A CRISPR / Cas-regulated DNA silver nanocluster sensor and its method and application for detecting meat adulteration

Abstract

This invention discloses a CRISPR / Cas-regulated DNA silver nanocluster sensor and its method and application for detecting adulteration in meat, belonging to the field of food safety testing technology. This DNA-templated silver nanocluster sensor introduces a novel tagless fluorescent probe, DNA-AgNCs, as a substrate for CRISPR / Cas12a. After the CRISPR / Cas12a-crRNA complex specifically recognizes the target nucleic acid, its trans-cleavage activity is activated, leading to the cleavage of the DNA template and a significant decrease in fluorescence signal. Detecting adulteration in meat using this DNA-templated silver nanocluster sensor can cleverly convert the concentration of adulteration into a quantifiable fluorescence signal output, achieving quantitative detection of adulterants. It exhibits high sensitivity and specificity, providing a universal and sensitive detection method for meat adulteration.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to a CRISPR / Cas-regulated DNA silver nanocluster sensor and its method and application for detecting adulteration in meat. Background Technology

[0002] In recent years, meat adulteration has gradually become a concern in the global food market. Although many laws have been introduced at home and abroad to ensure and supervise the quality of meat, meat adulteration still occurs frequently.

[0003] Existing methods for meat authenticity testing mainly include detection techniques using characteristic proteins, specific metabolites, and DNA as markers, such as mass spectrometry (MS), enzyme-linked immunosorbent assay (ELISA), spectroscopic methods, polymerase chain reaction (PCR), quantitative PCR (qPCR), and emerging non-destructive testing (NDT) techniques such as near-infrared spectroscopy (NIRS), hyperspectral imaging (HSI), and Raman spectroscopy (RS). Among these methods, mass spectrometry and spectroscopic techniques are highly versatile due to their ability to provide a wealth of structural information about the analytes. Non-destructive testing techniques are suitable for repeated measurements and automated detection methods, demonstrating advantages in high-throughput and automated analysis. However, proteins are easily degraded or denatured under heat processing or complex processing conditions, thus compromising the reliability of protein-targeted detection methods. Furthermore, metabolites are significantly affected by the animal's growth environment and meat storage and processing conditions, and the species specificity of metabolites remains controversial. Therefore, metabolite-based techniques are not yet sufficiently convincing in detecting meat adulteration. In contrast, nucleic acids possess recognized species specificity, interspecies polymorphism, and thermostability, making them specific and accurate biomarkers. Traditional PCR and qPCR techniques offer good specificity and sensitivity; however, their widespread application is limited by the requirements for sophisticated equipment, stringent experimental environments, and specialized personnel. Furthermore, due to complex food matrices, extremely low target DNA content, and various adulteration methods, more specific and sensitive detection technologies urgently need to be developed. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide a CRISPR / Cas-regulated DNA silver nanocluster sensor and its method and application for detecting meat adulteration, thereby providing a detection solution for meat adulteration detection.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This invention discloses a CRISPR / Cas-regulated DNA silver nanocluster sensor, comprising a CRISPR / Cas system and DNA-templated silver nanoclusters;

[0007] The CRISPR / Cas system consists of Cas12a protein, crRNA, RNase inhibitor, buffer, and amplified target adulterated sample DNA; the DNA-templated silver nanoclusters are synthesized from a solution containing silver ions, NaBH4, and a DNA template; the recognition sequence of the crRNA is complementary to the target adulterated sample DNA fragment; the DNA template sequence is shown in SEQ.ID.NO.9, SEQ.ID.NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12, SEQ.ID.NO.13, or SEQ.ID.NO.14.

[0008] Preferably, the adulterated sample is duck meat, and the nucleotide sequence of the duck meat sample DNA is shown in SEQ.ID.NO.8; the nucleotide sequence of the crRNA is shown in SEQ.ID.NO.7.

[0009] Preferably, the DNA template sequence is C-G4.

[0010] More preferably, the amplification is loop-mediated isothermal amplification, and the primers used for amplification include duck meat-FIP primers, duck meat-BIP primers, duck meat-F3 primers, duck meat-B3 primers, duck meat-LF primers, and duck meat-LB primers; wherein the nucleotide sequence of the duck meat-F3 primer is shown in SEQ.ID.NO.1, the nucleotide sequence of the duck meat-B3 primer is shown in SEQ.ID.NO.2, the nucleotide sequence of the duck meat-FIP primer is shown in SEQ.ID.NO.3, the nucleotide sequence of the duck meat-BIP primer is shown in SEQ.ID.NO.4, the nucleotide sequence of the duck meat-LF primer is shown in SEQ.ID.NO.5, and the nucleotide sequence of the duck meat-LB primer is shown in SEQ.ID.NO.6.

