Liquid phase chip method capable of simultaneously detecting 16 pathogenic pathogens in pork

Abstract

The invention relates to the technical field of biology, in particular to a liquid chip method capable of simultaneously detecting 16 pathogenic pathogens in pork. The invention provides a nucleic acid composition for simultaneously and rapidly detecting 16 pathogenic pathogens in pork. The nucleic acid composition comprises 16 primer pairs and corresponding probe sequences. The method provided by the invention can quickly lock infected pathogens when pork is polluted and causes are unknown, and has the advantages of rapidness, high sensitivity, wide detection range, good repeatability, low comprehensive detection cost, convenience in operation and the like.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a liquid chip method capable of simultaneously detecting 16 pathogenic pathogens in pork. Background Art

[0002] There are many types of zoonoses, among which foodborne zoonoses are closely related to human food safety. Pork is an important medium for the spread of foodborne zoonoses. According to monitoring data analysis, Staphylococcus aureus, Escherichia coli, Shigella, Salmonella, Yersinia enterocolitica, Bacillus cereus, Listeria monocytogenes, among which Trichinella spiralis, Toxoplasma gondii, cysticercus, etc. are also pathogens with greater harm to pork safety. These pathogenic pathogens not only affect the quality and safety of pork and its products, but also bring health risks to consumers and even endanger their lives. Therefore, pork quality and safety have a direct impact on the pig industry chain and are a prerequisite for product quality and safety.

[0003] Liquid chip technology divides microspheres into many different partitions through spectral fingerprints. Each microsphere can be coupled with different nucleic acids or proteins for specific biological detection targets, so different objects to be detected can be captured simultaneously in one sample. Due to the size of the microspheres and the low density in the reaction system, the biochip reaction system is changed from a liquid-solid phase reaction to a complete liquid phase reaction system close to the internal environment of the biological system. Up to 100 indicators can be detected in one operation. It also has the advantages of rapidity, high sensitivity, wide detection range, good repeatability, low cost, and easy operation. This study selected 16 pathogenic pathogens: Staphylococcus aureus, pathogenic Escherichia coli, Shigella, Salmonella, Yersinia enterocolitica, Bacillus cereus, Brucella, Listeria monocytogenes, Campylobacter coli, Clostridium perfringens, Erysipelothrix rhusiopathogenes, Streptococcus type 2, Trichinella, Toxoplasma, Cysticercus, and Sarcocystis. These 16 pathogens that require priority control were obtained based on the risk identification of multi-pathogen contamination or carriage in pork in accordance with the "Animal Epidemic Prevention Law of the People's Republic of China" and combined with literature research. The research aims to establish a rapid and high-throughput method for detecting multiple pathogenic pathogens through a liquid phase chip platform, which can provide a new method selection for laboratory testing of pork quality and safety, and better ensure the quality and safety of products and people's food safety. Summary of the invention

[0004] The purpose of the present invention is to provide a liquid chip method capable of simultaneously detecting 16 pathogenic pathogens in pork.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a nucleic acid composition for simultaneously and rapidly detecting 16 pathogenic pathogens in pork, comprising the following primer and probe sequences:

[0007] Table 2 Primer and probe sequences of 16 pathogenic pathogens

[0008]

[0009]

[0010] As shown in SEQ ID NO: 1-48.

[0011] In the present invention, W represents: A / T; Y represents: C / T; and R represents: A / G.

[0012] Preferably, the 5' end of the downstream primer of the nucleic acid composition is connected to biotin, and the 5' end of the probe is connected to NH2-C 12 .

[0013] The present invention also provides a kit for simultaneously and rapidly detecting 16 pathogenic pathogens in pork, comprising the nucleic acid composition and microspheres.

[0014] Preferably, it also includes multiple PCR Mix, MES solution, EDC solution, Tween 20 solution, SDS solution, TE solution and SAPE reporter solution.

