPV, MDPV, MDGPV, and SBDSV.

3. A quadruple TaqMan real-time fluorescent quantitative PCR detection method for detecting cGPV, MDPV, MDGPV, and SBDSV using the primer set and probe set of claim 1, which is not for the purpose of diagnosis, characterized in that: The total volume of the quadruple TaqMan-MGB real-time fluorescent PCR reaction system is 20 μL, which comprises: 10 μL of 2×All-Powerful qPCR PreMix, 0.4 μL of each of cGPV, MDPV, MDGPV, and SBDSV corresponding forward and reverse primers with a final concentration of 0.2 μM, 0.2 μL of each of the probes cGPV-PROBE, MDPV-PROBE, MDGPV-PROBE, and SBDSV-PROBE with a final concentration of 0.1 μM, 2 μL of mixed template DNA, and the volume is made up with nuclease-free water. PCR reaction conditions: contamination digestion 53℃ 10 min; pre-denaturation 95℃ 30 s; 95℃ denaturation 10 s, 60℃ annealing / extension 20 s, 45 cycles in total.

4. A quadruple TaqMan real-time fluorescence quantitative PCR kit for simultaneous detection of cGPV, MDPV, MDGPV, and SBDSV, characterized by: The kit comprises primers and probes for detecting cGPV, MDPV, MDGPV and SBDSV respectively; The primer and probe sequences for detecting cGPV are as follows: cGPV-F: 5'-TCCGAATCTCGGAGGA-3', cGPV-R: 5'-TGGTGCACGTATTCTACT-3'; cGPV-PROBE: 5'-ACCACCGCAGGTGTTCAT-3'; The primer and probe sequences for detecting MDPV are as follows: MDPV-F: 5'-AACCAGGTCCAGTAGC-3', MDPV-R: 5'-CTGACCTGTAGGGGTC-3'; MDPV-PROBE: 5'-AGGCTTCCAGTATACCGGCT-3'; The primer and probe sequences for detecting MDGPV are as follows: MDGPV-F: 5'-AACAGATTCCACTGCC-3', MDGPV-R: 5'-GGTATCACCTTGCAGAT-3'; MDGPV-PROBE: 5'-ACTTCTCTCCAAGAGA-3'; The primer and probe sequences for detecting SBDSV are as follows: SBDSV-F: 5'-GAAGCTTACTGACCATTACCCAGTAG-3', SBDSV-R: 5'-CCCGCACTGACTTCCTCG-3'; SBDSV-PROBE: 5'-AAGCCTAAACTCAC-3'; The 5'-end of the cGPV-PROBE is labeled with a fluorescent group Cy5, and the 3'-end is modified with MGB; The 5'-end of the MDPV-PROBE is labeled with a fluorescent group VIC, and the 3'-end is modified with MGB; The 5'-end of the MDGPV-PROBE is labeled with a fluorescent group Texas Red, and the 3'-end is modified with MGB; The 5'-end of the SBDSV-PROBE is labeled with a fluorescent group FAM, and the 3'-end is modified with MGB.

5. The kit of claim 4, wherein: The total volume of the quadruple TaqMan-MGB real-time fluorescence PCR reaction system established therefrom is 20 μL, comprising: 10 μL 2×All-Powerful qPCR PreMix, 0.4 μL of each of cGPV, MDPV, MDGPV and SBDSV corresponding forward and reverse primers with a final concentration of 0.2 μM, 0.2 μL of each of the probes cGPV-PROBE, MDPV-PROBE, MDGPV-PROBE and SBDSV-PROBE with a final concentration of 0.1 μM, 2 μL of mixed template DNA, and the volume is made up with nuclease-free water; PCR reaction conditions: Contamination digestion 53°C for 10 min; pre-denaturation 95°C for 30 s; 95°C denaturation for 10 s, 60°C annealing / extension for 20 s, 45 cycles.

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