Method for detecting salmonella typhimurium based on strand displacement reaction and CRISPR / Cas12a in combination with colorimetric sensor and dairy product detection method

Abstract

The invention discloses a method for detecting salmonella typhimurium based on strand displacement reaction and CRISPR / Cas12a in combination with a colorimetric sensor and a dairy product detection method, and belongs to the technical field of food safety rapid detection. The invention provides a method for detecting salmonella typhimurium based on strand displacement reaction and CRISPR (clustered regularly interspaced short palindromic repeats) / Cas12a combined with a colorimetric sensor in order to solve the problems of low sensitivity and inaccurate result of detection of salmonella typhimurium in dairy products by a traditional method in the prior art. According to the method, DNA-Ag / Pt NCs is used as a signal molecule to construct the aptamer colorimetric sensor for detecting salmonella typhimurium; the colorimetric sensor has relatively high sensitivity and specificity, and can quickly and conveniently realize on-site detection of salmonella typhimurium.

Description

Technical Field

[0001] This invention belongs to the field of rapid food safety detection technology, and specifically relates to a method for detecting Salmonella typhimurium based on chain displacement reaction and CRISPR / Cas12a combined with a colorimetric sensor, as well as a method for detecting dairy products. Background Technology

[0002] Salmonella Typhimurium is a common zoonotic pathogen that poses a serious threat to human and animal health. Milk and dairy products, due to their rich nutritional content, easily become breeding grounds for Salmonella Typhimurium. Therefore, timely detection and control of Salmonella Typhimurium contamination in milk is crucial to ensuring dairy product safety and protecting public health. Current detection methods for Salmonella Typhimurium, such as culture methods, still have limitations, including long detection times, insufficient sensitivity, and complex procedures. Therefore, there is an urgent need to develop a rapid detection method with high sensitivity and specificity for the immediate detection of Salmonella Typhimurium in milk.

[0003] The CRISPR / Cas12a system, composed of clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein (Cas12a), is a crucial CRISPR gene-editing tool. It boasts advantages such as high specificity, trans-cleavage activity, and multifunctionality, demonstrating significant advantages in nucleic acid detection. However, its sensitivity is limited at low concentrations of target substances. Isothermal amplification (AFA), a technique that rapidly amplifies nucleic acids at a constant temperature, can detect extremely low concentrations of target substances. Combining AFA with CRISPR / Cas12a can achieve high sensitivity and specificity in detection. Meanwhile, aptamer optical sensors, due to their high selectivity of aptamers and high sensitivity of optical detection, have become the preferred alternative to traditional detection methods due to their low cost and flexible design.

[0004] Therefore, this study constructed an aptamer colorimetric sensor for the detection of Salmonella Typhimurium using CRISPR / Cas12a combined with isothermal amplification as the detection method. Using the aptamer as the recognition element, Salmonella Typhimurium is captured, triggering a dual signal amplification reaction (SDA) combined with CRISPR / Cas12a, with DNA-silver / platinum nanoclusters (DNA-Ag / Pt NCs) as the signal molecule. When DNA-Ag / Pt NCs are cleaved, their peroxidase activity disappears, preventing the catalysis of 3,3',5,5'-tetramethylbenzidine (TMB) color development under hydrogen peroxide (H2O2). Therefore, the concentration of Salmonella Typhimurium can be determined by changes in color and absorbance within the system. Based on the advantages of this aptamer colorimetric sensor, such as ease of operation, high sensitivity, and strong specificity, it provides a new theoretical basis and research direction for the real-time detection of Salmonella Typhimurium in milk. Summary of the Invention

[0005] This invention addresses the problems of low sensitivity and inaccurate results in the detection of Salmonella Typhimurium in dairy products using traditional methods in the prior art. It provides a method for detecting Salmonella Typhimurium based on chain displacement reaction and CRISPR / Cas12a combined with a colorimetric sensor, as well as a method for detecting Salmonella Typhimurium in dairy products.

[0006] One objective of this invention is to provide a method for detecting Salmonella Typhimurium based on chain displacement reaction and a CRISPR / Cas12a colorimetric sensor, the method comprising the following steps: S1: Mix the DNA template strand, AgNO3 solution and K2PtCl4 solution, add PBS buffer and mix, react at room temperature in the dark for 15 min, then add NaBH4 solution and incubate at room temperature in the dark for 1.5 h to obtain DNA-Ag / Pt NCs; S2: Mix Salmonella Typhimurium aptamer Apt1 and cDNA1 and shake well. Place in a water bath at 95°C for annealing for 5 min. After the mixture cools down to 60°C in the water bath, remove it and cool to room temperature to obtain the Apt1-cDNA1 complex. S3: Mix the SMBs magnetic bead suspension with 1×B&W buffer in a siliconized tube, shake to mix, and then perform magnetic separation. Aspirate the supernatant. Repeat the above steps 3 times to obtain magnetic beads. Resuspend the magnetic beads in 2×B&W buffer to obtain magnetic bead resuspension. S4: Mix the bacterial culture to be tested with the Apt1-cDNA1 complex obtained in S2 and the magnetic bead resuspension obtained in S3, and shake in a shaker at 37℃ and 150 rpm for 45 min for magnetic separation. Transfer the supernatant to a new centrifuge tube, add the premixed solution, react at 37℃ for 2 h, and heat to 80℃ for enzyme inactivation reaction for 20 min to obtain SDA product. S5: Pre-assemble crRNA with LbCas12a at 37℃ for 20 min to obtain Cas12a / crRNA1 complex; S6: Add the Cas12a / crRNA1 complex from S5 to the SDA product obtained in S4, incubate at 37℃ and 200rpm for 20 min, add the DNA-Ag / Pt NCs obtained in S1, mix and shake for 60 min to obtain the mixture. S7: Add acetate buffer, TMB chromogenic solution and H2O2 to the mixture obtained in S6 and observe the color change. Within 30 min, use a multi-functional microplate reader to measure the fluorescence value at 652 nm. The infection status of Salmonella typhimurium is determined based on the fluorescence value.

