gRNA, detection system, and identification method for identifying Bacillus licheniformis in fermented mash

Abstract

The present invention belongs to the technical field of microbial detection, and discloses a gRNA, a detection system and an identification method for identifying Bacillus licheniformis in wine mash. The gRNA used to identify Bacillus licheniformis in wine mash consists of a conserved repeat sequence that binds to the Cas12a protein and a spacer sequence that specifically binds to the target, and the spacer sequence is SEQ ID NO: 4. The present invention combines ARMS-PCR technology with CRISPR / Cas12a technology to design a gRNA for identifying Bacillus licheniformis in wine mash, which can distinguish Bacillus licheniformis from other closely related species in wine mash. By quantitatively and qualitatively detecting Bacillus licheniformis, experimental goals such as rapid and efficient detection of the content and identification of the presence of Bacillus licheniformis in complex samples can be achieved. The present invention is simple to operate, has a shorter reaction time, good repeatability and high accuracy, and the results can be obtained in only 60 min; it has high sensitivity, with a detection limit of 1.32×10^2 CFU, and the detection results in complex samples are stable and have good specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and in particular to gRNA, a detection system and an identification method for identifying Bacillus licheniformis in fermented mash. Background Art

[0002] Currently, most researchers identify Bacillus licheniformis in mash through morphological, physiological, and biochemical analyses, as well as the construction of 16S rDNA phylogenetic trees. Traditional identification methods based on morphological, physiological, and biochemical analyses are time-consuming, and identification results are easily affected by environmental conditions and user experience. Furthermore, the mash contains numerous closely related Bacillus species, making them difficult to distinguish. 16S rDNA gene sequence identification is simple, fast, and highly efficient, but requires gel electrophoresis to obtain amplified fragments and sequence them. The 16S rDNA phylogenetic tree constructed from the sequencing results is then used to accurately identify the strain. Furthermore, due to the high interspecies homology of 16S rDNA sequences, reaching as high as 98.1-99.8%, closely related Bacillus strains can only be identified as groups, and species identification is difficult.

[0003] ARMS-qPCR technology uses specific primers to amplify and detect Bacillus licheniformis. This technology has strong specificity, high sensitivity, and high reproducibility. The experiment can be completed in the same centrifuge tube to avoid external contamination. However, the reagents used are expensive and the instrument usage requirements are high, resulting in high costs and is not conducive to promotion in practical applications.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to solve at least one problem in the background technology and provides gRNA, a detection system and an identification method for identifying Bacillus licheniformis in mash.

[0006] In order to achieve the above object, the first technical solution adopted by the present invention is:

[0007] The gRNA is used to identify Bacillus licheniformis in fermented mash, wherein the gRNA consists of a conserved repeat sequence that binds to the Cas12a protein and a target-specific binding spacer sequence, and the spacer sequence is SEQ ID NO: 4.

[0008] The second technical solution adopted in the present invention is:

[0009] The detection system for identifying Bacillus licheniformis in mash comprises: the gRNA, Cas12a protein and fluorescently modified single-stranded DNA reporter molecule described in the first technical solution, which is used to amplify the ARMS-PCR primer pair containing the 16S rRNA variable region V8 where SEQ ID NO: 4 is located.

[0010] Preferably, the upstream primer of the primer pair is SEQ ID NO: 8, and the downstream primer is SEQ ID NO: 9.

[0011] The third technical solution adopted in the present invention is:

[0012] The method for identifying Bacillus licheniformis in wine mash comprises using the detection system for identifying Bacillus licheniformis in wine mash described in the second technical solution to perform ARMS-PCR amplification and CRISPR / Cas12a fluorescence detection.

[0013] Preferably, the specific method is to extract the genomic DNA of the strain to be tested, use the DNA of the strain to be tested as a template, use the detection system for identifying Bacillus licheniformis in the mash to perform ARMS-PCR amplification and CRISPR / Cas12a fluorescence detection for quantitative analysis, and identify and determine the content of Bacillus licheniformis in the mash according to the fluorescence intensity.

[0014] Preferably, the ARMS-PCR amplification system comprises: genomic DNA of the strain to be tested, primers, and PCR premix.

[0015] Preferably, the PCR reaction program is: preheating at 95°C for 5 min, reaction at 95°C for 15 s, reaction at 60°C for 30 s, and reaction at 72°C for 30 s, for a total of 20 cycles.

