A specific composition, kit for detecting bacillus cereus and bacillus thuringiensis
By using primers and probes with specific compositions to distinguish between Bacillus cereus and Bacillus thuringiensis, the problems of difficulty in differentiation and long detection cycles in existing technologies have been solved, achieving rapid and accurate detection results.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENZHEN ACAD OF METROLOGY & QUALITY INSPECTION
- Filing Date
- 2026-05-09
- Publication Date
- 2026-07-14
AI Technical Summary
Existing technologies are difficult to effectively distinguish between Bacillus cereus and Bacillus thuringiensis, and the detection cycle is long. Conventional methods are prone to misjudgment, and the high resolution required by instruments is difficult to achieve.
A specific composition containing primer pairs and probes for detecting the isp and XRE genes was used to distinguish between Bacillus cereus and Bacillus thuringiensis by real-time fluorescent PCR. Primer A was used to narrow down the screening range, and primer B was used for further determination.
It enables rapid molecular detection of Bacillus cereus and Bacillus thuringiensis with good specificity and high sensitivity, and is suitable for conventional fluorescent PCR instruments.
Smart Images
Figure CN122382218A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of gene detection technology, specifically relating to a specific composition and kit for detecting Bacillus cereus and Bacillus thuringiensis. Background Technology
[0002] Bacillus cereus is a Gram-positive bacterium widely distributed in the environment and is one of the common foodborne pathogens. Bacillus cereus can cause food poisoning in infants and adults by contaminating dairy products, rice, unpackaged cooked meat products, and soy products. Symptoms of Bacillus cereus poisoning mainly include vomiting and diarrhea, and the resulting poisoning incidents are often quite severe.
[0003] Because Bacillus cereus and Bacillus thuringiensis have highly similar biochemical characteristics, existing national standard detection methods for Bacillus cereus still suffer from long detection cycles and potential for misdiagnosis. Currently, there are some studies both domestically and internationally on rapid molecular detection of Bacillus cereus and Bacillus thuringiensis, involving methods such as conventional PCR, multiplex PCR, fluorescent PCR, digital PCR, loop-mediated isothermal amplification (LAM), and recombinase polymerase amplification. However, most of these studies target two specific sequences: virulence genes and conserved sequences such as housekeeping genes. Additionally, some studies employ high-resolution melting curve analysis, a method based on PCR that uses high-resolution melting to detect single-base differences in target fragments.
[0004] Existing research on rapid molecular detection of Bacillus cereus and Bacillus thuringiensis faces several challenges: ① Bacillus cereus possesses numerous virulence genes, which are not unique to Bacillus cereus but may also exist in other microorganisms, making identification impossible through individual virulence genes; ② The conserved sequences of housekeeping genes (such as groEL, gyrB, vrrA, and rpoB) between Bacillus cereus and Bacillus thuringiensis share over 99% homology, making effective differentiation impossible; ③ High-resolution melting curves require instruments with high resolution (0.1-1.0℃ / s) to distinguish single-base differences, necessitating extremely high temperature uniformity. However, the interwell temperature difference of most real-time fluorescence PCR instruments fails to meet these requirements, limiting the applicability of this method.
[0005] Therefore, this invention aims to provide a more reliable and effective molecular detection scheme for Bacillus cereus and Bacillus thuringiensis. Summary of the Invention
[0006] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a specific composition and kit for detecting Bacillus cereus and Bacillus thuringiensis, which can realize rapid molecular detection of Bacillus cereus and Bacillus thuringiensis with good specificity and high sensitivity.
[0007] Specifically, the present invention provides a specific composition for detecting Bacillus cereus and Bacillus thuringiensis, comprising the following A and B: A. Primer pairs and probes used to detect the isp gene, the nucleotide sequences of which are shown in SEQ ID NO:1-3; B. Primer pairs and probes for detecting the XRE gene, the nucleotide sequences of which are shown in SEQ ID NO:4-6.
[0008] The specific composition of this invention comprises two sets of primers and probes. The primer pair and probe (designated as primer A) with nucleotide sequences as shown in SEQ ID NO:1-3 can distinguish *Bacillus cereus*, *Bacillus thuringiensis*, and other common pathogens, thus narrowing the screening scope. Then, the primer pair and probe (designated as primer B) with nucleotide sequences as shown in SEQ ID NO:4-6 are used for further detection and determination of suspected *Bacillus cereus* and *Bacillus thuringiensis* pathogens. The corresponding determination scheme can be found in Table 1.
[0009] Table 1 Judgment Scheme
[0010] Preferably, the probe in the specific composition is labeled with a fluorescent group at its 5' end and a quenching group at its 3' end.
