A primer probe set, chip, kit and application for detecting eight avian infectious disease pathogens

Abstract

The present invention belongs to the technical field of virus detection, and relates to a primer probe set, a chip, a kit and an application for detecting eight kinds of avian infectious disease pathogens. The present invention provides a primer probe set for detecting eight kinds of avian infectious disease pathogens, including primer probe sets for detecting one or more of the following eight kinds of avian infectious disease pathogens: avian influenza virus, Newcastle disease virus, avian leukemia virus type J, infectious bronchitis virus, infectious bursal disease virus, reticuloendotheliosis virus, Marek's disease virus and avian adenovirus type 4. The primer probe set of the present invention and the visualized gene chip prepared therefrom can quickly and efficiently detect eight kinds of major avian epidemic pathogens, and can be used for epidemiological surveys and disease diagnosis of poultry.

Description

Technical Field

[0001] The invention belongs to the technical field of virus detection, and specifically relates to a primer probe set, a chip, a kit and applications for detecting eight avian infectious disease pathogens. Background Art

[0002] With the rapid development of social economy, the demand for meat and egg products has also expanded rapidly. As the poultry industry has shifted to large-scale and intensive development, it has also led to frequent poultry infectious diseases, which are characterized by diverse pathogens and increased incidence. Among them, Newcastle disease, avian influenza, infectious bronchitis, infectious bursal disease, Marek's disease, avian leukosis, avian reticuloendotheliosis and hepatitis-pericardial effusion syndrome pose a serious threat to the poultry industry. Rapid clinical diagnosis and epidemiological investigation of these diseases are of great significance.

[0003] Avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), Marek's disease virus (MDV), avian leukosis virus-J (ALV-J), reticuloendotheliosis virus (REV) and fowl adenovirus serotype 4 (FAdV-4) are the main pathogens of the above diseases, among which AIV, NDV and IBV are common viral pathogens that cause respiratory diseases in birds. Common detection methods for AIV include hemagglutination test and molecular biological diagnosis, including RT-PCR and LAMP. NDV and IBV are mainly detected by molecular biological methods and serological methods, such as indirect immunofluorescence technology, enzyme-linked immunosorbent assay and neutralization test. IBDV, MDV, ALV-J and REV are the main viral pathogens that cause immunosuppression in poultry, while FAdV-4 is the main pathogen of hepatitis-pericardial effusion syndrome in my country in recent years. Its detection also relies on the same molecular biology and serological methods. Since the above detection methods all have one or more limitations such as complex operations, non-intuitive results, and single detection indicators, it is difficult for farms to screen for multiple viral infections on a large scale at the same time. Therefore, in order to promote the high-quality development of my country's poultry industry and reduce the economic losses of farmers, it is very necessary to establish a detection method that can quickly and efficiently detect these disease pathogens for the standardized management and disease prevention and control of farms. Summary of the invention

[0004] The purpose of the present invention is to provide a primer probe set, chip, kit and application for detecting eight kinds of avian infectious disease pathogens. The primer probe set of the present invention and the visualized gene chip prepared therefrom can quickly and efficiently detect eight kinds of major avian epidemic pathogens, and can be used for epidemiological investigation and disease diagnosis of avian diseases.

[0005] The invention provides a primer probe group for detecting eight avian infectious disease pathogens, comprising primer probe groups for detecting one or more of the following eight avian infectious disease pathogens: avian influenza virus, Newcastle disease virus, avian leukemia virus type J, infectious bronchitis virus, infectious bursal disease virus, reticuloendotheliosis virus, Marek's disease virus and avian adenovirus type 4. The primer probe group for detecting avian influenza virus comprises XPAIV-F, XPAIV-R and XPAIV-P, the primer probe group for detecting Newcastle disease virus comprises XPNDV-F, XPNDV-R and XPNDV-P, the primer probe group for detecting avian leukemia virus type J comprises XPALV-JF, XPALV-JR and XPALV-JP, the primer probe set for detecting infectious bronchitis virus includes XPIBV-F, XPIBV-R and XPIBV-P, the primer probe set for detecting infectious bursal disease virus includes XPIBDV-F, XPIBDV-R and XPIBDV-P, the primer probe set for detecting reticuloendotheliosis virus includes XPREV-F, XPREV-R and XPREV-P, the primer probe set for detecting fowl adenovirus type 4 includes XPFAdV-4-F, XPFAdV-4-R and XPFAdV-4-P, the primer probe set for detecting Marek's disease virus includes XPMDV-F, XPMDV-R and XPMDV-P;

[0006] The nucleotide sequence of the XPAIV-F is shown in SEQ ID NO.1, the nucleotide sequence of the XPAIV-R is shown in SEQ ID NO.2, and the nucleotide sequence of the XPAIV-P is shown in SEQ ID NO.3; the nucleotide sequence of the XPNDV-F is shown in SEQ ID NO.4, the nucleotide sequence of the XPNDV-R is shown in SEQ ID NO.5, and the nucleotide sequence of the XPNDV-P is shown in SEQ ID NO.6; the nucleotide sequence of the XPALV-JF is shown in SEQ ID NO.7, the nucleotide sequence of the XPALV-JR is shown in SEQ ID NO.8, and the nucleotide sequence of the XPALV-JP is shown in SEQ ID NO.9; the nucleotide sequence of the XPIBV-F is shown in SEQ ID NO.10, the nucleotide sequence of the XPIBV-R is shown in SEQ ID NO.11, and the nucleotide sequence of the XPIBV-P is shown in SEQ ID NO.12; the nucleotide sequence of the XPIBDV-F is shown in SEQ ID NO.13, and the nucleotide sequence of the XPIBDV-R is shown in SEQ ID NO. ID NO.14, the nucleotide sequence of the XPIBDV-P is shown in SEQ ID NO.15; the nucleotide sequence of the XPREV-F is shown in SEQ ID NO.16, the nucleotide sequence of the XPREV-R is shown in SEQ ID NO.17, and the nucleotide sequence of the XPREV-P is shown in SEQ ID NO.18; the nucleotide sequence of the XPFAdV-4-F is shown in SEQ ID NO.19, the nucleotide sequence of the XPFAdV-4-R is shown in SEQ ID NO.20, and the nucleotide sequence of the XPFAdV-4-P is shown in SEQ ID NO.21; the nucleotide sequence of the XPMDV-F is shown in SEQ ID NO.22, the nucleotide sequence of the XPMDV-R is shown in SEQ ID NO.23, and the nucleotide sequence of the XPMDV-P is shown in SEQ ID NO.24.

