Double-target portable detection method for Listeria monocytogenes by Argonaute-mediated reverse fluorescence enhanced lateral flow technology and application of double-target portable detection method for Listeria monocytogenes by Argonaute-mediated reverse fluorescence enhanced lateral flow technology

Abstract

The invention belongs to the technical field of microbiological detection, and discloses a method for realizing dual, rapid and portable detection of Listeria monocytogenes by integrating PfAgo cutting and LAMP amplification by virtue of a fluorescence and reverse fluorescence enhanced lateral flow technology, and the method is based on an Argonaute combined isothermal amplification detection technology, integrates a signal amplification system of PfAgo cutting and LAMP isothermal amplification, and realizes rapid and portable detection of Listeria monocytogenes by virtue of a fluorescence and reverse fluorescence enhanced lateral flow technology. And the reverse fluorescence lateral flow technology is combined, so that the listeria monocytogenes can be sensitively, selectively and accurately detected on site in a short time. According to the detection method based on the visual reverse fluorescence test strip, visual and reverse fluorescence mutual verification for rapid and ultra-sensitive detection and application of Listeria monocytogenes can be realized by combining visual colorimetry and a reverse fluorescence lateral flow technology, dependence on a large instrument is not needed, the cost is relatively low, the operation is simple and convenient, and the detection method can be applied to rapid and ultra-sensitive detection of Listeria monocytogenes. And portable on-site detection of the food-borne pathogenic bacteria can be realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, in particular to a dual-target portable detection method and application of Listeria monocytogenes by Argonaute-mediated reverse fluorescence enhanced lateral flow technology. Background Art

[0002] Foodborne illness is a type of disease caused by various toxic and harmful substances that enter the human body through food intake, usually with infectivity or toxicity. It is statistically shown that hundreds of thousands of people die from foodborne illness globally every year. Bacterial pathogens, due to their physiological characteristics and strong environmental adaptability, pose the most serious threat to foodborne illness. Food safety is a focus issue of national and social concern, attracting much attention from the government, academia, and the public. Microbial, chemical, and physical hazards can all lead to food safety problems, and these problems can occur at any stage of food production, processing, storage, transportation, and sales. Among them, food safety and medical health problems caused by foodborne pathogenic bacteria are particularly prominent, and the spread of diseases caused by them globally has seriously threatened human life and health.

[0003] Listeria monocytogenes (LM) is simply referred to as Listeria monocytogenes. It is a facultative anaerobic bacterium, belonging to Gram-positive bacteria and the phylum Firmicutes. It is also the only pathogenic bacterium that can cause zoonosis among the seven Listeria genera internationally. It is listed as one of the four important foodborne pathogenic bacteria together with Salmonella, Escherichia coli O157, and Shigella. Listeria monocytogenes often exists in raw and processed foods, such as dairy products, meats, vegetables, seafood, aquatic products, heat-treated meat products, and cheeses, and is usually the source of Listeria infection. It is widely distributed in nature, not easily affected by freezing and thawing, has strong tolerance to adverse environments, is salt-tolerant, acid-tolerant, and can tolerate relatively high osmotic pressure. It can survive even at 0°C - 45°C, especially can grow and reproduce at the refrigeration temperature of the refrigerator, and can even survive for 1 year in the freezer at -20°C, so it is known as the "refrigerator killer". It mostly occurs in summer and autumn. The transmission route of Listeria monocytogenes is mainly through oral-fecal transmission, and can also enter the body through the eyes, damaged skin, and mucous membranes to cause infection. Pregnant women, infants, the elderly, and those with weakened immune systems are susceptible to Listeria disease. The most common symptoms include fever, headache, muscle pain, nausea, vomiting, and diarrhea, with persistent high fever being common. Severe cases can cause diseases such as miscarriage, stillbirth, sepsis, pneumonia, and meningitis, and may even lead to death. Therefore, rapid detection of pathogenic Listeria monocytogenes helps to accurately assess and control the risk of pathogens in the food chain, and is of great significance for ensuring food safety and people's health.

[0004] There are many pain points in the detection of Listeria monocytogenes. The traditional plate culture and identification method takes a long time for detection. It takes several days or even a week from enrichment to biochemical identification, and the operation is complex, with high requirements for personnel skills and experimental environment, and the results are easily affected by operation errors. Immunological detection methods such as enzyme-linked immunosorbent assay have specificity, but are easily interfered by sample impurities, etc., resulting in false positives or false negatives, and the reliability is poor. Molecular biology detection methods such as PCR / qPCR rely on professional personnel and large instruments, with high costs, and the detection process is complex, unable to meet the needs of point-of-care diagnosis. Other detection methods based on isothermal amplification and nucleic acid probes also have their limitations. For example, isothermal amplification may have non-specific amplification, and the sensitivity may decrease when detecting mutant strains by nucleic acid probes. In addition, the samples themselves also pose challenges. There are many interfering substances in actual samples, which will affect the detection sensitivity and accuracy; the content of Listeria monocytogenes in different samples varies greatly, and low-content samples are prone to missed detection, threatening food safety. With the continuous progress of biosensor detection technology and the development of new programmable nucleases, the development of nucleic acid detection technology has been greatly promoted, making the rapid point-of-care detection of Listeria monocytogenes possible. Argonaute protein has become an ideal choice for nucleic acid detection sensors due to its programmability, sequence specificity, high sensitivity, and single-base resolution. Although this technology can achieve signal amplification, in order to ensure high sensitivity, isothermal amplification is usually combined with the amplification of the detection target.

[0005] Argonaute (abbreviated as Ago), as another programmable and target-activated nuclease, is a highly conserved protein widely present in natural organisms such as eukaryotes and prokaryotes. It can use single-stranded "guide RNA" or "guide DNA" to find invading RNA or DNA with complementary sequences. It can be divided into eukaryotic Argonaute protein (eAgos) and prokaryotic Argonaute protein (pAgos) according to the source. In recent years, nucleic acid detection technologies based on thermophilic pAgo (such as PfAgo and TtAgo) have been studied and established. Some studies have shown that under high-temperature conditions, the PfAgo protein can use 5'-phosphorylated ssDNA guidance to cleave single-stranded or double-stranded DNA targets. A new gene editing technology - PfAgo-based artificial restriction enzyme, multiple pairs of gDNA primers can mediate PfAgo to cleave double-stranded DNA, and compared with traditional endonucleases, PfAgo can recognize any region of the target sequence. The PfAgo-based nucleic acid diagnostic method has been successfully applied to the detection of the novel coronavirus, and a rapid, scalable, and portable detection system for COVID-19 has been developed by combining reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) with the target sequence of PfAgo. In summary, PfAgo has great advantages in in vitro diagnosis.

[0006] Lateral flow chromatography (LFC), also known as lateral immuno-chromatography or immunochromatographic strip technology, is a rapid detection technology based on immunoreaction and chromatography principles. It usually consists of a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad, a support plate, etc. With the nitrocellulose membrane as the carrier, parts such as the sample pad are demarcated, and bioactive substances are immobilized. During detection, the sample binds to the antibody / antigen of the labeled chromogenic substance and moves under capillary action. The target substance binds to the test line and shows color, and the control line is used to judge whether the detection is normal. This technology has the advantages of simple operation, no need for professional equipment and complex training; fast detection speed, with results available in 5 - 15 minutes; high flexibility, capable of detecting a variety of substances; low cost, with cheap consumables and no need for large-scale instruments, facilitating on-site detection, etc. It is widely used in the fields of medical diagnosis, food safety detection, environmental monitoring, animal husbandry, and agriculture.

[0007] The present invention for the first time uses PfAgo integrated with LAMP amplification-mediated reverse fluorescence-enhanced lateral flow technology for dual detection of drug-resistant bacteria, and has both high sensitivity and high specificity. In summary, the method of the present invention is beneficial to reducing reagent consumption, shortening the detection time, improving the detection reliability, and realizing ultra-sensitive portable on-site detection.

[0008] Through retrieval, no patent publication documents related to the patent application of the present invention have been found. Summary of the Invention

[0009] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a dual-target portable detection method and application of Argonaute-mediated reverse fluorescence-enhanced lateral flow technology for Listeria monocytogenes.

[0010] The technical solution adopted by the present invention to solve its technical problems is:

[0011] A method for dual, rapid, and portable detection of Listeria monocytogenes by integrating PfAgo cleavage and LAMP amplification with the aid of fluorescence and reverse fluorescence-enhanced lateral flow technology, which is not for the purpose of diagnosing and treating diseases. The method is based on the Argonaute-binding isothermal amplification detection technology, integrates the signal amplification system of PfAgo cleavage and LAMP isothermal amplification, and then combines the reverse fluorescence lateral flow technology to sensitively, selectively, and accurately detect Listeria monocytogenes on-site in a short time.

