A double-probe system, test strip and detection method for simultaneously detecting listeria and escherichia coli o157:h7

Abstract

The application discloses a double-probe system, a test strip and a detection method for simultaneously detecting listeria and escherichia coli O157:H7, wherein the constructed double-probe system comprises AIENPs-FAM fluorescent nano probes and AIENPs-digoxin fluorescent nano probes, and corresponding immunochromatography test strips, and further, the constructed RPA-CRISPR-LFCS method based on AIENPs can simultaneously realize high-sensitivity screening of listeria LM and escherichia coli O157:H7, the detection limit of LM is 29 fg / μL (223 CFU / mL), and the detection limit of escherichia coli O157:H7 is 25.1 fg / μL (195 CFU / mL).

Description

Technical Field

[0001] This invention belongs to the technical field, specifically relating to a dual-probe system, test strip, and detection method for simultaneously detecting Listeria and Escherichia coli O157:H7. Background Technology

[0002] Foodborne illnesses are among the most prevalent and widespread diseases globally. Of the various mechanisms of foodborne disease transmission, infection by foodborne pathogens is the most common. Pathogens can enter the human body and multiply or produce toxins by contaminating food, livestock, and water sources. Listeria monocytogenes (LM) and Escherichia coli O157:H7 are well-known pathogens causing foodborne illnesses and are commonly found in various foods such as dairy products, meats, fruits, and vegetables. Raw meat and ready-to-eat foods have the highest contamination rates. With the diversification and comprehensive development of human diets, food safety issues are becoming increasingly prominent. Rapid detection of pathogens is essential for effective disease diagnosis and biomonitoring.

[0003] Methods for identifying foodborne pathogens have evolved from traditional culture-based detection to those utilizing molecular biology and immunology techniques. Due to limitations of traditional methods, such as long processing times, complex pretreatment requirements, and demanding experimental conditions, various nucleic acid amplification techniques have been developed. These include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). However, the requirement for specialized equipment and skilled operators in PCR-based detection methods limits their application in field screening.

[0004] Lateral flow chromatography (LFCS) test strips have gained significant attention due to their ease of use, short detection time, and visualized results, making them an ideal choice for rapid on-site testing. Gold nanoparticles (AuNPs) are the main tracers in commercially available test strips, but test strips constructed with gold nanoparticles exhibit low detection sensitivity and limited quantitative potential. Aggregation-induced emission nanoparticles (AIENPs) are a novel class of fluorescent materials that emit virtually no fluorescence in solution but exhibit strong fluorescence in the aggregated state. This property overcomes the fluorescence quenching phenomenon observed in traditional fluorescent materials in the aggregated state. Therefore, AIENPs show potential as fluorescent tracers in test strip preparation, opening up new avenues for test strip development.

[0005] In recent years, clustered, regularly spaced short palindromic repeats and CRISPR-associated proteins (CRISPR / Cas) have been used for nucleic acid detection. The detection method relies on the non-specific endonuclease activity of Cas12a, which is activated when bound to a specific target using a programmable guide RNA. Detection signals can be observed when fragmented FAM or digoxigenin slices bind to their corresponding anti-FAM or digoxigenin antibodies. The bound complexes are then captured by goat anti-rabbit IgG or goat anti-mouse IgG antibodies, respectively. Integrating RPA with a CRISPR system and visualizing the results via LFCS allows for direct and rapid determination of detection results without the need for expensive equipment. Summary of the Invention

[0006] The purpose of this invention is to provide a dual-probe system, test strip, and detection method for the simultaneous detection of Listeria monocytogenes and Escherichia coli O157:H7, achieving highly sensitive screening for both Listeria monocytogenes LM and Escherichia coli O157:H7, with a detection limit of 29 fg / μL (223). The detection limit for Escherichia coli O157:H7 was 25.1 fg / μL (195 CFU / mL). (CFU / mL).

[0007] To achieve the above objectives, the technical solution of the present invention is as follows: A dual probe system for the simultaneous detection of Listeria and Escherichia coli O157:H7, comprising AIENPs-FAM nanoprobes and AIENPs-digoxigenin nanoprobes. The AIENPs-FAM nanoprobe has aggregation-induced emission nanoparticles as its core and an anti-fluorescein isothiocyanate antibody covalently coupled to the surface of the aggregation-induced emission nanoparticle core. The AIENPs-digoxigenin nanoprobes consist of aggregation-induced emission nanoparticles as the core, and anti-digoxigenin antibodies covalently coupled to the surface of the aggregation-induced emission nanoparticle core.