[0011] Preferably, the silver ion-containing solution is AgNO3; the molar ratio of DNA template, AgNO3 and NaBH4 is 1:(6~40):(6~40).

[0012] More preferably, the DNA template and Ag + The molar ratio is 1:30.

[0013] Preferably, the buffer solution is 10×NEBuffer 2.1.

[0014] Preferably, the concentration of the DNA template is 1, 10, 25, 50, 75 or 100 μM.

[0015] More preferably, the concentration of the DNA template is 50 μM.

[0016] This invention also discloses a method for detecting meat adulteration using a CRISPR / Cas-regulated DNA silver nanocluster sensor. First, the DNA of the target adulterated sample is extracted and LAMP amplified. Then, the amplification product is used to activate the Cas12a protein. The DNA-templated silver nanoclusters are used as the substrate for the Cas12a protease cleavage reaction. After cleavage, the sample is scanned by a fluorescence spectrometer. A standard curve is established using the fluorescence intensity difference between the blank group and the experimental group at 570 nm as the signal. The content of the target adulterated sample DNA in meat samples containing unknown concentrations is determined according to the standard curve.

[0017] Preferably, the preparation method of DNA-templated silver nanoclusters is as follows: after heating the silver nanocluster DNA template sequence at 95 °C, cooling it at room temperature, adding a solution containing silver ions, incubating it at 4 °C in the dark for 1 h, then adding NaBH4 and vortexing, incubating it at 4 °C in the dark for 0~12 h to obtain DNA-templated silver nanoclusters.

[0018] More preferably, the incubation time for the silver nanocluster DNA template sequence is 2 h.

[0019] Preferably, the LAMP amplification conditions are 65 °C for 30 min; the cleavage conditions are 37 °C for 30 min.

[0020] This invention also discloses the application of the aforementioned CRISPR / Cas-regulated DNA silver nanocluster sensor in detecting adulteration in meat.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides a CRISPR / Cas-regulated DNA silver nanocluster sensor, which utilizes the specific recognition and trans-cleavage activity of the gene editing tool CRISPR / Cas12a for sensitive detection of trace amounts of nucleic acids. This sensor overcomes the problems in the fluorescence signal output process of CRISPR / Cas technology based on organic dyes and quenchers, such as the relatively poor biocompatibility and high cost of organic chemical fluorescent materials, and fluorescence quenching and false positive results caused by various ions in complex matrices, prolonged exposure to excitation light, and background interference from scattered light and autofluorescence. The novel tagless fluorescent probe in the sensor, DNA-templated silver nanoclusters (DNA-AgNCs), is a natural integrated probe that requires no chemical modification or fluorescent group linkage during synthesis; it is synthesized simply by reducing Ag... +The synthesized nanomaterials consist of stable nanoclusters of several to dozens of Ag atoms and a DNA scaffold. The formation of the metal nucleus in the nucleation region has negligible impact on the chemical properties of DNA, while the degradation of the DNA template is accompanied by a significant decrease in fluorescence intensity. DNA-AgNCs are characterized by ease of synthesis, high photostability, low toxicity, and low cost. They also exhibit better biocompatibility, stable fluorescence, and resistance to background interference and false positives. They can serve as a direct signal output method, overcoming the cumbersome labeling process of traditional fluorescent probes. Nanomaterials that do not require chemical labeling or organic dyes have higher biocompatibility and are more suitable for CRISPR / Cas and other biological reaction systems. This biosensor detects dsDNA, possessing unique dsDNA recognition capabilities. Combined with the specific recognition capabilities and highly sensitive signal transduction characteristics of the CRISPR / Cas system, it provides a reliable detection method for trace dsDNA. This sensor was applied to the detection of adulteration in meat. In unadulterated samples, where the target nucleic acid was absent, the synthesized DNA-AgNCs exhibited strong fluorescence. In adulterated samples, the target nucleic acid was present, activating the trans-cleavage activity of CRISPR / Cas12a, which cleaved the DNA template surrounding the AgNCs. Consequently, the fluorescence intensity of the system decreased significantly with increasing adulteration concentration. Through this process, the nucleic acid signal of the adulterant in the sample was converted into a fluorescence signal.

