CRISPRCas12a fluorescent array sensor and application of CRISPRCas12a fluorescent array sensor in identifying vintage and adulteration of white spirit

Abstract

The invention relates to the technical field of white spirit identification, and discloses a fluorescent array sensor of CRISPRCas12a and an application of the fluorescent array sensor in identification of vintage and adulteration of white spirit, the fluorescent array sensor is composed of three groups of sensors, and each of the three groups of sensors comprises target nucleic acid, guide RNA and a nucleic acid probe; the three groups of sensors also respectively comprise different Cas12 proteins; the Cas12 protein is an LbCas12a protein, an AsCas12a protein and a BaCas12a protein, and the molecular weight of the Cas12 protein is 8000-9000; the amino acid sequence of the LbCas12a protein is as shown in SEQ ID NO. 1; the amino acid sequence of the AsCas12a protein is as shown in SEQ ID NO. 2; the amino acid sequence of the BaCas12a protein is as shown in SEQ ID NO. 3. A fluorescent array sensor adopting CRISPRCas12a can be used for successfully identifying the year and adulteration of the white spirit.

Description

Technical Field

[0001] The present invention relates to the technical field of liquor identification, and in particular to a CRISPR Cas12a fluorescence array sensor and an application thereof in identifying the year and adulteration of liquor. Background Art

[0002] The CRISPR-Cas system is a natural immune system of prokaryotes. In recent years, the CRISPR-Cas system has attracted widespread attention in the field of biological sciences as a revolutionary gene editing technology. The system uses the guidance of Cas nuclease and crRNA to achieve recognition and cutting of specific DNA sequences, and has extremely high target specificity. Once the CRISPR-Cas12a system detects the target DNA, the cutting activity of Cas12a will be activated, and the ssDNA labeled with the fluorescent reporter group will be cut arbitrarily, releasing the fluorescent signal. By capturing the fluorescent signal, the target DNA can be detected. The system has been used to detect various nucleic acids. The CRISPR-Cas system has the characteristics of high sensitivity, high specificity, rapid detection and low cost. Its reaction conditions are mild and the operation is simple. Baijiu is a unique distilled liquor in China. It is deeply loved by consumers and plays an important role in Chinese food culture. Baijiu contains 98%-99% ethanol and water, and 1%-2% flavor substances, mainly including a wide variety of trace organic compounds such as alcohol, aldehyde, acid, ester, etc., which have the effect of aroma and taste, giving Baijiu a unique style. With the rapid development of the liquor industry and the improvement of people's living standards, the monitoring and evaluation of liquor quality has become increasingly important for production process control, market management and consumer interest protection. Traditional monitoring methods include sensory evaluation, chromatography and spectroscopy. These methods rely on large instruments to study the analysis mode of a single sample component by component, which makes it difficult to accurately identify the overall characteristics of a variety of Chinese liquors. In addition, the high cost and complex operation methods also limit their application in real-time detection on site. Therefore, it is very important to develop a simple, fast and effective method for identifying different liquors.

[0003] There is no relevant research on identifying the year and adulteration of liquor by CRISPR-Cas system. In the preliminary experiments of the applicant, it was found that, first of all, the CRISPR-Cas system mainly relies on the cutting activity of a single Cas12a protein to achieve the cutting of the fluorescent group, thereby converting the target content into fluorescence intensity to achieve the purpose of detection. Because liquor contains ethanol, and the various trace organic compounds such as alcohol, aldehyde, acid, and ester in liquor are complex, ethanol causes the enzyme activity of Cas12a protein to decrease, and the organic compounds in liquor are complex and diverse, which further causes the single Cas12a protein detection sensitivity to decrease. Secondly, during the fermentation process of liquor, the ingredients of liquor are complex and diverse at each stage, and it is necessary to select a certain target to represent the year of liquor, which can improve the accuracy and convenience of detection. Based on the above two problems, the applicant conducted a large number of experiments and analyses. It is surprising that a lot of data was obtained under a lot of experimental work, not only the target was found, but also how to improve the sensitivity of CRISPR-Cas system in identifying liquor. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a CRISPR Cas12a fluorescence array sensor and its application in identifying the year and adulteration of liquor. The present invention uses the CRISPR Cas12a fluorescence array sensor to successfully identify the year and adulteration of liquor.

