A method for detecting Salmonella based on HCR-mediated multivalent aptamer and CRISPR-Cas12a system

Abstract

This invention discloses a method for detecting Salmonella based on HCR-mediated multivalent aptamers and the CRISPR-Cas12a system. The method includes the following steps: 1) mixing streptavidin nanobeads with biotinylated aptamers to obtain aptamer magnetic beads; 2) mixing trigger strands, hairpin DNA H1, and hairpin DNA H2 to obtain an HCR scaffold, followed by the addition of aptamers to obtain an HCR-based multivalent aptamer structure. The detection method involves isolating and enriching Salmonella using aptamer magnetic beads, adding the HCR-based multivalent aptamer to obtain a magnetic bead-Salmonella-HCR sandwich structure, and then adding it to the CRISPR-Cas12a system. After reacting at 37°C for 1 hour, an enhanced fluorescence signal is obtained, enabling the detection of Salmonella in the test solution. The advantages are high sensitivity, strong specificity, and good accuracy.

Description

Technical Field

[0001] This invention relates to a method for detecting Salmonella, and more particularly to a method for detecting Salmonella based on HCR-mediated multivalent aptamers and the CRISPR-Cas12a system. Background Technology

[0002] Salmonella is one of the most threatening foodborne pathogens, with high infection rates in a variety of foods, posing a significant threat to human health and having a wide-ranging impact on the global economy. Therefore, it is essential to develop simple and sensitive methods for detecting Salmonella to monitor and control its presence in food.

[0003] Traditional culture methods, nucleic acid-based methods, and immunological analysis methods are commonly used for detecting foodborne pathogens. Traditional culture methods involve culturing and biochemically identifying the bacterial strain. These methods are highly reliable and sensitive, enabling bacterial detection at the single-cell level. However, they are complex, labor-intensive, and time-consuming. Nucleic acid-based and immunological methods have seen widespread development in recent years, significantly reducing detection time and simplifying procedures. Nucleic acid-based methods offer high sensitivity but require complex DNA extraction steps, lengthy amplification processes, and expensive equipment. Immunological methods typically use antibody recognition to specifically detect whole bacterial cells, offering a simple, high-throughput, and rapid tool for detecting pathogens. However, antibodies are unstable and highly sensitive to their reaction environment, making detection conditions more demanding and results less consistent. Therefore, developing a simple, sensitive, specific, and stable method for detecting Salmonella is urgently needed.

[0004] Aptamers are short-chain oligonucleotides isolated from random nucleic acid libraries that can specifically bind to targets. Compared to antibodies, they offer significant advantages such as small molecular weight, low cost, simple synthesis, ease of modification, non-immunogenicity, good thermal stability, and pH stability, making them widely popular for the detection of foodborne pathogens. Many aptamer-based sensors have been developed for Salmonella detection; however, their practical application remains challenging due to the low affinity and specificity of aptamers in complex food matrices. Multivalent interactions, ubiquitous in nature, can increase the effective concentration of ligands at target sites, thereby reducing the entropy penalty of subsequent binding events and ultimately enhancing binding affinity. Furthermore, multivalent interactions also contribute to better selective target recognition. Inspired by this, multivalentity has become an effective strategy for improving binding affinity, specificity, and reaction kinetics in target recognition over the past decade. Therefore, developing aptamer sensors based on multivalent aptamers holds great potential for sensitive and specific detection of foodborne pathogens.

[0005] The CRISPR-Cas system is an adaptive immune system found in bacteria and archaea. CRISPR-Cas12a, guided by crRNA, binds to target nucleic acids, thereby stimulating its trans-cleavage activity, randomly cutting surrounding single-stranded DNA. However, using the CRISPR-Cas12a system alone for bacterial detection only reaches the pM level, which is insufficient for detecting low concentrations of foodborne pathogens. Therefore, to improve sensitivity, many CRISPR-Cas12a bacterial analysis techniques need to be combined with nucleic acid amplification techniques such as PCR, loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). While this approach can achieve highly sensitive pathogen detection, it requires extensive sample processing, time-consuming amplification, and carries the risk of contamination, limiting its application. Currently, there are no published research reports on HCR-mediated multivalent aptamer-binding CRISPR-Cas12a aptamer sensors for the detection of Salmonella. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for detecting Salmonella based on HCR-mediated multivalent aptamers and CRISPR-Cas12a system with high sensitivity, high specificity and good accuracy.