[0008] An immunochromatographic test strip for detecting Listeria and Escherichia coli O157:H7, characterized in that it includes a base plate and a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad disposed on the base plate; The nitrocellulose membrane is provided with a first detection line, a second detection line, and a quality control line sequentially arranged along the chromatography direction. The first and second detection lines are respectively coated with capture molecules capable of capturing aggregation-induced emission nanoparticle-labeled anti-fluorescein isothiocyanate antibody or aggregation-induced emission nanoparticle-labeled anti-digoxin antibody; The quality control line is coated with recombinant streptavidin; The binding pad is coated with AIENPs-FAM fluorescent nanoprobes and AIENPs-digoxigenin fluorescent nanoprobes.

[0009] Preferably, the first detection line is coated with BSA at a concentration of 1 mg / mL for IgG m+ and 1.25% concentration; the second detection line is coated with BSA at a concentration of 1 mg / mL for IgG r+ and 1% concentration; and the control line is coated with recombinant streptavidin at a concentration of 2 mg / mL.

[0010] Preferably, the amount of AIENPs-FAM fluorescent nanoprobe and AIENPs-digoxigenin fluorescent nanoprobe used is 5 μL.

[0011] An RPA-CRISPR-LFCS method for simultaneous detection of Listeria monocytogenes and Escherichia coli O157:H7 includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the genomic DNA as a template, the hly gene of Listeria monocytogenes and the gene of Escherichia coli O157:H7 were simultaneously amplified using recombinase polymerase amplification technology. rfb E gene, obtained hly Gene amplification products and rfb E gene amplification product; (3) Add the amplification product from step (2) to the CRISPR / Cas12a reaction system, wherein the reaction system contains LbCas12a protein and targets hly The first crRNA of the gene, targeting rfb The second crRNA of the E gene, the first paired-end labeled ssDNA reporter probe, and the second paired-end labeled ssDNA probe; wherein, one end of the first paired-end labeled ssDNA reporter probe is labeled with biotin and the other end is labeled with digoxigenin, and one end of the second paired-end labeled ssDNA probe is labeled with biotin and the other end is labeled with fluorescein isothiocyanate. (4) Load the CRISPR reaction product from step (3) onto the sample pad of the immunochromatographic test strip and perform chromatography along the test strip under the action of capillary force. (6) Observe the fluorescence signals of the first and second detection lines on the test strip under ultraviolet light to determine whether Listeria monocytogenes and / or Escherichia coli O157:H7 are present.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention developed different nanoprobes, namely AIENPs-FAM and AIENPs-digoxigenin. Furthermore, the constructed AIENPs-based RPA-CRISPR-LFCS method can be used for highly sensitive screening of Listeria LM and Escherichia coli O157:H7. This method utilizes RPA technology for exponential amplification of targets and combines it with a CRISPR / Cas12a trans-lysis reaction to achieve qualitative and quantitative analysis of multiple targets at different T-lines. It can simultaneously achieve highly sensitive screening for Listeria LM and Escherichia coli O157:H7, with a detection limit of 29 fg / μL (223 CFU / mL) for LM and 25.1 fg / μL (195 CFU / mL) for Escherichia coli O157:H7. Attached Figure Description

[0013] Figure 1 This diagram illustrates the process of nanoprobe synthesis, immunochromatographic strip assembly, and detection. Figure 2 This diagram illustrates the optimization results of LFCS. A shows TEM images of AIENPs (A1), AIENPs-digoxigenin antibody (A2), and AIENPs-FAM antibody (A3), scale bar: 100 nm; B shows probe concentration optimization; C shows optimized AIENPs-labeled antibody concentration; D shows r-SA concentration optimization; E shows optimization of IgGr and BSA concentrations under both positive and negative conditions; F shows optimization of IgGr and BSA concentrations under both positive and negative conditions. Figure 3 A schematic diagram for evaluating the sensitivity of PRA-CRISPR-LFCS, where A represents the results of multiplex LFCS and the corresponding LM linearity analysis; B represents the results of multiplex LFCS for E. coli O157:H7 and the corresponding linearity analysis; C represents the results of multiplex LFCS for simultaneous detection of two targets and the corresponding linearity analysis; N represents the negative control. Figure 4 This is a schematic diagram of the results of the study on the specificity of RPA-CRISPR-LFCS; the y-axis represents the ratio of the fluorescence intensity of the T1 / T2 line to that of the C line (T / C value); N, negative control; Figure 5 This is a schematic diagram of the actual sample test results; AC, the test results of the spiked sample; N, the negative control; D, the PCR and test strip results of the natural sample. Detailed Implementation

