Primer probe combination, gene chip, kit and method for detecting four bovine respiratory disease pathogens

Abstract

The invention discloses a gene chip detection kit for bovine respiratory diseases BPIV3, BRSV, IBRV and BVDV, which is characterized in that when a sample to be detected is amplified by the following primers: # imgabs0 #, every 25 mu L of reaction system comprises 1 mu L of 107 copies / mu L quality control, 0.6 mu L of 10 mu M primers, 0.5 mu L, 0.5 mu L, 0.7 mu L, 0.4 mu L, 12.5 mu L of 2 * One step MIX, 1.25 mu L of One step Enzyme MIX, 2 mu L of template and the balance of water, and a PCR product is obtained after a PCR reaction.

Description

Technical Field

[0001] The invention belongs to the technical field of gene chips, and in particular relates to a gene chip detection method for four respiratory system diseases of cattle. The detection method can detect BPIV3, BRSV, IBRV and BVDV. Background Art

[0002] Respiratory diseases have always been one of the major diseases that threaten cattle health. In recent years, the development of intensive farming models and the imbalance of regional scale farming, coupled with changes in the breeding environment, insufficient management level and emergency response to long-distance transportation, have led to a continuous increase in the incidence and mortality of respiratory diseases, causing significant economic losses to the cattle and sheep farming industry.

[0003] Bovine respiratory disease complex (BRDC) is the leading cause of respiratory disease and death in cattle worldwide. Cattle infected with BRDC will have symptoms such as fever, rapid breathing, repeated coughing, runny nose or tears, diarrhea, dehydration and loss of appetite, which will have a great impact on the health and production performance of cattle, and thus affect economic benefits.

[0004] Infectious bovinerhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV3), bovine respiratory syncytial virus (BRSV) and bovine viral diarrhea virus (BVDV) are the four main viral pathogens that cause bovine respiratory disease complex (BRDC).

[0005] Although traditional pathogen detection methods such as virus isolation and culture, immunofluorescence detection and enzyme-linked immunosorbent assay have been successfully applied, they are often helpless in dealing with the problem of differential diagnosis of various animal diseases. In recent years, gene chip technology has been widely used in the medical field, and has achieved outstanding results in research on gene function expression, pathogenic mechanism, clinical diagnosis, drug development, biological detection, etc. Gene chip (DNA chip, DNA microarray and oligonucleotide microarray) is a type of biochip. This technology fixes nucleotide probes with known sequence information on the surface of the support, and then specifically binds to the sample molecules of the amplified label, and analyzes the number of molecules and nucleotide sequence information of the sample by observing the hybridization signal intensity of each probe molecule and the sample. Gene chip technology can fix a large number of nucleic acid probes on the support at the same time, so as to detect and analyze a large number of nucleic acid sequences in the sample at one time. At the same time, compared with traditional chips, the biggest advantage of visual chips is that they do not require other complex and expensive equipment, and can directly observe whether the probe point responds to determine whether the sample contains the pathogenic nucleic acid to be detected.

[0006] The gene chip detection technology of this application is committed to establishing an efficient, rapid and accurate gene chip differential diagnosis detection method for bovine respiratory diseases, providing a useful tool for rapid detection of bovine respiratory diseases and their epidemiological investigation. Establish identification detection methods for IBRV, BPIV3, BRSV and BVDV, four important bovine respiratory diseases in BRDC, and develop a gene chip detection kit for differential diagnosis of IBRV, BPIV3, BRSV and BVDV based on Gaoling material biochips. It can quickly distinguish single or mixed infections of IBRV, BPIV3, BRSV and BVDV in clinical practice, and build a BRDC identification detection gene chip detection technology platform to achieve large-scale on-site detection in farmers or farms. Summary of the invention

[0007] The present invention provides the following technical solutions:

[0008] 1: Preparation of joint inspection chips: Using the sciFLEXARRAYER spotter from Scienion, Germany, ultra-micro dispensing and spotting are performed in a non-contact manner in combination with piezoelectric ceramic technology to prepare a gene (probe, including positive quality control probe) chip with a fixed arrangement pattern. Develop high-performance biochip nanomembrane technology based on Gaoling bioreaction materials, and develop a "0+X" medium- and low-density cattle high-incidence animal disease differential diagnosis gene chip method, which has the advantages of zero background capability, covalent fixation capability, ultra-wide load range and three-dimensional presentation capability, with strong specificity and high sensitivity.

[0009] Gaoling TM bioreaction substrate material is a new generation of high-performance biochip substrate material, which can replace traditional substrate materials such as nylon membranes and glass slides in the construction of chips such as genes, proteins and peptides (small molecules). It has a unique micro-nano structure and surface chemical properties, can covalently fix small molecules and proteins such as nucleic acids and peptides at high density, and realize three-dimensional spatial display; it can reduce detection background noise and improve sensitivity without BSA blocking; it simplifies cleaning steps and saves operation time.

[0010] The main advantages are as follows: ① Zero background capability: When the total concentration of protein in the solution is not higher than 500μg / mL, non-specific adsorption of protein can be prevented without blocking operations such as BSA. ② Covalent fixation capability: COOH can fix probes with amino groups under EDC / NHS activation conditions, including but not limited to nucleic acids, short peptides, proteins, cells, viruses, etc. ③ Ultra-wide loading range: Taking the fixation of a short peptide as an example, the monoclonal antibody corresponding to the short peptide has distinguishable signal values ​​under a series of spot concentrations. ④ Three-dimensional presentation capability: It is conducive to the interaction of prey-bait, reduces steric hindrance, and realizes rapid detection.

