A rapid quantitative detection method of salmonella by lfs-sers

Abstract

This invention discloses a rapid quantitative detection method for Salmonella using LFS-SERS, belonging to the field of food safety technology. The method includes the following steps: S1, lysing the sample to obtain a crude DNA solution; S2, using the crude DNA solution as a template, performing Salmonella-specific LAMP amplification to obtain a biotin-labeled product; S3, mixing dCas9 protein, sgRNA, SERS-labeled gold nanoparticle-probe conjugate, the biotin-labeled product, and a loading buffer solution, incubating at room temperature for 1-5 minutes, performing a colorimetric reaction using a lateral flow test strip, and using a portable Raman spectrometer connected to a smart terminal to detect the T line in the LFS using SERS, thus achieving quantitative detection of Salmonella in the sample. This method is highly specific, sensitive, and stable; the entire detection process can be completed within 40 minutes, and it is low-cost and easy to operate.

Description

Technical Field

[0001] This invention relates to the field of food safety technology, specifically to a rapid quantitative detection method for Salmonella using LFS-SERS. Background Technology

[0002] Rapid detection of foodborne pathogens is crucial for food safety and public health. Traditional detection methods are typically time-consuming and complex, making them unsuitable for on-site testing. The combination of CRISPR / dCas9 technology and lateral flow biosensors offers a new approach for the visual and rapid detection of foodborne pathogens such as Salmonella. For example, patent CN117025800A, "Preparation and Application of a Nucleic Acid Amplification Method Test Strip for Salmonella Detection Based on dCas9," proposes a CRISPR / dCas9-based lateral flow strip (LFS) method that achieves visual detection of Salmonella with a visual detection limit of 41 CFU / mL, demonstrating good performance in spiked samples. However, this method relies on ImageJ software for grayscale analysis of the T-line of the test strip for quantitative detection. This process is cumbersome and susceptible to interference from ambient light and other factors, leading to significant data errors and failing to meet the needs for rapid and stable on-site quantification. Therefore, there is an urgent need to develop a new method that combines visual rapid detection with accurate quantification.

[0003] In the field of biosensors, surface-enhanced Raman scattering (SERS) technology is widely used for the quantitative analysis of pathogenic bacteria due to its ultra-high sensitivity, high spectral resolution, and good photostability. Currently, SERS-based pathogen detection is mainly divided into two categories: (1) Label-free SERS detection: directly analyzing the inherent SERS signals of bacterial cell wall components. This method is simple to operate, but the inherent bacterial signals are weak and easily affected by environmental interference, resulting in low sensitivity and accuracy; (2) Labeled SERS detection: using SERS tags (such as noble metal nanomaterials combined with signal molecules) to enhance the characteristic peak signals, converting the pathogen concentration into SERS peak intensity. This method has strong anti-interference ability and higher sensitivity and accuracy.

[0004] Existing technologies have attempted to combine SERS with LFS for quantitative detection. For example, patent CN120539398A, "A Highly Sensitive Quantitative Detection Kit for Salmonella Enteritidis," designs a dual-signal probe integrating colorimetric and SERS signals using ZIF-8, combined with RPA amplification technology, to achieve quantitative detection while improving sensitivity. However, the core-shell structure SERS tag used in this solution has a complex and costly preparation process and faces reproducibility challenges, hindering large-scale application and rapid on-site detection.

[0005] Therefore, this invention aims to integrate the intuitive visualization advantages of LFS with the precise quantitative capabilities of SERS, combining CRISPR / dCas9-based LFS visualization detection with SERS quantitative analysis to develop a Salmonella detection method that is easy to operate, low in cost, suitable for field use, and can simultaneously achieve rapid visual screening and accurate, stable quantitative detection, in order to meet the wide application needs in fields such as food safety monitoring and clinical diagnosis. Summary of the Invention

[0006] Therefore, this invention provides a rapid quantitative detection method for Salmonella using LFS-SERS to address the shortcomings of existing technologies.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention proposes a rapid quantitative detection method for Salmonella using LFS-SERS, comprising the following steps: S1. Mix the sample to be tested with the lysis buffer evenly and perform thermal lysis to obtain crude DNA solution. S2. Using the crude DNA solution obtained in S1 as a template, Salmonella-specific primers were used to perform loop-mediated isothermal (LAMP) amplification to obtain biotin-labeled amplification products. S3. Mix dCas9 protein, sgRNA, SERS-labeled gold nanoparticles (GNPs)-probe conjugate, and the biotin-labeled amplification product obtained in S2 in a loading buffer solution and incubate at room temperature for 1-5 min to obtain a complex. Add the obtained complex to a lateral flow test strip and allow it to react for 1-5 min. After the reaction, use a portable Raman spectrometer connected to a smart terminal to measure the T line of the lateral flow test strip to obtain the SERS spectrum. Quantitative detection of Salmonella in the sample can be achieved based on the SERS spectrum.

[0008] Furthermore, the parameters for the thermal pyrolysis treatment in S1 are: temperature 80℃, treatment time 2min; the pyrolysis buffer contains magnesium chloride (MgCl2), Tris-HCl, triethanolamine dodecyl sulfate, potassium chloride (KCl) and methyl salicylate.

[0009] Further, the specific primer set described in S2 includes a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a loop primer LF, and a biotin-labeled loop primer LB. The nucleotide sequences of the forward inner primer FIP, the reverse inner primer BIP, the forward outer primer F3, the reverse outer primer B3, the loop primer LF, and the biotin-labeled loop primer LB are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. The 5' end of the biotin-labeled loop primer LB is labeled with biotin.