[0007] Preferably, in the primer probe set, the 5' end of the downstream primer is modified with biotin.

[0008] Preferably, in the primer probe set, the 5' end of the probe is amino-modified, and the amino group includes NH2C6.

[0009] The present invention also provides a gene chip for detecting eight avian infectious disease pathogens, on which the probes in the primer probe group described in the above technical solution are fixed.

[0010] The present invention also provides a method for preparing the gene chip described in the above technical solution, comprising the following steps:

[0011] The probes are spotted on the nano-membrane according to the chip arrangement order to obtain a membrane substrate, and the membrane substrate is air-dried under the conditions of a humidity of 40% to 60% and a temperature of 22 to 25° C. to obtain a gene chip.

[0012] Preferably, the chip arrangement includes spotting probes for detecting eight avian infectious disease pathogens at different positions.

[0013] The present invention also provides a kit for detecting eight avian infectious disease pathogens, comprising the primer probe set described in the above technical solution and the gene chip described in the above technical solution or the gene chip prepared by the preparation method described in the above technical solution.

[0014] In the present invention, when the kit amplifies the sample to be tested, the amplification conditions are 50°C, 30 min, 94°C, 4 min, 94°C, 30 s, 58°C, 30 s, 72°C, 20 s, 5 cycles, 94°C, 30 s, 56°C, 30 s, 72°C, 20 s, 35 cycles, 72°C, 10 min, 95°C, 5 min.

[0015] In the present invention, when the kit is used to amplify the sample to be tested, each 25 μL reaction system includes 0.05 μL of each primer of 100 μM, 12.5 μL of 2×One step MIX, 1.5 μL of One step Enzyme MIX, 5 μL of template and the balance of water.

[0016] The present invention also provides the use of the primer probe set described in the above technical scheme and / or the gene chip described in the above technical scheme or the gene chip prepared by the preparation method described in the above technical scheme in the preparation of a kit for diagnosing one or more avian infectious diseases selected from avian influenza, Newcastle disease, infectious bronchitis, infectious bursal disease, Marek's disease, avian leukosis, avian reticuloendotheliosis and hepatitis-pericardial effusion syndrome.

[0017] The present invention provides a primer probe set for detecting eight avian infectious disease pathogens. The primer probe set of the present invention detects eight avian infectious disease pathogens with good specificity and sensitivity. Combined with the gene chip detection method, it has a considerable detection sensitivity compared to the currently used single-plex and multiplex qPCR detection methods, and the detection sensitivity of some pathogens is higher. In addition, the present invention can simultaneously detect eight avian infectious disease pathogens, and there is no cross-response between the eight pathogens, and there is no response to healthy poultry serum tissues. The chip repetition rate can reach 100%, and the storage period at 2 to 8°C can reach 180 days. The infection of each virus in clinical samples is simultaneously detected by the gene chip method and the existing national standard qPCR detection method. The detection results of the two methods have a good coincidence rate, indicating that the gene chip developed in this study can meet the laboratory clinical sample detection and epidemiological monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The chip layout diagram provided by the present invention; wherein Biotin is the positive quality control of the chip; Buffer is the negative quality control of the chip;

[0020] Figure 2 The results of PCR reaction condition optimization provided by the present invention are shown in FIG. A, wherein: annealing temperature optimization results, M: Marker, 1: 54°C, 2: 56°C, 3: 58°C, 4: 60°C, 5: 56°C / 54°C, 6: 58°C / 54°C, 7: 58°C / 56°C, 8: 60°C / 56°C, 9: negative control; B: primer concentration optimization results, M: Marker, 1: 0.03 μL, 2: 0.04 μL, 3: 0.05 μL, 4: 0.06 μL, 5: 0.07 μL, 6: negative control; C: extension time optimization results, 1: 10s, 2: 15s, 3: 20s, 4: 30s, 5: negative control;

[0021] Figure 3 The chip reaction conditions optimization result diagram provided by the present invention; wherein, A: hybridization time optimization result diagram; B: SA-HRP working solution dilution concentration optimization result diagram;

[0022] Figure 4The specific result diagram of each virus probe under the eight-fold system provided by the present invention; wherein, A: AIV; B: NDV; C: IBV; D: IBDV; E: REV; F: ALV-J; G: MDV; H: FAdV-4; I: ALV-A; J: Serum of healthy chickens;

[0023] Figure 5 The sensitivity test results of each virus probe in the eight-fold system provided by the present invention are shown in FIG. 1 , wherein A: 3.39×10 2 copies / μL; B: 3.39×10 1 copies / μL; C: 3.39×10 0 copies / μL; D: 3.39×10 -1 copies / μL;

[0024] Figure 6 This is a sensitivity test result diagram of eight mixed samples provided by the present invention; where A: 3.39×10 4 copies / μL; B: 3.39×10 3 copies / μL; C: 3.39×10 2 copies / μL; D: 33.9copies / μL; E: 3.39copies / μL; F: 3.39×10 -1 copies / μL; G: 3.39×10 -2 copies / μL; H: 3.39×10 -3 copies / μL;