[0011] 2. Epidemiological survey: Conduct epidemiological surveys on respiratory diseases such as IBRV, BPIV3, BRSV and BVDV in intensive cattle farms in Xinjiang, including pathogenic antigen detection and serological antibody detection, to understand the epidemic situation of the above four bovine respiratory diseases in some areas of Xinjiang. Isolate and identify the above four bovine respiratory epidemic strains that are positive in pathogen detection, and use MAFFT, MEGA7, FastTree and other analysis software to perform multiple sequence alignment, evolutionary distance, phylogeny, conservation and other bioinformatics analysis to determine the target sequence.

[0012] Common PCR detection methods for IBRV, BPIV3, BRSV and BVDV were established respectively, and the reaction system was optimized for application in actual clinical sample detection. The obtained positive samples were subjected to whole genome sequencing analysis, and cultured and isolated using in vitro cells or culture media suitable for strain growth. Ten samples of IBRV, BPIV3, BRSV and BVDV were isolated and identified.

[0013] 3. Construction of the joint detection chip detection method: According to the above-mentioned epidemic strain identification results, the whole genome sequences of the target bacteria and strains were compared with the NCBI animal nt library and the bacterial nt library (the target strain sequence was removed), and the sequences that were not aligned to the database were screened, and the orthoml was further used to perform cluster analysis on the target genes. Finally, IBRV, BPIV3, BRSV and BVDV specific genes were obtained. Using Oligo6.0 primer-probe design software and DNAstar and other biological analysis software, primers and probes were designed for the above four viruses. Using the sciFLEXARRAYER spotter from Scienion, Germany, ultra-micro dispensing and spotting were performed in a non-contact manner combined with piezoelectric ceramic technology to prepare a gene (probe, including positive quality control probe) chip containing a fixed arrangement pattern. For each virus, 10 positive samples and 10 negative samples were first used for RT-PCR / probe hybridization tests, and the conditions such as RT-PCR, hybridization, probe, primer, etc. were optimized, the probe / primer combination was determined, and the chip preparation, nucleic acid extraction, RT-PCR, hybridization and other production processes, detection procedures and result determination were completed.

[0014] 3.1 Specificity test: The established gene chip test kits for the main viral pathogens of bovine respiratory disease syndrome, BPIV3, BRSV, IBRV and BVDV, and the test samples that are positive in clinical tests and have been verified by national standards or other test methods are used for testing. The compliance rate is more than 99%, and it can distinguish the above-mentioned single disease infection or multiple disease infection. It is confirmed that this method has no cross-reaction with other related viruses, has good specificity, and can distinguish whether multiple pathogens are infected while detecting and diagnosing a single disease.

[0015] 3.2 Sensitivity test: The plasmids of the major viral pathogens of bovine respiratory disease syndrome, BPIV3, BRSV, IBRV and BVDV, were diluted 10 times in a gradient, and then the sensitivity of the chip was calculated based on the chip reaction results using the gene chip detection kits of the major viral pathogens of bovine respiratory disease syndrome, BPIV3, BRSV, IBRV and BVDV. The Dalton copy number calculation formula [copy number = plasmid concentration 6.02 × 10 23 / (660×plasmid length)】The sensitivity limits of single disease detection and multi-disease detection are obtained. The copy number for single virus detection should be ≤5 copies / μL; the copy number for mixed joint detection should be ≤10 copies / μL.

[0016] 3.3 Preliminary application of differential diagnosis gene chip detection kit: About 54 clinical samples from Xinjiang (the positive rate of single or multiple diseases should be no less than 10%) were compared by PCR and gene chip detection methods for IBRV, BPIV3, BRSV and BVDV, and the consistency rate of the two detection methods should be no less than 99%. The constructed gene chip detection kit for the main viral pathogens of bovine respiratory disease syndrome, BPIV3, BRSV, IBRV and BVDV, can meet the clinical rapid diagnosis needs of the above-mentioned diseases. One sample can not only detect single virus infection, but also visually differentiate and diagnose multiple disease infections, providing a rapid and effective detection method for the epidemiological investigation of high-incidence animal diseases of the bovine respiratory tract, and also providing a scientific theoretical basis for the rapid differential diagnosis of other animal diseases.

[0017]

Beneficial Effects

[0018] High sensitivity, the minimum detection limit of a single chip can reach 1×10 0 copies / μL, the minimum detection limit of the quadruple chip can reach 1×10 1 The chip can be stored at 4°C for at least one year. The test can be completed within 2.5 hours, shortening the test time. One chip can test 48 samples at the same time, and can test 4 viruses at the same time, improving the test efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a single gene chip spotting probe arrangement.

[0020] Figure 2 It is a quadruple gene chip spotting probe arrangement.

[0021] Figure 3 It is the result of PCR amplification of the target gene.

[0022] Figure 4-1 is the optimization of PCR annealing temperature for BPIV3;

[0023] Figure 4-2 is the PCR annealing temperature optimization for BRSV;

[0024] Figure 4-3 It is the PCR annealing temperature optimization of IBRV;

[0025] Figure 4-4 It is the optimization of PCR annealing temperature for BVDV;

[0026] Note: M, DNA Marker Ⅰ; NC, negative control; 1-6, 54℃, 56℃, 60℃, 64℃, 68℃.