[0005] The present invention provides a CRISPRCas12a fluorescence array sensor, wherein the fluorescence array sensor is composed of three groups of sensors, each of which includes a target nucleic acid, a guide RNA, and a nucleic acid probe; each of the three groups of sensors also includes a different Cas12 protein; The Cas12 protein is LbCas12a protein, AsCas12a protein and BaCas12a protein; The amino acid sequence of the LbCas12a protein is shown in SEQ ID NO.1; The amino acid sequence of the AsCas12a protein is shown in SEQ ID NO.2; The amino acid sequence of the BaCas12a protein is shown in SEQ ID NO.3; The nucleotide sequence of the target nucleic acid is shown in SEQ ID NO.4.

[0006] Furthermore, the sequence of the guide RNA is shown as SEQ ID NO.5.

[0007] Furthermore, the sequence of the nucleic acid probe is TTAATT, the N-terminus of the nucleic acid probe is modified with a fluorescent group, and the C-terminus of the nucleic acid probe is modified with a quenching group; the fluorescent group is 5'-FAM; and the quenching group is 3'-BHQ1.

[0008] Furthermore, the three groups of sensors also include nuclease-free water and buffer.

[0009] The present invention also provides an application of the fluorescent array sensor in identifying the year and adulteration of liquor, and the application includes: S1: Select a Cas12 protein, disperse a solution containing the Cas12 protein, a solution containing the guide RNA, a solution containing the nucleic acid probe, and nuclease-free water in a buffer to obtain a mixed solution; S2: Repeat the method of S1 with the remaining two Cas12 proteins to obtain the remaining two groups of mixed solutions; S3: The liquor to be tested is incubated once with a solution containing the target nucleic acid, and then the same volume is transferred to three groups of mixed solutions respectively, incubated a second time, and fluorescence intensity detection is performed, and the detected fluorescence intensity is clustered and analyzed.

[0010] Furthermore, by volume, the ratio of the liquor transferred to the mixed solution, the solution containing Cas12 protein, the solution containing the guide RNA, the solution containing the nucleic acid probe transferred to the mixed solution, the nuclease-free water, the buffer solution, and the solution containing the target nucleic acid is 0.8:2:2:1:27.2:2:5.

[0011] Furthermore, the molar concentration of the solution containing Cas12 protein is 200nM.

[0012] Furthermore, the method for preparing the solution containing Cas12 protein is specifically as follows: dissolving and dispersing the Cas12 protein in nuclease-free water.

[0013] Furthermore, the molar concentration of the solution containing the target nucleic acid is 1 μM.

[0014] Furthermore, the method for preparing the solution containing the target nucleic acid is specifically as follows: dissolving and dispersing the target nucleic acid in nuclease-free water.

[0015] Furthermore, the molar concentration of the solution containing the guide RNA is 200 nM.

[0016] Furthermore, the method for preparing the solution containing the guide RNA is specifically as follows: dissolving and dispersing the guide RNA in nuclease-free water.

[0017] Furthermore, the molar concentration of the solution containing the nucleic acid probe is 200 nM.

[0018] Furthermore, the method for preparing the solution containing the nucleic acid probe is specifically: dissolving and dispersing the nucleic acid probe in nuclease-free water.

[0019] Furthermore, the preparation method of the buffer solution is as follows: 10 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 10 mM magnesium chloride, 10 mM sodium chloride, 100 g / mL bovine serum albumin solution (BSA), and the pH is adjusted to 7.4 using a hydrochloric acid solution.

[0020] Furthermore, the method for preparing the bovine serum albumin solution is specifically to disperse the bovine serum albumin solution in deionized water.

[0021] Furthermore, the incubation time is 9 min-11 min.

[0022] Furthermore, the temperature of the secondary incubation is 36° C.-38° C., and the time of the secondary incubation is 25 min-35 min.

[0023] Furthermore, the fluorescence intensity is detected using an enzyme marker with an excitation wavelength of 480nm-500nm and an emission wavelength of 510nm-540nm.

[0024] The embodiments of the present invention have the following technical effects: 1. In the present invention, three different Cas12 proteins are used in the fluorescent array sensor. The detection capabilities of the three Cas12 proteins can complement each other in detecting lactic acid content, thereby improving the sensitivity of the detection method.