[0010] Furthermore, the amplification conditions for loop-mediated isothermal amplification described in S2 are: temperature 61.3℃ and amplification time 30 min.

[0011] Further, the concentration of each primer in the Salmonella-specific primer set is 20-500 nM; the concentration of the dCas9 protein is 20-700 nM; the concentration of the sgRNA is 20-800 nM, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.7; the volume of the SERS-labeled gold nanoparticles (GNPs)-probe conjugate is 2-20 μL; and the volume of the biotin-labeled amplification product is 1-10 μL.

[0012] Furthermore, the preparation method of the dCas9 protein described in S3 is as follows: The dCas9 gene and vector were transformed into E. coli, and positive strains containing the dCas9 gene-vector were obtained through resistance selection. The positive strains were inoculated into LB liquid medium containing kanamycin and cultured with shaking. Isopropyl-β-D-thiogalactoside (IPTG) was added for low-temperature induction of expression. The bacterial cells were collected by centrifugation, resuspended with lysis buffer, and sonicated. The supernatant was collected by centrifugation and purified by immobilized metal ion affinity chromatography. The purified dCas9 was replaced with storage buffer using a desalting column to obtain the dCas9 protein, which was stored at -80°C.

[0013] Furthermore, the storage buffer contains 20 mM Tris-HCl, 150 mM sodium chloride (NaCl) and 10 wt.% glycerol, and the pH of the storage buffer is 8.0. The lysis buffer contains 20 mM Tris-HCl, 500 mM sodium chloride (NaCl) and 10 mM imidazole, and the pH of the lysis buffer is 7.0-8.5.

[0014] Furthermore, the preparation method of the SERS-labeled gold nanoparticles (GNPs)-probe conjugate described in S3 is as follows: A conical flask containing 0.5 wt.% chloroauric acid tetrahydrate solution and double-distilled water was placed on a magnetic stirrer and heated to boiling. 1 wt.% trisodium citrate solution was added while stirring continuously until the solution changed from colorless to red. The reaction was continued for 10 minutes while maintaining boiling. After the reaction was completed, the mixture was cooled to room temperature to obtain uniformly dispersed gold nanoparticles (GNPs). The probe, reducing agent, and buffer were mixed in proportion and incubated in the dark for 30 min to obtain the activated probe. The uniformly dispersed gold nanoparticles (GNPs) obtained above were mixed with SERS signal molecules and incubated for 30 min, and then concentrated by centrifugation to obtain pretreated gold nanoparticles (GNPs). The activated probe was mixed with the pretreated gold nanoparticles (GNPs) and incubated at room temperature for 2 h, and then blocked with 1 wt.% bovine serum albumin for 1 h. After the reaction was completed, the supernatant was discarded by centrifugation and the precipitate was resuspended to obtain the SERS-labeled gold nanoparticles (GNPs)-probe conjugate.

[0015] Further, the uniformly dispersed gold nanoparticles (GNPs) need to be analyzed by dynamic laser scattering (DLS), and the supernatant is concentrated 10-fold after centrifugation at 8500 r / min for 15 min; the probe is a thiolized probe, and the nucleotide sequence of the thiolized probe is shown in SEQ ID NO.8; the reducing agent is tris(2-carboxyethyl)phosphine (TCEP); the buffer is acetate buffer, and the pH of the acetate buffer is 5.2; the mixing ratio of the probe, reducing agent and buffer is 8:4:1; the volume ratio of the uniformly dispersed gold nanoparticles to the SERS signal molecule is 40:1, and the SERS signal molecule is 4-aminothiophenol (4-ATP); the volume ratio of the 0.5 wt.% chloroauric acid tetrahydrate solution to the 1 wt.% trisodium citrate solution is 16:15; the volume of double-distilled water is 50 mL; the volume of 1 wt.% bovine serum albumin is 10 μL; the buffer used for resuspending the precipitate is Tris-HCl buffer, and the Tris-HCl buffer contains 1 mM The buffer solution consisted of Tris-HCl, 5 wt.% bovine serum albumin (BSA), 0.25% (v / v) Tween-20, and 10 wt.% sucrose, with the pH of the Tris-HCl buffer being 7.0–8.5.

[0016] Furthermore, the settings parameters of the portable Raman spectrometer described in S3 are as follows: single exposure time is 1000ms, laser power is 100mW; the sample loading buffer solution contains phosphate buffer (PBS), 1wt.% polyethylene glycol 20000, 50mM magnesium chloride (MgCl2), 2wt.% bovine serum albumin (BSA) and 10wt.% sucrose, the phosphate buffer (PBS) contains 6.85mM sodium chloride (NaCl), 0.135mM potassium chloride (KCl), 0.5mM disodium hydrogen phosphate (Na2HPO4) and 0.1mM potassium dihydrogen phosphate (KH2PO4), and the pH value of the sample loading buffer is 7.0-8.5.