[0027] Figure 5-1is the optimization of PCR extension time for BPIV3;

[0028] Figure 5-2 It is the optimization of PCR extension time for BRSV;

[0029] Figure 5-3 It is the PCR extension time optimization of IBRV;

[0030] Figure 5-4 It is the optimization of PCR extension time for BVDV;

[0031] Note: M, DNA MarkerⅠ; NC, negative control; 1-4: 20, 30, 40, 50s.

[0032] Figure 6-1 It is the optimization of PCR primer concentration for BPIV3;

[0033] Figure 6-2 is the optimization of PCR primer concentration for BRSV;

[0034] Figure 6-3 It is the PCR primer concentration optimization of IBRV;

[0035] Figure 6-4 It is the optimization of PCR primer concentration for BVDV;

[0036] Note: M, DNA Marker Ⅰ; NC, negative control; 1-7: 0.3μL, 0.4μL, 0.5μL, 0.6μL, 0.7μL, 0.8μL, 0.9μL.

[0037] Figure 7 This is a SA-HRP dilution test.

[0038] Figure 8 It is a hybridization time optimization experiment.

[0039] Fig. 9 It is a specific test of gene chip.

[0040] Figure 10-1 It is a common PCR sensitivity test for BPIV3;

[0041] Figure 10-2 It is a common PCR sensitivity test for BRSV;

[0042] Figure 10-3 It is the IBRV common PCR sensitivity test;

[0043] Figure 10-4 It is a common PCR sensitivity test for BVDV;

[0044] Note: M, DNA MarkerⅠ; NC, negative control; 1~8, 108 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 and 10 1 copies / μL.

[0045] Fig.11 It is a gene chip sensitivity test.

[0046] Fig.12 It is a shelf life test of the chip. DETAILED DESCRIPTION

[0047] The above contents of the present invention are further described in detail below through embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] Embodiment 1:

[0049] 1 Materials

[0050] 1.1 Virus strains and clinical samples

[0051] Bovine parainfluenza virus type 3 (XJA13), bovine respiratory syncytial virus (USII / S1), bovine infectious rhinotracheitis virus (C36876-459), bovine viral diarrhea virus (No.12_E+), bovine coronavirus (BCoV4-6), bovine Pasteurella multocida (Pam250-03), bovine hemolytic Mannheimia (Mha58-72), and bovine Mycoplasma virus (MyP6-15) were all preserved and provided by the Animal Disease Prevention and Control Research Innovation Team of the State Key Laboratory of Sheep Genetic Improvement and Healthy Breeding, Xinjiang Academy of Agricultural Sciences. 54 bovine clinical samples were collected from cattle farms in some areas of Xinjiang, and all samples were stored in the laboratory at -80℃.

[0052] 1.2 Main reagents and instruments

[0053] Virus nucleic acid extraction kit, gel recovery kit, ordinary plasmid small-scale extraction kit, reverse transcriptase, DNA Marker were purchased from Bao Biotechnology (Dalian) Co., Ltd.; hot start enzyme was purchased from Kangwei Century Biological Co., Ltd.; pUC57 vector was purchased from Sangon Biotechnology Co., Ltd.; DH5α competent cells were purchased from Sangon Biotechnology Co., Ltd.; horseradish peroxidase-labeled streptavidin (SA-HRP) was purchased from Jackson Immuno Research. Sodium chloride, sodium citrate, SDS, trisodium citrate, etc. were purchased from Beijing Chemical Reagent Co., Ltd.

[0054] Nucleic acid and protein quantification instrument was purchased from Thermo Fisher Scientific Inc.; PCR instrument was purchased from Biometra, Germany; gel imaging system was purchased from BIO-RAD, USA; low-temperature high-speed centrifuge was purchased from Eppenddorf, Germany; agarose horizontal electrophoresis instrument was purchased from Beijing Liuyi Instrument Factory; high-throughput tissue grinding instrument was purchased from Shanghai Jingxin Industrial Development Co., Ltd.; constant temperature well plate chip incubator was purchased from Suzhou Abito Biotechnology Co., Ltd.; 48-well automatic imaging scanner was purchased from Suzhou Siju Biomaterials Co., Ltd.

[0055] 2 Methods

[0056] 2.1 Primer and probe design optimization

[0057] According to the P gene of bovine parainfluenza virus type 3 (KU198929.1), the N gene of bovine respiratory syncytial virus (NC_038272.1), the gI gene of bovine infectious rhinotracheitis virus (MK552112.1) and the 5'UTR gene of bovine viral diarrhea virus (LC068604.1) included in GenBank, five pairs of specific primers labeled with biotin and corresponding gene chip probes were designed using Primer5.0 software, which were used to amplify and detect BPIV3-P gene, BRSV-N gene, IBRV-gI gene and BVDV-5'UTR gene, respectively. The primers were synthesized by Takara Biotechnology (Dalian) Co., Ltd. PCR reaction quality control primers (th.ac-F / R), template (IC-AT1 plasmid) and chip probe (IC-AT1-P) were all provided by Suzhou Abito Biotechnology Co., Ltd. The primer information is shown in Table 1 and the probe information is shown in Table 2.

[0058] Table 1 Primer information

[0059]

[0060] Table 2 Probe information

[0061]

[0062] 2.2 Construction of recombinant positive plasmid

[0063] 2.3 Sample processing and nucleic acid extraction

[0064] Extract the nucleic acids of BPIV3, BRSV, IBRV and BVDV samples according to the kit instructions. After the nucleic acids of the extracted and processed clinical samples are stored at -80°C for future use. For specific operations, see steps A and B.

