Kit and method for detecting microorganisms of distiller's yeast
By using the RNA probes of LbuCas13a and TtCsm6 proteins that bind crRNA-13 and F-A4U6 in the Jiuko Microbial Detection Kit, a live bacteria detection without reverse transcription and nucleic acid amplification was achieved, solving the problems of cumbersome detection and low sensitivity in the prior art, and improving the detection limit and specificity.
Patent Information
- Application Number
- CN202411857530.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-02-25
AI Technical Summary
The prior art has problems such as high instrument and operator requirements, cumbersome operation, non-specific amplification and false positive results when detecting live bacteria. The detection methods based on CRISPR are not sensitive and are difficult to deal with micro-detection.
Jiuko microbial detection kit is provided, which includes LbuCas13a protein, TtCsm6 protein, primer crRNA-13, primer F-A4U6, fluorophore and fluorophore quenching groups. Through isothermal detection, no reverse transcription and nucleic acid amplification is required, and the output signal is cascaded amplified by the CRISPR/Cas system.
It realizes detection without reverse transcription and nucleic acid amplification, improves detection limit, reduces false positive errors, has high specificity and high sensitivity, and can quickly and easily detect the viable microbial content.
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Figure CN119506447A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microbial detection, and in particular to a kit and method for detecting koji microbial detection. Background Art
[0002] Microbial testing is very important in the food production process, especially live bacteria testing. Fermented foods account for one-third of the human diet, have huge market value and output, and are recognized to be beneficial to human health. Koji fermentation is a complex process involving the interaction between several microorganisms, which play a vital role in fermentation production and significantly affect the flavor and quality of fermented products. Detecting live bacteria in the koji fermentation process is crucial to understanding the fermentation process, understanding the fermentation mechanism, optimizing fermentation conditions, improving product quality and developing new products. At present, the main methods for detecting live bacteria are traditional methods (culture medium), molecular detection (PCR nucleic acid detection), and enzyme-linked immunosorbent assay (ELISA).
[0003] Nucleic acid-based detection directly targets specific regions of RNA, has high accuracy and sensitivity, and is an ideal method for molecular diagnosis. RNA is only stable in living bacterial cells. Along with cell apoptosis, bacteria will begin to degrade, resulting in leakage of contents, activation of intracellular RNA degradation enzymes, etc., and RNA will also be rapidly cleaved. Therefore, RNA is also an effective target for detecting living bacteria. In standard nucleic acid detection, the target RNA is reverse transcribed into complementary DNA (cDNA), and then amplified by PCR or isothermal amplification to produce a large number of copies of the target sequence. However, this method has high requirements for instruments and operators, cumbersome operation process, non-specific amplification, and prone to aerosol contamination and false positive results. The use of CRISPR-based detection methods further improves the specificity of RNA detection. In the CRISPR / Cas system, Cas13a was developed as a highly specific gene detection and editing tool, but its sensitivity is not high, and the detection limit can only reach the pM level, which is difficult to cope with trace detection. Summary of the invention
[0004] In order to solve the problems in the background technology, the present invention provides a koji microorganism detection kit and a detection method. The kit can calculate the content of live microorganisms by detecting microbial RNA nucleic acid, without amplifying the microbial nucleic acid, and improves the detection limit.
[0005] To achieve the above object, the first technical solution adopted by the present invention is: A koji microorganism detection kit, comprising: LbuCas13a protein, TtCsm6 protein, primer crRNA-13, primer F-A4U6, and RNA probes double-labeled with a fluorescent group and a fluorescent quenching group; Among them, the primer crRNA-13 is the guide RNA corresponding to the LbuCas13a protein, and the primer crRNA-13 sequence includes a fixed sequence and a binding sequence, wherein the binding sequence is a 20nt fragment that can be complementary to the microbial RNA, and the binding sequence is located at the 3' end of the fixed sequence; Preferably, the fermentation microorganisms include lactic acid bacteria, Bacillus, Pediococcus and any one of the microorganisms involved in the fermentation process of koji.