[0017] Furthermore, the smart terminal mentioned in S3 is a mobile device (such as a smartphone or tablet computer) with dedicated analysis software installed, which is connected to a portable Raman spectrometer wirelessly or via wired connection.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention constructs a rapid quantitative detection method for Salmonella LFS-SERS by combining a CRISPR / dCas9-mediated signal amplification mechanism with LAMP amplification and a 4-ATP-labeled GNPs-probe conjugate. This method integrates the high efficiency of LAMP amplification, the accurate identification of the CRISPR / dCas9 system, the convenience of LFS, and the quantitative analysis capability of SERS. Initial screening is performed by visually interpreting the T-line on the test strip, and SERS signals are acquired using a portable Raman spectrometer connected to a smart terminal. The SERS signal is determined by measuring the 4-ATP content at 1079 cm⁻¹ in the SERS spectrum. -1 The method utilizes the characteristic peak intensity at a specific location to achieve quantitative detection of Salmonella in the sample. While maintaining excellent specificity, the detection sensitivity is improved by one order of magnitude (10-fold) compared to naked-eye interpretation using LFS alone. The entire detection process is simple to operate, requiring only a common water bath and a portable Raman spectrometer, eliminating the need for specialized operators and complex equipment, significantly reducing application costs and technical barriers. This method combines high specificity, sensitivity, and stability, providing an efficient and economical solution for food safety monitoring and clinical bacterial infection diagnosis. Attached Figure Description

[0019] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0020] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0021] Figure 1 The working principle of the rapid quantitative detection method of Salmonella by LFS-SERS in this invention is shown in the figure: (A) Preparation of SERS-labeled gold nanoparticles (GNPs)-probe conjugates, (B) dCas9 protein-mediated specific nanoassembly, (C) LFS-SERS signal reading and analysis process based on smart terminal. Figure 2 The LFS-SERS rapid quantitative detection method for Salmonella of this invention yielded detection results for Salmonella under different combinations of conditions: Figure A shows the LFS colorimetric image, and Figure B shows the SERS characteristic peak histogram (1079 cm⁻¹). -1 (characteristic peaks); Figure 3 The sensitivity detection results of the LFS-SERS rapid quantitative detection method for Salmonella in this invention on three Salmonella samples are shown in Figure A: LFS colorimetric image, and SERS characteristic peak bar chart (taken at 1079 cm⁻¹). -1 (characteristic peaks); Figure 4 The LFS-SERS rapid quantitative detection method for Salmonella of this invention shows the specificity of different pathogenic bacteria and their combinations: Figure A is the LFS colorimetric image, and Figure B is the SERS characteristic peak bar chart (taken as 1079 cm⁻¹). -1 (characteristic peaks); Figure 5 The present invention provides a rapid quantitative detection method for Salmonella using LFS-SERS, which utilizes LFS colorimetric images to detect Salmonella in milk. Figure 6 The bar chart showing the characteristic peak values ​​of SERS in milk for the LFS-SERS rapid quantitative detection method of Salmonella in this invention (taken as 1079 cm⁻¹) is shown. -1 (characteristic peaks); Figure 7 The LFS colorimetric image of the rapid quantitative detection method for Salmonella by LFS-SERS in eggs is shown in this invention. Figure 8 The bar chart showing the characteristic peak values ​​of SERS in eggs for the LFS-SERS rapid quantitative detection method of Salmonella in this invention (taken as 1079 cm⁻¹) is shown.-1 (Characteristic peak). Detailed Implementation

[0022] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] This invention proposes a rapid quantitative detection method for Salmonella using LFS-SERS, comprising the following steps (see details of the working principle). Figure 1 ): S1. Mix the sample to be tested with the lysis buffer evenly and perform thermal lysis to obtain crude DNA solution. S2. Using the crude DNA solution obtained in S1 as a template, Salmonella-specific primers were used to perform loop-mediated isothermal (LAMP) amplification to obtain biotin-labeled amplification products. S3. Mix dCas9 protein, sgRNA, SERS-labeled gold nanoparticles (GNPs)-probe conjugate, and the biotin-labeled amplification product obtained in S2 in a loading buffer solution and incubate at room temperature for 1-5 min to obtain a complex. Add the obtained complex to a lateral flow test strip and allow it to react for 1-5 min. After the reaction, use a portable Raman spectrometer connected to a smart terminal to measure the T line of the lateral flow test strip to obtain the SERS spectrum. Quantitative detection of Salmonella in the sample can be achieved based on the SERS spectrum.

[0024] Furthermore, the parameters for the thermal pyrolysis treatment in S1 are: temperature 80℃, treatment time 2min; the pyrolysis buffer contains magnesium chloride (MgCl2), Tris-HCl, triethanolamine dodecyl sulfate, potassium chloride (KCl) and methyl salicylate.

[0025] Furthermore, the specific primer set in S2 includes the forward inner primer FIP, the reverse inner primer BIP, the forward outer primer F3, the reverse outer primer B3, the loop primer LF, and the biotin-labeled loop primer LB. The nucleotide sequences of the forward inner primer FIP, the reverse inner primer BIP, the forward outer primer F3, the reverse outer primer B3, the loop primer LF, and the biotin-labeled loop primer LB are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. The 5' end of the biotin-labeled loop primer LB is labeled with biotin.

[0026] Furthermore, the amplification conditions for LAMP in S2 were: temperature 61.3℃ and amplification time 30 min.

[0027] Furthermore, the concentrations of each primer in the Salmonella-specific primer set were 20-500 nM; the concentration of dCas9 protein was 20-700 nM; the concentration of sgRNA was 20-800 nM, and the nucleotide sequence of sgRNA was shown in SEQ ID NO.7; the volume of the SERS-labeled GNPs-probe conjugate was 2-20 μL; and the volume of the biotin-labeled amplification product was 1-10 μL.

