Rapid ratio colorimetric detection method for salmonella typhimurium based on hybridization chain reaction

Abstract

The invention provides a salmonella rapid ratio colorimetric detection method based on a hybridization chain reaction, and relates to the technical field of biosensing and nucleic acid detection, and the salmonella is adsorbed and enriched by Fe3O4 (at) PDA (at) PEI magnetic nanoparticles through electrostatic interaction. A salmonella specific hairpin aptamer is used for identifying and triggering a hybridization chain reaction (HCR), and a DNA sequence connected with urease is introduced as a signal detection unit. Phenol red is used as an indicator, and the absorbance and the solution color change in a ratio mode. The kit is rapid, sensitive, specific and easy to use, the sample treatment process is reduced, and the sample detection efficiency is improved; the method can be directly operated at room temperature without complex operation environment, and is convenient to use.

Description

Technical Field

[0001] This invention belongs to the fields of foodborne pathogen detection technology and biosensing and nucleic acid detection technology, specifically involving a rapid ratio colorimetric detection method for Salmonella typhimurium based on isothermal amplification. Background Technology

[0002] Salmonella is a Gram-negative facultative anaerobic bacterium belonging to the Enterobacteriaceae family. It is a globally recognized major foodborne pathogen, accounting for approximately 85% of all bacterial foodborne illnesses worldwide each year. It causes extremely low infectious doses, high morbidity, and high mortality, resulting in over 100,000 deaths and enormous economic losses. The World Health Organization has classified it as a foodborne pathogen of moderate to severe risk. Human infection may cause symptoms such as nausea, vomiting, abdominal pain, diarrhea, and fever. Therefore, developing and establishing rapid detection methods for Salmonella is crucial for ensuring food safety.

[0003] Currently, Salmonella detection methods mainly include culture methods, molecular biological methods, and immunological methods. While culture methods offer high specificity, they require long incubation times and lack sensitivity in samples with low initial bacterial counts. Molecular biological methods, such as polymerase chain reaction (PCR) and nucleic acid hybridization, are faster but involve expensive equipment, complex procedures, and are susceptible to contamination in the sample. Immunological methods rely on specific antibodies, which are complex and costly to prepare, and cross-reactivity can affect the accuracy of results. Therefore, these methods are difficult to widely apply in practical detection, necessitating the development of a new Salmonella detection method with high sensitivity, specificity, and stability. Summary of the Invention

[0004] To address some shortcomings in existing technologies, this invention provides a rapid ratiometric colorimetric detection method for Salmonella Typhimurium based on a hybridization chain reaction. This invention combines Fe3O4@PDA@PEI with an HCR reaction, utilizing the magnetic separation and photothermal properties of Fe3O4@PDA@PEI to achieve adsorption and enrichment of Salmonella Typhimurium. As the target triggers the hybridization chain reaction, changes in the colorimetric signal of the ratiometric colorimetric biosensor can be observed. It exhibits good selectivity for Salmonella Typhimurium and good anti-interference performance against other bacteria, making it suitable for rapid detection in the field of food safety.

[0005] The technical solution adopted in this invention is as follows:

[0006] A rapid colorimetric detection method for Salmonella Typhimurium based on hybridization chain reaction, specifically including the following steps:

[0007] 1) Design a hairpin-type trigger chain, wherein the 5' end contains a Salmonella typhimurium-specific aptamer (apt), and the 3' end contains its complementary blocking strand, which also serves as the trigger chain for hybridization chain reaction (HCR). The trigger chain is shown in SEQ ID No. 1. Design hairpins H1 and H2, which can be complementary and alternately open and extend to form HCR. H2 contains a binding site for an enzyme reporter (T3-urease). H1 is shown in SEQ ID No. 2 and SEQ ID No. 3. Design a T3 sequence, wherein T3 can connect to urease to form T3-urease, which then enters the HCR through partial complementarity with T2, serving as an enzyme reporter. T3 is shown in SEQ ID No. 4.

[0008] 2): Trigger, H1, and H2 were heated at 95°C for 5 minutes and then cooled to room temperature to form a hairpin structure. Salmonella typhimurium was then added to trigger the HCR reaction to obtain a long HCR chain containing a region complementary to T3-urease.

[0009] 3): Dissolve MBS in DMSO to obtain a 6.4 mM MBS solution. Dissolve urease powder in 1×PBS buffer (pH 7.2) to obtain a urease solution. Mix the MBS solution with the T3 solution, adjust the final reaction volume to 100 μL with 1×PBS buffer, incubate at room temperature, and then centrifuge the mixture using a 3 kDa ultrafiltration concentrator to remove excess MBS.