[0014] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0015] I. 1.1 Materials and Methods RNase inhibitors, LbCas12a nuclease, TIANamp bacterial DNA Kit DP302, and RPA kit were purchased from BBI (Shanghai, China), Tolo Biotech (Shanghai, China), Tiangen (Beijing, China), and TwistDx (Cambridge, Massachusetts, USA), respectively. Primers, crRNA, and reporter probes (double-labeled ssDNA) were synthesized by Sangon Biotech. Streptavidin (r-SA), bovine serum albumin (BSA), goat anti-mouse IgG antibody (IgGm), and goat anti-rabbit IgG antibody (IgGr) were obtained from Bosch Beijing, Beijing Biosynthetic Biotechnology Co., Ltd., and Merck Shanghai Co., Ltd., respectively. Aggregation-induced emission nanoparticles (AIENPs) were purchased from Nanjing Xianfeng Nanotechnology Co., Ltd. N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Sangon Biotech Shanghai. Nitrocellulose membranes, absorbent pads, sample pads, conjugate pads, and PVC backing plates were purchased from Millipore, USA.

[0016] 1.2 Preparation of AIENP-labeled antibodies See Figure 1 Add 5 μL of AIENPs to 1 mL of 0.1 M MES buffer (pH = 5.5–6.0), sonicate for 1 min, add 20 μL of EDC (0.1 M) and 50 μL of NHS (0.1 M), activate the mixture on a vibrating sieve at 250 rpm for 15 min, and centrifuge at 8000 rpm for 20 min. Resuspend the precipitate in 1 mL of PBS solution, vortex for 30 s, add 15 μg of antibody (anti-FAM or digoxin antibody), and react at 37°C for 2 h on a shaker. Finally, add bovine serum albumin to a final concentration of 2%, vortex for 1 h, centrifuge at 8000 rpm for 20 min, collect the supernatant, resuspend in 100 μL of storage solution (pH = 7.4, 10 mM Tris buffer, 1% BSA, 0.5% Tween-20), and store at 4°C.

[0017] 1.3. Preparation of Immunochromatographic Test Strips See Figure 1The sample pad was treated with buffer (0.01 M TB buffer, 0.5% BSA, 1% Tween-20, 1% sucrose). The NC membrane was fixed with IgGm + BSA (1 mg / mL + 1%) at T1 line, IgGr + BSA (1 mg / mL + 1.25%) at T2 line, and r-SA (2 mg / mL) at C line. The conjugate pad was coated with AIENPs-labeled FAM antibody and AIENPs-labeled digoxigenin antibody. The NC membrane, conjugate pad, sample pad, and absorbent pad were sequentially attached to a plastic base plate, overlapping by about 2 mm. The components were cut into widths of about 3 mm using a CM 4000 cutter.

[0018] 1.4 Bacterial Culture and Genome Extraction All RPA-CRISPR-LFCS experiments were performed using reference strains LM (ATCC43251) and Escherichia coli O157:H7 (ATCC43895). Bacteria were cultured in brain heart extract (BHI) broth at 37°C for 12 h. DNA extraction was performed according to the TIANamp bacterial DNA kit protocol. Genomic DNA concentration was detected using a NanoDrop 2000c (Thermo, Waltham, MA, USA), and extracted DNA was stored at -20°C.

[0019] 1.5 Design of primers, crRNA, and probes Choose LM hly Gene (GenBank MG922920.1) and Escherichia coli O157:H7 rfb The E gene (GenBank S83460) was used as the target gene. RPA and PCR primers were designed using Premier 5.0 software, and detailed information is shown in Table 1. In addition, the optimized crRNA was selected for CRISPR / Cas12a cleavage reaction, and the sequence information is listed in Table 1.