[0012] Further, when the target DNA is present, PfAgo can cleave the target DNA under the guidance of the guiding DNA, generating secondary ssDNA, which can bind to empty-PfAgo as a new guiding DNA and guide its specific cleavage of the fluorescent probe. The cleavage of PfAgo is based on strict base complementary pairing, and a dual detection platform is designed based on this; when only pathogenic and ciprofloxacin-resistant genes are present in the system, hly amplicons and lde amplicons can be obtained after LAMP amplification. PfAgo cleaves the hly amplicon and lde amplicon respectively under the guidance of the corresponding guiding DNA to obtain secondary ssDNA 1 and ssDNA2, which serve as new guiding DNAs to guide PfAgo to cleave fluorescent probe 1 (double-labeled with 5’FAM and 3’BHQ1, complementary to ssDNA1) and fluorescent probe 2 (double-labeled with 5’ROX and 3’BHQ2, complementary to ssDNA2); when only pathogenic Listeria monocytogenes is present in the system, only hly amplicons can be obtained after LAMP amplification. After PfAgo cleavage under the guidance of the corresponding guiding DNA, the resulting secondary ssDNA 1 guides PfAgo to cleave fluorescent probe 1; when ciprofloxacin-resistant Listeria monocytogenes is present in the system, only lde amplicons can be obtained after LAMP amplification. After PfAgo cleavage under the guidance of the corresponding guiding DNA, the resulting secondary ssDNA 2 guides PfAgo to cleave fluorescent probe 2; when none of the above three bacteria are present in the system, there are no amplicons and no fluorescent probes are cleaved, and no fluorescence is generated;

[0013] Similarly, in the reverse fluorescence lateral flow test strip, when pathogenic and ciprofloxacin-resistant genes exist in the system, the L1 and L2 reporter molecules replace FAM-ssDNA-BHQ1 and ROX-ssDNA-BHQ2 for PfAgo cleavage; Rhodamine 6G (R6G)-labeled streptavidin (SA) cannot form T1-Biotin-SA / R6G and T2-Biotin-SA / R6G on the T1 and T2 lines respectively to specifically bind to the probe-labeled AuNPs, namely AuNPs@DNA 1) and AuNPs@DNA 2, with the L1 and L2 reporter molecules. There is no visible red change on T1 and T2. At the same time, there is obvious R6G fluorescence on the T1 and T2 lines; when only pathogenic Listeria monocytogenes exists in the system, only the hly amplicon can be obtained after LAMP amplification. In the reverse fluorescence lateral flow test strip, the L2 reporter molecule is not cleaved, while the L1 reporter molecule is cleaved. A specific binding of AuNPs@DNA / L2 / T2-Biotin-SA / R6G will form on the T2 line, while this binding will not form on the T1 line, and a visible red change will form on the T2 line; at the same time, the R6G fluorescence on the T1 line is quenched; when only ciprofloxacin-resistant Listeria monocytogenes exists in the system, only the lde amplicon can be obtained after LAMP amplification. In the reverse fluorescence lateral flow test strip, a visible red change will form on the T1 line; at the same time, the R6G fluorescence on the T2 line is quenched; when none of the above three bacteria exist in the system, there is no amplicon. In the reverse fluorescence lateral flow test strip, there is no visible red change on the T1 and T2 lines; at the same time, the R6G fluorescence on the T1 and T2 lines is quenched. This method outputs fluorescence signals through an enzyme-labeled instrument and visual signals through a reverse fluorescence lateral flow test strip. This method can achieve highly sensitive detection of Listeria monocytogenes based on the R6G fluorescence intensity signal, and this signal is negatively correlated with the generation of visible AuNPs signals on the strip, providing a possibility for rapid and sensitive on-site detection.

[0014] Further, it includes the following steps:

[0015] The reverse fluorescence enhanced test strip consists of five main components, including a PVC backplane, a sample pad, a conjugate pad, an NC membrane, and an absorbent pad; for the T and C lines, different concentrations of SA, Biotin-modified DNA strands T1 and C1, and the fluorescent dye R6G are incubated in buffer MEST at 37 °C for 2 h, and then scribed onto the NC membrane at a rate of 1 μL / cm; the conjugate pad needs to be soaked in a specific buffer and then dried overnight; finally, the sample pad, the treated conjugate pad, the scribed NC membrane, and the absorbent pad are assembled on the backing card, cut into test strips 3.8 mm wide, and placed in a plastic card to obtain the reverse fluorescence enhanced test strip;

[0016] First, add the designed SH-DNA to TCEP and activate it in a metal heating block at 37°C for 2 hours. Then, add the colloidal gold solution with a particle size of 20 nm synthesized by reducing chloroauric acid with trisodium citrate. After pipetting and mixing evenly, place it in a -20°C refrigerator and freeze for 2 hours. After thawing at room temperature, centrifuge at 12,000 rpm for 20 minutes, discard the supernatant, resuspend and wash twice with ultrapure water. The red precipitate is the AuNPs@DNA probe. Resuspend it in ultrapure water and store it at 4°C;

[0017] Inoculate Listeria monocytogenes resistant to ciprofloxacin in TSB medium and culture it in a shaker at 37°C for 24 hours. Extract the genome of Listeria monocytogenes using a rapid extraction reagent. Add the genome to the LAMP dual-target amplification system and incubate at 65°C for 15 minutes. Add the obtained amplification product to the PfAgo system for cleavage reaction and incubate at 95°C for 15 minutes. Combine the incubated PfAgo system with the probe AuNPs@DNA at 37°C for 10 minutes;

[0018] Collect the fluorescence signal of the reaction solution after PfAgo cleavage using a microplate reader, or drop the mixed solution of the PfAgo system and the probe AuNPs@DNA incubated well on a plastic card equipped with a reverse fluorescence enhancement test strip, and observe the aggregation change of the colloidal gold on the test strip with the naked eye. Observe the fluorescence quenching of the fluorophore R6G on the test strip under ultraviolet light irradiation. To distinguish positive samples from negative samples.

[0019] Furthermore, the specific steps are as follows:

[0020] (1) Mix the test sample with the genomic rapid extraction reagent to release the genome of Listeria monocytogenes;

[0021] Mix the sample and the rapid extraction reagent at a ratio of 1:1, that is, add 100 μL of the sample and then add 100 μL of the rapid extraction reagent. The mixed solution of the sample and the rapid extraction reagent is shaken and mixed evenly and left standing for 30 s;

[0022] Among them, the formula of the rapid extraction reagent is: 20 mM Tris with pH = 8.0, 25 mM NaCl, 2.5 mM EDTA, SDS with a mass concentration of 0.05%, PVP-40 with a mass concentration of 2%, and the solvent is water;

[0023] The preparation steps for every 1 L of the rapid extraction reagent are: Dissolve 2.42 g of Tris, 14.6 g of NaCl and 20 g of PVP-40 in 900 mL of water. Then add 5 mL of EDTA with pH 8.0 and 500 mM and 5 mL of SDS with a mass concentration of 10%. Finally, adjust the pH to 8.0 with hydrochloric acid and add water to a final volume of 1 L;

[0024] (2) Primer design;

[0025] According to the whole genome sequence of the target strain of Listeria monocytogenes, specific genes hly and lde sequences in the sequence were selected to design LAMP primers for the detection of Listeria monocytogenes;

[0026] (3) Perform LAMP amplification on the extracted genome;

[0027] Place the mixed LAMP amplification system in a 65°C constant temperature metal bath and incubate for 15 min to obtain the LAMP amplification product; The specific amplification system of LAMP is as follows:

[0028]

[0029]

[0030] (4) Preparation of AuNPs

[0031] Reduce chloroauric acid with trisodium citrate to synthesize a colloidal gold solution with a particle size of 20 nm. The specific steps are as follows: Add a 250 mM HAuCl4·3H2O solution to ultrapure water and heat to boiling under constant magnetic stirring; Subsequently, quickly inject a 38.84 mM trisodium citrate solution into the solution; After the solution color changes from colorless to stable transparent red, continue stirring for 15 min and the color remains unchanged, indicating the successful synthesis of AuNPs; Finally, stir the obtained AuNPs solution at room temperature for 15 min, cool to room temperature, and centrifuge and concentrate 5 times at 12000 rp / min to obtain the AuNPs solution, which is stored at 4°C for further use;

[0032] Among them, the ratio of HAuCl4·3H2O solution: ultrapure water: trisodium citrate solution in μL: mL: mL is 100:100:3;

[0033] (5) Preparation of AuNPs@DNA probe

[0034] The preparation process of the nucleic acid strand probe AuNPs@DNA modified with AuNPs is achieved through the formation of gold-sulfur Au-S covalent bonds: First, mix a 100 μM nucleic acid strand SH-DNA solution containing thiol modification with a 0.5 M, pH 7.7 tris(2-carboxyethyl)phosphine TCEP solution; Incubate in the dark at room temperature for 2 h to activate the thiol group; Add the activated SH-DNA strand to the AuNPs solution and freeze at -20°C for 2 h; After thawing at room temperature, centrifuge at 12000 rpm for 20 min; After centrifugation, discard the supernatant, add ultrapure water to resuspend and wash, centrifuge at 12000 rpm for 20 min, and repeat 2 times. The red precipitate is the AuNPs@DNA probe, which is resuspended in 200 μL of ultrapure water and stored at 4°C for subsequent experiments;

[0035] Among them, the volume ratio of the nucleic acid strand SH-DNA solution: (2-carboxyethyl)phosphine TCEP solution: AuNPs solution: ultrapure water is 5:2:200:200;

