Liquid chip method for simultaneously detecting 11 pathogenic microorganisms or 10 drug-resistant genes in laying hens

Abstract

The invention relates to the technical field of biology. The invention provides a nucleic acid composition for simultaneously detecting 11 pathogenic microorganisms and 10 drug-resistant genes in laying hens. The nucleic acid composition comprises a specific upstream primer, a specific downstream primer and a probe of each pathogenic microorganism and drug-resistant gene. The invention also provides a detection method for simultaneously detecting 11 pathogenic microorganisms and 10 drug-resistant genes in laying hens. The liquid-phase chip detection method has the advantages of high efficiency, high sensitivity, good repeatability, wide detection range, low comprehensive detection cost and the like, 11 pathogenic microorganisms or 10 drug-resistant genes can be detected at the same time, and the detection efficiency and the detection range are greatly improved; the method has the advantages that pathogenic microorganisms and drug-resistant risk factors can be quickly identified, risk propagation points are locked, technical support is provided for prevention and control of microorganisms and drug-resistant risks of laying hens, and the quality safety of products and the diet safety of people are better guaranteed.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a liquid phase chip method for simultaneously detecting 11 pathogenic microorganisms or 10 drug resistance genes in laying hens. Background Art

[0002] Eggs are one of the important dietary foods for residents in China, and the quality and safety of eggs directly affect the dietary safety of consumers. With the continuous development of modern poultry industry, the circulation and consumption of eggs are becoming increasingly frequent, and the risks of disease occurrence and bacterial drug resistance transmission in laying hen flocks are continuously increasing, directly affecting the quality and safety of egg products and consumption safety. Monitoring the pathogenic microorganisms and bacterial drug resistance-related genes that need to be key controlled in the laying hen breeding process can quickly identify the pathogenic bacteria and drug resistance risks that affect the quality and safety of the produced egg products, and provide guidance and technical support for the prevention and control of related microorganisms and their drug resistance risks in the laying hen breeding process.

[0003] Liquid phase chip technology is a new type of biomolecule detection technology that integrates laser technology, flow cytometer, digital signal processing and traditional chemical technology. It is currently widely used in various immunoassays and nucleic acid detections. Liquid phase chip technology supports single and multiplex analyses, and can perform high-throughput detections on protein and nucleic acid targets in a variety of assay methods. It has the advantages of high throughput, simple operation, wide application range, good repeatability, high specificity, small sample volume required, more sensitive and stable, and low cost. It is gradually replacing traditional tools for detecting and quantifying pathogens, such as real-time fluorescence quantitative PCR amplification detection systems (qPCR), enzyme-linked immunosorbent assays (ELISA) and other detection methods. The breeding density of laying hens is relatively large, and the breeding cycle is long. During the breeding process, medication is inevitable. The occurrence of pathogenic bacteria and their drug resistance risks and their risk transmission among factors such as "animals - products - environment - humans" seriously endanger the healthy breeding and high-quality development of laying hens. The high-throughput, rapid and sensitive liquid phase chip detection technology for risk factors will help to quickly identify the pathogens and drug resistance risks in the laying hen breeding process, and is conducive to efficiently carrying out corresponding risk monitoring and assessment and early warning, providing a risk identification technology platform for the green and healthy breeding of laying hens.

[0004] By consulting relevant literature in the past five years, the pathogenic microorganisms with relatively high incidence rates in laying hens were known, including Pasteurella multocida (37%), Staphylococcus aureus (30%), Haemophilus paragallinarum (28.2%), Mycoplasma synoviae (20%), Avian pathogenic Escherichia coli (18%), Salmonella (9.53%). The above data vary due to regional and temporal differences, but all seriously endanger the production of laying hens. At the same time, referring to the actual clinical situation, the pathogenic microorganisms that pose a greater threat to the production and breeding of laying hens were known, including Clostridium perfringens, Campylobacter jejuni, Pseudomonas aeruginosa, Pullorum disease, and Mycoplasma gallisepticum. The dominant and harmful antibiotic drugs currently prevalent in laying hen flocks were also known, including β-lactams, aminoglycosides, tetracyclines, chloramphenicols, sulfonamides, macrolides, and colistin. If the above pathogenic microorganisms and the resistance genes of various drugs can be effectively monitored, the effective prevention and control of the risks of pathogenic microorganisms in laying hens can be achieved, and technical support can be provided for blocking the risk transmission of resistance genes in laying hens. Summary of the Invention

[0005] The purpose of the present invention is to provide a liquid-phase chip method for simultaneously detecting 11 pathogenic microorganisms or 10 resistance genes in laying hens. This detection method can simultaneously detect multiple pathogenic microorganisms or multiple resistance factors, and has the advantages of high efficiency, high sensitivity, good repeatability, wide detection range, and low comprehensive detection cost.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a nucleic acid composition for simultaneously detecting 11 pathogenic microorganisms or 10 resistance genes in laying hens. The 11 pathogenic microorganisms include Avian pathogenic Escherichia coli, Haemophilus paragallinarum, Clostridium perfringens, Pasteurella multocida, Salmonella, Pullorum disease, Staphylococcus aureus, Pseudomonas aeruginosa, Campylobacter jejuni, Mycoplasma synoviae, and Mycoplasma gallisepticum; the 10 resistance genes include sul1, ermB, blaSHV, blaCTX-M, aac(6’)-Ⅰb, aadA, tetA, tetM, catA1, and mcr-1; the nucleic acid composition includes specific upstream primers, specific downstream primers, and probe sequences.

