CRISPR / Cas12a-DNAzyme-based staphylococcus aureus colorimetric detection method and kit

Abstract

The invention discloses a colorimetric detection method and a colorimetric detection kit for staphylococcus aureus based on CRISPR (clustered regularly interspaced short palindromic repeats) / Cas12a-DNAzyme. According to the colorimetric detection method and the colorimetric detection kit, a specific nuc gene (an NCBI (National Center of Biotechnology Information) accession number V01281.1) of the staphylococcus aureus is taken as a target. Carrying out centrifugal impurity removal and boiling water cracking on the sample to obtain a nucleic acid template, and carrying out isothermal amplification on the nuc gene through RAA; cas12a protein is guided by targeting crRNA to recognize an amplification product, a trans-cleavage active cleavage CatG4R probe is activated, released CatG4 and hemin form DNAzyme, and ABTS-H2O2 is catalyzed to develop color. The detection limit is 100 CFU / mL, and the time consumed in the whole process is less than 2 hours. The specificity of the method is far better than that of an existing target; the pretreatment is simplified to be within 20 minutes, and an expensive kit is not needed; rAA does not need a thermal cycler, color comparison realizes naked eye interpretation, and equipment dependence is greatly reduced. The method is suitable for dairy product detection, and solves the problems of slow speed, poor immunoassay specificity and the like of a traditional method.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological detection, and relates to a Staphylococcus aureus colorimetric detection method and kit based on CRISPR / Cas12a-DNAzyme. BACKGROUND

[0002] Staphylococcus aureus is a typical foodborne pathogenic bacterium in dairy products, and its detection technology has been continuously developed with the increasing demand for food safety. At present, a diversified detection system has been formed based on traditional culture method, supplemented by molecular biology and immunology methods. However, there are still obvious limitations in the practical application of various technologies, and the specific development and defects are as follows: 1. Traditional microbial culture method The traditional microbial culture method is the gold standard for detection of foodborne pathogenic bacteria. It is based on the core process of enrichment-separation-identification: the target bacteria are enriched by selective medium, and qualitative and quantitative identification is achieved by combining Gram staining, biochemical reaction (such as plasma coagulase test) and serological identification. However, this technology has significant limitations in practical application: (1) The detection period is too long, from sample pretreatment to final identification, which takes 3-5 days, and cannot meet the rapid response needs of "raw material acceptance-production control-factory inspection" of perishable products such as dairy products and fresh foods. For example, if this method is used to detect Staphylococcus aureus in raw milk in dairy processing, by the time the results are available, the raw milk may have been processed or spoiled, resulting in economic losses and safety risks. (2) The operation is highly dependent on skilled personnel, and needs to be completed in a sterile environment such as a biological safety cabinet. Due to lack of skills and equipment, it is difficult for grassroots detection institutions or small food companies to apply it efficiently. (3) Poor adaptability to complex substrates. For samples containing complex microbial communities such as fermented milk beverages and high-fat meat products, problems such as colony morphology overlap and biochemical reaction interference may occur, leading to false negatives or identification errors.

[0003] 2. Immune detection technology Immune detection technology is based on the principle of specific binding of antigen-antibody, and has developed three major mainstream technologies: enzyme-linked immunosorbent assay (ELISA), immunochromatography test strip (ICA / LFIA), and immunomagnetic separation (IMS), which are widely used in food field rapid screening. ELISA technology amplifies the signal through enzyme reaction, and the detection limit can be as low as 10 0 -10 3CFU / mL, and can achieve quantitative analysis, has been used for batch detection of Salmonella and Escherichia coli O157:H7 in milk and fruit juice; ICA technology relies on colloidal gold, quantum dots and other markers, and completes the reaction through capillary action, and the results can be obtained within 1 hour, becoming the first choice for on-site scenes such as farms and processing workshops; IMS technology combines the targeted binding of magnetic beads and antibodies to achieve efficient separation of target bacteria, and can improve the detection sensitivity by 10-100 times when combined with other technologies. In recent years, the introduction of nanomaterials has further optimized the performance of immunodetection, for example, the ICA test strip prepared by using Cu2MoS4@PDA composite nanomaterials realizes high-sensitivity quantification of Salmonella through dual reading of color and thermal signals; the aptamer-based immunomagnetic beads (Apt-AuNPs) reduce the dependence on specific antibodies, and the detection cost is reduced by more than 40%.

[0004] However, the practical application of such technologies has limitations: (1) lack of specificity, cross-reactions occur frequently. Staphylococcus aureus and Staphylococcus epidermidis have common antigen epitopes, resulting in a false positive rate of ICA test strips as high as 15%-20%; proteins and fats in dairy products can also non-specifically bind to antibodies, further interfering with result interpretation. (2) The sensitivity is difficult to meet the requirements of national standards, and the detection limit of conventional ICA is mostly 10 1 -10 3 CFU / mL, and requires 6-8 hours of pre-enrichment to reach the detection threshold, making it impossible to achieve "zero enrichment" rapid detection; even the optimized IMS-ELISA combined technology has a detection limit of 6 CFU / mL when detecting pathogenic Escherichia coli in milk, which is higher than the trace detection requirement in some scenarios. (3) There is a contradiction between stability and cost, antibodies are sensitive to temperature and humidity, and ICA test strips need to be stored at 2-8°C, which can easily fail in high-temperature and high-humidity food processing workshops; high-end immunoreagents modified with nanomaterials (such as quantum dot-labeled antibodies) cost 3-5 times more than traditional reagents, making it difficult to popularize in small and medium-sized enterprises.

[0005] 3. Biosensor detection technology Biosensors integrate recognition and signal output through the combination of biological recognition elements and signal conversion modules, and have developed into optical, electrochemical, and magnetic sensors, showing great potential in rapid detection. For example, an electrochemical aptamer sensor based on UiO-66 metal organic framework (MOF) can detect pathogenic bacteria in various foods with a detection limit as low as 10 2 CFU / mL; a carbon nanofiber electrode modified with bacteriophage realizes portable detection of Escherichia coli with a response time of only 15 minutes; a magnetic bead-colorimetric combined sensor realizes visual detection of Staphylococcus aureus in pork through immune capture and enzyme catalysis, with a detection limit of 100 CFU / mL. In recent years, the integration of microfluidic chips and sensors has further reduced the size of the device, such as the "nucleic acid extraction-RAA amplification-signal detection" integrated microfluidic chip, which can simultaneously detect 3 kinds of pathogenic bacteria, and does not require large instruments.

