Liquid-phase gene chip method for simultaneously detecting listeria monocytogenes, staphylococcus aureus, salmonella and escherichia coli
The specific primers and plasmids were designed through the liquid phase gene chip method, combined with multiple PCR and fluorescence-encoded microsphere hybridization technology, high-throughput and simultaneous detection of Listeria, Staphylococcus aureus, Salmonella and E. coli were achieved, solving the problems of low sensitivity and cumbersome operation of existing detection methods, and meeting the needs of fast on-site real-time detection of foodborne pathogenic bacteria.
Patent Information
- Application Number
- CN202510410312.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-06-10
AI Technical Summary
The existing foodborne pathogenic bacteria detection methods are cumbersome, time-consuming, and have low sensitivity. They cannot achieve high-throughput detection and real-time rapid monitoring, and cannot meet the needs of fast on-site real-time detection of foodborne pathogenic bacteria.
Design the liquid phase gene chip method, design primers by downloading specific gene sequences, extracting bacterial DNA, constructing plasmids, and achieving simultaneous detection of Listeria, Staphylococcus aureus, Salmonella and E. coli by multiplex PCR and fluorescent-encoding microsphere hybridization technology.
It realizes high-throughput and simultaneous detection of a variety of food-borne pathogenic bacteria, shortens detection time, is simple to operate, has higher sensitivity, and is less specific and cost, meeting the needs of fast on-site real-time detection of food-borne pathogenic bacteria.
Smart Images
Figure CN120119014A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of bacteria detection, and specifically relates to a liquid-phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli. Background Art
[0002] Foodborne pathogens are one of the main causes of foodborne diseases. Pathogenic bacteria contamination in the food chain and the food safety problems caused by them are also common. Food safety problems caused by foodborne pathogens not only seriously threaten people's health, but also cause huge economic losses to consumers and food-related industries, and have an adverse impact on the country's social, economic and political stability.
[0003] Enterohemorrhagic Escherichia coli O157:H7, Salmonella, Staphylococcus aureus, and Listeria monocytogenes are the most common foodborne pathogens at home and abroad. At present, the gold standard for detecting foodborne pathogens is still the conventional bacterial isolation and culture method and the biochemical identification method of bacteria. However, these traditional detection methods require long-term bacterial culture and biochemical identification processes, which are cumbersome and time-consuming to operate, have low sensitivity, cannot achieve high-throughput detection, cannot achieve effective real-time rapid monitoring and early warning, and cannot meet the needs of rapid on-site real-time detection of foodborne pathogens. Summary of the invention
[0004] The object of the present invention is to provide a liquid phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli, so as to solve the problems raised in the above background technology.
[0005] In order to achieve the above object, the present invention provides the following technical solution: a liquid phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli, the specific steps are as follows:
[0006] Step 1: Design primers
[0007] Download the Hly gene sequence of Listeria monocytogenes, the FemA gene of Staphylococcus aureus, the invA gene of Salmonella and the rfbE gene of Escherichia coli O157:H7, and design a pair of specific primers based on the conserved sequences of these genes. The 5' end of the upstream primer is added with a TAG sequence through Spacer18, and the 5' end of the downstream primer is added with biotin;
[0008] Step 2: Extract bacterial DNA
[0009] 200 μL of virus solution was added to a 1.5 mL centrifuge tube, 20 μL of proteinase K and 200 μL of carrier RNA solution were added, the mixture was shaken and mixed, after 15 minutes in a 56°C water bath, 250 μL of anhydrous ethanol was added, transferred to a tube, and centrifuged at 8000 rpm for 1 minute, the washing solution was washed twice, and then TE was added, left at room temperature for 5 minutes and centrifuged to obtain DNA;
[0010] Step 3: Construct plasmid
[0011] Amplify the target gene using PCR, recover the PCR product by agarose gel electrophoresis, mix 4.5 μL of the recovered product with 0.5 μL of pMD-18T vector and 5 μL of solution I to a total volume of 10 μL, place at 16°C overnight and clone into the pMD-18T vector to construct a plasmid;
[0012] Transform 10 μL of the ligation product into DH5a E. coli competent cells: Add 10 μL of the ligation product to 50 μL of competent cells, mix gently, and place on ice for 30 minutes; heat shock in a metal bath at 42°C for 90 seconds and then place on ice for 2 minutes; add 500 mL of LB medium and shake on a shaker at 200 rpm and 37°C for 45 minutes; take 100 μL and spread on a plate containing ampicillin resistance, pick positive colonies and expand the culture;
[0013] Step 4: Establish a detection method