[0023] This invention provides a method for detecting adulteration in meat using a CRISPR / Cas-regulated DNA silver nanocluster sensor. This method exhibits a wide linear range and low LOD. Based on the specific recognition and rapid, sensitive trans-cleavage activity of CRISPR / Cas12a for nucleic acids, combined with the fluorescence enhancement characteristics of G-rich DNA sequences in DNA-AgNCs, it can detect adulteration in duck meat. This method can detect extremely low levels of adulterated samples and demonstrates good specificity and high sensitivity for trace nucleic acid detection in meat adulteration, detecting adulteration as low as 1.9 pM. Its environmentally friendly nature facilitates the detection of various biomarkers in a wider range of food samples and other complex biological samples. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the CRISPR / Cas-regulated DNA silver nanocluster sensor of the present invention.

[0025] Figure 2 This is an electrophoresis diagram of the duck meat gene of the present invention after loop-mediated isothermal amplification;

[0026] Figure 3 This is a diagram showing the optimized DNA sequence used in the synthesis of DNA-AgNCs according to the present invention;

[0027] Figure 4 This is a diagram showing the optimization of DNA concentration in the synthesis of DNA-AgNCs according to the present invention;

[0028] Figure 5 DNA and Ag in the synthesis of DNA-AgNCs of the present invention + Optimization diagram of molar ratio;

[0029] Figure 6 This is a diagram showing the optimization of incubation time in the synthesis of DNA-AgNCs according to the present invention;

[0030] Figure 7 The fluorescence spectra of the DNA-templated silver nanocluster sensor of the present invention for detecting target nucleic acids at different concentrations are shown.

[0031] Figure 8 This is a standard curve diagram showing the detection of different concentrations of target nucleic acids by the DNA templated silver nanocluster sensor of the present invention. Detailed Implementation

[0032] The following is in conjunction with the appendix Figures 1-8 The present invention will be described in further detail as follows:

[0033] 1. Materials and Equipment

[0034] (1) Main materials and reagents

[0035] DNA Marker, 4S Green nucleic acid staining agent, agarose, and MAGK-MK animal genomic DNA extraction kit (purchased from Shanghai Sangon Biotech Co., Ltd.). Cas12a was purchased from NEB Ltd. (Beijing, China). Sodium borohydride (NaBH4), silver nitrate (AgNO3), disodium hydrogen phosphate (Na2HPO4), and sodium dihydrogen phosphate (NaH2PO4) were purchased from Sigma-Aldrich (Shanghai, China). crRNA was designed using the CRISPOR program (…). http: / / crispor.tefor.net / The target site was supplemented with a 5' TTTN protospacer motif from the target DNA. The crRNA was synthesized by GenScript (Nanjing, China). Loop-mediated isothermal amplification (LAMP) primers (F3 / B3, FIP / BIP, LF / LB) and DNA templates for DNA-AgNCs (sequences shown as SEQ.ID.NO.9 to SEQ.ID.NO.14) were synthesized by Shanghai Sangon Biotech Co., Ltd. The oligonucleotides used in this invention are listed in Table 1. Lamb and duck samples were purchased from a local market (Xi'an, China).

[0036] (2) Main instruments

[0037] Transmission electron microscope (JEM-2100F, Japan electron Optics Laboratory Ltd, JPN), Zetasizer Nano (ZS-90, Malvern, UK), CD spectrometer (Chirascan, Applied phophysics Ltd, UK), fluorescence spectrophotometer (FluoroMax-4, FR), microplate reader (Multiskan GO, ThermoScientific, USA).

[0038] 2. CRISPR / Cas-regulated DNA silver nanocluster sensor for detecting meat adulteration

[0039] The present invention provides a CRISPR / Cas-regulated DNA silver nanocluster sensor, comprising a CRISPR / Cas system and DNA-templated silver nanoclusters;

[0040] The CRISPR / Cas system consists of Cas12a protein, crRNA, RNase inhibitor, buffer, and amplified target adulterated sample DNA. The DNA-templated silver nanoclusters are synthesized from a silver ion-containing solution, NaBH4, and a DNA template. The target adulterated sample is duck meat. The DNA template sequence is G, G3, G4, CG, C-G3, or C-G4. The recognition sequence of crRNA is complementary to the duck meat sample DNA fragment, and the nucleotide sequence of the duck meat sample DNA is shown in SEQ.ID.NO.8. The silver ion-containing solution is AgNO3. The molar ratio of DNA template, AgNO3, and NaBH4 is 1:(6~40):(6~40).

