TaqMan primer probe combination for detecting virulent and attenuated Newcastle disease as well as application and kit of TaqMan primer probe combination

Abstract

The invention belongs to the technical field of biology, and discloses a TaqMan primer probe combination for detecting virulent and attenuated Newcastle disease, which comprises a virulent primer RT, a virulent probe RT-probe, a universal primer SRT and a universal probe SRT-probe, the virulent primer RT comprises an upstream primer RT-F and a downstream primer RT-R, and the universal primer SRT comprises an upstream primer SRT-F and a downstream primer SRT-R; a TaqMan probe fluorescent quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection method for virulent and attenuated NDV (Newcastle Disease Virus) established by using the TaqMan primer probe combination has no cross reaction on amplification of common avian viruses AIV, FADV, IBV and IBDV and has good specificity; the lowest detection concentration of virulent and attenuated Newcastle disease virus is 101 copies / uL, which is 100 times higher than that of a conventional RT-PCR method, and the kit has good sensitivity; and compared with the conventional RT-PCR method, the detection rate is 100% and is superior to that of the conventional RT-PCR method, and the detection result is consistent with the sequencing result.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a TaqMan primer-probe combination for detecting virulent and attenuated Newcastle disease virus, its application, and a reagent kit. Background Technology

[0002] Newcastle disease (ND) is an acute, septicemic, and highly contagious poultry disease caused by Newcastle disease virus (NDV). It is highly pathogenic and spreads rapidly, posing a serious threat to poultry farming. Since its identification, the diagnostic system for ND has been continuously upgraded along with viral evolution, resulting in the isolation of eight type II genotypes: I, II, III, VI, VII, VIII, IX, and XII. Among these, type VII has gradually become the dominant genotype, and currently, the majority of ND cases are caused by type VII.

[0003] In recent years, the epidemiological characteristics of this disease have become increasingly complex. Co-infection with both virulent and weak strains frequently occurs in non-large-scale farms, exacerbating clinical symptoms and pathological damage, causing immunosuppression and immunization failure, and interfering with diagnosis and epidemic assessment. Therefore, identifying the pathogenicity of the strain has become a crucial aspect of epidemic prevention and control. Preliminary detection mainly uses serological testing due to its low cost and ease of operation, primarily through ELISA, HA, and HI tests. However, this method cannot further determine the specific NDV genotype for targeted prevention. Although current NDV control has achieved good results, a certain number of ND outbreaks still exist due to the expanding host range of the virus, viral evolution, vaccine failure, and mixed infections. Traditional detection methods, such as chicken embryo lethality time determination, intracerebral inoculation pathogenicity tests in chicks, and intravenous inoculation virulence index assessment, are cumbersome and time-consuming. Furthermore, the national standard GB / T 16550-2020 for routine RT-PCR detection only covers virulent strains VI and VII, and cannot effectively detect other prevalent strains; moreover, its sensitivity is only 10. 3 copies / μL.

[0004] Based on this, in order to detect strains with lower copy numbers and wider coverage, and to improve detection accuracy, the technical problem that this invention needs to solve is: how to provide a TaqMan primer-probe combination that can simultaneously detect strong and weak Newcastle disease virus strains, with a wider coverage of detectable strains, a lower minimum detectable copy number, and higher sensitivity, which can be used for clinical sample detection, providing technical reserves for the prevention and control of NDV, facilitating timely diagnosis and detection of NDV outbreaks, and enabling targeted measures to be taken based on the results of differential diagnosis of strong and weak NDV strains. Summary of the Invention

[0005] The purpose of this invention is to provide a TaqMan primer-probe combination for detecting virulent and attenuated Newcastle disease virus, including a virulent primer RT, a virulent probe RT-probe, and universal primers SRT and SRT-probe; the virulent primer RT includes an upstream primer RT-F and a downstream primer RT-R, and the universal primer SRT includes an upstream primer SRT-F and a downstream primer SRT-R.

[0006] In addition, the present invention also provides applications of the TaqMan primer-probe combination and kits containing the TaqMan primer-probe combination.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A TaqMan primer-probe combination for detecting virulent and attenuated Newcastle disease virus includes a virulent primer RT, a virulent probe RT-probe, a universal primer SRT, and a universal probe SRT-probe; the virulent primer RT includes an upstream primer RT-F and a downstream primer RT-R, and the universal primer SRT includes an upstream primer SRT-F and a downstream primer SRT-R.

[0009] The nucleotide sequence of the RT-F is shown in SEQ.ID NO.1; the nucleotide sequence of the RT-R is shown in SEQ.ID NO.2; and the nucleotide sequence of the highly toxic probe RT-probe is shown in SEQ.ID NO.3.