[0025] Figure 7 This is a graph of the shelf life experiment results provided by the present invention; wherein, A: 0 days; B: 30 days; C: 60 days; D: 90 days; E: 120 days; F: 180 days. DETAILED DESCRIPTION

[0026] The invention provides a primer probe group for detecting eight avian infectious disease pathogens, comprising primer probe groups for detecting one or more of the following eight avian infectious disease pathogens: avian influenza virus, Newcastle disease virus, avian leukemia virus type J, infectious bronchitis virus, infectious bursal disease virus, reticuloendotheliosis virus, Marek's disease virus and avian adenovirus type 4. The primer probe group for detecting avian influenza virus comprises XPAIV-F, XPAIV-R and XPAIV-P, the primer probe group for detecting Newcastle disease virus comprises XPNDV-F, XPNDV-R and XPNDV-P, the primer probe group for detecting avian leukemia virus type J comprises XPALV-JF, XPALV-JR and XPALV-JP, the primer probe set for detecting infectious bronchitis virus includes XPIBV-F, XPIBV-R and XPIBV-P, the primer probe set for detecting infectious bursal disease virus includes XPIBDV-F, XPIBDV-R and XPIBDV-P, the primer probe set for detecting reticuloendotheliosis virus includes XPREV-F, XPREV-R and XPREV-P, the primer probe set for detecting fowl adenovirus type 4 includes XPFAdV-4-F, XPFAdV-4-R and XPFAdV-4-P, the primer probe set for detecting Marek's disease virus includes XPMDV-F, XPMDV-R and XPMDV-P;

[0027] The nucleotide sequence of the XPAIV-F is shown in SEQ ID NO.1, the nucleotide sequence of the XPAIV-R is shown in SEQ ID NO.2, and the nucleotide sequence of the XPAIV-P is shown in SEQ ID NO.3; the nucleotide sequence of the XPNDV-F is shown in SEQ ID NO.4, the nucleotide sequence of the XPNDV-R is shown in SEQ ID NO.5, and the nucleotide sequence of the XPNDV-P is shown in SEQ ID NO.6; the nucleotide sequence of the XPALV-JF is shown in SEQ ID NO.7, the nucleotide sequence of the XPALV-JR is shown in SEQ ID NO.8, and the nucleotide sequence of the XPALV-JP is shown in SEQ ID NO.9; the nucleotide sequence of the XPIBV-F is shown in SEQ ID NO.10, the nucleotide sequence of the XPIBV-R is shown in SEQ ID NO.11, and the nucleotide sequence of the XPIBV-P is shown in SEQ ID NO.12; the nucleotide sequence of the XPIBDV-F is shown in SEQ ID NO.13, and the nucleotide sequence of the XPIBDV-R is shown in SEQ ID NO. ID NO.14, the nucleotide sequence of XPIBDV-P is shown in SEQ ID NO.15; the nucleotide sequence of XPREV-F is shown in SEQ ID NO.16, the nucleotide sequence of XPREV-R is shown in SEQ ID NO.17, and the nucleotide sequence of XPREV-P is shown in SEQ ID NO.18; the nucleotide sequence of XPFAdV-4-F is shown in SEQ ID NO.19, the nucleotide sequence of XPFAdV-4-R is shown in SEQ ID NO.20, and the nucleotide sequence of XPFAdV-4-P is shown in SEQ ID NO.21; the nucleotide sequence of XPMDV-F is shown in SEQ ID NO.22, the nucleotide sequence of XPMDV-R is shown in SEQ ID NO.23, and the nucleotide sequence of XPMDV-P is shown in SEQ ID NO.24. See Table 1 for details.

[0028] In the present invention, in the primer probe set, the 5' end of the downstream primer is preferably modified with biotin. Biotin has good affinity with streptavidin, and the primer with biotin labeled at the 5' end is used to amplify the target fragment. The probe is combined with the amplified product with biotin and then reacts with HRP-labeled streptavidin. Finally, under the action of the substrate color developing solution, the probe point can show a blue-purple response point visible to the naked eye. It is convenient for naked eye observation, and no complex instruments are required to record the experimental results.

[0029] In the present invention, in the primer probe set, the 5' end of the probe is preferably modified with an amino group, and the amino group preferably includes NH2C6, which is used to bind to the biotin label on the downstream primer.

[0030] The present invention also provides a gene chip for detecting eight avian infectious disease pathogens, on which the probes in the primer probe group described in the above technical solution are fixed. Gene chip detection technology generally includes three aspects: chip preparation, sample preparation, and signal detection and analysis of sample-chip hybridization reaction. The preparation method of the gene chip described in the present invention comprises the following steps: spotting each probe on a nanofilm according to the chip arrangement order to obtain a membrane substrate, and drying the membrane substrate under the conditions of a humidity of 40% to 60% and a temperature of 22 to 25° C. to obtain a gene chip. In the present invention, the concentration of the probe used for spotting is preferably 6 μmol / L. In terms of sample preparation, the gene chip method established by the present invention designs probe primers according to the specific conservative regions of each pathogen. Factors such as probe size and target DNA length will affect the hybridization signal. Generally, the oligonucleotide probe can take into account the sensitivity and specificity of the chip when it is 16 to 25 bp. At the same time, the smaller the target fragment, the higher the efficiency of amplification. However, when the length is too low, its poor stability may cause a decrease in the hybridization signal. The length of each probe of the present invention is 16-24 bp, the target fragment of the AIV target gene is 75 bp, the target fragment of the NDV target gene is 122 bp, the target fragment of the ALV-J target gene is 195 bp, the target fragment of the IBV target gene is 135 bp, the target fragment of the IBDV target gene is 129 bp, the target fragment of the REV target gene is 161 bp, the target fragment of the FAdV-4 target gene is 197 bp, and the target fragment of the MDV target gene is 120 bp, which ensures the stability and clarity of the response signal. Biotin and streptavidin have good affinity, and the primer with 5' end labeled with biotin is used to amplify the target fragment. The probe is combined with the amplification product with biotin and then reacts with HRP-labeled streptavidin. Finally, under the action of the substrate color developing solution, the probe point can show a blue-purple response point visible to the naked eye. It is convenient for naked eye observation, and no complex instrument is required to record the experimental results. At the same time, the method of the present invention sets three positive quality control points and one negative quality control point when spotting, so as to monitor whether each step of the operation process is effective and ensure the reliability of the test results. The eight-fold detection gene chip of the present invention has good specificity and sensitivity, and can detect eight avian infectious disease pathogens at the same time. There is no cross-response between the eight pathogens, and there is no response to healthy poultry serum tissue. The chip repetition rate can reach 100%, and the storage period at 2-8°C can reach 180 days, which can meet the laboratory clinical sample detection and epidemiological monitoring.