[0006] Preferably, when the microbial RNA is Bacillus licheniformis, the primer crRNA-13 sequence is as shown in SEQ ID NO.1.
[0007] Preferably, the molar ratio of LbuCas13 protein, TtCsm6 protein, primer crRNA-13, primer F-A4U6, fluorescent group and fluorescent quenching group double-labeled RNA probe is 4:10:4:100:(10 -6 ~0.1):20 Preferably, the sequence of the RNA probe is shown as SEQ ID NO.3.
[0008] Preferably, it further comprises 10×LbuCas13a buffer, which is composed of 200 mM HEPES, 500 mM KCl, 20% glycerol, 50 mM MgCl2, 100 μg ml-1 bovine serum albumin (BSA), and 0.1% Igepal CA630 at pH=7.0.
[0009] The second technical solution adopted by the present invention is: The method for detecting koji microorganisms for non-diagnostic purposes is carried out using the koji microorganism detection kit described in any one of the first technical solutions, and comprises the following steps: Provide LbuCas13a protein, TtCsm6 protein, primer crRNA-13, primer F-A4U6, a fluorescent group and a fluorescent quenching group double-labeled RNA probe; The LbuCas13a protein was mixed with the primer crRNA-13, and the first mixed solution was obtained after incubation at room temperature for 15 minutes; Add primer F-A4U6, TtCsm6 protein, RNA probe and RNA to be tested to the first mixed solution and incubate at 37° C. for 20 to 60 minutes to obtain a second mixed solution; measuring the peak fluorescence intensity of the second mixed solution; The presence of the target microbial RNA and the viable bacterial content of the target microorganism when the target microbial RNA is present are determined based on the peak value of the fluorescence intensity of the second mixed solution.
[0010] Preferably, the preparation method of primer crRNA-13 is as follows: 4 μL 10 μM crRNA-13 DNA template chain is mixed with 4 μL 10 μM promoter and 4 μL 10X phi 29 buffer, and then added to 10 μL ultrapure water. After incubation at 90°C for 5 min, incubate at 37°C for 30 min, continue to add 0.5 μL phi 29 pol and 2 μL dNTP and incubate at 37°C for 30 min, then add 8 μL 5X TransBuffer, 4 μL rNTP, and 1 μL 20 U / μL T7 pol and incubate at 37°C overnight for 12 to 18 h; after the incubation is completed, 8 μL 5X DNase I buffer and 4 μL DNase I pol are added to the system and incubated at 37°C for 0.5 to 4 h, and finally incubated at 75°C for 10 min.
[0011] When the fermentation microorganism is Bacillus licheniformis, the DNA template chain of crRNA-13 is MB-13, and the sequence is shown in SEQ ID NO.4.
[0012] Compared with the prior art, the present invention has the following beneficial effects: 1) The detection process of the kit of the present invention does not require a reverse transcription process, and isothermal detection can be performed.
[0013] 2) During the detection process of the kit of the present invention, there is no need to amplify the microbial nucleic acid, which effectively reduces false positive errors and outputs signals through the cascade amplification of the CRISPR / Cas system.
[0014] 3) The detection method of the present invention has high specificity and is designed according to the 16S rRNA of the detected microorganism, and will hardly produce a positive reaction to other non-target microorganisms.
[0015] 4) The detection method of the present invention has high sensitivity, and the minimum detection limit can be as low as 500 CFU / mL.
[0016] 5) The present invention can process complex fermentation samples and only requires simple pre-treatment, which is convenient and fast and can be applied to on-site rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 It is a schematic diagram of the principle of detecting the content of live bacteria of fermented microorganisms provided by the present invention; the green irregular ellipse represents LbuCas13a; the blue ellipse dimer represents TtCsm6; FAM, fluorescent dye group; BHQ1, quenching group; Figure 2 This is a schematic diagram of the principle of detecting the content of live bacteria of fermentation microorganisms in Comparative Example 2; Figure 3 It is the standard curve drawn in Example 2; Figure 4 This is the result of detecting Bacillus licheniformis during the fermentation process of koji in Example 3; Figure 5 This is the result of the selective test on other microbial RNA in Example 4. DETAILED DESCRIPTION
[0018] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments.It should be understood that these descriptions are exemplary only, and are not intended to limit the scope of the present invention.In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention.Unspecified conditions in the embodiments are carried out according to the conditions of normal conditions or manufacturer recommendations.Reagents used or instruments that do not specify manufacturers are conventional products that can be purchased and obtained commercially.