[0006] However, such technology has not been scaled up for application, and the core limitations are: (1) unstable detection performance. Batch differences of nanomaterials (such as quantum dots, carbon nanotubes) will cause fluctuations in sensor signals, for example, under the same conditions, the detection error of AuNPs labeled sensor for Salmonella can reach 30% between different batches; oil and pigment in food matrix will also interfere with optical signal reading, resulting in decreased sensitivity. (2) Contradiction between service life and cost. Biological recognition elements (such as antibodies, aptamers) are prone to inactivation, and the service life of the sensor is mostly 1-3 months, and repeated replacement increases the detection cost; while the sensor with reuse function (such as ultraviolet sterilization microfluidic chip), its preparation process is complex, and the unit price is 5-10 times that of traditional sensor. (3) Lack of industry verification. Most biological sensors are still in the laboratory research and development stage, and have not been systematically verified by national standard methods, and lack of unified performance evaluation standard, food enterprises are reluctant to adopt due to concerns about reliability.

[0007] 4. Molecular biology detection technology Molecular biology detection technology is based on nucleic acid target recognition, and has formed a technology system centered on PCR, isothermal amplification (IAT), and CRISPR / Cas system, which has significantly improved detection efficiency and sensitivity. PCR technology realizes target gene amplification through thermal cycling, and the detection limit of real-time fluorescent quantitative PCR (RT-PCR) can reach 10 0 CFU / mL, and can distinguish between dead and live bacteria, and has been used for simultaneous detection of multiple pathogenic bacteria in fresh meat and vegetables; isothermal amplification technology (such as LAMP, RAA) has broken away from the dependence on thermal cycler, and can complete amplification within 30 minutes at 37-42℃, among which RAA technology combined with lateral flow test strip can realize visual detection of Salmonella in pork, with a detection limit as low as 40 CFU / reaction; CRISPR / Cas system has become a research hotspot in recent years due to its high specificity and accompanying cleavage activity, and the DETECTR platform combined with Cas12a and RAA has a detection limit of 10 1 -10 2 CFU / mL for Staphylococcus aureus, and the detection period is shortened to 70 minutes, and visual interpretation can be achieved through DNAzyme-mediated colorimetric reaction.

[0008] However, such technologies face three major bottlenecks in practical application: first, the instrument and operation threshold are high. RT-PCR requires a ten-thousand-level fluorescence quantitative instrument, and although CRISPR / Cas detection can simplify the equipment, nucleic acid extraction still requires centrifugation, purification and other steps, which is complex and requires professional training for basic detection personnel to master. Second, the matrix interference is significant. Lactose and protein in dairy products can inhibit the activity of DNA polymerase, resulting in a decrease in PCR amplification efficiency of more than 50%, which requires complex pretreatment such as multiple centrifugation and protease digestion to eliminate interference, further prolonging the detection time. Third, false positives and cost issues are prominent. LAMP technology has complex primer design (requires 4-6 primers), which is prone to non-specific amplification, with a false positive rate of 10-15%; the synthesis cost of crRNA of the CRISPR / Cas system is high, and the cost of a single detection is 2-3 times that of traditional PCR, making it difficult to meet the demand for large-scale batch detection.

[0009] In summary, there is no comprehensive solution in the prior art that can simultaneously meet the requirements of rapid response, high specificity, high sensitivity, simple operation, cost control, etc. for Staphylococcus aureus detection in dairy matrix, therefore, it is urgent to develop a Staphylococcus aureus colorimetric detection method with excellent performance to fill the gap in the prior art for rapid and reliable detection of pathogenic bacteria in dairy products, and to meet the safety management and control needs of the food industry from raw material inspection to factory inspection. SUMMARY

[0010] Based on the above-mentioned shortcomings in the prior art, the purpose of the present application is to provide a Staphylococcus aureus colorimetric detection method and kit based on CRISPR / Cas12a-DNAzyme.

[0011] The detection principle is as shown in Figure 1 The present application takes the specific nuc gene (encoding heat-resistant nuclease, NCBI accession number: V01281.1) of Staphylococcus aureus as the specific target, and integrates the following three technologies to achieve detection: (1) RAA isothermal amplification technology: under the condition of constant temperature at 37℃, through the synergistic effect of recombinase, single-stranded binding protein, etc., the nuc gene target fragment is rapidly amplified, without the need for a thermal cycler, meeting the equipment requirements for on-site detection; (2) transcleavage activity of CRISPR / Cas12a system: design a target nucThe crRNA of the gene guides the Cas12a protein to recognize and bind the amplified target DNA, and activates the non-specific trans-cleavage activity of Cas12a to single-stranded DNA (ssDNA); (3) DNAzyme colorimetric signal output: taking CatG4R (the complementary strand of G-quadruplex DNA) as the cleavage substrate of Cas12a, the Cas12a is cleaved after being activated to release CatG4 single strand; CatG4 combines with hemin to form DNAzyme with peroxidase activity, which catalyzes the oxidation reaction of the substrate ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) and H2O2, so that the solution changes from colorless to green, and the color change is observed by naked eye or the absorbance value is detected by an enzyme-labeled instrument to realize quantitative detection.