[0031] In the present invention, the chip arrangement includes spotting probes for detecting eight avian infectious disease pathogens at different positions. The chip arrangement of the present invention preferably also includes setting positive quality control and negative quality control. In the present invention, the positive quality control is preferably Biotin. In the present invention, the negative quality control is preferably Buffer. In the present invention, the Buffer is preferably phosphate buffer.

[0032] The present invention also provides a kit for detecting eight avian infectious disease pathogens, comprising the primer probe set described in the above technical solution and the gene chip described in the above technical solution or the gene chip prepared by the preparation method described in the above technical solution. In the present invention, when the kit amplifies the sample to be tested, the amplification conditions are preferably 50°C, 30min, 94°C, 4min, 94°C, 30s, 58°C, 30s, 72°C, 20s, 5 cycles, 94°C, 30s, 56°C, 30s, 72°C, 20s, 35 cycles, 72°C, 10min, 95°C, 5min. In the present invention, when the kit amplifies the sample to be tested, each 25μL reaction system preferably includes 0.05μL of each primer of 100μM, 12.5μL of 2×One step MIX, 1.5μL of One step Enzyme MIX, 5μL of template and the balance of water. After the sample to be tested is amplified, an amplification product is obtained. After the amplification product is hybridized with the chip, it is washed, incubated, mixed with SA-HRP working solution, incubated, washed, and TMB color developing solution is added to achieve virus detection. In the present invention, the hybridization time of the chip is preferably 20 minutes. In the present invention, the SA-HRP working solution is preferably diluted 1000 times.

[0033] The present invention also provides the use of the primer probe set described in the above technical solution and / or the gene chip described in the above technical solution or the gene chip prepared by the preparation method described in the above technical solution in the preparation of a kit for diagnosing one or more avian infectious diseases among avian influenza, Newcastle disease, infectious bronchitis, infectious bursal disease, Marek's disease, avian leukosis, avian reticuloendotheliosis and hepatitis-pericardial effusion syndrome. Currently, the widely used diagnostic methods for these diseases are mainly serological methods and molecular biological methods, and the number of pathogens detected in a single test is small, which makes it difficult to meet the detection needs under the continuous development of the intensive model of my country's poultry industry. The primer probe set, the visualization gene chip and the kit described in the present invention can realize the high-throughput rapid joint detection of the pathogens of the above eight common diseases at the same time.

[0034] To further illustrate the present invention, a primer probe set, chip, kit and application for detecting eight avian infectious disease pathogens provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1

[0036] Materials and Methods

[0037] 1 Materials

[0038] The pathogens used in this experiment: avian influenza virus (FJ strain), Newcastle disease virus (Lasota strain), infectious bronchitis virus (H120 strain), infectious bursal disease virus (B87 strain), Marek's disease virus (CVI988 strain), J subgroup avian leukosis virus (RAV-J strain), reticuloendotheliosis virus (T strain), fowl adenovirus type 4 (FAV-4 strain), and A subgroup avian leukosis virus (RAV-1 strain) were all provided by the China Veterinary Drug Administration.

[0039] 2 Main reagents

[0040] Viral genomic DNA extraction kit, viral genomic RNA extraction kit, and rapid plasmid extraction kit were purchased from Tiangen Biochemical Technology Co., Ltd. DH5α competent cells were purchased from Novozymes, MiniBESTAgaroseGelDNAExtraction Kit, One Step PrimeScript TM III RT-qPCR Mix, with UNG, pMD 19-T Vector Cloning Kit were purchased from TAKARA.

[0041] 3 Method establishment

[0042] 3.1 Design of primers and probes

[0043] Primers and probes were designed based on the NP gene of AIV, the M gene of NDV, the env gene of ALV-J, the M gene of IBV, the VP2 gene of IBDV, the LTR gene of REV, the Hexon gene of FAdV-4, and the meq gene of MDV. The 5' end of the downstream primer was modified with biotin, and the 5' end of the probe was amino-modified by NH2C6 to connect biotin. The detailed information of the primers and probes is shown in Table 1.

[0044] Table 1 Primers and probes for eight viral gene chips

[0045]

[0046]

[0047] 3.2 Preparation of eight viral recombinant positive plasmids

[0048] 3.2.1 Sample nucleic acid extraction

[0049] According to the instructions of the Tiangen Virus Genome DNA / RNA Extraction Kit, nucleic acids of 8 virus samples were extracted respectively.

