A triple qPCR system for comprehensive evaluation of bacillus cereus toxin production capacity and application thereof

Abstract

This invention discloses a triple qPCR system for comprehensively evaluating the toxin-producing capacity of Bacillus cereus and its application. The triple qPCR system simultaneously detects key genes of Bacillus cereus vomitoxin. cesB Key genes for non-hemolytic enterotoxin nheB、 Key genes for hemolytic enterotoxin hblD The nucleic acid sequences of the primers and probes corresponding to each virulence gene are shown in SEQ ID NO. 1-9. This invention designs specific primer and probe sets for key virulence genes of *Bacillus cereus* and establishes a triple qPCR system, solving the problems of long detection time, low detection efficiency, and low detection accuracy in existing technologies. This enables rapid and accurate detection of key virulence genes in *Bacillus cereus*, thereby accurately assessing the toxin-producing capacity of *Bacillus cereus*. It can be applied to the screening of key virulence genes of *Bacillus cereus* in highly contaminated dairy products, thus effectively assessing the toxin-producing capacity of *Bacillus cereus* and providing technical support and application basis for the prevention and control of foodborne diseases.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically involving a triple qPCR system for comprehensively evaluating the toxin-producing ability of Bacillus cereus and its application. Background Technology

[0002] Bacillus cereus ( Bacillus cereus Bacillus cereus is a foodborne opportunistic pathogen widely distributed in the natural environment. When it contaminates food, it can produce vomitoxin and various enterotoxins, causing vomiting or diarrhea-type food poisoning, and in severe cases, liver damage or even death. With the rapid development of the food industry and changes in consumer habits, Bacillus cereus contamination incidents are frequent, becoming a major concern in the global food safety field.

[0003] The core pathogenicity of Bacillus cereus lies in the various toxins it produces, including cereulide, which induces vomiting, and non-hemolytic enterotoxins (Nhe) and hemolytic enterotoxins (Hbl), which cause diarrhea. The synthesis of these toxins is regulated by specific gene clusters, and some key virulence genes that have been identified include: the key gene encoding cereulide. cesB Key genes encoding Hbl hblD Key genes encoding Nhe nheB Furthermore, these three virulence genes are highly conserved in Bacillus cereus. Therefore, by detecting these virulence genes, the toxin-producing capacity of the strain can be accurately assessed.

[0004] Currently, the detection of Bacillus cereus mainly relies on traditional isolation and culture methods, which take several days and cannot distinguish between toxin-producing and non-toxin-producing strains. Existing molecular biology methods, such as conventional PCR, can detect virulence genes, but suffer from low sensitivity, inability to quantify, and poor accuracy. Therefore, there is a lack of rapid and accurate screening technologies for detecting the toxin-producing capacity of Bacillus cereus, resulting in a lack of effective technical support for assessing its toxin-producing ability.

[0005] Therefore, developing a detection technology system capable of comprehensively assessing the toxin-producing capacity of Bacillus cereus is of great significance for rapidly assessing food poisoning risks, guiding production process control, and improving food safety standards. This invention aims to address the aforementioned technical challenges by designing a specific primer and probe set and establishing a real-time quantitative qPCR system to achieve simultaneous, rapid, and accurate detection of key virulence genes in Bacillus cereus. This effectively assesses the toxin-producing capacity of Bacillus cereus, providing technical support and an application foundation for the prevention and control of foodborne diseases. Summary of the Invention

[0006] The purpose of this invention is to overcome at least one of the shortcomings of existing technologies in the detection of virulence genes of Bacillus cereus, such as complex operation, low detection efficiency, low accuracy, and difficulty in simultaneously reflecting the pathogenic risk of strains. The invention provides a triple qPCR system for simultaneously detecting different virulence genes of Bacillus cereus to determine its toxin production capacity, so as to achieve a simultaneous, rapid, and accurate assessment of the toxin production capacity of Bacillus cereus.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] First, this invention provides a triple qPCR system for comprehensively evaluating the toxin-producing capacity of Bacillus cereus. This triple qPCR system targets key genes of Bacillus cereus vomitoxin. cesB Key genes for non-hemolytic enterotoxin nheB, Key genes for hemolytic enterotoxin hblD Specific primers and TaqMan fluorescent probes were designed to simultaneously detect key genes of Bacillus cereus toxins in a single tube, in order to assess the toxin-producing capacity of Bacillus cereus and achieve a reasonable analysis of the toxin risk of the tested organism.

