A kit for detecting bacillus cereus and application thereof

Abstract

The present application relates to a kind of kit for Bacillus cereus detection and its application, the kit includes RPA amplification reaction system and CRISPR-Cas13a protease detection system;Wherein, RPA amplification reaction system includes RPA amplification primer pair, CRISPR-Cas13a protease detection system includes sgRNA, Cas13a protein and probe.The present application also provides corresponding detection method, by the kit under constant temperature condition, it can be realized to Bacillus cereus detection, with short detection time, easy operation, detection condition is mild, without the advantages such as expensive professional instrument auxiliary.This kit and corresponding detection method have good application prospect, have potential application in clinical detection.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, specifically to a kit for detecting Bacillus cereus and its application. Background Technology

[0002] Bacillus cereus is a facultative anaerobic, rod-shaped, Gram-positive bacterium belonging to the genus Bacillus in the family Bacillusaceae. It is abundant in soil, water, plants, feed, and various foods. This bacterium produces endospores and is highly adaptable to its environment, resisting adverse conditions such as high temperatures, ultraviolet radiation, electromagnetic radiation, and harmful chemicals. On the one hand, some strains, like many others in the genus such as Bacillus subtilis and Bacillus brevis, can secrete various active substances that antagonize bacteria, fungi, and nematodes, making them effective active bacteria for preventing plant diseases and killing pathogens. On the other hand, some strains of the genus Bacillus cereus are also major pathogens, easily causing food contamination and food poisoning. Their clinical manifestations are mainly diarrhea and vomiting.

[0003] Methods for detecting Bacillus cereus mainly include PCR, multiplex PCR (mPCR), real-time quantitative PCR (RT-qPCR), droplet digital PCR (ddPCR), loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), enzyme-linked immunosorbent assay (ELISA), and commercial toxin detection kits. All of these methods have drawbacks, such as low accuracy, susceptibility to non-specific amplification or cross-reactivity, high cost, false positives, difficulty in primer design, lack of real-time microbial detection capabilities, and the possibility of missed or false positives. Among these, RPA technology has low equipment requirements, good specificity, and significantly shortens detection time, making it widely applicable in clinical and field testing. It also provides new directions for food quality monitoring and disease detection. However, on the one hand, due to the difficulty in precisely controlling primer recombination and extension, the batch robustness of amplification remains limited, making it susceptible to environmental changes. On the other hand, low operating temperatures may generate multiple non-target amplifications, resulting in unexpected noise signals. Therefore, there is an urgent need for a simple, efficient, and sensitive detection technology.

[0004] Professor Zhang Feng's team has offered a novel interpretation of the detection capabilities of RPA technology, demonstrating how the combination of isothermal amplification and gene editing technologies has created a new platform for the development of nucleic acid isothermal amplification detection. Research shows that RPA can be combined with a gene editing system called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas13a, achieving attomolar-level detection sensitivity (attomolar = 10⁻³ fmol), capable of identifying single-base differences. The researchers have given this combination a new name: SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing).

[0005] Therefore, combining RPA technology with the CRISPR / Cas13a gene editing system can enable detection to have the advantages of low variability, high sensitivity, high accuracy, simple application scenarios, and no need for expensive instruments and equipment. Summary of the Invention

[0006] The purpose of this invention is to provide a kit for the detection of Bacillus cereus and its application. This kit enables the detection of Bacillus cereus under isothermal conditions, offering advantages such as short detection time, simple operation, mild detection conditions, and no need for expensive specialized instruments. This kit and corresponding detection method have promising application prospects and potential for clinical testing.

[0007] Therefore, in a first aspect, the present invention provides a reagent combination for the detection of Bacillus cereus, comprising an RPA amplification primer pair, sgRNA, Cas13a protein and a probe;

[0008] The RPA amplification primer pair is selected from at least one of the following groups: the primer pair shown in SEQ ID NO: 1 and SEQ ID NO: 2, the primer pair shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the primer pair shown in SEQ ID NO: 5 and SEQ ID NO: 6; the sequence of the sgRNA is shown in SEQ ID NO: 7.

[0009] sgRNA binds to Cas13a protein to form a complex. After adding the amplification product obtained by RPA amplification primer pair, the complex recognizes and binds to the target RNA sequence, and the Cas13a protein is activated. The activated Cas13a protein cleaves the probe, thereby emitting a detectable signal.

[0010] According to the technical solution of the present invention, those skilled in the art can design crRNA and tracrRNA that can be assembled into the single guide RNA (sgRNA) described in the present invention; if the sgRNA in the technical solution of the present invention is replaced with the corresponding crRNA and tracrRNA, the technical solution is still within the protection scope of the present invention.