[0065] Step A. Viral DNA / RNA extraction steps:

[0066] The virus sample to be separated was taken out from the -80°C low-temperature refrigerator and thawed in a 4°C refrigerator. Viral DNA / RNA extraction was performed according to the steps of the TaKaRa virus extraction kit (Cat: 9766).

[0067] 1) Take 200 μL of the treated sample solution and put it into a 1.5 mL EP tube, add 200 μL Buffer VGB, 20 uL Proteinase K and 1.0 μL Carrier RNA, mix thoroughly, and place in a 56°C water bath for 10 minutes.

[0068] 2) Add 200 μL of anhydrous ethanol and pipette thoroughly to mix well.

[0069] 3) Place the Spin Column on the Collection Tube, transfer the solution to the Spin Column, centrifuge at 12000 rpm for 2 min, and discard the filtrate.

[0070] 4) Add 500 μL of Buffer RWA to the Spin Column, centrifuge at 12000 rpm for 1 min, and discard the filtrate.

[0071] 5) Add 700 μL of Buffer RWB to the Spin Column along the tube wall, centrifuge at 12000 rpm for 1 min, discard the filtrate, and repeat the steps once.

[0072] 6) Place the Spin Column on the Collection Tube and centrifuge at 12000 rpm for 2 min.

[0073] 7) Place the Spin Column on a new 1.5 mL RNase free collection tube, add 30 μL of RNase free dd H2O to the center of the Spin Column membrane, let stand at room temperature for 5 minutes, and then centrifuge at 12000 rpm for 2 minutes to obtain RNA.

[0074] Step B. Reverse transcription step:

[0075] Use TaKaRa reverse transcription kit, the specific operation is as follows:

[0076] 1) DNA removal reaction

[0077]

[0078] Incubate in 42℃ water bath for 2min.

[0079] 2) Reverse transcription reaction

[0080]

[0081] 37℃ water bath for 15min

[0082] After instantaneous heat shock at 85°C for 5 seconds, the reverse transcription product was obtained.

[0083] 2.4 Amplification of target gene

[0084] Using a commercial RNA / DNA extraction kit, RNA was extracted from BPIV3, BRSV, and BVDV positive strains, and the RNA was reverse transcribed into cDNA. DNA was extracted from IBRV positive strains. The extracted cDNA and DNA were used as templates to perform conventional PCR amplification according to the four pairs of specific primers designed in this study, the system in Table 3, and the reaction conditions in Table 4. The products were subjected to 2% agarose gel electrophoresis.

[0085] Table 3 Conventional PCR reaction system

[0086]

[0087] Table 4 Conventional PCR reaction procedure

[0088]

[0089] 2.5 Purification of PCR products

[0090] Recover the PCR product from the gel according to the kit instructions. For specific operations, see step C.

[0091] Step C. Purification step:

[0092] Use the TIANGEN gel recovery kit, the specific steps are as follows:

[0093] (1) After gel excision and recovery of the target fragment, add an equal volume of Buffer BN to the PCR reaction product and mix evenly in a 56°C water bath.

[0094] (2) Place the adsorption column in the kit into the collection tube, add 500 μL of balancing solution Buffer BL to the adsorption column CA2, centrifuge at 12000 rpm / min for 1 minute, and discard the filtrate.

[0095] (3) Transfer the solution in operation (1) to adsorption column CA2. Place at room temperature for 2 minutes, centrifuge at 12000 rpm / min for 1 minute, and discard the filtrate.

[0096] (4) Add 600 μL of Buffer PW to CA2, centrifuge at 12,000 rpm / min for 30 s at room temperature, and discard the filtrate.

[0097] (5) Repeat (4).

[0098] (6) Place the adsorption column CA2 on the collection tube and centrifuge at 12000 rpm / min for 2 minutes at room temperature.

[0099] (7) Place the adsorption column CA2 in a new 1.5 ml centrifuge tube, add 25-30 μL of Buffer EB to the center of the filter column membrane, and let it stand at room temperature for 2 minutes.

[0100] (8) Centrifuge at 12000 rpm / min for 2 min at room temperature to elute the DNA.

[0101] 2.6 Construction of recombinant T vector

[0102] For specific operations, see step D, step E, step F, and step G.

[0103] Step D. Connect:

[0104] Ligation reaction system

[0105]

[0106] After incubation at room temperature for 1 h, the reaction system was incubated at 4°C overnight, and then the ligation product was transformed.

[0107] Step E. Transformation

[0108] (1) Place 100 μL of competent cells in a 1.5 mL sterile centrifuge tube and place in an ice bath.

[0109] (2) Add 10 μL of the ligation product to 100 μL of the competent cell DH5α suspension, flick to mix, and let stand in an ice bath for 30 min.

[0110] (3) Place the centrifuge tube in a 42°C water bath for 60-90 seconds, then quickly transfer the centrifuge tube to an ice bath to cool the cells for 2-3 minutes. Do not shake the centrifuge tube during the entire process.

[0111] (4) Add 900 μL of sterile LB (without antibiotics) to each centrifuge tube, mix well, and incubate at 37°C in a shaker at 180 rpm / min for 60 min.