[0008] Preferably, the specific upstream primer sequence of Avian pathogenic Escherichia coli is as shown in SEQ ID NO.1, the specific downstream primer sequence of Avian pathogenic Escherichia coli is as shown in SEQ ID NO.2, and the probe sequence of Avian pathogenic Escherichia coli is as shown in SEQ ID NO.3;

[0009] The specific upstream primer sequence of Haemophilus paragallinarum is shown as SEQ ID NO.4, the specific downstream primer sequence of Haemophilus paragallinarum is shown as SEQ ID NO.5, and the probe sequence of Haemophilus paragallinarum is shown as SEQ ID NO.6;

[0010] The specific upstream primer sequence of Clostridium perfringens is shown as SEQ ID NO.7, the specific downstream primer sequence of Clostridium perfringens is shown as SEQ ID NO.8, and the probe sequence of Clostridium perfringens is shown as SEQ ID NO.9;

[0011] The specific upstream primer sequence of Pasteurella multocida is shown as SEQ ID NO.10, the specific downstream primer sequence of Pasteurella multocida is shown as SEQ ID NO.11, and the probe sequence of Pasteurella multocida is shown as SEQ ID NO.12;

[0012] The specific upstream primer sequence of Salmonella is shown as SEQ ID NO.13, the specific downstream primer sequence of Salmonella is shown as SEQ ID NO.14, and the probe sequence of Salmonella is shown as SEQ ID NO.15;

[0013] The specific upstream primer sequence of Pullorum disease is shown as SEQ ID NO.16, the specific downstream primer sequence of Pullorum disease is shown as SEQ ID NO.17, and the probe sequence of Pullorum disease is shown as SEQ ID NO.18;

[0014] The specific upstream primer sequence of Staphylococcus aureus is shown as SEQ ID NO.19, the specific downstream primer sequence of Staphylococcus aureus is shown as SEQ ID NO.20, and the probe sequence of Staphylococcus aureus is shown as SEQ ID NO.21;

[0015] The specific upstream primer sequence of Pseudomonas aeruginosa is shown as SEQ ID NO.22, the specific downstream primer sequence of Pseudomonas aeruginosa is shown as SEQ ID NO.23, and the probe sequence of Pseudomonas aeruginosa is shown as SEQ ID NO.24;

[0016] The specific upstream primer sequence of Campylobacter jejuni is shown as SEQ ID NO.25, the specific downstream primer sequence of Campylobacter jejuni is shown as SEQ ID NO.26, and the probe sequence of Campylobacter jejuni is shown as SEQ ID NO.27;

[0017] The specific upstream primer sequence of Mycoplasma synoviae is shown as SEQ ID NO.28, the specific downstream primer sequence of Mycoplasma synoviae is shown as SEQ ID NO.29, and the probe sequence of Mycoplasma synoviae is shown as SEQ ID NO.30;

[0018] The specific upstream primer sequence of Mycoplasma gallisepticum is shown as SEQ ID NO.31, the specific downstream primer sequence of Mycoplasma gallisepticum is shown as SEQ ID NO.32, and the probe sequence of Mycoplasma gallisepticum is shown as SEQ ID NO.33;

[0019] The specific upstream primer sequence of sul1 is shown as SEQ ID NO.34, the specific downstream primer sequence of sul1 is shown as SEQ ID NO.35, and the probe sequence of sul1 is shown as SEQ ID NO.36;

[0020] The specific upstream primer sequence of ermB is shown as SEQ ID NO.37, the specific downstream primer sequence of ermB is shown as SEQ ID NO.38, and the probe sequence of ermB is shown as SEQ ID NO.39;

[0021] The specific upstream primer sequence of blaSHV is shown as SEQ ID NO.40, the specific downstream primer sequence of blaSHV is shown as SEQ ID NO.41, and the probe sequence of blaSHV is shown as SEQ ID NO.42;

[0022] The specific upstream primer sequence of blaCTX-M is shown as SEQ ID NO.43, the specific downstream primer sequence of blaCTX-M is shown as SEQ ID NO.44, and the probe sequence of blaCTX-M is shown as SEQ ID NO.45;

[0023] The specific upstream primer sequence of aac(6’)-Ⅰb is shown as SEQ ID NO.46, the specific downstream primer sequence of aac(6’)-Ⅰb is shown as SEQ ID NO.47, and the probe sequence of aac(6’)-Ⅰb is shown as SEQ ID NO.48;

[0024] The specific upstream primer sequence of aadA is shown as SEQ ID NO.49, the specific downstream primer sequence of aadA is shown as SEQ ID NO.50, and the probe sequence of aadA is shown as SEQ ID NO.51;

[0025] The specific upstream primer sequence of tetA is shown as SEQ ID NO.52, the specific downstream primer sequence of tetA is shown as SEQ ID NO.53, and the probe sequence of tetA is shown as SEQ ID NO.54;

[0026] The specific upstream primer sequence of tetM is shown as SEQ ID NO.55, the specific downstream primer sequence of tetM is shown as SEQ ID NO.56, and the probe sequence of tetM is shown as SEQ ID NO.57;

[0027] The specific upstream primer sequence of catA1 is shown as SEQ ID NO.58, the specific downstream primer sequence of catA1 is shown as SEQ ID NO.59, and the probe sequence of catA1 is shown as SEQ ID NO.60;

[0028] The specific upstream primer sequence of mcr-1 is shown as SEQ ID NO.61, the specific downstream primer sequence of mcr-1 is shown as SEQ ID NO.62, and the probe sequence of mcr-1 is shown as SEQ ID NO.63.

[0029] Preferably, biotin is linked to the 5'-end of the specific upstream primer, and NH2-C is linked to the 5'-end of the probe 12 .

[0030] The present invention also provides a kit for simultaneously detecting 11 pathogenic microorganisms or 10 drug resistance genes in laying hens, including the above nucleic acid composition and microspheres.

[0031] The present invention also provides a liquid phase chip method for simultaneously detecting 11 pathogenic microorganisms or 10 drug resistance genes in laying hens, comprising the following steps:

[0032] (1) Using the DNA sequence of the sample to be tested as a template, and performing multiplex PCR amplification using the specific upstream primer and the specific downstream primer to obtain a multiplex PCR product;

[0033] (2) Coupling the probe and the microspheres according to a volume ratio of (1-5):50 to obtain a working solution of the liquid phase chip;

[0034] (3) Mixing the multiplex PCR product and the working solution of the liquid phase chip according to a volume ratio of (3-6):33, hybridizing, then incubating with SAPE, detecting the MFI value and calculating the qualitative ratio result, and then making a result determination; the qualitative ratio result = MFI value of the sample to be tested / MFI value of the blank control; the judgment criterion is: when the MFI value ≥ 100 and the qualitative ratio result ≥ 3, the result is determined to be positive.