[0010] MBS-activated DNA. Urease solution was added to the activated T3, and after incubation at room temperature, the mixture was filtered through a 30 kDa ultrafiltration centrifuge column to obtain T3-urease;

[0011] 4): Add T3-urease to the HCR product; T3-urease is coupled to the long chain of the HCR product.

[0012] 5): Disperse 10 mg of Fe3O4 in 10 mL of Tris-HCl buffer (10 mM, pH 8.5). Add 10 mL of a solution containing 7 mg of DA to the suspension. Stir continuously at room temperature for 7 h, and then collect the obtained Fe3O4@PDA with a magnet. Add 30 mL of 25% PEI solution to Fe3O4@PDA and stir at room temperature for 10 h. After the reaction is complete, collect Fe3O4@PDA@PEI with a magnet. Aliquot the obtained Fe3O4@PDA@PEI into water (1 mg / mL) and store at 4 °C for later use.

[0013] 6): Add Fe3O4@PDA@PEI to the product obtained in step (4) and incubate. After Fe3O4@PDA@PEI captures Salmonella typhimurium, it forms Fe3O4@PDA@PEI / S. typhimurium / apt-HCR complex. Remove other unbound parts by magnetic separation.

[0014] 7): Add substrate buffer (3M NaCl, 60mM MgCl2, 50mM urea, 1mM HCl, adjust pH to 6.0) and phenol red to the complex obtained in step (4) for color development. Use a multi-functional microplate reader to measure the absorbance values ​​at A570 and A443, calculate A570 / A443, and complete the detection.

[0015] 8): Further, in step (2), the HCR reaction time is 60 min and the reaction temperature is 40℃;

[0016] 9): Further, in step (2), the addition ratio of H1 to H2 in the HCR is 1:1.5;

[0017] 10): Further, in step (2), the incubation time of Salmonella Typhimurium with the HCR system is 50 min;

[0018] 11): Further, in step (3), the concentration of T3 added is 40 nM, and the reaction time of T3-urease with HCR product is 50 min.

[0019] Beneficial technological achievements

[0020] This invention combines hybridization chain reaction (HCR) with Fe3O4@PDA@PEI to achieve rapid, highly specific, and highly sensitive detection of Salmonella Typhimurium in food. First, a functional hairpin probe is designed by utilizing the structural transformation capability of DNA and the competitive hybridization capability of the HCR as the energy source for signal amplification. The 5' end contains the aptamer sequence of Salmonella Typhimurium, and the 3' end contains a complementary blocking sequence, serving as the initiating strand for HCR. This hairpin structure allows HCR to be initiated only in the presence of Salmonella Typhimurium, continuously amplifying in cycles, exhibiting high specificity and significantly improving the sensitivity of the colorimetric nucleic acid aptamer sensor. Then, utilizing the magnetic and electrostatic interactions of Fe3O4@PDA@PEI, magnetic separation technology can capture and enrich Salmonella Typhimurium in the sample, effectively overcoming interference from complex matrices in food samples. Unlike traditional detection methods, pre-enrichment is unnecessary, significantly shortening the detection time and giving this method excellent timeliness. By introducing urease into the reaction using DNA as an enzyme reporter, urease catalyzes the hydrolysis of urea into ammonia and carbon dioxide. This enzymatic reaction increases the pH of the solution, causing the phenol red indicator to change color from yellow to pink, and the absorbance to change from 443 nm (yellow) to 570 nm (pink). The presence of target bacteria in the analyte can be determined based on this color change, enabling visual detection and reducing reliance on large instruments to some extent. In this technique, the detection limit for Salmonella Typhimurium reaches 2.34 CFU / mL. -1 The limit of visual detection is 10. 3 CFU mL -1 This provides a more comprehensive solution for on-site screening of foodborne pathogens.

[0021] This invention combines hybridization chain reaction with Fe3O4@PDA@PEI, which can not only detect Salmonella typhimurium, but also be applied to the detection of other foodborne pathogens such as Cronobacter sakazakii and Listeria monocytogenes, providing a new method for the detection of foodborne pathogens in food. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the present invention.

[0023] Figure 2 Characterization diagrams of Fe3O4@PDA@PEI of the present invention: a. Zeta potential diagram; b. TEM image of Fe3O4@PDA@PEI; c: hysteresis loop diagrams of Fe3O4 and Fe3O4@PDA@PEI.

[0024] Figure 3The following are images verifying the adsorption properties of Fe3O4@PDA@PEI of this invention: a. SEM image of Fe3O4@PDA@PEI adsorbing Salmonella typhimurium; b. Plate colony count image of Fe3O4@PDA@PEI after adsorbing Salmonella typhimurium.