[0019] The principle of the present invention for detecting nucleic acid of koji microorganisms is: crRNA-13 that can complement and pair with the specific 16S rRNA sequence of the microorganism is designed, and crRNA-13 can bind to LbuCas13a. When the specific RNA of the target microorganism is present, it is accurately recognized and bound by crRNA-13, changing the conformation of the LbuCas13a protein and activating the "bystander" activity of indiscriminate RNA cutting. Due to the influence of "bystander", the primer F-A4U6 in the detection system is cleaved to produce the primer F-A4>P with a phosphate group (>P) at the 3' end. The primer F-A4>P can bind to the specific domain of TtCsm6, change the conformation of TtCsm6, and activate the "bystander" activity of TtCsm6 indiscriminate RNA cutting. Then, TtCsm6 will cut the RNA probe with fluorescent groups and quenching groups modified at both ends, that is, C5-Reporter is cut, resulting in a significant increase in fluorescence. On the contrary, when there is no target microbial specific RNA present, C5-Reporter cannot be cut and displays low fluorescence values.
[0020] It should be noted that the RNA of the target microorganism to be detected is also referred to as Target-13 below, and its sequence can be artificially designed according to different microbial detection needs. The following specific embodiments only take Bacillus licheniformis as an example. The Target-13 used in the following specific embodiments is the total RNA extracted from Bacillus licheniformis.
[0021] The detection kit and the detection method of the present invention can be used to detect the content of live microorganisms in the fermentation process.
[0022] The first embodiment of the present invention provides a koji microorganism detection kit, comprising: LbuCas13a protein, TtCsm6 protein, primer crRNA-13, primer F-A4U6, and an RNA probe double-labeled with a fluorescent group and a fluorescent quenching group; Among them, the primer crRNA-13 is the guide RNA corresponding to the LbuCas13a protein, and the primer crRNA-13 sequence includes a fixed sequence and a binding sequence. The binding sequence is a 20nt fragment that can complement the microbial RNA, and the binding sequence is located at the 3' end of the fixed sequence.
[0023] Understandably, the sequence of primer crRNA-13 varies depending on the microorganism to be detected, and different microorganisms contain specific RNA fragments with different genetic information, and the binding site of primer crRNA-13 is designed according to these specific RNA fragments. The sequence of primer crRNA-13 is expressed as: 5'-fixed sequence-binding sequence-3'.
[0024] The detection kit of the present invention is suitable for detecting the content of live bacteria of all microorganisms, such as lactic acid bacteria (such as Lactobacillus; Lactobacillus bulgaricus; Lactobacillus plantarum), Bacillus (Bacillus licheniformis; Bacillus subtilis) and Pediococcus (Pediococcus pentosaceus), etc. When the microorganism is Bacillus licheniformis, the sequence of primer crRNA-13 is shown in SEQ ID NO.1.
[0025] crRNA is transcribed from a DNA template. After crRNA activates the corresponding LbuCas13a, the activated LbuCas13a can "side-cut" the intermediate, namely primer F-A4U6.
[0026] It should be noted that the fluorescent group and quenching group in the RNA probe (C5-Reporter) double-labeled with a fluorescent group and a fluorescent quenching group can be any common group that can be matched and modified on a nucleic acid molecule, and the excitation and emission wavelengths can be adaptively adjusted by technicians in this field according to the different groups. For example, the ROX red fluorescent group can be matched with the BHQ2 quenching group, with an excitation wavelength of 585nM and an emission wavelength of 605nM; the fluorescent group FAM can be matched with the BHQ1 quenching group, with an excitation wavelength of 450~500nM and an emission wavelength of 500~600nM.