[0016] Preferably, the CRISPR / Cas12a fluorescence detection system comprises: ARMS-PCR amplification product, Cas12a, gRNA, ssDNA reporter, Cas12a buffer; the reaction conditions are: reaction at 37°C for 45-60 minutes.

[0017] Preferably, the strains to be tested include Bacillus licheniformis, Bacillus subtilis, Bacillus aerobicus, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus thuringiensis, Bacillus pumilus, Bacillus ginseng and Bacillus methylotrophicus.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This study combines ARMS-PCR with CRISPR / Cas12a technology to design a guide RNA (gRNA) for identifying Bacillus licheniformis in fermented mash. This method can distinguish Bacillus licheniformis from other closely related species in fermented mash. This quantitative and qualitative detection of Bacillus licheniformis allows for rapid and efficient detection and identification of its presence in complex samples. This method offers simple operation, a shorter reaction time, excellent reproducibility, and high accuracy, with results available in just 60 minutes. It also demonstrates high sensitivity, with a limit of detection (LOD) of 1.32×10^2 CFU, consistent results in complex samples, and excellent specificity.

[0020] The present invention uses the ARMS-PCR coupled with CRISPR / Cas12a method to identify multiple Bacillus strains based on nucleic acid detection and detect the fluorescence intensity after nucleic acid amplification. Compared with qPCR, it reduces gel image processing, does not require expensive instruments and equipment, has high sensitivity, clear and rapid results, and has low technical requirements for operators, providing new ideas and methods for the detection of Bacillus licheniformis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the fluorescence curve of gRNA1 and gRNA2 in Example 2 for detecting Bacillus licheniformis and Bacillus amyloliquefaciens in the CRISPR / Cas12a system;

[0022] Figure 2 This is the fluorescence curve of gRNA3 and gRNA4 in Example 2 for detecting Bacillus licheniformis and Bacillus amyloliquefaciens in the CRISPR / Cas12a system;

[0023] Figure 3 This is the fluorescence curve of gRNA5 and gRNA6 in Example 2 for detecting Bacillus licheniformis and Bacillus amyloliquefaciens in the CRISPR / Cas12a system;

[0024] Figure 4 A is the fluorescence curve of gRNA7 in Example 2 for detecting Bacillus licheniformis and Bacillus amyloliquefaciens in the CRISPR / Cas12a system, and B is the ratio of the signal to the background of gRNA1-gRNA7 for detecting the CRISPR / Cas12a system;

[0025] Figure 5 Example 3 shows the fluorescence curves of the ARMS-PCR coupled CRISPR / Cas12a method in each reaction step;

[0026] Figure 6 A is the fluorescence intensity of Bacillus licheniformis, Bacillus amyloliquefaciens and water in Example 3, and B is the electrophoresis analysis verification of different steps in Example 3;

[0027] Figure 7 Where A is the fluorescence intensity and S / B ratio of the detection system at different ARMS-PCR cycle numbers in Example 4, B is the fluorescence intensity and S / B ratio of different Cas12a protein and gRNA molar ratios in the detection system in Example 4, C is the fluorescence intensity and S / B ratio of different Cas12a protein and reporter molecule molar ratios in Example 4, and D is the fluorescence intensity and S / B ratio of the detection system at different reaction times in Example 4;

[0028] Figure 8 A is the relationship between the number of Bacillus licheniformis and the fluorescence intensity in Example 5, and B is the fluorescence spectrum of Example 5 with different numbers of Bacillus licheniformis added;

[0029] Figure 9 This is the result of Cas12P doping determination in Example 6;

[0030] Figure 10 The fluorescence intensity changes of different Bacillus species determined by Cas12P in Example 7 are as follows: 1: Bacillus licheniformis, 2: Bacillus amyloliquefaciens, 3: Bacillus cereus, 4: Bacillus subtilis, 5: Bacillus aerobicus, 6: Bacillus methylotrophicus, 7: Bacillus thuringiensis, 8: Bacillus ginseng, and 9: Bacillus pumilus. DETAILED DESCRIPTION

[0031] It should be understood that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this application belongs. In order to enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below in conjunction with specific embodiments. The experimental materials used in the embodiments of the present invention and the comparative examples are all conventional experimental materials in the art and can be purchased through commercial channels.