[0025] Figure 4 The 15% PAGE electrophoresis diagram of this invention is as follows: 1: Trigger; 2: H1; 3: H2; 4: Trigger + H1; 5: H1 + H2; 6: Trigger + H1 + H2; 7: Salmonella Typhimurium + H1 + H2.

[0026] Figure 5 Feasibility analysis of the present invention: Figure a is the schematic diagram of Figure b; Figure b is the material feasibility verification diagram; Figure c is the schematic diagram of Figure d; Figure d is the schematic diagram of the feasibility of adding bacteria.

[0027] Figure 6 The following diagram shows the optimized conditions for this invention: a: bacterial culture time; b: HCR reaction time; c: HCR reaction temperature; d: ratio of H1 to H2 in HCR; e: concentration of T3; f: reaction time of T3-urease with HCR product.

[0028] Figure 7 For the performance analysis of this invention: a: Specificity analysis: 1. *Salmonella typhimurium*; 2. *Cronobacter sakazakii*; 3. *Staphylococcus aureus*; 4. *Staphylococcus epidermidis*; 5. *Citrobacter freundii*; 6. *Escherichia coli* O157:H7; 7. *Enterobacter cloacae*; b: Establishment of the standard curve. The error bar represents the standard deviation of three parallel measurements; c: Corresponding colorimetric assays for different concentrations of *Salmonella typhimurium*.

[0029] Figure 8 To compare the present invention with the national standard PCR method: a: Detection of Salmonella Typhimurium using the national standard PCR method at different enrichment times; b: Detection of Salmonella Typhimurium using the method of the present invention. Detailed Implementation

[0030] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0031] Example 1: A rapid ratio colorimetric detection method for Salmonella Typhimurium based on hybridization chain reaction. The experimental principle diagram of the detection method is shown below. Figure 1 (1) Preparation of Fe3O4@PDA@PEI

[0032] The preparation of Fe3O4@PDA@PEI mainly consists of three steps: synthesizing Fe3O4 magnetic nanoparticles, encapsulating a PDA layer with Fe3O4 to form Fe3O4@PDA nanoparticles, and bonding PEI to the surface of Fe3O4@PDA through C=O and CN bonds to form Fe3O4@PDA@PEI nanoparticles (2) Trigger, H1 and H2 form a hairpin structure

[0033] Trigger, Hairpin 1 (H1), and Hairpin 2 (H2) were heated at 95°C for 5 minutes and then gradually cooled to room temperature to form a hairpin structure. The sequence was stored at 4°C for later use.

[0034] (3) Hybridization chain reaction and ligation of enzyme reporter (T3-urease)

[0035] Take 1 mL of logarithmic-phase Salmonella Typhimurium bacterial suspension, centrifuge at 5000 rpm for 5 min at 4℃, remove the supernatant, resuspend the bacteria in 1 mL of PBS buffer, and add 100 μL of the bacterial suspension to the reaction solution containing 4 μM Trigger, 4 μM H1, and 6 μM H2, which has formed a hairpin structure in step (2). Incubate at 37℃ for 30 min to form a long chain of amplified product. Add T3-urease to the HCR amplified product and incubate at 37℃ in a shaker for 50 min to allow it to be linked to the product chain through base complementarity.

[0036] (4) Fe3O4@PDA@PEI enrichment of Salmonella typhimurium

[0037] Fe3O4@PDA@PEI was added to the mixture obtained in step (3), and the mixture was incubated in a shaker at 37°C for 1 hour to enrich Salmonella typhimurium and finally form the Fe3O4@PDA / PEI / S.typhimurium / apt-HCR structure.

[0038] (5) Colorimetric detection reaction

[0039] After magnetically separating the complex, phenol red and substrate buffer were added for color development. The absorbance values ​​were measured at wavelengths of 443 nm and 570 nm, and A570 / A443 was calculated to complete the display.

[0040] Example 2: Preparation of Fe3O4

[0041] 2.0 g of FeCl3·6H2O was dissolved in 20 mL of ethylene glycol and 20 mL of diethylene glycol to form a pale yellow transparent solution. Then, 2.0 g of polyethylene glycol (MW = 800 Da) and 3.0 g of sodium acetate were added. The mixture was stirred vigorously for 30 min to ensure thorough mixing, and then transferred to a tetrafluoroethylene high-pressure reactor. The reaction was carried out in an electrically heated constant-temperature drying oven at 200 °C for 6 h. The black precipitate (Fe3O4 NPs) was collected by magnetic separation, washed three times with ethanol, and then resuspended in 50 mL of ethanol. It was stored at 4 °C for later use.