[0027] The second embodiment of the present invention provides a method for detecting koji microorganisms for non-diagnostic purposes, using the koji microorganism detection kit described in any one of the first embodiments for detection, comprising the following steps: Provide LbuCas13a protein, TtCsm6 protein, primer crRNA-13, primer F-A4U6, a fluorescent group and a fluorescent quenching group double-labeled RNA probe; The LbuCas13a protein was mixed with the primer crRNA-13 and incubated at room temperature for 15 min to obtain a first mixed solution; Add primer F-A4U6, TtCsm6 protein, RNA probe and RNA to be tested to the first mixed solution and incubate at 37° C. for 20 to 60 minutes to obtain a second mixed solution; measuring the peak fluorescence intensity of the second mixed solution; The presence of the microorganism to be detected and the viable bacteria content of the microorganism when the microorganism to be detected is determined according to the peak value of the fluorescence intensity of the second mixed solution.
[0028] In some specific embodiments, taking Bacillus licheniformis as an example, the method for detecting koji microorganisms comprises the following steps: 1) 4μL primer and MB-13 (10μM) were mixed with 4μL primer promoter (10μM) and 4μL 10X phi 29 buffer, and then added to 10μL ultrapure water. After incubation at 90℃ for 5min, incubate at 37℃ for 30min, continue to add 0.5μL phi 29 pol and 2μL dNTP and incubate at 37℃ for 30min, then add 8μL 5X Trans Buffer, 4μL rNTP, 1μL T7pol (20 U / μL) and incubate at 37℃ overnight for 12~18h. After incubation, add 8μL 5X DNase I buffer and 4μL DNase I pol to the system and incubate at 37℃ for 0.5~4h to remove the DNA template, and finally incubate at 75℃ for 10min to inactivate the enzyme in the system to obtain the corresponding crRNA-13.
[0029] 2) LbuCas13a and crRNA-13 were mixed in a 1:1 molar ratio in 10×LbuCas13a buffer with a pH value of 7.5±0.5 and 17.1 μL ultrapure water, incubated at room temperature for 15 min, and then the mixture was incubated in a ratio of 4:4:10:100:20 (10 -6 ~0.1) was added to Bacillus licheniformis RNA, and the mixture was incubated at 37° C. for 20 to 60 min to obtain a first mixed solution.
[0030] 3) Take 20~50μL of the incubation product in step 2) into a black 384-well plate and read the fluorescence.
[0031] The standard curve can be obtained by adding different amounts of Bacillus licheniformis RNA and reading the fluorescence energy. The fluorescence peak value can be substituted into the standard curve by using the actual fermentation sample to test the fluorescence peak value through this method, and the corresponding live bacterial concentration of Bacillus licheniformis can be calculated, providing a reference for the detection of Bacillus licheniformis.
[0032] In the above steps, promoter is the promoter, and MB-13 is the DNA template chain of crRNA-13.
[0033] Preferably, the overnight incubation time in step 1) is 16 hours, and the enzyme inactivation incubation time is 2 hours.
[0034] Preferably, the pH value of the 10×LbuCas13a buffer in step 2) is 7.0, and the composition is 200 mM HEPES, 500 mM KCl, 20% glycerol, 50 mM MgCl2, 100 μg ml -1 Bovine serum albumin (BSA), 0.1% Igepal CA630. Specifically, the final concentrations of LbuCas13, crRNA-13, TtCsm6, F-A4U6, and RNA probe were 40nM, 40nM, 100nM, 1μM, and 200nM, respectively. The incubation time was 30min.
[0035] In order to make the technical solution of the present invention clearer, the koji microorganism detection kit, detection method and detection effect are described in detail through multiple specific embodiments below.