[0036] (6) Preparation of the reverse fluorescence-enhanced test strip

[0037] The reverse fluorescence-enhanced test strip consists of five main components, including a PVC bottom plate, a sample pad, a conjugate pad, an NC membrane, and an absorbent pad; for the T line, first dissolve rhodamine 6G (i.e., R6G) in PBS buffer at pH 7.4 to prepare a 100 mM solution. According to the molar ratio of R6G: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC): N-hydroxysuccinimide (NHS) of 1:5:5, slowly add EDC and NHS to the R6G solution while stirring, and continue stirring for 30 minutes to 1 hour to activate R6G and form an active intermediate capable of binding to streptavidin (SA); weigh SA and dissolve it in MEST buffer to prepare a 10 mg / mL solution; slowly drop the activated R6G solution into the SA solution while stirring to ensure sufficient contact; control the molar ratio of R6G to SA to be 5:1, and continue stirring and reacting at room temperature for 2 hours to allow the reaction of groups such as amino groups on R6G and SA molecules; then, add 100 μM of T1-Biotin or 100 μM of T2-Biotin to the mixed solution, and the molar ratio of SA to Biotin is 1:5. React at room temperature for 30 min. The obtained R6G-SA-Biotin-T1 or R6G-SA-Biotin-T2 solution is centrifuged at 12,000 rpm for 40 min at 4 °C using a 10 kD microporous ultrafiltration membrane, diluted to 1 mg / mL with MEST solution, and scribed onto the NC membrane at a rate of 1 μL / cm; for the C line, use the same method to prepare an SA-Biotin-C1 / C2 solution with a final concentration of SA of 3 mg / mL and scribe it onto the NC membrane at a rate of 1 μL / cm; the conjugate pad needs to be soaked in PBS buffer at pH 7.4 containing 5 mM PVP-1000, 0.15 M sucrose, 0.45 mM BSA, and 2% (mass concentration) Tween-20, and then dried overnight; finally, assemble the sample pad, the treated conjugate pad, the scribed NC membrane, and the absorbent pad on the backing card, cut it into test strips 3.8 mm wide, and place them in a plastic card; all the test strips are sealed in an aluminum foil bag with a desiccant for subsequent experiments;

[0038] Among them, the composition of the PBS buffer required for the reaction is: 10 mM NaCl, 10 mM KCl, 25 mM Na2HPO4, 7.8 mM K2HPO4, the solvent is water, and pH = 7.4;

[0039] The composition of the MEST buffer is as follows: 25 mM 2-(N-morpholino)ethanesulfonic acid MES, 0.05% (mass concentration) of Tween-20, and the solvent is water;

[0040] (7) The amplified product is added to the PfAgo system for cleavage reaction;

[0041] The LAMP amplification product obtained in step (3) is combined with the PfAgo reaction system, incubated at 95 °C for 15 min, and after the reaction, the fluorescence value is measured by a microplate reader, or the reaction solution is incubated with AuNPs@DNA probe at 37 °C for 10 min and then dropped on a reverse fluorescence enhanced test strip to output the result;

[0042] When the hly and lde targets are present, compared with the fluorescence value of the negative control group without targets, the fluorescence of the positive group with targets increases. Accordingly, the Linker-DNA is cleaved and cannot hybridize with the AuNPs@DNA probe and the T line, and thus cannot aggregate on the T line. At the same time, there is no aggregation of AuNPs on the T1 line and the T2 line, and the R6G fluorescence is not quenched, proving that the bacterium is Listeria monocytogenes with pathogenicity and ciprofloxacin resistance.

[0043] Furthermore, the total volume of the LAMP amplification system in step (3) is 25 μL per reaction, and the specific composition is as follows:

[0044] BufferⅠ 2.5 μL, 10 μM dNTPs mix 2.5 μL, 10× primer mix-hly 2.5 μL, 2% BSA 2.5 μL, Bst DNA polymerase 0.5 μL, genomic DNA to be detected 10 μL, ddH2O 4.5 μL;

[0045] Among them, the composition of BufferⅠ is: 200 mM Tris-HCl, pH 8.8, 500 mM KCl, 80 mM (NH4)2SO4, 20 mM MgSO4, 1% (mass concentration) of Tween-20, 600 mM betaine, and the solvent is water;

[0046] The 10× primer mix-hly gene amplification includes: 2 μM F3, 2 μM B3, 16 μM FIP, 16 μM BIP, 4 μM LoopF, 4 μM Loop B;

[0047] The dNTPs mix includes: 10 mM dATP, 10 mM dTTP, 10 mM dCTP, 10 mM dGTP, 10 mM dUTP.

[0048] Furthermore, the system of the PfAgo cleavage reaction is as follows:

[0049]

[0050] Furthermore, the PfAgo reaction system in step (7) is as follows: 1.0 μM PfAgo, 0.5 μM hly gene-guided DNAs, 0.5 μM lde gene-guided DNAs, 10 μL Target, 0.2 μM fluorescent probe A, 0.2 μM fluorescent probe 1, ddH2O, and reaction buffer;

[0051] Among them, the hly and lde gene Guide DNAs (Guide mix) include 3 corresponding guide DNAs, namely guide DNA1, guide DNA 2, and guide DNA 3. The total concentration of the three guide DNAs is 10 μM, and the molar concentration ratio of the three guide DNAs is 1:1:1;

[0052] The composition of the reaction buffer is: 200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 100 mM MgSO4, 800 mM betaine, 1% (mass concentration) Tween-20, pH = 8.8, and the solvent is water.

[0053] Furthermore, when pathogenic and ciprofloxacin-resistant genes are present in the system, hly amplicons and lde amplicons can be obtained through LAMP amplification. PfAgo cuts the hly amplicon and lde amplicon respectively under the guidance of the corresponding guide DNA to obtain secondary ssDNA 1 and ssDNA 2, which serve as new guide DNAs to respectively guide PfAgo to cut the fluorescent probe 1 (FAM-ssDNA-BHQ1, complementary to ssDNA 1) and the fluorescent probe 2 (ROX-ssDNA-BHQ2, complementary to ssDNA 2). In the reverse fluorescence lateral flow test strip, the L1 and L2 reporter molecules replace FAM-ssDNA-BHQ1 and ROX-ssDNA-BHQ2 for PfAgo cleavage; SA labeled with R6G cannot form T1-Biotin-SA / R6G and T2-Biotin-SA / R6G on the T1 and T2 lines to specifically bind to the probe-labeled AuNPs (AuNPs@DNA1) and AuNPs (AuNPs@DNA2) of the L1 and L2 reporter molecules respectively, and no visible red change occurs on the T1 and T2 lines. At the same time, there is obvious R6G fluorescence on the T1 and T2 lines, and red aggregation on the C line; when pathogenic Listeria monocytogenes is present in the system, only hly amplicons can be obtained through LAMP amplification. After PfAgo cleavage under the guidance of the corresponding guide DNA, the secondary ssDNA 1 is obtained to guide PfAgo to cut the fluorescent probe 1. In the reverse fluorescence lateral flow test strip, the L1 reporter molecule is cleaved, while the L2 reporter molecule is not cleaved. The T2 line will form a specific binding of AuNPs@DNA2 / L2 / T2-Biotin-SA / R6G, while the T1 line will not form this binding. A visible red change occurs on the T2 line. At the same time, there is obvious R6G fluorescence on the T1 line, the R6G fluorescence on the T2 line is quenched, and red aggregation occurs on the C line; when ciprofloxacin-resistant Listeria monocytogenes is present in the system, only lde amplicons can be obtained through LAMP amplification. After PfAgo cleavage under the guidance of the corresponding guide DNA, the secondary ssDNA B is obtained to guide PfAgo to cut the fluorescent probe 2. In the reverse fluorescence lateral flow test strip, L2 is cleaved while L1 is not cleaved. The T1 line will form a specific binding of AuNPs@DNA1 / L1 / T1-Biotin-SA / R6G, while the T2 line will not form this binding. A visible red change occurs on the T1 line. At the same time, the R6G fluorescence on the T1 line is quenched, there is obvious R6G fluorescence on the T2 line, and red aggregation occurs on the C line; when none of the above three bacteria are present in the system, there are no amplicons and no fluorescent probes are cleaved. In the reverse fluorescence lateral flow test strip, neither the L1 nor the L2 reporter molecule is cleaved.The R6G-labeled SA can form T1-Biotin-SA / R6G and T2-Biotin-SA / R6G on the T1 and T2 lines respectively, which specifically bind to the probe-labeled AuNPs, namely AuNPs@DNA1 and AuNPs@DNA2, to the L1 and L2 reporter molecules. Visible red changes occur in T1 and T2. At the same time, the R6G fluorescence on the T1 and T2 lines is quenched, and red aggregation appears on the C line.

[0054] Furthermore, the lowest detection limit of the method is 1 CFU / mL; the detection time of the method only takes 45 minutes from nucleic acid extraction, LAMP amplification, PfAgo cleavage to signal output.

[0055] Application of the method as described above in the preparation of drugs and / or kits for detecting Listeria monocytogenes.

[0056] The advantages and positive effects achieved by the present invention are as follows:

[0057] 1. The present invention designs a biosensor based on the Argonaute system to achieve signal transduction and amplification, and is used to detect effective targets in Listeria monocytogenes.

[0058] 2. The PfAgo protein used in the present invention is a thermophilic protein with thermal stability, which avoids reactions at room temperature during the sample addition process and reduces experimental errors.

[0059] 3. The method established by the present invention can simultaneously amplify two target genes in one tube, simultaneously identify and cleave the products of the two targets, and analyze the detection results using multiple signals such as fluorescence signals generated by fluorescence analysis (PfAgo-fluorescence, PfAgo-FL) and reverse fluorescence enhanced test strip detection method (PfAgo-reverse fluorescent enhancement lateral flow test strip, PfAgo-rLFTS), colorimetric signals generated by the reverse fluorescence enhanced test strip, and reverse fluorescence signals. Moreover, it only takes 45 minutes from the sample entering to signal output, which is superior to most existing detection technologies.

[0060] 4. The nucleic acid test strip in the present invention is based on the principle of base complementary pairing, with higher specificity than common antigen-antibody test strips, and uses visual and reverse fluorescence dual signal outputs to verify each other and enhance the accuracy of the detection results.