[0112] (5) Mix the contents of the centrifuge tube, pipette 100 μL of the transformed competent cells onto the LB solid medium containing the corresponding antibiotics, and gently spread the cells evenly with a sterile curved glass rod. Place the plate at room temperature until the liquid is absorbed, invert the plate, and culture at 37°C for 12-16 hours.

[0113] Step F. Bacteria selection and bacterial liquid PCR identification

[0114] (1) Pick a single colony and place it in 10 mL LB (containing Amp) and shake at 37°C, 220 rpm / min for 12-16 hours.

[0115] (2) After the bacterial solution has been shaken for approximately 5-6 hours, PCR identification of the bacterial solution is performed. The reaction system and reaction procedure are shown in the table below.

[0116] PCR reaction system

[0117]

[0118] Conventional PCR reaction procedure

[0119]

[0120] (3) 10 μL of the amplified product was taken and PCR identification was performed using 2% agarose gel. Target bands of approximately 117 bp, 111 bp, 75, 147 bp and 84 bp were amplified, respectively, which were consistent with the expected sizes.

[0121] (4) Take 10 μL of positive bacterial solution and add it to 5 ml of LB medium (containing Amp). Incubate at 37°C, 220 rpm / min for 12-16 hours. Extract plasmids the next day.

[0122] Step G. Plasmid extraction

[0123] (1) Take out the shaken bacterial solution, centrifuge it at 5000g for 10 min at room temperature, and discard the culture medium.

[0124] (2) Add 500 μL of solution 1 (containing RNase A) and vortex to suspend the cells. Transfer the suspension to a new 2 mL centrifuge tube.

[0125] (3) Add 500 μL of solution 2 and gently invert 5-7 times to mix. The reaction time should not exceed 5 min.

[0126] (4) 700 μL solution 3, gently invert several times until white flocculent precipitate is formed. Centrifuge at 13000 g for 10 min at room temperature. Dense white particles will form.

[0127] 2.7 Identification of recombinant positive plasmids

[0128] After the four recombinant plasmids were sequenced and identified correctly, their mass concentrations were determined by spectrophotometry, and the mass concentrations were converted to copy number concentrations using the formula: copy number concentration / (copy / μL) = [(6.02×10 23 )×mass concentration

[0129] / (ng / μL)×10 -9 ] / (DNA sequence length × 660). Mix the four recombinant plasmids in equal volumes as Quadruple Gene Chip detection Standard product of the law.

[0130] 2.8 Preparation of gene chips

[0131] 2.8.1 Schematic diagram of spotting on a single chip

[0132] 2.8.1.1 Schematic diagram of chip spotting

[0133] The probes were diluted with phosphate buffer to a final concentration of 6 μmol / L. According to the pre-set spotting pattern, Suzhou Abituo Biotechnology Co., Ltd. was commissioned to spot each probe on the nanomembrane. The spotting pattern of a single gene chip is shown in Figure 1 , quadruple gene chip spotting mode see Figure 2 , Biotin is the biotin staining control for chip hybridization reaction, BPIV3, BRSV, IBRV, BVDV1 and BVDV2 are detection probes, and IC-AT1 is the quality control probe for PCR reaction.

[0134] 2.8.2 Activation of gene chip membrane

[0135] See step H for specific operations.

[0136] Step H. Activation of GeneChip Membrane

[0137] Place 2% EDC and 1.21% NHS in ultrapure water and fully dissolve them to prepare the activation solution. Then place the 76mm×65mm nano-membrane in the activation tank, facing up, and evenly pour 15mL / piece of activation solution to make the membrane surface completely penetrated and soak for 30 minutes. After the activation is completed, rinse the membrane with ultrapure water 3 times and blow dry with nitrogen to ensure that the membrane surface is dry and free of foreign matter.

[0138] 2.8.3 Assembly of gene chips

[0139] See step I for specific operations.

[0140] Step I. Assembly of gene chip

[0141] Load the activated membrane on the membrane rack, and load the 48-well plate and the membrane rack with the membrane on the corresponding position of the spotter. Dilute the probe to 6μmol / L with phosphate buffer, add 4μL to each well, set the spotter parameter to 100drop, and spot the probe on the SJ modified silica gel membrane assembled in the 48 wells according to the pre-arranged spotting order. The temperature is 20-25℃, the humidity is 40%-70%, and click the program to start spotting. After the spotting is completed, take out the spotted membrane from the spotter and check whether there are defects or tailing on the membrane. Place the qualified membrane on the membrane base with double-sided tape, align the position and install the cover, assemble it into a gene chip, and ensure that the point of each well is in the well. Place the gene chip in an aluminum foil bag, put in a bag of desiccant, vacuum seal it, and store it in a refrigerator at 2-8℃.

[0142] 2.8.4 Hybridization reaction of gene chip

[0143] For preparation before reaction, see step J for details.

[0144] Step J. Preparation before hybridization reaction

[0145] Prepare hybridization solution A, elution solution B and eluent C; take 100 μL of hybridization solution A and add it to the well of the well plate incubator chip preheated at 47℃, 200 r / min, and react for 10 minutes; take 20 μL of PCR product and add it to the well of the well plate incubator chip preheated at 47℃, 200 r / min, react for 5 minutes, then rinse with elution solution B preheated at 47℃ for 3 times, using 150 μL each time; dilute SA-HRP with hybridization solution A at 1000:1, add 150 μL / well, 200 r / min, react at 47℃ for 10 minutes, then rinse with hybridization solution A and eluent C for 3 times at room temperature, add TMB substrate color development solution (90 μL / well) for color development for 5 minutes; wash with pure water 3 times to terminate the reaction, and blow dry gently with a compressed air can; observe and record the experimental results. (Hybridization solution A: Take 10mL 20×SSC and 1mL 10% SDS, add pure water to make up to 100mL, mix thoroughly and set aside; Elution solution B: Take 3mL 20×SSC and 1.2mL 10% SDS, add pure water to make up to 120mL, mix thoroughly and set aside; Eluent C: Take 10mL 1mol sodium citrate, add pure water to make up to 100mL, mix thoroughly and set aside.)