[0007] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a method for detecting Salmonella based on HCR-mediated multivalent aptamers and the CRISPR-Cas12a system. This method is not for diagnosis or treatment purposes and includes the following steps:

[0008] (1) Synthesis of aptamer magnetic beads

[0009] Mix 100 μL of 1 mg / mL streptavidin-modified magnetic nanobead solution and 100 μL of 1 μM biotinylated aptamer 1 (biotin-poly-Apt) solution, react at room temperature for 45 minutes, add 200 μL of 4 μM D-biotin, react at room temperature for 30 minutes to block the magnetic nanobeads, wash twice with phosphate buffer containing 0.01 wt% Tween-20, reconstitute in 100 μL phosphate buffer to obtain aptamer magnetic bead solution, store at 4℃ for later use; (2) Synthesis of HCR-based multivalent aptamers

[0010] After hairpin DNA1, hairpin DNA2 and aptamer 2 were pretreated by annealing, hairpin DNAH1, hairpin DNAH2 and trigger strand DNA were mixed and reacted to obtain HCR backbone solution. Then, annealed aptamer 2 was added to HCR backbone solution to obtain HCR-based multivalent aptamer solution.

[0011] (3) Detection of Salmonella

[0012] The aptamer magnetic bead solution prepared in step (1) was mixed with 200 μL of Salmonella bacterial suspension of different concentrations. After reacting at room temperature for 45 minutes and washing three times, 20 μL of 0.5 μM HCR-based multivalent aptamer prepared in step (2) was added. After reacting at room temperature for 45 minutes and washing three times, it was reconstituted in 5 μL of PBS solution to obtain a "magnetic bead-Salmonella-HCR" sandwich structure. This sandwich structure was added to the CRISPR-Cas12a system. Finally, the reaction system was reacted at 37°C for 1 hour. The fluorescence intensity was observed under blue light and the relative fluorescence intensity was measured using an ELISA reader. Based on the absorbance values ​​corresponding to different concentrations of Salmonella, the concentration of Salmonella in the test solution was quantitatively detected.

[0013] Furthermore, the nucleotide sequence of the biotinylated aptamer 1 (biotin-poly-Apt) described in step (1) is as follows: biotin-TTT TTT TTT TTT TTT CTC CTC TGA CTG TAA CCA CGG TGG TTT GAT CAC TATTGG GCC TTT GTG ATG TCG GTA GT.

[0014] Further, step (2) specifically involves heating hairpin DNAH1, hairpin DNAH2, and aptamer 2 at 95°C for 10 minutes, then immediately placing them on ice to cool for 10 minutes to obtain annealed hairpin DNA and aptamer 2; then mixing 2.5 μL of 10 μM hairpin DNAH1, 2.5 μL of 10 μM hairpin DNAH2, and 2.5 μL of 0.2 μM trigger strand DNA, adding 17.5 μL of phosphate buffer, and incubating overnight at 37°C to obtain the HCR backbone; then adding 1.5 μL of 25 μM aptamer 2 solution to the HCR backbone solution, and adjusting the volume to 50 μL with phosphate buffer, and reacting at 37°C for 2 hours to obtain the HCR-based multivalent aptamer.

[0015] Furthermore, the nucleotide sequence of the trigger chain DNA is as follows: GTA TGT TGT TGC GGA ATG GTCTAG GTG ATT GAG TGG; the nucleotide sequence of the hairpin DNA H1 is as follows: TTT CCC TTA TAT TCT CThe nucleotide sequence of hairpin DNA H2 is as follows: GAT AAC ACA CCA CTC AAT CAC CTA GTC AAA CCC GCA GTA TGT TGTTGC GGA ATG GTC TAG GTG ATT GAG TGG; The nucleotide sequence of aptamer 2 is as follows: GAG AGA GAA TAT AAG GGA AA A AAA ACT CCT CTG ACT GTA ACC ACG GTG GTT TGA TCA CTA TTG GGCCTT TGT GAT GTC GGT AGT (The underlined part is complementary to H1).