[0009] Secondly, this invention provides a triple qPCR-specific primer and probe set for comprehensively evaluating the toxin-producing capacity of Bacillus cereus, wherein the triple qPCR-specific primer and probe set includes a key gene for Bacillus cereus vomitoxin. cesB Key genes for non-hemolytic enterotoxin nheB, Key genes for hemolytic enterotoxin hblD The primer and probe set; the specific primer and probe set includes specific primers and TaqMan probes corresponding to these three virulence genes:

[0010] The detection of Bacillus cereus vomitoxin cesB The primer sequences and TaqMan probe sequences for the gene are as follows: upstream primer cesB -F, downstream primer cesB -R, TaqMan probe cesB -P;

[0011] The cesB The nucleic acid sequence of -F is shown in SEQ ID NO.1;

[0012] The cesB The nucleic acid sequence of -R is shown in SEQ ID NO.2;

[0013] The cesB The nucleic acid sequence of -P is shown in SEQ ID NO.3;

[0014] The detection of Bacillus cereus non-hemolytic enterotoxin nheBThe primer sequences and TaqMan probe sequences for the gene are as follows: upstream primer nheB -F, downstream primer nheB -R, TaqMan probe nheB -P;

[0015] The nheB The nucleic acid sequence of -F is shown in SEQ ID NO.4;

[0016] The nheB The nucleic acid sequence of -R is shown in SEQ ID NO.5;

[0017] The nheB The nucleic acid sequence of -P is shown in SEQ ID NO.6;

[0018] The detection of Bacillus cereus hemolytic enterotoxin hblD The primer sequences and TaqMan probe sequences for the gene are as follows: upstream primer hblD -F, downstream primer hblD -R, TaqMan probe hblD -P;

[0019] The hblD The nucleic acid sequence of -F is shown in SEQ ID NO.7;

[0020] The hblD The nucleic acid sequence of -R is shown in SEQ ID NO.8;

[0021] The hblD The nucleic acid sequence of -P is shown in SEQ ID NO.9.

[0022] In some instances, the triple qPCR-specific primer-probe set has each virulence gene labeled with a different fluorescent-quenching group:

[0023] The probe cesB The 5' end of the -P is labeled with the fluorescent reporter dye FAM, and the 3' end is labeled with the fluorescent quencher group BHQ1.

[0024] The probe nheB The 5' end of the -P is labeled with the fluorescent reporter dye ROX, and the 3' end is labeled with the fluorescent quencher group BHQ1;

[0025] The probe hblD The 5' end of the -P is labeled with the fluorescent reporter dye VIC, and the 3' end is labeled with the fluorescent quencher group BHQ1.

[0026] Thirdly, this invention provides a triple qPCR reaction system for comprehensively evaluating the toxin-producing capacity of Bacillus cereus. The triple qPCR reaction system comprises: 10 μL of 2 × qPCR Mix; 0.8 μL of [unspecified ingredient] with an initial concentration of 20 μmol / L. cesB -F、 cesB -R, 0.5 μL initial concentration of 10 μmol / L cesB -P; 0.2 μL initial concentration 20 μmol / L nheB -F、 nheB -R, 0.4 μL initial concentration of 10 μmol / L nheB -P; 0.4 μL initial concentration 20 μmol / L hblD -F、 hblD -R, 0.5 μL initial concentration of 10 μmol / L hblD -P; 2 μL template; 3.8 μL DEPC water.