[0036] Some of the sequences involved in the following examples are shown in Table 1, where FAM represents a modified fluorescent group and BHQ1 represents a modified quenching group. The nucleotide sequences in Table 1 were commissioned to a professional synthesis company, Shanghai Shenggong Biotechnology Co., Ltd.
[0037] Table 1 Related sequences
[0038] In this embodiment, the crRNA-13 solution is prepared in the following steps: Add 4 μL of 10 μM promoter stock solution, 4 μL of 10 μM crRNA-13 DNA template chain solution (MB-13) and 4 μL of 10X phi 29 buffer to 10 μL ultrapure water, shake well, anneal at 90°C for 5 minutes, and then place at 37°C for 30 minutes, add 0.5 μL phi 29 pol and 2 μL dNTP, shake well, incubate at 30°C for 30 minutes, add 1 μL T7 pol (20 U / μL), 8 μL 5X Trans Buffer and 4 μL rNTP mixture, mix well, incubate at 37°C for 16 hours, add 8 μL 5X DNase I buffer and 4 μL DNase I pol was incubated at 37°C for 2 h to remove the DNA template, and finally incubated at 75°C for 10 min to inactivate the enzyme in the system to obtain a crRNA-13 solution. The sequence of crRNA-13 was 5'-TAGACCACCCCAAAAATGAAGGGGACTAAAACAAAACCTTCATCACTCACGC-3', as shown in SEQ ID NO.1.
[0039] Example 2 Drawing a standard curve In this embodiment, the standard curve solution is drawn, and the steps are as follows: 1) Prepare Bacillus licheniformis standard solution Take 10 8 CFU / mL of Bacillus licheniformis was used as the stock solution to extract RNA, and the concentration was configured to 3×10 5 , 1×10 5 , 8×10 4 , 5×10 4 , 3×10 4 , 1×10 4 , 5×10 3 , 1×10 3 , 0.6 μL of 500 CFU / mL Bacillus licheniformis RNA standard solution.
[0040] 2) Draw a standard curve ① Take 0.6 μL of the 2 μM crRNA-13 solution prepared in Example 1, add 1.2 μL of 1 μM LbuCas13a, 3 μL of 10×LbuCas13a buffer and 17.1 μL of ultrapure water, mix well, and incubate at room temperature for 15 min to obtain a first mixed solution.
[0041] ② Add 3 μL of 10 μM TtCsm6, 3 μL of 10 μM primer F-A4U6, 1.5 μL of 4 μM C5-Reporter and 0.6 μL of the Bacillus licheniformis standard solution prepared in Example 2 to the first mixed solution, mix well, and incubate at 37°C for 30 min to obtain a second mixed solution. Take 30 uL of the second mixed solution, measure its fluorescence intensity, and record the peak fluorescence intensity corresponding to the Bacillus licheniformis standard solution.
[0042] ③ The concentrations used were 0, 3×10 5 , 1×10 5 , 8×10 4 , 5×10 4 , 3×10 4 , 1×10 4 , 5×10 3 , 1×10 3 , 500 CFU / mL Bacillus licheniformis standard solution was used to replace the Bacillus licheniformis standard solution in step ①, and the operations of steps ① and ② were repeated to obtain a series of fluorescence intensity peak values of the Bacillus licheniformis standard solution. The logarithm of the bacterial concentration of the Bacillus licheniformis standard solution was taken as the abscissa, and the ratio of the fluorescence intensity peak value of the Bacillus licheniformis standard solution to the fluorescence intensity of group 0 was taken as the ordinate to draw a standard curve. The regression equation of the fitted standard curve was y = 5.056x –15.62, R 2 = 0.93, where y represents the ratio of fluorescence intensity and x represents the concentration of Bacillus licheniformis. The standard curve is shown in Figure 3 shown.
[0043] According to the standard curve, the detection limit of this method was 500 CFU / mL.