[0010] The nucleotide sequence of the SRT-F is shown in SEQ.ID NO.4; the nucleotide sequence of the SRT-R is shown in SEQ.ID NO.5; and the nucleotide sequence of the universal probe SRT-probe is shown in SEQ.ID NO.6.

[0011] Preferably, the 5' end of the highly toxic probe RT-probe is labeled with the fluorescent group FAM, and the 3' end is labeled with the quenching group BHQ1; the 5' end of the universal probe SRT-probe is labeled with the fluorescent group Cy5, and the 3' end is labeled with the quenching group BHQ1.

[0012] Furthermore, this invention discloses the application of the TaqMan primer-probe combination described above in the preparation of RT-PCR detection kits.

[0013] Finally, the present invention also discloses a kit comprising a reaction solution containing the TaqMan primer-probe combination as described in any of the preceding claims;

[0014] The reaction solution contains a TaqMan primer-probe combination for detecting virulent and attenuated Newcastle disease virus.

[0015] Preferably, the concentration of primers and probes contained in the reaction solution is 0.2~0.25 μM / L.

[0016] Preferably, the kit has a minimum detection concentration of 10 for both potent and attenuated Newcastle disease virus strains. 1 copies / uL.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention provides a TaqMan primer-probe combination for detecting virulent and attenuated Newcastle disease (NDV) viruses. A quantitative real-time RT-PCR method for detecting NDV virulent and attenuated viruses using this TaqMan primer-probe combination is established. This method shows no cross-reactivity with common avian viruses AIV, FADV, IBV, and IBDV; its minimum detection concentration for virulent and attenuated NDV viruses is 10. 1 The number of copies / µL was 100-fold higher than that of conventional RT-PCR; the intra-assay coefficient of variation and inter-assay coefficient of variation were 0.36–0.59 and 0.42–1.71, respectively, both less than 3%; a dual-channel amplification curve with a Ct value ≤34 was required to confirm virulent NDV infection, while a single-channel amplification curve with a Ct value ≤35 was required to confirm attenuated NDV infection, allowing for simultaneous differentiation between virulent and attenuated virus infections. Compared with conventional RT-PCR, the detection rate was 100%, superior to conventional RT-PCR, and the detection results were consistent with sequencing results, indicating that this method can detect mixed infections of virulent and attenuated viruses. Attached Figure Description

[0019] Figure 1 The amino acids at positions 112-117 are the cleavage sites for the potent NDV toxicity.

[0020] Figure 2 The NDV highly virulent primer RT-F;

[0021] Figure 3 This refers to amino acids 147-153 of the NDV F gene;

[0022] Figure 4 The NDV highly virulent primer RT-R;

[0023] Figure 5 This refers to amino acids 408-416 of the NDV F gene;

[0024] Figure 6 The universal primer for NDV is SRT-F;

[0025] Figure 7 This refers to amino acids 432-439 of the NDV F gene;

[0026] Figure 8 The universal primer for NDV is SRT-R;

[0027] Figure 9 The image shows the RT-PCR identification results of the virulent NDV fragment. In the figure, M1: Marker 2000 bp; M2: Marker 2000 bp; 1: LaSota; 2: GM; 3: GX-2022-99; 4: E115; 5: AIV; 6: IBV; 7: FADV; 8: H2O.

[0028] Figure 10 The figure shows the RT-PCR identification results of the universal fragment of NDV. In the figure, M1: Marker 2000 bp; M2: Marker 2000 bp; 1: LaSota; 2: GM; 3: GX-2022-99; 4: E115; 5: AIV; 6: IBV; 7: FADV; 8: H2O.

[0029] Figure 11 The figure shows the amplification results of the target fragment. In the figure, M: Marker 2000 bp; 1: NDV virulent fragment; 2: NDV universal fragment.

[0030] Figure 12 The results are for bacterial culture PCR. In the figure, M: Marker 2000 bp; 1-5: NDV virulent fragments; 6-10: NDV universal fragments.

[0031] Figure 13 The standard curve for the high toxicity of NDV;

[0032] Figure 14 This is the general standard curve for NDV;

[0033] Figure 15 This is the result of the method specificity verification;

[0034] Figure 16 Amplification curves of highly virulent NDV primers;

[0035] Figure 17 This is a general amplification curve for NDV;

[0036] Figure 18 This is a mixed amplification curve;

[0037] Figure 19 The figure shows the RT-PCR sensitivity detection results for the NDV virulent standard plasmid. In the figure, M: Marker 2000 bp; 1~7: 1.32×10 8 ~1.32×10 4 Copies / uL of NDV highly toxic standard particles; 8: H2O;

[0038] Figure 20The results show the sensitivity detection of RT-PCR using the universal standard plasmid for NDV. In the figure, M: Marker 2000 bp; 1-8: 1.1 × 10⁻⁸. 8 ~1.1×10 3 Copies / µL NDV universal standard plasmid; 8: H2O;

[0039] Figure 21 Sequencing results for the virulent strain of GX-2022-99 infection;

[0040] Figure 22 Sequencing results for the attenuated virus group of GX-2022-99 infection;

[0041] Figure 23 These are the sequencing results of virulent GM infection groups;

[0042] Figure 24 These are sequencing results of attenuated GM virus groups.