[0016] Further, the CRISPR-Cas12a system described in step (3) consists of: 2.5 μL 2 μM Cas12a enzyme, 2.5 μL 2 μM crRNA, 2 μL 2 μM single-stranded DNA fluorescent reporter probe, 2 μL 10 U RNase inhibitor, and finally diluted to 20 μL with enzyme-free water.

[0017] Furthermore, the nucleotide sequence of the single-stranded DNA fluorescent reporter probe is as follows: FAM-TTA TT-BHQ; the nucleotide sequence of the crRNA is as follows: GAA UUU CUA CUG UUG UAG AAC UAG GUG AUU GAG UGG UGUGUU.

[0018] Inventive principle: such as Figure 1As shown, Salmonella-specific aptamers were attached to the surface of magnetic nanobeads via biotin-streptavidin interactions to prepare aptamer-based magnetic beads for the specific capture of Salmonella. Two hairpin DNA scaffolds were designed: H1 with a branched arm at its 5' end, and H2 containing a CRISPR targeting sequence. Under the action of the trigger strand, H1 and H2 hybridized to form a long double-stranded DNA scaffold containing numerous repetitive CRISPR targeting sequences, providing binding sites for the aptamers. After the aptamers were added, base complementarity pairing between the 5' extended sequence and the branched arm on the HCR scaffold resulted in an HCR-based multivalent aptamer structure. In the presence of Salmonella, the target bacteria were captured and separated by the aptamer-based magnetic beads and recognized with high affinity by the HCR-based multivalent aptamer. Both simultaneously attached to the Salmonella, resulting in a "magnetic bead-Salmonella-HCR" sandwich complex. The signal from the target bacteria is amplified to hundreds of repeating CRISPR targeting units via an HCR scaffold. Subsequently, the CRISPR targeting units on the HCR scaffold activate the trans-cleavage activity of Cas12a, randomly cleaving the surrounding single-stranded DNA reporter probe and generating an amplified fluorescent signal. High-sensitivity detection of Salmonella is achieved through the high affinity of the multivalent aptamer and the cascade signal amplification.

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] (1) The HCR-based multivalent aptamer in this invention connects the aptamer to the HCR scaffold through complementary base pairing, thereby obtaining a high-affinity multivalent aptamer;

[0021] (2) The present invention is based on a multivalent aptamer of HCR. The HCR scaffold contains a CRISPR targeting unit, which triggers the trans-cleavage activity of Cas12a to generate a fluorescent signal, which can convert bacterial signals into fluorescent signals.

[0022] (3) The HCR-based multivalent aptamer in this invention can improve the affinity of the aptamer and convert bacterial signals into fluorescent signals through DNA design.

[0023] (4) In this invention, the signal is amplified in one step by the cleavage of multiple single-stranded DNA fluorescent probes by multiple CRISPR targeting units and Cas12a on the HCR scaffold, and finally the detection limit of 2 CFU / mL is obtained.

[0024] (5) In this invention, aptamer magnetic beads capture and separate Salmonella in the sample, reducing the interference of complex sample matrix on the detection system.

[0025] In summary, this invention presents a bifunctional HCR-mediated multivalent aptamer-CRISPR-Cas12a aptamer sensor, combining aptamer magnetic separation technology with an HCR-based multivalent aptamer and the CRISPR-Cas12a system. Salmonella-specific aptamers are attached to the surface of magnetic nanobeads via biotin-streptavidin interactions to obtain aptamer beads. These aptamers are then assembled onto an HCR scaffold through base pairing to obtain an HCR-based multivalent aptamer. Under the influence of multivalent properties, the affinity of the multivalent aptamer is significantly enhanced. Simultaneously, the HCR scaffold contains numerous repeating CRISPR targeting units, which can trigger the trans-cleavage activity of Cas12a to generate a fluorescent signal. Through the multiple repeating units on the long HCR scaffold and the activity of Cas12a, the signal of the target bacteria is amplified in a step-by-step cascade, achieving highly sensitive and specific detection of Salmonella in food samples by combining aptamer magnetic separation technology. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the principle of the aptamer sensor for detecting Salmonella in this invention.

