Primer, kit and identification method for identifying bacillus cereus in fermented grains

Abstract

The invention belongs to the technical field of biological detection, and discloses primers, a kit and an identification method for identifying bacillus cereus in fermented grains. The primers for identifying the bacillus cereus in the fermented grains are a pair of specific ARMS primers: C3F and CR, and the nucleotide sequences of the primers are respectively shown as SEQ ID NO: 13 and SEQ ID NO: 15. According to different 23sRNA sequences of the bacillus cereus and other related bacillus, primers are designed by adopting a qPCR technology mutation amplification retarding system, and an ARMS-qPCR detection method of the bacillus cereus is established, so that the bacillus cereus in a complex sample can be rapidly and efficiently detected and identified, the experimental period is shortened, and the detection efficiency is improved. Specificity, sensitivity and doping ratio verification shows that the method can be used for rapid and accurate identification, and compared with traditional morphology and physiological and biochemical identification means which have the defects of large error and low efficiency, the method is simple and convenient to operate, rapid in reaction and capable of achieving accurate qualitative identification.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly to primers, kits and identification methods for identifying Bacillus cereus in fermented grains for liquor brewing. Background Art

[0002] Bacillus is one of the main dominant bacteria in high-temperature stacking. Bacillus cereus, Bacillus subtilis, Bacillus licheniformis, and Bacillus amyloliquefaciens are the main types of Bacillus. These Bacillus have strong abilities to hydrolyze starch and protein, and can produce various flavor compounds during the brewing process of Maotai-flavor liquor to generate complex and rich flavor and aroma characteristics, which play an important role in forming the unique strong aroma and full taste of Maotai-flavor liquor. Among them, Bacillus cereus is a common Bacillus with high production of tetramethylpyrazine during the high-temperature stacking process. This strain participates in the formation of flavor substances such as tetramethylpyrazine during the saccharification and fermentation process, and thus plays a very important role. Distinguishing Bacillus cereus from other strains can judge the growth situation of the strains with high production of tetramethylpyrazine in fermented grains for liquor brewing.

[0003] In view of this, the present invention is specifically proposed. Summary of the Invention

[0004] To solve the problems in the background art, the present invention provides primers, kits and identification methods for identifying Bacillus cereus in fermented grains for liquor brewing, and the primers can quickly and accurately identify Bacillus cereus and other related species.

[0005] To achieve the above object, the first technical solution adopted by the present invention is: Primers for identifying Bacillus cereus in fermented grains for liquor brewing, which are a pair of specific ARMS primers: C3F and CR, and their nucleotide sequences are shown in SEQ ID NO:13 and SEQ ID NO:15 respectively.

[0006] The second technical solution adopted by the present invention is: A kit for identifying Bacillus cereus in fermented grains for liquor brewing, which contains the primers for identifying Bacillus cereus in fermented grains for liquor brewing described in the first technical solution.

[0007] The third technical solution adopted by the present invention is: A method for identifying Bacillus cereus in fermented grains for liquor brewing, which includes the following steps: Extracting the genomic DNA of the strain to be tested; Using the DNA of the strain to be tested as a template, performing a real-time fluorescence quantitative PCR reaction with the primers for identifying Bacillus cereus in fermented grains for liquor brewing described in the first technical solution, and identifying Bacillus cereus from the strains to be detected through the difference in the number of amplification cycles.

[0008] Preferably, the real-time fluorescence quantitative PCR reaction system comprises the following components: the primer, qPCR Mix, and DNA template.

[0009] Preferably, the dosage of the primer is 2.5 µmol / L, 0.8 - 4 μL.

[0010] Preferably, the dosage of the DNA template is 1 - 2 μL.

[0011] Preferably, the real-time fluorescence quantitative PCR reaction conditions are as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, extension at 75°C for 45 s, for 40 cycles, and fluorescence signals are collected at 60°C.

[0012] Preferably, the strains to be detected include Bacillus cereus, Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus.