[0014] Multiplex PCR: Preparation of upstream primer mixture: the upstream primers of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes were mixed at a molar ratio of 1:1:1:0.7; Preparation of downstream primer mixture: the downstream primers of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes were mixed at a molar ratio of 1:1:1:0.7; using the specific templates of these four pathogens and the quadruple template, the atopic regions of the above four viruses were amplified, and the obtained PCR products were hybridized with the fluorescent encoded microsphere working solution and the streptavidin phycoerythrin (SA-PE) working solution;
[0015] Prepare fluorescent coded microsphere working solution:
[0016] Dilute 2500 / μL fluorescent coded microspheres with 1.1×Tm Hybrdization Buffer to 1 μL containing approximately 125 / type fluorescent coded microspheres;
[0017] Preparation of SA-PE working solution:
[0018] Dilute 1 mg / ml SA-PE to 10 μg / μL with 1×Tm Hybrdization Buffer, fully resuspend the fluorescent coded microsphere working solution, add 20 μL of microsphere working solution to each sample well and background well, add 5 μL of PCR product to the sample well, add 5 μL of PCR blank product to the background well, and then add 75 μL of SA-PE working solution, mix well, and incubate at 37°C in a metal heater for 30 min;
[0019] Analyze and judge the samples to be tested:
[0020] When the MFI value of the sample to be tested is greater than 1000, it is judged as a positive sample; when the MFI value of the sample to be tested is ≤1000, it is judged as negative and needs to be repeated or further verified by other detection methods;
[0021] Step 5: Specificity test
[0022] The specificity of the method was tested using the established detection method with nucleic acids of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Vibrio cholerae, and Edwardsiella tarda as templates;
[0023] Step 6: Sensitivity test
[0024] The four plasmid standards were diluted 10-fold with diluent to a concentration of 1×10 8 ~1×10 1 copies / μL of plasmid was used as the standard template to conduct sensitivity analysis of the method;
[0025] Step 7: Repeatability test
[0026] Three 10-fold serial dilutions of DNA standards (1×106-1×102 copies / μL) were measured three times in the same reaction, and the MFIA values of each dilution were statistically analyzed to calculate the intra-assay coefficient of variation (CV%) between each reaction tube of each sample; the above samples were measured three times respectively, and the inter-assay coefficient of variation (CV%) between the results of each measurement of the same sample was calculated;
[0027] Step 8: Testing clinical samples
[0028] Artificially contaminated pork samples and 30 samples stored in the laboratory were tested. Positive and negative controls were set up for each reaction. The amplified products were hybridized with fluorescent coded microspheres and SA-PE, read on the Luminex 200 detector, and compared using fluorescent quantitative PCR method.
[0029] As a preferred technical solution of the present invention, the amplification system for amplifying the target gene by the PCR method described in step three is: 95°C / 5min pre-denaturation, 94°C / 30Sec, 55°C / 30Sec and 72°C / 20Sec 35 cycles, and 72°C extension for 10 minutes.
[0030] As a preferred technical solution of the present invention, the method for preparing the quadruple template in step 4 is to mix the four plasmids in a volume ratio of 1:1:1:1.
[0031] As a preferred technical solution of the present invention, the PCR product described in step 4 is hybridized with the fluorescent encoded microsphere working solution and the streptavidin phycoerythrin (SA-PE) working solution in the following manner: four types of microspheres each carry a specific anti-tag sequence, wherein the anti-tag sequence can be complementary paired with the tag sequences on the four pathogen primers of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes.
[0032] As a preferred technical solution of the present invention, the judgment standard of the preparation result of the SA-PE working solution described in step 4 is determined by the determination of the minimum detection threshold value (cutoff value): 10 healthy animal tissue samples are selected (each sample is repeated 3 times in parallel), the MFI values are read respectively, and their average and standard deviation are calculated, and the MIF value of the average value plus 3 times the standard deviation is set as the cutoff value.
[0033] As a preferred technical solution of the present invention, the reaction system for detecting clinical samples described in step eight is as follows: 2 μL of DNA template, 10 μL of 2×Premix Ex Taq Mix (containing Mg2+, dNTP, and rTaq enzyme), 1 μL of upstream primer (8 μM), 1 μL of downstream primer (8 μM), 1 μL of probe (4 μM), 0.4 μL of Rox, and H2O supplemented to 20 μL; reaction conditions: 94°C for 30 sec; 94°C for 5 sec, 60°C for 34 sec (fluorescence signal collection), for a total of 40 cycles; the baseline adjustment takes 3-15 cycles of fluorescence signal, the threshold line is set to exceed the highest point of the negative control amplification curve, and the result is determined after reading the Ct value.