[0027] Figure 2 This is an agarose gel electrophoresis image of the multivalent aptamer constructed based on HCR in Specific Embodiment 2;

[0028] Figure 3 This is an atomic force microscope image of the HCR-based multivalent aptamer in Specific Embodiment 2;

[0029] Figure 4 This is a comparison diagram of the binding ability of the multivalent and monovalent aptamers based on HCR of this invention to Salmonella.

[0030] Figure 5 Fluorescence microscopy images of the binding of the multivalent and monovalent aptamers based on HCR of this invention to Salmonella;

[0031] Figure 6 The bar graph (a) shows the activation of CRISPR-Cas12a trans-cleavage by the multivalent aptamer based on HCR in this invention, and the agarose gel electrophoresis graph (b) shows the activation of CRISPR-Cas12a cis-cleavage.

[0032] Figure 7 Different concentrations (1-10) of PBS in this invention 7 Linear relationship between relative fluorescence intensity of Salmonella (CFU / mL) and Salmonella concentration;

[0033] Figure 8 This invention relates to different concentrations (1-10) of [materials] in milk, eggs, and chicken. 7Linear relationship between relative fluorescence intensity of Salmonella (CFU / mL) and Salmonella concentration; (a) milk, (b) eggs, (c) chicken.

[0034] Figure 9 A specific embodiment of a detection method is used to detect 10 5 CFU / mL Staphylococcus aureus, 10 5 CFU / mL Listeria monocytogenes, 10 5 CFU / mL E. coli, 10 5 CFU / mL Salmonella, 10 5 CFU / mL Staphylococcus aureus: 10 5 CFU / mL Listeria monocytogenes: 10 5 CFU / mL E. coli: 10 5 The relative fluorescence intensity for detection was determined by a CFU / mL Salmonella ratio of 1:1:1:1. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Specific Implementation Example 1

[0037] Synthesis of aptamer magnetic beads

[0038] Aptamer magnetic beads are formed by linking magnetic nanobeads and aptamers together via biotin-streptavidin interactions. The specific steps are as follows: 100 μL of 1 mg / mL streptavidin-modified magnetic nanobead solution and 100 μL of 1 μM biotinylated aptamer 1 (biotin-poly-Apt) solution are mixed and reacted at room temperature for 45 minutes. Then, 200 μL of 4 μM D-biotin is added, and the mixture is reacted at room temperature for 30 minutes to block the magnetic nanobeads. The nanobeads are then magnetically separated and washed twice with phosphate buffer (containing 0.01% Tween-20), reconstituted in 100 μL of phosphate buffer, and stored at 4°C for later use. The nucleotide sequence of biotinylated aptamer 1 (biotin-poly-Apt) is as follows: biotin-TTT TTT TTT TTT TTT CTC CTC TGACTG TAA CCA CGG TGG TTT GAT CAC TAT TGG GCC TTT GTG ATG TCG GTA GT. Specific Implementation Example 2

[0040] Synthesis of HCR-based multivalent aptamers

[0041] HCR-based multivalent aptamers are obtained by ligating aptamers to long double-stranded DNA obtained through hybridization chain reaction (HCR) via base complementarity pairing. The specific steps are as follows: First, two hairpin DNAs (H1, H2) and aptamer 2 are heated separately at 95°C for 10 minutes, and then immediately placed on ice to cool for 10 minutes to obtain annealed hairpin DNA and aptamers. Next, 2.5 μL of 10 μM hairpin DNA (H1, H2) is mixed with 2.5 μL of 0.2 μM trigger strand, and 17.5 μL of phosphate buffer is added. The mixture is incubated overnight at 37°C to obtain the HCR backbone. Finally, 1.5 μL of 25 μM aptamer solution is added to the HCR backbone solution, and the volume is adjusted to 50 μL with phosphate buffer. The mixture is reacted at 37°C for 2 hours to obtain the HCR-based multivalent aptamer. The nucleotide sequence of the trigger strand DNA is as follows: GTA TGT TGT TGC GGA ATG GTC TAG GTG ATT GAG TGG; the nucleotide sequence of the hairpin DNA H1 is as follows: TTT CCC TTA TAT TCT C The nucleotide sequence of hairpin DNA H2 is as follows: TC TCT CTC CTG CGG GTT TGA CTA GGT GATTGA GTG GTG TGT TAT CCC ACT CAA TCA CCT AGA CCA TTC CGC AAC AAC ATA C (underlined parts are complementary to the aptamer); GAG AGA GAA TAT AAG GGA AA A AAA ACT CCT CTG ACT GTA ACC ACGGTG GTT TGA TCA CTA TTG GGC CTT TGT GAT GTC GGT AGT (The underlined part is complementary to H1).