[0027] Fourthly, the present invention provides a triple qPCR detection kit for comprehensively evaluating the toxin-producing ability of Bacillus cereus, the qPCR detection kit comprising the triple qPCR specific primer and probe set described in the second aspect and the triple qPCR reaction system described in the third aspect;

[0028] In some instances, the Bacillus cereus virulence genes include key genes for vomitoxin. cesB Key genes for non-hemolytic enterotoxin nheB, Key genes for hemolytic enterotoxin hblD ;

[0029] In some instances, the triple qPCR detection kit also includes a negative control, a positive control, and a lysis buffer;

[0030] In some instances, the positive control is a key gene of Bacillus cereus vomitoxin. cesB Key genes for non-hemolytic enterotoxin nheB Key genes for hemolytic enterotoxin hblD Plasmids constructed for the target fragment;

[0031] In some instances, the negative control is DEPC water;

[0032] In some instances, the lysis buffer is composed of 4.9–5.1 mmol / L tris(hydroxymethyl)aminomethane and 0.02–0.12% sodium dodecyl sulfate solution.

[0033] Fifthly, this invention provides a triple qPCR detection method for comprehensively evaluating the toxin-producing capacity of Bacillus cereus, comprising the following steps:

[0034] (1) Extract genomic DNA from the sample to be tested for later use;

[0035] (2) The genomic DNA of the sample to be tested is added as a template to the triple qPCR reaction system for amplification;

[0036] (3) Set up the qPCR program, collect the fluorescence signal during the amplification process, and determine the results.

[0037] This invention has the following outstanding advantages:

[0038] 1. The key gene targeting Bacillus cereus vomitoxin provided by this invention. cesB Key genes for non-hemolytic enterotoxin nheB Key genes for hemolytic enterotoxin hblD The primer and probe set exhibits extremely high specificity; a fluorescent signal can only be observed in the corresponding fluorescence detection channel when the sample contains the corresponding virulence gene, effectively avoiding false positives or false negatives and resulting in more accurate detection results. By detecting these virulence genes, the toxin-producing capacity of Bacillus cereus can be accurately assessed.

[0039] 2. The detection method established in this invention provides a reliable basis for the prevention and control of toxin-producing Bacillus cereus, and also reduces economic costs to a certain extent. Compared with traditional biochemical identification (7 days) and whole-genome sequencing based on pure cultures, the qPCR reaction time in this detection method is only 1.5 hours; compared with PCR detection of virulence genes, the detection method is more accurate due to probe design. Therefore, this detection method significantly reduces detection time, improves detection accuracy, and greatly enhances work efficiency. This significant advantage of being economical and efficient makes it particularly suitable for large-scale sample screening and actual detection. It is especially suitable for the detection of key virulence genes of Bacillus cereus in dairy products with high Bacillus cereus contamination, thereby accurately assessing the toxin-producing capacity of Bacillus cereus in samples.

[0040] 3. The triple qPCR detection kit provided in this invention contains a detection system for three virulence genes, which can be operated in a single tube to achieve simultaneous detection of three virulence genes. This detection kit is simple to operate, using a premixed solution system; only the premixed solution and DNA template are needed to complete the reaction, eliminating the need for manual preparation of probes, primers, dNTPs, Mg²⁺, and other components, thus reducing operational errors. It is also compatible with mainstream qPCR instruments (such as ABI, Roche, Bio-Rad, etc.), supports high-throughput automated detection, and can process 96-384 samples per batch, making it suitable for large-scale food screening scenarios. Attached Figure Description

[0041] Figure 1This is a gel image of the positive control plasmid construction. M stands for Marker; lanes 1-9 represent 9 single colonies randomly selected from the ampicillin-resistant plate, with the successful plasmid construction band size being 3944 bp; N is the blank control group of the PCR system.