[0015] The present invention also provides a method for simultaneously and rapidly detecting 16 pathogenic pathogens in pork, comprising the following steps:

[0016] (1) using the sample DNA sequence as a template, performing multiplex PCR amplification using the upstream primer and the downstream primer to obtain multiplex PCR products;

[0017] (2) coupling the probe and the microspheres at a volume ratio of 2 to 4:50 to obtain a liquid chip working solution;

[0018] (3) The multiplex PCR product is hybridized with the liquid chip working solution at a volume ratio of 1:8-10, and then incubated with SAPE to detect the MFI value; if the qualitative ratio result is ≥6, the result is positive.

[0019] Preferably, the total system of the multiplex PCR amplification is 50 μL, including 25 μL of multiplex PCR Mix, 7 μL of upstream primers, 7 μL of downstream primers, 2 μL of template DNA, and then ddH2O is added to 50 μL.

[0020] Preferably, the multiplex PCR amplification program is: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 1 min; 35 cycles; and finally extension at 72°C for 10 min.

[0021] Preferably, the qualitative ratio result = MFI value of the test sample / MFI value of the blank control.

[0022] Preferably, the concentrations of the upstream primers for Staphylococcus aureus, pathogenic Escherichia coli, Shigella, Listeria monocytogenes, Erysipelothrix rhusiopathiae, and Cysticercus are all 0.35-0.45 pmol / μL; the concentration of the primers for Sarcocystis is 0.5-1.5 pmol / μL; the concentrations of the primers for Salmonella, Yersinia enterocolitica, Bacillus cereus, Brucella, Campylobacter coli, Clostridium perfringens, Streptococcus type 2, Trichinella spiralis, and Toxoplasma gondii are all 0.07-0.09 pmol / μL;

[0023] The concentrations of the downstream primers for Staphylococcus aureus, pathogenic Escherichia coli, Shigella, Listeria monocytogenes, Erysipelothrix rhusiopathiae, and cysticercosis are all 0.35-0.45 pmol / μL; the concentration of the primers for Sarcocystis is 0.5-1.5 pmol / μL; the concentrations of the primers for Salmonella, Yersinia enterocolitica, Bacillus cereus, Brucella, Campylobacter coli, Clostridium perfringens, Streptococcus type 2, Trichinella spiralis, and Toxoplasma gondii are all 0.07-0.09 pmol / μL.

[0024] Preferably, the hybridization procedure in step (3) is 93-97°C for 5-10 min; 45-54°C for 15-20 min; the incubation temperature is 45-54°C for 5-20 min.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides a liquid chip method that can simultaneously detect 16 pathogenic pathogens in pork. When the pork is contaminated and the cause is unknown, the infected pathogens can be quickly identified. The method has the advantages of being fast, highly sensitive, having a wide detection range, good repeatability, low comprehensive detection cost, and being easy to operate.

[0027] Compared with the fluorescent quantitative PCR method, the method of the present invention can detect 4 to 5 signals in a single hole (limited by the principle of fluorescence detection technology), that is, it can detect 4 to 5 pathogens at the same time. The scheme of the present invention solves the problem of the existing fluorescent quantitative PCR detection method with few pathogen detection types, and can detect 16 common pathogens in pork in a single hole at the same time, greatly improving the detection efficiency and the range of pathogen detection.

[0028] It can be seen from the experiment provided by the present invention that compared with the detection sensitivity of the fluorescent quantitative PCR method, the method of the present invention has a lower limit for the copy number of 11 pathogens detected by the liquid phase chip, and the lower limit of 4 detection copies is the same as the fluorescent quantitative PCR method, indicating that the detection sensitivity of the method of the present invention is higher. Compared with the negative nucleic acid, the detection result of the present invention is judged as positive by the qualitative ratio ≥ 6, which reduces the detection error and strengthens the judgment of negative and positive, making the result more accurate. In terms of nucleic acid detection, the solid phase chip is a fixed-point capture sequencing based on the complementary binding of the target probe and the target sequence, while the liquid phase chip uses the microsphere suspension chip technology to perform the binding reaction of antigen-antibody, enzyme substrate, ligand receptor and nucleic acid hybridization reaction. The solid phase chip can partially replace the liquid phase chip, but the detection method of the liquid phase chip provided by the present invention has a wider range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0030] Figure 1 The results are from liquid chip specific experiments;

[0031] Figure 2 The results of the liquid chip sensitivity experiment are shown in Figure 2.