[0042] To verify the synthesis of the HCR-based multivalent aptamer in the second specific embodiment of the present invention, hairpin DNA H1 (annealed hairpin DNA H1), hairpin DNA H2 (annealed hairpin DNA H2), trigger strand (trigger strand stock solution), hairpin DNA H1 + hairpin DNA H2 (0.5 μL of 10 μM annealed hairpin DNA H1 and H2 mixed, with 4 μL of PBS added, reacted overnight at 37°C), HCR backbone (HCR backbone prepared by the above method), HCR-based multivalent aptamer (HCR-based multivalent aptamer prepared by the above method), and aptamer 2 (annealed aptamer 2) were sequentially added to a 1% agarose gel for separation (4 SGelRed nucleic acid dye pre-stained, electrophoresis buffer 1×TAE, constant voltage 130V for 30 minutes) and analyzed by gel imaging. The results are as follows: Figure 2 As shown, lanes 1-3 and lane 7 represent hairpin DNA H1, hairpin DNA H2, trigger strand, and aptamer 2, respectively, exhibiting different bands under different molecular weights. In the absence of a trigger strand, the two hairpin DNAs remain stable in solution (lane 4). With the addition of a trigger strand, the molecular weight of the HCR product (lane 5) increases significantly, and with the addition of an aptamer (lane 6), the molecular weight further increases, indicating the successful synthesis of HCR-based multivalent aptamers.

[0043] To further demonstrate the construction of the HCR-based multivalent aptamer in Specific Example 2, biotin was modified onto the aptamer. After synthesizing the HCR-based multivalent aptamer, three times the concentration of streptavidin was added, allowing it to attach to the HCR-based multivalent aptamer by binding to the biotin on the aptamer. The results were then observed under an atomic force microscope. Figure 3 As shown, there are many highlights on the linear HCR backbone, namely streptavidin, which indicates that the construction of the HCR-based multivalent aptamer is complete. Specific Implementation Example 3

[0045] The binding affinity of HCR-based multivalent and monovalent aptamers to Salmonella was compared using ELISA. The specific steps were as follows: First, 50 μL of 10... 5CFU / mL Salmonella bacterial suspension was added to a high-adsorption ELISA plate and incubated at 37°C for 2 hours. The plate was then washed three times with phosphate-buffered saline (containing 0.01% Tween-20), followed by the addition of 150 μL of 1 wt% bovine serum albumin solution and incubation at 37°C for 30 minutes to block excess binding sites. After washing three times in the same manner, 50 μL of different concentrations (25, 50, 100, 250, 500, 800 nM) of HCR-based multivalent aptamers (constructed according to Example 2 using 5' biotin-modified aptamers) and monovalent aptamers (annealed 5' biotin-modified aptamers) were added and incubated at 37°C for 45 minutes. After three washes, 50 μL of streptavidin-modified horseradish peroxidase was added and incubated at room temperature for 25 minutes. After three washes, 50 μL of TMB chromogenic solution was added, and the reaction was terminated with 50 μL of 2M H2SO4 after 15 minutes of incubation in the dark. The absorbance at 450 nm was measured using a microplate reader. Figure 4 As shown, at low concentrations, the signal of the monovalent aptamer is very low, and the signal gradually increases with increasing concentration. In contrast, the HCR-based multivalent aptamer has a strong signal even at low concentrations, which indicates that the HCR-based multivalent aptamer has superior target binding performance.