[0042] Figure 2 It is a key gene for Bacillus cereus vomitoxin. cesB The amplification curve of qPCR detection.

[0043] Figure 3 It is a key gene for non-hemolytic enterotoxin of Bacillus cereus. nheB The amplification curve of qPCR detection.

[0044] Figure 4 It is a key gene for hemolytic enterotoxin in Bacillus cereus. hblD The amplification curve of qPCR detection.

[0045] Figure 5 This is the triple qPCR amplification curve for the virulence gene of Bacillus cereus. Detailed Implementation

[0046] The following disclosure provides many different implementations or examples of different ways to achieve the present invention.

[0047] Example 1: Design of specific primers and probes for key virulence genes in Bacillus cereus.

[0048] Bioinformatics analysis was performed based on the whole genome DNA sequences of *Bacillus cereus* from the GenBank database and our team's own sequencing. Key genes in each virulence gene cluster were selected, and primers and probes were designed using Primer 3. The specificity of the designed primers and probes across all bacterial species was confirmed using the Primer BLAST tool on the NCBI website. While adhering to primer and probe design principles, the amplified fragments were ensured to be within 200 bp. After a series of preliminary screenings and validations, the optimal primers and probes were determined, as detailed in Table 1.

[0049] Table 1. qPCR-specific primer and probe sequences for three virulence genes in Bacillus cereus

[0050] Example 2: Preparation of positive control plasmid for key virulence genes in Bacillus cereus

[0051] (1) Target strain selection: Bacillus cereus ATCC14579 is widely used as a positive control strain for Bacillus cereus detection because it contains nheB and hblDGenes were used to target this strain for the two virulence genes; Bacillus cereus F4810 (also Bacillus cereus F4810 / 72, identical to Bacillus cereus NCTC 11143) was widely used as a positive control strain for Bacillus cereus vomitoxin detection because it contains cesB Genes, using this strain as cesB The target strain of the gene.

[0052] (2) Construction of virulence gene plasmid

[0053] DNA extraction: DNA was extracted from activated Bacillus cereus ATCC14579 and F4810 using a DNA extraction kit.

[0054] Target fragment amplification and vector linearization: Three virulence gene target fragments and the linearized vector pUC19 were amplified using primers listed in Table 7. The amplification system and procedure are shown in Tables 2 and 3. The purified DNA fragments were recovered using an agarose gel DNA recovery kit. The DNA concentration was measured using a nucleic acid concentration analyzer and stored at -20°C.

[0055] Recombination of target fragment and linearized vector: Amount used per fragment = 0.02 × number of fragment bases in ng. The reaction system is shown in Table 4. It was prepared on ice. After preparation, the system was mixed and centrifuged to the bottom of the tube. The tube was then placed in a PCR instrument and reacted at 50°C for 50 min. After the reaction was completed, the recombinant product was placed on ice to cool for 5 min to prevent the temperature from being too high and reducing the transformation efficiency of competent cells.

[0056] Transformation: Thaw competent cells on ice, add the recombinant product to competent cells and mix gently. Incubate on ice for 30 min, then heat shock at 42°C for 90 s, incubate on ice for 2 min, add 900 μL LB liquid medium, and recover in a shaker at 37°C and 200 rpm for 1 h. After recovery, 5000 samples were transferred to the appropriate volume. g Centrifuge for 3 min, remove 900 μL of supernatant, resuspend the bacterial cells in the remaining culture medium, spread on a 100 mg / L ampicillin-resistant plate, and incubate upside down at 37°C overnight.