[0007] In a preferred embodiment of the present invention, the mixing ratio of DNA template strand: AgNO3 solution: K2PtCl4 solution: PBS buffer: NaBH4 solution in S1 is 7.2 μL: 11.3 μL: 22.5 μL: 214 μL: 45 μL; the nucleotide sequence of the DNA template strand is shown in SEQ ID NO.5 and the concentration is 50 μM; the concentration of the AgNO3 solution is 0.4 mM; the concentration of the K2PtCl4 solution is 0.4 mM; and the concentration of the NaBH4 solution is 2.0 mM.

[0008] In a preferred embodiment of the present invention, the mixing volume ratio of the Salmonella Typhimurium aptamer Apt1 to cDNA1 is 1:1; the nucleotide sequence of the Salmonella Typhimurium aptamer Apt1 is shown in SEQ ID NO.1 and the concentration is 4 μM; the nucleotide sequence of the cDNA1 is shown in SEQ ID NO.2.

[0009] In a preferred embodiment of the present invention, the mixing volume ratio of the bacterial culture to be tested, the Apt1-cDNA1 complex, and the magnetic bead resuspension in S4 is 40 μL:40 μL:10 μL.

[0010] In a preferred embodiment of the present invention, the premixed solution in S4 consists of: 1 μM HP1, 2 μL DNA polymerase, 3 μL Nb.BbvCI, 25 μL dNTP 2.5 mM and 10 μL 10×NEB buffer 2.1; the nucleotide sequence of the HP1 is shown in SEQ ID NO.3.

[0011] In a preferred embodiment of the present invention, the volume ratio of crRNA to LbCas12a in S5 is 1:1; the nucleotide sequence of the crRNA is shown in SEQ ID NO.4 and the concentration is 200 nM, and the concentration of LbCas12a is 200 nM.

[0012] In a preferred embodiment of the present invention, the mixing ratio of the Cas12a / crRNA1 complex, SDA product and DNA-Ag / Pt NCs complex in S6 is 6 μL:6 μL:6 μL.

[0013] In a preferred embodiment of the present invention, the mixing volume ratio of the mixture, acetate buffer, TMB colorimetric solution and H2O2 in S7 is 12 μL:180 μL:40 μL:20 μL; and the concentration of the acetate buffer is 5 mM.

[0014] In a preferred embodiment of the present invention, the judgment criterion mentioned in S7 is: when the fluorescence value at 652 nm is >0.2, it is judged as positive, and when the fluorescence value at 652 nm is ≤0.2, it is judged as negative.

[0015] The second objective of this invention is to provide a method for detecting dairy products, wherein the method for detecting Salmonella typhimurium is the method described above.

[0016] Compared with the prior art, the beneficial effects of the present invention are: the present invention provides a method for detecting Salmonella Typhimurium based on chain displacement reaction and CRISPR / Cas12a combined with a colorimetric sensor, which triggers chain displacement reaction after capturing Salmonella Typhimurium ( Strand Displacement Amplification A colorimetric sensor for detecting Salmonella Typhimurium was constructed using DNA-Ag / Pt NCs as signal molecules, combining dual signal amplification with SDA and CRISPR / Cas12a. This colorimetric sensor has high sensitivity and specificity, enabling rapid and convenient on-site detection of Salmonella Typhimurium.

[0017] This invention provides a successful construction of a dual-signal amplification DNA-Ag / PtNCs colorimetric sensor based on SDA and CRISPR / Cas12a. The optimal conditions for the optimized reaction system are as follows: 2 μM Apt1-cDNA1 is ligated to magnetic beads for 45 min to obtain an SMBs-Apt1-cDNA1 complex. Salmonella typhimurium is added for incubation, followed by magnetic separation. Then, 1 μM HP1, 20 I / U DNA polymerase, 20 U Nb.BbvCI restriction enzyme, and 2.5 mM dNTPs are added to the supernatant sequentially for complete reaction. Finally, acetate buffer at pH 4.5, 50 mM H2O2, and TMB colorimetric solution are added for further reaction.

[0018] This invention utilizes a dual-signal amplification DNA-Ag / Pt NCs colorimetric sensor based on SDA and CRISPR / Cas12a, exhibiting excellent specificity and allowing for direct visual observation of detection results. The colorimetric sensor provided by this invention achieves a detection limit as low as 2.5 CFU / mL for pure bacterial culture of *Salmonella typhimurium*; and as low as 3 CFU / mL and 5 CFU / mL for artificially contaminated skim milk and whole milk samples, respectively. In spiked recovery analysis, the recovery rate for spiked skim milk is 93.94%-100.67%, with an RSD of 2.30%-2.76%. The recovery rate for spiked whole milk is 88.90%-105.39%, with an RSD of 2.07%-2.43%. Furthermore, in both pure bacterial culture and artificially contaminated actual samples, the colorimetric sensor provided by this invention achieves detection limits for *Salmonella typhimurium* within 10... 1 -10 7 The CFU / mL concentration showed good linearity. Attached Figure Description