[0028] Furthermore, the preparation method of dCas9 protein in S3 is as follows: The dCas9 gene and vector were transformed into E. coli, and positive strains containing the dCas9 gene-vector were obtained through resistance selection. The positive strains were inoculated into LB liquid medium containing kanamycin and cultured with shaking. Isopropyl-β-D-thiogalactoside (IPTG) was added for low-temperature induction of expression. The bacterial cells were collected by centrifugation, resuspended with lysis buffer, and sonicated. The supernatant was collected by centrifugation and purified by immobilized metal ion affinity chromatography. The purified dCas9 was replaced with storage buffer using a desalting column to obtain the dCas9 protein, which was stored at -80°C.

[0029] Furthermore, the storage buffer contains 20 mM Tris-HCl, 150 mM sodium chloride (NaCl) and 10 wt.% glycerol, with a pH of 8.0, and the lysis buffer contains Tris-HCl, sodium chloride (NaCl) and imidazole, with a pH of 8.0.

[0030] Furthermore, the preparation method of the SERS-labeled GNPs-probe conjugate in S3 is as follows: A conical flask containing 0.5 wt.% chloroauric acid tetrahydrate solution and double-distilled water was placed on a magnetic stirrer and heated to boiling. 1 wt.% trisodium citrate solution was added while stirring continuously until the solution changed from colorless to red. The reaction was continued for 10 min while maintaining boiling. After the reaction was completed, the mixture was cooled to room temperature to obtain uniformly dispersed GNPs. The probe, reducing agent, and buffer were mixed in proportion and incubated in the dark for 30 min to obtain the activated probe. The uniformly dispersed GNPs obtained above were mixed with SERS signal molecules and incubated for 30 min, and then concentrated by centrifugation to obtain pretreated GNPs. The activated probe was mixed with the pretreated GNPs, incubated at room temperature for 2 h, and then blocked with 1 wt.% BSA for 1 h. After the reaction was completed, the supernatant was discarded by centrifugation and the precipitate was resuspended to obtain the SERS-labeled GNPs-probe conjugate.

[0031] Furthermore, the uniformly dispersed GNPs were analyzed using a dynamic laser scattering (DLS) analyzer, followed by centrifugation at 8500 rpm for 15 min, removal of supernatant, and 10-fold concentration. The probe was a thiolized probe, and its nucleotide sequence is shown in SEQ. As shown in ID NO. 8; the reducing agent is tris(2-carboxyethyl)phosphine (TCEP); the buffer is acetate buffer with a pH of 5.2; the mixing ratio of probe, reducing agent, and buffer is 8:4:1; the volume ratio of uniformly dispersed gold nanoparticles to SERS signal molecules is 40:1, and the SERS signal molecule is 4-aminothiophenol (4-ATP); the volume ratio of 0.5 wt.% chloroauric acid tetrahydrate solution to 1 wt.% trisodium citrate solution is 16:15; the volume of double-distilled water is 50 mL; the volume of 1 wt.% bovine serum albumin is 10 μL; the buffer used to resuspend the precipitate is Tris-HCl buffer, which contains Tris-HCl, bovine serum albumin (BSA), Tween-20, and sucrose, and the pH of Tris-HCl buffer is 7.0-8.5.

[0032] Furthermore, the settings for the portable Raman spectrometer in S3 are as follows: single exposure time is 1000ms, laser power is 100mW; the loading buffer solution contains phosphate buffer (PBS), 1 wt.% polyethylene glycol 20000, 50mM magnesium chloride (MgCl2), 2 wt.% bovine serum albumin (BSA) and 10 wt.% sucrose; the phosphate buffer (PBS) contains 6.85mM sodium chloride (NaCl), 0.135mM potassium chloride (KCl), 0.5mM disodium hydrogen phosphate (Na2HPO4) and 0.1mM potassium dihydrogen phosphate (KH2PO4); and the pH of the loading buffer is 7.0-8.5.

[0033] Furthermore, the smart terminal in S3 is a mobile device (such as a smartphone or tablet) with dedicated analysis software installed, which connects to the portable Raman spectrometer wirelessly or via wired connection.

[0034] Example 1 bacterial culture 1.1 Experimental Materials and Methods The strains used in this experiment are as follows:

[0035] It also includes mixed strains of different combinations of these strains.

[0036] 1.2 Strain resuscitation Take the target bacterial strain from the -80℃ glycerol storage tube, thaw it on ice, and inoculate 10 μL of the bacterial suspension into 5 mL of TSB liquid medium. Incubate at 37℃ and 150 r / min for 6-12 h with shaking. After the bacterial suspension becomes turbid, use a sterile inoculation loop to pick up the bacterial suspension, streak it in three zones on TSA solid medium, and incubate it upside down in a 37℃ incubator for 16-18 h. Select single colonies with uniform morphology and clear edges for positive strain screening.

[0037] 1.3 Microbial Culture The selected positive strains were inoculated into 5 mL of TSB liquid medium and cultured with shaking at 37°C and 150 rpm for 6–12 h. Using a sterile pipette, 10 μL of the bacterial culture was inoculated into a fresh 5 mL TSB liquid medium and cultured under the same conditions, with OD measured every 30 min. 600 When OD 600 When the concentration reaches approximately 0.6, stop the culture and obtain the bacterial culture of the target strain.

[0038] 1.4 Microbial Count The traditional plate count method was used. The bacterial culture of the target strain obtained above was serially diluted with sterile PBS at a ratio of 1:9. Three parallel experiments were set up for each concentration. Plates with colony counts of 30-300 CFU / plate were selected for counting.