[0013] Compared with the prior art, the present invention has the following beneficial effects: Based on the differences in the 23sRNA sequences between Bacillus cereus and other related Bacillus species, the present invention designs primers using the qPCR technology mutant amplification blocking system, and establishes an ARMS-qPCR detection method for Bacillus cereus, which can realize the rapid and efficient detection and identification of Bacillus cereus in complex samples, shorten the experimental period, and improve the detection efficiency. After specificity, sensitivity, and admixture ratio verification, it is shown that the present invention can be used for rapid and accurate identification. Moreover, compared with traditional morphological, physiological, and biochemical identification methods, which have the disadvantages of large errors and low efficiency, this method is specifically characterized by simple operation, rapid reaction, and can achieve accurate qualitative identification. Description of the Drawings

[0014] Figure 1 Ct values of the specific primers C3F and CR for 6 related Bacillus species in Example 4; Figure 2 Standard curve of Ct values and DNA concentration for the detection limit of Bacillus cereus by the specific primers C3F and CR in Example 5; Figure 3 Ct values of the specific primers C3F and CR for different ratios of Bacillus cereus admixture in Example 6. Detailed Embodiments

[0015] It should be understood that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs. In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments. The experimental materials used in the embodiments and comparative examples of the present invention are all conventional experimental materials in the art and can be obtained through commercial channels.

[0016] There are many types of Bacillus in the fermented grains. Genes such as gyrB commonly used can identify Bacillus cereus in common food detections, but there are abundant related species in the fermented grains. For example, when using the gyrB universal primer for amplification, the base sequence of Bacillus cereus cannot be obtained, and a large amount of time is required for the screening of universal primers. The present application discovers that directly obtaining specific primers by using the base mismatch of 23sRNA can directly omit the step of using universal primers for amplification, with a simpler operation method and more accurate detection results.

[0017] The embodiment of the present invention uses the 23sRNA sequence of Bacillus cereus in the NCBI GenBank public database for comparison and screening to obtain the required base sequence; designs specific ARMS primers under the guidance of the amplification refractory mutation system (ARMS) technology; extracts the genomic DNA of the sample to be tested by using the Ezup column bacterial gene DNA extraction kit; uses real-time fluorescence quantitative PCR (Quantitative Real-time PCR, qPCR) to identify the target strain from other related strains and common strains in the fermented grains. The present invention combines the real-time fluorescence quantitative PCR technology with the ARMS technology, and designs specific primers C3F and CR for identifying Bacillus cereus in the fermented grains, and their nucleotide sequences are shown in SEQ ID NO:13 and SEQ ID NO:15 respectively, which can distinguish Bacillus cereus in the fermented grains from other related strains. By quantitatively and qualitatively detecting Bacillus cereus, experimental objectives such as quickly and efficiently detecting the content and identifying the presence of Bacillus cereus in complex samples can be achieved.

[0018] The following details the primer design method, identification method and effect for identifying Bacillus cereus in the fermented grains through multiple specific embodiments. In the following specific examples of the present invention, specific ARMS primers are designed using Primer Premier 5, and the real-time fluorescence quantitative PCR primers used in the present invention are synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0019] Example 1 Primer Design The Bacillus cereus 23sRNA sequence with the sequence name NR-076204.1 was screened on the official website of the National Center for Biotechnology Information (NCBI). It was compared with the 23sRNA of other related strains, and 3 segments of sequences were selected to design specific ARMS primers under the guidance of ARMS technology, as shown in Table 1 specifically.

[0020] Table 1 Specific ARMS primer design sites 。

[0021] In the above table, Query is Bacillus cereus, Sbjct is related strains. The base at the 3' end of Site 1 can be used to design the specific base of the ARMS primer, Site 2 can be used as the specific recognition site of the ARMS primer, and Site 3 can be used to design highly specific primers.

[0022] Based on Table 1, a total of 16 pairs (20 pieces) of primers were designed, and the primer sequences are shown in Table 2.

[0023] Table 2 Numbers and sequences of 16 pairs of ARMS-qPCR specific primers 。

[0024] Example 2 Extraction of genomic DNA of strains (1)Liquid culture of strains: Inoculate the beads of 6 experimental strains from the bead preservation tubes and place them into the liquid medium under sterile conditions. Liquid medium: 5 g of yeast extract, 10 g of peptone, 10 g of sodium chloride, pH 7.0 - 7.2, 1 L of distilled water, sterilized at 121 °C for 30 min.