[0032] The present invention uses ARMS-PCR to amplify a large number of target genes in Bacillus licheniformis to activate the CRISPR / Cas12a protein. Comparison of bacterial 16S rRNA sequences revealed a single base difference between Bacillus licheniformis and other Bacillus species. Therefore, using Primer 5.0, specific primers were designed for the SNP site. ARMS-PCR was then used to amplify the specific gene in Bacillus licheniformis, allowing for specific differentiation between different Bacillus species.

[0033] ARMS-PCR amplification using upstream primer 2-2 and downstream primer 2-R (located in the variable region V8 of the 16S rRNA) generates a large amount of target sequence. Double-stranded PCR products containing the CRISPR / Cas12a spacer sequence are recruited to the Cas12a-gRNA ribonucleoprotein complex through specific hybridization. Hybridization of the gRNA with the target DNA activates the trans-cleavage activity of Cas12a, which allows it to cleave single-stranded DNA molecules. This allows it to cleave the fluorescently modified ssDNA reporter molecule in the system (two segments modified with a fluorophore (6-carboxy-fluorescein, FAM) and a quencher (Black hole quencher 1, BHQ1), respectively), resulting in a fluorescent signal. In the absence of the target sequence, the Cas12a-gRNA complex exhibits no cleavage activity, resulting in quenching of the 6-FAM fluorophore by BHQ1, resulting in low fluorescence. In this way, the presence of Bacillus licheniformis in the system will cause a fluorescence change, which can be conveniently measured using a fluorometer.

[0034] By combining ARMS-PCR with CRISPR / Cas12a technology, a guide RNA (gRNA) for identifying Bacillus licheniformis in fermented mash (Zhang et al., 2017) was designed. Its nucleotide sequence is shown in SEQ ID NO: 4, which can distinguish Bacillus licheniformis from other closely related species in fermented mash. By quantitatively and qualitatively detecting Bacillus licheniformis, the experimental goal is to rapidly and efficiently detect and identify its presence in complex samples.

[0035] Example 1 Preparation of reagent samples and DNA extraction of test strains

[0036] LB medium: Add 1 g of peptone, 0.5 g of yeast extract, and 1 g of sodium chloride to neutralize the pH. Then add 0.1 L of distilled water and sterilize at 121°C for 20 min.

[0037] Seed solution preparation: Place 2-3 Bacillus bacteria stored in magnetic beads into sterilized culture medium cooled to room temperature, mix well, and culture on a shaker at 37°C and 120 rpm / min for 24 h.

[0038] DNA extraction: Take 0.5-1 mL of the overnight culture of the test strain and add it to a 2.5 mL centrifuge tube. Centrifuge at 8000 rpm / min for 1 min at room temperature. Discard the supernatant and retain the bacterial precipitate. Extract DNA according to the instructions of the Ezup column-based bacterial genomic DNA extraction kit.

[0039] The strains tested included Bacillus licheniformis, Bacillus subtilis, Bacillus aerobicus, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus thuringiensis, Bacillus pumilus, Bacillus ginseng, and Bacillus methylotrophicus. In subsequent DNA extraction and testing, the Bacillus amyloliquefaciens strain was treated in the same manner as Bacillus licheniformis.

[0040] Example 2 Design of gRNA and Optimization of Recognition Sites

[0041] The NCBI database (http: / / www.ncbi.nlm.nih.gov / ) was used to search for 16S rRNA sequences of different Bacillus species, and DNAstar was imported for sequence alignment to find the base differences between Bacillus licheniformis and other common Bacillus species. Primer Premier 5.0 was used to design upstream and downstream primers for amplification and sequencing based on the position of the bases to verify the NCBI alignment results. The sequencing results showed that Bacillus licheniformis differed from other Bacillus species by a single base. ARMS-PCR primers could be designed for specific amplification to distinguish Bacillus licheniformis from other Bacillus species, and the amplified products were sequenced to obtain relevant sequences for gRNA design. The gRNA consists of a repeat sequence (the fixed part that binds to Cas12a) and a spacer sequence (the part that binds to the double-stranded hybridization). In the Cas12a system, a stem-loop structure is formed by the repeat sequence "UAAUUUCUA CUAAGUGUAGAU", and the spacer sequence is designed to be complementary to the target sequence. A T-rich protospacer adjacent motif (PAM) (TTN, where N represents A, T, G, or C) was found in the sequence of the Bacillus licheniformis ARMS-PCR amplification product, which is used to activate the Cas12a protein. Therefore, the gRNA consists of two parts: a 21nt conserved repeat sequence at the front end that is used to recognize and bind to the Cas12a protein, and a 20nt spacer region at the back end that is complementary to the target DNA. Based on the gRNA design principles, seven gRNAs were obtained, which contain target binding sites and guide sites. The gRNA encoding DNA sequence information is shown in Table 1. The ssDNA reporter molecules are modified with FAM and BHQ1 at both ends, and the sequence information is shown in Table 2.