[0039] Example 2 Preparation of 4-ATP-labeled GNPs-probe conjugates Add 800 μL of 0.5 wt.% chloroauric acid tetrahydrate solution and 50 mL of double-distilled water to an Erlenmeyer flask and heat to boiling on a magnetic stirrer. Add 750 μL of 1 wt.% trisodium citrate solution while stirring continuously until the solution changes from colorless to red. Continue the reaction at boiling for 10 min. After the reaction is complete, cool to room temperature to obtain uniformly dispersed GNPs. Analyze them using dynamic laser scattering (DLS) and centrifuge at 8500 r / min for 15 min to remove the supernatant and concentrate it 10 times for later use. 20 μL of thiolated probe (5 μM), 10 μL of TCEP (1 mM), and 2.5 μL of acetate buffer (pH=5.2) were mixed and incubated in the dark for 30 min to obtain activated thiolated probe. 1 mL of the uniformly dispersed GNPs obtained above were mixed with 25 μL of 4-aminobenzylthiophenol (4-ATP) and incubated for 30 min. The mixture was then centrifuged at 8500 rpm for 15 min, the supernatant was discarded, and the concentration was increased 10-fold to obtain pretreated GNPs. The activated thiolated probe was mixed with the pretreated GNPs and incubated at room temperature for 2 h. A blocking reaction was then performed using 10 μL of 1 wt.% BSA for 1 h. After the reaction, the supernatant was discarded by centrifugation, and the solution was mixed with 100 μL of Tris-HCl buffer (composed of 1 mM Tris-HCl, 5 wt.% BSA, and 1 wt.% BSA). The precipitate was resuspended in BSA, 0.25% (v / v) Tween-20 and 10 wt.% sucrose in Tris-HCl buffer (pH 8.0) to obtain a 4-ATP-labeled GNPs-probe conjugate. The nucleotide sequence of this thiolated probe is as follows: 5´-SH-TTTTTTTTTTTTTTTTTTTTCCTCAGTTTTTCAACGTTTCCTG-3´ (SEQ ID NO. 8).

[0040] Example 3 LFS preparation LFS consists of four parts: sample pad, NC membrane, absorbent pad, and backing plate. The preparation method is as follows: (1) Sample pad treatment The glass fiber membrane was saturated with a buffer solution (composed of 50 mM Tris-HCl, 150 mM NaCl, 0.25% (v / v) Triton X-100 and 0.1 wt.% BSA, pH=8.0) and dried at 45°C for 2 h to improve sample permeability. (2) NC membrane treatment (T line & C line) C-line probe pretreatment: 2.5 μL of biotinylated C-line probe (100 μM) was mixed with 2.5 μL of streptavidin (1 mg / mL) and 15 μL of 3×PBS buffer (composed of 20.55 mM NaCl, 0.405 mM KCl, 1.5 mM Na2HPO4 and 0.3 mM KH2PO4) and incubated at room temperature for 30 min; the nucleotide sequence of the biotinylated C-line probe is as follows: 5´-Biotin-AAAAAAAAAAAAAAAAAAAAA-3´ (SEQ ID NO.9); T-line spraying: Spraying 0.5 mg / mL streptavidin (containing 0.5 wt.% trehalose and 0.1% (v / v) Tween-20 in 1×PBS (composed of 6.85 mM NaCl, 0.135 mM KCl, 0.5 mM Na2HPO4 and 0.1 mM KH2PO4)) to capture target nucleic acid complexes; C-line spraying: Spraying streptavidin-C-line probe complex to verify the effectiveness of the test strip; Drying conditions: Store at 30℃ and a vacuum of 0.1MPa; (3) LFS assembly Assemble the LFS in the following order: sample pad, NC membrane, absorbent pad, and backing plate. (4) Storage The assembled LFS is placed in a sealed container and stored at room temperature for later use.

[0041] Example 4 Preparation of dCas9 protein and sgRNA Preparation of dCas9 protein The dCas9 gene containing *Streptococcus pyogenes* and the pET-28a vector with a 6×His tag were transformed into *Escherichia coli* Rosetta2 (DE3) competent cells (purchased from Shanghai Sangon Biotech Co., Ltd.) to obtain a strain containing the dCas9 gene-pET-28a vector. This strain was inoculated into 5 mL of LB liquid medium containing kanamycin (50 µg / mL) and cultured at 37°C with shaking at 200 rpm for 12–16 h as a seed culture. The seed culture was then inoculated into 50 mL of fresh LB liquid medium (containing 50 µg / mL kanamycin) at a ratio of 1:10 and cultured with shaking at 37°C and 200 rpm. When OD... 600When the concentration was >0.6, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and expression was induced at 18°C ​​for 16 h. Cells were collected by centrifugation at 6000 r / min for 10 min and lysed with pre-cooled lysis buffer (composed of 20 mM Tris-HCl, 500 mM... The bacterial cells were resuspended in a solution of NaCl and 10 mM imidazole (pH=8.0) and sonicated (300W, 10 s working time / 10 s interval, 60 min total). The supernatant was collected by centrifugation at 16000 r / min for 30 min and purified by affinity chromatography using Ni-NTA agarose (purchased from Shanghai Sangon Biotech Co., Ltd.). The specific steps were as follows: the medium was equilibrated with 10 mM imidazole equilibration buffer (pH=8.0) and incubated with the supernatant. The medium was then washed sequentially with 10 mM, 20 mM, and 50 mM imidazole equilibration buffer (pH=8.0), and finally eluted with 250 mM imidazole equilibration buffer (pH=8.0). Finally, the purified dCas9 was replaced with storage buffer (20 mM Tris-HCl, 150 mM NaCl, and 10 wt.% glycerol, pH=8.0) using a desalting column for later use.