[0146] 2.9 Determination of results

[0147] The result determination method is: the Biotin spot and the th.ac-P probe spot should be a distinct dark blue, indicating that the test is established; if the probe spot where the tested sample is located does not show color, it is negative; if the probe spot where the tested sample is located is blue, it is positive. In this experiment, if BPIV3-P, BRSV-P, IBRV-P, BVDV-P1 and BVDV-P2 show blue-black, it indicates that the tested sample is infected with the four pathogens BPIV3, BRSV, IBRV and BVDV.

[0148] 2.10 Establishment and optimization of quadruple PCR amplification reaction

[0149] The extracted nucleic acid was used as a template, and four pairs of primers were placed in the same PCR reaction system. The annealing temperature, extension time and primer dosage of the reaction were optimized to explore the best target gene amplification conditions. The reaction system was 25μL: 2X Onestep MIX 12.5μL, One step Enzyme MIX 1.25μL, BPIV3, BRSV, IBRV, BVDV and th.ac primers were mixed with 1.2μL, 1μL, 1μL, 1μL, 1.4μL and 0.8μL, IC-AT1 plasmid 1μL, template nucleic acid 2μL, sterile double distilled water 2.85μL. The reaction conditions were 50℃30min; 94℃5min; (94℃30s, 56℃30s, 72℃20s); 72℃10min, 35 cycles, and stored at -20℃.

[0150] 4.2.9.1 Optimization of PCR reaction annealing temperature

[0151] Using Primer5 software, we can see that the primer annealing temperature of each pathogen is different, ranging from 54 to 68°C. In order to maintain uniformity and ease of operation, the annealing temperature of each pathogen was optimized. Using BPIV3-P strain nucleic acid as a template, the primer annealing temperature of the PCR system was optimized according to the above amplification system. The annealing temperatures were set to 54°C, 56°C, 60°C, 64°C, and 68°C, respectively.

[0152] 4.2.9.2 Optimization of PCR reaction extension time

[0153] Using BPIV3-P strain nucleic acid as template, the extension time in the PCR process was optimized and set to 72°C (60s, 40s, 30s, 20s) for PCR amplification, and the amplified products were analyzed by 2% agarose gel electrophoresis to compare the results. The optimal reaction extension time was determined.

[0154] 4.2.9.3 Optimization of primer concentration in PCR reaction

[0155] Using BPIV3-P strain nucleic acid as template, dilute the primer concentration to 10 μmol / L, optimize the volume of upstream and downstream primers. Perform PCR amplification at different primer pair dosages of 0.5 μL-1 μL, analyze the amplified products by 2% agarose gel electrophoresis, and determine the dosage of each pair of specific upstream and downstream primers.

[0156] 4.2.10 Optimization of Gene Chip Hybridization Reaction Conditions

[0157] 4.2.10.1 Optimization of SA-HRP dilution

[0158] Use the recombinant plasmid standard to amplify the target fragment according to the above reaction conditions, and then hybridize the product with SA-HRP diluted 1:500, 1:1000, 1:2000, 1:4000, 1:8000, and 1:16000 in hybridization solution A, and observe the results after chip reaction (taking BPIV3-P as an example).

[0159] 4.2.10.2 Optimization of hybridization time

[0160] The length of hybridization is an important factor affecting the hybridization effect of the chip. The hybridization time was set to 5min, 10min, 15min, and 20min in sequence, and the results were analyzed and compared to determine the optimal hybridization time (taking BPIV3-P as an example).

[0161] 4.2.11 Chip specificity test

[0162] The nucleic acid of BPIV3-XJA13, BRSV-USII / S1, IBRV-US7 and BVD V-No.12_E+ strains as well as common cattle viruses and bacteria such as BCoV, M-bov, Mh and Pm were extracted using a nucleic acid extraction kit. Sterile double-distilled water was used as the negative control. According to the gene chip detection method established in this experiment, gene chip detection was performed after quadruple RT-PCR amplification to evaluate the specificity of this method.

[0163] 4.2.12 Chip sensitivity test

[0164] 4.2.12.1 Single chip sensitivity test

[0165] Four recombinant plasmid standards, pUC57-BPIV3-P, pUC57-BRSV-N, pUC57-IBRV-gI and pUC57-BVDV-5'URT, were diluted 10-fold and tested according to the gene chip detection method established in this experiment to evaluate the single chip sensitivity of this method.

[0166] 4.2.12.2 Sensitivity test of quadruple gene chip

[0167] Four recombinant plasmid standards, pUC57-BPIV3-P, pUC57-BRSV-N, pUC57-IBRV-gI, and pUC57-BVDV-5'URT, were diluted to 1 ng / μL and then diluted 10-fold. Then, pUC57-BPIV3-P, pUC57-BRSV-N, pUC57-IBRV-gI, and pUC57-BVDV-5'URT were mixed in a ratio of 1:1:1:1 and detected according to the gene chip detection method established in this experiment to evaluate the sensitivity of the method.