[0061] 5. The sensor in the present invention is sensitive in detection, with a lowest detection limit of 1 CFU / mL, and can detect up to 10 8 CFU / mL, with a relatively wide detection range and high sensitivity, meeting the specified threshold.

[0062] 6. The detection method based on the visual reverse fluorescence test strip in the present invention combines visual colorimetry and reverse fluorescence lateral flow technology to achieve visual and reverse fluorescence mutual verification for rapid and ultrasensitive detection and application of Listeria monocytogenes. It does not rely on large instruments, has low costs, is easy to operate, and can realize portable on-site detection of foodborne pathogenic bacteria.

[0063] 7. The method of the present invention has good accuracy and applicability in complex food matrices. The biosensor based on the Argonaute system has been confirmed by detecting spiked real samples. The detection technology of the present invention based on Argonaute combined with isothermal amplification has high sensitivity and high specificity, provides a signal amplification system integrating PfAgo cleavage and LAMP isothermal amplification, and combined with reverse fluorescence lateral flow technology, makes it possible to sensitively, selectively and accurately detect foodborne pathogenic bacteria on-site in a short time. This method provides a new idea for accurately detecting pathogenic bacteria. The foodborne pathogenic bacteria detected in this experiment are Listeria monocytogenes.

[0064] 8. The detection technology of the present invention based on Argonaute combined with isothermal amplification has high sensitivity and high specificity, provides a signal amplification system integrating PfAgo cleavage and LAMP isothermal amplification, and combined with reverse fluorescence lateral flow technology, makes it possible to sensitively, selectively and accurately detect foodborne pathogenic bacteria on-site in a short time. This method provides a new idea for accurately detecting pathogenic bacteria. The foodborne pathogenic bacteria detected in this experiment are Listeria monocytogenes. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 It is a schematic diagram for detecting Listeria monocytogenes by the method proposed in the present invention; wherein 1A: is a schematic diagram for detecting Listeria monocytogenes by the established rapid extraction of sample genome and LAMP-PfAgo reaction platform; 1B: is a flow chart for detecting by the reverse fluorescence enhanced test strip; 1C: is a diagram for outputting detection results under different conditions;

[0066] Figure 2 It is a preparation process and characterization diagram of the probe required in the present invention; wherein, 2A: is a particle size diagram of AuNPs; 2B: is a characterization diagram of the hydrodynamic diameter of AuNPs; 2C: is a schematic diagram for preparing the AuNPs@DNA probe; 2D: is a Zeta potential characterization diagram; 2E: is an ultraviolet absorption characterization diagram of AuNPs and AuNPs@DNA;

[0067] Figure 3This is the feasibility diagram explored in the present invention; among which 3A: Schematic diagram of the cleavage of the single target sequence of the hly gene; 3B: Fluorescence image of a single component deletion; 3C: Fluorescence scanning image of a single component deletion; 3D: Single gene detection result diagram of the reverse fluorescence enhanced test strip with a single component deletion, top: macroscopic photograph, bottom: R6G fluorescence photograph; 3E: Single gene detection result diagram of the reverse fluorescence enhanced test strip, top: macroscopic photograph, bottom: R6G fluorescence photograph; 3F: Schematic diagram of the cleavage activity of PfAgo; 3G: PAGE gel verification diagram of the cleavage activity of PfAgo;

[0068] Figure 4 This is the key factor optimization diagram in the present invention; among which 4A: Optimization of the PfAgo concentration; 4B: Optimization of the guide DNA concentration; 4C: Optimization of the reaction time; 4D: Optimization of the reaction temperature; 4E: Optimization of the magnesium ion concentration; 4F: Influence of additives on the reaction system;

[0069] Figure 5 This is the key factor optimization and stability verification diagram of the test strip designed in the present invention; among which 5A: Visualization diagram of the optimization of the SA - Biotin - T1 concentration; 5B: Grayscale diagram of the optimization analysis of the SA - Biotin - T1 concentration using ImageJ software; 5C: Visualization diagram of the optimization of the SA - Biotin - C1 concentration; 5D: Grayscale diagram of the optimization analysis of the SA - Biotin - C1 concentration using ImageJ software; 5E: Visualization diagram of the optimization of the AuNPs@DNA addition amounts of the T1 and C lines; 5F: Grayscale diagram of the optimization analysis of the AuNPs@DNA addition amounts of the T1 and C lines using ImageJ software; 5G: Stability diagram of the output results of the reverse fluorescence enhanced test strip, top: macroscopic photograph, bottom: R6G fluorescence photograph; 5H: Grayscale diagram and reverse fluorescence diagram of the stability analysis of the output results of the reverse fluorescence enhanced test strip using ImageJ software; 5I: Stability diagram of the storage of the reverse fluorescence enhanced test strip, top: macroscopic photograph, bottom: R6G fluorescence photograph; 5J: Grayscale diagram and reverse fluorescence diagram of the stability analysis of the storage of the reverse fluorescence enhanced test strip using ImageJ software;

[0070] Figure 6 This is the detection performance analysis diagram of the method of the present invention; among which 6A: Fluorescence diagrams of different concentrations of the target; 6B: Result output diagrams of the reverse fluorescence enhanced test strip for different concentrations of the target, top: macroscopic photograph, bottom: R6G fluorescence photograph; 6C: Visualization grayscale diagram and result output diagram of the fluorescence of the reverse fluorescence test strip analyzed by ImageJ software for different concentrations of the target; 6D: Fluorescence intensity of specific detection; 6E: Result output diagrams of the reverse fluorescence enhanced test strip for specific detection, top: macroscopic photograph, bottom: R6G fluorescence photograph; 6F: Visualization grayscale diagram and result output diagram of the fluorescence of the reverse fluorescence test strip analyzed by ImageJ software for specific detection;

[0071] Figure 7 Reproducibility and repeatability diagrams in the present invention; 7A: Repeatability diagram of fluorescence detection; 7B: Repeatability diagram of reverse fluorescence enhanced test strip detection; 7C: Gray-scale and fluorescence analysis diagram of the repeatability of reverse fluorescence enhanced test strip detection using ImageJ software; 7D: Reproducibility diagram of fluorescence detection; 7E: Reproducibility diagram of reverse fluorescence enhanced test strip detection; 7F: Gray-scale and fluorescence analysis diagram of the reproducibility of reverse fluorescence enhanced test strip detection using ImageJ software;

[0072] Figure 8 Method comparison diagrams in the present invention; wherein 8A: Heat map analysis of detecting the hly gene in 30 spiked samples by comparing the methods of the present invention (output modes: colorimetric PfAgo-CMC, reverse fluorescence PfAgo-rLFTS-FL, fluorescence PfAgo-FL), qPCR (SG), and qPCR (TaqMan), wherein the PfAgo-CMC value is analyzed by gray scale using ImageJ and the PfAgo-rLFTS-FL value is analyzed by fluorescence using ImageJ; 8B: Visual colorimetric and fluorescence result diagram of reverse fluorescence enhanced test strip detection, top: macroscopic photograph; bottom: R6G fluorescence diagram; 8C: Output result diagram of the method PfAgo-FL of the present invention; 8D: ROC curve analysis; 8E: Radar diagram comparing the sensitivity, specificity, and accuracy of each method; 8F: Significant differences (paired two-tailed Student's t-test, ****P<0.0001) in detecting 30 randomly spiked samples by the methods of the present invention (output modes PfAgo-CMC, PfAgo-rLFTS-FL, PfAgo-FL), qPCR (SG), and qPCR (TaqMan);

[0073] Figure 9 Bland-Altman analysis diagrams of the method of the present invention and the qPCR method; wherein 9A-B: Bland-Altman analysis of the method PfAgo-CMC of the present invention and qPCR (SG), qPCR (TaqMan); 9C-D: Bland-Altman analysis of the method PfAgo-rLFTS-FL of the present invention and qPCR (SG), qPCR (TaqMan); 9E-F: Bland-Altman analysis of the method PfAgo-FL of the present invention and qPCR (SG), qPCR (TaqMan);

[0074] Figure 10This is the correlation analysis diagram between the method of the present invention and the qPCR detection method; among them, 10A-B: correlation analysis between PfAgo-CMC of the method of the present invention and the qPCR detection method; 10C-D: correlation analysis between PfAgo-rLFTS-FL of the method of the present invention and the qPCR detection method; 10E-F: correlation analysis between PfAgo-FL of the method of the present invention and the qPCR detection method;

[0075] Figure 11 This is the detection result diagram of the traditional method and the LAMP amplification method for Listeria monocytogenes detected by the present invention; among them, 11A: detecting aqueous solutions contaminated with different concentrations of Listeria monocytogenes by the plate counting method; 11B: LAMP amplification combined with polyacrylamide gel electrophoresis analysis, where M: standard DNA molecular weight marker Marker;

[0076] Figure 12 This is the detection diagram of real samples in the present invention; among them, 12A: fluorescence intensity diagram of detecting different concentrations of Listeria monocytogenes in spiked milk by the method of the present invention; 12B: reverse fluorescence enhanced test strip detection diagram of different concentrations of Listeria monocytogenes in spiked milk; 12C: fluorescence intensity diagram of detecting different concentrations of Listeria monocytogenes in spiked beef by the method of the present invention; 12D: reverse fluorescence enhanced test strip detection diagram of different concentrations of Listeria monocytogenes in spiked beef; 9E: fluorescence intensity diagram of detecting different concentrations of Listeria monocytogenes in spiked shrimp by the method of the present invention; 12F: reverse fluorescence enhanced test strip detection diagram of different concentrations of Listeria monocytogenes in spiked shrimp;

[0077] Figure 13 This is the result diagram of the method of the present invention for dual-gene detection; among them, 13A: schematic diagram of the cleavage of the double-target sequences of the hly gene and the lde gene; 13B: fluorescence scanning diagram of single-gene and dual-gene detection; 13C: measurement diagram of the fluorescence intensity end value of single-gene and dual-gene detection; 13D: feasibility result diagram of reverse fluorescence enhanced test strip dual-gene detection, N represents the double-detection negative result, P1 represents the result where only the hly gene exists in the double-detection, P2 represents the result where only the lde gene exists in the double-detection, and P represents the result where both the hly gene and the lde gene exist in the double-detection. Detailed implementation manners

[0078] The following further illustrates the present invention in conjunction with embodiments. The following embodiments are narrative and not restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0079] All kinds of experimental operations involved in the specific embodiments are conventional techniques in the art. For parts not specifically annotated in this article, those of ordinary skill in the art can refer to various commonly used reference books, scientific and technological literatures, or relevant specifications, manuals, etc. before the filing date of the present invention application for implementation.