[0042] Example 3: Preparation of Fe3O4 and Fe3O4@PDA@PEI

[0043] 10 mg of Fe3O4 NPs (2 mL, 5 mg mL) -1 Fe3O4@PDA was dispersed in 10 mL of Tris-HCl buffer (10 mM, pH 8.5). 10 mL of a solution containing 7 mg DA was added to the suspension, and the mixture was stirred continuously at room temperature for 7 h. The resulting Fe3O4@PDA was collected using a magnet, washed alternately with ethanol and ultrapure water, and then redispersed in 30 mL of water. 30 mL of 25% PEI solution was added to Fe3O4@PDA and stirred at room temperature for 10 h. After the reaction was complete, Fe3O4@PDA@PEI was collected using a magnet and washed with ethanol and ultrapure water. Finally, the obtained Fe3O4@PDA@PEI was distributed in water (1 mg / mL). -1 Store at 4℃ for later use.

[0044] Characterization of Fe3O4@PDA@PEI

[0045] The synthesis of Fe3O4@PDA@PEI was demonstrated by using Zeta potential. The Zeta potentials of Fe3O4, Fe3O4@PDA, and Fe3O4@PDA@PEI were compared. Figure 2 As can be seen, the potential of unmodified Fe3O4 is -26.23 eV, while the Zeta potential of the modified Fe3O4@PDA@PEI turns positive at 22.96 eV. This change is due to the presence of negatively charged carboxyl groups and other functional groups on the Fe3O4 surface. PEI is a cationic polymer with the cationic properties of nitrogen atoms, exhibiting a high positive charge density. The synthesized Fe3O4@PDA@PEI has a PEI coating, thus displaying a positive charge. These results demonstrate the successful preparation of Fe3O4@PDA@PEI. The synthesized Fe3O4@PDA@PEI was characterized using TEM. Figure 2TEM images of b show that Fe3O4@PDA@PEI has a typical spherical structure with a diameter of approximately 200 nm. Magnetic analysis reveals that the saturation magnetic susceptibility of Fe3O4@PDA@PEI is 6.58 emu / g, significantly lower than that of Fe3O4 (34.79 emu / g). Figure 2 (As shown in c). However, it can still meet the experimental requirements for rapid separation under the action of an external magnetic field (as shown in the illustration).

[0046] Example 4: Scanning electron microscopy imaging of bacteria

[0047] 10 8 CFU mL -1 Salmonella typhimurium was mixed with Fe3O4@PDA@PEI magnetic nanocomposite material and incubated at 37℃ for 1 h. Then, Salmonella typhimurium was magnetically separated and enriched. The unbound parts were washed three times with sterile water to remove the unbound parts. Then, it was fixed overnight with glutaraldehyde (2.5%) to prepare Fe3O4@PDA@PEI and bacterial adsorption samples.

[0048] 10 8 CFU mL -1 Salmonella typhimurium was mixed with Fe3O4@PDA@PEI magnetic nanocomposite material, and after incubation at 37°C for 1 hour, it was incubated at 1W cm⁻¹. 2 After irradiation with 808 nm near-infrared light for 10 min, *Salmonella typhimurium* was collected by magnetic separation. The samples were washed three times with sterile water and then fixed overnight with glutaraldehyde (2.5%) to prepare Fe3O4@PDA@PEI photothermal sterilization samples. After washing with sterile water, each group of samples was dehydrated sequentially with gradient concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and anhydrous ethanol) for 10 min each time. 10 μL of the treated bacterial suspension was dropped onto a coverslip, allowed to dry, and then imaged using a field emission scanning electron microscope (SEM).

[0049] This invention verifies the enrichment effect of Fe3O4@PDA@PEI on bacteria using SEM imaging, and obtains... Figure 3 The results showed that, compared with the PBS group, the Fe3O4@PDA@PEI group clearly showed Fe3O4@PDA@PEI particles adsorbed around Salmonella typhimurium, indicating that Fe3O4@PDA@PEI can successfully bind to and enrich bacteria through electrostatic interactions. Figure 3 b shows the plate colony count results of Fe3O4@PDA@PEI after magnetic separation following incubation at 37°C for 1 h with Salmonella Typhimurium. The colony count indicates that approximately 84.5% of the bacteria were captured and enriched by Fe3O4@PDA@PEI, demonstrating its effectiveness in bacterial capture and enrichment.