[0050] 3.2.2 Amplification of each viral target fragment

[0051] Use the fragments with a length of 350-600 bp containing the fragments amplified by the primers of each pathogen in Table 1 as the target fragment to be inserted into the T vector, and design the corresponding primers. The primer information is shown in Table 2. Use the nucleic acid extracted in 3.2.1 as the template and amplify according to the reaction system in Table 3. The reaction procedure is: (reverse transcription 50℃, 30min); 94℃, 3min; 94℃, 30s, 57℃, 30s, 72℃, 30s, 35 cycles; 72℃, 7min, 4℃ storage.

[0052] Table 2 Gene chip positive plasmid primer information

[0053]

[0054]

[0055] Table 3 Gene chip positive plasmid target fragment reaction system

[0056] Components RT-PCR dosage (μL / unit) PCR volume (μL / unit) <![CDATA[Double-distilled water ddH2O]]> 14.5 17 Upstream primer F (10 μM) 2 2 Downstream primer R (10 μM) 2 2 2×TaqMasterMix / 25 2×OnestepMIX 25 / OnestepEnzymeMIX 2.5 / template 4 4 Total volume 50 50

[0057] 3.2.3 Purification of PCR products

[0058] Use TAE buffer to make agarose gel, and then perform agarose gel electrophoresis on the target DNA. Cut out the agarose gel containing the target DNA under UV light, minimize the gel volume, and absorb the liquid on the surface of the gel with a paper towel. Put the cut agarose gel into EP tubes respectively, and purify the PCR product according to the instructions of TAKARA's MiniBESTAgaroseGelDNAExtractionKitVer.4.0 gel recovery kit.

[0059] 3.2.4 Construction of recombinant T vector

[0060] According to TARAKA's pMD TM19-T Vector Cloning Kit instructions, add 1μL of pMD19-T Vector, 1μL of purified PCR product, 3μL of double distilled water and 5μL of SolutionI to a centrifuge tube, react at 16℃ for 30min, heat shock in a 42℃ water bath for 45-60s, quickly transfer to an ice bath and let stand for 2min. Add 700μL of non-resistant LB culture medium to the centrifuge tube, and resuscitate at 37℃, 200rpm for 60min. Pipette 80μL of the resuscitation solution and evenly spread it on the Amp-resistant culture medium, and incubate it in an incubator at 37℃ overnight.

[0061] 3.2.5 Extraction of recombinant positive plasmid

[0062] Pick a single colony, add it to 5 mL of Amp-resistant liquid culture medium, and culture it at 37°C, 200 rpm for 12 h. Select PCR-positive bacterial samples for sequencing, use NCBI Nucleotide Blast to align the sequencing results, and extract the recombinant positive plasmids of each virus according to the instructions of Tiangen's rapid plasmid extraction kit.

[0063] 3.3 Chip preparation

[0064] The probe was diluted with phosphate buffer to a final concentration of 6 μmol / L, and the spotter parameters were set to 100 drops. Each probe was spotted on the nanofilm according to the chip layout order. After spotting, the membrane substrate was placed in a humidity of 45% and a temperature of 22-25°C to dry overnight, and stored at 4°C. The chip layout is shown in Figure 1 The target fragment carrying the Biotin label can specifically bind to the probe of the virus at the corresponding position, and the Biotin binds to horseradish peroxidase-labeled streptavidin (SA-HRP) and catalyzes 3,3',5,5'-tetramethylbenzidine (TMB) to form a blue-purple product.

[0065] Figure 1 The diagram shows the layout of the chip; Biotin is the positive quality control of the chip; Buffer is the negative quality control of the chip.

[0066] 3.4 Amplification of target fragments in mixed samples

[0067] The mixed sample nucleic acid was amplified by RT-PCR method, and the annealing temperature, primer concentration and extension time were optimized. When the annealing temperature was 58°C, 5 cycles were followed by 5°C, and 35 cycles, the band of the target product was the brightest; when the concentration of each primer in the reaction system was 0.2 μmol / L, i.e., 0.05 μL / reaction, the band of the target product was the brightest; when the extension time was 20 s, the band of the target product was the brightest (see Figure 2, PCR reaction condition optimization result diagram; A: annealing temperature optimization result diagram, M: Marker, 1: 54°C, 40 cycles, 2: 56°C, 40 cycles, 3: 58°C, 40 cycles, 4: 60°C, 40 cycles, 5: 56°C, 54°C after 5 cycles, 35 cycles, 6: 58°C, 54°C after 5 cycles, 35 cycles, 7: 58°C, 56°C after 5 cycles, 35 cycles, 8: 60°C, 56°C after 5 cycles, 35 cycles; B: primer concentration optimization result diagram, M: Marker; 1: 0.05μmol / L; 2: 0.1μ mol / L; 3: 0.2μmol / L; 4: 0.4μmol / L; 5: 0.6μmol / L; C: extension time optimization result diagram, 1: 60s, 2: 40s, 3: 20s, 4: 10s), select the two-step cycle to increase the total amount of product, and react according to the following system. The reaction conditions are 50℃, 30min; 94℃, 4min; 94℃, 30s, 58℃, 30s, 72℃, 20s, 5 cycles; 94℃, 30s, 56℃, 30s, 72℃, 20s, 35 cycles; 72℃, 10min; 95℃, 5min. The reaction system is shown in Table 4.

[0068] Table 4 Eight-fold mixed sample amplification system

[0069]

[0070]

[0071] 3.5 Amplification of target fragments from a single sample

[0072] The mixed sample nucleic acid was amplified using the RT-PCR method. The reaction conditions were the same as in 3.4. The reaction system is shown in Table 5.

[0073] The reaction system is shown in Table 5.