[0020]

[0021] Different paired-end labeled ssDNA probes were applied to the CRISPR system. Reporter probe 1 (5'-biotin-TTATT-Dig-3') was used for LM detection, and reporter probe 2 (5'-biotin-TTATT-FAM-3') was used for the identification of Escherichia coli O157:H7.

[0022] 1.6, RPA, CRISPR / Cas12a reaction and LFCS See Figure 1The total RPA reaction mixture was 50 μL, containing 2.4 μL (10 μM) forward primer, 2.4 μL (10 μM) reverse primer, 2 μL DNA extraction buffer, 29.5 μL RPA buffer, 2.5 μL MgOAc, and 11.2 μL ddH2O. All components were thoroughly mixed and incubated at 37°C for 20 min. The results were verified by agarose gel electrophoresis.

[0023] The CRISPR / Cas12a system was carried out in a 20 μL reaction mixture, which included 2 μL of RPA product, 50 nM LbCas12a, 500 nM crRNA, 500 nM reporter probe, 1 U RNase inhibitor, 10 × Tolo buffer, and DEPC water, and then reacted at 37 °C for 20 min.

[0024] In LFCS, 5 μL of CRISPR product was spread onto the sample pad. The test strip was then immersed in 100 μL of flow buffer (0.9% NaCl) for chromatography. The detection results were visible after 5 min under a 365 nm UV lamp.

[0025] 1.7 Sensitivity and Specificity Evaluation The sensitivity of the detection platform was evaluated using different concentrations of genomic DNA and bacterial cultures. A DNA gradient dilution was prepared using ddH2O, with the E. coli O157:H7 range being 2.51 × 10⁻⁶. 8 ~2.51×10 0 fg / μL, LM range is 2.90×10 8 ~ 2.90×10 0 fg / μL, the concentration of Escherichia coli O157:H7 was 1.95×10⁻⁶ fg / μL. 8 ~1.95×10 0 CFU / mL, LM concentration is 2.23×10 8 ~2.23×10 0 CFU / mL. DNA was extracted from bacterial solutions of different concentrations for subsequent detection. The specificity of the platform was evaluated using six common foodborne pathogens; Table 2 provides detailed information on bacterial sources and culture conditions.

[0026]

[0027] 1.8 Detection of natural samples Mince 5 g of pork and mix thoroughly with 10 mL of ddH2O. Mix 0.1 mL of broth with different concentrations of E. coli O157:H7 (1 mL) and LM (1 mL) bacterial cultures. Finally, take 1 mL of the mixture to extract DNA for further analysis.

[0028] Different food varieties, including lettuce, cucumber, tomato, fresh milk, orange juice, apple, pear, grape, pork, and beef, were randomly purchased from a supermarket for natural sample testing. Each sample (25 g) was added to 225 mL of BHI broth and incubated overnight at 37°C. Then, 1 mL of the culture medium was used to extract DNA for subsequent testing.

[0029] II. Results and Discussion 2.1 Working principle of RPA-CRISPR-LFCS Figure 1 A illustrates the synthesis process of AIENP-labeled antibodies. In short, the labeling of anti-FAM and anti-digoxigenin antibodies is achieved through a condensation reaction between the carboxyl groups of AIENPs and the amino groups of the antibodies. NHS and EDC activate the carboxylated AIENPs to form active esters, promoting their binding to the antibody amino groups. BSA is used to block excess NHS active esters.

[0030] Figure 1 B describes the fabrication and application of AIENP-based test strips.

[0031] Figure 1 C describes the detection principle of RPA-CRISPR-LFCS for detecting LM and Escherichia coli O157:H7. First, the target DNA extracted from the pathogenic microorganism is exponentially amplified by RPA. A large amount of target DNA is transferred to the CRISPR system, where it binds to crRNA and Cas protein to form a ribonucleoprotein complex (RNP), activating its trans-cleavage activity and indiscriminately cleaving reporter probe 1 (5'-biotin-TTATT-Dig-3') or reporter probe 2 (5'-biotin-TTATT-FAM-3').

[0032] To differentiate bacteria, reporter probe 1 was matched with the LM CRISPR system, while reporter probe 2 was matched with the E. coli O157:H7 CRISPR system. A mixture of AIENP-labeled antibodies (AIENPs-FAM and AIENPs-digoxigenin) and reaction products was loaded onto a sample pad, which migrated along the test strip under capillary forces. Once the target appeared in the system, the trans-cleavage capability of Cas12a was triggered. A large number of probes were cleaved, resulting in the formation of a binary complex between the digoxigenin fragment (FAM fragment) and AIENPs-digoxigenin (AIENPs-FAM), which was captured by pre-coated IgGm (IgGr) on the T1 (T2) line. Without the presence of the target, the CRISPR system lost its activity, and all probes remained intact. The labeled antibody-probes bound to biotin via r-SA affinity and were captured on the C line.