[0035] Preferably, taking the total system of the multiplex PCR amplification in step (1) as 25 μL, it includes 12.5 μL of multiplex PCR Mix, 0.14 - 0.15 μL of each specific upstream primer for each pathogenic microorganism or each drug resistance gene, 0.14 - 0.15 μL of each specific downstream primer for each pathogenic microorganism or each drug resistance gene, 2 μL of template DNA, and ddH2O is added to make up to 25 μL.

[0036] Preferably, the concentration of each specific upstream primer for each pathogenic microorganism is 9 - 11 μM, and the concentration of each specific downstream primer is 9 - 11 μM; except that the concentrations of the specific upstream primers for the drug resistance genes tetM and catA1 are 4 - 6 μM respectively, the concentrations of each specific upstream primer for the remaining drug resistance genes are 9 - 11 μM; except that the concentrations of the specific downstream primers for the drug resistance genes tetM and catA1 are 4 - 6 μM respectively, the concentrations of each specific downstream primer for the remaining drug resistance genes are 9 - 11 μM.

[0037] Preferably, the reaction conditions of the multiplex PCR amplification in step (1) are: pre - denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, for 35 cycles; finally, extension at 72°C for 10 min.

[0038] Preferably, the concentration of the probe in step (2) is 18 - 22 μM.

[0039] Preferably, the hybridization procedure in step (3) is: denaturation at 95°C for 5 min, hybridization incubation at 57°C for 15 min.

[0040] Preferably, the temperature for incubation with SAPE in step (3) is 57°C and the time is 5 min.

[0041] By adopting the above - mentioned technical solution, the present invention has the following beneficial effects:

[0042] The technical solution of the present invention uses the liquid - phase chip technology to establish a rapid, high - throughput multiplex pathogen and multiplex drug resistance factor detection method for 11 common bacterial diseases and 10 drug resistance genes in the process of laying hen breeding. It has the advantages of high efficiency, high sensitivity, good repeatability, wide detection range, and low comprehensive detection cost. It provides technical support for the risk screening of pathogenic microorganisms and their drug resistance and the prevention and control of risk transmission in the process of laying hen breeding, realizes the effective prevention and control of pathogenic microorganisms and drug resistance risk factors in laying hens, improves the biosecurity level in the process of laying hen breeding, promotes the green and healthy breeding and high - quality development of laying hens, and better ensures the quality safety of products and the food safety of people. Brief Description of the Drawings

[0043] Figure 1 It is the specific experiment result diagram of the liquid-phase chip for 11 pathogen-specific genes;

[0044] Figure 2 It is the specific experiment result diagram of the liquid-phase chip for 10 drug-resistant genes;

[0045] Figure 3 It is the sensitivity experiment result diagram of the liquid-phase chip for 11 pathogen-specific genes;

[0046] Figure 4 It is the sensitivity experiment result diagram of the liquid-phase chip for 10 drug-resistant genes;

[0047] Figure 5 It is the experiment result diagram of different hybridization temperatures of the liquid-phase chip ( Figure 5 in which A represents the experiment result of 11 pathogen-specific genes, and B represents the experiment result of 10 drug-resistant genes);

[0048] Figure 6 It is the optimized result diagram of the hybridization incubation system for Pullorum disease. Detailed implementation mode

[0049] The present invention provides a nucleic acid composition for simultaneously detecting 11 pathogenic microorganisms or 10 drug-resistant genes in laying hens. The 11 pathogenic microorganisms include pathogenic Escherichia coli, Haemophilus paragallinarum, Clostridium perfringens, Pasteurella multocida, Salmonella, Pullorum disease, Staphylococcus aureus, Pseudomonas aeruginosa, Campylobacter jejuni, Mycoplasma synoviae, and Mycoplasma gallisepticum; the 10 drug-resistant genes include sul1, ermB, blaSHV, blaCTX-M, aac(6’)-Ⅰb, aadA, tetA, tetM, catA1, and mcr-1; the nucleic acid composition includes specific upstream primers, specific downstream primers, and probe sequences.

[0050] In the present invention, the specific sequence information of the nucleic acid composition for the 11 pathogenic microorganisms and 10 drug-resistant genes is shown in Table 1.

[0051] Table 1 Sequence information of primers and probes

[0052]

[0053]

[0054]

[0055]

[0056] In the present invention, the 5’ end of the specific upstream primer is linked with biotin, and the 5’ end of the probe is linked with NH2-C 12 .

[0057] The present invention also provides a kit for simultaneously detecting 11 pathogenic microorganisms or 10 drug resistance genes in laying hens, comprising the above nucleic acid composition and microspheres.

[0058] The present invention also provides a liquid chip method for simultaneously detecting 11 pathogenic microorganisms or 10 drug resistance genes in laying hens, comprising the following steps:

[0059] (1) Using the DNA sequence of the sample to be tested as a template, and performing multiplex PCR amplification with the specific upstream primer and the specific downstream primer described above to obtain a multiplex PCR product;

[0060] (2) Coupling the probe and the microspheres according to a volume ratio of (1 - 5):50 to obtain a working solution of the liquid chip;

[0061] (3) Mixing and hybridizing the multiplex PCR product and the working solution of the liquid chip according to a volume ratio of (3 - 6):33, then incubating with SAPE, detecting the MFI value and calculating the qualitative ratio result, and then making a result determination.

[0062] In the present invention, the total system of the multiplex PCR amplification described in step (1) is calculated as 25 μL, including 12.5 μL of multiplex PCR Mix, 0.14 - 0.15 μL of each specific upstream primer for each pathogenic microorganism or each drug resistance gene, 0.14 - 0.15 μL of each specific downstream primer for each pathogenic microorganism or each drug resistance gene, 2 μL of template DNA, and adding ddH2O to make up to 25 μL. In the present invention, when detecting pathogenic microorganisms, the dosage of each specific upstream primer for each pathogenic microorganism is 0.14 μL, and the dosage of each specific downstream primer is 0.14 μL; when detecting drug resistance genes, the dosage of each specific upstream primer for each drug resistance gene is 0.15 μL, and the dosage of each specific downstream primer is 0.15 μL.