[0011] In some embodiments of the present invention, the probe is a fluorescent probe, such as a single-stranded RNA with a fluorescent group and a quenching group attached to both ends.

[0012] Furthermore, the 5' end of the fluorescent probe is modified with a fluorescent group selected from the group consisting of FAM, HEX, ROX, CY5, Cy3, TET, JOE, and VIC; the 3' end of the fluorescent probe is modified with a quenching group selected from the group consisting of BHQ, Dabcy1, TAMRA, and MGB.

[0013] In some embodiments, the fluorescent probe is 5'-FAM-AUAGCUAC-BHQ1-3'.

[0014] In a second aspect, the present invention provides a kit for detecting Bacillus cereus, comprising an RPA amplification reaction system and a CRISPR-Cas13a protease detection system;

[0015] The RPA amplification reaction system includes RPA amplification primer pairs, wherein the RPA amplification primer pairs are selected from at least one of the following groups: primer pairs shown in SEQ ID NO: 1 and SEQ ID NO: 2, primer pairs shown in SEQ ID NO: 3 and SEQ ID NO: 4, and primer pairs shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0016] The CRISPR-Cas13a protease detection system includes sgRNA, Cas13a protein, and probe; the sequence of the sgRNA is shown in SEQ ID NO: 7.

[0017] Furthermore, the RPA amplification reaction system also includes RPA amplification reaction buffer and polymerase.

[0018] In some embodiments, the RPA amplification reaction buffer comprises RPA Buffer, Mg(CH3COO)2. For example, appropriate reagents provided in RPA kits available from Twist DX, UK, can be used.

[0019] Furthermore, in the PRA amplification reaction system, the concentration of each RPA amplification primer pair is 10-20 μM.

[0020] Furthermore, the CRISPR-Cas13a protease detection system also includes a CRISPR-Cas13a protease buffer and an RNase inhibitor.

[0021] Furthermore, the CRISPR-Cas13a protease buffer includes Fncas13a Buffer. For example, it can be prepared using a 10×Fncas13a Bufffer purchased from New England Biolabs (NEB).

[0022] Furthermore, the kit also includes a negative control and a positive control, wherein the positive control is a T-vector containing the target sequence for Bacillus cereus detection; and the negative control is ultrapure water.

[0023] A third aspect of the present invention provides a method for detecting Bacillus cereus using a kit provided by the present invention, comprising:

[0024] (1) Extract nucleic acid from the sample to be tested as a template for testing;

[0025] (2) The template to be tested is added to the RPA amplification reaction system for amplification to obtain the amplification product;

[0026] (3) The amplification product was added to the CRISPR-Cas13a protease detection system, and the detection signal was collected under isothermal conditions.

[0027] The detection method for Bacillus cereus according to the present invention is for non-diagnostic purposes.

[0028] Furthermore, when the probe is a fluorescent probe, the detection signal is a fluorescent signal.

[0029] Furthermore, the amplification reaction temperature is 35-45℃, such as 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, etc.; the amplification inversion time is 15-30min, such as 15min, 20min, 25min, 30min, etc.

[0030] Furthermore, the isothermal conditions are 35-45℃, such as 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, etc.

[0031] Based on the detection results of the fluorescence signal, if an amplification curve appears, the result is considered positive; if no amplification curve appears, the result is considered negative.

[0032] Compared with the prior art, the present invention has the following advantages:

[0033] (1) The kit and detection method provided by this invention combine RPA technology with the CRISPR / Cas13a system. This kit achieves high-sensitivity detection through isothermal amplification of Cas13a. Specifically, sgRNA binds to the Cas13a protein to form a complex. After adding the amplification product obtained from the RPA amplification primer pair, the complex recognizes and binds to the target RNA sequence, activating the Cas13a protein. The activated Cas13a protein cleaves the probe, thereby emitting a detectable signal. This detection method features low variability and high sensitivity.

[0034] (2) The reagent kit and detection method provided by the present invention can be used for detection under constant temperature conditions, and have the advantages of short detection time, simple operation, mild detection conditions and no need for expensive professional instruments.

[0035] (3) The kit and detection method provided by the present invention have good application prospects and have the potential to be applied to clinical testing. Attached Figure Description

[0036] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. In the drawings:

[0037] Figure 1 A flowchart of the Bacillus cereus detection method provided by the present invention;

[0038] Figure 2 The results show the comparison of amplification efficiencies of different RPA amplification primer pairs;

[0039] Figure 3 The results show the specificity analysis of the RPA amplification primer pairs.