[0011] The present invention also provides the use of the above-mentioned specific composition in the preparation of products for detecting Bacillus cereus and Bacillus thuringiensis.
[0012] The present invention also provides a kit for detecting Bacillus cereus and Bacillus thuringiensis, comprising the above-mentioned specific composition.
[0013] This invention also provides a method for detecting Bacillus cereus and Bacillus thuringiensis for non-diagnostic purposes, comprising the following steps: (1) Extract DNA from the sample to be tested; (2) Using the extracted DNA as a template, perform real-time fluorescent PCR reaction using the above-mentioned specific composition; (3) Collect fluorescence signals to obtain the detection results of the sample to be tested.
[0014] It should be noted that the A primer probe and the B primer probe in the above specific composition should be added to different reaction systems for reaction, but they can be detected on the same instrument (i.e., corresponding to different detection units).
[0015] Preferably, the reaction program for the real-time fluorescence PCR reaction in step (2) is: 95℃ for 30s, 95℃ for 5s, 60℃ for 34s, for a total of 40 cycles.
[0016] Preferably, the reaction system of the real-time fluorescence PCR reaction in step (2) includes: real-time fluorescence PCR reaction reagent, template, the above-mentioned specific composition, and ddH2O.
[0017] More preferably, in the reaction system, the concentration of each primer is 10 μmol / L and the concentration of each probe is 10 μmol / L.
[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: The specific composition of this invention comprises two sets of primer probes. Primer probe A can distinguish Bacillus cereus and Bacillus thuringiensis from other foodborne pathogens, thus narrowing down the screening scope. Primer probe B can further detect and determine suspected Bacillus cereus and Bacillus thuringiensis pathogens. Using the specific composition proposed in this invention, rapid molecular detection of Bacillus cereus and Bacillus thuringiensis can be achieved with good specificity and high sensitivity. Attached Figure Description
[0019] Figure 1 The results of real-time fluorescence PCR detection of strains numbered 1-6 using primer A probe in Example 4; Figure 2 The results of real-time fluorescent PCR detection of strains numbered 1-6 using primer probe B in Example 4; Figure 3 The results of real-time fluorescent PCR detection of Bacillus cereus at different bacterial concentrations using primer A probe in Example 6; Figure 4 The results of real-time fluorescent PCR detection of Bacillus thuringiensis at different bacterial concentrations using primer A probe in Example 6; Figure 5 The results are shown in Example 6, which uses the B primer probe to detect Bacillus thuringiensis at different bacterial concentrations using real-time fluorescent PCR. Detailed Implementation
[0020] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention. Unless otherwise specified, the raw materials, reagents, or apparatus used in the following embodiments can be obtained from conventional commercial sources or by existing known methods.
[0021] Example 1: Specific composition for detecting Bacillus cereus and Bacillus thuringiensis This embodiment provides a specific composition for detecting Bacillus cereus and Bacillus thuringiensis, comprising the following A and B: A. Primer pairs and probes (referred to as primer A and probe A) used to detect the isp gene, the nucleotide sequences of which are shown in SEQ ID NO: 1-3; B. Primer pairs and probes (referred to as B primers and probes) used to detect the XRE gene, the nucleotide sequences of which are shown in SEQ ID NO:4-6.
[0022] isp-F: 5'-CGCTGTACAAACACCACAAGGA-3' (SEQ ID NO: 1); isp-R: 5'-ACGTTCCACGAGACTTGCAT-3' (SEQ ID NO: 2); isp-P: 5'-AGAAGCTCATAGAAGYGCRAAGCARAGT-3' (SEQ ID NO: 3); XRE-F: 5'-AGCGGTAAGATCACAGAGGAAG-3' (SEQ ID NO: 4); XRE-R: 5'-AATTCCGAAATAAGTTCCACCAT-3' (SEQ ID NO: 5); XRE-P: 5'-AAAGCTGGAACATTAGAACCTTGGGAAGA-3' (SEQ ID NO: 6).
[0023] Both the isp-P probe and the XRE-P probe mentioned above are labeled with the fluorescent group FAM at the 5' end and the quenching group BHQ at the 3' end.
[0024] Example 2: Kit for detecting Bacillus cereus and Bacillus thuringiensis This embodiment provides a kit for detecting Bacillus cereus and Bacillus thuringiensis, comprising the specific composition of Example 1.