[0019] Figure 1 A schematic diagram of a dual-signal amplification DNA-Ag / Pt NCs colorimetric sensor based on SDA and CRISPR / Cas12a combined; Figure 2 Transmission electron microscopy image of DNA-Ag / Pt NCs; Figure 3 A is the XPS spectrum analysis diagram; B is the Ag XPS spectrum analysis diagram; C is the Pt XPS spectrum analysis diagram. Figure 4 A is the UV absorption spectrum; A is the UV absorption spectrum of different systems; B is the effect of different concentrations of DMSO on the hydroxyl radicals generated by the catalytic system. Figure 5 This is a graph showing the analysis of EPR results; Figure 6The diagram shows the kinetic analysis of DNA-Ag / Pt NCs peroxidase activity; A represents the change in TMB concentration; B represents the change in H2O2 concentration. Figure 7 The image shows the gel electrophoresis analysis. Lane 1: cDNA1; Lane 2: HP1; Lane 3: cDNA1+HP1; Lane 4: cDNA1+HP1+DNA polymerase+dNTP enzyme; Lane 5: cDNA1+HP1+DNA polymerase+dNTP enzyme+Nb.BbvCI endonuclease. Figure 8 The images show ultraviolet absorption spectra analysis; A represents DNA-Ag / Pt NCs; B represents the absence of HP1; C represents the absence of Cas12a; D represents the absence of Salmonella typhimurium; and E represents the presence of HP1, Cas12a, and Salmonella typhimurium. Figure 9 This is a graph showing the optimized concentration of pt1-cDNA1; different letters for the same indicator indicate significant differences (P<0.05). Figure 10 Optimization diagram for incubation time of SMBs and Apt1-cDNA1; different letters for the same indicator indicate significant differences (P<0.05); Figure 11 The HP1 concentration optimization plot is shown; different letters for the same indicator indicate significant differences (P<0.05). Figure 12 Figure 1 shows the optimization of enzyme concentration; A represents the optimization of DNA polymerase concentration; B represents the optimization of Nb.BbvCI endonuclease concentration; C represents the optimization of dNTP enzyme concentration; different letters for the same indicator indicate significant differences (P<0.05). Figure 13 The graph shows the optimization of H2O2 concentration; different letters for the same indicator indicate significant differences (P<0.05). Figure 14 This is a graph showing the optimization of acetate pH; different letters for the same indicator indicate significant differences (P<0.05). Figure 15 This is a verification diagram of the specificity of the color sensor; Figure 16 The sensitivity detection graph for the colorimetric sensor in detecting Salmonella typhimurium under pure bacterial culture conditions is shown in Figure A; A represents the UV absorption spectrum of the system in the presence of different concentrations of Salmonella typhimurium; B represents the relationship between absorbance value and logarithm of Salmonella typhimurium concentration. Figure 17The graphs show the sensitivity of colorimetric sensors in detecting Salmonella Typhimurium contaminated milk samples at different concentrations. A represents the UV absorption spectra of skim milk contaminated with different concentrations of Salmonella Typhimurium. B shows the relationship between the absorbance of skim milk contaminated with Salmonella Typhimurium and the logarithm of the Salmonella Typhimurium concentration. C represents the UV absorption spectra of whole milk contaminated with different concentrations of Salmonella Typhimurium. D shows the relationship between the absorbance of whole milk contaminated with Salmonella Typhimurium and the logarithm of the Salmonella Typhimurium concentration. Detailed Implementation

[0020] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0022] A schematic diagram of the DNA-Ag / Pt NCs colorimetric sensor based on strand displacement reaction (SDA) and CRISPR / Cas12a combined, provided by this invention, is shown below. Figure 1 As shown.

[0023] Unless otherwise specified, all strains tested in the following examples are Salmonella Typhimurium ATCC 14028, which was purchased from the Guangdong Institute of Microbiology.

[0024] Example 1: Synthesis of DNA-silver / platinum nanoclusters (DNA-Ag / Pt NCs) The DNA template strand (nucleotide sequence shown in SEQ ID NO.5, concentration 50 μM), AgNO3 solution (concentration 0.4 mM), and K2PtCl4 solution (concentration 0.4 mM) were mixed, and PBS buffer was added. The mixture was reacted at room temperature in the dark for 15 min, and then NaBH4 solution (concentration 2.0 mM) was added. The mixture was incubated at room temperature in the dark for 1.5 h to obtain DNA-Ag / Pt NCs. The mixing ratio of DNA template strand:AgNO3 solution:K2PtCl4 solution:PBS buffer:NaBH4 solution was 7.2 μL:11.3 μL:22.5 μL:214 μL:45 μL.

[0025] Effect Experiment: 1. Characterization of DNA-Ag / Pt NCs and verification of hydroxyl radicals (1) Transmission electron microscopy analysis Using transmission electron microscopy (TEM) Transmission Electron Microscope The DNA-Ag / Pt NCs obtained above were characterized by TEM to determine their morphology and dispersion. Specifically, the DNA-Ag / Pt NCs were pre-frozen in a -80°C freezer and then freeze-dried for 24 h in a freeze dryer to obtain powder. The powder sample was then dissolved in a small amount of ethanol to remove salt ions and directly dropped into a copper grid.

[0026] like Figure 2 As shown, the C-rich DNA framework has a significant impact on the formation of nanoclusters. The DNA-Ag / Pt material synthesized in this invention can be roughly divided into large spheres and small spheres in terms of morphology, and has good dispersibility with an average particle size of about 5 nm.

[0027] (2) X-ray photoelectron spectroscopy analysis X-ray photoelectron spectroscopy (XPS) was used X-ray photoelectron spectroscopy The chemical composition and valence state of the DNA-Ag / Pt NCs obtained above were characterized by XPS. Specifically, the DNA-Ag / Pt NCs were pre-frozen in a -80℃ freezer and then freeze-dried for 24 h in a freeze dryer to obtain powder. The powder was then pressed into tablets and placed in the instrument to determine the Ag and Pt elements in the sample.

[0028] like Figure 3 As shown, the Ag 3d (5 / 2) peak is located at 368.54 eV (Ag 0 ) and 371.54 eV (Ag) + At point ) Pt2 + The peak binding energies are 72.32 eV and 75.68 eV, while Pt 0The peak binding energies are 71.56 eV and 74.87 eV; Pt 2+ and Ag + The presence of these elements may be due to the coordination of DNA-Ag / Pt NCs with electron-rich groups (including purines and pyrimidines) in nucleic acids; this demonstrates that Ag and Pt elements exist in different valence states in the synthesized material.

[0029] (3) Ultraviolet absorption spectra of different systems To verify whether DNA-Ag / Pt NCs possess peroxidase activity to catalyze the oxidation of TMB to ox-TMB by H2O2, the absorbance values ​​of the following five systems were measured and their colors compared: (a) DNA-Ag / Pt NCs + H2O2 + TMB; (b) H2O2 + TMB; (c) DNA-Ag / Pt NCs + TMB; (d) DNA-Ag / Pt NCs + H2O2; (e) DNA-Ag / Pt NCs. Furthermore, to investigate the catalytic mechanism, 5, 10, 15, and 20 μL of DMSO were added to the catalytic system, and the absorbance values ​​and color changes at 652 nm were recorded for each group.

[0030] like Figure 4 As shown, systems (c), (d), and (e) contain only DNA-Ag / Pt NCs, DNA-Ag / Pt NCs and TMB, and DNA-Ag / Pt NCs and H2O2, respectively. The absorbance values ​​of these solutions are all around 0.04, and the color is colorless and transparent. When system (b) contains only H2O2 and TMB, the absorbance value of the solution is also very low, around 0.12, slightly higher than that of systems (c), (d), and (e), and the color is a very pale blue. This is because a large amount of H2O2 will undergo a small amount of decomposition, catalyzing the oxidation of TMB. When DNA-Ag / Pt NCs, H2O2, and TMB are present in system (a) at the same time, H2O2 can be catalyzed and decomposed, oxidizing TMB into a bright blue ox-TMB product, which has a high absorbance value of 0.57 at 652 nm.

