A droplet digital PCR method for simultaneously detecting the number of four foodborne pathogens.
The simultaneous detection of four foodborne pathogens using multiplex droplet digital PCR technology solves the problems of long detection time and low accuracy of traditional detection methods, achieving efficient and accurate food safety testing, and is suitable for food safety testing and immediate assurance for major events.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUANGZHOU INST FOR FOOD INSPECTION(GUANGZHOU INSPECTION CENT FOR WINE & SPIRITS)
- Filing Date
- 2022-08-19
- Publication Date
- 2026-06-30
AI Technical Summary
Existing technologies for detecting foodborne pathogens suffer from problems such as long detection time, low accuracy, cumbersome operation, and high cost. In particular, the workload is large when multiple pathogens coexist, and traditional methods are difficult to meet the requirements for rapid and accurate detection.
Using multiplex droplet digital PCR technology, specific primers and probes were designed to establish a method for the simultaneous detection of four foodborne pathogens (Staphylococcus aureus, Listeria monocytogenes, Salmonella typhi, and Bacillus cereus). By optimizing the ddPCR amplification reaction system and procedure, a highly sensitive and accurate absolute quantitative detection method was achieved.
It enables high-throughput, rapid, and accurate detection of four pathogenic bacteria, shortens detection time, improves detection efficiency, and is suitable for food safety testing, especially providing immediate assurance in emerging economic formats such as online food ordering and fresh food e-commerce, thereby enhancing the safety assurance capabilities for major events.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology, specifically to a droplet digital PCR method for simultaneously detecting the quantity of four pathogenic bacteria. Background Technology
[0002] Food safety issues caused by foodborne pathogens have always been a major social concern. According to a 2020 report by the World Health Organization, 550 million people worldwide suffer from diarrhea annually due to consuming contaminated food, with approximately 230,000 deaths. Currently, the most common foodborne pathogens globally include Salmonella Typhimurium (…). Salmonella typhi Staphylococcus aureus Staphylococcus aureus Listeria monocytogenes ( Listeria monocytogenes Vibrio parahaemolyticus Vibrio parahemolyticus ), pathogenic Escherichia coli ( Escherichia coli ), Clostridium botulinum ( Clostridium botulinum Among them, Salmonella typhi, Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus are the most important representative pathogenic microorganisms.
[0003] Currently, the national standard method for pathogen detection in China is the "National Food Safety Standard for Microbiological Examination of Food GB4789" series. This method primarily relies on traditional enrichment, selective culture, and a combination of morphological and biochemical identification to qualitatively and quantitatively detect pathogenic microorganisms. However, problematic foods often contain multiple coexisting pathogens. Conventional methods require different selective enrichment techniques for different microorganisms, significantly increasing workload, time, and efficiency. In short, traditional microbiological testing methods suffer from long testing times, low accuracy, cumbersome operation, and high technical requirements for testing personnel. Traditional methods for detecting and identifying foodborne pathogens are time-consuming and labor-intensive, failing to meet the current demands for rapid and accurate food testing.
[0004] Methods for detecting foodborne pathogens have evolved through several stages, from classical biochemical detection and immunological detection to molecular biological detection. The basic goal is to develop methods that are rapid, high-throughput, highly specific, and accurate. Molecular biological detection is currently a hot topic in pathogen detection, but commercially available single pathogen detection kits based on PCR technology are expensive and costly to use.
[0005] Droplet digital PCR (ddPCR) is a third-generation PCR technology, a novel nucleic acid detection technique based on Poisson distribution for absolute quantification. It eliminates the need for standard curves, directly detecting the absolute copy number of target genes in a sample through extreme dilution analysis and Poisson distribution analysis. It boasts advantages such as high sensitivity, high accuracy, high tolerability, and absolute quantification. Compared to qPCR, ddPCR provides higher accuracy and sensitivity. Multiplex ddPCR can detect the copy number of multiple target genes in the same PCR reaction system, ensuring accuracy and sensitivity while simultaneously detecting multiple targets, significantly reducing detection time and improving efficiency. Currently, multiplex ddPCR technology is maturely applied in gene mutation detection, copy number variation analysis, prenatal diagnosis, pathogen detection of viral diseases such as the novel coronavirus, and genetically modified food detection. With the continuous expansion of the technology, multiplex ddPCR technology also provides a new solution for quantitative detection of food safety, and its impact in the field of food safety testing is gradually becoming apparent, showing broad application prospects. However, there are currently no reports in China of triple or higher multiplex ddPCR detection methods for foodborne pathogens. Summary of the Invention
[0006] The purpose of this invention is to propose a droplet digital PCR method for the simultaneous detection of four foodborne pathogens. This method can simultaneously detect four pathogenic microorganisms: Staphylococcus aureus, Listeria monocytogenes, Salmonella typhi, and Bacillus cereus. It establishes an accurate, rapid, and absolute quantitative detection system for the simultaneous detection of these four pathogens using multiplex ddPCR technology, which features high specificity, high sensitivity, and strong repeatability.