[0044] Example 3 Detection of Bacillus licheniformis during koji fermentation In this embodiment, samples were taken at different days during the fermentation of koji and the amount of live bacteria of Bacillus licheniformis was detected, and the steps were as follows: 1) Take the fermentation samples on the third, fifth, and eighth days of fermentation, add 50 mL of saline solution respectively, mix well, filter out the lees with filter paper, keep the filtrate, extract RNA using a DNA / RNA extraction kit, quantify to 0.6 μL with saline solution, and record them as 2#~4# sample solutions respectively. Take ultrapure water as 1# sample solution.
[0045] 2) Detection of Bacillus licheniformis in fermentation sample solution ① Take the 2 μM crRNA-13 solution prepared in Example 1, add 1.2 μL of 1 μM LbuCas13a, 3 μL of 10×LbuCas13a buffer and 17.1 μL of ultrapure water, mix well, and incubate at room temperature for 15 min to obtain a first mixed solution.
[0046] ② Add 3 μL of 10 μM TtCsm6, 3 μL of 10 μM primer F-A4U6, 1.5 μL of 4 μM C5-Reporter and 0.6 μL of 1# sample solution to the first mixed solution, mix well, and incubate at 37°C for 30 min to obtain a second mixed solution. Take 30 μL of the second mixed solution to measure its fluorescence intensity and record the peak fluorescence intensity corresponding to the sample.
[0047] ③ Use 2#~4# sample solutions to replace 1# sample solution in step ②, repeat steps ① and ②, obtain the peak fluorescence intensity of 2#~4# sample solutions and record them, divide the peak fluorescence intensity of 2#~4# sample solutions by the peak fluorescence intensity of 1# sample solution, substitute the ratio into the standard curve, and calculate the content of Bacillus licheniformis in 2#~4# sample solutions. The results are as follows Figure 4 shown.
[0048] Example 4 Specificity of the method of the present invention In this example, the selectivity of the detection method provided by the present invention for microorganisms is investigated, and the steps are as follows: (1) Prepare 0.6 μL of 1 ng / μL Bacillus licheniformis RNA, 0.6 μL of Lactobacillus RNA, and 0.6 μL of Pediococcus pentosaceus solution in ultrapure water.
[0049] (2) Take the 2 μM crRNA-13 solution prepared in Example 1, add 1.2 μL of 1 μM LbuCas13a, 3 μL of 10×LbuCas13a buffer and 17.1 μL of ultrapure water, mix well, and incubate at room temperature for 15 min to obtain a first mixed solution.
[0050] (3) Add 3 μL of 10 μM TtCsm6, 3 μL of 10 μM primer F-A4U6, 1.5 μL of 4 μM C5-Reporter to the first mixed solution, and add 0.6 μL of the above-mentioned Bacillus licheniformis RNA, Lactobacillus RNA and Pediococcus pentosaceus solution, mix well, and incubate at 37°C for 30 min to obtain a second mixed solution. Take 30 μL of the second mixed solution to measure its fluorescence intensity and record the peak fluorescence intensity corresponding to the sample.
[0051] Depend on Figure 5It can be seen that the peak fluorescence intensity of the Lactobacillus group and the Pediococcus pentosaceus group is equivalent to that of the blank control group, while the peak fluorescence intensity of the Bacillus licheniformis group is significantly higher than the fluorescence intensity of the Lactobacillus group, the Pediococcus pentosaceus group and the blank control group (Blank), indicating that the detection method provided by the present invention is highly specific and selective for Bacillus licheniformis.
[0052] Example 5 Signal-to-noise ratio determination In this example, the signal-to-noise ratio of the method for detecting Bacillus licheniformis provided by the present invention was investigated, and the steps were as follows: (1) Prepare a 1 ng / μL Bacillus licheniformis standard solution. Take 0.6 μL of the 2 μM crRNA-13 solution prepared in Example 1, add 1.2 μL of 1 μM LbuCas13a, 3 μL of 10×LbuCas13a buffer and 17.1 μL of ultrapure water, mix well, and incubate at room temperature for 15 min to obtain a first mixed solution. Add 3 μL of 10 μM TtCsm6, 3 μL of 10 μM primer F-A4U6, 1.5 μL of 4 μM C5-Reporter to the first mixed solution, and add 0.6 μL of the prepared Bacillus licheniformis RNA, mix well, and incubate at 37°C for 30 min to obtain a second mixed solution. Take 30 μL of the second mixed solution, use a fluorescence spectrometer to measure its fluorescence intensity under the conditions of an excitation wavelength of 480 nm and an emission wavelength range of 510-600 nm, and record the peak fluorescence intensity corresponding to the Bacillus licheniformis standard solution.