[0046] In addition, the binding strength of HCR-based multivalent and monovalent aptamers to Salmonella was compared using fluorescence microscopy. The 5' end of the aptamer was modified with a fluorescent group (FAM), and an HCR-based multivalent aptamer was constructed according to Example 2. 2.5 μL of 1 μM monovalent aptamer or HCR-based multivalent aptamer was mixed with 100 μL of 10⁹ CFU / mL Salmonella bacterial suspension and incubated at 37°C for 2 hours. After centrifugation at 6000 rpm for 5 minutes, the mixture was washed twice with phosphate buffer to remove the supernatant, and the resulting mixture was reconstituted in 1 mL of phosphate buffer and observed under a fluorescence microscope. The results are as follows: Figure 5 As shown in the superimposed image of the light microscope and fluorescence image, fluorescence was observed on both Salmonella treated with monovalent and polyvalent aptamers, with a more obvious fluorescence signal on the bacteria treated with polyvalent aptamers. This indicates that the aptamers can bind to the target bacteria, and that the polyvalent aptamers have a stronger binding ability to Salmonella compared to the monovalent aptamers. Specific Implementation Example 4

[0048] CRISPR-Cas12a activity was stimulated using HCR-based multivalent aptamers.

[0049] 5 μL of the 1 μM HCR-based multivalent aptamer constructed in Example 2 was mixed with the CRISPR-Cas12a system, which consisted of 2.5 μL 2 μM Cas12a enzyme, 2.5 μL 2 μM crRNA, 2 μL 2 μM single-stranded DNA fluorescent reporter probe, and 2 μL 10 U RNase inhibitor. The volume was then adjusted to 20 μL with enzyme-free water. The reaction mixture was incubated at 37°C for 1 hour. The fluorescence intensity was then observed under blue light, and the relative fluorescence intensity was measured using a microplate reader to verify the activation of CRISPR-Cas12a trans-cleavage activity by the HCR-based multivalent aptamer. Finally, the HCR-based multivalent aptamer, reaction solution (a mixture of the HCR-based multivalent aptamer and the CRISPR-Cas12a system), and control solution (a mixture of the HCR-based multivalent aptamer and the CRISPR-Cas12a system without crRNA) were added to a 1% agarose gel for separation (pre-stained with 4S GelRed nucleic acid dye, electrophoresis buffer 1×TAE, constant voltage 130V for 30 minutes) and analyzed by gel imaging. The nucleotide sequences of the single-stranded DNA fluorescent reporter probe are as follows: FAM-TTA TT-BHQ.

[0050] from Figure 6 As shown in (a), the fluorescence intensity was greatly improved after the HCR-based multivalent aptamer was mixed with the CRISPR-Cas12a system. This indicates that the HCR-based multivalent aptamer successfully activated the trans-cleavage activity of CRISPR-Cas12a, non-specifically cleaving single-stranded DNA fluorescent reporter probes in the solution.

[0051] from Figure 6 As shown in (b), no multivalent aptamer bands appeared in the reaction solution, indicating that the HCR-based multivalent aptamer successfully activated the cis-cleavage activity of CRISPR-Cas12a and cleaved the HCR backbone. Specific Implementation Example 5

[0053] Aptamer sensor for detecting Salmonella

[0054] The aptamer magnetic beads prepared in Specific Example 1 were mixed with 200 μL of Salmonella bacterial suspensions of different concentrations. After reacting at room temperature for 45 minutes and washing three times, 20 μL of the 0.5 μM HCR-based multivalent aptamer prepared in Specific Example 2 was added. After reacting at room temperature for 45 minutes and washing three times, the mixture was reconstituted in 5 μL of PBS to obtain a "magnetic bead-Salmonella-HCR" sandwich structure. This sandwich structure was added to the CRISPR-Cas12a system, which consisted of: 2.5 μL 2 μM Cas12a enzyme, 2.5 μL 2 μM crRNA, 2 μL 2 μM single-stranded DNA fluorescent reporter probe, and 2 μL 10 U RNase inhibitor. Finally, the volume was adjusted to 20 μL with enzyme-free water. The reaction system was incubated at 37°C for 1 hour. Subsequently, the fluorescence intensity of the reaction solution was observed under blue light, and the relative fluorescence intensity was measured using an ELISA reader. The detection principle is as follows: Figure 1 As shown.