[0063] In the present invention, the concentration of each specific upstream primer for each pathogenic microorganism is preferably 9 - 11 μM, and more preferably 10 μM; the concentration of each specific downstream primer for each pathogenic microorganism is preferably 9 - 11 μM, and more preferably 10 μM.

[0064] In the present invention, the concentrations of the specific upstream primers for the drug-resistant genes tetM and catA1 are preferably 4 - 6 μM respectively, and more preferably 5 μM; except for the drug-resistant genes tetM and catA1, the concentrations of the specific upstream primers for each of the remaining drug-resistant genes are preferably 9 - 11 μM, and more preferably 10 μM. The concentrations of the specific downstream primers for the drug-resistant genes tetM and catA1 in the present invention are preferably 4 - 6 μM respectively, and more preferably 5 μM; except for the drug-resistant genes tetM and catA1, the concentrations of the specific downstream primers for each of the remaining drug-resistant genes are preferably 9 - 11 μM, and more preferably 10 μM.

[0065] In the present invention, the reaction conditions for the multiplex PCR amplification in step (1) are: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, for 35 cycles; and finally extension at 72°C for 10 min.

[0066] In the present invention, the volume ratio of the coupling of the probe with the microspheres is preferably (1 - 5):50, more preferably (2 - 4):50, and even more preferably 3:50; the concentration of the probe is preferably 18 - 22 μM, more preferably 19 - 21 μM, and even more preferably 20 μM.

[0067] In the present invention, the volume ratio of the mixing of the multiplex PCR product with the liquid chip working solution is preferably (3 - 6):33, more preferably (4 - 5.5):33, and even more preferably 5:33. The hybridization procedure after mixing in the present invention is preferably: the hybridization procedure is: denaturation at 95°C for 5 min, hybridization incubation at 57°C for 15 min.

[0068] In the present invention, the temperature for incubation with SAPE is preferably 57°C, and the time is preferably 5 min.

[0069] In the present invention, the MFI value is the median fluorescence intensity. In the present invention, the qualitative ratio result = MFI value of the test sample / MFI value of the blank control. The qualitative ratio result can be expressed as LQRR, the MFI value of the test sample can be expressed as MFIS, and the MFI value of the blank control can be expressed as MFIB.

[0070] In the present invention, the judgment criterion is: when the MFI value ≥ 100 and the qualitative ratio result ≥ 3, the result is determined to be positive; otherwise, the sample is determined to be negative for the detection of this gene.

[0071] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0072] Example 1

[0073] (I) Strains and drug resistance genes

[0074] Pathogenic Escherichia coli ATCC25922, Clostridium perfringens ATCC13124, Salmonella ATCC14028, Staphylococcus aureus ATCC29213, Pseudomonas aeruginosa ATCC27853, Campylobacter jejuni ATCC33560. The above standard strains were all provided by the China Animal Health and Epidemiology Center. Pullorum disease, Haemophilus paragallinarum, Pasteurella multocida, Mycoplasma synoviae, Mycoplasma gallisepticum are all clinical isolates preserved by the China Animal Health and Epidemiology Center.

[0075] By searching the literature in recent years, 10 drug resistance genes with relatively high drug resistance levels in laying hens were determined, namely: β-lactam drug resistance genes blaSHV, blaCTX-M; tetracycline tetA, tetM; aminoglycoside aac(6’)-Ib, aadA; macrolide ermB; sulfonamide sul1; amide alcohol catA1; polypeptide mcr-1. Among them, the plasmids of tetA and ermB drug resistance genes were synthesized by Beijing Tsingke Biotechnology Co., Ltd., and the remaining 8 drug resistance genes were provided by the China Animal Health and Epidemiology Center.

[0076] (II) Design of primers and probes

[0077] Retrieve and download the sequences of pathogen-specific genes and drug resistance genes in about 30 different species of strains from GenBank. Analyze through bioinformatics software such as Laser gene, and select the most conserved segment as the region with relatively high homology as the target sequence for designing multiplex PCR primers. The corresponding NCBI sequence numbers are shown in Table 1.

[0078] Using the PrimerPlex primer design software, according to the primer and design principles, set appropriate parameters to ensure that the melting temperature of the primer pair is between 51 - 60°C and the size of the PCR amplification product is between 100 - 200bp, and determine the optimal 2 sets of 21 pairs of primer and probe sequences. Considering the polymorphism of the target sequence, degenerate bases are designed for the probes with relatively low specificity for pathogenic Escherichia coli, effectively avoiding non-specific amplification and increasing the detection specificity and accuracy. The specific sequence information is shown in Table 1.

[0079] (III) Construction of positive plasmids

[0080] The target genes of 11 pathogenic microorganisms and 10 drug resistance genes were amplified respectively with specific primers by PCR method, and the reaction conditions were as follows: pre-denaturation at 95°C for 10 min; 35 cycles of amplification (denaturation at 95°C for 30 s; annealing at 60°C for 30 s; extension at 72°C for 30 s); and finally extension at 72°C for 10 min. The PCR reaction system was: 12.5 μL of PCR Mix, 1 μL of specific upstream primer (specific upstream primer concentration was 10 μM), 1 μL of specific downstream primer (specific upstream primer concentration was 10 μM), 2 μL of template DNA, and ddH2O was added to make up to 25 μL.

[0081] The PCR amplification products were identified by 2% agarose gel electrophoresis. The products were rapidly purified and recovered according to the instructions of the gel recovery kit (purchased from TaKaRa Biotechnology Co., Ltd.). Then the target fragment was ligated with pMD18-T vector (purchased from TaKaRa Biotechnology Co., Ltd.), and then transformed into DH5α chemically competent cells. After culturing at 37°C for 1 hour, the bacterial solution was spread on LB plate medium containing sodium ampicillin and cultured overnight. Some positive clones were selected and the size of the inserted fragment in the vector was confirmed by PCR method. Some were inoculated into LB liquid medium containing Amp, and an appropriate amount of positive recombinant plasmid bacterial solution with the size of the target gene fragment was sent to Beijing Tsingke Biotechnology Co., Ltd. for sequencing. Except for the tetA and ermB drug resistance genes, the plasmids of which were synthesized by Beijing Tsingke Biotechnology Co., Ltd., the plasmid construction of the other 19 genes was completed according to the above process.