[0032] Figure 3 The results of the liquid chip hybridization temperature experiment;

[0033] Figure 4 The results of the liquid chip hybridization time experiment. DETAILED DESCRIPTION

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1

[0036] 1. Strain source

[0037] The Shigella ATCC29903, Salmonella ATCC14028, Staphylococcus aureus ATCC29213, Listeria monocytogenes ATCC19111, pathogenic Escherichia coli ATCC24187, Clostridium perfringens ATCC13124, Campylobacter coli ATCC33559, Bacillus cereus ATCC11778, Yersinia enterocolitica ATCC23715 used in the present invention are all provided by the Pathogenic Microorganism Monitoring Office of the China Animal Health and Epidemiology Center. Brucella nucleic acid is provided by the Zoonotic Disease Monitoring Office of the China Animal Health and Epidemiology Center. Streptococcus type 2 nucleic acid is provided by the Key Laboratory of Biosafety of the China Animal Health and Epidemiology Center. The attenuated G4T10 strain vaccine strain of Erysipelothrix rhizogenes is provided by Qilu Animal Health Products Co., Ltd. Trichinella nucleic acid, Sarcocystis nucleic acid, and Toxoplasma nucleic acid are provided by the Shanghai Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. Cysticercus nucleic acid was provided by the Institute of Zoonoses, Jilin University.

[0038] 2. Probe Design

[0039] The sequences of pathogenic genes in 30 strains of different species were retrieved and downloaded from GenBank. The most conservative segment was selected as the region with high homology through analysis by bioinformatics software such as Lasergene, and used as the target sequence for designing multiple PCR primers. The corresponding NCBI sequence number is shown in Table 2. Using PrimerPlex primer design software, according to the primer and design principles, appropriate parameters were set to ensure that the melting temperature of the primer pair was between 51 and 60°C and the size of the PCR amplification product was between 100 and 200 bp. The optimal set of 16 pairs of primers and probe sequences was determined, and the results are shown in Table 2. Considering the polymorphism of the target sequence, degenerate bases were designed for probes with relatively low specificity for Salmonella, Listeria monocytogenes, Yersinia enterocolitica, and Erysipelothrix rhusiopathiae, which effectively avoided nonspecific amplification and increased the detection specificity and accuracy.

[0040] 3. Construction of pathogenic microorganism positive plasmid

[0041] The target genes were amplified by PCR using specific primers of 16 pathogenic pathogens, respectively, using the primer and probe sequences of the 16 pathogenic pathogens in Table 2, the same below.

[0042] The reaction conditions were as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s; annealing at 58°C for 30 s; 72°C for 1 min; 35 cycles; final extension at 72°C for 10 min;

[0043] The reaction system is as follows: the reaction system is PCR Mix 12.5 μL, upstream primer 1 μL (primer amount is 0.4 pmol / μL), downstream primer 1 μL (primer amount is 0.4 pmol / μL), template DNA 2 μL, and add water to make up to 25 μL.