[0055] Based on the curves obtained from the relative fluorescence intensity values ​​corresponding to different concentrations of Salmonella bacterial solutions, the concentration of Salmonella in the test solution was quantitatively analyzed. The higher the concentration of Salmonella added, the more multivalent aptamers it specifically binds, and the easier it is to activate CRISPR-Cas12a activity, causing cleavage of the surrounding single-stranded DNA fluorescent reporter probe.

[0056] like Figure 7 As shown, different concentrations of Salmonella (1-10 μL) in PBS were detected. 7 The linear relationship between relative fluorescence intensity (CFU / mL) and Salmonella concentration was calculated, and the standard curve equation was y = 0.446 × 10⁻⁶. 6 x + 0.0719 × 10 6 Correlation coefficient R 2 =0.993, detection limit is 2 CFU / mL, can be used for the detection of Salmonella at unknown concentrations. Specific Implementation Example Six

[0058] To verify the application of the detection method in specific embodiment five of the present invention in actual sample detection, Salmonella standard solution was added to processed samples of milk, eggs, and chicken, respectively, as actual samples. Different concentrations of Salmonella in the three samples were then detected, and the results are as follows: Figure 8 As shown, different concentrations (1-10) of substances in milk, eggs, and chicken... 7 The linear relationship between the relative fluorescence intensity of Salmonella (CFU / mL) and Salmonella concentration was shown in the standard curve equation: y = 0.409 × 10⁻⁶. 6 x + 0.0648 × 10 6 (R 2 =0.998), y =0.380×10 6x + 0.0813 × 10 6 (R 2 =0.997), y =0.345×10 6 x+0.0901×10 6 (R 2 =0.995) The detection limit is 2 CFU / mL. Specific Implementation Example 7

[0060] To verify the detection specificity of the detection method in specific embodiment five of the present invention, 10 5 CFU / mL Staphylococcus aureus, 10 5 CFU / mL Listeria monocytogenes, 10 5 CFU / mL E. coli, 10 5 CFU / mL Salmonella, 10 5 CFU / mL Staphylococcus aureus, 10 5 CFU / mL Listeria monocytogenes, 10 5 CFU / mL E. coli, 10 5 The detection was performed using a CFU / mL Salmonella ratio of 1:1:1:1. Results are as follows: Figure 9 As shown, the relative fluorescence intensity detected in the presence of Salmonella is much greater than that of other foodborne pathogens, indicating that the detection method is specific to Salmonella. Specific Implementation Example 8

[0062] To verify the accuracy and stability of the detection method in Specific Embodiment 5 of the present invention, different concentrations of Salmonella in milk, eggs, and chicken were detected, and the recovery rates were calculated. The results are shown in Table 1.

[0063] Table 1 shows the recovery rates of Salmonella at different concentrations in milk, eggs, and chicken.

[0064]

[0065]

[0066] Table 1 shows that different concentrations (5×10⁻⁶) in milk, eggs, and chicken... 2 -5×10 4 The average recoveries of Salmonella (CFU / mL) were 95.81%, 95.53%, and 99.26%, respectively, indicating that the detection method can be applied to the detection of Salmonella in food samples.