[0033] 2.2 Characterization of the nanoprobe and optimization of the test strip The size distribution and morphology of the nanoprobes were analyzed using transmission electron microscopy (TEM). Figure 2 As shown in A1, AIENPs have a smooth surface, are spherical, and have a uniform particle size and distribution. Using AIENPs as the core, two fluorescent probes, AIENPs-digoxigenin and AIENPs-FAM, were successfully obtained by covalently binding a large number of AIENPs-labeled anti-digoxigenin and FAM antibodies. Figure 2 As shown in A2 and A3.

[0034] System optimization of reaction parameters can eliminate false positives in negative samples and improve the detection performance of RPA-CRISPR-LFCS. In negative cases, the T line disappears when the probe concentration, AIENPs-labeled antibody volume, and r-SA concentration are all appropriate. First, the probe concentration is optimized ( Figure 2 (B) The final concentrations of the two reporter probes were screened separately in the CRISPR system. Results showed that at concentrations of 500 nM and 1000 nM, reporter probe 1 and reporter probe 2 could bind to the labeled antibody and be completely captured by r-SA. When the probe concentration deviated from the optimal value, strong false-positive bands appeared in negative samples. At low concentrations, the number of probes was insufficient, and few AIENPs-labeled antibodies could be biotinylated and captured by the C-line. However, blindly increasing the probe concentration could easily lead to a high-dose hook effect, reducing the formation of the sandwich complex and causing false positives in the T-line of negative samples.

[0035] Next, the volume of the AIENP-labeled antibody was optimized. Figure 2 C). Both AIENPs-digoxin and AIENPs-FAM showed optimal values ​​at a concentration of 5 μL, and no false positives were observed in the T-line under negative conditions. Figure 2 As shown in Figure D, the T line gradually weakens and eventually disappears as the r-SA concentration increases. At low r-SA concentrations, it cannot completely capture the probe bound to the labeled antibody, and excess labeled antibody continues to migrate towards the T line, causing it to become visible. At higher r-SA concentrations, only a very small amount of r-SA is needed to completely capture the complex; therefore, the colorimetric position appears at the bottom of the C line, and the band narrows. When the r-SA concentration reaches 2 mg / mL, the complex is almost completely captured on the C line. Finally, 2 mg / mL r-SA was used as the condition for the C line.

[0036] The T-line was optimized under both negative and positive conditions. The optimization results for different secondary antibody concentrations during test strip preparation are as follows: Figure 3As shown in E and 3F. Furthermore, BSA is added to the secondary antibody to eliminate false positives at the T line. Optimal detection is achieved at a concentration of 1 mg / mL + 1.25% for IgGm and BSA coating on the T1 line, and at a concentration of 1 mg / mL + 1% for IgGr and BSA coating on the T2 line. Unlike traditional test strips that use a secondary antibody as a control line (C line), this test strip uses the secondary antibody as the T line and r-SA as the C line.

[0037] 2.3 Performance Evaluation of RPA-CRISPR-LFCS The method for simultaneously detecting LM and Escherichia coli O157:H7 was evaluated using different concentrations of DNA and bacteria.