[0044] The PCR amplification product was identified by 2% agarose gel electrophoresis. The product was quickly purified and recovered according to the instructions of the gel recovery kit, and then the target fragment was connected to the pMD18-T vector and transformed into DH5 α After culturing chemically competent cells at 37°C for 1 hour, the bacterial solution was spread on LB plate medium containing ampicillin sodium and cultured overnight. A single white colony was selected and inoculated on a new LB plate medium containing ampicillin sodium. After 18 hours, the nucleic acid was extracted by water bath method, and the size of the inserted fragment in the vector was confirmed by PCR method. The nucleic acid that matched the size of the target gene fragment was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0045] 4. Synthesis and modification of liquid chip primers and probes

[0046] According to the information provided in the Luminex cookbook, select the appropriate TM Magnetic microspheres of the liquid chip detector. The 16 pathogen-specific primers and probes were modified as follows: biotin was connected to the 5' of the downstream primer, and NH2-C 12 The modified primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0047] 5. Multiplex PCR Reaction

[0048] The DNA of the positive strain was extracted by water bath method and used as a template for amplification. The target gene was amplified using the corresponding primers and positive strain. The PCR reaction conditions were 94℃ pre-denaturation for 4min; 94℃ denaturation for 30s; annealing at 58℃ for 30s; 72℃ for 1min; 35 cycles; and finally extension at 72℃ for 10min. The reaction system was 25 μL of PCRMix, 7 μL of upstream mixed primers (the primer amounts of Staphylococcus aureus, pathogenic Escherichia coli, Shigella, Listeria monocytogenes, Erysipelothrix rhusiopathiae, and cysticercus were all 0.4 pmol / μL, the primer amounts of Sarcocystis were 1 pmol / μL, and the primer amounts of other pathogens were all 0.08 pmol / μL), 7 μL of downstream mixed primers (the primer amounts of Staphylococcus aureus, pathogenic Escherichia coli, Shigella, Listeria monocytogenes, Erysipelothrix rhusiopathiae, and cysticercus were all 0.4 pmol / μL, the primer amounts of Sarcocystis were 1 pmol / μL, and the primer amounts of other pathogens were all 0.08 pmol / μL), 2 μL of template DNA, and water was added to make up to 50 μL.

[0049] 6. Probe and microsphere coupling

[0050] (1) Take 50 μL of microspheres and transfer them to a 1.5 ml centrifuge tube. Place the tube on a magnetic rack for 2 min and discard the supernatant.

[0051] (2) Add 50 μL of 0.1 M MES (pH 4.5) buffer to resuspend the microspheres, vortex and discard the supernatant.

[0052] (3) Add 7 μL of 0.1 M MES (pH 4.5) buffer and vortex to mix.

[0053] (4) Add 3 μL of diluted oligonucleotide probe (probe concentration is 20 μM).

[0054] (5) Add 2.5 μL of freshly prepared EDC solution (10 mg / mL) to the microspheres and shake immediately after addition.

[0055] (6) Hybridize at room temperature in the dark for 30 min, shaking and mixing every 10 min.

[0056] (7) Add 2.5 μL of freshly prepared EDC solution (10 mg / mL) to the microspheres and shake immediately after addition.

[0057] (8) Hybridize at room temperature in the dark for 30 min, shaking and mixing every 10 min.

[0058] (9) Add 1 mL of 0.02% Tween 20 solution, vortex to mix, place on a magnetic rack for 2 min, and discard the supernatant.

[0059] (10) Add 1 mL of 0.1% SDS solution, vortex to mix, place on a magnetic stand for 2 min, and discard the supernatant.

[0060] (11) Add 100 μL of TE solution and shake to mix.

[0061] (12) Store at 4°C away from light.

[0062] 7. Microsphere on-machine testing

[0063] The multiple PCR products (5 μL) and microsphere reaction solution (45 μL) were mixed, then oscillated and mixed, and then placed in a PCR instrument, set at 95°C for 5 min and 48°C for 15 min to allow the products and microspheres to hybridize. After the hybridization, 25 μL of SAPE reporter solution (5 μg / mL) was added, oscillated and mixed, and then placed in a PCR instrument, at 48°C for 5 min to allow hybridization, and then tested on the machine. The results were judged according to the MFI value.

[0064] The qualitative ratio result (LQRR) = MFI value of the test sample (MFIS) / MFI value of the blank control (MFIB). LQRR ≥ 6, the result is positive.