[0042] Preparation of sgRNA The target-specific sgRNA was synthesized using the T7 sgRNA synthesis kit (catalog number: E3322S) from New England Biolabs (NEB) according to the instructions. The nucleotide sequence of the sgRNA is as follows: 5´-GGCACGUUGAAAAACUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCUUUU-3´ (SEQ ID NO.7).

[0043] Example 5 Detection of bacterial strains LAMP amplification Different concentrations of bacterial suspensions were prepared by serial dilution. Before the LAMP reaction, 2 μL of bacterial suspension was mixed with 50 μL of lysis buffer (purchased from Shanghai Sangon Biotech Co., Ltd.) in a clean reaction tube, treated at 80°C for 2 min, and then used directly as a DNA template. LAMP amplification of the target bacterial strain was performed using Salmonella-specific primers to obtain biotin-labeled amplification products. The specific Salmonella-specific primer sequences are as follows: Forward inner primer F3: 5´-GACCTGAATTACTGATTCTGGT-3´ (SEQ ID NO.1); Reverse inner primer B3: 5´-ATCGGCTTCAATCAAGATAAGA-3´ (SEQ ID NO.2); Forward outer primer FIP: 5´-AACCAATATCGCCAGTACGATCGTCATTCCATTACCCTA-3´ (SEQ ID NO. 3); Reverse outer primer BIP: 5´-TTTACGTTTCCTGCGGTACTACTGGTACTGATCGATGC-3´ (SEQ ID NO. 4); Loop primer LF: 5´-TGCGATCAGGAAATCAACCA-3´ (SEQ ID NO.5); Biotin-labeled loop primer LB: 5´-Biotin-ACCACGCTCTTTCGTCTG-3´ (SEQ ID NO.6); The specific experimental parameters are as follows: The 50 μL LAMP reaction system contains: 5 μL 10×Bst buffer, 0.4 mM dNTP mixture, 1 μL Bst DNA polymerase (8 U / μL), 320 nM forward inner primer FIP, 320 nM reverse inner primer BIP, 80 nM forward outer primer F3, 80 nM reverse outer primer B3, 160 nM loop primer LF, 160 nM biotinylated loop primer LB, 1 μL DNA template, 3 mM MgSO4, and DEPC H2O to make up to 50 μL; Amplification conditions: 61.3℃, 30 min.

[0044] LFS-SERS assay The 35 μL LFS-SERS reaction system contained: 12 μL of mixed solution (containing 670 nM dCas9 and 750 nM sgRNA), 7 μL of 4-ATP-labeled GNPs-probe conjugate, 1 μL of biotin-labeled amplification product, and 15 μL of loading buffer (1×PBS (composed of 6.85 mM NaCl, 0.135 mM KCl, 0.5 mM Na2HPO4 and 0.1 mM KH2PO4), 1 wt.% polyethylene glycol 20000, 50 mM MgCl2, 2 wt.% BSA and 10 wt.% sucrose, pH=8.0). The components of the LFS-SERS reaction system were mixed thoroughly and incubated at room temperature for 5 min to obtain the complex. The obtained complex was then added dropwise onto a lateral flow test strip, and the color reaction was allowed to proceed for 1-5 min. After the reaction, a portable Raman spectrometer connected to a smartphone was used, with the parameters set (single exposure time of 1000 ms, laser power of 100 mW), to measure the T line of LFS and obtain the SERS spectrum. 4-ATP was extracted at 1079 cm⁻¹. -1 The intensity value of the characteristic Raman peak at a certain location is used to achieve quantitative detection of Salmonella in the sample.

[0045] Test Example 1 Feasibility test To verify the feasibility of the LFS-SERS rapid quantitative detection method for Salmonella of the present invention, the ability of this method to detect Salmonella was evaluated by reacting different combinations of biotin-labeled amplification products, dCas9, sgRNA, primer dimers, and 4-ATP-labeled GNPs-probe conjugates. Specific combinations included the following: 1. 4-ATP-labeled GNPs-probe conjugates; 2. Biotin-labeled amplification product + 4-ATP-labeled GNPs-probe conjugate; 3. dCas9+4-ATP-labeled GNPs-probe conjugates; 4. sgRNA + 4-ATP labeled GNPs-probe conjugate; 5. dCas9+sgRNA+4-ATP-labeled GNPs-probe conjugate; 6. Biotin-labeled amplification product + dCas9 + 4-ATP-labeled GNPs-probe conjugate; 7. Biotin-labeled amplification product + sgRNA + 4-ATP-labeled GNPs-probe conjugate; 8. Biotin-labeled amplification product + dCas9 + sgRNA + 4-ATP-labeled GNPs-probe conjugate; 9. Biotin-labeled amplification product + sgRNA + primer dimer + 4-ATP-labeled GNPs-probe conjugate.

[0046] from Figure 2 As shown in Figure A, only the group containing the amplification product, dCas9, sgRNA, and gold nanoprobe (combination 8) produced a clearly visible purple-red band on the T line of the test strip; the other combinations did not produce a visible band on the T line. These results demonstrate the feasibility of LFS-SERS for Salmonella detection.