[0050] Example 5: DNA and urease coupling

[0051] 2 mg of MBS was dissolved in 1 mL of dimethyl sulfoxide (DMSO) to obtain a 6.4 mM MBS solution. 1.5 mg of urease (10 KU) powder was dissolved in 1 mL of 1×PBS buffer (pH 7.2) to obtain a urease solution. 3.2 μL of 6.4 mM MBS solution was mixed with 1 mM T3 solution, and the final reaction volume was adjusted to 100 μL with 1×PBS buffer. After reacting at room temperature for 2 h, the mixture was centrifuged using a 3000 Dalton ultrafiltration concentrator column (NANOSEP OMEGA, Pall Incorporation) to remove excess MBS, yielding MBS-activated DNA. 1 mL of urease solution was added to the activated T3, and after reacting at room temperature for 1 h, the mixture was filtered using a 300,000 Dalton ultrafiltration concentrator column to obtain the T3-urease conjugate.

[0052] Example 6 15% Polyacrylamide Gel Electrophoresis

[0053] Gel electrophoresis is used to verify the reactions between DNA molecules during the detection process. First, the glass plates are wiped unidirectionally with ultrapure water and anhydrous ethanol, dried, and assembled before gel preparation (15% polyacrylamide gel). The specific procedure is as follows: 3.45 mL of ultrapure water, 1 mL of 10×TBE, 5 mL of 30% polyacrylamide, 50 μL of 10% ammonium persulfate, and 5 μL of TEMED are added to a beaker and stirred until homogeneous. The mixture is then poured into a pre-treated gel plate (to prevent air bubbles). The comb teeth are slowly inserted to create an injection port. After standing, the comb teeth are slowly removed, completing the gel preparation. The glass gel chamber in the gel plate is removed and fixed in the electrophoresis tank, and 1×TBE is added to immerse the gel. Next, the sample is loaded. The loading buffer and sample solution are mixed at a 1:3 ratio. Air bubbles are removed from the injection port, and the pipette tip is held perpendicular to the injection port. After loading the sample, the electrophoresis apparatus is turned on and run for 1.5 hours. After completion, the gel was removed and stained with ethidium bromide (EtBr), and finally scanned with an ultraviolet transilluminator for imaging.

[0054] Results of 15% polyacrylamide gel electrophoresis

[0055] The HCR reaction process was verified by 15% polyacrylamide gel electrophoresis (15% PAGE). Figure 4As shown, lanes 1, 2, and 3 show single bright bands, corresponding to the hairpin aptamer Trigger, Hairpin1 (H1), and Hairpin2 (H2), respectively. Lane 4, Trigger + H1, shows two single bright bands, indicating that Trigger cannot activate H1 alone in the absence of a target. Lane 5, H1 + H2, shows two single bright bands, indicating that H1 and H2 cannot bind to initiate the HCR reaction without a target and trigger. Lane 6 represents Trigger + H1 + H2 in the absence of a target, and lane 7 represents Salmonella Typhimurium + Trigger + H1 + H2. The comparison between lanes 6 and 7 clearly shows that the HCR reaction can only be successfully triggered in the presence of Salmonella Typhimurium; the reaction cannot proceed without the target bacteria. This demonstrates that the sequence designed in this invention is successful and feasible.

[0056] Table 1 shows the oligonucleotide chain sequences used.

[0057]

[0058] Example 7: Verification of Material Feasibility and Bacterial Addition Feasibility

[0059] from Figure 5 As shown in b, when Fe3O4@PDA@PEI and phenol red were added to the substrate buffer, a single peak was observed at a wavelength of 443 nm, and the solution appeared yellow to the naked eye. When T3-urease and phenol red were added to the substrate buffer, a peak appeared at a wavelength of 570 nm under the action of T3-urease and phenol red, and the solution appeared pink. This is because the T3 sequence, after successfully linking with urease, can catalyze the hydrolysis of urea in the substrate buffer into ammonia and carbon dioxide, thus causing an increase in pH. Phenol red, as an acid-base indicator, appears yellow under acidic conditions (pH below 6.8), orange under neutral conditions (pH between 6.8 and 8.0), and pink under alkaline conditions (pH above 8.0). Changes in pH cause changes in the observed color. This further proves that the color change is caused by the catalytic activity of urease. Fe3O4@PDA@PEI itself has no catalytic activity and cannot catalyze the hydrolysis of urea; its presence alone cannot cause a color change. Figure 5 (a is the corresponding experimental principle diagram)

[0060] In the absence of Salmonella typhimurium ( Figure 5d) A single peak was observed at a wavelength of 443 nm, and the solution color was yellow. However, in the presence of Salmonella typhimurium, a decrease in the peak at 443 nm was observed, while a new peak appeared at 570 nm, and the solution color changed from yellow to orange-red. This indicates that the present invention can be successfully used for the detection of Salmonella typhimurium. In the presence of the target bacteria, the reaction can be successfully triggered, and the solution color undergoes a significant change visible to the naked eye. Figure 5 d represents the corresponding experimental principle diagram.