[0034] As a preferred technical solution of the present invention, when recovering the PCR target product by agarose gel electrophoresis in step 3, agarose gel electrophoresis analysis is simultaneously performed.
[0035] As a preferred technical solution of the present invention, the specific test results described in step 5 are that only Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes are positive, and the others are negative.
[0036] As a preferred technical solution of the present invention, when the sensitivity test described in step 6 is performed with a 10-fold dilution, it is necessary to dilute to 10 1 copies / μL.
[0037] The beneficial effects of the present invention are as follows:
[0038] After the preparation work of designing primers, extracting bacterial DNA and constructing plasmids, the detection method established in step four can realize simultaneous and high-throughput detection of Listeria, Staphylococcus aureus, Salmonella and Escherichia coli, thereby effectively shortening the detection time and making the operation simpler. At the same time, the specificity test, sensitivity test, repeatability test and clinical sample test results show that the detection method of the present invention has higher sensitivity, lower specificity and cost, and meets the needs of rapid on-site real-time detection of foodborne pathogens. BRIEF DESCRIPTION OF THE DRAWINGS
[0039] Figure 1 is a primer sequence table of the present invention;
[0040] Figure 2 This is a table of the PCR amplification reaction system of the present invention;
[0041] Figure 3 The present invention is a fluorescent quantitative PCR primer and probe table;
[0042] Figure 4 This is the electrophoresis detection result diagram of the present invention;
[0043] Figure 5 It is a graph of the specificity test results of the present invention;
[0044] Figure 6 This is a sensitivity test result diagram of the present invention. DETAILED DESCRIPTION
[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of 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.
[0046] like Figures 1 to 6 As shown, the embodiment of the present invention provides a liquid phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli, and the specific steps are as follows:
[0047] Step 1: Design primers
[0048] Download the Hly gene sequence of Listeria monocytogenes, the FemA gene of Staphylococcus aureus, the invA gene of Salmonella and the rfbE gene of Escherichia coli O157:H7, and design a pair of specific primers based on the conserved sequences of these genes. The 5' end of the upstream primer is added with a TAG sequence through Spacer18, and the 5' end of the downstream primer is added with biotin;
[0049] Step 2: Extract bacterial DNA
[0050] 200 μL of virus solution was added to a 1.5 mL centrifuge tube, 20 μL of proteinase K and 200 μL of carrier RNA solution were added, the mixture was shaken and mixed, after 15 minutes in a 56°C water bath, 250 μL of anhydrous ethanol was added, transferred to a tube, and centrifuged at 8000 rpm for 1 minute, the washing solution was washed twice, and then TE was added, left at room temperature for 5 minutes and centrifuged to obtain DNA;
[0051] Step 3: Construct plasmid
[0052] Amplify the target gene using PCR, recover the PCR product by agarose gel electrophoresis, mix 4.5 μL of the recovered product with 0.5 μL of pMD-18T vector and 5 μL of solution I to a total volume of 10 μL, place at 16°C overnight and clone into the pMD-18T vector to construct a plasmid;
[0053] Transform 10 μL of the ligation product into DH5a E. coli competent cells: Add 10 μL of the ligation product to 50 μL of competent cells, mix gently, and place on ice for 30 minutes; heat shock in a metal bath at 42°C for 90 seconds and then place on ice for 2 minutes; add 500 mL of LB medium and shake on a shaker at 200 rpm and 37°C for 45 minutes; take 100 μL and spread on a plate containing ampicillin resistance, pick positive colonies and expand the culture;
[0054] Step 4: Establish a detection method
[0055] Multiplex PCR: Preparation of upstream primer mixture: the upstream primers of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes were mixed at a molar ratio of 1:1:1:0.7; Preparation of downstream primer mixture: the downstream primers of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes were mixed at a molar ratio of 1:1:1:0.7; using the specific templates of these four pathogens and the quadruple template, the atopic regions of the above four viruses were amplified, and the obtained PCR products were hybridized with the fluorescent encoded microsphere working solution and the streptavidin phycoerythrin (SA-PE) working solution;
[0056] Prepare fluorescent coded microsphere working solution:
[0057] Dilute 2500 / μL fluorescent coded microspheres with 1.1×Tm Hybrdization Buffer to 1 μL containing approximately 125 / type fluorescent coded microspheres;
[0058] Preparation of SA-PE working solution:
[0059] Dilute 1 mg / ml SA-PE to 10 μg / μL with 1×Tm Hybrdization Buffer, fully resuspend the fluorescent coded microsphere working solution, add 20 μL of microsphere working solution to each sample well and background well, add 5 μL of PCR product to the sample well, add 5 μL of PCR blank product to the background well, and then add 75 μL of SA-PE working solution, mix well, and incubate at 37°C in a metal heater for 30 min;
[0060] Analyze and judge the samples to be tested:
[0061] When the MFI value of the sample to be tested is greater than 1000, it is judged as a positive sample; when the MFI value of the sample to be tested is ≤1000, it is judged as negative and needs to be repeated or further verified by other detection methods;
[0062] Step 5: Specificity test
[0063] The specificity of the method was tested using the established detection method with nucleic acids of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Vibrio cholerae, and Edwardsiella tarda as templates;
[0064] Step 6: Sensitivity test
[0065] The four plasmid standards were diluted 10-fold with diluent to a concentration of 1×10 8 ~1×10 1 copies / μL of plasmid was used as the standard template to conduct sensitivity analysis of the method;
[0066] Step 7: Repeatability test
[0067] Three 10-fold serial dilutions of DNA standards (1×106-1×102 copies / μL) were measured three times in the same reaction, and the MFIA values of each dilution were statistically analyzed to calculate the intra-assay coefficient of variation (CV%) between each reaction tube of each sample; the above samples were measured three times respectively, and the inter-assay coefficient of variation (CV%) between the results of each measurement of the same sample was calculated;
[0068] Step 8: Testing clinical samples
[0069] Artificially contaminated pork samples and 30 samples stored in the laboratory were tested. Positive and negative controls were set up for each reaction. The amplified products were hybridized with fluorescent coded microspheres and SA-PE, read on the Luminex 200 detector, and compared using fluorescent quantitative PCR method.
[0070] After the preparation work of designing primers, extracting bacterial DNA and constructing plasmids, the detection method established in step 4 is used for detection, and then specificity tests, sensitivity tests, repeatability tests and verification of clinical samples are carried out to achieve high-throughput and simultaneous detection of Listeria, Staphylococcus aureus, Salmonella and Escherichia coli. Compared with traditional detection methods, the method proposed in the present invention is simpler to operate, has higher sensitivity, lower specificity and cost, and meets the needs of rapid on-site real-time detection of foodborne pathogens.
[0071] The amplification system for amplifying the target gene by the PCR method in step 3 is: 95°C / 5min pre-denaturation, 94°C / 30Sec, 55°C / 30Sec and 72°C / 20Sec 35 cycles, and 72°C extension for 10 minutes.
[0072] The PCR method for amplifying target genes has the advantages of high efficiency, high sensitivity, high specificity, simple operation, low requirements for starting materials, good repeatability and wide application.
[0073] The quadruple template preparation method in step 4 is to mix the four plasmids in a volume ratio of 1:1:1:1.
[0074] The reaction procedures for the specific templates of the four pathogens and the quadruple template for amplifying the atopic regions of four viruses, namely Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes, were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 90 s; 35 cycles; and extension at 72°C for 10 min.
[0075] Among them, the PCR product in step 4 is hybridized with the fluorescent encoded microsphere working solution and the streptavidin phycoerythrin (SA-PE) working solution in the following manner: 4 types of microspheres are respectively equipped with specific anti-tag sequences, wherein the anti-tag sequences can be complementary paired with the tag sequences on the four pathogen primers of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes.
[0076] The fluorescent coded microsphere numbers corresponding to Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes are MTAG-A012, MTAG-A029, MTAG-A046 and MTAG-067, respectively.
[0077] Among them, the judgment standard of the SA-PE working solution preparation result in step 4 adopts the determination of the minimum detection threshold (cutoff value): select 10 healthy animal tissue samples (each sample is repeated 3 times in parallel), read the MFI value respectively and calculate its mean value and standard deviation, and set the MFI value of the mean value plus 3 times the standard deviation as the cutoff value.
[0078] The cutoff value obtained in the present invention is 753, so the cutoff value of the present invention is set to 1000. Only when the MFI value of the detected sample is higher than 1000, the experimental data can be effectively analyzed.