[0007] The objective of this invention can be achieved through the following technical solutions:
[0008] A droplet digital PCR method for simultaneously detecting the quantities of four foodborne pathogens, characterized by comprising the following steps:
[0009] Step 1: Prepare the DNA sample to be tested.
[0010] The DNA sample to be tested was measured by an ultra-micro spectrophotometer, and its A260 / A280 was between 1.7 and 2.0, and the DNA concentration was >10 ng / μL. It was then stored at -20℃ for later use.
[0011] Step 2: Prepare primers and probes for four types of bacteria to be tested; these four bacteria are Staphylococcus aureus, Listeria monocytogenes, Salmonella typhi, and Bacillus cereus; among which:
[0012] The primer and probe sequences for Staphylococcus aureus are as follows:
[0013] Primer F: 5'-TTCTTCACGACTAAATAAACGCTCA-3'
[0014] Primer R: 5'-GGTACTACTAAAGATTATCAAGACGGCT-3'
[0015] Probe: 5'FAM-CAGAACACAATGTTTCCGATGCAACGT-3'BHQ1
[0016] The probe for Staphylococcus aureus is labeled with FAM at the 5' end and BHQ1 at the 3' end;
[0017] The primer and probe sequences for Listeria monocytogenes are as follows:
[0018] Primer F: 5'-CTGAATCTCAAGCAAAACCTGGT-3'
[0019] Primer R: 5'-CGCGACCGAAGCCAACTA-3'
[0020] Probe: 5'HEX-ATACGATAACATCCACGGCTCTGGCTGG-3'BHQ1
[0021] The probe for Listeria monocytogenes was labeled with HEX at the 5' end and BHQ1 at the 3' end;
[0022] The primer and probe sequences for Salmonella Typhi are as follows:
[0023] Primer F: 5'-CTCACCAGGAGATTACAACATGG-3'
[0024] Primer R: 5'-AGCTCAGACCAAAAGTGACCATG-3'
[0025] Probe: 5'FAM-CACCGACGGCGAGACCGACTTT-3'TAMRA
[0026] The probe for Salmonella typhi is labeled with FAM at the 5' end and TAMARA at the 3' end;
[0027] The primer and probe sequences for Bacillus cereus are as follows:
[0028] Primer F: 5'-CAGAACAACCAGAATTTATGAAAGAGT-3'
[0029] Primer R: 5'-ATCATCTACAACACCACTCGGTTT-3'
[0030] Probe: 5'FAM-CAGCGCGTATCGGTCGTTCACTCGG-3'BHQ1
[0031] The probe of Bacillus cereus was labeled with FAM at the 5' end and BHQ1 at the 3' end;
[0032] Step 3: Use the ddPCR amplification reaction system, which includes primers and probes for the four types of bacteria to be tested in Step 2, to amplify the DNA sample to be tested in Step 1, detect the amplification products, and obtain the detection results.
[0033] In the optimized scheme of this invention, the ddPCR amplification reaction system in step 3 is 20 μL, including 10 μL of ddPCR reaction premix, 0.72 μL each of primer F (25 μmol / L) for the four types of bacteria to be tested, 0.72 μL each of primer R (25 μmol / L) for the four types of bacteria to be tested, 0.2 μL each of probes (25 μmol / L) for the four types of bacteria to be tested, 2 μL of template DNA, and ddH2O is added to bring the ddPCR amplification reaction system to 20 μL.
[0034] In the optimized scheme of this invention, step 3 uses a ddPCR amplification reaction system, and the amplification program is a droplet digital PCR amplification program, specifically: first step pre-denaturation at 95℃ for 10 min, second step denaturation at 94℃ for 30 s, third step annealing at 61℃ for 2 min, and return to the second step for a total of 40 cycles; enzyme heat inactivation at 98℃ for 10 min, and storage at 4℃.