[0041] This invention provides a method for detecting adulteration in meat using a CRISPR / Cas-regulated DNA silver nanocluster sensor, such as... Figure 1 First, the genome was extracted from the sample and amplified to obtain a large amount of target nucleic acid. Second, using a solution containing silver ions, NaBH4, and a DNA template as raw materials, the NaBH4 was used to treat Ag... + The reduction process involved synthesizing DNA-templated silver nanoclusters using DNA as a scaffold and storing them at 4°C in the dark. After establishing a CRISPR / Cas system (including Cas12a protein, crRNA, and NEBuffer 2.1), DNA-templated silver nanoclusters were added as substrates for the Cas12a nuclease. The target nucleic acid was then added to activate the cleavage activity of the Cas12a nuclease, promoting the cleavage of the DNA-templated silver nanoclusters by Cas12a. This resulted in the DNA template surrounding the silver nanoclusters being cleaved into DNA fragments, thereby causing a decrease in the fluorescence emission intensity of the entire system.

[0042] The specific testing methods are as follows:

[0043] The first step is to extract the genome from the purchased duck meat according to the instructions of the MAGK-MK Animal Genomic DNA Extraction Kit.

[0044] The specific steps are as follows: 50 mg of fresh duck tissue was added to liquid nitrogen and ground into powder using a mortar and pestle. Then, 400 μL of Buffer MACL, 200 μL of Buffer MCL, and 20 μL of Protease K were added to a clean test tube. The mixture was heated in a 65 °C water bath for 20 min, centrifuged, and the supernatant was retained. Buffer MA and Magic Mag Beads were added, and the mixture was incubated at room temperature for 1 min. After magnetic separation, the mixture was washed twice with 70% ethanol. The target genomic DNA was eluted with TE buffer and stored in a clean centrifuge tube.

[0045] The second step is to amplify the genome extracted in the first step.

[0046] The specific steps are as follows: ND2 (The target genomic DNA extracted in the first step) was amplified using LAMP primers (F3 / B3, FIP / BIP, LF / LB) at 65 ℃ for 30 min. The amplified products were quantified using a B-500 UV spectrophotometer (Metash, Shanghai, China) and stored at -20 ℃ for later use. Agarose gel electrophoresis results are shown below. Figure 2 ;

[0047] The primers (6 primers) for loop-mediated isothermal amplification were selected according to industry standard SN / T 4419.21-2016, and their nucleotide sequences are shown in Table 1. The amplification system is shown in Table 2.

[0048] Table 1: Nucleotide Sequences

[0049]

[0050] Table 2: Loop-mediated isothermal amplification system

[0051]

[0052] The third step is the design of the crRNA sequence.

[0053] Selected duck meat mitochondrial genes ND2 For the target DNA, crRNA and selected duck mitochondrial genes ND2 (The nucleotide sequence is shown in SEQ.ID.NO.8) complements the duck meat mitochondrial gene. ND2 Nucleotide sequence input http: / / crispor.tefor.net / The website filters for the fragment with the highest cutting efficiency score. Based on this fragment, the crRNA backbone sequence is combined to obtain the final complete crRNA sequence. The obtained crRNA (synthesized by Genscript Biotech Co., Ltd.) sequence is shown in SEQ.ID.NO.7.

[0054] The fourth step is to synthesize DNA-AgNCs.

[0055] 10 μL of 50 μM DNA template was heated at 95 °C for 5 min, then cooled to room temperature for 10 min. 90 μL of phosphate buffer (PB, 10 μM, pH 7.4) and 50 μL of 60 μM AgNO3 were added. This mixture was incubated at 4 °C in the dark for 1 h. Then, 100 μL of freshly prepared 60 μM NaBH4 was added, vortexed for 1 min, and incubated at 4 °C in the dark for 2 h to obtain DNA-AgNCs. Store at 4 °C in the dark before use.

[0056] The fifth step is the construction and testing of the CRISPR / Cas12a sensing system.

[0057] Enzyme digestion system: 1 μL of pre-synthesized DNA-AgNCs was added to a 20 μL Cas12a cleavage system containing 0.03 μM Cas12a, 1.2 μM crRNA, 0.4 U RNase inhibitor, and 1×NEBuffer 2.1. Finally, 5 μL of target DNA was added to the cleavage system. The control group was identical to the Cas12a cleavage system except for the target DNA. The cleavage system was incubated at 37 ℃ for 30 min.