[0040] Figure 4 The sensitivity analysis results of the Bacillus cereus detection method provided by the present invention;

[0041] Figure 5 The results show the specificity analysis of the Bacillus cereus detection method provided by this invention. Detailed Implementation

[0042] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0043] Reagents and materials sourced as follows: RPA kit (RPA Buffer, Mg(CH3COO)2) was purchased from Twist DX, UK; Fncas13a enzyme and 10×Fncas13a Bufffer were purchased from New England Biolabs (NEB); RNase inhibitors were purchased from Shanghai Sangon Biotech Co., Ltd.

[0044] Example 1

[0045] This embodiment investigated the amplification efficiency of RPA amplification primer pairs for Bacillus cereus. The amplification efficiency of three pairs of Bacillus cereus RPA primers was studied while maintaining the composition of each component in the reaction system.

[0046] The amplification reaction system consisted of: 1 μL BC-F (10 μM), 1 μL BC-R (10 μM), 0.3 μL 1×syto-9, 20.5 μL LPA Buffer, 1.2 μL Mg(CH3COO)2 (280 mM), 50 ng template (T vector containing the target sequence for Bacillus cereus detection), and ultrapure water to a final volume of 25 μL. The amplification program was set to 42℃ for 30 s pre-denaturation, followed by 15 s denaturation at 42℃, annealing and extension at 37℃ for 15 s, for a total of 40 cycles.

[0047] The RPA amplification primer pairs for Bacillus cereus are shown in Table 1.

[0048] Table 1. Primer pairs for Bacillus cereus RPA amplification.

[0049]

[0050] Amplification results as follows Figure 2 As shown, according to Figure 2 As can be seen, all three RPA amplification primer pairs provided in Table 1 can specifically amplify the target sequence. Among them, the BC-F1 and BC-R1 primer pairs have the best amplification rate and will be used in subsequent examples.

[0051] Example 2

[0052] This embodiment uses 50 clinically negative Bacillus cereus samples as a specificity evaluation index to analyze the specificity of the Bacillus cereus RPA amplification primer pairs provided in Example 1. The 50 clinically negative Bacillus cereus samples were amplified according to the RPA amplification reaction system and amplification procedure described in Example 1, with both positive and negative controls included. The template for the positive control was a T-vector containing the target sequence for Bacillus cereus detection; no template was added to the negative control.

[0053] Test results as follows Figure 3 As shown, according to Figure 3 Only the positive control showed an "S"-shaped amplification curve, while the negative control and 50 clinically negative samples did not show amplification. This indicates that the RPA amplification primer pair provided by this invention has good specificity.

[0054] Example 3

[0055] This embodiment provides a kit for detecting Bacillus cereus, including an RPA reaction system and a CRISPR-Cas13a protease detection system.

[0056] The RPA reaction system is shown in Table 2.

[0057] Table 2 RPA Reaction System

[0058] reagent components Dosage (μL) RPA Buffer 20.5 BC-F 1 BC-R 1 Syto-9 0.3 <![CDATA[Mg(CH3COO)2]]> 1.2 template 2≤X≤10 <![CDATA[ddH2O]]> Up to 25

[0059] The CRISPR-Cas13a protease detection system is shown in Table 4.

[0060] Table 3. Base sequences required for CRISPR-Cas13a protease detection

[0061]

[0062]

[0063] Table 4 CRISPR-Cas13a protease detection system

[0064] reagent components Dosage (μL) NTP Buffer Mix 10 T7 transcriptase 3 sgRNA 1 Report sequence 5 <![CDATA[Mg(CH3COO)2]]> 3 RPA reaction products 1 RNase-free Up to 25

[0065] Example 4

[0066] This embodiment provides a method for detecting Bacillus cereus, including the following steps:

[0067] I. Nucleic Acid Sample Extraction

[0068] (1) Take 200 μL of the sample to be tested, add 400 μL of lysis buffer, and heat in a water bath at 80℃ for 10 min;

[0069] (2) Add 20 μL of nucleic acid adsorption magnetic beads, vortex for 30 s, let stand for 5 min, transfer the reaction tube to the magnetic rack for adsorption for 1-5 min, and discard or aspirate the solution.

[0070] (3) After adding 1000 μL of 80% ethanol solution and vortexing, transfer the reaction tube to a magnetic rack for adsorption for 5 min, and then discard the solution;

[0071] (4) Repeat the previous step;

[0072] (5) Use a magnetic rod to collect the magnetic beads in the tube, transfer them to a new centrifuge tube, and add 150 μL of nucleic acid elution buffer. Use the prepared nucleic acid sample as a template for subsequent steps.

[0073] II. Recombinase Polymerase Isothermal Amplification Reaction

[0074] The RPA reaction system shown in Table 2 of Example 3 was prepared, and an isothermal amplification reaction was carried out under the following conditions: incubation at 37°C for 15 min. The amplification product was then obtained.