[0012] Based on the above principle, the application provides a Staphylococcus aureus colorimetric detection method based on CRISPR / Cas12a-DNAzyme, comprising the following steps: (1) Sample pretreatment: centrifugal impurity removal and bacterial lysis are performed on the sample to be detected under sterile conditions, and the supernatant is collected as a nucleic acid amplification template; The sample to be detected in the application is generally dairy products, such as milk / dairy beverages, but other detectable samples are not excluded; In some specific embodiments of the application, step (1) is specifically: Under sterile conditions, 1 mL of the sample to be detected is taken into a sterilized centrifuge tube, centrifuged at 12000 rpm for 2 min, and the supernatant is discarded; 1 mL of sterilized deionized water is added to resuspend the precipitate, centrifuged at 12000 rpm for 2 min, and the supernatant is discarded, repeated once; 100 μL of sterilized deionized water is added to resuspend, boiled in 100°C water bath for 10 min to lyse the bacteria; centrifuged at 12000 rpm for 2 min, and the supernatant is collected as a nucleic acid amplification template. The parameters used in the above centrifugation, water bath, etc. are not necessarily limited to the specific values mentioned, such as 12000 rpm, which can be replaced by 11500 rpm or 12500 rpm in actual application, which can be adjusted according to the model of the centrifuge actually used, and cannot be used to limit the protection scope of the application. The following related parameters are the same.

[0013] (2) RAA isothermal amplification: the supernatant obtained in step (1) is used as a template to amplify the specific nuc gene of Staphylococcus aureus under constant temperature conditions, to obtain nuc gene amplification product; The nuc gene is a heat-resistant nuclease encoding gene with NCBI accession number V01281.1; In some specific embodiments of the application, step (2) is specifically: Take a tube of RAA lyophilized enzyme particles, add 36 μL of nuclease-free water, 2 μL of upstream primer, and 2 μL of downstream primer, mix well, then add 10 μL of sample template and 2 magnesium ion lyophilized particles, and mix by inverting; incubate at 37℃ for 30 min to complete the process. nuc Gene amplification, with the amplification products used directly for subsequent detection.

[0014] (3) CRISPR / Cas12a-DNAzyme colorimetric detection: The result obtained in step (2) nuc Gene amplification products are added with Cas12a protein and targeted nuc In the reaction system of gene crRNA and CatG4R probe, after incubation, CatG4 and Annealing Buffer were added for treatment, then hemin and MES Buffer were added for incubation in the dark, and finally ABTS solution and H2O2 were added for reaction. The target nuc The crRNA of the gene can guide the Cas12a protein to specifically recognize and bind to it. nuc The gene amplification product then activates the trans-cleavage activity of the Cas12a protein on the CatG4R probe. Furthermore, the target nuc The sequence of the crRNA of the gene is shown in SEQ.ID.NO.11 or SEQ.ID.NO.12; the sequence of the CatG4R probe is shown in SEQ.ID.NO.8; the sequence of the CatG4 is shown in SEQ.ID.NO.7. In some specific embodiments of the present invention, step (3) specifically includes: Prepare a 100 μL reaction solution containing 1×HOLMES Buffer, 250 nM Cas12a protein, 250 nM crRNA, 250 nM CatG4R probe, and 5 μL RAA amplification product. Incubate at 37°C for 20 min. Add 5 μL CatG4 and 5 μL Annealing Buffer, boil at 100°C for 5 min, and then slowly cool to room temperature. Add 2 μL hemin and 72 μL MES Buffer, and incubate in the dark for 30 min. Add 2 μL ABTS solution and 2 μL 3% H2O2, and react at room temperature for 5 min.

[0015] (4) Signal interpretation: The color change of the solution after the reaction is observed by the naked eye. A positive result is green and a negative result is colorless. Alternatively, the absorbance value can be measured at a wavelength of 414 nm using an enzyme-linked immunosorbent assay (ELISA) reader to detect Staphylococcus aureus.

[0016] Furthermore, the detection limit of this method for Staphylococcus aureus is 10. 0The sample concentration was CFU / mL, and the entire process from dairy sample pretreatment to signal interpretation took less than 2 hours.

[0017] This invention also provides a colorimetric detection kit for Staphylococcus aureus based on CRISPR / Cas12a-DNAzyme, the kit comprising: RAA lyophilized enzyme particles, targeting Staphylococcus aureus. nuc Upstream and downstream primers for the gene (NCBI accession number: V01281.1), magnesium ion lyophilized particles, Cas12a protein, and targeted... nuc The contents include: crRNA of the gene, CatG4R probe, CatG4, hemin, ABTS solution, 3% H2O2, 1×HOLMES Buffer, MES Buffer, Annealing Buffer; also includes nuclease-free water, sterile deionized water, elution buffer, sterile centrifuge tubes, adsorption column, Buffer BS, lysozyme, Buffer L, Proteinase K, Nase A, Buffer GB, anhydrous ethanol, Buffer WA, and Buffer WB.

[0018] The upstream and downstream primers are selected from the upstream primer shown in sequence SEQ.ID.NO.5 and the downstream primer shown in sequence SEQ.ID.NO.6.

[0019] The present invention also provides the application of the above-mentioned detection method and kit in the detection of food pathogens, wherein the food pathogen is Staphylococcus aureus.

[0020] Compared with the prior art, this application has at least the following improvements and beneficial effects: (1) Enhanced target specificity: Select targets with a conservation rate >98%. nuc Using genes (specific to Staphylococcus aureus only) as targets, combined with the sequence specificity of CRISPR / Cas12a, improves detection specificity and solves the problems of existing methods. spa, coa False negatives are caused by low conservation of gene targets. (2) Simplified sample pretreatment: dairy samples are treated by a two-step method of centrifugation to remove impurities and boiling water lysis (time < 20 min), without the need for expensive kits, effectively removing matrix interference and directly obtaining amplifiable templates. This method is significantly more efficient than the traditional proteinase K digestion + phenol-chloroform extraction (time > 1 h). (3) Reduced equipment dependence: RAA isothermal amplification (37℃ constant temperature, no need for thermal cycler) is used, which can be completed with a portable temperature block, solving the problem of large size and high cost of PCR technology equipment; (4) Convenient signal output: Combined with the DNAzyme colorimetric system, it enables visual interpretation without the need for a fluorescence quantitative instrument, and the sensitivity reaches 10. 0 CFU / mL (consistent with fluorescence method), result reading time <5 min, solving the problems of existing CRISPR detection relying on instruments and low sensitivity (>1000 CFU / mL) of colloidal gold test strips; (5) Optimized detection efficiency: The entire process takes less than 2 hours, and each step is seamlessly connected (RAA amplification is directly followed by CRISPR detection without product purification). Compared with traditional culture methods (3-5 days) and PCR + fluorescence detection (4-6 hours), it is more suitable for real-time quality control in the dairy production chain. Attached Figure Description