[0058] Fluorescence signal detection: After the enzyme cleavage reaction was completed, 20 μL of the reaction solution was diluted to 170 μL, and the fluorescence emission intensity of the system at 570 nm was measured using a fluorescence spectrometer.

[0059] 1) Establish a standard curve for detecting DNA-templated silver nanoclusters.

[0060] The fluorescence intensity at 570 nm of the experimental group (where 1 ng / μL of duck meat DNA corresponds to 100% duck meat content) and the blank group (i.e., 5 μL of ultrapure water replaces the target DNA in the enzyme digestion system) containing different concentrations of duck meat DNA were measured according to the above method. The fluorescence intensity difference (ΔI) between the blank group and the experimental group was obtained. A standard curve was established with the fluorescence intensity difference (ΔI) as the ordinate and the target DNA concentration as the abscissa.

[0061] 2) Calculate the amount of duck meat mixed into the mutton.

[0062] The target DNA content in mutton samples containing unknown concentrations of duck meat was determined based on the established standard curve (only one standard curve for the fluorescence sensor needs to be established for multiple determinations).

[0063] The specific calculation method for the adulteration content of duck meat in mutton samples is as follows: Substitute the ΔI value of the blank group and the mutton sample to be tested at 570nm into the standard curve to obtain the corresponding target DNA concentration value. The proportion of the corresponding target DNA concentration value to the total target DNA concentration value (i.e. the concentration value of pure duck meat DNA) is the adulteration content of duck meat in mutton.

[0064] The above steps constitute the method for detecting adulteration in meat using a CRISPR / Cas-regulated DNA silver nanocluster sensor.

[0065] To improve the detection performance of this DNA-templated silver nanocluster sensor, this invention also investigated the DNA sequence, DNA concentration, and the relationship between DNA and Ag in DNA-AgNCs synthesis. + The effects of molar ratio, incubation time, and stability of DNA-AgNCs nanomaterials on the sensor performance were investigated. Figures 3 to 6 As shown. Among them, Figure 3 The study verified that different nucleotide sequences and lengths affect the fluorescence properties of DNA-AgNCs, with cytosine contributing to AgNC formation and guanine enhancing fluorescence intensity. Among the six designed DNA sequences containing different cytosine sequences and guanine amounts, the sequences G, G3, G4, CG, C-G3, and C-G4 are shown in SEQ.ID.NO.9 to SEQ.ID.NO.14. G4 (i.e., the G-quadruplex) showed better enhancement than G and G3. CG, C-G3, and C-G4, containing cytosine sequences, exhibited significantly enhanced fluorescence emission, with C-G4 showing the best performance. This may be due to the effect of cytosine on AgNC formation. + Its affinity is much higher than that of guanine, which is beneficial for the synthesis of DNA-AgNCs. Figure 4 The optimized concentration range of the DNA template was 1, 10, 25, 50, 75, and 100 μM. DNA-AgNCs synthesized with a 50 μM DNA template showed the strongest fluorescence emission; therefore, 50 μM DNA was selected for the synthesis of DNA-AgNCs. Figure 5 As shown, DNA and Ag + The optimal molar ratio range was 1:6 to 1:40. Results showed that fluorescence intensity increased with increasing molar ratio, reaching a maximum at 1:30 and then decreasing. Therefore, DNA and Ag were selected as the optimal molar ratios. + The molar ratio is 1:30. Figure 6The optimization of incubation time during DNA-AgNCs synthesis was demonstrated. The results showed that the fluorescence intensity reached its maximum value after 2 h of incubation, so an incubation time of 2 h was selected.

[0066] 3. Evaluate the detection performance of the CRISPR / Cas-regulated DNA silver nanocluster sensor.

[0067] To further illustrate the detection performance of the CRISPR / Cas-regulated DNA silver nanocluster sensor in meat adulteration detection, the present invention conducted the following evaluation: Under the optimal conditions described above, 1 μL of pre-synthesized DNA-AgNCs was added to a 20 μL Cas12a cleavage system containing 0.03 μM Cas12a, 1.2 μM crRNA, 0.4 U RNase inhibitor, and 1×NEBuffer 2.1. Then, 5 μL of target DNA (diluted 10-fold from 1 μM to 10 pM) was added, followed by the gene extracted from the sample and amplified by LAMP. After incubation at 37 °C, the fluorescence intensity at 570 nm was measured. The results are as follows: Figure 7 As shown, with the increase of adulterant concentration, the concentration of the reaction system gradually decreases, and the fluorescence intensity ΔI of the control group gradually increases.