[0025] 2. After the lactic acid molecules in the liquor and the target nucleic acid of known concentration are incubated once to form a complex in the method of the present invention, part of the target nucleic acid can be consumed, and the content of lactic acid molecules can be reacted by the remaining target nucleic acid. And the liquor solution containing less is diluted by the solution containing the target nucleic acid, so it is in the solution containing Cas12 protein, the solution containing guide RNA, the solution containing nucleic acid probe and the mixed solution obtained in nuclease-free water and buffer, and the influence of ethanol on the activity of Cas12 protein in liquor can be reduced. And the remaining target nucleic acid can form a ternary complex with guide RNA and Cas12a protein, the cutting activity of Cas12a protein will be activated, and the fluorescent group on the nucleic acid probe will be cut by Cas12a protein, and the present invention uses three different Cas12a proteins at the same time to form a fluorescent array sensor, and the liquor of different years is classified and identified.

[0026] 3. The method of the present invention, first, has high sensitivity and high specificity: due to the high specificity of lactic acid target nucleic acid and Cas12a protein, the method has high detection sensitivity and specificity. Second, it is simple and easy to implement: the detection method of the present invention does not require the use of complex instruments and equipment, only a constant temperature water bath or metal bath is required, making on-site detection more convenient and easy. Third, the cost is low: the detection method of the present invention does not require the use of precision instruments such as real-time fluorescence PCR instruments, which reduces the detection cost and can also avoid errors caused by instrument failure or operating errors. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 It is the activity of different Cas12 proteins provided in the embodiments of the present invention in the presence or absence of liquor.

[0029] Figure 2 This is a protein electrophoresis diagram provided by an embodiment of the present invention.

[0030] Figure 3 It is the test result of the incubation time of liquor and target nucleic acid in the present invention.

[0031] Figure 4 It is the test result of the added volume of liquor in the present invention.

[0032] Figure 5 This is the classification result of liquors of different years by the fluorescence array sensor of the present invention.

[0033] Figure 6 This is the result of adulteration of liquor by the fluorescent array sensor of the present invention.

[0034] Figure 7 It is the classification result of liquors of different years in the comparative example of the present invention. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0036] In the first aspect, some embodiments of the present invention provide a CRISPRCas12a fluorescence array sensor, wherein the fluorescence array sensor is composed of three groups of sensors, each of which includes a target nucleic acid, a guide RNA, and a nucleic acid probe; each of the three groups of sensors also includes a different Cas12 protein; The Cas12 protein is LbCas12a protein, AsCas12a protein and BaCas12a protein; The amino acid sequence of the LbCas12a protein is shown in SEQ ID NO.1; The amino acid sequence of the AsCas12a protein is shown in SEQ ID NO.2; The amino acid sequence of the BaCas12a protein is shown in SEQ ID NO.3; The nucleotide sequence of the target nucleic acid is shown in SEQ ID NO.4.

[0037] In some embodiments, the sequence of the guide RNA is shown as SEQ ID NO.5.

[0038] In some embodiments, the sequence of the nucleic acid probe is TTAATT, the N-terminus of the nucleic acid probe is modified with a fluorescent group, and the C-terminus of the nucleic acid probe is modified with a quenching group; the fluorescent group is 5'-FAM; and the quenching group is 3'-BHQ1.

[0039] In some embodiments, the three sets of sensors also include nuclease-free water and buffer.

[0040] In a second aspect, the present invention further provides an application of the fluorescent array sensor in identifying the year and adulteration of liquor, and the application includes: S1: Select a Cas12 protein, disperse a solution containing the Cas12 protein, a solution containing the guide RNA, a solution containing the nucleic acid probe, and nuclease-free water in a buffer to obtain a mixed solution; S2: Repeat the method of S1 with the remaining two Cas12 proteins to obtain the remaining two groups of mixed solutions; S3: The liquor to be tested is incubated once with a solution containing the target nucleic acid, and then the same volume is transferred to three groups of mixed solutions respectively, incubated a second time, and fluorescence intensity detection is performed, and the detected fluorescence intensity is clustered and analyzed.