[0031] To further study its catalytic mechanism, this invention adds different volumes of DMSO to the catalytic system. Since ·OH is easily captured by DMSO to form methanesulfinic acid, the absorbance decreases as the volume of DMSO increases.

[0032] (4) Electron paramagnetic resonance (EPR) analysis To verify that the catalytic performance of DNA-Ag / Pt NCs is achieved by decomposing H2O2 to generate ·OH, DMPO was introduced into the system and EPR analysis was performed on the following three systems: (a) DNA-Ag / Pt NCs + DMPO + H2O2; (b) DNA-Ag / Pt NCs + DMPO; (c) H2O2 + DMPO.

[0033] like Figure 5 As shown, when only DMPO and H2O2 are present in the system, H2O2 cannot generate ·OH radicals, and DMPO lacks a capture agent, resulting in a weak EPR signal. Similarly, when only DMPO and DNA-Ag / Pt NCs are present, ·OH radicals are also lacking, and DMPO lacks a capture agent. However, when DMPO, H2O2, and DNA-Ag / Pt NCs are present simultaneously, DNA-Ag / Pt NCs can catalyze the decomposition of H2O2 to produce ·OH. In this case, DMPO in the solution can capture ·OH, forming a DMPO-·OH adduct, resulting in a strong quartet signal. These EPR results indicate that the peroxidase activity of DNA-Ag / Pt NCs is mainly attributed to the ·OH generated from the decomposition of H2O2 by DNA-Ag / Pt NCs.

[0034] (5) Peroxidase kinetics analysis of DNA-Ag / Pt NCs DNA-Ag / Pt NCs possess peroxidase activity, capable of catalyzing the oxidation of TMB to ox-TMB by H2O2. Therefore, this experiment evaluated the peroxidase activity of DNA-Ag / Pt NCs by using the kinetic parameters of the catalytic system. By varying the concentrations of TMB and H2O2 within a certain range, steady-state kinetics were used to analyze the kinetic parameters of the enzymatic reaction, resulting in Michaelis-Menten curves.

[0035] Based on the formula 1 / V0=1 / Vmax(1+KmB / (B)+KmA / (A)) and the obtained curve, the Michaelis-Menten constant (Km) and the maximum reaction rate (Vmax) of the DNA-Ag / PtNCs catalytic reaction were calculated.

[0036] like Figure 6 As shown, the Km of DNA-Ag / Pt NCs against TMB is 0.076 mM, and the Vmax is 5.52 × 10⁻⁶ mM. -8 mol L -1s-1 The Km for H2O2 is 36.65 mM, and the Vmax is 1.113 × 10⁻⁶. -7 mol L⁻¹s -1 The Km of HPR relative to TMB is approximately 0.434 mM, and the Vmax is approximately 10 × 10⁻⁶ mM.-8 mol L -1 s -1 The Km for H2O2 is approximately 3.7 mM, and the Vmax is approximately 8.71 × 10⁻⁶. -8 mol L -1 s -1 Compared to HRP, TMB has a Km value approximately 7 times smaller, while DNA-Ag / Pt NCs exhibit a higher Vmax value for H2O2. This is because the Km value represents the enzyme's affinity for the substrate (a lower Km value indicates a stronger affinity), while a higher Vmax value indicates higher catalytic activity. Therefore, DNA-Ag / Pt NCs demonstrate high catalytic activity and possess significant advantages as a peroxidase mimic.

[0037] Example 2: A colorimetric sensor based on chain displacement reaction and CRISPR / Cas12a S1: The Salmonella typhimurium aptamer Apt1 (nucleotide sequence shown in SEQ ID NO.1, concentration 4 μM) and cDNA1 (nucleotide sequence shown in SEQ ID NO.2) were mixed at a volume ratio of 1:1 and shaken evenly. The mixture was then placed in a water bath at 95°C for 5 min to anneal. After the mixture was cooled to 60°C in the water bath, it was removed and cooled to room temperature to obtain the Apt1-cDNA1 complex. S2: Mix the SMBs magnetic bead suspension with 1×B&W buffer in a siliconized tube, shake to mix, and then perform magnetic separation. Aspirate the supernatant. Repeat the above steps 3 times to obtain magnetic beads. Resuspend the magnetic beads in 2×B&W buffer to obtain magnetic bead resuspension. S3: The bacterial culture to be tested was mixed with the Apt1-cDNA1 complex obtained in S1 and the magnetic bead resuspension obtained in S2 at a volume ratio of 40 μL:40 μL:10 μL. The mixture was shaken at 37°C and 150 rpm for 45 min for magnetic separation. The supernatant was transferred to a new centrifuge tube, and a premix was added (the premix consisted of 1 μM HP1, 2 μL DNA polymerase, 3 μL Nb.BbvCI, 25 μL dNTP 2.5 mM, and 10 μL 10×NEB buffer 2.1; the HP1 nucleotide sequence is shown in SEQ ID NO.3). The mixture was reacted at 37°C for 2 h, and then heated to 80°C for enzyme inactivation for 20 min to obtain the SDA product. S4: At 37°C, crRNA (nucleotide sequence shown in SEQ ID NO.4, concentration 200 nM) and LbCas12a (concentration 200 nM) were pre-assembled at a volume ratio of 1:1 for 20 min to obtain the Cas12a / crRNA1 complex. S5: Add the Cas12a / crRNA1 complex from S4 to the SDA product obtained in S3, incubate at 37°C and 200 rpm for 20 min, add the DNA-Ag / Pt NCs obtained in Example 1, mix and vortex for 60 min to obtain a mixture; the mixing ratio of the Cas12a / crRNA1 complex, SDA product and DNA-Ag / Pt NCs complex is 6 μL: 6 μL: 6 μL; S6: Add acetate buffer (concentration of 5 mM), TMB chromogenic solution and H2O2 to the mixture obtained in S5, observe the color change, and measure the fluorescence value at 652 nm using a multi-functional microplate reader within 30 min. Determine the infection status of Salmonella typhimurium based on the fluorescence value. The mixing volume ratio of the mixture, acetate buffer, TMB chromogenic solution and H2O2 is 12 μL:180 μL:40 μL:20 μL.