[0042] Table 1 Coding DNA sequences of 7 gRNAs

[0043] .

[0044] Table 2 Other DNA sequences

[0045] .

[0046] The choice of gRNA site can greatly affect the fluorescence response of the CRISPR-Cas12a system, so we first optimized it and selected gRNA4 with the highest signal-to-noise ratio among gRNA1-gRNA7. Figure 1-4 As shown in the figure, when gRNA4 is selected as the recognition site, the signal / background (S / B) ratio is the highest. Compared with other gRNA sites, the S / B ratio of gRNA4 is significantly improved to 8.95, while the S / B ratios of other sites range from 1 to 3, all lower than gRNA4. This optimization result significantly improves the signal-to-noise ratio. Therefore, gRNA4 was finally selected as the recognition site, that is, the spacer sequence of the gRNA used to identify Bacillus licheniformis in fermented grains is shown in SEQ ID NO: 4.

[0047] Example 3 Principle feasibility verification

[0048] To confirm the feasibility of the working principle of the ARMS-PCR coupled CRISPR / Cas12a method, the applicant performed fluorescence analysis at each step of the detection method.

[0049] Detection system and method: A 25 µL PCR mixture contained 1 µL of total DNA, 1 µL of upstream primer 2-2 (2.5 µM), 1 µL of downstream primer 2-R (2.5 µM), and 12.5 µL of PCR premix, prepared in 9.5 µL of H₂O. The PCR reaction program consisted of 95°C preheating for 5 minutes, followed by 20 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds to obtain amplified products. 1 µL of ARMS-PCR product, 4 µL of Cas12a protein (1 µM), 4 µL of gRNA4 (10 µM), 4 µL of ssDNA reporter (10 µM), 4 µL of Cas12a buffer, and 21 µL of H₂O were mixed and incubated at 37°C for 60 minutes. The fluorescence spectrum was measured using a microplate reader Synergy H1. The slit width of the emission light and the excitation light were both 2 nm. During the detection process, the excitation wavelength was 488 nm, and the emission wavelength range was 510 nm to 600 nm.

[0050] like Figure 5As shown, first, adding the target sequence, gRNA, reporter and Cas12a protein alone will not increase the fluorescence signal value. It can be seen from the detection of the microplate reader that the fluorescence intensity is about 20~60, which shows that adding only a single component cannot enhance the fluorescence intensity. Further adding gRNA or gRNA and reporter to the target sequence will still result in a lower fluorescence intensity. The former has a fluorescence intensity of 22, and the latter is 671, indicating that adding gRNA or gRNA and reporter on the basis of the spacer sequence cannot activate the cleavage degree of the reporter gene. When the target sequence, gRNA, reporter and Cas12a protein are all added to the system, the fluorescence intensity is significantly enhanced from 671 to 24800. The addition of the target sequence can activate the cleavage activity of the Cas12 / gRNA system, thereby shearing the fluorescently modified ssDNA reporter molecules in the system and emitting a fluorescent signal. The results show that this method is more effective in activating the Cas12a / gRNA system, and shearing the reporter gene leads to an increase in fluorescence intensity.

[0051] like Figure 6 As shown in A, the specificity of the ARMS-PCR coupled CRISPR / Cas12a method was further verified by adding equal amounts of Bacillus licheniformis, Bacillus amyloliquefaciens and water for detection. The results showed that only Bacillus licheniformis could cause an increase in the fluorescence signal.

[0052] like Figure 6 As shown in B, reagents were added according to different steps for electrophoresis analysis. The results showed that the addition of target sequence, gRNA, and signal molecule alone would produce corresponding bands. When Cas12a was added to the system and compared with the system without Cas12a, it was found that only the addition of Cas12a could bind to gRNA and activate its trans-cleavage activity to shear the single strand in the system, thereby weakening the added signal molecule band.

[0053] Therefore, the ARMS-PCR coupled CRISPR / Cas12a method can be used for the specific detection of Bacillus licheniformis.