[0041] In some embodiments, the ratio of the liquor transferred to the mixed solution, the solution containing Cas12 protein, the solution containing guide RNA, the solution containing nucleic acid probe transferred to the mixed solution, nuclease-free water, the buffer and the solution containing target nucleic acid is 0.8:2:2:1:27.2:2:5 by volume.

[0042] In some embodiments, the molar concentration of the solution containing Cas12 protein is 200nM.

[0043] In some embodiments, the method for preparing the solution containing Cas12 protein is specifically: dissolving and dispersing the Cas12 protein in nuclease-free water.

[0044] In some embodiments, the molar concentration of the solution containing the target nucleic acid is 1 μM.

[0045] In some embodiments, the method for preparing the solution containing the target nucleic acid is specifically: dissolving and dispersing the target nucleic acid in nuclease-free water.

[0046] In some embodiments, the molar concentration of the solution containing the guide RNA is 200 nM.

[0047] In some embodiments, the method for preparing the solution containing the guide RNA is specifically: dissolving and dispersing the guide RNA in nuclease-free water.

[0048] In some embodiments, the molar concentration of the solution containing the nucleic acid probe is 200 nM.

[0049] In some embodiments, the method for preparing the solution containing the nucleic acid probe is specifically: dissolving and dispersing the nucleic acid probe in nuclease-free water.

[0050] In some embodiments, the buffer solution is prepared by: 10 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 10 mM magnesium chloride, 10 mM sodium chloride, 100 g / mL bovine serum albumin solution (BSA), and the pH is adjusted to 7.4 using hydrochloric acid solution.

[0051] In some embodiments, the method for preparing the bovine serum albumin solution is specifically to disperse the bovine serum albumin solution in deionized water.

[0052] In some embodiments, the incubation time is 9 min-11 min.

[0053] In some embodiments, the secondary incubation temperature is 36° C.-38° C., and the secondary incubation time is 25 min-35 min.

[0054] In some embodiments, the fluorescence intensity is detected using an enzyme reader with an excitation wavelength of 480nm-500nm and an emission wavelength of 510nm-540nm.

[0055] The following is described in conjunction with specific embodiments and comparative examples: (1) Preparation method of three Cas12a proteins in the present invention: Large-scale expression of Cas12a protein: Thaw a BL-21 competent cell on ice for 5 minutes, then add 1µL of 50ng / µL Cas12a protein expression plasmid. Incubate on ice for 30 minutes. Heat shock the competent cells at 42℃ (preheated) for 45s, then place on ice for 3 minutes. Add 1mL of LB medium without antibiotics to the competent cells, culture on a shaker at 37℃ for 1h, centrifuge, and apply 100µL of the cell suspension on a pre-warmed LB agar plate (containing kanamycin). Culture in an incubator at 37℃ overnight. The next day, pick a single colony and inoculate it in 3mL of LB liquid medium containing kanamycin, and culture it in a biological shaker at 37℃ and 220rpm overnight. The starting culture is inoculated into 100mL of LB medium containing kanamycin at a volume ratio of 1:100 and the optical density is determined. Culture at 37℃ and 220rpm. Monitor the OD value every hour until the OD value reaches 0.6-0.8, transfer to 4℃ for 30min, and let it cool before induction. Take 1mL of uninduced culture for SDS-PAGE analysis. Induce expression with 0.5M IPTG (isopropyl-β-D-thiogalactoside) and culture at 160rpm for 20h in a pre-cooled 16℃ bio-oscillator. Centrifuge the culture at 4℃ for 15min to harvest the cells. Take a small portion and resuspend it in 500µL PBS buffer, perform SDS-PAGE electrophoresis with the uninduced culture, and then perform Coomassie Brilliant Blue staining to detect whether the target protein is successfully induced.