[0038] Effect Experiment: 1. Agarose gel electrophoresis analysis Agarose gel electrophoresis was used to verify the feasibility of the strand displacement amplification (SDA) amplification process after capturing Salmonella typhimurium. A 4% agarose gel was prepared using 1×TBE buffer and pre-stained with gelred. The following systems were mixed with 6× loading buffer and loaded into the wells of the agarose gel at a loading volume of 5 μL: (a) 10 bp DNA marker; (b) cDNA1; (c) HP1; (d) cDNA1 + HP1; (e) cDNA1 + HP1 + dNTP + DNA Polymerase I; (f) cDNA1 + HP1 + dNTP + DNA Polymerase I + Nb.BbvCI nicking endonuclease. The final nucleic acid concentration in each component was 5 μM. The gel electrophoresis program was performed at 120 V for 40 min, and the electrophoresis images were analyzed using a nucleic acid gel imaging system.

[0039] To verify whether the SDA amplification process was triggered, agarose gel electrophoresis analysis was performed on systems containing different components. Figure 7As shown, lane 1 represents the band of cDNA1, lane 2 represents the band of HP1, and lane 3 represents the product of cDNA1 and HP1 binding. Because there is a complementary base pairing sequence between the 5' ends of cDNA1 and HP1, cDNA1 can open the HP1 stem, allowing the ssDNA portion at the 3' end to hybridize with a portion of the original circular sequence, resulting in a new hairpin structure. When cDNA1, HP1, DNA polymerase, and dNTPs are present in the system (lane 4), only one darker band is observed. This indicates that after the addition of DNA polymerase and dNTPs, the newly generated hairpin extends at the 3' end, and the 5' and 3' ends hybridize to generate HP1 due to complementary base pairing, displacing cDNA1. However, the concentration of the displaced cDNA1 is low, only enough to regenerate HP1 in the newly generated hairpin, insufficient to trigger subsequent experiments. As shown in lane 5, when Nb.BbvCI endonuclease is added to the system, specific cleavage occurs at a specific site of HP1 to generate more ssDNA with the same sequence as cDNA1. Multiple rounds of polymerization and strand displacement then occur to trigger subsequent reactions. The lighter band at the bottom represents the generated ssDNA product. These results further confirm the successful construction of the SDA amplification process in this invention.

[0040] 2. Ultraviolet absorption spectroscopy analysis The absorbance of the following five systems was measured: (a) DNA-Ag / Pt NCs; (b) Apt1-cDNA1 + Salmonella Typhimurium + SMBs + Cas12a + DNA-Ag / Pt NCs; (c) Apt1-cDNA1 + Salmonella Typhimurium + SMBs + HP1 + DNA-Ag / Pt NCs; (d) Apt1-cDNA1 + SMBs + HP1 + Cas12a + DNA-Ag / Pt NCs; (e) Apt1-cDNA1 + Salmonella Typhimurium + SMBs + HP1 + Cas12a + DNA-Ag / Pt NCs; the color and absorbance value of each system were recorded.

[0041] To further demonstrate the feasibility of the entire system construction, the absorbance values ​​of the following five systems were measured. For example... Figure 8As shown, when only DNA-Ag / Pt NCs are added to the system (A), the solution is blue, and the absorbance is 0.52. When HP1 is absent from the system (B), SDA cycle amplification cannot proceed normally, thus preventing the subsequent Cas12a trans-cleavage activity from being triggered to cleave DNA-Ag / Pt. In the first two cases (C and D), the system solution was blue with an absorbance of 0.47. When Cas12a was absent (C), although the first part of the amplification process proceeded normally, the second part lacked Cas12a cleavage, resulting in a blue solution with an absorbance of 0.46. When Salmonella Typhimurium was absent (D), Apt1 could not capture Salmonella Typhimurium to release cDNA1, and SDA cycle amplification could not proceed normally, resulting in a blue solution with an absorbance of approximately 0.44, slightly lower than systems a, b, and c. When HP1, Cas12a, and Salmonella Typhimurium were all present (E), the entire reaction proceeded smoothly, and the solution was almost colorless with an absorbance of 0.15. This indicates that a dual-signal amplification DNA-Ag / Pt NCs colorimetric biosensor based on SDA and CRISPR / Cas12a has been successfully constructed.

[0042] 3. Optimization of Apt1-cDNA1 concentration The concentration of Apt1-cDNA1 directly determines the concentration of cDNA1 in triggering SDA cycle amplification. Apt1 and cDNA1 were diluted to 2, 4, 6, 8, and 10 μM, respectively. After mixing different concentrations of Apt1 and cDNA1, the mixture was thoroughly shaken and annealed in a 95°C water bath for 5 min. After the mixture was cooled to 60°C in the water bath, it was removed and gradually cooled to room temperature to form a stable Apt1-cDNA1. Subsequently, samples containing Salmonella typhimurium and blank samples were tested, and the optimal concentration of the Apt1-cDNA1 complex was determined by measuring the absorbance and color change using a microplate reader.

[0043] like Figure 9As shown, when the concentration of Apt1-cDNA1 is 1 μM, the concentration of cDNA1 released after Apt1 fully captures Salmonella typhimurium is low, which is insufficient to trigger the subsequent SDA cycle amplification process. At this time, the absorbance value in the system is high, and the A0-A value is small but shows a gradual upward trend. When the concentration of Apt1-cDNA1 is 2 μM, the A0-A value reaches its maximum. At this time, the cDNA1 released by the control group can just fully bind with HP1 to trigger the subsequent SDA cycle amplification process. When the concentration of Apt1-cDNA1 exceeds 2 μM, the excessive Apt1-cDNA1 exceeds the loading capacity of SMBs and will remain in the supernatant during the subsequent magnetic separation process. Since the binding ability of cDNA1 to HP1 is stronger, a small portion of cDNA1 will detach from Apt1 and bind to HP1. At this time, the absorbance value in the blank group system decreases more, so the A0-A value shows a downward trend. Once the detached cDNA1 has fully bound to HP1, the A0-A phase becomes stable. Therefore, the optimal concentration of Apt1-cDNA1 in the system is 2 μM.