[0079] Among them, the clinical sample detection reaction system in step eight is as follows: DNA template 2μL, 2×Premix Ex TaqMix (containing Mg2+, dNTP, rTaq enzyme) 10μL, upstream primer (8μM) 1μL, downstream primer (8μM) 1μL, probe (4μM) 1μL, Rox 0.4μL, supplemented with H2O to 20μL; reaction conditions: 94℃30sec; 94℃5sec, 60℃34sec (fluorescence signal collection), a total of 40 cycles; baseline adjustment takes 3-15 cycles of fluorescence signal, the threshold line setting should exceed the highest point of the negative control amplification curve, and the result is determined after reading the Ct value.
[0080] By evaluating the detection of clinical samples, the feasibility of the established xTAG method in actual sample detection can be evaluated.
[0081] Wherein, when recovering the PCR target product by agarose gel electrophoresis in step 3, agarose gel electrophoresis analysis is simultaneously performed.
[0082] When performing agarose gel electrophoresis analysis, M was set to DL2000bp DNA marker, 1 to Escherichia coli O157:H7, 2 to Staphylococcus aureus, 3 to Salmonella, 4 to Listeria monocytogenes, 5 to quadruple PCR for these four pathogens, and 6 to PCR blank control. The electrophoresis test results showed that the amplification product size of Salmonella was approximately 93bp, the amplification product size of Staphylococcus aureus was approximately 114bp, the amplification product size of Escherichia coli O157:H7 was approximately 140bp, and the amplification product size of Listeria monocytogenes was approximately 134bp. Since the amplification products of these four pathogens were similar in size, the electrophoresis bands of the quadruple PCR amplification products could not be distinguished.
[0083] Among them, the specific test results in step five were that only Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes were positive, and the others were negative.
[0084] The results of the specificity test showed that the detection system had good specificity.
[0085] Among them, when the sensitivity test in step 6 is performed with a 10-fold dilution, it needs to be diluted to 10 1 copies / μL.
[0086] The experimental results showed that the sensitivity detection limit of all pathogens was 10 2 copies / μL.
[0087] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.
[0088] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A liquid phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli, characterized in that: The specific steps are as follows: Step 1: Design primers Download the Hly gene sequence of Listeria monocytogenes, the FemA gene of Staphylococcus aureus, the invA gene of Salmonella and the rfbE gene of Escherichia coli O157:H7, and design a pair of specific primers based on the conserved sequences of these genes. The 5' end of the upstream primer is added with a TAG sequence through Spacer18, and the 5' end of the downstream primer is added with biotin; Step 2: Extract bacterial DNA 200 μL of virus solution was added to a 1.5 mL centrifuge tube, 20 μL of proteinase K and 200 μL of carrier RNA solution were added, the mixture was shaken and mixed, after 15 minutes in a 56°C water bath, 250 μL of anhydrous ethanol was added, transferred to a tube, and centrifuged at 8000 rpm for 1 minute, the washing solution was washed twice, and then TE was added, left at room temperature for 5 minutes and centrifuged to obtain DNA; Step 3: Construct plasmid Amplify the target gene using PCR, recover the PCR product by agarose gel electrophoresis, mix 4.5 μL of the recovered product with 0.5 μL of pMD-18T vector and 5 μL of solution I to a total volume of 10 μL, place at 16°C overnight and clone into the pMD-18T vector to construct a plasmid; Transform 10 μL of the ligation product into DH5a E. coli competent cells: Add 10 μL of the ligation product to 50 μL of competent cells, mix gently, and place on ice for 30 minutes; heat shock in a metal bath at 42°C for 90 seconds and then place on ice for 2 minutes; add 500 mL of LB medium and shake on a shaker at 200 rpm and 37°C for 45 minutes; take 100 μL and spread on a plate containing ampicillin resistance, pick positive colonies and expand the culture; Step 4: Establish a detection method Multiplex PCR: Preparation of upstream primer mixture: the upstream primers of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes were mixed at a molar ratio of 1:1:1:0.7; Preparation of downstream primer mixture: the downstream primers of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes were mixed at a molar ratio of 1:1:1:0.7; using the specific templates of these four pathogens and the quadruple template, the atopic regions of the above four viruses were amplified, and the obtained PCR products were hybridized with the fluorescent encoded microsphere working solution and the streptavidin phycoerythrin (SA-PE) working solution; Prepare fluorescent coded microsphere working solution: Dilute 2500 / μL fluorescent coded microspheres with 1.1×Tm Hybrdization Buffer to 1 μL containing approximately 125 / type fluorescent coded microspheres; Preparation of SA-PE working solution: Dilute 1 mg / ml SA-PE to 10 μg / μL with 1×Tm Hybrdization Buffer, fully resuspend the fluorescent coded microsphere working solution, add 20 μL of microsphere working