[0082] (IV) Synthesis and modification of primers and probes for liquid chip

[0083] According to the information provided in the Luminex cookbook, magnetic microspheres suitable for the Luminex 200 TM liquid chip detector were selected. The specific primers and probes for 11 pathogenic microorganisms and 10 drug resistance genes were modified as follows: biotin was linked to the 5' of the specific upstream primer, and NH2-C 12 was linked to the 5' of the probe. The modified primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0084] (V) Liquid chip detection

[0085] 1. Multiplex PCR reaction

[0086] The DNA of the positive strain was crudely extracted using the water bath method and used as an amplification template. The corresponding primers and the positive strain were used to amplify its target gene. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 10 min; 35 cycles of amplification (denaturation at 95°C for 30 s; annealing at 60°C for 30 s; extension at 72°C for 30 s); and finally extension at 72°C for 10 min. The reaction system was: 12.5 μL of PCR Mix, 0.14 μL of the specific upstream primer for each pathogenic microorganism or 0.15 μL of the specific upstream primer for each drug resistance gene (except that the concentrations of tetM and catA1 were 5 μM, and the concentrations of the remaining primers were 10 μM), 0.14 μL of the specific downstream primer for each pathogenic microorganism or 0.15 μL of the specific downstream primer for each pathogenic microorganism (except that the concentrations of tetM and catA1 were 5 μM, and the concentrations of the remaining primers were 10 μM), 2 μL of template DNA, and ddH2O was added to make up to 25 μL.

[0087] In the present invention, except that the concentrations of the specific upstream primers for the drug resistance genes tetM and catA1 were 5 μM respectively, the concentrations of the specific upstream primers for each of the remaining drug resistance genes were 10 μM; except that the concentrations of the specific downstream primers for the drug resistance genes tetM and catA1 were 5 μM respectively, the concentrations of the specific downstream primers for each of the remaining drug resistance genes were 10 μM.

[0088] 2. Probe and microsphere coupling

[0089] After thoroughly vortexing the microsphere stock solution with a vortex mixer and sonicating for 10 - 20 s, 50 μL of the microspheres were taken into a low-binding centrifuge tube. The centrifuge tube was placed on a magnetic stand for 2 min, and the supernatant was aspirated. 50 μL of 0.1 M MES (pH 4.5) buffer was added to resuspend the microspheres, vortexed and mixed evenly, and the supernatant was aspirated. 7 μL of 0.1 M MES (pH 4.5) buffer was added, vortexed and mixed evenly. 3 μL of the diluted 5'-end amino-modified oligonucleotide probe (probe concentration was 20 μM) was added, vortexed and mixed evenly. 2.5 μL of freshly prepared EDC solution (10 mg / mL) was added to the microspheres and immediately oscillated. Hybridization was carried out at room temperature in the dark for 30 min, and mixed evenly every 10 minutes. Another 2.5 μL of freshly prepared EDC solution (10 mg / mL) was added to the microspheres and immediately oscillated. Hybridization was carried out at room temperature in the dark for 30 min, and mixed evenly every 10 minutes. 1 mL of 0.02% Tween-20 solution was added, vortexed and mixed evenly, placed on a magnetic stand for 2 min, and the supernatant was aspirated. 1 mL of 0.1% SDS solution was added, vortexed and mixed evenly, placed on a magnetic stand for 2 min, and the supernatant was aspirated. 100 μL of TE (pH 8.0) solution was added, vortexed and mixed evenly. It was stored for standby at 4°C in the dark.

[0090] 3. Hybridization incubation

[0091] Adjust the microsphere concentration to 75 microspheres / μL with 1.5×TMAC hybridization buffer. Take the mixed microsphere working solution (33 μL) and mix it with the PCR product (5 μL), and make up to a total volume of 50 μL with TE (pH = 8) buffer. After mixing well by oscillation, put it into a PCR instrument. For 11 pathogenic microorganisms and 10 drug resistance genes, set it at 95 °C for denaturation for 5 min and hybridize and incubate at 57 °C for 15 min.

[0092] Dilute SAPE to a working concentration of 4 μg / mL with 1×TMAC hybridization buffer. After the first incubation ends, add 25 μL of SAPE reporting solution (when detecting 11 pathogenic microorganisms, place the eight-well row on a magnetic plate and let it stand for 2 min, aspirate the supernatant, and add 75 μL of SAPE containing a solution with a final concentration of 0.1% BSA), and hybridize and incubate at 57 °C for 5 min.

[0093] 4. Machine analysis

[0094] Turn on the liquid phase chip detection device, preheat the copper plate to the incubation temperature in advance, set the program, and perform machine detection. Judge the results according to the MFI value. Judgment criterion: The qualitative ratio result (LQRR) is equal to the median fluorescence intensity MFI (MFIS) of the sample divided by the median fluorescence intensity MFI (MFIB) of the blank control, that is, LQRR = MFIS / MFIB. When the MFI value of a specific coding microsphere associated with a certain gene in the liquid phase chip detector is ≥100 and LQRR ≥ 3, the sample is judged to be positive for the nucleic acid detection of a certain gene; otherwise, the sample is judged to be negative for the detection of this gene.

[0095] Example 2

[0096] (1) Liquid phase chip specificity experiment

[0097] Use the water bath method to roughly extract the DNA of the positive strain and use it as the amplification template. Use the corresponding primers and the positive strain to amplify its target gene, and perform a multiplex PCR reaction. The specific operation is the same as that in Example 1.