[0054] Example 4 Optimization of the detection system for identifying Bacillus licheniformis in fermented grains

[0055] The concentrations of gRNA and fluorescent reporter genes can significantly affect the fluorescence reaction in CRISPR / Cas12a nucleic acid detection, while optimizing the number of PCR cycles and reaction time can minimize time and increase detection speed. Therefore, the number of PCR cycles, gRNA concentration, fluorescent reporter gene concentration, and Cas12a reaction time were optimized, and the fluorescence intensity under different reaction conditions was measured to obtain an optimal reaction system.

[0056] like Figure 7 As shown in A, as the number of PCR cycles increases, the fluorescence intensity also increases. However, when the number of cycles exceeds a certain number, the control group Bacillus amyloliquefaciens also produces amplification products, and the fluorescence intensity increases, which leads to a decrease in the signal-to-noise ratio of the two. Therefore, when the number of cycles is 18x, the signal-to-noise ratio is the highest. Figure 7 As shown in Figure B, gRNA4 is conducive to the enhancement of fluorescence intensity within a certain concentration range. When the ratio of Cas12 protein to gRNA4 is 1:4, the maximum S / B ratio reached is 18.66; subsequently, the background fluorescence intensity increases sharply, resulting in a decrease in the S / B ratio. Figure 7 As shown in Figure C, the increase in reporter gene concentration can significantly increase the fluorescence intensity. When the ratio of Cas12 protein to reporter gene is 1:10, the maximum S / B ratio reached is 25.80. Figure 7 As shown in Figure D, increasing the reaction time is beneficial to the increase of fluorescence intensity within a certain range. However, when the reaction time exceeds 30 minutes, the fluorescence intensity of the control group also increases, causing the signal-to-noise ratio of the two to drop rapidly. Therefore, when the reaction time is 30 minutes, the signal-to-noise ratio is 26.48.

[0057] Therefore, by optimizing the reaction conditions, the optimal identification system was an ARMS-PCR cycle of 18x, a Cas12 protein to gRNA ratio of 1:4, a Cas12 protein to reporter gene ratio of 1:10, and a reaction time of 30 min, at which time the fluorescence intensity was the strongest and the signal-to-noise ratio was the highest at 26.48.

[0058] Example 5 Sensitivity detection of ARMS-PCR coupled CRISPR / Cas12a method

[0059] The ARMS-PCR coupled CRISPR / Cas12a detection method is referred to as Cas12P below. The applicant tested the detection performance of the method of the present invention under the optimal experimental conditions. The detection system and method were the same as those in Example 3. A series of different amounts of total DNA extracted from Bacillus licheniformis (0-6×10^7 CFU, 0, 6×10 2 , 6×10 3 , 6×10 4 , 6×10 5 , 6×10 6 , 1.2×10 7 , 2.4×10 7 , 3.6×10 7 , 4.8×10 7 , 6×10 7 CFU) were added to the reaction system ( Figure 8). As the total DNA concentration increases, the fluorescence intensity gradually increases. The total DNA concentration range of 6×10^2 -6×10^7 CFU shows a good linear relationship with the fluorescence intensity. Figure 8 As shown in Figures A and B, ARMS-PCR coupled with CRISPR / Cas12a was used to detect different numbers of added Bacillus licheniformis. A linear relationship was observed between 6×10^2 and 6×10^7 CFU. The linear regression equation for Bacillus licheniformis detection was y=389.7x+1010.9 (R²=0.982), where x and y represent the number of Bacillus licheniformis and the fluorescence intensity of the CRISPR / Cas12a system, respectively. The limit of detection (LOD) obtained by the present method was 1.32×10^2 CFU. This limit of detection was calculated from the concentration corresponding to the fluorescence intensity at three times the standard deviation of the no-DNA control. The measured fluorescence intensity can then be used to identify and calculate the content of Bacillus licheniformis in the mash.