[0068] Example 1

[0069] The following is a detailed procedure for detecting pure mutton products using a constructed CRISPR / Cas-regulated DNA silver nanocluster sensor:

[0070] The first step was to extract the genome from the purchased mutton according to the instructions of the MAGK-MK Animal Genomic DNA Extraction Kit.

[0071] The operation method is the same as the first step of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanoclusters.

[0072] The second step is to amplify the extracted genome.

[0073] The operation method is the same as the second step of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanocluster sensors.

[0074] The third step is the design of the crRNA sequence.

[0075] The operation method is the same as step three of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanocluster sensors.

[0076] The fourth step is to synthesize DNA-AgNCs.

[0077] The operation method is the same as step four of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanocluster sensors.

[0078] Step 5: Construction and testing of the CRISPR / Cas12a sensing system.

[0079] The operation method is the same as step five of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanoclusters.

[0080] Fluorescence signal detection: After the enzyme cleavage reaction was completed, 20 μL of the reaction solution was diluted to 170 μL, and the fluorescence emission intensity of the system at 570 nm was measured using a fluorescence spectrometer.

[0081] 1) Establish a standard curve for detecting the fluorescence sensor.

[0082] The fluorescence intensity at 570 nm of the experimental group (where 1 ng / μL of duck meat DNA corresponds to 100% duck meat content) and the blank group (i.e., 5 μL of ultrapure water replaces the target DNA in the enzyme digestion system) containing different concentrations of duck meat DNA were measured according to the above method. The fluorescence intensity difference (ΔI) between the blank group and the experimental group was obtained. A standard curve was established with the fluorescence intensity difference (ΔI) as the ordinate and the target DNA concentration as the abscissa.

[0083] 2) Calculate the amount of duck meat mixed into the mutton.

[0084] Based on the established standard curve (e.g.) Figure 8 (As shown) The content of target DNA in mutton samples containing an unknown concentration of duck meat was determined. The experiment was repeated three times, and the average fluorescence intensity at 570 nm and the error bar were calculated.

[0085] The obtained fluorescence intensities were all within the error range of the blank signal, indicating that the mutton sample did not contain duck meat.

[0086] Example 2

[0087] The following is a detailed procedure for detecting adulteration in commercially available mutton samples using a constructed CRISPR / Cas-regulated DNA silver nanocluster sensor:

[0088] The first step was to extract the genome from the purchased mutton according to the instructions of the MAGK-MK Animal Genomic DNA Extraction Kit.

[0089] The operation method is the same as the first step of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanoclusters.

[0090] The second step is to amplify the extracted genome.

[0091] The operation method is the same as the second step of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanocluster sensors.

[0092] The third step is the design of the crRNA sequence.

[0093] The operation method is the same as step three of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanocluster sensors.

[0094] The fourth step is to synthesize DNA-AgNCs.

[0095] The operation method is the same as step four of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanocluster sensors.

[0096] Step 5: Construction and testing of the CRISPR / Cas12a sensing system.

[0097] The operation method is the same as step five of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanoclusters.

[0098] Fluorescence signal detection: After the enzyme cleavage reaction was completed, 20 μL of the reaction solution was diluted to 170 μL and the fluorescence emission intensity of the system at 570 nm was measured using a fluorescence spectrometer.

[0099] 1) Establish a standard curve for detecting the fluorescence sensor.

[0100] The fluorescence intensity at 570 nm of the experimental group (where 1 ng / μL of duck meat DNA corresponds to 100% duck meat content) and the blank group (i.e., 5 μL of ultrapure water replaces the target DNA in the enzyme digestion system) containing different concentrations of duck meat DNA were measured according to the above method. The fluorescence intensity difference (ΔI) between the blank group and the experimental group was obtained. A standard curve was established with the fluorescence intensity difference (ΔI) as the ordinate and the target DNA concentration as the abscissa.

[0101] 2) Calculate the amount of duck meat mixed into the mutton.

[0102] Based on the established standard curve (e.g.) Figure 8 (As shown) The content of target DNA in mutton samples containing an unknown concentration of duck meat was determined. The experiment was repeated three times, and the average fluorescence intensity at 570 nm and the error bar were calculated.

[0103] The obtained fluorescence intensities were all within the error range of the blank signal, indicating that the commercially available mutton sample did not contain duck meat.