[0080] A method for dual, rapid, and portable detection of *Listeria monocytogenes* by integrating PfAgo cleavage and LAMP amplification with fluorescence and reverse fluorescence enhanced lateral flow technology. The method is based on the Argonaute-binding isothermal amplification detection technology, integrating a signal amplification system of PfAgo cleavage and LAMP isothermal amplification, and then combining with reverse fluorescence lateral flow technology to sensitively, selectively, and accurately detect *Listeria monocytogenes* on-site in a short time.

[0081] When the target DNA is present, PfAgo can cleave the target DNA under the guidance of the guide DNA, generating secondary ssDNA, which can serve as a new guide DNA to bind to empty-PfAgo and guide its specific cleavage of the fluorescent probe. The cleavage of PfAgo is based on strict base complementary pairing, and a dual detection platform is designed based on this. When only pathogenic and ciprofloxacin-resistant genes are present in the system, hly amplicons and lde amplicons can be obtained after LAMP amplification. PfAgo cleaves the hly amplicon and lde amplicon respectively under the guidance of the corresponding guide DNA to obtain secondary ssDNA 1 and ssDNA 2, which serve as new guide DNAs to guide PfAgo to cleave fluorescent probe 1 (double-labeled with 5’FAM and 3’BHQ1, complementary to ssDNA 1) and fluorescent probe 2 (double-labeled with 5’ROX and 3’BHQ2, complementary to ssDNA 2) respectively. When only pathogenic *Listeria monocytogenes* is present in the system, only hly amplicons can be obtained after LAMP amplification. After cleavage by PfAgo under the guidance of the corresponding guide DNA, the resulting secondary ssDNA 1 guides PfAgo to cleave fluorescent probe 1. When ciprofloxacin-resistant *Listeria monocytogenes* is present in the system, only lde amplicons can be obtained after LAMP amplification. After cleavage by PfAgo under the guidance of the corresponding guide DNA, the resulting secondary ssDNA 2 guides PfAgo to cleave fluorescent probe 2. When none of the above three bacteria are present in the system, there are no amplicons and no fluorescent probes are cleaved, and no fluorescence is generated.

[0082] Similarly, in the reverse fluorescence lateral flow test strip, when pathogenic and ciprofloxacin-resistant genes exist in the system, the L1 and L2 reporter molecules replace FAM-ssDNA-BHQ1 and ROX-ssDNA-BHQ2 for PfAgo cleavage; R6G-labeled SA cannot form T1-Biotin-SA / R6G and T2-Biotin-SA / R6G on the T1 and T2 lines to specifically bind to the probe-labeled AuNPs, namely AuNPs@DNA 1) and AuNPs@DNA2, respectively, with the L1 and L2 reporter molecules. No visible red change occurs on the T1 and T2 lines. Meanwhile, there is obvious R6G fluorescence on the T1 and T2 lines, and red aggregation on the C line. When only pathogenic Listeria monocytogenes exists in the system, only the hly amplicon can be obtained after LAMP amplification. In the reverse fluorescence lateral flow test strip, the L2 reporter molecule is not cleaved, while the L1 reporter molecule is cleaved. A specific binding of AuNPs@DNA / L2 / T2-Biotin-SA / R6G will form on the T2 line, while such binding will not form on the T1 line. A visible red change occurs on the T2 line. Meanwhile, the R6G fluorescence on the T1 line is quenched, and red aggregation occurs on the C line. When only ciprofloxacin-resistant Listeria monocytogenes exists in the system, only the lde amplicon can be obtained after LAMP amplification. In the reverse fluorescence lateral flow test strip, a visible red change occurs on the T1 line. Meanwhile, the R6G fluorescence on the T2 line is quenched, and red aggregation occurs on the C line. When none of the above three bacteria exist in the system, no amplicon is produced. In the reverse fluorescence lateral flow test strip, no visible red change occurs on the T1 and T2 lines. Meanwhile, the R6G fluorescence on the T1 and T2 lines is quenched, and red aggregation occurs on the C line. This method outputs fluorescence signals through an enzyme-labeled instrument and visual signals through a reverse fluorescence lateral flow test strip. This method can achieve highly sensitive detection of Listeria monocytogenes based on the R6G fluorescence intensity signal, and this signal is negatively correlated with the generation of the visible AuNPs signal on the strip, providing a possibility for rapid and sensitive on-site detection.

[0083] It includes the following steps:

[0084] The reverse fluorescence enhanced test strip consists of five main components, including a PVC backplane, a sample pad, a conjugate pad, an NC membrane, and an absorbent pad; for the T and C lines, different concentrations of SA, Biotin-modified DNA strands T1 and C1, and the fluorescent dye R6G are incubated in buffer MEST at 37 °C for 2 h, and then scribed onto the NC membrane at a rate of 1 μL / cm; the conjugate pad needs to be soaked in a specific buffer and then dried overnight; finally, the sample pad, the treated conjugate pad, the scribed NC membrane, and the absorbent pad are assembled on the backing card, cut into test strips 3.8 mm wide, and placed in a plastic card to obtain the reverse fluorescence enhanced test strip;

[0085] First, add the designed SH-DNA to TCEP and activate it in a metal heating block at 37°C for 2 h. Then, add the colloidal gold solution with a particle size of 20 nm synthesized by reducing chloroauric acid with trisodium citrate. After pipetting and mixing evenly, place it in a -20°C refrigerator and freeze for 2 h. After thawing at room temperature, centrifuge at 12,000 rpm for 20 min. Resuspend and wash twice with ultrapure water. The red precipitate is the AuNPs@DNA probe. Resuspend it in ultrapure water again and store it at 4°C;

[0086] Inoculate Listeria monocytogenes resistant to ciprofloxacin in TSB medium and culture it with shaking at 37°C for 24 h. Extract the genome of Listeria monocytogenes using a rapid extraction reagent. Add the genome to the LAMP dual-target amplification system and incubate at 65°C for 15 min. Add the obtained amplification product to the PfAgo system for cleavage reaction and incubate at 95°C for 15 min. Combine the incubated PfAgo system with the probe AuNPs@DNA at 37°C for 10 min;

[0087] Collect the fluorescence signal of the reaction solution after PfAgo cleavage using a microplate reader, or drop the mixed solution of the PfAgo system and the probe AuNPs@DNA incubated well on a plastic card equipped with a reverse fluorescence enhancement test strip, and observe the change in colloidal gold aggregation of the test strip with the naked eye. Observe the fluorescence quenching of the fluorophore R6G on the test strip under ultraviolet light irradiation. To distinguish positive samples from negative samples.

[0088] The specific steps are as follows:

[0089] (1) Mix the test sample with the genomic rapid extraction reagent to release the genome of Listeria monocytogenes;

[0090] Mix the sample and the rapid extraction reagent at a ratio of 1:1, that is, add 100 μL of the sample and then add 100 μL of the rapid extraction reagent. The mixed solution of the sample and the rapid extraction reagent is shaken and mixed evenly and left standing for 30 s;

[0091] Among them, the formula of the rapid extraction reagent is: 20 mM Tris with pH = 8.0, 25 mM NaCl, 2.5 mM EDTA, SDS with a mass concentration of 0.05%, PVP-40 with a mass concentration of 2%, and the solvent is water;

[0092] The preparation steps for every 1 L of the rapid extraction reagent are: Dissolve 2.42 g of Tris, 14.6 g of NaCl, and 20 g of PVP-40 in 900 mL of water. Then add 5 mL of EDTA with pH 8.0 and 500 mM and 5 mL of SDS with a mass concentration of 10%. Finally, adjust the pH to 8.0 with hydrochloric acid and add water to a final volume of 1 L;

[0093] (2) Primer design;

[0094] Based on the complete genome sequence of the target strain of Listeria monocytogenes, specific genes hly and lde sequences in the sequence were selected to design LAMP primers for the detection of Listeria monocytogenes;

[0095] (3) Perform LAMP amplification on the extracted genome;

[0096] Place the mixed LAMP amplification system in a 65°C constant-temperature metal bath and incubate for 15 min to obtain the LAMP amplification product; The specific amplification system of LAMP is as follows:

[0097]

[0098]

[0099] (4) Preparation of AuNPs

[0100] Reduce chloroauric acid with trisodium citrate to synthesize a colloidal gold solution with a particle size of 20 nm. The specific steps are as follows: Add 250 mM HAuCl4·3H2O solution to ultrapure water and heat to boiling under constant magnetic stirring; Subsequently, quickly inject 38.84 mM trisodium citrate solution into the solution; After the solution color changes from colorless to stable transparent red, continue stirring for 15 min and keep the color unchanged, indicating the successful synthesis of AuNPs; Finally, stir the obtained AuNPs solution at room temperature for 15 min, cool to room temperature, and centrifuge and concentrate 5 times at 12000 rp / min to obtain the AuNPs solution, which is stored at 4°C for further use;