[0025] (2)After culturing for 18 - 24 hours, aspirate 1 mL of the bacterial liquid, centrifuge to obtain the bacterial cells, then extract DNA using the Ezup column bacterial genomic DNA extraction kit, and store it at -20 °C for standby.

[0026] The experimental strains include: Bacillus cereus, Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus.

[0027] Example 3 Verification of the effectiveness of 16 pairs of primers and primer screening Establish a real-time fluorescence quantitative reaction system: The total volume is 20 μL, including 10 μL of qPCR Mix, 0.8 μL each of the upstream and downstream primers (2.5 μmol / L), 1 μL of DNA template, 7.4 μL of ddH2O; establish a real-time fluorescence reaction program: pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 10 s, annealing at 60 °C for 30 s, extension at 75 °C for 45 s, 40 cycles, and collect fluorescence signals at 60 °C.

[0028] The determination criterion for the real-time fluorescence quantitative PCR method is as follows: when the difference in Ct values between the target strain and the non-target strain > 4, it indicates that the ARMS primer can achieve a discrimination effect; when the difference in Ct values between the target strain and the non-target strain ≤ 4, it indicates that the ARMS primer cannot distinguish.

[0029] Since, ① the common strains producing tetramethylpyrazine in fermented grains are mainly Bacillus cereus, Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis, etc.; ② the applicant compared the base sequences from the NCBI gene bank in the early stage and found that only the base sequences of Bacillus cereus and Bacillus thuringiensis are very similar and are the most difficult to distinguish; the base sequences of other strains such as Bacillus licheniformis and Bacillus cereus have relatively large differences and are relatively easy to distinguish. Therefore, in the first step, to verify the effectiveness of the primer, only the most difficult-to-distinguish Bacillus cereus and Bacillus thuringiensis need to be identified. In the first step, only Bacillus cereus and Bacillus thuringiensis are used to verify the effectiveness of 16 pairs of primers. The applicant verified all 16 pairs of primers with 6 kinds of bacteria specifically in the next step, and the results also proved to be feasible.

[0030] Verify the effectiveness of the 16 pairs of ARMS primers in Table 2 with Bacillus cereus and Bacillus thuringiensis and screen out a pair of primers with the best effect. The results are shown in Table 3.

[0031] Table 3 Effectiveness of 16 pairs of ARMS primers .

[0032] Note: NA indicates the undetected data It can be seen from Table 3 that the amplification cycle numbers of primers B1 (B1F + BR), C1 (C1F + CR), C2 (C2F + CR), C3 (C3F + CR), C4 (C4F + CR), D1 (D1F + DR), D4 (D4F + DR) for Bacillus cereus < 15 and the Ct difference from Bacillus thuringiensis is significantly > 4, while A1 (A1F + AR), A2 (A2F + AR), A3 (A3F + AR), A4 (A4F + AR), B2 (B2F + BR), B3 (B3F + BR), B4 (B4F + BR), D2 (D2F + DR), D3 (D3F + DR) do not meet the requirements.

[0033] Determine the amplification efficiency of the 7 pairs of primers B1, C1, C2, C3, C3, D1, D4 screened out. The results are shown in Table 4. It can be obtained from Table 4 that the amplification efficiency of primer C3 is the highest and R^2 > 0.99. Combining Figure 1This pair of primers requires fewer cycles to amplify the gene and takes less time, so it can be used as specific primers to distinguish between *Bacillus cereus* and *Bacillus thuringiensis*. Therefore, the effectiveness of this pair of primers, C3F and CR, can be demonstrated, and this pair of specific ARMS primers is selected to continue with the subsequent experiments.

[0034] Table 4 Amplification efficiency of 7 pairs of specific primers after primary screening 。

[0035] Example 4 Specific detection Select the pair of primers C3F and CR obtained in Example 3, and dilute the DNA of 6 species of bacilli, namely *Bacillus cereus*, *Bacillus thuringiensis*, *Bacillus amyloliquefaciens*, *Bacillus licheniformis*, *Bacillus aerophilus*, and *Bacillus methylotrophicus*, to the same concentration of 10^7 for ARMS-qPCR experiments.