[0067] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A method for detecting Salmonella based on HCR-mediated multivalent aptamer and CRISPR-Cas12a system, the method is not for the purpose of diagnosis or treatment, characterized in that Comprising the following steps: (1) Synthesis of aptamer magnetic beads Mix 100 μL of 1 mg / mL streptavidin-coated nanomagnetic beads solution and 100 μL of 1 μM biotinylated aptamer 1 solution, react at room temperature for 45 minutes, then add 200 μL of 4 μM D-biotin, react at room temperature for 30 minutes to block the nanomagnetic beads, wash twice with phosphate buffer containing 0.01wt% Tween-20 by magnetic separation, resuspend in 100 μL of phosphate buffer to obtain the aptamer magnetic bead solution, store at 4 ℃ for standby, wherein the nucleotide sequence of the biotinylated aptamer 1 is as follows: biotin-TTT TTT TTT TTT TTT CTC CTC TGA CTG TAA CCA CGG TGG TTT GAT CACTAT TGG GCC TTT GTG ATG TCG GTA GT; (2) Synthesis of multivalent aptamer based on HCR The hairpin DNA H1, the hairpin DNA H2 and the aptamer 2 are respectively treated by annealing pretreatment, the hairpin DNA H1, the hairpin DNA H2 and the trigger chain DNA are mixed to obtain a HCR skeleton solution, then the annealing treated aptamer 2 is added into the HCR skeleton solution to obtain a multivalent aptamer solution based on HCR, wherein the nucleotide sequence of the trigger chain DNA is as follows: GTA TGT TGT TGC GGA ATG GTC TAG GTG ATT GAG TGG; the nucleotide sequence of the hairpin DNA H1 is as follows: TTT CCC TTA TAT TCT C TCT CTC CTG CGG GTT TGA CTA GGT GAT TGA GTG GTG TGT TAT CCC ACT CAA TCA CCT AGA CCA TTC CGC AAC AAC ATA C; the nucleotide sequence of the hairpin DNA H2 is as follows: GAT AAC ACA CCA CTC AAT CAC CTA GTC AAA CCC GCA GTA TGT TGT TGC GGA ATG GTC TAG GTG ATT GAG TGG; and the nucleotide sequence of the aptamer 2 is as follows: GAG AGA GAA TAT AAG GGA AA A AAA ACT CCT CTG ACT GTA ACC ACG GTG GTT TGA TCA CTA TTG GGC CTT TGT GAT GTC GGT AGT; (3) Detection of Salmonella Mix the aptamer magnetic bead solution prepared in step (1) with 200 μL of different concentrations of Salmonella solution, react at room temperature for 45 minutes and wash three times, then add 20 μL of 0.5 μM multivalent aptamer based on HCR prepared in step (2), react at room temperature for 45 minutes and wash three times, then resuspend in 5 μL of PBS solution to obtain the sandwich structure of magnetic beads-Salmonella-HCR, then add the CRISPR-Cas12a system, finally react the reaction system at 37 ℃ for 1 hour, observe the fluorescence intensity under blue light and measure the relative fluorescence intensity with a microplate reader, according to the curve obtained by detecting the absorbance values corresponding to different concentrations of Salmonella, the concentration of Salmonella in the test solution is quantitatively detected, wherein the CRISPR-Cas12a system is: 2.5 μL of 2 μM Cas12a enzyme, 2.5 μL of 2 μM crRNA, 2 μL of 2 μM single-stranded DNA fluorescent reporter probe, 2 μL of 10 U RNAase inhibitor, finally add enzyme-free water to 20 μL, the nucleotide sequence of the single-stranded DNA fluorescent reporter probe is as follows: FAM-TTA TT-BHQ; the nucleotide sequence of the crRNA is as follows: GAA UUU CUA CUG UUG UAG AACUAG GUG AUU GAG UGG UGU GUU. 2.The method for detecting Salmonella based on the HCR-mediated multivalent aptamer and CRISPR-Cas12a system according to claim 1, which is not for the purpose of diagnosis or treatment, characterized in that Step (2) is specifically as follows: the hairpin DNA H1, the hairpin DNA H2 and the aptamer 2 are heated at 95 ℃ for 10 minutes, and then immediately inserted into ice to cool for 10 minutes to obtain the annealed hairpin DNA and the aptamer 2; 2.5 μL of 10 μM hairpin DNA H1, 2.5 μL of 10 μM hairpin DNA H2 and 2.5 μL of 0.2 μM trigger strand DNA are mixed, 17.5 μL of phosphate buffer is added, and incubation is carried out at 37 ℃ overnight to obtain an HCR skeleton; then 1.5 μL of 25 μM aptamer 2 solution is added to the HCR skeleton solution, and the volume is adjusted to 50 μL with the phosphate buffer, and reaction is carried out at 37 ℃ for 2 hours to obtain the HCR-based multivalent aptamer.

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