[0101] Among them, the ratio of HAuCl4·3H2O solution: ultrapure water: trisodium citrate solution in μL: mL: mL is 100:100:3;

[0102] (5) Preparation of AuNPs@DNA probe

[0103] The preparation process of the nucleic acid strand probe AuNPs@DNA modified with AuNPs is achieved through the formation of gold-sulfur Au-S covalent bonds: First, mix 100 μM SH-DNA solution containing thiol-modified nucleic acid strand with 0.5 M, pH 7.7 tris(2-carboxyethyl)phosphine TCEP solution; Incubate in the dark at room temperature for 2 h to activate the thiol group; Add the activated SH-DNA strand to the AuNPs solution and freeze at -20°C for 2 h; After thawing at room temperature, centrifuge at 12000 rpm for 20 min; After centrifugation, discard the supernatant, add ultrapure water to resuspend and wash, centrifuge at 12000 rpm for 20 min, and repeat 2 times. The red precipitate is the AuNPs@DNA probe, which is resuspended in 200 μL of ultrapure water and stored at 4°C for subsequent experiments;

[0104] Among them, the volume ratio of the nucleic acid strand SH-DNA solution: (2-carboxyethyl)phosphine TCEP solution: AuNPs solution: ultrapure water is 5:2:200:200;

[0105] (6) Preparation of the reverse fluorescence-enhanced test strip

[0106] The reverse fluorescence-enhanced test strip consists of five main components, including a PVC bottom plate, a sample pad, a conjugate pad, an NC membrane, and an absorbent pad; for the T line, first dissolve rhodamine 6G (i.e., R6G) in a PBS buffer solution with a pH of 7.4 to prepare a 100 mg / mL solution. According to the molar ratio of R6G:1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC):N-hydroxysuccinimide (NHS) of 1:5:5, slowly add EDC and NHS to the R6G solution while stirring, and continue stirring for 30 minutes to 1 hour to activate R6G and form an active intermediate capable of binding to streptavidin (SA); weigh SA and dissolve it in MEST buffer solution to prepare a 10 mg / mL solution; slowly drop the activated R6G solution into the SA solution while stirring to ensure sufficient contact; control the molar ratio of R6G to SA to be 5:1, and continue stirring and reacting at room temperature for 2 hours to make the amino groups and other groups on the R6G and SA molecules react; then, add 100 μM of T1-Biotin or 100 μM of T2-Biotin to the mixed solution, and the molar ratio of SA to Biotin is 1:5. React at room temperature for 30 min. The obtained R6G-SA-Biotin-T1 or R6G-SA-Biotin-T2 solution is centrifuged at 12,000 rpm for 40 min at 4 °C using a 10 kD microporous ultrafiltration membrane, diluted to 1 mg / mL with MEST solution, and scribed onto the NC membrane at a rate of 1 μL / cm; for the C line, use the same method to prepare an SA-Biotin-C1 / C2 solution with a final SA concentration of 3 mg / mL and scribe it onto the NC membrane at a rate of 1 μL / cm; the conjugate pad needs to be soaked in a PBS buffer solution with a pH of 7.4 containing 5 mM PVP-1000, 0.15 M sucrose, 0.45 mM BSA, and 2% (mass concentration) Tween-20, and then dried overnight; finally, assemble the sample pad, the treated conjugate pad, the scribed NC membrane, and the absorbent pad on the backing card, cut it into test strips 3.8 mm wide, and place them in a plastic card; all the test strips are sealed in an aluminum foil bag with a desiccant for subsequent experiments;

[0107] Among them, the composition of the PBS buffer solution required for the reaction is: 10 mM NaCl, 10 mM KCl, 25 mM Na2HPO4, 7.8 mM K2HPO4, the solvent is water, and pH = 7.4;

[0108] The composition of the MEST buffer is as follows: 25 mM 2-(N-morpholino)ethanesulfonic acid MES, 0.05% (mass concentration) of Tween-20, and the solvent is water;

[0109] (7) The amplified product is added to the PfAgo system for cleavage reaction;

[0110] The LAMP amplification product obtained in step (3) is combined with the PfAgo reaction system, incubated at 95 °C for 15 min, and after the reaction is completed, the fluorescence value is measured by a microplate reader, or the reaction solution is incubated with AuNPs@DNA probe at 37 °C for 10 min and then dropped on a reverse fluorescence enhancement test strip to output the result;

[0111] When the hly and lde targets are present, compared with the fluorescence value of the negative control group without targets, the fluorescence of the positive group with targets increases. Accordingly, the Linker-DNA is cleaved and cannot hybridize with the AuNPs@DNA probe and the T line, and thus cannot aggregate on the T line. At the same time, there is no aggregation of AuNPs on the T1 line and the T2 line, and the R6G fluorescence is not quenched, proving that the bacterium is Listeria monocytogenes with pathogenicity and ciprofloxacin resistance.

[0112] The specific volume of the LAMP amplification system in step (3) is 25 μL per reaction as follows:

[0113] BufferⅠ 2.5 μL, 10 μM dNTPs mix 2.5 μL, 10× primer mix-hly 2.5 μL, 2% BSA 2.5 μL, Bst DNA polymerase 0.5 μL, genomic DNA to be detected 10 μL, ddH2O 4.5 μL;

[0114] Among them, the composition of BufferⅠ is: 200 mM Tris-HCl, pH 8.8, 500 mM KCl, 80 mM (NH4)2SO4, 20 mM MgSO4, 1% (mass concentration) of Tween-20, 600 mM betaine, and the solvent is water;

[0115] 10× primer mix-hly gene amplification includes: 2 μM F3, 2 μM B3, 16 μM FIP, 16 μM BIP, 4 μM LoopF, 4 μM Loop B;

[0116] dNTPs mix includes: 10 mM dATP, 10 mM dTTP, 10 mM dCTP, 10 mM dGTP, 10 mM dUTP.

[0117] Furthermore, the system of the PfAgo cleavage reaction is as follows:

[0118] Component Volume μL PfAgo 1.5 Guide DNAs (10 μM) 5 Reporter / Linker (2 μM) 10 Target 10 10×PfAgo buffer 10 <![CDATA[ddH2O]]> 63.5 Total volume 100

[0119] In step (7), the PfAgo reaction system is as follows: 1.0 μM PfAgo, 0.5 μM hly gene-guided DNAs, 0.5 μM lde gene-guided DNAs, 10 μL Target, 0.2 μM fluorescent probe A, 0.2 μM fluorescent probe 1, ddH2O, and reaction buffer;

[0120] Among them, the hly and lde gene Guide DNAs (Guide mix) include 3 corresponding guide DNAs, namely guide DNA1, guide DNA 2, and guide DNA 3. The total concentration of the three guide DNAs is 10 μM, and the molar concentration ratio of the three guide DNAs is 1:1:1;

[0121] The composition of the reaction buffer is: 200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 100 mM MgSO4, 800 mM betaine, 1% (mass concentration) Tween-20, pH = 8.8, and the solvent is water.

[0122] When pathogenic and ciprofloxacin-resistant genes exist in the system, hly amplicons and lde amplicons can be obtained through LAMP amplification. PfAgo cuts the hly amplicon and lde amplicon respectively under the guidance of the corresponding guide DNA to obtain secondary ssDNA 1 and ssDNA 2, which serve as new guide DNAs to guide PfAgo to cut fluorescence probe 1 (FAM-ssDNA-BHQ1, complementary to ssDNA 1) and fluorescence probe 2 (ROX-ssDNA-BHQ2, complementary to ssDNA 2). In the reverse fluorescence lateral flow strip, L1 and L2 reporter molecules replace FAM-ssDNA-BHQ1 and ROX-ssDNA-BHQ2 for PfAgo cleavage; SA labeled with R6G cannot form T1-Biotin-SA / R6G and T2-Biotin-SA / R6G on T1 and T2 lines to specifically bind to probe-labeled AuNPs (AuNPs@DNA1) and AuNPs (AuNPs@DNA2) with L1 and L2 reporter molecules respectively, and no visible red change occurs on T1 and T2. At the same time, there is obvious R6G fluorescence on T1 and T2 lines, and red aggregation on the C line; when pathogenic Listeria monocytogenes exists in the system, only hly amplicons can be obtained through LAMP amplification. After PfAgo cleavage under the guidance of the corresponding guide DNA, secondary ssDNA A is obtained to guide PfAgo to cut fluorescence probe 1. In the reverse fluorescence lateral flow strip, the L1 reporter molecule is cleaved, while the L2 reporter molecule is not cleaved. The T2 line will form a specific binding of AuNPs@DNA2 / L2 / T2-Biotin-SA / R6G, while such binding will not form on T1. A visible red change occurs on T2. At the same time, there is obvious R6G fluorescence on the T1 line, the R6G fluorescence on the T2 line is quenched, and red aggregation occurs on the C line; when ciprofloxacin-resistant Listeria monocytogenes exists in the system, only lde amplicons can be obtained through LAMP amplification. After PfAgo cleavage under the guidance of the corresponding guide DNA, secondary ssDNA B is obtained to guide PfAgo to cut fluorescence probe 2. In the reverse fluorescence lateral flow strip, L2 is cleaved while L1 is not cleaved. The T1 line will form a specific binding of AuNPs@DNA1 / L1 / T1-Biotin-SA / R6G, while such binding will not form on T2. A visible red change occurs on T1. At the same time, the R6G fluorescence on the T1 line is quenched, there is obvious R6G fluorescence on the T2 line, and red aggregation occurs on the C line; when none of the above three bacteria exist in the system, there are no amplicons and no fluorescence probes are cleaved. In the reverse fluorescence lateral flow strip, neither the L1 nor the L2 reporter molecule is cleaved.The R6G-labeled SA can form T1-Biotin-SA / R6G and T2-Biotin-SA / R6G on T1 and T2 lines respectively, which specifically bind to the probe-labeled AuNPs, namely AuNPs@DNA1 and AuNPs@DNA2, with the L1 and L2 reporter molecules. T1 and T2 show a visible red change. At the same time, the R6G fluorescence on T1 and T2 lines is quenched, and there is a red aggregation on the C line.