[0021] Figure 1 This describes the detection principle of this detection method; Figure 2 The image shows the screening results for crRNA. Figure 3 Feasibility verification of DNAzyme-assisted CRISPR / Cas12a colorimetric bioassay: (A) Verification of trans-cleavage activity of fluorescence signal; (B) Fluorescence intensity at a reaction time of 90 min; (C) Fluorescence intensity and... nuc (D) Linear relationship of gene concentration; (E) Verification of trans-cleavage activity by colorimetric signal; (F) Absorbance at 90 min reaction time; (G) Absorbance and... nuc Linear relationship of gene concentration; Figure 4 Sensitivity of DNAzyme-assisted CRISPR / Cas12a colorimetric bioassay for Staphylococcus aureus: (A) Characterization of fluorescence signal kinetics; (B) Fluorescence intensity at a reaction time of 40 min; (C) Linear relationship between fluorescence intensity and Staphylococcus aureus concentration; (D) Full-spectrum scan of colorimetric signal; (E) Absorbance at 415 nm; (F) Linear relationship between absorbance and Staphylococcus aureus concentration. Figure 5 To determine the specificity of Staphylococcus aureus against other common bacteria using a DNAzyme-assisted CRISPR / Cas12a colorimetric bioassay: (A) fluorescence signal kinetic characterization; (B) fluorescence intensity at a reaction time of 40 min; (C) full-spectrum colorimetric signal scanning; (D) absorbance at 415 nm. Figure 6 Sensitivity characterization: (A) shows the test strip reaction results; (B) shows the ImageJ grayscale analysis of (A). Detailed Implementation

[0022] For those skilled in the art to better understand the technical solutions of the present application, the present application will be described in detail below in combination with specific examples. It should be pointed out that the following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be pointed out that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made. These all belong to the protection scope of the present application.

[0023] All raw materials of the present application, if not specially specified, represent that there is no special restriction on their source, and all can be purchased on the market or prepared according to the conventional method well known to those skilled in the art.

[0024] The reagents used in the experiment and their manufacturers are as follows: tryptone, beef heart infusion powder, yeast extract powder, disodium hydrogen phosphate, glucose, agar, sodium chloride, sodium hydroxide, MES buffer (0.1M, pH 4.7) and 2,2'-diamino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt are all purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.; hydrochloric acid is purchased from Shengong Biological Engineering (Shanghai) Co., Ltd.; 2x Phanta Max Master Mix (Dye Plus) is purchased from Nanjing Novizen Biological Technology Co., Ltd.; DNA marker, loading buffer and bacterial genome extraction kit are purchased from Baori Medical Biological Technology (Beijing) Co., Ltd.; AxyPrep PCR clean kit is purchased from Axygen Biotechnology (Hangzhou) Co., Ltd.; basic RAA nucleic acid amplification reagent (lyophilized particles) is purchased from Shanghai Huicheng Biological Technology Co., Ltd.; LbCas12a protein and crRNA synthesis and purification kit are purchased from Shanghai Tuolu Gang Biological Technology Co., Ltd.; Hemin is purchased from Shanghai Titan Science and Technology Co., Ltd. Hydrogen peroxide (30%, v / v), dimethyl sulfoxide, DNA annealing buffer (5x), needle type filter 0.45 μm nylon membrane, needle type filter 0.22 μm hydrophilic and 15 mL ultrafiltration centrifuge tube are all purchased from Shanghai Titan Science and Technology Co., Ltd.; kanamycin sulfate, IPTG, 2-(N-morpholino)ethanesulfonic acid monohydrate, polyoxyethylene lauryl ether and silica gel desiccant are all purchased from Shengong Biological Engineering (Shanghai) Co., Ltd.; 10-250 kDa three-color pre-stained protein marker, 12.5% SDS-PAGE gel preparation kit, 6x protein loading buffer and coomassie brilliant blue rapid staining solution are all purchased from Shanghai Yezheng Biological Medicine Technology Co., Ltd.; BCA protein quantification kit, magnesium chloride hexahydrate and magnesium chloride, Tris, imidazole, glycine, dithiothreitol (DTT) and sodium dodecyl sulfate (SDS) are all purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.; 5% BSA blocking solution is purchased from Beijing Solabio Technology Co., Ltd.; 90 type NC membrane, 120 type NC membrane, sample pad, water absorption pad and PVC bottom plate are all purchased from Shanghai Jieyi Biological Technology Co., Ltd.

[0025] The instruments used in the experiment and their manufacturers are as follows: PX-150B-Z biochemical incubator purchased from Shanghai Boxun Industry Co., Ltd. Medical Equipment Factory; constant temperature metal bath purchased from Hangzhou Ruicheng Instrument Co., Ltd.; high-speed refrigerated centrifuge purchased from Sigma Company, USA; super-clean workbench purchased from Suzhou Purification Equipment Co., Ltd.; PB-10 pH meter purchased from Sartorius Company, Germany; PCR instrument, Powerpac basic electrophoresis instrument and GelDocXR gel imaging instrument are all purchased from Bole Life Medicine Product Co., Ltd.; electronic balance and analytical balance are purchased from Shanghai Precision Instrument Co., Ltd.; Nanodrop 2000 micro-nucleic acid protein quantification instrument is purchased from Thermo Fisher Company, USA; Spectra Max i3x type enzyme label instrument is purchased from Austria Meigu Molecular Instrument Co., Ltd.; high-pressure sterilizer is purchased from Shanghai Renzhe Instrument and Equipment Co., Ltd.; biological dot sample instrument is purchased from BIO-DOT Company, USA; plate sealer and automatic cutter are all purchased from Shanghai Jinbiao Biotechnology Co., Ltd.