[0043] Figure 25 These are the sequencing results of the virulent strain of E115 infection.

[0044] Figure 26 These are the sequencing results of the attenuated virus from the E115 infection group. Detailed Implementation

[0045] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0046] The following are the main experimental materials and reagents used in the examples:

[0047] Strain:

[0048] The following strains were preserved by the Department of Animal Infectious Diseases, College of Veterinary Medicine, South China Agricultural University: NDV attenuated strain II LaSota, NDV virulent strain VI Herts33, NDV virulent strain VI GX-2022-99, NDV virulent strain VII GM, NDV virulent strain IX F48E9, NDV virulent strain XII E115, AIV H5, AIV H7, AIV H9, FADV, IBV, and IBDV.

[0049] Animals and Chicken Embryos:

[0050] Both SPF and regular chicken embryos used in the experiment were provided by Guangdong Dahua Agricultural Biotechnology Co., Ltd., and Guangdong Huasheng Biotechnology Co., Ltd. provided the incubation site for hatching SPF chicken embryos. The embryos were raised to 8 days old to meet the experimental requirements. All animal experiments involving highly virulent NDV strains were conducted in the Animal Biosafety Level 3 (ABSL-3) laboratory of South China Agricultural University.

[0051] Main reagents and consumables:

[0052] Reagents: PBS phosphate powder was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.; the Fejie total RNA rapid extraction kit was purchased from Guangzhou Zhuxiang Biotechnology Co., Ltd.; DNA purification and recovery kit, Mix Tap, rTap enzyme, DL 500 DNA Marker, DL 2000 DNA Marker, M-MLV reverse transcriptase, reverse transcription kit, etc., were all purchased from Shanghai Taraka Biotechnology Co., Ltd.; sodium penicillin and streptomycin sulfate were products of Guangzhou Jetway Biotechnology Co., Ltd.; plasmid extraction kit was purchased from OMEGA Biotechnology Co., Ltd.; qPCR probe Master Mix, homologous recombinase pMD19-T, and T4 ligase were purchased from Nanjing Novizan Biotechnology Co., Ltd.; DH5α competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd. Primers and probes were purified by HPLC and were synthesized by Shanghai Sangon Biotech Co., Ltd., and stored at -20℃; red blood cells were prepared from healthy chicken venous blood.

[0053] Consumables: EP tubes, pipette tips, syringes of various sizes, 96-well microplates, sterile sampling swabs, foot tags, centrifuge tubes of various sizes, masks, shoe covers, gloves, etc.

[0054] Preparation of common reagents:

[0055] Preparation procedure for 1% chicken red blood cell suspension: Collect blood from adult SPF chickens and inject it into an anticoagulant tube. Centrifuge at 1000 r / min to obtain red blood cells. Add an appropriate amount of PBS buffer to the red blood cells, mix well, and centrifuge at low speed to complete the washing. Repeat this washing step 3 times, add an appropriate amount of PBS buffer, and shake thoroughly to prepare a 1% chicken red blood cell suspension.

[0056] Preparation method of double-antibiotic phosphate buffer: Take autoclaved PBS buffer and add streptomycin sulfate, sodium penicillin, and glycerol in the following proportions: final concentration of glycerol 10%, final concentration of streptomycin 2 mg / mL, and final concentration of penicillin 2000 U / mL. Use immediately after preparation or store at 4°C.

[0057] PBS buffer preparation procedure: Dissolve one packet of PBS powder in 2L of DEPC H2O, stir thoroughly until completely dissolved, and autoclave. After the solution cools to room temperature, seal the package and store at 4℃ for later use.

[0058] Main instruments and equipment:

[0059] The main instruments and equipment used in this invention are shown in Table 1.

[0060] Table 1. Information on Main Instruments and Equipment

[0061] Instruments and equipment source AQUELIX100 Pure Water System Millipore, USA Low-temperature high-speed centrifuge Eppendorf, Germany micropipettes Eppendorf, Germany Real-time PCR instrument Bio-Rad, Inc. (USA) Thermostatic water bath (Germany) Qixin Technology Co., Ltd. PCR amplification instrument WhatmanBiometra, Germany Gel Imaging Analysis System WhatmanBiometra, Germany Chicken embryo incubator Huida Incubation Equipment Factory Vortex oscillator Wiggens, Germany Electronic balance Shanghai Precision Scientific Instruments Co., Ltd. Chicken stainless steel isolator Suzhou Fengshi Experimental Animal Equipment Co., Ltd. High temperature and high pressure sterilizer Sanyo Company of Japan ABSL-2 Biosafety Cabinet Singapore ESCO Company

[0062] Data collection, processing, and analysis:

[0063] The required software and websites include: Oligoarchitect, Bioedit, Megalign, SnapGene, Bio-Rad CFX Maestro, GraphPad, Python, and Excel. The F gene sequences of each NDV genotype were downloaded from the GenBank database as reference sequences. Oligoarchitect, Bioedit, Megalign, and SnapGene were used for sequence alignment and primer design. Bio-Rad CFX Maestro, GraphPad, Python, and Excel were used for quantitative real-time PCR data analysis.