[0056] (2) Purification of Cas12a protein: Resuspend the bacterial precipitate in Buffer A (formula: 50mM Tris-HCl and 300mM NaCl), then perform subsequent high-pressure crushing, centrifuge to obtain the supernatant, and collect 100µL of the fraction for SDS-PAGE electrophoresis analysis. The crushed bacterial suspension was filtered through an aqueous phase filter to obtain a crude enzyme solution, which was purified using a Ni-NTA (agarose purification resin can be used for the purification of histidine-tagged proteins expressed in Escherichia coli, purchased from Shanghai Biotechnology (Cat. No.: C600791-0001)) affinity chromatography column. Add the crude enzyme solution to the balanced nickel column, add a low concentration of imidazole buffer to wash the impurities, then add a high concentration of imidazole buffer to elute the target protein, and collect each flow-through for subsequent SDS-PAGE detection. Perform SDS-PAGE analysis on all collected protein fractions to verify the target protein.

[0057] (3) Determination of Cas12a protein concentration after purification: The concentration of purified Cas12a protein was determined using a Bradford protein concentration assay kit. When preparing the sensor, the concentrations of the three Cas12a proteins can be made consistent by dilution.

[0058] (4) Determination of Cas12a protein activity after purification: For CRISPR detection, a 40 µL reaction system includes 2 µL of a solution containing Cas12a protein (200 nM), 2 µL of a solution containing guide RNA (200 nM), 2 µL of a buffer, 5 µL of a solution containing target nucleic acid (1 μM), 1 µL of a solution containing a nucleic acid probe (8 μM), and 28 µL of nuclease-free water, and incubated in a 37 °C water bath for 30 min.

[0059] (5) Lactate aptamer DNA search: Chinese liquor contains a variety of trace components. Currently, more than 1,870 volatile compounds have been identified, including alcohols, acids, esters, aldehydes, ketones, phenols, etc. Lactic acid accounts for 20% of the total acid content and 3% of the total flavor components in Luzhou-flavor liquor. The present invention searches for the target nucleic acid of lactic acid molecules in the literature, as shown in SEQ ID NO.4.

[0060] The nucleotide sequence of the target nucleic acid of lactic acid (SEQ ID NO.4): 5′-GACGACGAGTAGCGCGTATGAATGCTTTTCTATGGAGTCGTC-3′.

[0061] (6) Design of guide RNA: According to the nucleotide sequence of the target nucleic acid of lactic acid, a corresponding guide sequence is designed for use in the detection system, as shown in SEQ ID NO.5.

[0062] Guide RNA sequence (SEQ ID NO.5): 5'-UAAUUUCUACUAAGUGUAGAUAAAGCAUUCAUACGCGCUACUC-3'.

[0063] (7) Design of nucleic acid probes: The base sequence of the nucleic acid probe provided by the present invention is TTAATT, the 5' end of the nucleic acid probe is labeled with a FAM fluorescent reporter group, and the 3' end of the nucleic acid probe is labeled with a BHQ1 fluorescent quencher group.

[0064] (8) The selection of 10 different vintages of Luzhou Laojiao liquor is shown in Table 1: Table 1 Luzhou Laojiao liquor in different years

[0065] (9) Selection of Cas12a protein: The present invention uses three different Cas12a proteins to form a sensing element, wherein the LbCas12a protein is derived from Lachnospiraceae bacteria ND2006 strain, AsCas12a protein is derived from Acidaminococcus sp .BV3L6 strain, BaCas12a protein is derived from Butyrivibrio _Asp 000431815 strain, in the present invention, can be obtained by the above method, or commercially available LbCas12a and AsCas12a proteins can be directly used: Amino acid sequence of LbCas12a protein (SEQ ID NO.1):

[0066] Amino acid sequence of AsCas12a protein (SEQ ID NO.2):

[0067] Amino acid sequence of BaCas12a protein (SEQ ID NO.3):

[0068] (10) Determination of one group of sensors in the fluorescence array sensor: According to Table 2, lactic acid target nucleic acid and liquor stock solution were added to the reaction tube. After incubation at room temperature for 10 minutes, nuclease-free water, a solution containing one of the Cas12a proteins, 1× buffer, a solution containing a nucleic acid probe, and a solution containing a guide RNA were added in sequence, and the mixture was mixed by low-speed rotation on a vortexer and reacted at 37°C for 30 minutes. The fluorescence value of the reacted system was measured in a multifunctional microplate reader with an excitation wavelength of 480nm-500nm and an emission wavelength of 510nm-540nm to detect the fluorescence value of the sample to be tested.