[0038] When only LM is present in the sample, the fragmentation of reporter probe 1 causes fluorescence in the T1 line. Increased LM concentration leads to more fragmentation of reporter probe 1, resulting in enhanced T1 line fluorescence and a gradual decrease in C line fluorescence. In the absence of *E. coli* O157:H7 in the sample, reporter probe 2 remains intact, forming an AIENPs-FAM-reporter probe 2 complex, which is captured only by the C line, and no fluorescence is observed in the T2 line. Figure 3 A can directly observe that the visual detection limit (vLOD) is vLOD. DNA = 29 fg / μL, vLOD CFU =223 CFU / mL. Furthermore, by measuring the ratio of fluorescence signal values ​​of the T1 line to the C line (T1 / C value), the linear range of the DNA fluorescence signal was 2.90 × 10⁻⁶. 0 ~2.90×10 5 fg / μL, the linear range of bacterial fluorescence signal is 2.23× 10 ~2.23×10 5 The limits of quantitation (LOQ) were 3.48 fg / μL and 3.12 CFU / mL, respectively. When the sample contained only *E. coli* O157:H7, the number of lysed reporter probe 2 molecules increased with increasing concentration, leading to a gradual enhancement of the T2 detection line. Reporter probe 1 remained intact, forming a fluorescent complex at the C line, with visually obtained vLODs of 25.1 fg / μL and 195 CFU / mL, respectively. The logarithm of DNA and bacterial concentrations was found to be 2.51 × 10⁻⁶ CFU / mL when the sample contained only *E. coli* O157:H7. 0 ~2.51×10 5 fg / μL and 1.95× 10 ~1.95×10 5 A linear correlation was observed within the range of CFU / mL. Figure 3 B), with LOQs of 3.31 fg / μL and 15.48 CFU / mL, respectively.

[0039] The effectiveness of multiplex LFCS in detecting different targets was evaluated by simultaneously introducing LM and Escherichia coli O157:H7 into samples. The concentrations of both pathogens showed a consistent upward trend and remained at similar levels. As shown in Figure C, decreasing bacterial concentrations led to a reduction in the amount of probe cleaved, resulting in a weaker T-line and a gradually stronger C-line. The vLODs of LM and E. coli O157:H7 were identical based on both DNA and bacterial concentration. The LOQ, derived from the T / C values, was 3.10 fg / μL (2.39 CFU / mL) for LM and 2.76 fg / μL (2.94 CFU / mL) for E. coli O157:H7.

[0040] Unlike the stable C-line on traditional test strips, the intensity of the C-line in this study varied. The C-line fluorescence signal weakened with increasing target concentration in the sample, which may reflect the effectiveness of Cas12a cleavage.

[0041] 2.4 RPA-CRISPR-LFCS Specificity Assessment To verify the specificity of this method, several foodborne pathogens, including LM, Escherichia coli O157:H7, Staphylococcus xylosus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio hemolyticus, and Staphylococcus aureus, were tested. Figure 4 This indicates that RPA-CRISPR-LFCS can effectively identify targets. The specificity of the CRISPR system mainly depends on the specificity of the crRNA for the target. The crRNA transcribed and processed from the CRISPR repeat spacer sequence array guides the Cas protein sequence to specifically recognize invading DNA. Targeting sequences with abundant 3'-t protospacer neighbor motifs (PAMs) is a necessary condition for achieving lysis. This method has strong specificity and applicability, and can rapidly screen for LM and E. coli O157:H7 in samples.

[0042] 2.5 Practical Application Different concentrations of pure culture LM and Escherichia coli O157:H7 were introduced into pork homogenate. Figure 5 AC analysis showed that increasing the bacterial concentration resulted in significant fluorescence on both the T1 and T2 detection lines. This color change reduced the detection concentrations of LM and E. coli O157:H7 to as low as 2.23 × 10⁻⁶. 3 CFU / mL and 1.95×10 3 CFU / mL. The vLOD in the sample extract was lower compared to the sensitivity test. Because pork is a complex food sample containing high concentrations of lipids and proteins, the detection sensitivity may be affected.

[0043] LFCS was used to analyze actual food samples to further test the capability of the established method. Results showed that all samples were free from bacterial contamination, and PCR analysis confirmed the LFCS results. Figure 5 (D). The results confirm the accuracy and reliability of this method in the analysis of actual food samples. The results show that RPA-CRISPR-LFCS can effectively identify targets in different food samples.

[0044] III. Conclusion In summary, the AIENPs-based RPA-CRISPR-LFCS method constructed in this invention can be used for highly sensitive screening of LM and Escherichia coli O157:H7. This invention has developed different nanoprobes, namely AIENPs-FAM and AIENPs-digoxigenin. This method uses RPA technology to exponentially amplify the target sites and combines it with CRISPR / Cas12a trans cleavage reaction to achieve qualitative and quantitative analysis of multiple target sites on different T lines.