[0064] Example 1: Primer and probe design

[0065] The NDV reference strain F gene sequence downloaded in Table 2 was compared using Clustal W in SnapGene software to find the most conserved sequence. Primers for real-time PCR were designed using Oligo. The designed primers were comprehensively evaluated by the online website Primer-BLAST to ensure that the design avoided structures that were detrimental to the amplification of the target gene, such as the formation of dimers between primers or between primers and probes.

[0066] The virulent and attenuated NDV sequences were identified based on sequence differences in the F gene of synvirulent strains. The cleavage site of virulent NDV is RRQR(K)RF. Amino acid residues 112-117 of the cleavage region in all virulent strains are RRQR(K)RF, while the corresponding region in some pigeon virulent strains is GRQKRF, and the corresponding region in attenuated strains is GR(K)QGRL. Primer RT-F was designed for virulent NDV detection. Alignment using SnapGene software revealed that amino acids 147-153 of the F gene in virulent NDV subtypes were relatively conserved. Based on this, primer RT-R and probe RT-probe were designed for virulent NDV detection. Amino acids 408-416 of the F gene in all NDV subtypes were relatively conserved. Primer SRT-F and probe SRT-probe were designed for universal NDV detection. The specific design and alignment are as follows: Figures 1-8 As shown.

[0067] The fluorescent group of the NDV highly toxic primer RT probe is FAM, and the quenching group is BHQ1. The fluorescent group of the NDV universal primer SRT probe is Cy5, and the quenching group is BHQ1. The primer and probe sequences, Tm values, and product sizes are shown in Table 3.

[0068] Table 2 Genotypes and Accession Numbers of Viruses

[0069]

[0070] Table 3. Primer and probe design results

[0071]

[0072] Example 2: RNA extraction

[0073] Viral RNA was extracted from allantoic fluid using the Flyjie extraction kit. The specific steps are as follows:

[0074] Take 200 μL of chicken embryo allantoic fluid sample that has been vortexed and placed in a 1.5 mL centrifuge tube, add 500 μL of RA2 lysis buffer, and mix thoroughly by inverting 15 times. Incubate at room temperature for 1 min. Transfer the mixture to a nucleic acid adsorption column and centrifuge at 12000 rpm for 1 min to bind RNA to the silica membrane. Discard the filtrate and add 500 μL of Wash Buffer for two washes, centrifuging at 12000 rpm for 1 min each time. After 1 min of air centrifugation, remove residual ethanol completely. Transfer the adsorption column to a sterile RNase-free centrifuge tube, add 25-50 μL of Elution Buffer directly to the center of the membrane, and let stand for 1 min to allow RNA to be fully eluted. Finally, centrifuge at 12000 rpm for 2 min to obtain high-purity total RNA. Store at -20℃ for short-term storage and at -80℃ for long-term storage.

[0075] Example 3: Reverse transcription and PCR amplification

[0076] Reverse transcription: cDNA was prepared by transcription using the M-MLV two-step reverse transcriptase purchased from Takara. The total reverse transcription volume was 20 μL, and the sample was added according to the recommended steps in the manufacturer's instructions. The specific steps are shown in Table 4.

[0077] Table 4 Reverse Transcription System

[0078] reagents Volume (μL) M-MLV Buffer 4 10 mol dNTP 1 Random primers R:O 1 RNase inhibitors RRI 0.5 M-MLV reverse transcriptase 1 Extracted RNA 9.5 <![CDATA[DEPC H2O]]> 3

[0079] Add the above components to a clean bench, vortex to mix, briefly incubate for 10-20 seconds, then immediately incubate in a 42℃ water bath for 1 hour, and finally let stand at room temperature for 5 minutes to terminate the reaction. Place the prepared reaction system in a PCR amplification instrument and execute the program. The initial stage is maintained at 95℃ for 3 minutes to fully activate the hot-start Taq DNA polymerase; subsequently, 30 amplification cycles are performed, each cycle including 95℃ for 30 seconds of denaturation, gradient annealing at 55℃ for 30 seconds to optimize primer binding, and extension at 72℃ at 30 seconds / kb according to product length; the final extension stage is at 72℃ for 5 minutes to ensure product integrity. After the reaction, store at 4℃ for a short period. Separate the amplified products by electrophoresis on a 3% agarose gel at a constant voltage of 120V for 15 minutes.