[0098] After the PCR amplification of 11 pathogenic microorganisms and 10 drug resistance genes in two systems, gel electrophoresis imaging was performed respectively. After confirmation, the multiplex PCR products (5 μL) were mixed with the microsphere reaction solution (45 μL, including 33 μL microsphere working solution and 12 μL TE buffer), and then shaken and mixed evenly. After mixing, it was placed in a PCR instrument. For the detection of 11 pathogens and 10 drug resistance genes, denaturation was set at 95 °C for 5 min, and hybridization incubation was carried out at 57 °C for 15 min to hybridize the PCR products and microspheres. After the hybridization was completed, 25 μL of SAPE reporting solution (4 μg / mL) was added. When detecting 11 pathogens, the eight-well row was placed on the magnetic plate and left standing for 2 min, the supernatant was aspirated, and 75 μL of SAPE containing BSA solution with a final concentration of 0.1% was added. Hybridization incubation was carried out at 57 °C for 5 min for hybridization, and then detected by machine, and the results were judged according to the MFI value.

[0099] Table 2 Specificity experimental results of liquid-phase chip for specific genes of 11 pathogenic microorganisms

[0100]

[0101] Table 3 Specificity experimental results of liquid-phase chip for 10 drug resistance genes

[0102]

[0103] The results are shown in Table 2, Table 3 and Figure 1 、 Figure 2 as shown, indicating that the detection results of the target genes by this method are all positive, while the detection values of the amplification products of non-target bacteria are all negative, indicating that the established method has good specificity.

[0104] (2) Liquid-phase chip sensitivity experiment

[0105] The DNA of positive strains was extracted roughly by the water bath method and used as the amplification template. The nucleic acid concentration was measured, and the nucleic acid copy number was calculated. The nucleic acid was diluted ten-fold from 10 7 copies to 10 0Copy and dilute to 8 nucleic acid concentration gradients in total. Perform PCR amplification experiments on the above 21 kinds of positive plasmids with different concentrations. The specific process is the same as that in Example 1. Mix the PCR product (5 μL) with a single kind of microsphere reaction solution (45 μL), then mix well by oscillation. After mixing, put it into a PCR instrument. For the detection of 11 pathogens and 10 drug resistance genes, set the denaturation at 95 °C for 5 min and the hybridization incubation at 57 °C for 15 min. Hybridize the PCR product with the microspheres. After the hybridization ends, add 25 μL of SAPE reporting solution (4 μg / mL. After the first hybridization of Salmonella pullorum pathogen, place the eight-well row on a magnetic plate and let it stand for 2 min. Aspirate and discard the supernatant, and add 75 μL of SAPE containing BSA solution with a final concentration of 0.1%). Incubate at 57 °C for 5 min for hybridization, then detect on the machine, and judge the results according to the MFI value.

[0106] Table 4 Results of the sensitivity experiment of the liquid-phase chip for 11 pathogen-specific genes

[0107]

[0108] Table 5 Results of the sensitivity experiment of the liquid-phase chip for 10 drug resistance genes

[0109]

[0110] The results are shown in Table 4, Table 5 and Figure 3 、 Figure 4 It shows that the sensitivity detected for Pseudomonas aeruginosa is 1 copy / reaction, and the sensitivities for pathogenic Escherichia coli, Mycoplasma gallisepticum, and ermB are 10 2 copies / reaction, the sensitivities for Pasteurella multocida, Mycoplasma synoviae, Salmonella, sul1, and aadA are 10 3 copies / reaction, the sensitivities for Clostridium perfringens, Haemophilus paragallinarum, Staphylococcus aureus, Salmonella pullorum, tetM, catA1, and aac(6’)-Ib are 10 4 copies / reaction, and the sensitivities for Campylobacter jejuni, blaCTX-M, tetA, blaSHV, and mcr-1 are 10 5 copies / reaction.

[0111] (III) Control experiment between liquid-phase chip and fluorescence quantitative PCR

[0112] Collect 20 actual samples from the cloacas of laying hens, extract nucleic acids using a genomic DNA extraction kit, and perform liquid-phase chip and fluorescence quantitative PCR experiments, where the fluorescence quantitative PCR experiment is used as the gold standard.

[0113] The PCR reaction system is as follows: 12.5 μL of PCR Mix, 0.14 μL of the specific upstream primer for each pathogenic microorganism or 0.15 μL of the specific upstream primer for each drug resistance gene (except that the concentrations of tetM and catA1 are 5 μM, and the concentrations of the remaining primers are 10 μM), 0.14 μL of the specific downstream primer for each pathogenic microorganism or 0.15 μL of the specific downstream primer for each pathogenic microorganism (except that the concentrations of tetM and catA1 are 5 μM, and the concentrations of the remaining primers are 10 μM), 2 μL of template DNA, and ddH2O is added to make up to 25 μL. Other steps are the same as in Example 1.

[0114] The reaction conditions for fluorescence quantitative PCR are as follows: pre-denaturation at 95 °C for 30 s; 40 cycles (denaturation at 95 °C for 5 s; annealing at 56 °C for 30 s). The fluorescence quantitative PCR reaction system is as follows: 5 μL of PCR Mix, 0.4 μL of each upstream primer (the primer concentration is 10 μM), 0.4 μL of each downstream primer (the primer concentration is 10 μM), 0.4 μL of each probe, 0.5 μL of each template DNA, and ddH2O is added to make up to 20 μL. The core criterion for positive determination of fluorescence quantitative PCR is that the CT value ≤ 35.

[0115] Table 6 Results of the control experiment between liquid-phase chip and fluorescence quantitative PCR

[0116]

[0117] By consulting the literature, conserved genes unique to pathogenic microorganisms that are widely used were selected. The 11 genes recorded in Table 6 correspond to 11 pathogenic microorganisms respectively.

[0118] As can be seen from Table 6, the coincidence rate between liquid-phase chip detection and fluorescence quantitative PCR detection is 100% for 15 genes, and the lowest coincidence rate between liquid-phase chip detection and fluorescence quantitative PCR detection is 53.8%. That is, it shows that the liquid-phase chip detection method has a high consistency with the fluorescence quantitative PCR detection method during the multiplex detection process.