[0065] Experimental Example 2

[0066] 1. Liquid phase chip specificity experiment

[0067] Each positive plasmid obtained in Example 1 was used as a template, and the multiple PCR amplification products were respectively taken for specific detection using the liquid chip detection method; at the same time, a negative control group (with the same volume of DEPC water) was set up for the same specific detection.

[0068] The experimental process is the same as that of Experiment 1. The test results are as follows Figure 1 And as shown in Table 3.

[0069] Table 3 Liquid phase chip specificity experimental results

[0070]

[0071]

[0072] It can be seen that the detection results of this method for target bacteria are all positive, while the detection values ​​of the amplified products of non-target bacteria are all negative, indicating that the established method has good specificity.

[0073] Experimental Example 3

[0074] Each positive plasmid obtained in Example 1 was diluted 10 times (10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 ), using different concentrations of positive plasmids as templates, multiple PCR amplification was performed, and then the liquid chip detection method was used for sensitivity detection; at the same time, a negative control group (with the same volume of DEPC water) was set up for the same sensitivity detection. The experimental process was the same as that of Experimental Example 1, and the results are shown in Tables 4 and Figure 2 shown.

[0075] Table 4 Liquid phase chip sensitivity test results

[0076]

[0077]

[0078] The results showed that the sensitivity for detecting Toxoplasma gondii, Erysipelothrix rhusiopathiae, pathogenic Escherichia coli, Campylobacter coli, Shigella, Salmonella, and Staphylococcus aureus was 1 copy / reaction; the sensitivity for Brucella, Clostridium perfringens, and Yersinia enterocolitica was 10 copies / reaction; the sensitivity for cysticercus, Bacillus cereus, Listeria monocytogenes, Streptococcus type 2, and Trichinella spiralis was 100 copies / reaction; and the sensitivity for Sarcocystis was 1000 copies / reaction.

[0079] Experimental Example 4

[0080] The DNA of the positive strains crudely extracted in Experimental Example 1 was used as the amplification template for PCR experiments. The PCR reaction conditions were 94°C pre-denaturation for 4 min; 94°C denaturation for 30 s; 58°C annealing for 30 s; 72°C 1 min; 35 cycles; and finally extension at 72°C for 10 min. The reaction system was as follows: 12.5 μL of PCRMix, 1 μL of upstream primers (the amount of primers was 0.4 pmol / μL), 1 μL of downstream primers (the amount of primers was 0.4 pmol / μL), 2 μL of template DNA, and water was added to make up to 25 μL.

[0081] The probe and microsphere coupling method is the same as that in Experimental Example 1. The PCR product (5 μL) and the single microsphere reaction solution (45 μL) were mixed, then oscillated and mixed, and then placed in a PCR instrument, set at 95°C for 5 min, and the hybridization temperature was set at 45°C, 48°C, 51°C, and 54°C for 15 min to allow the product and microsphere to hybridize. After the hybridization, 25 μL of SAPE reporter solution (5 μg / mL) was added, and then oscillated and mixed, and then placed in a PCR instrument, and the hybridization temperature was set at 45°C, 48°C, 51°C, and 54°C for 5 min to allow the product and microsphere to hybridize, and then the results were shown in Tables 5 and 6. Figure 3 shown.

[0082] Table 5 Liquid chip hybridization temperature experimental results

[0083]

[0084] The results showed that at a hybridization temperature of 48°C, the detection signal values ​​(MFI) of 9 pathogens were the highest. The most suitable hybridization temperature should keep the values ​​of the entire system in a relatively balanced state, which is conducive to increasing the lower positive values. Finally, 48°C was selected as the hybridization temperature on the liquid chip.