[0060] Example 6 ARMS-PCR coupled CRISPR / Cas12a method for doping detection

[0061] Bacillus licheniformis and Bacillus amyloliquefaciens with a total bacterial count of 6×10^6 CFU were mixed in a certain proportion (the volume ratio of the Bacillus licheniformis solution added ranged from 0% to 100%, namely 0.5%, 1%, 10%, 20%, 40%, 60%, 80% and 100% respectively). The nucleic acid test of DNA with different mixing ratios was performed in triplicate using the ARMS-PCR coupled CRISPR / Cas12a method (same as Example 3). The results showed that as the proportion of Bacillus licheniformis increased, the fluorescence intensity gradually increased. There was a good linear relationship between the number of Bacillus licheniformis added in the doping range of 20%-100% and the fluorescence intensity. Figure 9 As shown, the ARMS-PCR coupled with CRISPR / Cas12a method was used to detect the addition of different proportions of Bacillus licheniformis, and a linear relationship was found between 20% and 100%. The linear regression equation for the total number of Bacillus licheniformis was y=1854.9x+2894.3, R2=0.997.

[0062] Example 7 Specific detection of ARMS-PCR coupled CRISPR / Cas12a method

[0063] Nine Bacillus species were selected as specific detection targets, namely Bacillus licheniformis, Bacillus subtilis, Bacillus aerobicus, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus thuringiensis, Bacillus pumilus, Bacillus ginseng, and Bacillus methylotrophicus. DNA extracted from these Bacillus species according to the method of Example 3 was detected and analyzed by ARMS-PCR coupled with CRISPR / Cas12a. The results are shown in Figure 3. Figure 10 As shown, the Cas12P method was used to detect different Bacillus species. When the target sequence was introduced into the system, the fluorescence intensity increased dramatically to 28,900. The fluorescence intensity of non-target strains was far less than 10% of that of Bacillus licheniformis, approaching a state of no signal. The specific detection of Bacillus licheniformis among nine Bacillus strains allowed its growth to be monitored during actual fermentation, providing data guidance for subsequent process optimization.

[0064] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A gRNA for identifying Bacillus licheniformis in fermented grains, characterized in that: The gRNA consists of a promoter, a conserved repeat sequence that binds to the Cas12a protein, and a target-specific binding spacer sequence, the spacer sequence is SEQ ID NO: 4, the sequence of the promoter is SEQ ID NO: 10, and the conserved repeat sequence is SEQ ID NO:

12.

2. A detection system for identifying Bacillus licheniformis in fermented grains, characterized in that: Comprising: the gRNA according to claim 1, the Cas12a protein and the fluorescently modified single-stranded DNA reporter molecule, for amplifying an ARMS-PCR primer pair comprising the 16S rRNA variable region V8 where SEQ ID NO: 4 is located, the upstream primer of the primer pair is SEQ ID NO: 8, the downstream primer is SEQ ID NO: 9, and the fluorescently modified single-stranded DNA reporter molecule is SEQ ID NO:

11.

3. A method for identifying Bacillus licheniformis in fermented grains, characterized in that: The method comprises using the detection system for identifying Bacillus licheniformis in mash according to claim 2 to perform ARMS-PCR amplification and CRISPR / Cas12a fluorescence detection.

4. The method for identifying Bacillus licheniformis in fermented grains according to claim 3, wherein: The specific method is to extract the genomic DNA of the strain to be tested, use the DNA of the strain to be tested as a template, use the detection system for identifying Bacillus licheniformis in the mash to perform ARMS-PCR amplification and CRISPR / Cas12a fluorescence detection for quantitative analysis, and identify and determine the content of Bacillus licheniformis in the mash based on the fluorescence intensity.

5. The method for identifying Bacillus licheniformis in fermented grains according to claim 4, wherein: The ARMS-PCR amplification system includes: genomic DNA of the strain to be tested, primers, and PCR premix.

6. The method for identifying Bacillus licheniformis in fermented grains according to claim 4, wherein: The PCR reaction program was as follows: preheating at 95°C for 5 min, reaction at 95°C for 15 s, reaction at 60°C for 30 s, and reaction at 72°C for 30 s, for a total of 20 cycles.

7. The method for identifying Bacillus licheniformis in fermented grains according to claim 4, wherein: The CRISPR / Cas12a fluorescence detection system includes: ARMS-PCR amplification product, Cas12a, gRNA, single-stranded DNA reporter molecule, and Cas12a buffer; the reaction conditions are: reaction at 37°C for 30 min.

8. The method for identifying Bacillus licheniformis in fermented grains according to claim 4, wherein: The strains to be tested include Bacillus licheniformis, Bacillus subtilis, Bacillus aerobicus, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus thuringiensis, Bacillus pumilus, Bacillus ginseng and Bacillus methylotrophicus.

Images