[0074] Table 5 Single sample amplification system

[0075] Single system components μL / portion <![CDATA[Double distilled water ddH2O]]> 5.9 Upstream primer F (100 μM) 0.05 Downstream primer R (100 μM) 0.05 2×OnestepMIX 12.5 OnestepEnzymeMIX 1.5 template 5 Total volume 25

[0076] 3.6 Hybridization, washing and color development of the chip

[0077] By optimizing the hybridization time and the dilution concentration of SA-HRP working solution, the detection time can be reduced and clear color development results can be obtained (see Figure 3). The final chip reaction conditions are as follows: the chip is preheated in an incubator at 47°C and 200rpm for 5 to 10 minutes, 100μL of solution A is added to each well first, and then 20μL of mixed sample amplification product is added, and hybridization is carried out at 47°C and 200rpm for 20 minutes. The hybridization solution is aspirated, and 150μL of solution B preheated to 47°C is added to each well for washing, and then the hybridization solution is aspirated, washed twice, and 150μL of solution B preheated to 47°C is added to each well, and incubated at 47°C and 200rpm for 10 minutes in the incubator. After the vacuum pump sucks the liquid dry, 150μL of SA-HRP working solution is added to each well, and incubated at 47°C and 200rpm for 10 minutes in the incubator. The hybridization time and the dilution concentration of the SA-HRP working solution were optimized (see Figure 3 ), aspirate the SA-HRP working solution, add 150μL of solution A to each well and wash 3 times, then add 150μL of solution C to each well and wash 3 times, then aspirate the liquid. Add 85μL of TMB colorimetric solution to each well and let stand at room temperature and away from light for 5-10 minutes. Aspirate the TMB colorimetric solution, add 150μL of pure water to each well and wash 3 times, then observe the results.

[0078] Figure 3 The results of chip reaction condition optimization are shown in Figure 1. A: hybridization time optimization results; B: SA-HRP working solution dilution concentration optimization results. When the hybridization time is extended to 20 minutes, a clear color development result appears. When the hybridization time reaches 30 minutes, the background color of the color development result is strong and the reaction time increases. Therefore, 20 minutes is selected as the optimal hybridization time for chip detection. At 1:250 and 1:500 dilutions, the chip hybridization signal dissipates and contaminates the background; at 1:1000 dilution, the signal is clear and stable; at 1:2000 and 1:4000 dilutions, the hybridization signal is significantly attenuated. Therefore, a SA-HRP working solution with a dilution of 1:1000 should be selected for chip testing.

[0079] 3.7 Determination of test results

[0080] The negative control point does not show color, and the color of the positive control point is blue-purple, which is obviously stronger than the negative control point. At least one of the three positive control points shows color, which is a valid experimental result. If the probe point where the sample is tested does not show color, it is negative; if the probe point where the sample is tested is blue-purple, it is positive.

[0081] 3.8 Chip sensitivity test

[0082] After measuring the concentration of each positive plasmid using an ultra-micro UV spectrophotometer, the average value was taken and each positive plasmid was diluted to 3.39×10 9 The sensitivity of the chip was determined by PCR after 10-fold dilution. The diluted eight pathogen-positive plasmids were mixed in equal volumes to make the plasmid concentration of each pathogen 3.39×108 copies / μL, 10-fold gradient dilution, hybridization with the chip after PCR amplification, and chip sensitivity determination of the mixed sample.

[0083] 3.9 Specificity detection of chip

[0084] The templates of AIV, NDV, IBV, IBDV, REV, ALV-J, MDV, FAdV-4, ALV-A and healthy chicken serum were amplified according to method 3.4 and then detected using the chip.

[0085] 3.10 Chip repeatability test

[0086] Different people used the diluted concentration of 3.39×10 5 , 3.39×10 3 and 3.39×10 1 The mixed quality control sample (positive recombinant plasmid) with 100 copies / μL was used for chip experiments according to the methods in 3.4 and 3.6 to verify the repeatability of chips within the same batch and between different batches.

[0087] 3.11 Chip Stability Test

[0088] The amplification products of the same quality control sample were tested by different personnel at different times using different chips in the same batch and chips from different batches. The gene chip and substrate colorimetric solution were stored at 2-8°C; SA-HRP was stored below -20°C; hybridization solution, washing solution, and eluent were stored at room temperature. The stability of the chip was tested using eight mixed samples at 0 days, 30 days, 60 days, 90 days, 120 days, and 180 days after the chip was made.

[0089] 3.12 Quality evaluation of clinical samples detected by microarray

[0090] Amplify the target fragments from clinical samples according to the method in 3.4 and use the chip for detection. Refer to the national standards for various virus diagnostic technologies [1-3] The qPCR detection methods of each virus were established using primers from the literature. [4-8] , determine its specificity and sensitivity, and compare the quality with the chip detection method established by the present invention.

[0091] Results and Analysis

[0092] 1. Evaluation of chip specificity

[0093] Each pathogen probe site responded specifically, the negative quality control site had no response, and the positive quality control site responded. Except for the positive quality control site response of ALV-A and healthy chicken blood, there was no response at other probe sites, indicating that the specificity of each virus probe used in the chip was good. The results are shown in Figure 4 .

[0094] Figure 4 This is a graph showing the specific results of each virus probe under the eight-fold system; among them, A: AIV; B: NDV; C: IBV; D: IBDV; E: REV; F: ALV-J; G: MDV; H: FAdV-4; I: ALV-A; J: healthy chicken serum.

[0095] 2. Chip sensitivity evaluation

[0096] 2.1 Single positive sample sensitivity

[0097] The detection sensitivity of eight viruses in the mixed system is shown in Figure 5 The single detection sensitivity of 6 viruses including AIV, NDV, IBDV, ALV-J, MDV, and FAdV-4 was 3.39 copies / μL, and the single detection sensitivity of IBV and REV was 33.9 copies / μL.