[0026] Example 1: Design of crRNA sequence Figure 1 The principle diagram of the detection method based on DNAzyme (deoxyribozyme) designed in the present application is shown in the figure. The Staphylococcus aureus nuc The specific crRNA sequence is designed for the Staphylococcus aureus gene, and high-activity crRNA is obtained by in vitro transcription and purification. nuc The gene is a specific target of Staphylococcus aureus (encoding heat-resistant nuclease), and the amplification product activates the reverse cleavage activity of Cas12a protein through crRNA guidance, and specifically cleaves the CatG4R (the sequence is shown as SEQ.ID.NO.8, see Table 1) probe, which is the complementary strand of CatG4 (the sequence is shown as SEQ.ID.NO.7, see Table 1). After CatG4R is cut, single-stranded CatG4 is released, which combines with hemin to form G-quadruplex DNAzyme with peroxidase activity, catalyzing ABTS-H2O2 oxidation color development. nuc The presence of the gene is a sufficient condition to trigger the Cas12a cleavage of CatG4R. CatG4R as the substrate of Cas12a reverse cleavage, whether it is cleaved or not directly determines the formation of DNAzyme and the generation of colorimetric signal, both of which realize the indirect coupling of “target recognition→signal triggering” through the CRISPR / Cas12a system. Based on the reverse cleavage characteristics of Cas12a, the detection system is constructed by combining RAA isothermal amplification technology: the signal is enhanced by amplifying the target nucleic acid through RAA, and the fluorescence signal detection system is established by using the reverse cleavage characteristics of Cas12a, and the DNAzyme-mediated colorimetric signal detection system is also developed.

[0027] Figure 2In Staphylococcus aureus nuc In the CDS sequence of the gene, 5'-TTTN was selected as the PAM site (original interval adjacent motif), and then the DNA sequence of the 20 nucleotides (nt) immediately downstream of the PAM site was selected as the spacer sequence of the crRNA. The spacer sequence was fused with the inherent scaffold sequence to assemble a complete crRNA molecule with function. Six PAM sites were selected before the experiment, and the corresponding crRNAs (denoted as 1, 2, 3, 4, 5, and 6, corresponding to the sequences shown as SEQ.ID.NO.10-15 in Table 1, respectively) were designed and verified in a system containing all necessary components for trans-cleavage to screen the best crRNA. The sequences of the transcription primers and the crRNAs are shown in Table 1.

[0028] In this embodiment, the crRNA was synthesized by T7 in vitro transcription and purified by magnetic bead method, and the specific method was as follows: 1) After preparing the annealing system according to the instructions of the kit, the transcription primer was reacted at 95°C for 5 min, and then slowly cooled to room temperature at a speed of 0.2°C / s to prepare the transcription template. The sequences of the transcription primers are shown as SEQ.ID.NO.1-4 in Table 1, wherein Nuc-PCR-F1 and Nuc-PCR-R1 are a pair of upstream and downstream primers, and Nuc-PCR-F2 and Nuc-PCR-R2 are a pair of upstream and downstream primers. The transcription template is the Nuc-CDS shown in SEQ.ID.NO.9.

[0029] 2) The reagents were sequentially added according to the instructions of the T7 in vitro transcription kit, and the in vitro transcription reaction system was prepared. After thorough mixing, the system was incubated at 37°C for 4 h.

[0030] 3) After incubation, DNA template was removed from the transcription system using DNase I reaction solution.

[0031] 4) The magnetic beads were mixed with the crRNA, and incubated at room temperature to allow them to bind to the magnetic beads. Then, the impurities were removed by ethanol rinsing, and the crRNA was eluted using nucleic acid-free water.

[0032] 5) The purified crRNA was treated at 70°C for 10 min to inactivate the residual DNase I.

[0033] The experimental steps for observing fluorescence in this embodiment are as follows: (1) Extraction of genomic DNA: In this embodiment, Staphylococcus aureus bacterial solution was used as the sample to be detected, and the template was extracted from Staphylococcus aureus genomic DNA, specifically as follows: Take 1 mL of logarithmic phase S. aureus bacterial solution, centrifuge at 12000 rpm for 1 min, discard the supernatant; add 500 μL Buffer BS to resuspend the precipitate, then add 50 μL lysozyme, incubate at 37°C for 60 min, then centrifuge and discard the supernatant; add 180 μL Buffer L, 20 μL Proteinase K and 10 μL Nase A, incubate at 56°C for 30 min to lyse the cells; add 200 μL Buffer GB and 200 μL anhydrous ethanol, mix well and transfer to the adsorption column, centrifuge at 12000 rpm for 1 min to discard the waste liquid; wash the adsorption column with 500 μL Buffer WA, 700 μL Buffer WB (2 times) in turn, and centrifuge for 2 min to remove residual ethanol; add 50 μL Elution Buffer, stand at room temperature for 5 min, then centrifuge and elute, collect the genomic DNA and store at -20°C.

[0034] (2) RAA isothermal amplification Take the RAA lyophilized enzyme pellet tube, add 36 μL nuclease-free water, 2 μL upstream primer, 2 μL downstream primer, mix well, then add 10 μL nucleic acid amplification template obtained in step (1) and 2 magnesium ion lyophilized pellets, invert and mix well; incubate at 37°C for 30 min, complete nuc gene amplification, and the amplification product is directly used for subsequent detection. The sequences of the upstream and downstream primers for RAA amplification are shown in SEQ.ID.NO.5-6.

[0035] (3) CRISPR / Cas12a-DNAzyme colorimetric detection Prepare the reaction solution according to a 20 μL system: containing 1×HOLMES Buffer, 250 nM Cas12a protein, 250 nM crRNA, 250 nM CatG4R probe and 5 μL RAA amplification product, incubate at 37°C for 20 min; add 5 μL CatG4 and 5 μL Annealing Buffer, boil at 100°C for 5 min, then slowly cool to room temperature; add 2 μL hemin and 72 μL MES Buffer, incubate in the dark for 30 min; add 2 μL ABTS solution and 2 μL 3% H2O2, react at room temperature for 5 min, observe the color change (positive is green, negative is colorless), or measure the absorbance value at 414 nm with a microplate reader.