[0057] Cloning identification: Multiple single colonies were randomly selected and colony PCR was performed using the vector identification primer pairs in Table 7 to identify positive clones. Single colonies from the antibody-resistant plate were dipped into the PCR reaction system and mixed thoroughly. A PCR system without template was used as a quality control. The PCR reaction system and procedure are shown in Tables 5 and 6. Agarose gel electrophoresis was performed at 120 V for 25 min to detect the amplified products. The appearance of a band of 3944 bp indicated successful recombination. Figure 1Single colonies that successfully recombined were picked and placed in 3 mL of ampicillin-resistant LB liquid medium and incubated overnight at 37°C and 200 rpm. The bacterial culture was then subjected to first-generation sequencing using vector identification primers to determine whether the plasmid was successfully constructed.

[0058] Plasmid extraction: Extract the constructed plasmid using a plasmid extraction kit and store at -20℃ for later use.

[0059] The primers and their uses are shown in Table 7.

[0060] Table 2. Target fragment amplification and vector linearization reaction system

[0061] Table 3. Procedure for target fragment amplification and vector linearization

[0062] Table 4 Recombination Reaction System

[0063] Note: X and Y are the amount of target fragment and linearized vector used, respectively, calculated according to the above formula.

[0064] Table 5 Colony PCR Reaction System

[0065] Table 6 Colony PCR Reaction Procedure

[0066] Table 7 Primer sequences for plasmid construction and their uses

[0067] Example 3: Triple qPCR Detection Method for Key Virulence Genes in Bacillus cereus

[0068] (1) Crude extraction of DNA from the sample to be tested: After picking suspected colonies and culturing them continuously for 14 h, take 1 mL of the enrichment broth into a 1.5 mL sterile centrifuge tube, centrifuge at 6000 r / min for 5 min, and completely remove the supernatant; add 30 μL of lysis buffer prepared with 4.9~5.1 mmol / L tris(hydroxymethyl)aminomethane and 0.02~0.12% sodium dodecyl sulfate solution, fully dissolve the precipitate at the bottom of the tube with the lysis buffer, gently tap the tube wall to remove air bubbles, heat at 99 ℃ for 10 min; centrifuge at 12000 r / min for 15 min, the supernatant is the DNA of the sample to be tested, place it on ice, and centrifuge again if not used for a long time. All obtained DNA samples are stored at -20 ℃ for later use.

[0069] (2) qPCR detection: Three sets of qPCR primers and their corresponding probes were used for detection. The DNA obtained in (1) was used as a template, and qPCR amplification was performed using the primers and probes described in Example 1. Fluorescent signals were collected.

[0070] The components of the qPCR reaction system are: 10 μL 2 × qPCR Mix, 0.6 μL forward and reverse primers with an initial concentration of 20 μmol / L, 0.4 μL TaqMan probe with an initial concentration of 10 μmol / L, 2 μL template, and 6.4 μL DEPC water.

[0071] The qPCR amplification reaction program is as follows: the fluorescence channel is set to temperature control; the program is set to 94 ℃ for 60 s, with 40 cycles including 95 ℃ for 20 s, 57 ℃ for 20 s, and 72 ℃ for 30 s; the fluorescence channels of the qPCR instrument are set as follows: channel 1 is FAM, channel 2 is ROX, and channel 3 is VIC; the qPCR instrument automatically collects fluorescence signals in the set fluorescence channels after each cycle.

[0072] (3) Result determination: Based on the fluorescence signal and Ct value collected in (2), determine whether the sample contains one or more of the three virulence genes. When the Ct value is >35 or the system determines it to be negative, the result is determined to be negative.

[0073] Example 4: Construction and optimization of triple qPCR system

[0074] (1) Construction of a single qPCR system: Genomic DNA of the extracted target strains (Bacillus cereus ATCC14579, F4810) was used as a positive control template, and DEPC water was used as a blank control. qPCR reactions were performed using the corresponding primers and probes (refer to the qPCR reaction system and qPCR amplification reaction procedure in Example 3) to preliminarily demonstrate that the designed primer and probe set can detect the corresponding fluorescence after the set procedure. Figures 2 - 4 ).