[0104] Example 3

[0105] The following is a detailed procedure for detecting adulteration in commercially available mutton products (numbers 1-5) using a constructed CRISPR / Cas-regulated DNA silver nanocluster sensor:

[0106] The first step was to extract the genome from the purchased mutton according to the instructions of the MAGK-MK Animal Genomic DNA Extraction Kit.

[0107] The operation method is the same as the first step of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanoclusters.

[0108] The second step is to amplify the extracted genome.

[0109] The operation method is the same as the second step of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanocluster sensors.

[0110] The third step is the design of the crRNA sequence.

[0111] The operation method is the same as step three of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanocluster sensors.

[0112] The fourth step is to synthesize DNA-AgNCs.

[0113] The operation method is the same as step four of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanocluster sensors.

[0114] Step 5: Construction and testing of the CRISPR / Cas12a sensing system.

[0115] The operation method is the same as step five of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanoclusters.

[0116] Fluorescence signal detection: After the enzyme cleavage reaction was completed, 20 μL of the reaction solution was diluted to 170 μL and the fluorescence emission intensity of the system at 570 nm was measured using a fluorescence spectrometer.

[0117] 1) Establish a standard curve for detecting the fluorescence sensor.

[0118] The fluorescence intensity at 570 nm of the experimental group (where 1 ng / μL of duck meat DNA corresponds to 100% duck meat content) and the blank group (i.e., 5 μL of ultrapure water replaces the target DNA in the enzyme digestion system) containing different concentrations of duck meat DNA were measured according to the above method. The fluorescence intensity difference (ΔI) between the blank group and the experimental group was obtained. A standard curve was established with the fluorescence intensity difference (ΔI) as the ordinate and the target DNA concentration as the abscissa.

[0119] 2) Calculate the amount of duck meat mixed into the mutton.

[0120] Based on the established standard curve (e.g.) Figure 8 (As shown) The content of target DNA in mutton samples containing an unknown concentration of duck meat was determined. The experiment was repeated three times, and the average fluorescence intensity at 570 nm and the error bar were calculated.

[0121] The obtained fluorescence intensities were all within the error range of the blank signal, indicating that commercially available mutton samples 1-5 did not contain duck meat.

[0122] Example 4

[0123] The following is a detailed procedure for detecting artificially adulterated commercially available mutton products using a constructed CRISPR / Cas-regulated DNA silver nanocluster sensor:

[0124] The first step was to extract the genome from the purchased mutton according to the instructions of the MAGK-MK Animal Genomic DNA Extraction Kit.

[0125] The operation method is the same as the first step of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanoclusters.

[0126] The second step is to amplify the extracted genome.

[0127] Lamb samples were prepared with 1%, 5%, and 10% duck meat added artificially. The genome was extracted from both the commercially available lamb samples and the prepared adulterated samples using the MAGK-MK animal genomic DNA extraction kit. The remaining procedures were the same as the second step of the construction method of the CRISPR / Cas regulated DNA silver nanocluster sensor described above.

[0128] The third step is the design of the crRNA sequence.

[0129] The operation method is the same as step three of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanocluster sensors.

[0130] The fourth step is to synthesize DNA-AgNCs.

[0131] The operation method is the same as step four of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanocluster sensors.

[0132] Step 5: Construction and testing of the CRISPR / Cas12a sensing system.

[0133] The operation method is the same as step five of the above-described method for constructing CRISPR / Cas-regulated DNA silver nanoclusters.

[0134] Fluorescence signal detection: After the enzyme cleavage reaction was completed, 20 μL of the reaction solution was diluted to 170 μL and the fluorescence emission intensity of the system at 570 nm was measured using a fluorescence spectrometer.

[0135] 1) Establish a standard curve for detecting the fluorescence sensor.

[0136] The fluorescence intensity at 570 nm of the experimental group (where 1 ng / μL of duck meat DNA corresponds to 100% duck meat content) and the blank group (i.e., 5 μL of ultrapure water replaces the target DNA in the enzyme digestion system) containing different concentrations of duck meat DNA were measured according to the above method. The fluorescence intensity difference (ΔI) between the blank group and the experimental group was obtained. A standard curve was established with the fluorescence intensity difference (ΔI) as the ordinate and the target DNA concentration as the abscissa.

[0137] 2) Calculate the amount of duck meat mixed into the mutton.

[0138] Based on the established standard curve (e.g.) Figure 8 (As shown) The content of target DNA in mutton samples containing artificially added duck meat was determined. The experiment was repeated three times, and the average fluorescence intensity at 570 nm and the error bar were calculated.