[0080] Observation under UV transilluminator and verification by comparison with DNA molecular weight standards were performed. Gel blocks containing the target fragment were precisely cut using a sterile blade. Gel recovery was performed according to the DNA purification kit specifications: dissolving buffer was used, followed by incubation in a 65°C oven for 10 min. After column adsorption, impurities were removed by two washes. Finally, high-purity DNA was obtained by elution with 30 μL of DEPC H2O. For short-term storage, the DNA was kept at -20°C; for long-term storage, it needed to be aliquoted and stored at -80°C.

[0081] The results of electrophoresis of the obtained target gene fragment on an agarose gel are as follows: Figures 9-10 As shown, the size of the amplified fragment of the highly virulent NDV target gene is 125 bp; the size of the amplified fragment of the universal NDV target gene is 89 bp, which is consistent with the size of the designed target band. No primer dimers were found, which is in line with the expected results.

[0082] Example 4: Preparation of Standard Plasmids

[0083] The purified product was ligated with the pMD19-T vector. 2.5 μL of the PCR gel-recovered product, 0.5 μL of 19T and 0.3 μL of T4 ligase were added to the same tube, mixed thoroughly, and then placed in a ligator at 16℃ overnight for ligation.

[0084] The conversion will proceed as follows:

[0085] (1) Take out the DH5α competent cells from the -80℃ freezer and place them on ice to thaw. Take 50 μL of the thawed DH5α competent cells and place them in a 1.5 mL sterile centrifuge tube. Then take 5 μL of the ligation product and slowly add it to the competent cells in a spiral manner with a pipette. Gently pipette the mixture several times to mix it. Place the tube in ice for 30 min.

[0086] (2) Transfer the conversion system to a 42°C water bath for 1 min of heat shock, then quickly place it back on ice and let it stand for 5 min;

[0087] (3) Add 400 μL of antibiotic-free LB to the shaker tube and shake at 200 r / min for 1 h at 37℃;

[0088] (4) The shaken bacteria appear turbid. Centrifuge, remove 40 μL of supernatant, and then thoroughly mix the transformant. Add the transformant to a culture plate containing Amp. Invert the culture plate and incubate at 37°C for 12 h. Using the OMEGA plasmid extraction kit, pick well-grown single colonies from the cultured agar plate and inoculate them into LB medium containing Amp resistance. Pick 5 single colonies for each virus and incubate overnight at 37°C with shaking. Plasmid extraction was performed according to the OMEGA plasmid extraction kit. The specific steps are as follows:

[0089] (1) Take 1.5~5 mL of the cultured bacterial solution and put it into a 10 mL centrifuge tube. Centrifuge at 10000 r / min for 1 min at room temperature, let the bacterial cells precipitate and then remove the supernatant.

[0090] (2) Add 250 μL of Solution I / RNase A to the kit and pipette thoroughly to suspend the bacterial precipitate. Note: If small bacterial clumps remain on the tube wall during pipetting, shake vigorously with a shaker to mix them.

[0091] (3) After mixing evenly, add 350 μL of Solution I, gently turn it upside down to mix it until you see a white flocculent precipitate, and centrifuge at 10000 r / min at room temperature for 1 min.

[0092] (4) Slowly aspirate the supernatant, being careful not to aspirate the precipitate, and add it to a Minicolumn with a 2mL collection tube. Centrifuge at 10000r / min for 1min at room temperature to completely separate the bacterial lysis products and then discard the liquid.

[0093] (5) Perform primary washing with 500 μL HB buffer, centrifuge at 10000 r / min for 1 min and discard the liquid to remove residual protein impurities.

[0094] (6) Perform two treatments with 700 μL of ethanol-modified washing buffer, centrifuge at 10000 r / min for 1 min each time and discard the liquid to completely remove salt ions and organic solvent residues;

[0095] (7) At room temperature, the column is air-displaced at 10000 r / min for 1 min to dry the column, which can remove ethanol;

[0096] (8) The adsorption column matrix was dried by centrifugation at 10000r / min for 2min to eliminate ethanol interference; finally, the adsorption column was transferred to a sterile centrifuge tube, 50~100μL of elution buffer was added, and high-purity DNA solution was obtained by centrifugation at 10000r / min for 1min.

[0097] Sequence determination of recombinant plasmid: The proposed recombinant plasmid was identified by PCR amplification. 10 μL of the PCR product of plasmid DNA was run on a 3% agarose gel for electrophoresis to obtain the results.

[0098] The results of the identification are as follows Figures 11-12 As shown, the recombinant plasmid amplification fragment is identical to the RT-PCR identification result.