[0168] 4.2.13 Chip Repeatability

[0169] Three different batches of gene chips (CH240401101, CH240718001, and CH240910001) were selected, and the 10-fold diluted recombinant plasmid standards pUC57-BPIV3-P, pUC57-BRSV-N, pUC57-IBRV-gI, and pUC57-BVDV-5'URT were detected according to the gene chip detection method established in this experiment to evaluate the repeatability of this method.

[0170] 4.2.14 Shelf life of chips

[0171] The same batch of gene chips stored at 4°C for 30d, 60d, 120d, 180d, 240d, 360d, and 400d were tested according to the above method, and the shelf life of the chips was evaluated based on the results.

[0172] 4.2.15 Comparison of compliance rate with standard test methods

[0173] After nucleic acid was extracted from 54 clinical samples, the amplified products were subjected to chip hybridization reaction according to the above conditions and the results were interpreted. At the same time, each clinical sample was tested for BVDV using the national standard method (GB / T 18637-2018), IBRV using the national standard method (GB / T 26875-2018), and BPIV3 and BRSV using the patented qPCR method (CN115044686A). The test results of the two were compared and the coincidence rate was calculated. Since the national standard method did not identify the four viruses, the samples positive for the national standard test needed to be sequenced. The sequencing results were compared with the national standard strain sequence using DNA star (version 7.2) sequence analysis software and the gene evolution tree was drawn for analysis.

[0174] 4.3 Results

[0175] 4.3.1 Construction of recombinant plasmid standards

[0176] Using viral RNA from BPIV3-XJA13, BRSV-USII / S1, and BVDV-No.12_E+, and DNA from IBRV-US7 as templates, RT-PCR amplification yielded a 117 bp BPIV3-P gene fragment, 112 bp and 73 bp BRSV-N gene fragments, 75 bp IBRV-gI gene fragment, and 147 bp BVDV-5'URT gene fragment, which were consistent with their expected sizes ( Figure 3 ). The recombinant plasmids pUC57-BPIV3-P, pUC57-BR SV-N, pUC57-IBRV-gI and pUC57-BVDV-5'URT were constructed and sequenced after PCR identification. The results showed that the gene sequences amplified were consistent with the corresponding sequences registered in NCBI, indicating that the five recombinant plasmids were correctly constructed and their concentrations were 1.86×10 10 Copies / μL, 1.65×10 10 Copies / μL, 2.03×10 10 copies / μL and 2.72×10 10 copies / μL.

[0177] 4.3.2 Optimization of PCR reaction conditions

[0178] According to the results of each optimization condition, the optimal annealing temperature is 56°C, the optimal annealing time is 20s, and the amount of the best primers used for BPIV3, BRSV, IBRV and BVDV is 0.6ul, 0.5ul, 0.5ul and 0.7ul for the upstream and downstream primers respectively, and the reaction system with the best target gene amplification conditions is determined (Table 5). The reaction conditions are 50°C 30min, 94°C 4min; (94°C 30s, 56°C 30s, 72°C 20s) 35 cycles, 72°C 10min, 4°C storage, if not used immediately, store at -20°C.

[0179] Table 5 Quadruple RT-PCR reaction system

[0180]

[0181]

[0182] 4.3.2.1 Annealing temperature optimization

[0183] According to the experimental results, the amplification effect is best when the annealing temperature is 56°C (Figure 4), so it is determined that the annealing temperature in the reaction system should be set to 56°C.

[0184] 4.3.2.2 Extension time optimization

[0185] According to the experimental results (Figure 5), when the extension time is 20s, the amplification effect can also be guaranteed. Therefore, it is determined that the extension time in the reaction system should be set to 20s, which shortens the time required for the reaction.

[0186] 4.3.2.3 Primer concentration optimization

[0187] According to the experimental results (Figure 6), the amplification effect was best when the primer amounts were 0.6, 0.5, 0.5 and 0.7 μL, respectively. Therefore, the primer amounts of BPIV3, IBRV, BVDV and BRSV in the reaction system were determined to be 0.6, 0.5, 0.5 and 0.7 μL, respectively.

[0188] 4.3.3 Optimization of gene chip hybridization reaction conditions

[0189] 4.3.3.1 Optimization of SA-HRP dilution

[0190] The results show that Figure 7 ), at a dilution of 1:500, the chip hybridization signal was scattered, at a dilution of 1:1000 and 1:2000, the signal was clear and stable, and at a dilution of 1:4000, 1:8000 and 1:16000, the hybridization signal was significantly attenuated. It can be concluded that a better experimental result can be obtained when the dilution factor of SA-HRP is 1:1000.

[0191] 4.3.3.2 Optimization of hybridization time

[0192] The results showed that the color development was successful when the hybridization time was 5 min ( Figure 8 ), so the optimal hybridization time was determined to be 5 min.

[0193] 4.3.3 Specificity of gene chips

[0194] After amplification according to the quadruple RT-PCR method established in this experiment, the specificity test was performed using the prepared gene chip. The results showed that all positive quality control points were blue, and the negative quality control points were not colored, and the test results were established. The designed BPIV3-P1 probe can specifically bind to and color the BPIV3 amplification product, the BRSV-P probe can specifically bind to and color the BRSV N amplification product, the IBRV-P probe can specifically bind to and color the IBRV gI amplification product, and the BVDV-P probe can specifically bind to and color the BVDV5'URT amplification product. There is no cross-reaction phenomenon. When the chip detects BCoV, M-bov, Mh and Pm, the detection probe points are not colored ( Fig. 9 ). This indicates that the chip has good specificity and achieves the purpose of detecting and diagnosing BPIV3, BRSV, IBRV, and BVDV1.