[0139] The method was used to test five commercially available mutton samples in the experimental group, and the obtained ΔI was substituted into the result of Example 1. Figure 8 The standard curve was used to test commercially available mutton. The test results were within the error range of the blank group, and no duck meat was detected. The amount of duck meat detected in commercially available mutton samples with 1% duck meat was 0.97~1.01%, the amount of duck meat detected in commercially available mutton samples with 5% duck meat was 4.85~4.90%, and the amount of duck meat detected in commercially available mutton samples with 10% duck meat was 10.07~10.40%. The ratio of the detected adulteration amount to the actual adulteration amount was 97.09%~104.00%, close to 100%, confirming that it has good accuracy.

[0140] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A CRISPR / Cas regulated DNA silver nanocluster sensor for detecting adulteration of mutton with duck meat, characterized in that, The CRISPR / Cas system comprises a Cas12a protein, crRNA, an RNA nuclease inhibitor, a buffer, and amplified duck meat sample DNA; and the DNA template silver nanocluster is synthesized from a silver ion-containing solution, NaBH4, and a DNA template. The nucleotide sequence of the crRNA is shown in SEQ.ID.NO.7; the amplification is loop-mediated isothermal amplification, and the primers used for the amplification comprise duck meat-FIP primers, duck meat-BIP primers, duck meat-F3 primers, duck meat-B3 primers, duck meat-LF primers, and duck meat-LB primers; the nucleotide sequence of the duck meat-F3 primers is shown in SEQ.ID.NO.1, the nucleotide sequence of the duck meat-B3 primers is shown in SEQ.ID.NO.2, the nucleotide sequence of the duck meat-FIP primers is shown in SEQ.ID.NO.3, the nucleotide sequence of the duck meat-BIP primers is shown in SEQ.ID.NO.4, the nucleotide sequence of the duck meat-LF primers is shown in SEQ.ID.NO.5, and the nucleotide sequence of the duck meat-LB primers is shown in SEQ.ID.NO.6; the nucleotide sequence of the duck meat sample DNA is shown in SEQ.ID.NO.8; the silver ion-containing solution is AgNO3; the sequence of the DNA template is shown in SEQ.ID.NO.9, SEQ.ID.NO.10, SEQ.ID.NO.11, SEQ.ID.NO.12, SEQ.ID.NO.13, or SEQ.ID.NO.14; the concentration of the DNA template is 1, 10, 25, 50, 75, or 100 μM; and the molar ratio of the DNA template, AgNO3, and NaBH4 is 1:(6-40):(6-40).

2. The CRISPR / Cas regulated DNA silver nanoclusters sensor for detecting adulteration of mutton with duck meat according to claim 1, wherein, The buffer is 10×NEBuffer 2.

1.

3. The method for detecting adulteration of mutton with duck meat by using the CRISPR / Cas regulated DNA silver nanocluster sensor according to claim 1 or 2, characterized in that, First, the target adulterated sample DNA is extracted and subjected to LAMP amplification, and then the Cas12a protein is activated by the amplification product, and the DNA template silver nanocluster is used as the substrate for the Cas12a proteinase cleavage reaction, and after the cleavage is completed, the fluorescence spectrometer is scanned, and the difference in the fluorescence intensity of the blank group and the experimental group at 570 nm is used as the signal to establish a standard curve, and the content of the target adulterated sample DNA in the meat sample with an unknown concentration is determined according to the standard curve.

4. The method for detecting adulteration of mutton with duck meat by CRISPR / Cas regulated DNA silver nanoclusters sensor according to claim 3, characterized in that, The preparation method of the DNA template silver nanocluster is as follows: after the silver nanocluster DNA template sequence is heated at 95℃, it is cooled at room temperature, a silver ion-containing solution is added, and incubated at 4℃ in the dark for 1 h, then NaBH4 is added and vortexed, and incubated at 4℃ in the dark for 0-12 h to obtain the DNA template silver nanocluster.

5. The method for detecting adulteration of mutton with duck meat using CRISPR / Cas regulated DNA silver nanoclusters sensor according to claim 3, characterized in that, The LAMP amplification conditions are 65℃ for 30 min, and the cleavage conditions are 37℃ for 30 min.

6. The use of the CRISPR / Cas regulated DNA silver nanocluster sensor of claim 1 in detecting adulterated duck meat in mutton.

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