[0044] 4. Optimization of incubation time between SMBs and Apt1-cDNA1 The prepared Apt1-cDNA1 double strands were incubated with SMBs for 0, 15, 30, 45, and 60 min, respectively. After incubation in a shaker at 12000 rpm and 37℃, magnetic separation was performed. 2 μL of the supernatant was aspirated and the DNA content was measured using an ultra-micro spectrophotometer. The most suitable incubation time was selected by comparison.

[0045] like Figure 10 As shown, when the incubation time of SMBs and Apt1-cDNA1 is between 0 and 45 min, the concentration of Apt1-cDNA1 in the supernatant decreases with increasing incubation time, indicating that the concentration of Apt1-cDNA1 loaded on SMBs gradually increases. When the incubation time is 45 min, the concentration of Apt1-cDNA1 in the supernatant no longer increases, indicating that SMBs have completely bound to the double strand of Apt1-cDNA1. When the incubation time is greater than 45 min, the concentration of Apt1-cDNA1 in the supernatant no longer changes significantly. Therefore, the optimal incubation time of SMBs and Apt1-cDNA1 in this invention is 45 min.

[0046] 5. Optimization of HP1 concentration The concentration of HP1 directly affects the reaction efficiency of the SDA cyclic amplification process. HP1 was diluted to 0.5, 1, 1.5, 2, and 2.5 μM, and annealed in a water bath at 95°C for 5 min. After the mixture was cooled to 60°C in the water bath, it was removed and gradually cooled to room temperature. Then, samples containing Salmonella typhimurium and blank samples were tested. The optimal HP1 concentration was determined by measuring the absorbance and color change using an ELISA reader.

[0047] like Figure 11 As shown, when the HP1 concentration is below 1 μM, the more ssDNA is generated during SDA cyclic amplification with increasing HP1 concentration, triggering subsequent Cas12a cleavage. Since DNA-Ag / Pt NCs in the control group are cleaved, the absorbance value in the system gradually decreases, thus A0-A gradually increases. When the HP1 concentration is above 1 μM, the excessively high concentration exceeds the needs of SDA cyclic amplification in the system, increasing the possibility of binding between a few unbound hairpins. The hairpins open, and under the action of various enzymes, SDA cyclic amplification also occurs in the blank group, resulting in a greater decrease in absorbance value and a gradual decrease in A0-A. Therefore, the optimal HP1 concentration is 1 μM.

[0048] 6. Optimization of enzyme concentration in the system DNA Polymerase I, Nb.BbvCI endonuclease, and dNTP enzyme are the main driving forces for SDA cyclic amplification in the system and play a crucial role in its amplification efficiency. Therefore, this experiment needs to select the optimal concentration of the three enzymes to achieve the best SDA amplification efficiency.

[0049] To optimize the detection performance of the colorimetric biosensor, this experiment optimized the concentrations and dosages of enzymes used in the SDA process, including the concentrations of DNA Polymerase I, dNTPs, and Nb.BbvCI nicking endonuclease. The enzyme activities of DNA Polymerase I were 5, 10, 15, 20, 25, and 30 I / U, the dNTP concentrations were diluted to 1, 1.5, 2, 2.5, 3, and 3.5 mM, and the Nb.BbvCI nicking endonuclease activities were 10, 15, 20, 25, and 30 U. Samples containing Salmonella typhimurium and blank samples were tested. The optimal concentrations and dosages of each enzyme were determined by measuring the absorbance and color changes using a microplate reader, thus maximizing the performance of the colorimetric sensor.

[0050] DNA Polymerase I enzyme is used to extend the 3' end of a hairpin to complete both ends. To maximize the polymerase's effectiveness while avoiding waste, this experiment first optimized the polymerase concentration. Figure 12As shown, when the polymerase concentration was 5, 10, and 15 I / U, the SDA cyclic amplification reaction became more and more complete with increasing enzyme concentration, and the absorbance value in the control group gradually decreased, thus A0-A showed an upward trend; when the polymerase concentration was 20 I / U, the A0-A value reached its maximum, at which point the SDA cyclic amplification reaction was complete; when the polymerase concentration was greater than 20 I / U, the A0-A value tended to stabilize. To avoid waste, the optimal concentration of DNA Polymerase I was determined to be 20 I / U for subsequent experiments. Figure 12 A).

[0051] Nb.BbvCI endonuclease is used to recognize and cleave specific sequences, playing a crucial role in SDA cycle amplification, such as... Figure 12 As shown, with increasing endonuclease concentration, more ssDNA sequences are cleaved, the SDA cycle amplification process is more complete, and the A0-A value gradually increases, reaching a maximum at a concentration of 20 U, after which the absorbance value tends to stabilize. Therefore, this invention selects 20 U of Nb.BbvCI endonuclease for subsequent experiments to obtain optimal sensing performance and avoid waste. Figure 12 B).

[0052] dNTP enzymes are composed of four different types of bases: dATP, dGTP, dTTP, and dCTP. They are the raw materials required for polymerase to synthesize DNA fragments, such as... Figure 12 As shown, when the dNTP concentration is below 2.5 mM, the A0-A value increases significantly with increasing enzyme concentration; when the enzyme concentration reaches 2.5 mM, the A0-A value reaches its highest value, and then the absorbance value tends to stabilize. Therefore, this invention selects 2.5 mM dNTP enzyme as the optimal dosage. Figure 12 C).

[0053] 7. Optimization of H2O2 concentration H2O2 solution was diluted to 10, 20, 30, 40, 50, and 60 mM, respectively. After Cas12a cleaved DNA-Ag / Pt NCs, the solution was added to the system. Samples containing Salmonella Typhimurium and blank samples were tested. The optimal hydrogen peroxide concentration was determined by measuring the absorbance and color change using an ELISA reader.

[0054] When DNA-Ag / Pt NCs act as peroxidases, they can only oxidize TMB to TMB in the presence of H2O2. + .like Figure 13As shown, when the H2O2 concentration is below 50 mM, with increasing concentration, the oxidation of TMB under the catalysis of DNA-Ag / Pt NCs becomes increasingly complete, resulting in a continuous upward trend in absorbance values ​​within the system. When the H2O2 concentration exceeds 50 mM, the continued oxidation of TMB by excess H2O2 leads to a further increase in background signals in both the blank and control groups. The background signal value in the control group is excessively high, resulting in a decrease in A0-A. Therefore, the optimal H2O2 concentration in this invention is 50 mM.