[0075] (2) Construction of triple qPCR system: The positive control plasmid constructed in Example 2 was used as the positive control template, and DEPC water was used as the blank control. Three pairs of primers and probes were placed in the same tube for triple qPCR reaction. The reaction system consisted of 10 μL 2 × qPCR Mix, 0.6 μL of forward and reverse primers with an initial concentration of 20 μmol / L, 0.4 μL of TaqMan probe with an initial concentration of 10 μmol / L, 2 μL of template, and 3.2 μL of DEPC water. The reaction procedure for qPCR amplification was followed as described in Example 3 to demonstrate that the designed primer and probe set could detect three fluorescences simultaneously without interference or non-specific binding.Figure 5 ).

[0076] (3) Triple qPCR system optimization: First, the single qPCR detection system was optimized by selecting primers with different final concentrations (0.2, 0.4, 0.6, 0.8, 1.0 μmol / L concentration gradients) and probes with concentration gradients (0.10, 0.15, 0.20, 0.25 μmol / L concentration gradients) and performing single-factor optimization experiments (Tables 8-9). Based on the single qPCR method, appropriate primer and probe concentrations were selected and combined, taking into account the cycle threshold (Ct) and fluorescence signal value (Rn) of the amplification curve, so that the three key virulence genes could be detected simultaneously in a single tube system, and finally, the appropriate concentration was selected (Table 10). The triple qPCR detection reaction system is shown in Table 11.

[0077] Table 8. Optimization of primer concentrations (Ct values) for singleton qPCR detection of key virulence genes in Bacillus cereus.

[0078] Table 9. Optimization of probe concentrations for singleton qPCR detection of key virulence genes in Bacillus cereus (Ct values)

[0079] Table 10 Primer and probe concentrations for triple qPCR detection of key virulence genes in Bacillus cereus

[0080] Table 11 Triple qPCR Detection Reaction System for Key Virulence Genes in Bacillus cereus

[0081] Example 5: Specificity Validation of the Triple qPCR System

[0082] To verify the specificity of the triple qPCR system constructed in this invention for comprehensively assessing the toxin-producing ability of Bacillus cereus, genomic DNA from different Bacillus cereus strains was extracted as templates based on the method described in Example 3. Triple qPCR detection was performed according to the triple qPCR detection reaction system in Table 11 and the reaction procedure in Example 3. Each template was tested in triplicate. The positive control plasmid served as the positive control, and DEPC water served as the negative control. The specificity verification results of the triple qPCR system are shown in Table 12. These results demonstrate the high specificity of the qPCR system constructed in this invention.

[0083] Table 12. Strains and Validation Results for Specificity Verification of the Triple qPCR System

Claims

1. A triple qPCR system for comprehensively evaluating the toxin-producing capacity of Bacillus cereus and its application, characterized in that, The triple qPCR system was constructed by targeting the key gene of Bacillus cereus vomitoxin. cesB Key genes for non-hemolytic enterotoxin nheB、 Key genes for hemolytic enterotoxin hblD Specific primers and TaqMan fluorescent probes were designed to simultaneously detect key virulence genes of Bacillus cereus in a single tube, in order to assess the toxin-producing capacity of Bacillus cereus and achieve a reasonable analysis of the toxin risk of the tested organism.