solution to each sample well and background well, add 5 μL of PCR product to the sample well, add 5 μL of PCR blank product to the background well, and then add 75 μL of SA-PE working solution, mix well, and incubate at 37°C in a metal heater for 30 min; Analyze and judge the samples to be tested: When the MFI value of the sample to be tested is greater than 1000, it is judged as a positive sample; when the MFI value of the sample to be tested is ≤1000, it is judged as negative and needs to be repeated or other detection methods are used for further verification; Step 5: Specificity test The specificity of the method was tested using the established detection method with nucleic acids of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes, Vibrio parahaemolyticus, Pseudomonas aeruginosa, Vibrio cholerae, and Edwardsiella tarda as templates; Step 6: Sensitivity test Dilute the four plasmid standards 10-fold with diluent to a concentration of 1×10 8 ~1×10 1 copies / μL of plasmid was used as the standard template to conduct sensitivity analysis of the method; Step 7: Repeatability test Three 10-fold serial dilutions of DNA standards (1×106-1×102 copies / μL) were measured three times in the same reaction, and the MFIA values of each dilution were statistically analyzed to calculate the intra-assay coefficient of variation (CV%) between each reaction tube of each sample; the above samples were measured three times respectively, and the inter-assay coefficient of variation (CV%) between the results of each measurement of the same sample was calculated; Step 8: Testing clinical samples Artificially contaminated pork samples and 30 samples stored in the laboratory were tested. Positive and negative controls were set up for each reaction. The amplified products were hybridized with fluorescent coded microspheres and SA-PE, read on the Luminex 200 detector, and compared using fluorescent quantitative PCR method.
2. A liquid phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli according to claim 1, characterized in that: The amplification system for amplifying the target gene by the PCR method described in step 3 is: 95°C / 5min pre-denaturation, 94°C / 30Sec, 55°C / 30Sec and 72°C / 20Sec 35 cycles, and 72°C / 10min extension.
3. The liquid phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli according to claim 1, characterized in that: The method for preparing the quadruple template described in step 4 is to mix the four plasmids in a volume ratio of 1:1:1:
1.
4. The liquid phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli according to claim 1, characterized in that: The PCR product described in step 4 is hybridized with the fluorescent encoded microsphere working solution and the streptavidin phycoerythrin (SA-PE) working solution in the following manner: 4 types of microspheres are respectively provided with specific anti-tag sequences, wherein the anti-tag sequences can be complementary paired with the tag sequences on the primers of the four pathogens of Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes.
5. The liquid phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli according to claim 1, characterized in that: The judgment standard for the preparation result of the SA-PE working solution described in step 4 is determined by the determination of the minimum detection threshold (cutoff value): 10 healthy animal tissue samples are selected (each sample is repeated 3 times in parallel), the MFI values are read respectively, and their average and standard deviation are calculated, and the MIF value of the average plus 3 times the standard deviation is set as the cutoff value.
6. The liquid phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli according to claim 1, characterized in that: The clinical sample detection reaction system described in step eight is as follows: 2 μL DNA template, 10 μL 2×Premix Ex Taq Mix (containing Mg2+, dNTP, rTaq enzyme), 1 μL upstream primer (8 μM), 1 μL downstream primer (8 μM), 1 μL probe (4 μM), 0.4 μL Rox, supplemented with H2O to 20 μL; reaction conditions: 94°C 30 sec; 94°C 5 sec, 60°C 34 sec (fluorescence signal collection), a total of 40 cycles; baseline adjustment takes 3-15 cycles of fluorescence signal, the threshold line should be set to exceed the highest point of the negative control amplification curve, and the result is determined after reading the Ct value.
7. The liquid phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli according to claim 1, characterized in that: When the PCR target product is recovered by agarose gel electrophoresis as described in step 3, agarose gel electrophoresis analysis is simultaneously performed.
8. The liquid phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli according to claim 1, characterized in that: The specific test results described in step 5 are that only Salmonella, Staphylococcus aureus, Escherichia coli O157:H7 and Listeria monocytogenes are positive, and the others are negative.
9. The liquid phase gene chip method for simultaneously detecting Listeria, Staphylococcus aureus, Salmonella and Escherichia coli according to claim 1, characterized in that: When performing a 10-fold dilution in the sensitivity test described in step 6, the dilution should be 10 1 copies / μL.