[0099] Based on the size of the target band in the plasmid DNA, positive recombinant plasmids are selected. After confirmation by testing, the successfully constructed recombinant plasmids are analyzed for concentration, and the copy number is calculated using the following formula. These plasmids are then stored at -80℃ for later use as standard plasmids.

[0100] Plasmid copy number (copies / µL) = plasmid concentration (ng / µL) × (6.02 × 10²³) × 10 -6 / Plasmid molecular weight (660 × base pairs).

[0101] The copy numbers of the two plasmids were calculated using the formula. The concentration of the NDV virulent standard plasmid was 1318.6 ng / µL, and the copy number of the NDV virulent plasmid was 9.62 × 10⁻⁶. 12The concentration of the NDV universal standard plasmid was 1113.8 ng / µL, and the copy number of the NDV universal plasmid was 11.41 × 10⁻⁶. 12 copies / uL.

[0102] Example 5: Exploring Primer and Probe Concentrations

[0103] Using a virulent NDV standard plasmid and a universal NDV standard plasmid as templates, and keeping the template and other reagent loading amounts constant, the primer and probe concentrations were optimized. The concentrations of the upstream and downstream primers were explored within the range of 0.2–0.4 μM, with an interval of 0.1 μM, and the probe concentration was explored within the range of 0.15–0.3 μM, with an interval of 0.05 μM. Each sample was tested in triplicate, and the mean Ct value was used.

[0104] The optimal final concentration of the NDV virulent probe (0.25 μM / L) and the optimal final concentration of the NDV universal probe (0.2 μM / L) resulted in the lowest mean Ct value and the best amplification efficiency. The final reaction system is shown in Table 5. The components were thoroughly mixed according to the following system, briefly incubated, and then placed in a real-time quantitative PCR environment for amplification. The amplification curves were then analyzed.

[0105] Table 5. Real-time PCR reaction system

[0106] Element Volume (μL) Concentration (µM / L) cDNA 2 qPCR probe Master Mix 10 - RT-F 0.4 0.2 RT-R 0.4 0.2 RT-probe 0.5 0.25 SRT-F 0.4 0.2 SRT-R 0.4 0.2 SRT-probe 0.4 0.2 <![CDATA[DEPC H2O]]> 5.5 -

[0107] Example 6: Establishment of the Standard Curve

[0108] The NDV virulent strain and the universal standard plasmid were first diluted to 10. 8 Copies / µL, then subjected to a 10-fold serial dilution, with a gradient range of 10... 1 ~10 8 Copies / μL, with 3 replicates for each dilution. The instrument reaction program was set to 95℃ for 5 min, 55℃ for 10 s, and 60℃ for 30 s, for a total of 38 cycles. Amplification was performed according to the optimized amplification system and conditions described above. Fluorescence signals were collected after the reaction. After the reaction, a standard curve was constructed using the five-point method based on the derived Ct values ​​and their corresponding dilution log values.

[0109] The results show that the standard curve equation for the highly toxic NDV is: y = -3.495x + 41.688, R0 2 =0.9997, E=93.1%, y=-3.524x+42.27, R 2 =0.9998, E=89.9%; NDV universal standard curve equation y=-3.524x+42.27, R²=0.9998, E=89.9%, as Figures 13-14 As shown, this indicates that the two standard plasmids at 103 ~10 7 The coefficient of performance (Ct) shows a good linear relationship with the Ct value within the range of copies / μL.

[0110] Example 7: Specificity Test

[0111] The established TaqMan probe-based quantitative RT-PCR method was used to amplify the nucleic acids of five virulent NDV strains (GX-2022-99, GM, E115, F48E9, Herts33), and attenuated NDV strains LaSota, AIV (H5, H7, H9), FADV, IBV, and IBDV using quantitative RT-PCR as templates. DEPC H2O was used as a negative control. Figure 15 As shown, the results indicate that two expected amplification curves were observed for the virulent NDV strains VI, VII, IX, and XII, while one expected amplification curve was observed for the attenuated NDV strain II, LaSota. Specificity tests on common poultry viruses AIV, FADV, IBV, and IBDV did not produce any amplification curves, indicating that this method has good specificity.

[0112] Example 8: Sensitivity Test

[0113] A serial dilution system of 10-fold serial dilutions was constructed using NDV virulent virus and universal standard plasmid, with the dilution range being 10^10. 1 ~10 8 Copies / μL, and the same sample was subjected to parallel RT-PCR and real-time RT-PCR experiments. Using two serially diluted standard plasmids as templates, each sample was tested in triplicate to compare the sensitivity of the two methods and determine the detection limit of each method.