2. The triple qPCR system according to claim 1, characterized in that, The triple qPCR-specific primer and probe set used to comprehensively assess the toxin-producing capacity of Bacillus cereus includes the key gene for Bacillus cereus vomitoxin. cesB Key genes for non-hemolytic enterotoxin nheB、 Key genes for hemolytic enterotoxin hblD Primer and probe set: The detection of Bacillus cereus vomitoxin cesB The primer sequences and TaqMan probe sequences for the gene are as follows: upstream primer cesB -F, downstream primer cesB -R, TaqMan probe cesB -P; The cesB The nucleic acid sequence of -F is shown in SEQ ID NO.1; The cesB The nucleic acid sequence of -R is shown in SEQ ID NO.2; The cesB The nucleic acid sequence of -P is shown in SEQ ID NO.3; The detection of Bacillus cereus non-hemolytic enterotoxin nheB The primer sequences and TaqMan probe sequences for the gene are as follows: upstream primer nheB -F, downstream primer nheB -R, TaqMan probe nheB -P; The nheB The nucleic acid sequence of -F is shown in SEQ ID NO.4; The nheB The nucleic acid sequence of -R is shown in SEQ ID NO.5; The nheB The nucleic acid sequence of -P is shown in SEQ ID NO.6; The detection of Bacillus cereus hemolytic enterotoxin hblD The primer sequences and TaqMan probe sequences for the gene are as follows: upstream primer hblD -F, downstream primer hblD -R, TaqMan probe hblD -P; The hblD The nucleic acid sequence of -F is shown in SEQ ID NO.7; The hblD The nucleic acid sequence of -R is shown in SEQ ID NO.8; The hblD The nucleic acid sequence of -P is shown in SEQ ID NO.

9.

3. The triple qPCR specific primer and probe set according to claim 2, characterized in that, Each virulence gene marker has a different fluorescent quencher group: The probe cesB The 5' end of the -P is labeled with the fluorescent reporter dye FAM, and the 3' end is labeled with the fluorescent quencher group BHQ1. The probe nheB The 5' end of the -P is labeled with the fluorescent reporter dye ROX, and the 3' end is labeled with the fluorescent quencher group BHQ1; The probe hblD The 5' end of the -P is labeled with the fluorescent reporter dye VIC, and the 3' end is labeled with the fluorescent quencher group BHQ1.

4. A triple qPCR reaction system for comprehensively assessing the toxin-producing ability of Bacillus cereus, characterized in that, The triple qPCR reaction system comprises the qPCR-specific primer and probe set as described in any one of claims 2 to 3.

5. The triple qPCR reaction system according to claim 4, characterized in that, The triple qPCR reaction system consisted of: 10 μL 2 × qPCR Mix; 0.8 μL of a solution with an initial concentration of 20 μmol / L. cesB- F, cesB- R, 0.5 μL initial concentration of 10 μmol / L cesB- P; 0.2 μL of an initial concentration of 20 μmol / L nheB- F, nheB- R, 0.4 μL initial concentration of 10 μmol / L nheB- P; 0.4 μL of an initial concentration of 20 μmol / L hblD- F, hblD- R, 0.5 μL initial concentration of 10 μmol / L hblD- P; 2 μL template; 3.8 μL DEPC water.

6. A triple qPCR detection kit for comprehensively evaluating the toxin-producing ability of Bacillus cereus, characterized in that, The triple qPCR detection kit includes the qPCR-specific primer and probe set as described in any one of claims 2 to 3, and further includes a negative control, a positive control, and a lysis buffer.

7. The triple qPCR detection kit according to claim 6, characterized in that, The positive control was based on the key gene of Bacillus cereus vomitoxin. cesB Key genes for non-hemolytic enterotoxin nheB、 Key genes for hemolytic enterotoxin hblD The plasmid was constructed for the target fragment; the negative control was DEPC water; the lysis buffer consisted of 4.9-5.1 mmol / L tris(hydroxymethyl)aminomethane and 0.02-0.12% sodium dodecyl sulfate solution.

8. A triple qPCR detection method for comprehensively evaluating the toxin-producing capacity of Bacillus cereus, characterized in that, The detection method includes the following steps: (1) Extract genomic DNA from the sample to be tested for later use; (2) The genomic DNA of the sample to be tested is added as a template to the triple qPCR reaction system for amplification; (3) Set up the qPCR program, collect the fluorescence signal during the amplification process, and determine the results.

9. A triple qPCR system for comprehensively evaluating the toxin-producing capacity of Bacillus cereus and its application, characterized in that, Applications of the triple qPCR system in food, clinical, and environmental sample testing.

Images