[0025] Example 3: Real-time fluorescence PCR detection method for Bacillus cereus and Bacillus thuringiensis This embodiment provides a real-time fluorescent PCR detection method for Bacillus cereus and Bacillus thuringiensis, including the following steps: (1) A commercially available bacterial genomic DNA extraction kit was used to extract DNA from the sample to be tested; (2) Using the extracted DNA as a template, real-time fluorescent PCR detection was performed using the specific composition in Example 1; The following reaction systems were constructed using primer A and primer B, respectively, and the reaction procedure was executed: Reaction system: 10 μL of real-time fluorescent PCR reaction reagent (2X), 0.4 μL of upstream primer (10 μmol / L), 0.4 μL of downstream primer (10 μmol / L), 0.2 μL of probe (10 μmol / L), 1-10 ng of DNA template (Qubit fluorescence method), and sterile ultrapure water to 20 μL; Reaction program: 95℃ for 30s, 95℃ for 5s, 60℃ for 34s, for a total of 40 cycles.
[0026] (3) Collect fluorescence signals to obtain the detection results of the sample to be tested; The judgment scheme is shown in Table 2.
[0027] Table 2 Judgment Scheme
[0028] Example 4: Detection of Bacillus cereus and Bacillus thuringiensis standard bacteria This embodiment targets the following 6 bacterial strains (see Table 3 for details) and uses the real-time fluorescence PCR detection method described in Example 3 for detection.
[0029] Table 3 Information on the 6 bacterial strains in Example 4
[0030] Test results as follows Figure 1-2 As shown. When using primer A, strains numbered 1-6 were all detected; when using primer B, only strain number 5 was detected.
[0031] Example 5: Specificity Detection This embodiment targets the following 28 bacterial strains (see Table 4 for details), and uses the real-time fluorescence PCR detection method described in Example 3 for detection.
[0032] Table 4 Information on the 28 bacterial strains in Example 5
[0033] The test results showed that none of the 28 bacterial strains showed an amplification curve, meaning they were not detected, regardless of whether primer A or primer B was used.
[0034] Example 6: Sensitivity Detection In this embodiment, after overnight culture of Bacillus cereus and Bacillus thuringiensis, 1 mL of bacterial suspension was serially diluted 10-fold with 0.85% physiological saline and detected using the real-time fluorescence PCR detection method in Example 3 to verify the bacterial suspension sensitivity of the primer probe.
[0035] Test results as follows Figure 3-5 As shown (where numbers 1 to 6 represent bacterial concentrations of 10⁻⁶), 7 CFU / mL, 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 (CFU / mL), the results showed that the detection limit of primer A probe for Bacillus cereus was 10. 2 The limit of detection for Bacillus thuringiensis in bacterial suspension was 10 CFU / mL. 2 CFU / mL; the detection limit of primer B for Bacillus thuringiensis culture is 10. 2 CFU / mL.
[0036] Example 7: Actual Sample Testing This embodiment uses 43 actual samples obtained by the applicant in a food safety sampling inspection as the testing objects. The real-time fluorescence PCR detection method in Embodiment 3 of this invention and the national standard GB 4789.14-2014 "Food Microbiology Examination - Bacillus cereus Examination" were used for testing to verify the applicability and accuracy of the method of this invention. The test results are shown in Table 5.
[0037] Table 5. Detection results of 43 actual samples in Example 7
[0038] As can be seen from the above, the detection results of the two methods are consistent, indicating that the method established in this invention can accurately and reliably detect and identify Bacillus cereus and Bacillus thuringiensis.
[0039] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.
Claims
1. A specific composition for detecting Bacillus cereus and Bacillus thuringiensis, characterized in that, Including the following A and B: A. Primer pairs and probes used to detect the isp gene, the nucleotide sequences of which are shown in SEQ ID NO:1-3; B. Primer pairs and probes for detecting the XRE gene, the nucleotide sequences of which are shown in SEQ ID NO:4-6.
2. The use of the specific composition according to claim 1 in the preparation of products for detecting Bacillus cereus and Bacillus thuringiensis.
3. A kit for detecting Bacillus cereus and Bacillus thuringiensis, characterized in that, It comprises the specific composition according to claim 1.
4. A method for detecting Bacillus cereus and Bacillus thuringiensis for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Extract DNA from the sample to be tested; (2) Using the extracted DNA as a template, perform real-time fluorescent PCR reaction using the specific composition described in claim 1; (3) Collect fluorescence signals to obtain the detection results of the sample to be tested.
5. The detection method according to claim 4, characterized in that, The reaction program for the real-time fluorescence PCR reaction described in step (2) is: 95℃ for 30s, 95℃ for 5s, 60℃ for 34s, for a total of 40 cycles.
6. The detection method according to claim 4, characterized in that, The reaction system for the real-time fluorescence PCR reaction in step (2) includes: real-time fluorescence PCR reaction reagents, template, the specific composition described in claim 1, and ddH2O.
7. The detection method according to claim 6, characterized in that, In the reaction system, the concentration of each primer is 10 μmol / L, and the concentration of each probe is 10 μmol / L.