[0114] The results are as follows Figures 16-18 As shown, the minimum copy number of the highly virulent NDV and universal standard plasmid is 10. 1 copies / uL. NDV virulent strain and universal standard plasmid were detected by routine RT-PCR, and the results are as follows: Figures 19-20 As shown. Their detection limits are 10. 4 10 3 The copies / uL indicates that the detection limit of this method is more than 100 times higher than that of the conventional RT-PCR method.

[0115] Example 9: Repeatability Test

[0116] This method was used to perform intra-batch and inter-batch repeatability tests, and the standard deviation and coefficient of variation (CV) for both groups were calculated. The NDV virulent plasmid and the universal standard plasmid were serially diluted to a concentration of 10-1.5 ~10 8 Copies / μL. In intra-batch repeatability assessment, each concentration gradient of the same batch of samples was replicated three times; in inter-batch repeatability assessment, the same template was used for three independent experiments, spaced 72 h apart, with independently prepared reaction systems. Based on the dynamic changes in Ct values ​​at different concentration gradients, intra-batch and inter-batch standard deviations and CVs were calculated. The CVs were required to meet the following requirements: intra-batch CVs were all less than 3%, and inter-batch CVs were all less than 5%, to ensure the reliability of the detection method in clinical sample analysis.

[0117] By constructing 10 5 ~10 8 Repeatability tests were conducted using copies / μL of highly virulent NDV and universal standard plasmids, with each gradient performed in triplicate. The results are shown in Tables 6 and 7. The results indicate that the intra-batch CV values ​​were all below 0.6%, and the inter-batch CV values ​​were below 1.8%. This data demonstrates excellent repeatability characteristics within the detection system.

[0118] Table 6. Repeatability Validation of NDV Virulence Standard Plasmid

[0119]

[0120] Table 7. Repeatability Validation of NDV Universal Standard Plasmid

[0121]

[0122] Example 10: Ct value determination

[0123] Three allantoic fluid samples each of the virulent strain GX-2022-99, GM strain, E115 strain, and the attenuated strain LaSota were selected. Nucleic acid was extracted and serially diluted 10-fold. 10 samples were then taken. -8 ~10 -1 Diluted samples were amplified using a real-time PCR instrument, with each dilution performed in triplicate. The nucleic acid sample with the highest Ct value detected in all three replicates was selected. Each dilution was then performed in triplicate, and the average Ct value was calculated to determine the Ct determination value for virulent and attenuated strains using the TaqMan probe-based real-time RT-PCR method established in this invention.

[0124] The results showed that: in each experimental group 10 -4 ~10 -1 All samples showed typical S-shaped amplification curves as detected by real-time quantitative RT-PCR. To optimize the positive threshold, 10... -4For critical concentration samples with Ct values ​​≤ 40, three repeated tests were performed, and the average Ct value was used as the standard for determining virulent and attenuated NDV strains, as shown in Table 8. The average Ct values ​​for virulent NDV strains at the highest dilution were 33.96 and 35.00, and the average Ct value for attenuated NDV strains at the highest dilution was 35.44. Based on statistical analysis, standardized interpretation criteria were established to ensure the reliability and stability of Ct value determination using the TaqMan probe-based quantitative RT-PCR detection method: NDV virulent infection determination requires a dual-channel amplification curve and a Ct value ≤ 34; NDV attenuated infection determination requires a single-channel amplification curve and a Ct value ≤ 35; for critical samples with Ct values ​​between 35 and 40, repeated testing is required for confirmation.

[0125] Table 8 Ct Judgment Values

[0126]

[0127] Example 11: Animal Experiment

[0128] Each screened strain was used in 10 3 Nine-day-old SPF chicks were challenged with an EID50 dose of 0.15 mL via eye drops or nasal drops. The animals were grouped as follows:

[0129] The challenge group refers to the group of animals in an infection group that were challenged with the corresponding virulent strain before being housed together with the infected group; the cohabitation group refers to the group of animals in an infection group that were not challenged before being housed together with the infected group; the attenuated virus group refers to the group of animals in an infection group that were challenged with the LaSota strain before being housed together with the infected group.

[0130] One hundred and five 9-day-old SPF chicks were divided into five groups: a virulent virus infection group (GX-2022-99), a GM group, an E115 group, an attenuated virus infection group (LaSota), and a negative control group. Each virulent virus infection group consisted of 28 chicks, including 10 challenged chicks, 8 cohabiting chicks, and 10 attenuated virus chicks. The three virulent virus infection groups were challenged with virulent NDV strain VI (GX-2022-99), virulent NDV strain VII (GM), and virulent NDV strain XII (E115), respectively. The cohabiting chicks were not challenged. The attenuated virus infection group (n=11) was challenged with attenuated NDV strain II (LaSota). The negative control group (n=10) received an equal dose of double-antibody PBS buffer.