[0061] Example 8: Optimization of Experimental Conditions

[0062] (1) Optimization of bacterial incubation time: bacterial incubation time was set to 20, 30, 40, 50 and 60 min respectively. The absorbance value was measured at 443 nm and 570 nm respectively and A570 / A443 was calculated. Three parallel groups were set for each incubation time.

[0063] (2) HCR reaction time optimization: HCR reaction times of 20, 30, 40, 50, 60 and 70 min were set, and absorbance values ​​were measured at 443 nm and 570 nm respectively, and A570 / A443 was calculated. Three parallel groups were set for each reaction time.

[0064] (3) HCR reaction temperature optimization: The HCR reaction temperatures were set to 20, 25, 30, 35 and 40℃, and the absorbance values ​​were measured at 443nm and 570nm respectively and A570 / A443 was calculated. Three parallel groups were set for each temperature.

[0065] (4) Optimization of the ratio of H1 to H2 in HCR: The ratio of H1 to H2 in HCR was set to 1:0.5, 1:1, 1:1.5, 1:2, and 1:2.5, respectively. The absorbance values ​​were measured at 443 nm and 570 nm and A570 / A443 were calculated. Three parallel groups were set for each ratio.

[0066] (5) Optimization of T3 concentration: The concentrations of T3 were set to 10, 20, 30, 40 and 50 nM, and the absorbance values ​​were measured at 443 nm and 570 nm respectively and A570 / A443 was calculated. Three parallel groups were set for each concentration.

[0067] (6) Optimization of reaction time between T3-urease and HCR product: The reaction time between adding T3-urease and HCR product was set to 20, 30, 40, 50 and 60 min respectively. The absorbance values ​​were measured at 443 nm and 570 nm respectively and A570 / A443 was calculated. Three parallel groups were set for each time.

[0068] Results of Optimization of Experimental Conditions

[0069] The reaction time between Salmonella Typhimurium and Trigger was optimized, with an incubation time of 20-60 min selected as the experimental range. Figure 6 As shown in Figure a, the A570 / A443 ratio reaches its maximum when the incubation time is extended to 40 min; the selected reaction time of 20 min–70 min and temperature range of 25 °C–45 °C are the reaction conditions for HCR. Figure 6 As shown in b and 6c, the HCR reaction efficiency was highest when the reaction time was 60 min and the reaction temperature was 40℃, and the A570 / A443 ratio reached its maximum value; the H1:H2 ratio range of 1:0.5-1:2.5 was selected as the experimental conditions. Figure 6 As shown in d, when H1:H2 increases to 1:1.5, the A570 / A443 ratio reaches its maximum value; Figure 6 For the optimization of T3 concentration, the experimental concentration range was selected as 10 nM-50 nM. When the T3 concentration was gradually increased to 40 nM, the A570 / A443 ratio increased to 0.9. However, as the T3 concentration further increased from 40 nM to 50 nM, the A570 / A443 ratio increased slightly but was almost negligible. Considering economic cost and efficiency, 40 nM was ultimately chosen as the optimal experimental condition; the reaction time between T3-urease and the HCR product was optimized, and the experimental range was determined to be 20-60 min. Figure 6 As shown in f, when the reaction time is extended from 20 min to 50 min, the A570 / A443 value increases, and then tends to stabilize at 60 min. Therefore, a reaction time of 50 min is selected as the optimal reaction condition.

[0070] In summary, the following experimental conditions were selected: incubation time of Salmonella Typhimurium and Trigger of 40 min, reaction time of HCR of 60 min, reaction temperature of HCR of 40℃, H1:H2 ratio of 1:1.5 in HCR, concentration of T3 sequence of 40 nM, and reaction time of T3-urease with HCR of 50 min.

[0071] Example 9 Specificity Experiment

[0072] Seven typical foodborne pathogens (including Salmonella typhimurium, Cronobacter sakazakii, Staphylococcus aureus, Staphylococcus epidermidis, Citrobacter freundii, Staphylococcus saprophyticus, and Enterobacter cloacae) were selected to verify the detection specificity of this method. The concentration of all experimental strains was set at 10. 8 CFU mL -1 Each group was divided into three parallel groups, and the error bar represents the standard deviation of three parallel experiments. The selective recognition ability of the present invention for target bacterial species was evaluated by comparing the colorimetric differences produced by different strains reacting with the sensing system.