[0047] Test Example 2 Sensitivity test The sensitivity of the LFS-SERS rapid quantitative detection method for Salmonella of the present invention was evaluated. Figure 3 It can be seen that all three Salmonella samples showed good detection responses. The color intensity of the T line on the test strip and the corresponding SERS signal intensity both showed a significant positive correlation with the bacterial concentration: as the concentration increased (from 10... 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 (CFU / mL), the T line gradually increases, and the SERS signal also gradually increases. The detection limit for LFS visual interpretation is 10. 2 CFU / mL, while the detection limit of LFS-SERS can be as low as 10. 1 The CFU / mL concentration is significantly better than visual analysis. This is because the inherent high sensitivity of SERS, when combined with LFS, further enhances the overall detection sensitivity of the LFS-SERS method.

[0048] Test Example 3 Specificity test Since multiple bacteria may coexist in actual food samples, specificity is a key indicator for evaluating the application potential of the LFS-SERS rapid quantitative detection method for Salmonella in this invention. Therefore, under the same experimental conditions, the specificity of the target bacterial culture obtained in Example 1 was tested using this method. For single bacteria, combinations of two or more coexisting bacteria, the concentration of each bacterium was maintained at 10. 8 CFU / mL. Specific combinations include the following: 1. Salmonella ( Salmonella ); 2. Enterococcus faecalis ( E. faecalis ); 3. Cronobacter ( Cronobacter sakazakii ); 4. Staphylococcus aureus ( Staphylococcus aureus ); 5. Vibrio parahaemolyticus ( Vibrio parahaemolyticus ); 6. Bacillus subtilis ( B. subtilis ); 7. Escherichia coli ( Escherichia coli ); 8. Bacillus cereus ( Bacillus cereus ); 9. Serratia marcescens ( Serratia marcescens ); 10. Escherichia coli ( Escherichia coli O157:H7); 11. Salmonella ( Salmonella ) + Enterococcus faecalis ( E . faecalis ); 12. Salmonella ( Salmonella ) + Cronobacter ( C . sakazakii ); 13. Salmonella ( Salmonella ) + Staphylococcus aureus ( S . aureus ); 14. Salmonella ( Salmonella ) + Vibrio parahaemolyticus ( V . parahaemolyticus ); 15. Salmonella ( Salmonella ) + Bacillus subtilis ( B . subtilis ); 16. Salmonella ( Salmonella ) + E. coli ( E . coli ); 17. Salmonella ( Salmonella ) + Bacillus cereus ( B . cereus ); 18. Salmonella ( Salmonella ) + Serratia marcescens ( S . marcescens ); 19. Salmonella ( Salmonella ) + Escherichia coli ( E . coli O157:H7); 20. Salmonella ( Salmonella ) + Enterococcus faecalis ( E . faecalis ) + Cronobacter ( C . sakazakii ) + Staphylococcus aureus ( S . aureus ) + Vibrio parahaemolyticus ( V . parahaemolyticus ) + Bacillus subtilis ( B. subtilis ) + E. coli ( E . coli ) + Bacillus cereus ( B . cereus ) + Serratia marcescens ( S . marcescens ) + Escherichia coli ( E . coli O157:H7); 21. Enterococcus faecalis ( E . faecalis ) + Cronobacter ( C . sakazakii ) + Staphylococcus aureus ( S . aureus ) + Vibrio parahaemolyticus ( V . parahaemolyticus ) + Bacillus subtilis ( B. subtilis ) + E. coli ( E . coli ) + Bacillus cereus ( B . cereus ) + Serratia marcescens ( S . marcescens ) + Escherichia coli ( E . coli O157:H7); NC(N). An equal volume of deionized water was used as a negative control.

[0049] from Figure 4 It can be seen that only Salmonella or combinations containing Salmonella (combinations 1 and 11-20) showed a distinct T-line and a 1079 cm⁻¹. -1 The target bacteria showed a characteristic SERS peak at the T-line, while other non-target bacteria and their mixtures showed no T-line color development or characteristic SERS signal. This result indicates that the LFS-SERS method of this invention maintains high sensitivity while exhibiting excellent specificity for Salmonella detection, effectively distinguishing between target and non-target bacteria.

[0050] Test Example 4 Actual sample application To verify the practical performance of the LFS-SERS rapid quantitative detection method for Salmonella in real food samples, egg and milk samples were selected for spiked recovery experiments. Different concentrations of Salmonella were added to the samples for detection. Figure 5 and Figure 6 It is known that the LFS visual detection limit for Salmonella in milk is 10. 3 CFU / mL, LFS-SERS detection limit is 10 2 CFU / mL. From Figure 7 and Figure 8 It is known that the LFS visual detection limit for Salmonella in eggs is 10. 4 CFU / mL, LFS-SERS detection limit is 10 3CFU / mL. This result indicates that the LFS-SERS rapid quantitative detection method for Salmonella established in this invention still has high sensitivity when detecting real samples, and has great application potential in the field of on-site detection.

[0051] In summary, this invention establishes a rapid quantitative detection method for Salmonella using LFS-SERS. By integrating the advantages of precise gene recognition by CRISPR / dCas9, LAMP isothermal amplification, lateral flow strip (LFS) instant detection, and surface-enhanced Raman spectroscopy (SERS) quantitative analysis, it achieves visualized rapid screening and highly sensitive quantitative analysis of Salmonella. This method, through the design of specific sgRNAs and optimization of the detection system, achieves accurate identification of Salmonella and can effectively distinguish between single and mixed infections of Salmonella and common foodborne pathogens. The detection sensitivity is improved by one order of magnitude (10-fold) compared to LFS, providing a new, efficient, and reliable rapid detection strategy for food safety monitoring.