[0053] (2) Replace the Bacillus licheniformis standard solution in step (1) with an equal amount of ultrapure water, repeat the operation in step (1), and record the peak fluorescence intensity.
[0054] (3) Calculate the ratio of the peak fluorescence intensity measured in step (2) to that measured in step (1), that is, the signal-to-noise ratio. The results are shown in Table 2.
[0055] Comparative Example 1 In this comparative example, the crRNA-13 solution prepared in Example 1 was used for Bacillus licheniformis detection, and the LbuCas13a-TtCsm6 enzyme cleavage time was 15 min to examine its signal-to-noise ratio.
[0056] The experimental steps are as follows: (1) Take 0.6 μL of a 2 μM crRNA-13 solution, add 1.2 μL of 1 μM LbuCas13a, 3 μL of 10×LbuCas13a buffer and 17.1 μL of ultrapure water, mix well, and incubate at room temperature for 15 min to obtain a first mixed solution.
[0057] (2) Add 3 μL of 10 μM TtCsm6, 3 μL of 10 μM primer F-A4U6, 1.5 μL of 4 μM C5-Reporter, and 0.6 μL of Bacillus licheniformis RNA to the first mixed solution, mix well, and incubate at 37°C for 15 min to obtain a second mixed solution. Take 30 μL of the second mixed solution and use a fluorescence spectrometer to measure its fluorescence intensity under the conditions of an excitation wavelength of 480 nm and an emission wavelength range of 510-600 nm, and record the peak fluorescence intensity corresponding to the Bacillus licheniformis standard solution.
[0058] (3) Replace the Bacillus licheniformis standard solution in step (1) with an equal amount of ultrapure water, repeat steps (1) and (2), and record the peak fluorescence intensity.
[0059] (4) Calculate the ratio of the peak fluorescence intensity measured in step (3) to that measured in step (2), that is, the signal-to-noise ratio. The results are shown in Table 2.
[0060] Comparative Example 2 In this comparative example, the crRNA-13 solution prepared in Example 1 was used to perform a cascade-free, single LbuCas13a detection experiment on Bacillus licheniformis to examine its signal-to-noise ratio. Figure 2 shown.
[0061] The experimental steps are as follows: (1) Take 0.6 μL of the 2 μM crRNA-13 solution prepared in Example 1, add 1.2 μL of 1 μM LbuCas13a, 1.5 μL of 4 μM U5-reporter, whose sequence is shown in SEQ ID NO.5, 3 μL of 10×LbuCas13a buffer and 26.1 μL of ultrapure water, mix well, and incubate at room temperature for 15 min to obtain a first mixed solution.
[0062] (2) Take 0.6 μL of the Bacillus licheniformis standard solution and mix it with the solution in step (1), shake it well, and incubate it at 37°C for 30 min. Then measure the fluorescence intensity of the reaction mixture and record the peak fluorescence intensity corresponding to the Bacillus licheniformis standard solution.
[0063] (3) Replace the Bacillus licheniformis standard solution in step (2) with an equal amount of ultrapure water, repeat steps (1) and (2), and record the peak fluorescence intensity.
[0064] (4) Calculate the ratio of the peak fluorescence intensity measured in step (3) to that measured in step (2), that is, the signal-to-noise ratio. The results are shown in Table 2.
[0065] Table 2 Signal-to-noise ratio of Example 5 and Comparative Examples 1 and 2 As can be seen from Table 2, the signal-to-noise ratio of the detection method provided by the present invention is significantly higher than that of the comparative example, indicating that the detection method of the present invention has high performance such as sensitivity.