[0131] The grouping is shown in Table 9. Immediately after the challenge experiment, the challenge group, cohabitation group, and attenuated virus group in the GX-2022-99 group, GM group, and E115 group were mixed and housed together. The attenuated virus group LaSota group and the negative control group were housed separately. The animal experiment lasted for 14 days.

[0132] Table 9 Animal Grouping Table

[0133]

[0134] Pharyngeal and cloacal swabs were collected from all chickens in each infection group on days 3, 5, 7, and 9. Swabs from each swab sample in different experimental groups were detected using both the TaqMan probe-based quantitative RT-PCR method and the RT-PCR method established in this invention. The primers for the RT-PCR method were the NDV national standard virulent primers and the national standard universal primers. Simultaneously, five swabs from each group were sequenced to determine the type of virus infection in that swab group, thus assessing the accuracy of the two methods. The PCR products from the sampled swabs were sent for sequencing. The sequencing results were used to determine whether mixed infection occurred and to verify the accuracy of the two methods. The method comparison results are shown in Table 10.

[0135] Table 10 Comparison Results of Experimental Methods

[0136]

[0137] As shown in Table 10, the detection rate of the virulent, cohabiting, and attenuated virus groups in the three infection groups of the TaqMan probe quantitative RT-PCR of this invention was 100%, which was consistent with the submitted test results. Mixed infection occurred in each group. For specific sequencing results, please refer to the appendix. Figures 21-26 In conventional RT-PCR, the virulent primers failed to detect cohabiting infections in the GX-2022-99 infection group, and the virulent primers failed to detect attenuated infections in the E115 infection group. Therefore, the detection accuracy is not as high as that of the TaqMan probe-based quantitative RT-PCR detection method of this invention.

[0138] Five days after challenge, three chickens from each infection group (challenge group, cohabitation group, and attenuated virus group) were euthanized. Their proventriculus, spleen, and trachea were collected, ground, and centrifuged. The supernatants were then used for fluorescent RT-PCR detection, as shown in Table 11. Results showed that out of 30 samples from the trachea, proventriculus, and spleen of different infection groups, the GX-2022-99 challenge group, GM challenge group, E115 challenge group, and their cohabitation group all showed virulent infection in all tissues, with the Ct value of the virulent primer significantly lower than 34. The GX-2022-99 attenuated virus group, GM attenuated virus group, E115 attenuated virus group, and LaSota challenge group all met the attenuated virus criteria, with the Ct value of the universal primer lower than 35. The TaqMan probe-based quantitative RT-PCR method provided by this invention can detect mixed infections of NDV types II, VI, VII, and XII in animal samples with 100% accuracy and no missed detections.

[0139] Table 11. Results of quantitative real-time RT-PCR identification

[0140]

[0141] Note: The data in the table are the qPCR Ct value detection results of organ grinding samples from different challenge test groups. - indicates no Ct value.

[0142] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A TaqMan primer-probe combination for detecting virulent and attenuated Newcastle disease virus, characterized in that, It includes highly virulent primer RT, highly virulent probe RT-probe, and universal primer SRT, universal probe SRT-probe; the highly virulent primer RT includes upstream primer RT-F and downstream primer RT-R, and the universal primer SRT includes upstream primer SRT-F and downstream primer SRT-R. The nucleotide sequence of the RT-F is shown in SEQ.ID NO.1; the nucleotide sequence of the RT-R is shown in SEQ.ID NO.2; and the nucleotide sequence of the highly toxic probe RT-probe is shown in SEQ.ID NO.

3. The nucleotide sequence of the SRT-F is shown in SEQ.ID NO.4; the nucleotide sequence of the SRT-R is shown in SEQ.ID NO.5; and the nucleotide sequence of the universal probe SRT-probe is shown in SEQ.ID NO.

6.

2. The TaqMan primer-probe combination for detecting virulent and attenuated Newcastle disease virus according to claim 1, characterized in that, The highly toxic probe RT-probe has a 5' end labeled with the fluorescent group FAM and a 3' end labeled with the quenching group BHQ1; the universal probe SRT-probe has a 5' end labeled with the fluorescent group Cy5 and a 3' end labeled with the quenching group BHQ1.

3. The application of the TaqMan primer-probe combination as described in any one of claims 1 to 2 in the preparation of an RT-PCR detection kit.

4. A reagent kit, characterized in that, The kit includes a reaction solution containing the TaqMan primer-probe combination as described in any one of claims 1 to 2; The reaction solution contains a TaqMan primer-probe combination for detecting virulent and attenuated Newcastle disease virus.

5. The reagent kit according to claim 4, characterized in that, The concentration of primers and probes contained in the reaction solution is 0.2~0.25μM / L.

6. The reagent kit according to claim 4, characterized in that, The kit has a minimum detectable concentration of 10 for both potent and attenuated Newcastle disease virus. 1 copies / uL.

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