[0052] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A rapid quantitative detection method for Salmonella using LFS-SERS, characterized in that, Includes the following steps: S1. Mix the sample to be tested with the lysis buffer evenly and perform thermal lysis to obtain crude DNA solution. S2. Using the crude DNA solution obtained in S1 as a template, Salmonella-specific primers were used to perform loop-mediated isothermal amplification to obtain biotin-labeled amplification products. S3. Mix dCas9 protein, sgRNA, SERS-labeled gold nanoparticle-probe conjugate, and the biotin-labeled amplification product obtained in S2 in a loading buffer solution and incubate at room temperature for 1-5 min to obtain a complex. Add the obtained complex to a lateral flow test strip and allow it to react for 1-5 min. After the reaction, use a portable Raman spectrometer connected to a smart terminal to measure the T line of the lateral flow test strip to obtain the SERS spectrum. Quantitative detection of Salmonella in the sample can be achieved based on the SERS spectrum.

2. The rapid quantitative detection method for Salmonella using LFS-SERS as described in claim 1, characterized in that, The parameters for the pyrolysis treatment described in S1 are: temperature 80℃, treatment time 2min.

3. The rapid quantitative detection method for Salmonella using LFS-SERS as described in claim 1, characterized in that, The specific primer set described in S2 includes a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a loop primer LF, and a biotin-labeled loop primer LB. The nucleotide sequences of the forward inner primer FIP, the reverse inner primer BIP, the forward outer primer F3, the reverse outer primer B3, the loop primer LF, and the biotin-labeled loop primer LB are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively. The 5' end of the biotin-labeled loop primer LB is labeled with biotin.

4. The rapid quantitative detection method for Salmonella using LFS-SERS as described in claim 1, characterized in that, The amplification conditions for loop-mediated isothermal amplification described in S2 are: temperature 61.3℃ and amplification time 30 min.

5. The rapid quantitative detection method for Salmonella using LFS-SERS as described in claim 1, characterized in that, The concentration of each primer in the Salmonella-specific primer set is 20-500 nM; the concentration of the dCas9 protein is 20-700 nM; the concentration of the sgRNA is 20-800 nM, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO.7; the volume of the SERS-labeled gold nanoparticle-probe conjugate is 2-20 μL; and the volume of the biotin-labeled amplification product is 1-10 μL.

6. The rapid quantitative detection method for Salmonella using LFS-SERS as described in claim 1, characterized in that, The preparation method of the dCas9 protein described in S3 is as follows: The dCas9 gene and vector were transformed into E. coli, and positive strains containing the dCas9 gene-vector were obtained through resistance selection. The positive strains were inoculated into LB liquid medium containing kanamycin and cultured with shaking. Isopropyl-β-D-thiogalactoside was added for low-temperature induction of expression. The bacterial cells were collected by centrifugation, resuspended with lysis buffer, and sonicated. The supernatant was collected by centrifugation and purified by immobilized metal ion affinity chromatography. The purified dCas9 was replaced with storage buffer using a desalting column to obtain the dCas9 protein.

7. The rapid quantitative detection method for Salmonella using LFS-SERS as described in claim 6, characterized in that, The storage buffer contains 20 mM Tris-HCl, 150 mM sodium chloride and 10 wt.% glycerol, and the pH of the storage buffer is 8.

0.

8. The rapid quantitative detection method for Salmonella using LFS-SERS as described in claim 1, characterized in that, The preparation method of the SERS-labeled gold nanoparticle-probe conjugate described in S3 is as follows: A conical flask containing 0.5 wt.% chloroauric acid tetrahydrate solution and double-distilled water was placed on a magnetic stirrer and heated to boiling. 1 wt.% trisodium citrate solution was added while stirring continuously until the solution changed from colorless to red. The reaction was continued for 10 min while maintaining boiling. After the reaction was completed, the mixture was cooled to room temperature to obtain uniformly dispersed gold nanoparticles. The probe, reducing agent, and buffer were mixed in proportion and incubated in the dark for 30 min to obtain the activated probe. The uniformly dispersed gold nanoparticles obtained above were mixed with SERS signal molecules and incubated for 30 min, and then concentrated by centrifugation to obtain pretreated gold nanoparticles. The activated probe was mixed with the pretreated gold nanoparticles, incubated at room temperature for 2 h, and then blocked with 1 wt.% bovine serum albumin for 1 h. After the reaction was completed, the supernatant was discarded by centrifugation and the precipitate was resuspended to obtain the SERS-labeled gold nanoparticle-probe conjugate.

9. The rapid quantitative detection method for Salmonella using LFS-SERS as described in claim 8, characterized in that, The uniformly dispersed gold nanoparticles need to be analyzed by dynamic laser scattering, and then concentrated 10-fold by centrifugation at 8500 r / min for 15 min to remove the supernatant; the probe is a thiolized probe, and the nucleotide sequence of the thiolized probe is shown in SEQ ID NO.8; the reducing agent is tris(2-carboxyethyl)phosphine; the buffer is acetate buffer, and the pH of the acetate buffer is 5.2; the mixing ratio of the probe, reducing agent and buffer is 8:4:1; the volume ratio of the uniformly dispersed gold nanoparticles to the SERS signal molecule is 40:1, and the SERS signal molecule is 4-aminobenzylthiophenol; the volume ratio of the 0.5 wt.% tetrahydrate chloroauric acid solution to the 1 wt.% trisodium citrate solution is 16:

15.

10. The rapid quantitative detection method for Salmonella using LFS-SERS as described in claim 1, characterized in that, The settings for the portable Raman spectrometer described in S3 are: single exposure time of 1000ms and laser power of 100mW.

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