[0075] III. CRISPR-Cas13a protease isothermal detection reaction and fluorescence detection

[0076] Take the amplification product obtained in step two and prepare the CRISPR-Cas13a protease detection system shown in Table 4 of Example 3. Place the prepared CRISPR-Cas13a protease isothermal detection reaction system in an ABI 7500 real-time PCR instrument and set the program as follows: 37℃ pre-denaturation for 30s, 37℃ denaturation for 45s, 37℃ annealing and extension for 15s, for 40 cycles.

[0077] IV. Result Interpretation

[0078] According to the experimental results, those showing an amplification curve are positive, while those without an amplification curve are negative.

[0079] Example 5

[0080] This embodiment analyzes the sensitivity of the detection method for Bacillus cereus provided by the present invention. The specific steps are as follows:

[0081] Using a T-vector containing the target sequence for Bacillus cereus detection as a positive plasmid, the plasmid was extracted and quantified using NanoDrop One. The positive plasmid was diluted to 1 pg / μL, 100 fg / μL, 10 fg / μL, 1 fg / μL, 100 ag / μL, and 10 ag / μL as templates. The detection method described in Example 4 was used to detect different concentrations of the templates. The detection results are as follows: Figure 4 As shown.

[0082] Figure 4 In the figure, the curves from left to right represent the amplification results of positive plasmids at concentrations of 1 pg / μL, 100 fg / μL, 10 fg / μL, 1 fg / μL, 100 ag / μL, and 10 ag / μL, respectively. It can be seen that the detection method for Bacillus cereus of the present invention has a sensitivity of up to 100 ag / μL, and the detection kit and detection method for Bacillus cereus of the present invention have high sensitivity for the diagnosis of Bacillus cereus.

[0083] Example 6

[0084] This embodiment analyzes the specificity of the detection method for Bacillus cereus provided by the present invention. The specific steps are as follows:

[0085] Fifty clinically negative Bacillus cereus samples were used as specificity evaluation indicators, and the samples were tested according to the detection method in Example 4. The test results are as follows: Figure 5 As shown, only the positive plasmid exhibited an "S"-shaped amplification curve, while the negative control and 50 clinically negative Bacillus cereus samples showed no amplification curve. This indicates that the detection method for Bacillus cereus of the present invention has high specificity.

[0086] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A reagent combination for detection of Bacillus cereus, characterized in that, The RPA amplification primer pair, sgRNA, Cas13a protein and probe are included. The RPA amplification primer pair is shown in SEQ ID NO: 1 and SEQ ID NO:

2. The sequence of the sgRNA is shown in SEQ ID NO:

7. The sequence of SEQ ID NO: 1 is CAGAATTCCCCTTTTCCGTCG. The sequence of SEQ ID NO: 2 is ACCCCAAAATAGAGGTCACCAT. The sequence of SEQ ID NO: 7 is: UAAUUUCUACUAAGUGUAGAUAUAUGCAUAAUUGCAUAAUAAAAC.

2. A kit for detection of Bacillus cereus comprising, The RPA amplification reaction system and the CRISPR-Cas13a proteinase detection system are included. The RPA amplification reaction system includes the RPA amplification primer pair, and the RPA amplification primer pair is shown in SEQ ID NO: 1 and SEQ ID NO:

2. The CRISPR-Cas13a proteinase detection system includes sgRNA, Cas13a protein and probe, and the sequence of the sgRNA is shown in SEQ ID NO:

7. The sequence of SEQ ID NO: 1 is CAGAATTCCCCTTTTCCGTCG. The sequence of SEQ ID NO: 2 is ACCCCAAAATAGAGGTCACCAT. The sequence of SEQ ID NO: 7 is: UAAUUUCUACUAAGUGUAGAUAUAUGCAUAAUUGCAUAAUAAAAC.

3. The kit of claim 2, wherein The RPA amplification reaction system further includes RPA amplification reaction buffer and polymerase.

4. The kit of claim 3, wherein The RPA amplification reaction buffer includes RPA Buffer and Mg(CH3COO)2.

5. The kit of claim 3, wherein In the RPA amplification reaction system, the concentration of the RPA amplification primer pair is 10-20 μM for each primer.

6. The kit of claim 2, wherein The CRISPR-Cas13a proteinase detection system further includes CRISPR-Cas13a proteinase buffer and RNase inhibitor.

7. The kit of claim 2, wherein The kit further includes negative control and positive control.

8. The kit of claim 7, wherein The positive control is a T vector containing the target sequence of Bacillus cereus detection, and the negative control is ultrapure water.

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