[0195] 4.3.4 Sensitivity of a single chip

[0196] The conventional PCR sensitivity results showed (Figure 10) that the sensitivity of single detection of recombinant plasmid standards pUC57-BPIV3-P, pUC57-BRSV-P, pUC57-IBRV-gI, and pUC57-BVDV-5'UTR could reach 1.86×10 4 Copies / μL, 1.65×10 3 Copies / μL, 2.03×10 4 copies / μL and 2.72×10 3 copies / μL; the sensitivity results of the chip show ( Fig.11 ), the sensitivity of single detection of recombinant plasmid standards pUC57-BPIV3-P, pUC57-BRSV-P, pUC57-IBRV-gI, and pUC57-BVDV-5'UTR can reach 1.86×10 0 Copies / μL, 1.65×10 0 Copies / μL, 2.03×10 0 copies / μL and 2.72×10 0 copies / μL; the detection sensitivity for mixed joint testing is 1.0×10 1 copies / μL, indicating that the method established in this study has high sensitivity.

[0197] 4.3.6 Chip Repeatability

[0198] According to the method established in this experiment, the repeatability test was conducted on gene chips of three different batches (CH240401101, CH240718001, and CH240910001). The results showed that for the same plasmid standard, the detection results of different batches of chips were the same, the repeatability of the chip was 100%, and the Kappa value was 1 (Table 6), indicating that the method had good repeatability.

[0199] Table 6 Chip repeatability

[0200]

[0201]

[0202] 4.3.7 Shelf life of chips

[0203] The probes of the same batch of gene chips stored at 4℃ for 30d, 60d, 120d, 180d, 240d, and 360d can successfully detect the target gene, but the response signal of the chip probe stored for 360d is weaker, which means that the chip can be stored for at least 360d. After 360d, the response ability of the probe will decrease. Fig.12 ).

[0204] 4.3.8 Comparison of the results with those of the standard test method

[0205] 54 known clinical samples were tested according to the above gene chip detection method. The chip detection method detected 10 samples infected with BPIV3 virus, 10 samples infected with BRSV virus, 10 samples infected with IBRV virus, 10 samples infected with BVDV virus, and 14 samples were negative; the national standard detection method bovine viral diarrhea-mucosal disease PCR detection method (GB / T18637-2018), bovine infectious rhinotracheitis virus (GB / T 27981-2011), bovine parainfluenza virus type 3 (CN115044686A), bovine respiratory syncytial virus (CN115044686A) combined with sequence evolution tree analysis, the results showed that 10 samples infected with BPIV3 virus, 10 samples infected with BRSV virus, 10 samples infected with IBRV virus, 10 samples infected with BVDV virus, and 14 samples were negative. The positive coincidence rate of the two detection methods was 100%, the negative coincidence rate was 100%, the overall coincidence rate was 100%, and the Kappa value was 1. It shows that the BPIV3, BRSV, IBRV and BVDV bovine respiratory disease quadruple gene chip differential diagnosis method established in this study can be used for the detection of clinical samples, and can simultaneously distinguish BPIV3, BRSV, IBRV and BVDV strains.

Claims

1. A gene chip detection kit for bovine respiratory diseases BPIV3, BRSV, IBRV and BVDV, characterized by: The following primers: When amplifying the sample to be tested, each 25 μL reaction system includes 10 7 1μL of quality control (10μM copies / μL), 0.6μL, 0.5μL, 0.5μL, 0.7μL, 0.4μL of each primer at 10μM, 12.5μL of 2×One step MIX, 1.25μL of One step Enzyme MIX, 2μL of template and the balance of water, at 50℃30min, 94℃4min; 35 cycles of (94℃30s, 56℃30s, 72℃20s), 72℃10min, store at 4℃, if not used immediately, store at -20℃ to obtain the PCR product.

2. A gene chip detection method for bovine respiratory diseases BPIV3, BRSV, IBRV and BVDV, characterized by: Take 100 μL of hybridization solution A and add it to the well of the well plate incubator chip preheated at 47°C, 200 r / min, and react for 10 minutes; take 20 μL of the PCR product prepared according to claim 1 and add it to the well of the well plate incubator chip preheated at 47°C, 200 r / min, react for 5 minutes, and then use the elution solution B preheated at 47°C to rinse 3 times, using 150 μL each time; dilute SA-HRP with hybridization solution A at 1000:1 and add 150 μL / well, 200 r / min, react at 47°C for 10 minutes, then rinse 3 times with hybridization solution A and elution solution C at room temperature, add TMB substrate color development solution (90 μL / well) for color development for 5 minutes; wash with pure water 3 times to terminate the reaction, and blow dry gently with a compressed air tank; observe the color development results; Hybridization solution A: Take 10 mL 20×SSC and 1 mL 10% SDS, add pure water to make up to 100 mL; The eluent B: take 3 mL 20×SSC and 1.2 mL 10% SDS, add pure water to make up to 120 mL; The eluent C: take 10 mL of 1 mol sodium citrate and add pure water to make up to 100 mL.

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