[0035] This invention has the following outstanding substantive features and significant progress:
[0036] 1. This invention provides a method for simultaneously detecting four foodborne pathogens, namely Staphylococcus aureus, Listeria monocytogenes, Salmonella typhi, and Bacillus cereus. The method of this invention has good specificity and reproducibility, and the results are accurate and reliable.
[0037] 2. The method of the present invention can achieve high-throughput and rapid detection of the above four foodborne pathogens, avoiding the problems of low detection accuracy, easy false negatives, cumbersome detection procedures and long time consumption of traditional testing methods.
[0038] 3. The method of this invention is applicable to the sampling and testing of various foods, which can greatly shorten the detection time of microorganisms in food and promote the rapid development of emerging economic formats such as online food ordering and fresh food e-commerce. As a necessary means for port quarantine or self-inspection by fresh food enterprises, it can drive the development of related industries. At the same time, in terms of major event security and emergency rescue, the rapid, accurate and quantifiable characteristics of the technical method of this project can provide immediate food safety assurance measures and improve the security assurance capabilities of major events. Attached Figure Description
[0039] Figure 1 This is a droplet digital PCR map of the four pathogenic microorganisms of the present invention.
[0040] Figure 2 This is a droplet digital PCR pattern of the food sample in Example 2.
[0041] Figure 3 This is a qPCR amplification pattern of Salmonella typhi.
[0042] Figure 4 This is a qPCR amplification pattern of Listeria monocytogenes.
[0043] Figure 5 This is a qPCR amplification pattern of Staphylococcus aureus.
[0044] Figure 6 This is a qPCR amplification pattern of Bacillus cereus.
[0045] Figure 7 This is the standard curve for Salmonella typhi.
[0046] Figure 8 This is the standard curve for Staphylococcus aureus.
[0047] Figure 9 The standard curve for Listeria monocytogenes.
[0048] Figure 10 This is the standard curve for Bacillus cereus. Detailed Implementation
[0049] The invention will now be further described with reference to the accompanying drawings. Example 1
[0050] A droplet digital PCR method for simultaneously detecting the quantities of four foodborne pathogens includes the following steps:
[0051] Step 1: Prepare the DNA sample to be tested.
[0052] Four standard strains of the four test bacteria were inoculated into four nutrient broth cultures, with one test bacteria inoculated into each nutrient broth culture. The cultures were incubated at 36℃ for 18-24 hours. The four test bacteria were Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus Abbreviated as Sa), Listeria monocytogenes ( Listeria monocytogenes Abbreviated as Lm), Salmonella typhi ( Salmonella typhi, Abbreviated as Sal) and Bacillus cereus ( Bacillus cereus, (abbreviated as Bc)
[0053] Take appropriate amounts of nutrient broth cultures containing the four types of bacteria to be tested, mix them separately, and take 1 ml of the mixture for bacterial DNA extraction. Use a bacterial DNA extraction kit (Tiangen Company: catalog number: DP302-02) to extract bacterial DNA. For the extraction method, please refer to the above kit operation instructions to obtain the DNA sample to be tested.
[0054] The DNA sample to be tested was measured by an ultra-micro spectrophotometer. The A260 / 280 was between 1.7 and 2.0, and the DNA concentration was >10 ng / μL. It was then stored at -20℃ for later use.
[0055] Step 2: Prepare primers and probes for the four types of bacteria to be tested. Among them, select the primer and probe sequences for Staphylococcus aureus and Listeria monocytogenes in the standard "Real-time Fluorescent PCR Method for Detection of Foodborne Pathogenic Bacteria in Exported Food" (SN / T 1870-2016), and select the primer and probe sequences for Salmonella typhi in the standard "Real-time Fluorescent PCR Method for Detection of Salmonella in Imported and Exported Food" (SN / T 1059.7-2010). Consult the literature (Ke Zhenhua, Research on Real-time Fluorescent PCR Detection Method of Bacillus cereus in Catering Food, Fujian Light Textile, 2020(4):19-23.), and select the essC gene and its primer and probe sequences for Bacillus cereus, as follows:
[0056] The primer and probe sequences for Staphylococcus aureus are as follows:
[0057] Primer F (Sa-F): 5'-TTCTTCACGACTAAATAAACGCTCA-3'
[0058] Primer R (Sa-R): 5'-GGTACTACTAAAGATTATCAAGACGGCT-3'
[0059] Probe (Sa-P): 5'FAM-CAGAACACAATGTTTCCGATGCAACGT-3'BHQ1
[0060] The probe for Staphylococcus aureus is labeled with FAM at the 5' end and BHQ1 at the 3' end;
[0061] The primer and probe sequences for Listeria monocytogenes are as follows:
[0062] Primer F (Lm-F): 5'-CTGAATCTCAAGCAAAACCTGGT-3'
[0063] Primer R (Lm-R): 5'-CGCGACCGAAGCCAACTA-3'
[0064] Probe (Lm-P): 5'HEX-ATACGATAACATCCACGGCTCTGGCTGG-3'BHQ1
[0065] The probe for Listeria monocytogenes was labeled with HEX at the 5' end and BHQ1 at the 3' end;
[0066] The primer and probe sequences for Salmonella Typhi are as follows:
[0067] Primer F (Sal-F): 5'-CTCACCAGGAGATTACAACATGG-3'
[0068] Primer R (Sal-R): 5'-AGCTCAGACCAAAAGTGACCATG-3'
[0069] Probe (Sal-P): 5'FAM-CACCGACGGCGAGACCGACTTT-3'TAMRA
[0070] The probe for Salmonella typhi is labeled with FAM at the 5' end and TAMARA at the 3' end;
[0071] The primer and probe sequences for Bacillus cereus are as follows:
[0072] Primer F (Bc-F): 5'-CAGAACAACCAGAATTTATGAAAGAGT-3'
[0073] Primer R (Bc-R): 5'-ATCATCTACAACACCACTCGGTTT-3'
[0074] Probe (Bc-P): 5'FAM-CAGCGCGTATCGGTCGTTCACTCGG-3'BHQ1
[0075] The probe of Bacillus cereus was labeled with FAM at the 5' end and BHQ1 at the 3' end;
[0076] Step 3: Use the ddPCR amplification reaction system, which includes primers and probes for the four types of bacteria to be tested in Step 2, to amplify the DNA sample to be tested in Step 1, detect the amplification products, and obtain the detection results.
[0077] The ddPCR amplification reaction system is 20 μL, including 10 μL of ddPCR reaction premix, 0.72 μL each of primer F (25 μmol / L) for the four test bacteria, 0.72 μL each of primer R (25 μmol / L) for the four test bacteria, 0.2 μL each of probes (25 μmol / L) for the four test bacteria, 2 μL of template DNA, and ddH2O added to bring the ddPCR amplification reaction system to 20 μL.
[0078] The specific testing process is as follows:
[0079] 1. Droplet formation;
[0080] Adjust the pipette to 20 μL and transfer the ddPCR amplification reaction system to the sample wells of the droplet generation card. Add 70 μL of droplet generation oil to the oil wells, and then transfer the droplet generation card to the droplet generator (QX200). TM Droplet generation is automatically completed in Droplet Generator;
[0081] 2. PCR amplification;
[0082] Adjust the pipette to 40 μL and transfer the generated droplets into a 96-well plate. The plate is then sealed using an automated sealing device (PX1). TM After sealing with a PCR PlateSealer, the sample was transferred to a PCR instrument (T100). TM The microdroplet digital PCR amplification program was performed using a Thermal Cycler, specifically: Step 1: pre-denaturation at 95℃ for 10 min; Step 2: denaturation at 94℃ for 30 s; Step 3: annealing at 61℃ for 2 min; and then returning to Step 2 for a total of 40 cycles; Enzyme was heat-inactivated at 98℃ for 10 min and stored at 4℃.
[0083] 3. Reading of microdroplet fluorescence signals
[0084] Remove the 96-well plate after PCR amplification and use a droplet detector (QX200). TM Droplet Reader was used to read and count droplets, and heatmap analysis was performed to determine the fluorescence threshold to calculate the number of positive and negative droplets, resulting in a quadruple droplet digital PCR pattern for four pathogenic microorganisms. Figure 1 As shown.
[0085] The droplet digital PCR method according to this embodiment can be used to identify four pathogenic microorganisms in a test sample, determine whether the test sample contains one or more of the four pathogenic microorganisms, and the quantity of the pathogenic microorganisms contained therein. Example 2
[0086] The droplet digital PCR method for simultaneously detecting four types of foodborne pathogens in this embodiment differs in its steps as follows: In step 1 of this embodiment, the sample to be tested is a sampled food product, specifically rice noodles. 25g of the food sample is diluted in 225ml of sterile physiological saline to prepare a 1:10 dilution. Then, 1 to 5ml of this 1:10 dilution (1ml in this embodiment) is taken and bacterial DNA is extracted using the bacterial DNA extraction kit from step 1 of Example 1 to obtain the DNA sample to be tested. The subsequent steps of step 1 and step 2 of Example 1 are then performed.