[0055] 8. Optimization of acetate pH Prepare a 5 mM acetate buffer solution and adjust its pH to 3, 3.5, 4, 4.5, 5, 5.5, and 6 using NaOH or HCl, respectively. Test samples containing Salmonella typhimurium and blank samples. Determine the optimal hydrogen peroxide concentration by measuring the absorbance and color change using an ELISA reader.

[0056] The pH of the acetate buffer has a significant impact on the DNA-Ag / Pt NCs-catalyzed oxidation of TMB to TMB+ by H2O2; excessively acidic or alkaline environments will affect the catalytic colorimetric effect. Figure 14 As shown, when the pH of acetate is less than 4.5, the excessively acidic environment leads to a low TMB colorimetric reaction rate, but A0-A shows a significant upward trend; when the pH is 4.5, the value of A0-A is the largest; when the pH is greater than 4.5, the weakly acidic environment also affects the TMB colorimetric reaction rate, indicating that the catalytic activity of DNA-Ag / PtNCs is affected by the pH environment. Therefore, the optimal pH of acetate in this invention is 4.5.

[0057] 9. Evaluation of colorimetric sensors based on chain substitution reaction and CRISPR / Cas12a combination (1) Specificity test of colorimetric sensors To verify the specificity of the colorimetric sensor for detecting Salmonella typhimurium, 10 preserved common foodborne pathogens (as shown in Table 1) were selected for specificity evaluation. The strains were washed three times with PBS buffer, then streaked in three zones, and the strain concentration was determined by plate counting. All bacterial solutions were diluted to the same concentration (10⁻⁶) with PBS buffer. 7 The colorimetric sensor provided by this invention (CFU / mL) was used to detect different strains. Each sample was measured three times. The specificity of the colorimetric sensor was analyzed by measuring the absorbance value and color change using an enzyme-linked immunosorbent assay (ELISA) reader.

[0058] Table 1

[0059] like Figure 15As shown, in the specificity verification of the colorimetric sensor, the color changes of seven different strains were measured. The absorbance value for *Salmonella typhimurium* was only about 0.15, indicating a very pale color, while the absorbance values ​​for other non-*Salmonella typhimurium* strains were around 0.6, indicating a blue color. Comparing these measured values ​​with the blank group, it can be determined that the colorimetric sensor has high specificity in distinguishing *Salmonella typhimurium* from other non-target bacteria.

[0060] (2) Sensitivity test of colorimetric sensor To verify the sensitivity of the colorimetric sensor for detecting Salmonella Typhimurium, the sensitivity of pure Salmonella Typhimurium culture was first determined. The concentration of Salmonella Typhimurium was determined to be 1.93 × 10⁻⁶ using the three-zone streak plating method and the dilution plating method. 9 CFU / mL. It was then serially diluted with PBS buffer to 1.93 × 10⁰ CFU / mL – 1.93 × 10⁰ CFU / mL. 8 The colorimetric sensor provided by this invention was used to detect different concentrations of Salmonella typhimurium at CFU / mL. Each sample was measured three times. The sensitivity of the colorimetric sensor to pure Salmonella typhimurium culture was analyzed by measuring the absorbance value and color change using an enzyme-linked immunosorbent assay (ELISA) reader.

[0061] The sensitivity of the colorimetric sensor for detecting pure Salmonella typhimurium culture is as follows: Figure 16 As shown, in pure cultures of Salmonella typhimurium, the absorbance value in the system increases with increasing bacterial concentration, and the detection concentration can reach 10. 8 CFU / mL. The absorbance value is correlated with the logarithm of the concentration of Salmonella Typhimurium (10⁻¹⁰). 1 ~10 7 The colorimetric sensor exhibits a good linear relationship with the concentration of Salmonella Typhimurium (CFU / mL), with the linear equation being y = 0.72424 - 0.07104x (R² = 0.9971). The detection limit is 2.5 CFU / mL (S / N = 3), indicating that the colorimetric sensor provided by this invention has good sensitivity in detecting pure Salmonella Typhimurium culture.

[0062] To further determine the sensitivity of the colorimetric sensor, commercially available whole milk and skim milk, both of which had undergone ultra-high temperature sterilization, were selected as test samples. One mL of *Salmonella typhimurium* was centrifuged and washed three times to obtain bacterial sludge. Whole milk and skim milk were then added to the bacterial sludge, and the mixture was shaken thoroughly. The concentrations of *Salmonella typhimurium* in the whole milk and skim milk were determined to be 1.16 × 10⁻⁶ and 1.16 × 10⁻⁶, respectively, using the streak plating method and dilution plating. 9 CFU / mL and 2.37×10 9 CFU / mL, then serially diluted with PBS buffer to 1.16 × 100 CFU / mL - 1.16 × 108 CFU / mL and 2.37×100 CFU / mL-2.37×10 8 The colorimetric sensor provided by this invention was used to detect different concentrations of Salmonella typhimurium in milk at CFU / mL. Each sample was measured three times. The absorbance value and color change were measured by an enzyme-linked immunosorbent assay (ELISA) reader to analyze the sensitivity of the colorimetric sensor to Salmonella typhimurium in real milk samples.

[0063] like Figure 17 As shown, in both whole milk and skim milk, the absorbance values ​​decreased with increasing bacterial concentration, reaching a minimum at a concentration of 10. 7 After reaching CFU / mL, the concentration gradually stabilized, and the absorbance value correlated with the logarithm of the concentration of Salmonella typhimurium (10⁻¹⁰). 1 ~10 7 The equations (CFU / mL) showed a good linear relationship, with the linear equations being: y = 0.68176 - 0.06165x (R² = 0.9968) and y = 0.67569 - 0.07208x (R² = 0.9968). 2 =0.992), with detection limits of 3 CFU / mL and 5 CFU / mL, respectively. The trend of these results is similar to that of the detection results of pure Salmonella typhimurium culture, indicating that the colorimetric sensor constructed in this invention also has good sensitivity for detecting different types of actual milk samples.