[0085] Experimental Example 5

[0086] The DNA of the positive strains crudely extracted in Experimental Example 1 was used as the amplification template for PCR experiments. The PCR reaction conditions were 94°C pre-denaturation for 4 min; 94°C denaturation for 30 s; 58°C annealing for 30 s; 72°C 1 min; 35 cycles; and finally extension at 72°C for 10 min. The reaction system was as follows: PCR Mix 12.5 μL, upstream primer 1 μL (primer amount was 0.4 pmol / μL), downstream primer 1 μL (primer amount was 0.4 pmol / μL), template DNA 2 μL, and water was added to make up to 25 μL.

[0087] The probe and microsphere coupling method is the same as in Experimental Example 1. The PCR product (5 μL) and the single microsphere reaction solution (45 μL) were mixed, then oscillated and mixed, and then placed in a PCR instrument, set at 95°C for 5 min, set the hybridization temperature to 48°C for 15 min, and hybridize the product and microsphere. After the hybridization, 25 μL SAPE reporter solution (5 μg / mL) was added, oscillated and mixed, and then placed in a PCR instrument, set the hybridization temperature to 48°C for 5 min, 10 min, 15 min, and 20 min, respectively, and tested on the machine. The results are shown in Table 6 and Figure 4 shown.

[0088] Table 6 Liquid phase chip hybridization time experimental results

[0089]

[0090] The results showed that when the hybridization temperature and time were 20 minutes, the detection signal values ​​(MFI) of 12 pathogens were the highest. The most appropriate hybridization time should keep the values ​​of the entire system in a relatively balanced state, which is conducive to increasing the lower positive values. Finally, 20 minutes was selected as the hybridization time on the liquid chip.

[0091] Experimental Example 6

[0092] The DNA of the positive strains was extracted from Experimental Example 1 and used as a template for amplification. The nucleic acid concentration was determined and the nucleic acid copy number was calculated. 7 The copies were diluted tenfold to 100 copies, and a total of 8 nucleic acid concentration gradients were diluted. The diluted nucleic acids were subjected to PCR and fluorescence quantitative PCR experiments respectively.

[0093] Liquid chip detection method: PCR reaction conditions are 94℃ pre-denaturation for 4min; 94℃ denaturation for 30s; 58℃ annealing for 30s; 72℃ 1min; 35 cycles; final extension at 72℃ 10min. Reaction system: PCR Mix 12.5μL, upstream primer 1μL (primer amount is 0.4pmol / μL), downstream primer 1μL (primer amount is 0.4pmol / μL), template DNA 2μL, add water to 25μL.

[0094] The probe and microsphere coupling method is the same as that in Experimental Example 1. The PCR product (5 μL) and the single microsphere reaction solution (45 μL) were mixed, then oscillated and mixed, and then placed in a PCR instrument, set at 95°C for 5 min, and the hybridization temperature was set at 48°C for 15 min to allow the product and microsphere to hybridize. After the hybridization, 25 μL SAPE reporter solution (5 μg / mL) was added, and then oscillated and mixed, and then placed in a PCR instrument, and the hybridization temperature was set at 48°C for 20 min to allow the product and microsphere to hybridize, and then tested on the instrument.

[0095] The reaction conditions of fluorescence quantitative PCR were as follows: 95℃ pre-denaturation for 4min; 95℃ denaturation for 30s; 58℃ annealing for 30s; 72℃ 1min; 40 cycles; and final extension at 72℃ for 10min. The reaction system of fluorescence quantitative PCR was as follows: PCR Mix 12.5μL, upstream primer 0.5μL (primer amount was 0.25pmol / μL), downstream primer 0.5μL (primer amount was 0.25pmol / μL), probe 0.5μL (probe amount was 0.25pmol / μL), template DNA 2μL, and water was added to 20μL. The core standard for positive determination of fluorescence quantitative PCR was CT value ≤35.

[0096] Table 7 Liquid chip sensitivity test results

[0097]

[0098]

[0099] Table 8 Fluorescence quantitative PCR sensitivity test results

[0100]

[0101]

[0102] The results showed that the sensitivity of liquid-phase chip detection for 11 pathogens was higher than that of fluorescent quantitative PCR, and the sensitivity of liquid-phase chip detection for 4 pathogens was the same as that of fluorescent quantitative PCR. It can be concluded that the sensitivity of liquid-phase chip detection method is generally better than that of fluorescent quantitative PCR method.