[0098] Figure 5 The sensitivity test results of each virus probe in the eight-fold system are shown in Figure 2. Among them, A: 3.39×10 2 copies / μL; B: 3.39×10 1 copies / μL; C: 3.39×10 0 copies / μL; D: 3.39×10 -1 copies / μL.

[0099] 2.1 Sensitivity of mixed positive samples

[0100] The plasmid concentration of each pathogen in the mixed sample was diluted to 3.39×10 8 copies / μL, 10-fold gradient dilution to 3.39×10 -1 The results show that the sensitivity of the eight-fold mixed sample is 33.9 copies / μL. Figure 6 .

[0101] Figure 6 This is the sensitivity test result of eight mixed samples; A: 3.39×10 4 copies / μL; B: 3.39×10 3 copies / μL; C: 3.39×10 2 copies / μL; D: 33.9copies / μL; E: 3.39copies / μL; F: 3.39×10 - 1 copies / μL; G: 3.39×10-2 copies / μL; H: 3.39×10 -3 copies / μL.

[0102] 3. Repeatability test of the chip

[0103] To verify the repeatability of the chip method, the amplification products of different dilutions of the same quality control sample (positive recombinant plasmid) were used to verify the chips within batch P20230730001 and between batches P20230522001, P20230730001 and P20231018001. The results are shown in Table 6: The consistency rate of the test results within and between batches was 100%, with good repeatability.

[0104] Table 6 Intra-batch and inter-batch reproducibility of gene chips

[0105]

[0106] 4. Chip Stability Evaluation

[0107] The results of the eight-fold mixed samples at 0, 30, 60, 90, 120, and 180 days after chip production are shown in Figure 7 .

[0108] Figure 7 This is a graph of the shelf life experiment results; A: 0 days; B: 30 days; C: 60 days; D: 90 days; E: 120 days; F: 180 days.

[0109] 5. Clinical sample testing

[0110] The present invention collected 138 clinical samples from chickens that were positive or suspected of being sick with various pathogens to test the actual application effect of gene chips, and used the diagnostic techniques of qPCR methods of various pathogens in national standards and related literature to jointly detect [1-8] , to evaluate the accuracy of the gene chip, the consistency rates of the two detection methods are shown in Table 7.

[0111] Table 7 Clinical sample test results

[0112]

[0113]

[0114] Conformity rate (%) = (total number of clinical samples tested - number of samples with different results by two methods) number of different results / total number of clinical samples tested × 100%

[0115] discuss

[0116] In recent years, visualized gene chip technology has been widely used in the medical field due to its outstanding achievements in gene function expression, pathogenic mechanism, clinical diagnosis, etc. At present, the detection methods for the pathogens of these eight common infectious diseases in poultry are generally molecular diagnostic methods such as fluorescent quantitative PCR. Such methods can usually only detect a few pathogens at a time, resulting in a lot of time and manpower, and the construction cost and operating cost of the standard qPCR laboratory are both high, which is difficult for general breeding companies to bear independently. The gene chip detection method designed by the present invention can detect eight poultry infectious diseases at the same time, and has the characteristics of simple operation, rapidity, convenience, and high throughput.

[0117] By selecting the primer-probe combination and optimizing the reaction conditions, the eight-fold detection gene chip of the present invention has achieved good specificity and sensitivity. Compared with the sensitivity of the eight-kind poultry gene chip detection technology reported by Ye Ranfeng et al. in 2014 (10 4 Copy to 10 5 Between copies) has been greatly improved [9] .

[0118] The application of gene chip detection technology improves the quality of molecular diagnosis of infectious diseases and shortens the analysis time. It is an independent method that can screen multiple genes in a large number of pathological specimens. The method established by the present invention can simultaneously detect eight avian infectious pathogens, and there is no cross-response between the eight pathogens, and there is no response to healthy poultry serum tissue. The chip repetition rate can reach 100%, and the storage period at 2-8°C can reach 180 days. The infection of each virus in clinical samples was simultaneously detected by the gene chip method and the existing national standard qPCR detection method. The detection results of the two methods have a good coincidence rate, indicating that the gene chip developed in this study can meet the laboratory clinical sample detection and epidemiological monitoring.

[0119] [1] GB / T18936-2020 Diagnostic technology for highly pathogenic avian influenza[S].

[0120] [2] GBT26436-2010 Diagnostic technology of avian leukosis[S].

[0121] [3] GB-T19167-2020 Diagnostic technology of infectious bursal disease[S].

[0122] [4]CALLISONSA,HILTDA,BOYNTONTO,etal.Developmentandevaluationofareal-time Taqman RT-PCR assay for the detection of infectious bronchitis virusfrom infected chickens[J].J VirolMethods,2006,138(1-2):60-65.

[0123] [5]ZHANGX,YAOM,TANGZ,etal.Developmentandapplicationofatriplexreal-timePCRassay forsimultaneousdetectionofavianinfluenzavirus,Newcastlediseasevirus,andduckTembusuvirus[J].BMCVetRes,2020,16(1):203.

[0124] [6]LI X,ZHANG K,PEI Y,et al.Development and Application of an MRT-qPCR Assay for Detecting Coinfection of Six Vertically Transmitted orImmunosuppressive Avian Viruses[J].

[0125] FrontMicrobiol,2020,11:1581.

[0126] [7]LI K,GAO H,GAO L,et al.Development of TaqMan real-time PCR assayfor detection and quantitationofreticuloendotheliosisvirus[J].JVirolMethods,2012,179(2):402-408.

[0127] [8] HENNIGH, OSTERRIEDERN, MULLER-STEINHARDTM, etal. Detection of Marek's disease virus DNAinchickenbutnotinhumanplasma [J]. J Clin Microbiol, 2003, 41 (6): 2428-2432.