[0069] Preparation method of buffer: 10 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride), 10 mM magnesium chloride, 10 mM sodium chloride, 100 g / mL bovine serum albumin solution (BSA), and adjust the pH to 7.4 using hydrochloric acid solution.

[0070] Table 2 Composition of one group of sensors in the fluorescence array sensor

[0071] Preparation of fluorescence array sensor: Take three groups of 5 μL solutions containing target nucleic acids with a molar concentration of 1 μM, add 0.8 μL of liquor stock solution and incubate at room temperature for 10 min respectively, add 2 μL of LbCas12a protein solution, AsCas12a protein solution and BaCas12a protein solution with a molar concentration of 200 nM to the three groups respectively, and add 2 μL of guide RNA solution with a molar concentration of 200 nM, 2 μL of buffer, 1 μL of nucleic acid probe solution with a molar concentration of 200 nM and 27.2 μL of nuclease-free water (ddH2O) to all three groups to obtain a fluorescent array sensor.

[0072] Comparative Example 1: 5 μL of a solution containing the target nucleic acid at a molar concentration of 1 μM and 0.8 μL of liquor stock solution were added to the reaction tube. After incubation at room temperature for 10 minutes, 27.2 μL of nuclease-free water, 2 μL of a solution containing BaCas12a protein at a molar concentration of 200 nM, 2 μL of buffer, 1 μL of a solution containing a nucleic acid probe at a molar concentration of 200 nM, and 2 μL of a solution containing a guide RNA at a molar concentration of 200 nM were added in sequence, and the mixture was mixed by rotating at low speed on a vortex instrument and reacted at 37 ° C for 30 minutes. The experiment was repeated 6 times, and the fluorescence value of the reacted system was measured in a multifunctional microplate reader, the excitation wavelength of the microplate reader was 480 nm-500 nm, and the emission wavelength was 510 nm-540 nm, and the fluorescence value of the sample to be tested was detected. The fluorescence value was clustered using Origin 2022 to obtain a principal component analysis diagram.

[0073] Results and Analysis: Figure 1 It is a data graph of the cutting activity of three different Cas12a proteins in the presence or absence of liquor. The three purified Cas12a proteins were applied to the detection system. The reaction temperature was 37°C. They were transferred to the ELISA plate after 30 minutes of water bath at the set temperature. The fluorescence intensity values ​​with excitation wavelengths of 494 nm and emission wavelengths of 525 nm were measured using an ELISA instrument.

[0074] The results are as follows Figure 1 As shown, it was found that the fluorescence intensity value in the liquor system was significantly lower than that in the system without liquor, indicating that a part of the target nucleic acid DNA of lactic acid added to the reaction system first specifically binds to the lactic acid in the liquor, and the remaining target nucleic acid DNA will form a ternary complex with the added guide RNA and Cas12a protein, activating the trans-cutting activity of Cas12a, and arbitrarily cutting the nucleic acid probe with a fluorescent reporter group to generate a fluorescent signal.

[0075] The plasmids of different Cas12a proteins were transferred into BL21 competent cells, and then spread on LB solid agar plates for overnight culture after heat shock ice bath. The next day, a single colony was picked and inoculated into LB liquid medium containing kanamycin, and cultured at 37°C overnight for 12 hours, inoculated into fresh LB liquid medium, and cultured at 37°C for 2-3 hours. IPTG was added to induce expression, and the cells were centrifuged and collected after culturing at 16°C and 160 rpm for 20 hours. The cells were resuspended by adding buffer, ultrasonically broken or added with bacterial lysis solution, and the supernatant was collected by centrifugation to obtain the crude enzyme solution. After passing through the membrane, the supernatant was passed through a Ni-NTA affinity chromatography column, the impurities were washed with a low concentration of imidazole buffer, and the target protein was eluted with a high concentration of imidazole buffer. The obtained eluate was placed in an ultrafiltration centrifuge tube for concentration.

[0076] According to the literature, the molecular weight of Cas12a protein is about 150kDa. Figure 2It is the SDS-PAGE verification diagram of BaCas12a protein before and after purification. Band M is the band of Marker, band 1 is the band of the supernatant after bacterial crushing, band 2 is the band of the precipitate after bacterial crushing, band 3 is the band of the supernatant after nickel column purification, and band 4 is the band after the supernatant is purified and concentrated. It can be seen from the figure that each component has a band at 150 kDa, and the concentrated band is wider than the unconcentrated band, that is to say, the protein concentration after concentration is increased.