[0064] (3) Accuracy analysis of colorimetric sensors To evaluate the accuracy of the colorimetric sensor provided by this invention, a spiked recovery test was conducted on actual milk samples. 1 mL of *Salmonella typhimurium* was taken, and the bacterial concentration was calculated to be 1.8 × 10⁻⁶ using the dilution plating method. 8 CFU / mL, then diluted to 10 with PBS buffer. 3 10 4 10 5 CFU / mL, whole milk and skim milk were added to the samples respectively. The fluorescence intensity of the spiked whole milk and skim milk samples at three concentration gradients was measured using the colorimetric sensor provided by this invention. The linear relationship between the logarithm of the bacterial concentration and the absorbance value A0-A in the two milk samples was plotted. The absorbance value was converted into bacterial concentration and compared with the known spiked concentration in the bacterial solution to calculate the recovery rate.

[0065] As shown in Table 2, this experiment selected skim milk and whole milk to conduct three concentration gradients of Salmonella typhimurium (1.80 × 10⁻⁶). 3 1.80×10 5 1.80×10 7Spiking tests (CFU / mL) showed that the recovery rate of spiked skim milk was 93.94%-100.67%, with an RSD of 2.30%-2.76%. The recovery rate of spiked whole milk was 88.90%-105.39%, with an RSD of 2.07%-2.43%. The recovery rates of both groups of milk samples were between 80% and 120%, indicating that the colorimetric sensor provided by this invention has good accuracy and reliability in detecting Salmonella typhimurium in different types of milk.

[0066] Table 2

[0067] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A method for detecting Salmonella typhimurium based on chain displacement reaction and CRISPR / Cas12a combined with a colorimetric sensor, characterized in that, The method includes the following steps: S1: Mix the DNA template strand, AgNO3 solution and K2PtCl4 solution, add PBS buffer and mix, react at room temperature in the dark for 15 min, then add NaBH4 solution and incubate at room temperature in the dark for 1.5 h to obtain DNA-Ag / Pt NCs; S2: Mix Salmonella Typhimurium aptamer Apt1 and cDNA1 and shake well. Place in a water bath at 95°C for annealing for 5 min. After the mixture cools down to 60°C in the water bath, remove it and cool to room temperature to obtain the Apt1-cDNA1 complex. S3: Mix the SMBs magnetic bead suspension with 1×B&W buffer in a siliconized tube, shake to mix, and then perform magnetic separation. Aspirate the supernatant. Repeat the above steps 3 times to obtain magnetic beads. Resuspend the magnetic beads in 2×B&W buffer to obtain magnetic bead resuspension. S4: Mix the bacterial culture to be tested with the Apt1-cDNA1 complex obtained in S2 and the magnetic bead resuspension obtained in S3, and shake in a shaker at 37℃ and 150 rpm for 45 min for magnetic separation. Transfer the supernatant to a new centrifuge tube, add the premixed solution, react at 37℃ for 2 h, and heat to 80℃ for enzyme inactivation reaction for 20 min to obtain SDA product. S5: Pre-assemble crRNA with LbCas12a at 37℃ for 20 min to obtain Cas12a / crRNA1 complex; S6: Add the Cas12a / crRNA1 complex from S5 to the SDA product obtained in S4, incubate at 37℃ and 200 rpm for 20 min, add the DNA-Ag / Pt NCs obtained in S1, mix and shake for 60 min to obtain the mixture. S7: Add acetate buffer, TMB chromogenic solution and H2O2 to the mixture obtained in S6 and observe the color change. Within 30 minutes, use a multi-functional microplate reader to measure the fluorescence value at 652 nm. The infection status of Salmonella typhimurium is determined based on the fluorescence value.

2. The method according to claim 1, characterized in that, The mixing ratio of DNA template strand: AgNO3 solution: K2PtCl4 solution: PBS buffer: NaBH4 solution in S1 is 7.2 μL: 11.3 μL: 22.5 μL: 214 μL: 45 μL; the nucleotide sequence of the DNA template strand is shown in SEQ ID NO.5, and the concentration is 50 μM; the concentration of the AgNO3 solution is 0.4 mM; the concentration of the K2PtCl4 solution is 0.4 mM; and the concentration of the NaBH4 solution is 2.0 mM.

3. The method according to claim 1, characterized in that, The volume ratio of the Salmonella Typhimurium aptamer Apt1 to cDNA1 is 1:1; the nucleotide sequence of the Salmonella Typhimurium aptamer Apt1 is shown in SEQ ID NO.1, and the concentration is 4 μM; the nucleotide sequence of the cDNA1 is shown in SEQ ID NO.

2.

4. The method according to claim 1, characterized in that, The mixing volume ratio of the bacterial culture to be tested, the Apt1-cDNA1 complex, and the magnetic bead resuspension in S4 is 40 μL:40 μL:10 μL.

5. The method according to claim 1, characterized in that, The premixed solution in S4 consists of: 1 μM HP1, 2 μL DNA polymerase, 3 μL Nb.BbvCI, 25 μL dNTP 2.5 mM and 10 μL 10×NEB buffer 2.1; the nucleotide sequence of HP1 is shown in SEQ ID NO.

3.

6. The method according to claim 1, characterized in that, The volume ratio of crRNA to LbCas12a in S5 is 1:1; the nucleotide sequence of crRNA is shown in SEQ ID NO.4 and the concentration is 200 nM; the concentration of LbCas12a is 200 nM.

7. The method according to claim 1, characterized in that, The mixing ratio of the Cas12a / crRNA1 complex, SDA product, and DNA-Ag / Pt NCs complex in S6 is 6 μL: 6 μL: 6 μL.

8. The method according to claim 1, characterized in that, The mixing volume ratio of the mixture, acetate buffer, TMB colorimetric solution and H2O2 in S7 is 12 μL:180 μL:40 μL:20 μL; the concentration of the acetate buffer is 5 mM.

9. The method according to claim 1, characterized in that, The judgment criteria described in S7 are: A fluorescence value at 652 nm > 0.2 is considered positive, and a fluorescence value at 652 nm ≤ 0.2 is considered negative.

10. A method for detecting dairy products, characterized in that, The detection method described herein is the method described in any one of claims 1 to 9.

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