[0128] [9] Ye Ranfeng. Preliminary study on gene chip detection technology of eight avian viruses[D]. Central China Normal University, 2012.

[0129] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A primer probe set for detecting eight avian infectious disease pathogens, wherein the eight avian infectious disease pathogens are: avian influenza virus, Newcastle disease virus, avian leukemia virus type J, infectious bronchitis virus, infectious bursal disease virus, reticuloendotheliosis virus, Marek's disease virus and avian adenovirus type 4, characterized in that: The primer probe sets for detecting avian influenza virus include XPAIV-F, XPAIV-R and XPAIV-P, the primer probe sets for detecting Newcastle disease virus include XPNDV-F, XPNDV-R and XPNDV-P, the primer probe sets for detecting avian leukemia virus type J include XPALV-JF, XPALV-JR and XPALV-JP, the primer probe sets for detecting infectious bronchitis virus include XPIBV-F, XPIBV-R and XPIBV-P, the primer probe sets for detecting infectious bursal disease virus include XPI-JF, XPI-JR and XPI-JP The primer probe set for detecting the disease virus includes XPIBDV-F, XPIBDV-R and XPIBDV-P, the primer probe set for detecting the reticuloendotheliosis virus includes XPREV-F, XPREV-R and XPREV-P, the primer probe set for detecting avian adenovirus type 4 includes XPFAdV-4-F, XPFAdV-4-R and XPFAdV-4-P, and the primer probe set for detecting Marek's disease virus includes XPMDV-F, XPMDV-R and XPMDV-P; The nucleotide sequence of the XPAIV-F is shown in SEQ ID NO.1, the nucleotide sequence of the XPAIV-R is shown in SEQ ID NO.2, and the nucleotide sequence of the XPAIV-P is shown in SEQ ID NO.3; the nucleotide sequence of the XPNDV-F is shown in SEQ ID NO.4, the nucleotide sequence of the XPNDV-R is shown in SEQ ID NO.5, and the nucleotide sequence of the XPNDV-P is shown in SEQ ID NO.6; the nucleotide sequence of the XPALV-JF is shown in SEQ ID NO.7, the nucleotide sequence of the XPALV-JR is shown in SEQ ID NO.8, and the nucleotide sequence of the XPALV-JP is shown in SEQ ID NO.9; the nucleotide sequence of the XPIBV-F is shown in SEQ ID NO.10, the nucleotide sequence of the XPIBV-R is shown in SEQ ID NO.11, and the nucleotide sequence of the XPIBV-P is shown in SEQ ID NO.12; the nucleotide sequence of the XPIBDV-F is shown in SEQ ID NO.13, and the nucleotide sequence of the XPIBDV-R is shown in SEQ ID NO. ID NO.14, the nucleotide sequence of the XPIBDV-P is shown in SEQ ID NO.15; the nucleotide sequence of the XPREV-F is shown in SEQ ID NO.16, the nucleotide sequence of the XPREV-R is shown in SEQ ID NO.17, and the nucleotide sequence of the XPREV-P is shown in SEQ ID NO.18; the nucleotide sequence of the XPFAdV-4-F is shown in SEQ ID NO.19, the nucleotide sequence of the XPFAdV-4-R is shown in SEQ ID NO.20, and the nucleotide sequence of the XPFAdV-4-P is shown in SEQ ID NO.21; the nucleotide sequence of the XPMDV-F is shown in SEQ ID NO.22, the nucleotide sequence of the XPMDV-R is shown in SEQ ID NO.23, and the nucleotide sequence of the XPMDV-P is shown in SEQ ID NO.

24.

2. The primer probe set according to claim 1, characterized in that: In the primer probe set, the 5' end of the downstream primer is biotin-modified.

3. The primer probe set according to claim 1, characterized in that: In the primer probe set, the 5' end of the probe is modified with an amino group, and the amino group includes NH2C6.

4. A gene chip for detecting eight avian infectious disease pathogens, characterized in that: The probes in the primer probe set according to any one of claims 1 to 3 are fixed on the gene chip.

5. The method for preparing the gene chip according to claim 4, comprising the following steps: The probes are spotted on the nano-membrane according to the chip arrangement order to obtain a membrane substrate, and the membrane substrate is air-dried under the conditions of a humidity of 40% to 60% and a temperature of 22 to 25° C. to obtain a gene chip.

6. The preparation method according to claim 5, characterized in that: The chip arrangement includes spotting probes for detecting eight avian infectious disease pathogens at different positions.

7. A kit for detecting eight avian infectious disease pathogens, characterized in that: The invention comprises the primer probe set according to any one of claims 1 to 3 and the gene chip according to claim 4 or the gene chip prepared by the preparation method according to claim 5.

8. The kit according to claim 7, characterized in that When the kit amplifies the sample to be tested, the amplification conditions are 50°C, 30 min, 94°C, 4 min, 94°C, 30 s, 58°C, 30 s, 72°C, 20 s, 5 cycles, 94°C, 30 s, 56°C, 30 s, 72°C, 20 s, 35 cycles, 72°C, 10 min, 95°C, 5 min.

9. The kit according to claim 7, characterized in that When the kit is used to amplify the sample to be tested, each 25 μL reaction system includes 0.05 μL of each primer of 100 μM, 12.5 μL of 2×One step MIX, 1.5 μL of One step Enzyme MIX, 5 μL of template and the balance of water.

10. Use of the primer probe set according to any one of claims 1 to 3 and / or the gene chip according to claim 4 or the gene chip prepared by the preparation method according to claim 5 in preparing a kit for diagnosing avian infectious diseases; the avian infectious diseases are avian influenza, Newcastle disease, infectious bronchitis, infectious bursal disease, Marek's disease, avian leukosis, avian reticuloendotheliosis and hepatitis-pericardial effusion syndrome.

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