[0036] Specifically, for the DNA of the 6 strains extracted in Example 2, using the DNA of each strain as a template, a real-time fluorescence quantitative reaction system was established according to Example 3 for real-time fluorescence quantitative PCR reactions. The results are as Figure 1 shown.

[0037] From Figure 1 it can be seen that the Ct value difference of *Bacillus cereus* detection is far > 4 compared with the Ct values of other strains. This indicates that this pair of specific ARMS primers, C3F and CR, can effectively distinguish *Bacillus cereus* from other strains and has specificity.

[0038] Example 5 Sensitivity detection The DNA extracted from *Bacillus cereus* was measured for DNA concentration using a microplate reader and serially diluted with deionized water to colony counts (10^7, 10^6, 10^5, 10^4, 10^3 CFU) for ARMS-qPCR experiments. The establishment of the real-time fluorescence quantitative PCR method and the reaction program were the same as in Example 3, and a colony count - Ct value standard curve was made at five points.

[0039] The results are as Figure 2 shown. From Figure 2 it can be seen that the ARMS-qCPR method shows a good linear relationship in the range of DNA concentration from 10^3 to 10^7 CFU, y = -3.69x + 39.15, and the correlation coefficient R^2 = 0.9934. The lowest detection limit LOD is 21.35 CFU. LOD is defined as the concentration at three times the standard deviation subtracted from the CT value when no *Bacillus cereus* DNA is added. This indicates that this method has the ability for quantitative detection and good sensitivity.

[0040] Example 6 Spiking detection of the ARMS-qPCR method Using Bacillus cereus as the target strain, Bacillus thuringiensis with the highest genetic affinity to the target strain was selected as the background bacterium. The DNAs of the two bacteria were mixed at a certain ratio (the number of DNA colonies was 10^7 for both), and the volume ratios of Bacillus cereus were 0%, 0.5%, 1%, 10%, 30%, 50%, 70%, 90%, and 100% respectively, and the ARMS-qPCR experiment was carried out. The establishment of the real-time fluorescence quantitative PCR method and the reaction procedure were the same as those in Example 3.

[0041] The results are as Figure 3 shown. It can be seen from Figure 3 the figure that as the ratio of Bacillus cereus increases, the Ct value becomes smaller and smaller. When the content of Bacillus cereus is greater than 0.5%, the Ct difference is greater than 4. Based on this, it is determined that this method can detect Bacillus cereus with a volume ratio of more than 0.5%.

[0042] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, various changes and modifications can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Primers for identifying Bacillus cereus in fermented grains, characterized in that, A pair of specific ARMS primers: C3F and CR, whose nucleotide sequences are shown in SEQ ID NO:13 and SEQ ID NO:15 respectively.

2. Kit for identifying Bacillus cereus in fermented grains, characterized in that, Comprising the primers for identifying Bacillus cereus in fermented grains as described in claim 1.

3. Method for identifying Bacillus cereus in fermented grains, characterized in that, Comprising the following steps: Extracting genomic DNA of the strain to be tested; Using the DNA of the strain to be tested as a template, performing a real-time fluorescence quantitative PCR reaction with the primers for identifying Bacillus cereus in fermented grains as described in claim 1, and identifying Bacillus cereus from the strains to be detected through the difference in the number of amplification cycles.

4. The method for identifying Bacillus cereus in fermented grains according to claim 3, wherein The real-time fluorescence quantitative PCR reaction system comprises the following components: the primers, qPCR Mix, and DNA template.

5. The method for identifying Bacillus cereus in fermented grains as claimed in claim 4, characterized in that, The dosage of the primers is 2.5 μmol / L, 0.8 - 4 μL.

6. The method for identifying Bacillus cereus in fermented grains as claimed in claim 4, wherein The dosage of the DNA template is 1 - 2 μL.

7. The method for identifying Bacillus cereus in fermented grains according to claim 3, wherein The real-time fluorescence quantitative PCR reaction conditions are: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, extension at 75°C for 45 s, 40 cycles, and collecting fluorescence signals at 60°C.

8. The method for identifying Bacillus cereus in fermented grains according to claim 3, characterized in that, The strains to be tested include Bacillus cereus, Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis, and Bacillus pumilus.

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