[0077] The incubation time of the target nucleic acid of liquor and lactic acid at room temperature needs to be determined by experiments. The incubation time of liquor and lactic acid aptamer DNA is set to 10min, 20min, 30min, 40min, and 50min, respectively. After the incubation is completed, the CRISPR / Cas12a detection system (guide RNA, BaCas12a protein, buffer, fluorescent probe, nuclease-free water) is added to the reaction system. After incubation at 37°C for 30min, the fluorescence intensity value is determined using an enzyme reader.

[0078] The results are as follows Figure 3 As shown in the figure, after incubation of liquor and lactic acid target nucleic acid for 10 minutes, the relative fluorescence value decreased significantly to about 60%. As time went on, the fluorescence intensity tended to equilibrium. That is to say, after incubation for 10 minutes, the lactic acid in the liquor had completely combined with the target nucleic acid DNA. Therefore, the incubation time of liquor and lactic acid target nucleic acid DNA was selected to be 10 minutes for subsequent experiments.

[0079] The final concentration of liquor in the detection system needs to be determined by experiment. If the final concentration of liquor is too high, it will affect the activity of Cas12a protein. Therefore, a suitable final concentration of liquor needs to be selected in the reaction system. The volume fraction of liquor in the reaction system is set to 1% (the total volume of the reaction system is 40μL, then the added volume of liquor is 0.4μL, and the remainder is supplemented with nuclease-free water to a reaction system volume of 40μL), 2% (0.8μL), 3% (1.2μL), 4% (1.6μL), 5% (2μL). After incubating different volumes of liquor with lactic acid target nucleic acid for 10 minutes, the CRISPR / Cas12a detection system (guide RNA, BaCas12a protein, buffer, fluorescent probe, nuclease-free water) is added to the reaction system. After incubation at 37°C for 30 minutes, the fluorescence intensity value is measured using an enzyme reader.

[0080] The results are as follows Figure 4As shown in the figure, as the final concentration of liquor in the reaction system increases, the relative fluorescence intensity value becomes lower and lower, that is, the cutting activity of Cas12a protein becomes smaller and smaller. When the final concentration of liquor is 1%, there are fewer targets in the detection system; when the final concentration of liquor is 5%, the relative fluorescence intensity value is the lowest, and the cutting activity of Cas12 protein is low; when the final concentration of liquor is 2%, the relative fluorescence intensity value decreases to 50%. Although the cutting activity of Cas12a protein is reduced at this time, its fluorescence intensity value still reflects that Cas12a protein still has relatively good cutting activity and can perform cutting reaction. In summary, the volume fraction of liquor in the reaction system is 2%.

[0081] The above conditions were used for subsequent experiments, and three Cas12a proteases were used as sensing elements to construct a fluorescence array sensor to identify 10 different years of Luzhou Laojiao. The data obtained by the fluorescence array sensor were clustered using Origin 2022 to obtain the principal component analysis diagram (PCA).

[0082] Specifically: take three groups of 5 μL solutions containing target nucleic acids with a molar concentration of 1 μM, add 0.8 μL of liquor stock solution and incubate at room temperature for 10 minutes, add 2 μL of LbCas12a protein solution, AsCas12a protein solution and BaCas12a protein solution with a molar concentration of 200 nM to each group, and add 2 μL of guide RNA solution with a molar concentration of 200 nM, 2 μL of buffer, 1 μL of nucleic acid probe solution with a molar concentration of 200 nM and 27.2 μL of nuclease-free water (ddH2O) to each group to obtain a fluorescent array sensor. The fluorescent array sensor performs 6 parallel experiments on each liquor to be tested. Prepare 10 groups of liquors of different years, and use the fluorescent array sensor of the present invention to detect these 10 groups of liquors. The 10 groups of liquors are shown in Table 1 (LZ1, LZ2, LZ3, LZ4, LZ5, LZ6, LZ7, LZ8, LZ9, LZ10). After incubation at 37°C for 30 min, the results were recorded on a microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Cluster analysis was performed based on the detected fluorescence intensity values. The clustering results are shown in Figure 2. Figure 5 shown.