[0119] (IV) Selection of the hybridization temperature of the liquid-phase chip

[0120] Perform multiplex PCR reaction and probe and microsphere coupling according to the liquid-phase chip detection part in Example 1. Then adjust the microsphere concentration to 75 microspheres / μL with 1.5×TMAC hybridization buffer. Take the mixed microsphere working solution (33 μL) and mix it with the PCR product (5 μL), and make up to a total volume of 50 μL with TE (pH = 8) buffer. After shaking and mixing evenly, put it into a PCR instrument. For 11 pathogenic microorganisms and 10 drug resistance genes, set denaturation at 95°C for 5 min, and the hybridization temperatures are 42°C, 47°C, 52°C, and 57°C respectively, and incubate for 15 min to hybridize the product and microspheres. After hybridization, add 25 μL of SAPE reporting solution (4 μg / mL. When detecting 11 pathogenic microorganisms, place the eight-well row on a magnetic plate and let it stand for 2 min, aspirate the supernatant, and add 75 μL of SAPE containing a BSA solution with a final concentration of 0.1%). After shaking and mixing evenly, put it into a PCR instrument, and hybridize at the corresponding hybridization temperature for 5 min, then perform on-machine detection, and judge the results according to the MFI value.

[0121] Table 7 Experimental results of different liquid-phase chip hybridization temperatures

[0122]

[0123] As can be seen from Table 7, when the hybridization temperature is 57°C, the detection signal values (MFI) of 7 pathogenic microorganisms and 6 drug resistance genes are the highest for 11 pathogen-specific genes and 10 drug resistance genes respectively. And the most suitable hybridization temperature should keep the values of the whole system in a relatively balanced state, which is beneficial to increasing the values of lower positives. Therefore, 57°C is finally selected as the liquid-phase chip on-machine hybridization temperature.

[0124] (V) Optimization of the liquid-phase chip system

[0125] The MFI value of the specific gene of Pullorum disease is too high in the sensitivity detection, so the secondary hybridization incubation system is optimized, and a total of 2 systems are made.

[0126] ① Secondary hybridization system: After the first hybridization incubation is completed, place the eight-well row on a magnetic plate and let it stand for 2 min, aspirate the supernatant, add 75 μL of SAPE reporting solution (in which a BSA solution with a final concentration of 0.1% is added), shake and mix evenly, and hybridize at 57°C for 5 min to make it hybridize, then perform on-machine detection.

[0127] ② Secondary hybridization system: After the first hybridization incubation is completed, add 25 μL of SAPE reporting solution (4 μg / mL), shake and mix evenly, and hybridize at 57°C for 5 min to make it hybridize, then perform on-machine detection, and judge the results according to the MFI value. Other steps are the same as in Example 1.

[0128] Table 8 Optimization results of the hybridization incubation system for Pullorum disease

[0129]

[0130] The results are shown in Table 8 and Figure 6 as shown, it is advisable to use the first hybrid incubation reaction system optimization scheme.

[0131] In summary, it can be seen that the technical solution of the present invention has established a rapid, high-throughput multiplex pathogen and multiplex drug resistance factor detection method for 11 common bacterial diseases and 10 drug resistance genes in the process of laying hen breeding by using liquid phase chip technology. It has the advantages of high efficiency, high sensitivity, good repeatability, wide detection range, and low comprehensive detection cost, providing technical support for the risk screening of pathogenic microorganisms and their drug resistance and the prevention and control of risk transmission in the process of laying hen breeding, and realizing the effective prevention and control of pathogenic microorganisms and drug resistance risk factors in laying hens.