[0103] It can be seen from the above embodiments and test examples that the detection method of the present invention, which designs specific primers and probes for 16 pathogenic strains and uses liquid phase chip technology to identify pathogenic strains, can simultaneously detect 16 pathogenic pathogens, quickly lock the infected pathogens, require a small amount of sample, and has high sensitivity and strong specificity.

[0104] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nucleic acid composition for rapid simultaneous detection of 16 pathogenic pathogens in pork, characterized in that: The following primer and probe sequences were included: Table 1 2. The nucleic acid composition according to claim 1, characterized in that The 5' end of the downstream primer of the nucleic acid composition is connected to biotin, and the 5' end of the probe is connected to NH2-C 12 .

3. A kit for rapid simultaneous detection of 16 pathogenic pathogens in pork, characterized in that: The method comprises the nucleic acid composition and microspheres as described in claim 1 or 2.

4. The kit according to claim 3, characterized in that It also includes multiplex PCR Mix, MES solution, EDC solution, Tween 20 solution, SDS solution, TE solution and SAPE reporter solution.

5. A method for simultaneously and rapidly detecting 16 pathogenic pathogens in pork, characterized in that: The steps include: (1) using the sample DNA sequence as a template, performing multiplex PCR amplification using the upstream primer and the downstream primer described in claim 1 or 2 to obtain multiplex PCR products; (2) coupling the probe described in claim 1 or 2 with the microspheres at a volume ratio of 2 to 4:50 to obtain a liquid chip working solution; (3) The multiplex PCR product is hybridized with the liquid chip working solution at a volume ratio of 1:8-10, and then incubated with SAPE to detect the MFI value; if the qualitative ratio result is ≥6, the result is positive.

6. The detection method according to claim 5, characterized in that: The total system of the multiplex PCR amplification is 50 μL, including 25 μL of multiplex PCR Mix, 7 μL of upstream primers, 7 μL of downstream primers, 2 μL of template DNA, and then ddH2O is added to 50 μL.

7. The detection method according to claim 5, characterized in that: The multiplex PCR amplification program was as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 1 min; 35 cycles; and finally extension at 72°C for 10 min.

8. The detection method according to claim 5, characterized in that: The qualitative ratio result = MFI value of the test sample / MFI value of the blank control.

9. The detection method according to claim 6, characterized in that: The concentrations of the upstream primers for Staphylococcus aureus, pathogenic Escherichia coli, Shigella, Listeria monocytogenes, Erysipelothrix rhusiopathiae, and Cysticercus are all 0.35-0.45 pmol / μL; the concentration of the primers for Sarcocystis is 0.5-1.5 pmol / μL; the concentrations of the primers for Salmonella, Yersinia enterocolitica, Bacillus cereus, Brucella, Campylobacter coli, Clostridium perfringens, Streptococcus type 2, Trichinella spiralis, and Toxoplasma gondii are all 0.07-0.09 pmol / μL; The concentrations of the downstream primers for Staphylococcus aureus, pathogenic Escherichia coli, Shigella, Listeria monocytogenes, Erysipelothrix rhusiopathiae, and cysticercosis are all 0.35-0.45 pmol / μL; the concentration of the primers for Sarcocystis is 0.5-1.5 pmol / μL; the concentrations of the primers for Salmonella, Yersinia enterocolitica, Bacillus cereus, Brucella, Campylobacter coli, Clostridium perfringens, Streptococcus type 2, Trichinella spiralis, and Toxoplasma gondii are all 0.07-0.09 pmol / μL.

10. The detection method according to claim 5, characterized in that: The hybridization procedure in step (3) is 93-97° C. for 5-10 min; 45-54° C. for 15-20 min; the incubation temperature is 45-54° C. for 5-20 min.

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