[0066] In summary, the detection method of the present invention has high sensitivity and specificity, can be used to detect the content of live microorganisms in all fermentations, and has good application prospects.
[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A koji microorganism detection kit, characterized in that: Comprising: LbuCas13a protein, TtCsm6 protein, primer crRNA-13, primer F-A4U6, and RNA probe double-labeled with a fluorescent group and a fluorescent quenching group; Among them, the primer crRNA-13 is the guide RNA corresponding to the LbuCas13a protein, and the primer crRNA-13 sequence includes a fixed sequence and a binding sequence, wherein the binding sequence is a 20nt fragment that can be complementary to the microbial RNA, and the binding sequence is located at the 3' end of the fixed sequence; Primer F-A4U6 is the main target substrate of LbuCas13 protein, which is a fluorine-modified RNA sequence at position 2, and its sequence is shown in SEQ ID NO.
2.
2. The yeast microorganism detection kit according to claim 1, characterized in that: The molar ratio of LbuCas13 protein, TtCsm6 protein, primer crRNA-13, primer F-A4U6, RNA to be tested, and RNA probe is 4:10:4:100:(10 -6 ~0.1):
20.
3. The yeast microorganism detection kit according to claim 1, characterized in that The sequence of the RNA probe is shown as SEQ ID NO.
3.
4. The koji microorganism detection kit according to claim 1, characterized in that It also contains 10×LbuCas13a buffer, consisting of 200 mM HEPES, pH=7.0, 500 mM KCl, 20% glycerol, 50 mM MgCl2, 100 μg ml-1 bovine serum albumin (BSA), and 0.1% Igepal CA630.
5. The yeast microorganism detection kit according to claim 1, characterized in that: The microorganisms include lactic acid bacteria, bacillus, pediococcus and any one of the microorganisms involved in the fermentation process.
6. The yeast microorganism detection kit according to claim 5, characterized in that: When the microorganism is Bacillus licheniformis, the primer crRNA-13 sequence is shown as SEQ ID NO.
1.
7. A method for detecting distiller's yeast microorganisms for non-diagnostic purposes, characterized in that: The detection is performed using the koji microorganism detection kit as described in any one of claims 1 to 5, comprising the following steps: Provide LbuCas13a protein, TtCsm6 protein, primer crRNA-13, primer F-A4U6, a fluorescent group and a fluorescent quenching group double-labeled RNA probe; The LbuCas13a protein was mixed with the primer crRNA-13, and the first mixed solution was obtained after incubation at room temperature for 15 minutes; Add primer F-A4U6, TtCsm6 protein, RNA probe and RNA to be tested to the first mixed solution and incubate at 37° C. for 20 to 60 minutes to obtain a first mixed solution; Determining the peak fluorescence intensity of the second mixed solution; The presence of the target microbial RNA and the viable bacterial content of the target microorganism when the target microbial RNA is present are determined based on the peak value of the fluorescence intensity of the second mixed solution.
8. The non-diagnostic method for detecting koji microorganisms according to claim 7, characterized in that: The preparation method of primer crRNA-13 is as follows: 4 μL 10 μM crRNA-13 DNA template chain is mixed with 4 μL 10 μM promoter and 4 μL 10X phi 29 buffer, and then added to 10 μL ultrapure water. After incubation at 90°C for 5 minutes, incubation at 37°C for 30 minutes, 0.5 μL phi 29 pol and 2 μL dNTP are added and incubated at 37°C for 30 minutes, and then 8 μL 5X Trans Buffer, 4 μL rNTP, and 1 μL 20U / μL T7 pol are added and incubated at 37°C overnight for 12~18 hours; after the incubation is completed, 8 μL 5X DNase I buffer and 4 μL DNase I pol are added to the system and incubated at 37°C for 0.5~4 hours, and finally incubated at 75°C for 10 minutes.