[0036] According to Figure 2The fluorescence curve results show that only No. 2 and No. 3 crRNA produce fluorescence signals. The crRNA labeled as "2" as shown in SEQ. ID. NO. 11 (crRNA-M2) should be selected. The reason is that: the curve (red curve) corresponding to the crRNA rises fastest in fluorescence value and finally reaches the highest fluorescence intensity, indicating that it can most efficiently activate the transcleavage activity of Cas12a in the system containing all necessary components for transcleavage, and the recognition and signal amplification effect of the target (Staphylococcus aureus nuc gene related) is optimal. The crRNA labeled as "3" as shown in SEQ. ID. NO. 12 (crRNA-M3) can be the second choice.

[0037] Table 1 primer sequence Example 2: feasibility of the kit in nucleic acid detection In order to verify the feasibility of the DNAzyme-assisted CRISPR / Cas12a colorimetric biological detection method in nucleic acid detection, a series of experiments were carried out to verify it.

[0038] The operation method of the fluorescence detection method based on CRISPR / Cas12a technology: the reaction system of CRISPR / Cas12a transcleavage is as follows: 1xHOLMES Buffer, 250 nM Cas12a, 250 nM crRNA, 250 nM ssDNA reporter, 10 nM target DNA, and the total volume is 20 μL. The optimal conditions for subsequent experiments were screened by replacing crRNA and the ratio of Cas12a and crRNA. The reaction solution was added to a 384-well plate, and the fluorescence intensity was measured every 2-3 min at 37°C using the "Fluorescence kinetics" program in the enzyme marker, and the excitation and emission wavelengths were 492 nm and 518 nm, respectively.

[0039] The operation steps of the colorimetric biological detection method based on the combination of CRISPR / Cas12a technology and DNAzyme are as follows: the ssDNA reporter in the reaction system of CRISPR / Cas12a trans-cleavage is replaced with CatG4R, 20 μL of the above-mentioned Cas12a reaction product is mixed with a CatG4 (10 μM) solution and an Annealing buffer, boiled at 100°C for 5 min, and cooled to room temperature to anneal CatG4 and the probe CatG4R. Next, 2 μL of hemin (100 μM) and 72 μL of MES buffer (0.1 M, pH 4.7) are added to the annealing solution, and the solution is incubated in the dark for 30 min. After the reaction is completed, 2 μL of ABTS solution is added to the solution, and finally 2 μL of H2O2 (v / v = 3%) is added, the color change is observed, and the absorbance value is measured by an enzyme marker.

[0040] First, the Staphylococcus aureus genomic DNA is extracted, and then detected by the CRISPR / Cas12a system with the probe replaced by CatG4. When the amplified nucleic acid sequence is successfully recognized by the specific crRNA, the trans-cleavage activity of Cas12a is activated, thereby inducing the non-specific degradation of the fluorescent reporter probe. To verify the trans-cleavage activity of CRISPR / Cas12a, four groups of variable experiments are designed. The experiments use a fluorescence method for signal output, and the fluorescence intensity of each group within 90 minutes is detected by an enzyme marker, and the fluorescence signal kinetics results are plotted. Among them, the experimental group contains all the necessary components for fluorescent cleavage, and the four negative control groups are respectively Cas12a-free group, crRNA-free group, target DNA-free group, and fluorescent reporter probe-only group.

[0041] As shown in Figure 3 A, only the fluorescence value of the experimental group increases with time, and the fluorescence values of the other four negative control groups do not show a significant increasing trend. This result fully shows that Cas12a has the trans-cleavage activity of non-specifically cleaving ssDNA reporter triggered by specific recognition of target DNA. In the absence of specific crRNA or target DNA, the trans-cleavage activity of Cas12a is in a silent state, at which time the fluorescent reporter probe is not degraded and the fluorescence signal is quenched. Only in the presence of target DNA, crRNA and Cas12a protein, the fluorescent reporter probe will be degraded, thereby generating a fluorescence signal. Further observation shows that the fluorescence intensity shows a concentration-dependent increasing trend (see Figure 3 B), and a good linear relationship is established between the fluorescence intensity and the concentration of target DNA (R 2 = 0.967, see Figure 3C)。

[0042] Compared with fluorescence signal detection, colorimetric signal output is more convenient and does not require complex instrument equipment. In this study, a colorimetric bioassay technology based on DNAzyme assisted CRISPR / Cas12a was successfully developed, and relevant tests were carried out. When target DNA exists, CatG4R will be degraded by activated Cas12a, and then DNAzyme is formed to catalyze ABTS to generate ABST⁻, thereby causing obvious color change and producing specific light absorption at 415 nm wavelength. On the contrary, if there is no target DNA, CatG4R remains intact and cannot catalyze ABTS to react. Under the condition that target DNA, crRNA and Cas12a exist at the same time, significant color change can be observed, and the absorbance at 414 nm wavelength increases significantly (see Figure 3 D). In addition, the absorbance value gradually increases with the increase of target DNA concentration, and a good linear relationship is shown between them (R 2 =0.975, see Figure 3 E, F).

[0043] The above results show that the kit has both fluorescence detection and colorimetric detection modes: the fluorescence mode realizes high-sensitivity quantification of trace targets through the enzyme label instrument, and the colorimetric mode can be completed by naked eye or ordinary spectrophotometer for rapid qualitative / semi-quantitative. The experimental results of the two modes are verified with each other, and both are enhanced with the increase of target concentration, which further embodies the stability and reliability of the detection system, and meets the needs of multi-dimensional verification, flexible adaptation scene of nucleic acid detection.

[0044] Example 3: Sensitivity of DNAzyme assisted CRISPR / Cas12a colorimetric biological detection method to Staphylococcus aureus The specific operation method of this example is the same as that of Example 2.