[0083] The lactic acid content in liquors of different years is different, and the amount of target nucleic acid DNA added to the system is different. Therefore, the amount of aptamer DNA bound to the remaining target nucleic acid and the guide RNA and Cas12a protease is different, and the final fluorescence intensity is different. Cluster analysis of the fluorescence intensity of liquors of the same year is performed to generate Figure 5 ,It can be found that liquors of different years are clustered at different positions in the figure, thus realizing the clustering and distinction of liquors of different years through fluorescence intensity.

[0084] Only BaCas12a protein was used to classify liquors of different years. The results are as follows Figure 7 As shown, the results showed that when one Cas12a protein was used, it was impossible to classify liquors of different years, and there was an overlap between LZ2 and LZ4.

[0085] The fluorescent array sensor constructed by the present invention was used to measure the adulterated liquor. The results are as follows: Figure 6 As shown, industrial ethanol is doped into the three liquors LZ1, LZ2 and LZ3, which will cause the fluorescence intensity of the doped liquor to deviate from that of the undoped liquor after cluster analysis. It can be seen that the method of the present invention can successfully identify adulterated liquor.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present invention.

Claims

1. A fluorescence array sensor of CRISPRCas12a, characterized in that The fluorescent array sensor is composed of three groups of sensors, each of which includes a target nucleic acid, a guide RNA, and a nucleic acid probe; each of the three groups of sensors also includes a different Cas12 protein; The Cas12 protein is LbCas12a protein, AsCas12a protein and BaCas12a protein; The amino acid sequence of the LbCas12a protein is shown in SEQ ID NO.1; The amino acid sequence of the AsCas12a protein is shown in SEQ ID NO.2; The amino acid sequence of the BaCas12a protein is shown in SEQ ID NO.3; The nucleotide sequence of the target nucleic acid is shown in SEQ ID NO.

4.

2. The fluorescence array sensor according to claim 1, characterized in that: The sequence of the guide RNA is shown as SEQ ID NO.

5.

3. A fluorescence array sensor, characterized in that: The sequence of the nucleic acid probe is TTAATT, the N-terminus of the nucleic acid probe is modified with a fluorescent group, and the C-terminus of the nucleic acid probe is modified with a quenching group; the fluorescent group is 5'-FAM; and the quenching group is 3'-BHQ1; All three sets of sensors also included nuclease-free water and buffer.

4. Application of the fluorescence array sensor according to any one of claims 1 to 3 in identifying the vintage and adulteration of liquor, characterized in that: The applications include: S1: Select a Cas12 protein, disperse a solution containing the Cas12 protein, a solution containing the guide RNA, a solution containing the nucleic acid probe, and nuclease-free water in a buffer to obtain a mixed solution; S2: Repeat the method of S1 with the remaining two Cas12 proteins to obtain the remaining two groups of mixed solutions; S3: The liquor to be tested is incubated once with a solution containing the target nucleic acid, and then the same volume is transferred to three groups of mixed solutions respectively, incubated a second time, and fluorescence intensity detection is performed, and the detected fluorescence intensity is clustered and analyzed.

5. The use according to claim 4, characterized in that In terms of volume, the ratio of the liquor transferred to the mixed solution, the solution containing Cas12 protein, the solution containing the guide RNA, the solution containing the nucleic acid probe transferred to the mixed solution, the nuclease-free water, the buffer, and the solution containing the target nucleic acid is 0.8:2:2:1:27.2:2:

5.

6. Use according to claim 5, characterized in that The molar concentration of the solution containing Cas12 protein is 200nM; The molar concentration of the solution containing the target nucleic acid is 1 μM; The molar concentration of the solution containing the guide RNA is 200 nM; The molar concentration of the solution containing the nucleic acid probe is 200 nM.

7. The use according to claim 4, characterized in that The time of one incubation is 9 min-11 min.

8. The use according to claim 4, characterized in that The temperature of the secondary incubation is 36° C.-38° C., and the time of the secondary incubation is 25 min-35 min.

9. The use according to claim 4, characterized in that The fluorescence intensity was detected using an enzyme marker, with an excitation wavelength of 480nm-500nm and an emission wavelength of 510nm-540nm.

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