[0123] The lowest detection limit of the method is 1 CFU / mL; the detection time of the method only requires 45 minutes from nucleic acid extraction, LAMP amplification, PfAgo cleavage to signal output.

[0124] Specifically, the related preparation and detection are as follows:

[0125] Example 1. Detection principle of the method of the present invention

[0126] PfAgo is a novel nucleic acid-guided endonuclease with nucleic acid targeting, cleavage ability and programmability, which plays an important role in biosensors. PfAgo realizes precise target recognition and cleavage functions through a 5'-phosphorylated single-stranded DNA (ssDNA) guide strand. The detection system established in the present invention innovatively integrates a dual detection platform of a test strip method (PfAgo-rLFTS) and a fluorescence method (PfAgo-FL). The core workflow is as Figure 1 shown in A. First, the genomic DNA of Listeria monocytogenes is extracted, and the conserved sequences of its virulence gene hly and resistance gene lde are specifically amplified by LAMP technology to provide a primary recognition target for subsequent enzymatic cleavage reactions. First, under the synergistic action of three specific guide DNAs, the hly gene and lde gene are targeted for primary cleavage to generate 16-nt 5'-phosphorylated ssDNA 1 and ssDNA2. Subsequently, the ssDNA serves as a novel guide strand to base-pair with the fluorescence-labeled probe respectively to trigger secondary cleavage. For the fluorescence method (PfAgo-FL) dual detection platform, the dual-labeled fluorescence reporter probe (FAM-ssDNA-BHQ1, ROX-ssDNA-BHQ2) is specifically cleaved. Through this cascade signal amplification mechanism, the molecular conformational change is efficiently converted into a detectable signal. In the fluorescence detection mode, enzymatic cleavage causes the spatial separation of the fluorophore and the quencher group, thereby generating a fluorescence signal that can be detected by a microplate reader.

[0127] Figure 1 Figure B describes the schematic diagram of the color development of the reverse fluorescence-enhanced test strip method (PfAgo-rLFTS). In this method, the Linker DNA 1 (L1) reporter molecule replaces the fluorescence reporter probe FAM-ssDNA-BHQ1, Linker DNA 1 (L 2)The reporter molecule replaces the fluorescent reporter probe ROX-ssDNA-BHQ2, and triggers signal conversion through PfAgo-mediated cascade cleavage combined with a reverse fluorescence-enhanced test strip. Figure 1 C shows the sequence and complementary cleavage schematic diagram of guide DNAs and detection targets during the detection of the pathogenic gene hly gene of Listeria monocytogenes and the lde gene resistant to ciprofloxacin. In the reverse fluorescence-enhanced test strip, the cleavage product realizes specific recognition and dual interpretation of pathogenic genes through a reverse signal conversion strategy. The fluorescence quenching effect of AuNPs on rhodamine 6G (R6G) mainly works through the synergistic action of two mechanisms: fluorescence resonance energy transfer (FRET). When the distance between R6G and AuNPs is between 1 and 10 nm, non-radiative energy transfer occurs between the donor (R6G) and the acceptor (AuNPs), resulting in fluorescence attenuation; plasmon resonance energy transfer (PRET) - the strong scattered light generated by the surface plasmon resonance of AuNPs overlaps with the excitation spectrum of R6G, and the fluorescence signal is suppressed through competitive light absorption interference. Based on this phenomenon, a reporter molecule (AuNPs@DNA) using DNA-functionalized AuNPs was designed for the PfAgo reverse fluorescence-enhanced test strip detection system.

[0128] For positive samples, the L1 and L2 reporter molecules are specifically cleaved by PfAgo, resulting in the inability to form a complete capture complex at the corresponding test lines T1 and T2 of the test strip - the colloidal gold-labeled probes (AuNPs@DNA 1, AuNPs@DNA2) cannot bind to T1-Biotin-SA / R6G and T2-Biotin-SA / R6G immobilized on the test line due to the loss of the L1 and L2 binding sites. At this time, no visible red bands (absence of colloidal gold aggregation signal) appear on the T1 and T2 lines, but the R6G fluorescence signal is significantly manifested in the chemiluminescence / fluorescence imaging analysis system due to the intact molecular conformation. And because SH-DNA 1 / 2 on AuNPs@DNA is complementary to the bases of C1-Biotin-SA / R6G and C2-Biotin-SA / R6G on the C line respectively, colloidal gold aggregates to present a red band on the C line; conversely, for negative samples, when there is no target strain in the sample, after LAMP amplification, the absence of amplicons of the hly gene and lde gene keeps the L1 and L2 reporter molecules intact. The corresponding AuNPs@DNA, Linker DNA and T-Biotin-SA / R6G form a stable complex. The T line shows a red band due to the enrichment of colloidal gold. At the same time, the R6G fluorophore is quenched, and the remaining colloidal gold-labeled probes that do not aggregate on the T line aggregate on the C line to present a red band. This technical system realizes exponential signal amplification through a two-stage cascade cleavage design, combines the dual-mode output of fluorescence quantification and reverse fluorescence-enhanced test strip for rapid screening, and fully utilizes the target-compatible advantage of the programmability of the guide DNA, providing a modular technical platform for the multiplex detection of foodborne pathogenic bacteria. Its integrated design not only meets the accurate quantification requirements of the laboratory, but also opens up a new path for on-site point-of-care testing.

[0129] The system of the PfAgo cleavage reaction is as follows:

[0130] Component Volume μL PfAgo 1.5 Guide (10 μM) 5 Reporter / Linker (2 μM) 10 Target 10 10×PfAgo buffer 10 <![CDATA[ddH2O]]> 63.5 Total volume 100

[0131] Among them, the nucleic acid sequences used in the system are as follows:

[0132] Three specific guide DNA sequences of the hly gene: Guide1 / 2 / 3 are shown in SEQ ID NO.07-09;

[0133] Three specific guide DNA sequences of the lde gene: Guide1 / 2 / 3 are shown in SEQ ID NO.24-26;

[0134] FAM-ssDNA-BHQ: The nucleic acid sequence is shown in SEQ ID NO.10;

[0135] ROX-ssDNA-BHQ2: The nucleic acid sequence is shown in SEQ ID NO.27;

[0136] Linker DNA 1: The nucleic acid sequence is as shown in SEQ ID NO.11;

[0137] Linker DNA2: The nucleic acid sequence is as shown in SEQ ID NO.28.

[0138] The composition of 10×PfAgo buffer is: 200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 100 mM MgSO4, 800 mM betaine, 1% (mass concentration) Tween-20, pH = 8.8, and the solvent is water.

[0139] The hly and lde gene Guide DNAs (Guide DNAs) include 3 corresponding guide DNAs, namely guide DNA 1, guide DNA2, and guide DNA 3. The total concentration of the three guide DNAs is 10 μM, and the molar concentration ratio of the three guide DNAs is 1:1:1.

[0140] Example 2: Bacterial culture and colony forming unit (CFU) test

[0141] Listeria monocytogenes grows in Tryptic Soy Broth (TSB) medium. Liquid culture is carried out in a flask with a shaker at 37 °C for 24 h. For the colony forming unit (CFU) test, serial dilution samples of bacteria were prepared. 100 μL of the diluted sample was taken and spread on a Listera Chromogenic Plate (purchased from Haibo Biotechnology Co., Ltd., Qingdao High-Tech Industrial Park), and kept at 37 °C for 24 - 28 h, and the number of colonies on each plate was recorded.

[0142] Example 3: Rapid extraction of the genome of Listeria monocytogenes

[0143] The present invention selects a prepared rapid genomic extraction reagent (20 mM Tris (pH = 8.0), 25 mM NaCl, 2.5 mM EDTA, 0.05% (wt / vol) SDS, 2% (wt / vol) PVP-40 (polyvinylpyrrolidone), and the solvent is water) to rapidly extract the genome of Listeria monocytogenes in the bacterial liquid or food sample. 100 μL of the Listeria monocytogenes culture of the sample to be detected was added to a 1.5 mL centrifuge tube, and then 100 μL of the rapid genomic extraction reagent was added, shaken and mixed evenly, and left standing for 30 s.

[0144] Example 4: Primer design and LAMP amplification

[0145] On the website of the American Type Culture Collection (ATCC), download the whole genome sequence of the target strain of Listeria monocytogenes (ATCC19115), and select the specific gene hly and lde sequences in the sequence to design LAMP primers for the detection of Listeria monocytogenes. The LAMP primer sequences of the hly gene are shown in SEQ ID NO.01 - 06, and the LAMP primer sequences of the lde gene are shown in SEQ ID NO.18 - 23.