[0045] As shown in Figure 4 A, with the increase of Staphylococcus aureus colony concentration, the fluorescence signal gradually increases, which shows that the CRISPR / Cas12a system can effectively recognize and respond to different concentrations of Staphylococcus aureus. Further analysis found that within the range of 10 0 -10 8 CFU / mL, a very strong linear relationship between fluorescence signal and Staphylococcus aureus colony concentration was obtained, with a linear correlation coefficient as high as R 2 =0.988, as shown in Figure 4 B and C. This shows that the fluorescence detection method has high sensitivity and accuracy for Staphylococcus aureus. In addition, the detection limit of the fluorescence readout method for Staphylococcus aureus is 10 0CFU / mL, which provides an important reference for the subsequent detection method optimization.

[0046] In order to adapt to the needs of detection visualization and simplicity, a colorimetric bio-detection method based on DNAzyme-assisted CRISPR / Cas12a was used for the detection of S. aureus. After amplification and detection of different concentrations of S. aureus, obvious color changes were observed, as shown in Figure 4 D. With the increase of S. aureus concentration, more CatG4R was degraded to form DNAzyme, which in turn catalyzed the ABTS-H2O2 reaction to enhance, leading to more significant color changes. The corresponding absorbance spectrum and absorbance at 415 nm are shown in Figure 4 E. The absorbance at 415 nm increased in a S. aureus concentration-dependent manner, indicating that the colorimetric bio-detection method had good quantitative ability for S. aureus detection. Further analysis found that in the range of 10 0 -10 8 CFU / mL, a very strong linear relationship between absorbance and S. aureus colony concentration was also obtained, with a linear correlation coefficient of R 2 =0.908 ( Figure 4 F).

[0047] Example 4: Selectivity of the colorimetric bio-detection method based on DNAzyme-assisted CRISPR / Cas12a for S. aureus The specific operation method of this embodiment is the same as that of Example 2.

[0048] As shown in Figure 5 A and B, when detecting 4 strains of interfering bacteria: Salmonella, Escherichia coli (abbreviated as E. coli), Listeria monocytogenes (abbreviated as L. monocytogenes), and Lactobacillus plantarum (abbreviated as L. plantarum), the fluorescence signals obtained were significantly lower than those of S. aureus samples. However, when detecting S. aureus samples alone and mixing S. aureus with the above interfering bacteria (marked as Mixture), the fluorescence signals were significantly increased. This indicates that the method has good specificity, and even in the presence of interfering bacteria, S. aureus can still be effectively detected.

[0049] Further, when the colorimetric bio-detection method of DNAzyme-assisted CRISPR / Cas12a was used for detection, the relative absorbance at 415 nm increased significantly compared with other interfering bacteria as long as S. aureus existed, which was consistent with the result obtained by using the fluorescence method, as shown in Figure 5 C and D. This fully proves that the influence of these interfering bacteria on the detection of S. aureus by the DNAzyme-assisted CRISPR / Cas12a can be ignored. Therefore, it can be concluded that the colorimetric bio-detection method of DNAzyme-assisted CRISPR / Cas12a proposed in the present study has good selectivity for the detection of S. aureus, and can effectively distinguish S. aureus from other interfering bacteria, thereby providing a reliable method for the accurate detection of S. aureus.

[0050] Example 5: Detection limit of S. aureus by the colorimetric bio-detection method of DNAzyme-assisted CRISPR / Cas12a The specific operation method of the present embodiment is the same as that of Example 2.

[0051] The constructed CRISPR / Cas12a-POCT detection method was applied to the detection of S. aureus. The test strip detection results and the ImageJ gray scale analysis results are shown in Figure 6 A and Figure 6 B, respectively. As shown in Figure 6 A, within the range of 10 0 -10 8 CFU / mL, the color of the T line gradually deepened with the increase of the concentration of the bacterial solution; and when the concentration of the bacterial solution was lower than 10 3 CFU / mL, the color of the T line was difficult to distinguish by naked eye. Figure 6 B shows that the results of gray scale analysis by using ImageJ have a lower detection limit than naked eye observation, and can detect a higher gray scale value at a concentration of 10 1 CFU / mL, which is significantly different from the negative group. Therefore, the detection limit of the method is 10 1 CFU / mL.

[0052] Example 6: Application of a CRISPR / Cas12a-DNAzyme-based colorimetric detection kit for S. aureus in dairy product samples Based on the research and optimization design in the above examples, the kit provided in the present embodiment includes the following components: RAA lyophilized enzyme particles, upstream primers and downstream primers for S. aureus nuc gene (NCBI accession number: V01281.1), magnesium ion lyophilized particles, Cas12a protein, and target nuccrRNA of gene, CatG4R probe, CatG4, hemin, ABTS solution, 3% H2O2, 1xHOLMES Buffer, MES Buffer, Annealing Buffer, nuclease-free water, sterilized deionized water, Elution Buffer, sterilized centrifuge tube, adsorption column, Buffer BS, lysozyme, Buffer L, Proteinase K, NaseA, Buffer GB, anhydrous ethanol, Buffer WA, Buffer WB.

[0053] This example is based on the above-mentioned kit, and a dairy product sample is taken as a sample to be detected, and the steps are as follows: (1) Pretreatment of dairy product sample: Under sterile conditions, 1 mL of the dairy product sample to be detected (milk / dairy beverage) was taken into a sterilized centrifuge tube, centrifuged at 12000 rpm for 2 min, and the supernatant was discarded; 1 mL of sterilized deionized water was added to resuspend the precipitate, centrifuged at 12000 rpm for 2 min, and the supernatant was discarded, repeated once; 100 μL of sterilized deionized water was added to resuspend, boiled in 100°C water bath for 10 min to lyse the bacterial body; centrifuged at 12000 rpm for 2 min, and the supernatant was collected as the nucleic acid amplification template.