[0087] Test results are shown Figure 2 The DNA sample to be tested contained 97.5 copies / ul of Salmonella Typhi; 12 copies / ul of Bacillus cereus; and 41.2 copies / ul of Listeria monocytogenes. Converted to the levels of pathogenic bacteria in the sample, these are: 975,000 copies / g of Salmonella Typhi; 120,000 copies / g of Bacillus cereus; and 412,000 copies / g of Listeria monocytogenes. Example 3
[0088] This embodiment tests the specificity of primers and probes for the four types of bacteria to be tested in this invention. Specifically, 21 standard strains tested in this embodiment (the names of the 21 standard strains are shown in Table 1) and the standard strains of the four types of bacteria to be tested in Example 1, totaling 25 strains, were inoculated into 25 portions of nutrient broth culture medium and cultured at 36°C for 24 hours for later use.
[0089] Using a bacterial DNA extraction kit (Tiangen Biotech: Catalog No.: DP302-02), 2 ml of each of 25 cultured nutrient broths was taken and DNA was extracted using the bacterial DNA extraction kit described in Example 1 and the extraction method in its instructions, resulting in DNA samples of 25 different strains.
[0090] Using quantitative real-time PCR, the genomic DNA components of the 25 strains tested in this example were detected in DNA samples of Salmonella Typhi, Bacillus cereus, Staphylococcus aureus, and Listeria monocytogenes, respectively. The specificity of the four pathogenic bacteria detection primers / probes was verified.
[0091] The fluorescence PCR reaction system in this embodiment was as follows: 12.5 µL of 2×qPCR Mix, 0.75 µL each of 10 µmol / L forward and reverse primers, 0.5 µL of 10 µmol / L probe, 2 µL of template DNA, and deionized water to a final volume of 25 µL. The fluorescence PCR amplification program was: 95℃ for 15 min; 95℃ for 15 s, 60℃ for 1 min, for 40 cycles. The fluorescence signal of the amplified products was detected; the detection results are shown in Table 1. Figures 3 to 6 .
[0092]
[0093] from Figures 3 to 6 It can be seen that the upstream and downstream primers and probes in Example 1 amplified positively for the four types of bacteria to be tested, with obvious S-shaped amplification curves, while they amplified negatively for other bacterial DNA samples, with no obvious S-shaped amplification curves.
[0094] Conclusion: The upstream and downstream primers and probes used in the detection method of this invention have good specificity. Example 4
[0095] This embodiment tests the linear range, accuracy, and detection limit of the droplet digital PCR method for simultaneously detecting four foodborne pathogens established in this invention. Specifically:
[0096] In this embodiment, the DNA samples used were based on the following initial concentrations: Salmonella Typhimurium 884.3 pg / ul; Bacillus cereus 446.0 pg / ul; Staphylococcus aureus 308.5 pg / ul; Listeria monocytogenes 245.3 pg / ul. A total of four DNA samples were prepared, and each sample was serially diluted seven times in a 4-fold gradient. This resulted in eight DNA samples for each type of foodborne pathogen, including samples from the initial concentrations to the serially diluted samples. Each DNA sample contained all four foodborne pathogens. Subsequently, the samples were tested three times according to step 2 in Example 1, and the results correspond to Experiments 1 to 3 in Table 2.
[0097] In Table 2, "DNA Dilution 1" represents the DNA in Group 1, corresponding to the "initial DNA concentration of the DNA sample to be tested." Except for Group 1, where the DNA concentration was too high and the positive droplets could not be effectively separated, and Group 8, where the DNA concentration was too low for accurate quantification, the results for the other six groups showed good linearity. See details below. Figures 7 to 10The linear range for *Salmonella typhi* was 0.22–221.08 pg / ul; for *Listeria monocytogenes*, it was 0.06–61.33 pg / ul; for *Staphylococcus aureus*, it was 0.075–77.13 pg / ul; and for *Bacillus cereus*, it was 0.109–111.5 pg / ul (corresponding to copy number linear ranges of 60–43,120 copies / 20ul; 46–53,560 copies / 20ul; 50–37,160 copies / 20ul; and 34–30,880 copies / 20ul, respectively). 2 All values were greater than 999, and the parallelism of the three tests in each group was good, with RSDs all less than 25% (European Network of GMO Laboratories (ENGL). Definition of minimum performance requirements for analytical methods of GMO testing [EB / OL]. 2015, 9. http: / / gmo-crl.jrc.ec.europa.eu / .). Group 8 was used as the method detection limit, and the detection results for the four bacteria were as follows: Salmonella typhi 321 copies / mL; Listeria monocytogenes 315 copies / mL; Staphylococcus aureus 288 copies / mL; Bacillus cereus 194 copies / mL.