[0073] like Figure 7 As shown in Figure a, the presence of *Salmonella typhimurium* leads to a significant increase in the A570 / A443 ratio, resulting in a visually pink solution. In contrast, the change in the A570 / A443 ratio is negligible in the presence of several other common foodborne pathogens, and the solution remains yellow to the naked eye. These results demonstrate that the present invention exhibits good selectivity against *Salmonella typhimurium*.

[0074] Example 10 Sensitivity Experiment

[0075] The sensitivity of this method was analyzed by varying the CFU value of Salmonella typhimurium, with the concentration range set at 10. 1 -10 5 CFU mL -1 Five concentrations were measured colorimetrically to determine their detection limits and linear equations. Three parallel groups were set up for each group.

[0076] The sensitivity of the invention was studied under optimal conditions. For example... Figure 7 As shown in b, the A570 / A443 ratio increases with increasing Salmonella Typhimurium concentration (from 10...). 1 CFU mL -1 Increase to 10 5 CFU mL -1 The ratio increases proportionally. A standard curve was plotted between the A570 / A443 ratio and the logarithm of the Salmonella typhimurium concentration, yielding the linear regression equation: y = 0.1538x - 0.0716 (R²). 2 =0.9927), where y represents A570 / A443, and x represents the logarithm of the Salmonella Typhimurium concentration. The detection limit for this system was calculated to be 2.34 CFU / mL using the formula 3SD / m (where SD is the standard deviation of the A570 / A443 ratio in the absence of Salmonella Typhimurium, i.e., the standard deviation of the blank group; m is the slope of the linear regression equation). -1 This indicates that the present invention has high sensitivity. Figure 7 c shows the colorimetric results for different concentrations of Salmonella typhimurium, revealing a detection limit of 10 for visual detection. 3 CFU mL -1 .

[0077] Example 11 Detection of Salmonella Typhimurium in actual samples

[0078] To verify the practicality of the Salmonella Typhimurium detection system developed in this invention, commercially available milk and eggs were selected as detection matrices for validation. Salmonella Typhimurium was serially diluted and then inoculated into milk and egg samples. Milk sample preparation: 1 mL of milk was added to 9 mL of PBS and diluted 10-fold, and different concentrations of Salmonella Typhimurium were added until the final concentration was 10. 1 10 3 and 10 5 CFU mL -1 Milk free of Salmonella typhimurium served as a blank control, with three replicates per group. Egg sample processing: 1 mL of egg white was mixed thoroughly with 9 mL of PBS and filtered through a 0.22 μm membrane filter to remove bacteria. Different concentrations of Salmonella typhimurium were added to a final concentration of 10. 1 10 3 and 10 5 CFU mL -1 Samples free of Salmonella typhimurium were used as blank controls, with three replicates in each group. Bacterial detection was performed on the samples, and Table 1 shows that the method based on the invention exhibits high reliability in detecting Salmonella typhimurium, with recoveries ranging from 87.3% to 108% and relative standard deviations (RSDs) below 13%.

[0079] Table 2 shows the recovery rates of Salmonella typhimurium in milk and eggs using this method.

[0080]

[0081]

[0082] Example 12 Comparison with National Standard Method

[0083] 25g of chicken sample was incubated in 225mL of selenite cystine enrichment broth (SC) at 37±1℃. A blank group (no bacteria added) and experimental group 1 (10g of chicken sample) were set up. 1 CFU mL -1 Salmonella typhimurium) and experimental group 2 (10 3 CFU mL -1 Selective enrichment of *Salmonella typhimurium* was performed, and samples were collected hourly for each group. The samples were then tested using PCR and compared with the method established in this study.

[0084] Forward primers (SEQ ID NO. 5) and reverse primers (SEQ ID NO. 6) were designed based on the specific gene of *Salmonella typhimurium*. DNA from *Salmonella typhimurium* was extracted using a kit, and 25 μL of Premix Taq buffer, 1 μL of primer (F), 1 μL of primer (R), 4 μL of template, and 19 μL of deionized water were added, for a total volume of 50 μL. The reaction was then incubated at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. Finally, a 331 bp PCR product was obtained by incubation at 72 °C for 7 min. The product was then detected by 1% agarose gel electrophoresis. Primers SalF and SalR used are shown in SEQ ID NO. 5 and SEQ ID NO. 6.