[0132] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A nucleic acid composition for simultaneously detecting 11 pathogenic microorganisms or 10 drug resistance genes in laying hens, characterized in that, The 11 pathogenic microorganisms include pathogenic Escherichia coli, Haemophilus paragallinarum, Clostridium perfringens, Pasteurella multocida, Salmonella, Pullorum disease, Staphylococcus aureus, Pseudomonas aeruginosa, Campylobacter jejuni, Mycoplasma synoviae, and Mycoplasma gallisepticum; the 10 resistance genes include sul1, ermB, blaSHV, blaCTX-M, aac(6’)-Ib, aadA, tetA, tetM, catA1, and mcr-1; the nucleic acid composition includes a specific upstream primer, a specific downstream primer, and a probe sequence; The specific upstream primer sequence of the pathogenic Escherichia coli is shown in SEQ ID NO.1, the specific downstream primer sequence of the pathogenic Escherichia coli is shown in SEQ ID NO.2, and the probe sequence of the pathogenic Escherichia coli is shown in SEQ ID NO.3; The specific upstream primer sequence of the Haemophilus paragallinarum is shown in SEQ ID NO.4, the specific downstream primer sequence of the Haemophilus paragallinarum is shown in SEQ ID NO.5, and the probe sequence of the Haemophilus paragallinarum is shown in SEQ ID NO.6; The specific upstream primer sequence of the Clostridium perfringens is shown in SEQ ID NO.7, the specific downstream primer sequence of the Clostridium perfringens is shown in SEQ ID NO.8, and the probe sequence of the Clostridium perfringens is shown in SEQ ID NO.9; The specific upstream primer sequence of the Pasteurella multocida is shown in SEQ ID NO.10, the specific downstream primer sequence of the Pasteurella multocida is shown in SEQ ID NO.11, and the probe sequence of the Pasteurella multocida is shown in SEQ ID NO.12; The specific upstream primer sequence of the Salmonella is shown in SEQ ID NO.13, the specific downstream primer sequence of the Salmonella is shown in SEQ ID NO.14, and the probe sequence of the Salmonella is shown in SEQ ID NO.15; The specific upstream primer sequence of the Pullorum disease is shown in SEQ ID NO.16, the specific downstream primer sequence of the Pullorum disease is shown in SEQ ID NO.17, and the probe sequence of the Pullorum disease is shown in SEQ ID NO.18; The specific upstream primer sequence of the Staphylococcus aureus is shown in SEQ ID NO.19, the specific downstream primer sequence of the Staphylococcus aureus is shown in SEQ ID NO.20, and the probe sequence of the Staphylococcus aureus is shown in SEQ ID NO.21; The specific upstream primer sequence of the Pseudomonas aeruginosa is shown in SEQ ID NO.22, the specific downstream primer sequence of the Pseudomonas aeruginosa is shown in SEQ ID NO.23, and the probe sequence of the Pseudomonas aeruginosa is shown in SEQ ID NO.24; The specific upstream primer sequence of Campylobacter jejuni is shown as SEQ ID NO.25, the specific downstream primer sequence of Campylobacter jejuni is shown as SEQ ID NO.26, and the probe sequence of Campylobacter jejuni is shown as SEQ ID NO.27; The specific upstream primer sequence of Mycoplasma synoviae is shown as SEQ ID NO.28, the specific downstream primer sequence of Mycoplasma synoviae is shown as SEQ ID NO.29, and the probe sequence of Mycoplasma synoviae is shown as SEQ ID NO.30; The specific upstream primer sequence of Mycoplasma gallisepticum is shown as SEQ ID NO.31, the specific downstream primer sequence of Mycoplasma gallisepticum is shown as SEQ ID NO.32, and the probe sequence of Mycoplasma gallisepticum is shown as SEQ ID NO.33; The specific upstream primer sequence of sul1 is shown as SEQ ID NO.34, the specific downstream primer sequence of sul1 is shown as SEQ ID NO.35, and the probe sequence of sul1 is shown as SEQ ID NO.36; The specific upstream primer sequence of ermB is shown as SEQ ID NO.37, the specific downstream primer sequence of ermB is shown as SEQ ID NO.38, and the probe sequence of ermB is shown as SEQ ID NO.39; The specific upstream primer sequence of blaSHV is shown as SEQ ID NO.40, the specific downstream primer sequence of blaSHV is shown as SEQ ID NO.41, and the probe sequence of blaSHV is shown as SEQ ID NO.42; The specific upstream primer sequence of blaCTX-M is shown as SEQ ID NO.43, the specific downstream primer sequence of blaCTX-M is shown as SEQ ID NO.44, and the probe sequence of blaCTX-M is shown as SEQ ID NO.45; The specific upstream primer sequence of aac(6’)-Ⅰb is shown as SEQ ID NO.46, the specific downstream primer sequence of aac(6’)-Ⅰb is shown as SEQ ID NO.47, and the probe sequence of aac(6’)-Ⅰb is shown as SEQ ID NO.48; The specific upstream primer sequence of aadA is shown as SEQ ID NO.49, the specific downstream primer sequence of aadA is shown as SEQ ID NO.50, and the probe sequence of aadA is shown as SEQ ID NO.51; The specific upstream primer sequence of tetA is shown as SEQ ID NO.52, the specific downstream primer sequence of tetA is shown as SEQ ID NO.53, and the probe sequence of tetA is shown as SEQ ID NO.54; The specific upstream primer sequence of tetM is shown as SEQ ID NO.55, the specific downstream primer sequence of tetM is shown as SEQ ID NO.56, and the probe sequence of tetM is shown as SEQ ID NO.57; The specific upstream primer sequence of catA1 is shown as SEQ ID NO.58, the specific downstream primer sequence of catA1 is shown as SEQ ID NO.59, and the probe sequence of catA1 is shown as SEQ ID NO.60; The specific upstream primer sequence of mcr-1 is shown as SEQ ID NO.61, the specific downstream primer sequence of mcr-1 is shown as SEQ ID NO.62, and the probe sequence of mcr-1 is shown as SEQ ID NO.

63.

2. The nucleic acid composition according to claim 1, wherein The 5'-end of the specific upstream primer is linked with biotin, and the 5'-end of the probe is linked with NH2-C 12 .

3. A kit for simultaneously detecting 11 pathogenic microorganisms or 10 drug resistance genes in laying hens, characterized in that, Comprising the nucleic acid composition and microspheres according to claim 1 or 2.

4. A liquid-phase chip method for simultaneously detecting 11 pathogenic microorganisms or 10 drug-resistant genes in laying hens, characterized in that, Comprising the following steps: (1) Using the DNA sequence of the sample to be tested as a template, and performing multiplex PCR amplification with the specific upstream primer and specific downstream primer according to claim 1 or 2 to obtain a multiplex PCR product; (2) Coupling the probe according to claim 1 or 2 with the microspheres at a volume ratio of (1-5):50 to obtain a liquid chip working solution; (3) Mixing and hybridizing the multiplex PCR product with the liquid chip working solution at a volume ratio of (3-6):33, then incubating with SAPE, detecting the MFI value and calculating the qualitative ratio result, and then making a result determination; The qualitative ratio result = MFI value of the sample to be tested / MFI value of the blank control; The judgment criterion is: when the MFI value ≥ 100 and the qualitative ratio result ≥ 3, the result is determined to be positive.

5. The liquid-phase chip method according to claim 4, wherein The total system of the multiplex PCR amplification in step (1) is calculated as 25 μL, including 12.5 μL of multiplex PCR Mix, 0.14-0.15 μL of the specific upstream primer for each pathogenic microorganism or each drug resistance gene, 0.14-0.15 μL of the specific downstream primer for each pathogenic microorganism or each drug resistance gene, 2 μL of template DNA, and adding ddH2O to make up to 25 μL.

6. The liquid-phase chip method according to claim 5, wherein The concentration of the specific upstream primer for each pathogenic microorganism is 9-11 μM, and the concentration of the specific downstream primer is 9-11 μM; Except that the concentrations of the specific upstream primers of the drug resistance genes tetM and catA1 are 4-6 μM respectively, the concentrations of the specific upstream primers of the remaining drug resistance genes are all 9-11 μM; Except that the concentrations of the specific downstream primers of the drug resistance genes tetM and catA1 are 4-6 μM respectively, the concentrations of the specific downstream primers of the remaining drug resistance genes are all 9-11 μM.

7. The liquid-phase chip method according to claim 4, wherein The reaction conditions of the multiplex PCR amplification in step (1) are: pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 30 s, for 35 cycles; and finally extension at 72 °C for 10 min.

8. The liquid-phase chip method according to claim 4, wherein The concentration of the probe in step (2) is 18-22 μM.

9. The liquid-phase chip method according to claim 4, wherein The procedure of the hybridization described in step (3) is as follows: denaturation at 95°C for 5 min and hybridization incubation at 57°C for 15 min.

10. The liquid phase chip method according to claim 4, characterized in that, The temperature for incubation with SAPE in step (3) is 57°C and the time is 5 min.

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