[0098] In addition, DNA mixed samples were used, and amplification was performed using both singlet and quadruple systems in a ddPCR system.
[0099] The test results were analyzed using a paired t-test with SPSS, and no significant differences were found. DNA was extracted from a mixture of the four bacterial strains, and the copy number of each strain was detected using quadruple ddPCR; simultaneously, the number of added bacteria was determined using plate counting, and the results were consistent. This indicates that the established quadruple ddPCR detection system is accurate.
[0100]
Claims
1. A droplet digital PCR method for simultaneously detecting the quantities of four foodborne pathogens for purposes other than disease diagnosis and treatment, characterized in that... It includes the following steps: Step 1: Prepare the DNA sample to be tested. The DNA sample to be tested was measured by an ultra-micro spectrophotometer, and its A260 / A280 was between 1.7 and 2.0, and the DNA concentration was >10 ng / μL. It was then stored at -20℃ for later use. Step 2: Prepare primers and probes for four types of bacteria to be tested; these four types of bacteria are Staphylococcus aureus. 、 Listeria monocytogenes, Salmonella typhi, and Bacillus cereus; among which: The primer and probe sequences for Staphylococcus aureus are as follows: Primer F: 5'-TTCTTCACGACTAAATAAACGCTCA-3' Primer R: 5'-GGTACTACTAAAGATTATCAAGACGGCT-3' Probe: 5'FAM-CAGAACACAATGTTTCCGATGCAACGT-3'BHQ1 The 5' end of the Staphylococcus aureus probe was labeled with FAM, and the 3' end was labeled with BHQ1. The primer and probe sequences for Listeria monocytogenes are as follows: Primer F: 5'-CTGAATCTCAAGCAAAACCTGGT-3' Primer R: 5'-CGCGACCGAAGCCAACTA-3' Probe: 5'HEX-ATACGATAACATCCACGGCTCTGGCTGG-3'BHQ1 The 5' end of the Listeria monocytogenes probe was labeled with HEX, and the 3' end was labeled with BHQ1; The primer and probe sequences for Salmonella Typhi are as follows: Primer F: 5'-CTCACCAGGAGATTACAACATGG-3' Primer R: 5'-AGCTCAGACCAAAAGTGACCATG-3' Probe: 5'FAM-CACCGACGGCGAGACCGACTTT-3'TAMRA The 5' end of the Salmonella typhi probe is labeled with FAM, and the 3' end is labeled with TAMARA. The primer and probe sequences for Bacillus cereus are as follows: Primer F: 5'-CAGAACAACCAGAATTTATGAAAGAGT-3' Primer R: 5'-ATCATCTACAACACCACTCGGTTT-3' Probe: 5'FAM-CAGCGCGTATCGGTCGTTCACTCGG-3'BHQ1 The 5' end of the Bacillus cereus probe was labeled with FAM, and the 3' end was labeled with BHQ1; Step 3: Use the ddPCR amplification reaction system, which includes primers and probes for the four types of bacteria to be tested in Step 2, to amplify the DNA sample to be tested in Step 1, detect the amplification products, and obtain the detection results.
2. The droplet digital PCR method for simultaneously detecting the quantities of four foodborne pathogens for purposes other than disease diagnosis and treatment, as described in claim 1, is characterized in that: The ddPCR amplification reaction system in step 3 is 20 μL, including 10 μL of ddPCR reaction premix, 0.72 μL each of primer F (25 μmol / L) for the four test bacteria, 0.72 μL each of primer R (25 μmol / L) for the four test bacteria, 0.2 μL each of probes (25 μmol / L) for the four test bacteria, 2 μL of template DNA, and ddH2O added to bring the ddPCR amplification reaction system to 20 μL.
3. The droplet digital PCR method for simultaneously detecting the quantities of four foodborne pathogens for purposes other than disease diagnosis and treatment, as described in claim 2, is characterized in that: In step 3, the ddPCR amplification reaction system is used, and the amplification program is the droplet digital PCR amplification program, which is as follows: first step, pre-denaturation at 95℃ for 10 min; second step, denaturation at 94℃ for 30 s; third step, annealing at 61℃ for 2 min; return to the second step and repeat for a total of 40 cycles; enzyme heat inactivation at 98℃ for 10 min; and storage at 4℃.