[0085] The results are as follows Figure 8 As shown, Figure 8 a shows the results of detecting Salmonella Typhimurium using the national standard PCR method at different enrichment times. The electrophoresis image shows the effect of adding 10... (The sentence is incomplete in the original text, so I cannot provide a direct translation.) 3 CFU mL -1 When detecting Salmonella typhimurium, a 4-hour enrichment period is required before it can be detected. Add 10... 1 CFU mL -1 When detecting Salmonella typhimurium, it requires 5 hours of enrichment before it can be detected, while this invention can detect it after 3-4 hours of enrichment. Figure 8 b). This demonstrates that the detection method established in this study has excellent detection performance.

[0086] In summary, the rapid ratio colorimetric detection method for Salmonella Typhimurium based on isothermal amplification described in this invention can detect Salmonella Typhimurium with high sensitivity and high specificity, and its accuracy is within 10... 1 Up to 10 5 CFU mL -1 It exhibits a wide linear response within a certain range, with a detection limit as low as 2.34 CFU / mL. -1 The detection limit for visual observation is 10. 3 CFU mL -1 It exhibits good resistance to interference from other bacteria or common contaminants, enabling rapid detection in clinical and food safety fields. It provides a novel strategy for the detection of foodborne pathogens and has significant practical value.

[0087] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A rapid ratio colorimetric detection method of Salmonella typhimurium based on hybridization chain reaction, characterized in that, The method comprises the following steps: (a) providing a Salmonella typhi specific recognition and signal amplification system, which comprises: (i) a hairpin trigger chain Trigger, the sequence of which is shown in SEQ ID NO. 1, and the 5' end of which comprises a Salmonella typhi aptamer sequence; (ii) a hairpin probe H1, the sequence of which is shown in SEQ ID NO. 2; (iii) a hairpin probe H2, the sequence of which is shown in SEQ ID NO. 3, and the H2 comprises an enzyme reporter binding site; (iv) a T3-urease complex, wherein the T3 sequence is shown in SEQ ID NO. 4, and the urease is covalently linked thereto; (b) mixing the Trigger, H1, H2 with the sample to be tested, and when Salmonella typhi is present, the Trigger recognizes Salmonella typhi and opens its hairpin structure, triggering a hybridization chain reaction (HCR) of H1 and H2 to form an HCR long chain product comprising a region complementary to the T3 sequence; (c) adding the T3-urease complex to the product of step (b) to allow the T3-urease complex to bind to the HCR long chain product through the complementary region; (d) adding Fe3O4@PDA@PEI magnetic nanoparticles and incubating to allow the Fe3O4@PDA@PEI to capture and enrich Salmonella typhi through electrostatic interaction, forming a Fe3O4@PDA@PEI / Salmonella typhi / apt-HCR complex; (e) performing magnetic separation on the mixture of step (d) to remove unbound components and retain the complex; (f) adding a substrate buffer containing urea and phenol red to the complex obtained in step (e), and the T3-urease complex catalyzes the hydrolysis of urea to cause the solution pH to rise, causing the color of phenol red to change from yellow to pink and the absorbance ratio to change (A570 / A443 increases), thereby achieving the ratio colorimetric detection of Salmonella typhi by detecting the change in absorbance ratio or observing the color change with the naked eye.

2. The method of claim 1, wherein, The HCR reaction temperature in step (b) is 40°C, and the reaction time is 60 min.

3. The method of claim 1, wherein, The molar addition ratio of the hairpin probes H1 and H2 in step (b) is 1:1.

5.

4. The method of claim 1, wherein, The reaction time of the T3-urease complex with the HCR product in step (c) is 50 min.

5. The method of claim 1, wherein, The preparation method of the Fe3O4@PDA@PEI magnetic nanoparticles in step (d) comprises: (1) dispersing Fe3O4 nanoparticles in Tris-HCl buffer; (2) adding dopamine at a final concentration of 0.7 mg / mL, stirring at room temperature for 7 h, and magnetically separating to obtain Fe3O4@PDA; (3) dispersing Fe3O4@PDA in a 25% polyethyleneimine (PEI) solution, stirring at room temperature for 10 h, and magnetically separating to obtain Fe3O4@PDA@PEI.

6. The method of claim 1, wherein, The substrate buffer in step (f) comprises 3M NaCl, 60mM MgCl2, 50mM urea, 1mM HCl, and the pH is adjusted to 6.

0.

7. The method according to any one of claims 1 to 6, characterized in that: The limit of detection of the method was 2.34 CFU mL -1 .

8. Use of the method according to any one of claims 1 to 7 for the manufacture of a kit for the detection of Salmonella typhimurium.

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