[0045] The sensitivity of the method described in this invention was evaluated, and the results showed that the detection limit for Listeria monocytogenes (LM) was 29 fg / μL (223 CFU / mL), and the detection limit for Escherichia coli O157:H7 was 25.1 fg / μL (195 CFU / mL). Quantitative detection was performed using grayscale values ​​obtained from ImageJ, with a LOQ of 3.10 fg / μL (2.39 CFU / mL) for LM and 2.76 fg / μL (2.94 CFU / mL) for Escherichia coli O157:H7. Furthermore, this platform exhibits strong specificity and can effectively detect the presence of Listeria monocytogenes (LM) and Escherichia coli O157:H7 in real food products.

Claims

1. A dual-probe system for simultaneously detecting Listeria and Escherichia coli O157:H7, characterized in that, Includes AIENPs-FAM fluorescent nanoprobes and AIENPs-digoxigenin fluorescent nanoprobes; The AIENPs-FAM fluorescent nanoprobe has aggregation-induced emission nanoparticles as its core and an anti-fluorescein isothiocyanate antibody covalently coupled to the surface of the aggregation-induced emission nanoparticle core. The AIENPs-digoxigenin fluorescent nanoprobes consist of aggregation-induced emission nanoparticles as the core, and anti-digoxigenin antibodies covalently coupled to the surface of the aggregation-induced emission nanoparticle core.

2. An immunochromatographic test strip for detecting Listeria and Escherichia coli O157:H7, characterized in that, Includes a base plate and a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad disposed on the base plate; The nitrocellulose membrane is provided with a first detection line, a second detection line, and a quality control line sequentially arranged along the chromatography direction. The first and second detection lines are respectively coated with capture molecules capable of capturing aggregation-induced emission nanoparticle-labeled anti-fluorescein isothiocyanate antibody or aggregation-induced emission nanoparticle-labeled anti-digoxin antibody; The quality control line is coated with recombinant streptavidin; The binding pad is coated with AIENPs-FAM fluorescent nanoprobes and AIENPs-digoxigenin fluorescent nanoprobes.

3. The immunochromatographic test strip as described in claim 2, characterized in that, The first detection line is coated with 1 mg / mL of IgG m+ and 1.25% BSA; the second detection line is coated with 1 mg / mL of IgG r+ and 1% BSA; and the control line is coated with 2 mg / mL of recombinant streptavidin.

4. The immunochromatographic test strip as described in claim 2, characterized in that, The sample pad is pretreated with a buffer solution containing 0.01 M TB buffer, 0.5% bovine serum albumin, 1% Tween-20 and 1% sucrose.

5. The immunochromatographic test strip as described in claim 2, characterized in that, The amount of AIENPs-FAM fluorescent nanoprobes and AIENPs-digoxigenin fluorescent nanoprobes used is 5 μL.

6. An RPA-CRISPR-LFCS method for simultaneous detection of Listeria monocytogenes and Escherichia coli O157:H7, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the genomic DNA as a template, Listeria monocytogenes was simultaneously amplified using recombinase polymerase amplification technology. hly Genes and E. coli O157:H7 rfb E gene, obtained hly Gene amplification products and rfb E gene amplification product; (3) Add the amplification product from step (2) to the CRISPR / Cas12a reaction system, wherein the reaction system contains LbCas12a protein and targets hly The first crRNA of the gene, targeting rfb The E gene has a second crRNA, a first paired-end labeled ssDNA reporter probe, and a second paired-end labeled ssDNA reporter probe; wherein, one end of the first paired-end labeled ssDNA reporter probe is labeled with biotin and the other end is labeled with digoxigenin, and one end of the second paired-end labeled ssDNA reporter probe is labeled with biotin and the other end is labeled with fluorescein isothiocyanate. (4) Load the CRISPR reaction product from step (3) onto the sample pad of the immunochromatographic test strip and perform chromatography along the test strip under the action of capillary force. (5) Observe the fluorescence signals of the first and second detection lines on the test strip under ultraviolet light to determine whether Listeria monocytogenes and / or Escherichia coli O157:H7 are present.

7. The method as described in claim 6, characterized in that, The sequence of the first paired-end labeled ssDNA probe is 5'-biotin-TTATT-Dig-3', and the sequence of the second paired-end labeled ssDNA probe is 5'-biotin-TTATT-FAM-3'.

8. The method as described in claim 6, characterized in that, In step 4), the concentration of the first paired-end labeled ssDNA reporter probe is 500 nM, and the concentration of the second paired-end labeled ssDNA reporter probe is 1000 nM.

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