[0054] (2) RAA isothermal amplification The RAA freeze-dried enzyme granule tube was taken, 36 μL of nuclease-free water, 2 μL of upstream primer, and 2 μL of downstream primer were added, mixed, and then 10 μL of the nucleic acid amplification template obtained in step (1) and 2 magnesium ion freeze-dried particles were added, and mixed by inversion; incubated at 37°C for 30 min, and the nuc gene amplification was completed, and the amplification product was directly used for subsequent detection.

[0055] (3) CRISPR / Cas12a-DNAzyme colorimetric detection The reaction solution was prepared according to a 20 μL system: containing 1xHOLMES Buffer, 250 nM Cas12a protein, 250 nM crRNA, 250 nM CatG4R probe, and 5 μL RAA amplification product, incubated at 37°C for 20 min; 5 μL of CatG4 and 5 μL of Annealing Buffer were added, boiled at 100°C for 5 min, and then slowly cooled to room temperature; 2 μL of hemin and 72 μL of MES Buffer were added, and incubated in the dark for 30 min; 2 μL of ABTS solution and 2 μL of 3% H2O2 were added, and reacted at room temperature for 5 min, the color change was observed (positive for green color, negative for colorless), or the absorbance value was measured at 414 nm using an enzyme marker.

[0056] The foregoing description of the embodiments has been presented for the purpose of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed. Modifications and variations are possible in light of the above teachings or can be acquired from practice of the application. As well, the description is presented in the context of the preferred embodiments as a number of alternatives. It is not intended to limit the application to the precise form described.

Claims

1. A colorimetric detection method for Staphylococcus aureus based on CRISPR / Cas12a-DNAzyme, characterized in that, Includes the following steps: (1) Sample pretreatment: under aseptic conditions, the sample to be tested is centrifuged to remove impurities and the bacterial cells are lysed. The supernatant is collected as a template for nucleic acid amplification. (2) RAA isothermal amplification: Using the supernatant obtained in step (1) as a template, Staphylococcus aureus-specific amplification was performed under isothermal conditions. nuc Genes, obtained nuc Gene amplification products; (3) CRISPR / Cas12a-DNAzyme colorimetric detection: The result obtained in step (2) nuc Gene amplification products are added with Cas12a protein and targeted nuc In the reaction system of the gene crRNA and CatG4R probe, after incubation, CatG4 and Annealing Buffer were added for treatment, then hemin and MES Buffer were added for incubation in the dark, and finally ABTS solution and H2O2 were added for reaction. (4) Signal interpretation: The color change of the solution after the reaction is observed by the naked eye. A positive result is green and a negative result is colorless. Alternatively, the absorbance value can be measured at a wavelength of 414 nm using an enzyme-linked immunosorbent assay (ELISA) reader to detect Staphylococcus aureus.

2. The colorimetric detection method for Staphylococcus aureus based on CRISPR / Cas12a-DNAzyme according to claim 1, characterized in that, The sample to be tested in step (1) is a dairy product.

3. The colorimetric detection method for Staphylococcus aureus based on CRISPR / Cas12a-DNAzyme according to claim 1, characterized in that, Step (1) Sample pretreatment is as follows: Under sterile conditions, take the sample to be tested, centrifuge and discard the supernatant; add sterile deionized water to resuspend the precipitate, centrifuge and discard the supernatant, repeat several times; add sterile deionized water to resuspend, lyse the bacterial cells in a boiling water bath; centrifuge and collect the supernatant as a template for nucleic acid amplification.

4. The colorimetric detection method for Staphylococcus aureus based on CRISPR / Cas12a-DNAzyme according to claim 1, characterized in that, Step (2) nuc The gene encodes a thermostable nuclease, and its NCBI accession number is V01281.

1.

5. The colorimetric detection method for Staphylococcus aureus based on CRISPR / Cas12a-DNAzyme according to claim 1, characterized in that, Step (2) RAA isothermal amplification is as follows: Take a tube of RAA lyophilized enzyme particles, add nuclease-free water, upstream primer, and downstream primer, mix well, then add the sample template and magnesium ion lyophilized particles obtained in step (1), invert and mix well; after constant temperature incubation, the process is complete. nuc Gene amplification yields amplification products.

6. The colorimetric detection method for Staphylococcus aureus based on CRISPR / Cas12a-DNAzyme according to claim 1, characterized in that, The target described in step (3) nuc The sequence of the gene's crRNA is shown in SEQ.ID.NO.11, or in SEQ.ID.NO.12; The sequence of the CatG4R probe is shown in SEQ.ID.NO.8; The sequence of CatG4 is shown in SEQ.ID.NO.

7.

7. The colorimetric detection method for Staphylococcus aureus based on CRISPR / Cas12a-DNAzyme according to claim 1, characterized in that, Step (3) CRISPR / Cas12a-DNAzyme colorimetric detection is as follows: Prepare a reaction solution containing 1×HOLMES Buffer, Cas12a protein, crRNA, CatG4R probe and RAA amplification product obtained in step (2). After incubation, add CatG4 and Annealing Buffer, boil and cool to room temperature; add hemin and MES Buffer, incubate in the dark; add ABTS solution and H2O2, and react at room temperature.

8. A colorimetric detection kit for Staphylococcus aureus based on CRISPR / Cas12a-DNAzyme, characterized in that, The kit includes: RAA lyophilized enzyme particles, targeting Staphylococcus aureus. nuc Upstream and downstream primers for genes, magnesium ion lyophilized particles, Cas12a protein, targeting nuc crRNA of the gene, CatG4R probe, CatG4, hemin, ABTS solution, H2O2.

9. The Staphylococcus aureus colorimetric detection kit based on CRISPR / Cas12a-DNAzyme according to claim 8, characterized in that, The upstream and downstream primers are selected from the upstream primer shown in sequence SEQ.ID.NO.5 and the downstream primer shown in sequence SEQ.ID.NO.

6.

10. The application of the detection method according to any one of claims 1-7 or the kit according to any one of claims 8-9 in the detection of food pathogens, characterized in that, The foodborne pathogen is Staphylococcus aureus, and the detection limit for Staphylococcus aureus by the detection method or kit is 10-1. 0 CFU / mL, detection time < 2 h.

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