[0146] Place the mixed LAMP amplification system in a 65°C constant-temperature metal bath and incubate for 15 min to obtain the LAMP amplification product. The specific amplification system of LAMP is as follows:

[0147] Component Volume (μL) 10× Primer mix 2.5 dNTPs mix (10 μM) 2.5 Buffer Ⅰ 2.5 2% BSA 2.5 Bst DNA polymerase 0.5 Genome 10 <![CDATA[ddH2O]]> 4.5 Total volume 25

[0148] *Note:

[0149] 1. The components of BufferⅠ are: 200 mM Tris-HCl, pH 8.8, 500 mM potassium chloride, 100 mM ammonium sulfate, 20 mM magnesium sulfate, 1% Tween-20, 600 mM betaine, and the solvent is water.

[0150] 2. 10× primer mix includes: 16 μM FIP, 16 μM BIP, 2 μM F3, 2 μM B3, 4 μM Loop F, 4 μM LoopB.

[0151] 3. dNTPs mix includes: 10 mM dATP, 10 mM dTTP, 10 mM dCTP, 10 mM dGTP, 10 mM dUTP.

[0152] Example 5: Preparation of AuNPs, AuNPs@DNA and reverse fluorescence-enhanced test strips

[0153] Reduce chloroauric acid with trisodium citrate to synthesize a colloidal gold solution with a particle size of 20 nm. Add 100 μL of 250 mM HAuCl4·3H2O to 100 mL of ultrapure water and heat to boiling under constant magnetic stirring. Subsequently, quickly inject 3 mL of 38.84 mM trisodium citrate into the solution. After the solution color changes from colorless to a stable transparent red and remains unchanged after continuous stirring for 15 min, it indicates the successful synthesis of AuNPs. Finally, stir the obtained AuNPs solution for 15 min without heating, cool to room temperature, and centrifuge and concentrate 5 times at 12,000 rpm to obtain the AuNPs solution, which is stored at 4°C for further use.

[0154] The preparation process of the nucleic acid strand probe modified with AuNPs (AuNPs@DNA 1) is achieved through the formation of gold-sulfur (Au-S) covalent bonds. First, 5 μL of the nucleic acid strand modified with a mercapto group (SH-DNA 1, 100 μM, the sequence is shown in SEQ ID NO. 12) is mixed with 2 μL of tris(2-carboxyethyl)phosphine (TCEP, 0.5 M, pH 7.7). Incubate in the dark at room temperature for 2 h to activate the mercapto group. The activated SH-DNA 1 strand is added to 200 μL of the AuNPs solution and frozen at -20 °C for 2 h. After thawing at room temperature, centrifuge at 12,000 rpm for 20 min. After centrifugation, discard the supernatant, add 200 μL of ultrapure water to resuspend and wash twice. The red precipitate is the AuNPs@DNA 1 probe for the hly gene. Resuspend it in 200 μL of ultrapure water and store it at 4 °C for subsequent experiments. As Figure 2 shown, the prepared AuNP, SH-DNA, and AuNPs@DNA were characterized. The AuNPs@DNA2 probe for the lde gene was prepared by the same method, and its SH-DNA2 sequence is shown in SEQ ID NO. 29.

[0155] The reverse fluorescence enhanced test strip consists of five main components, including a PVC bottom plate, a sample pad, a conjugate pad, an NC membrane, and an absorbent pad. For the T line, first dissolve rhodamine 6G (R6G) in PBS (pH 7.4) buffer to prepare a solution of about 100 mM. According to the molar ratio of R6G, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) of 1:5:5, slowly add EDC and NHS to the R6G solution while stirring, and continue stirring for 30 minutes to 1 hour to activate R6G and form an active intermediate capable of binding to streptavidin (SA). Weigh an appropriate amount of SA and dissolve it in MEST buffer to prepare a 10 mg / mL solution. Slowly drip the activated R6G solution into the SA solution while stirring to ensure sufficient contact. Control the molar ratio of R6G to SA to be 5:1, and continue stirring and reacting at room temperature for 2 hours to allow the reaction of R6G with amino groups and other groups on the SA molecule. Then, add T1-Biotin (100 μM) or T2-Biotin (100 μM) to the mixed solution (the molar ratio of SA to Biotin is 1:5), and react at room temperature for 30 min. The obtained R6G-SA-Biotin-T1 or R6G-SA-Biotin-T2 solution is centrifuged at 12,000 rpm for 40 min at 4 °C using a 10 kD microporous ultrafiltration membrane, diluted to 1 mg / mL with MEST solution, and scribed onto the NC membrane at a rate of 1 μL / cm. For the C line, use the same method to prepare an SA-Biotin-C1 / C2 solution with a final SA concentration of (3 mg / mL), and scribe it onto the NC membrane at a rate of 1 μL / cm. The glass fiber conjugate pad needs to be pre-soaked in PBS buffer at pH 7.4 (containing 5 mM PVP-1000, 0.15 M sucrose, 0.45 mM BSA, 2% Tween-20, and the solvent is water), and then dried overnight in a 37 °C forced air drying oven. Finally, stack the sample pad, the treated conjugate pad, the scribed nitrocellulose membrane (NC membrane), and the absorbent pad precisely on the PVC backplane in the chromatography flow direction, ensuring that the edges of each component overlap by 1.5 mm to form a capillary siphon channel. Use an automatic strip cutter to cut the chromatography plate into 3.8 mm wide test strips and place them in prefabricated plastic card slots. All test strips are sealed in an aluminum foil bag with a desiccant for subsequent experiments.

[0156] Among them, the nucleic acid sequences used in the system are as follows:

[0157] T1-Biotin: The nucleic acid sequence is as shown in SEQ ID NO.13;

[0158] C1-Biotin: The nucleic acid sequence is as shown in SEQ ID NO.14;

[0159] T2-Biotin: The nucleic acid sequence is as shown in SEQ ID NO.30;

[0160] C2-Biotin: The nucleic acid sequence is as shown in SEQ ID NO.31;

[0161] The composition of PBS buffer is: 10 mM NaCl, 10 mM KCl, 25 mM Na2HPO4, 7.8 mM K2HPO4, pH = 7.4, and the solvent is water;

[0162] The composition of MEST buffer is: 25 mM 2-(N-morpholino)ethanesulfonic acid (MES), 0.05% (v / v) Tween-20, and the solvent is water.

[0163] Example 6. Feasibility verification experiment

[0164] To prove the ability of the present invention to simultaneously detect two targets of Listeria monocytogenes based on specific recognition and cleavage, the feasibility of the platform of the present invention was verified. The specific detection system is as follows:

[0165]

[0166] As Figure 3 shown, first, a single-gene detection of the pathogenic gene hly of Listeria monocytogenes was performed. When the reaction system completely contains PfAgo protein, Guide DNA, target DNA, reaction Buffer containing Mg 2+ and the fluorescent reporter molecule (FAM-ssDNA-BHQ1), a significant specific fluorescent response signal was detected at an excitation wavelength of 484 nm and an emission wavelength of 529 nm, while no significant fluorescence enhancement signal was detected in the negative control group (lacking PfAgo protein, Guide DNA, target DNA, reaction Buffer containing Mg 2+ and the fluorescent reporter molecule). The background fluorescence intensity of the blank control group (only containing the reporter molecule) remained at a low level, excluding the interference of the spontaneous fluorescence of the reporter molecule on the detection result. Similarly, in the reverse fluorescence-enhanced test strip, when the reaction system is complete, the L1 reporter molecule is specifically cleaved by PfAgo, resulting in the inability to form a complete capture complex at the corresponding T line of the test strip - the colloidal gold-labeled probe (AuNPs@DNA 1) cannot bind to the T1-Biotin-SA / R6G immobilized on the test line due to the loss of the L1 binding site. At this time, there is no visible red band at the T line (the absence of colloidal gold aggregation signal), but the R6G fluorescence signal is significantly manifested due to the intact molecular conformation, and the C line shows a red band; while in the negative control group (lacking PfAgo protein, Guide DNA, target DNA and reaction Buffer containing Mg 2+When in the reaction Buffer, the L1 reporter molecule remains intact and can form a stable complex with the corresponding AuNPs@DNA 1 and T1-Biotin-SA / R6G. The T line shows a red band due to the enrichment of colloidal gold, while the fluorescence group of R6G is quenched and the C line shows a red band. In addition, PAGE-electrophoresis verification proves that the reporter probe can only be cleaved and degraded when Ago, guide DNAs, the target, and the reaction Buffer are present simultaneously.

[0167] The above experiments preliminarily prove the feasibility of the method of the present invention, which has high specific recognition and cleavage capabilities.

[0168] Example 7. Optimize the reaction conditions

[0169] To obtain the best performance, the present invention optimizes some key factors. Based on test tubes and using fluorescence intensity as an index, the present invention optimizes the reaction conditions of PfAgo cleavage combined with the LAMP technique to improve the detection performance of the system, which is more conducive to subsequent loading onto a reverse fluorescence lateral flow test strip for detecting Listeria monocytogenes. The protein concentration of PfAgo, the concentration of guide DNA, the optimal reaction temperature, the concentration of magnesium ions, and the reaction time required will all affect the cleavage system. Therefore, the present invention optimizes these conditions. First, under the fixed reaction conditions: 0.5 μM Guide DNAs, 10 μL Target DNA (amplified from 10 8 copies of the Listeria monocytogenes genome), 200 nM Reporter, 10 μL reaction Buffer, and a reaction duration of 30 min at 95 °C, the effect of protein concentration on detection sensitivity is explored by establishing a continuous final concentration gradient (0 - 3.0 μM). Similarly, we optimize the concentration of Guide DNAs, the reaction time, the optimal reaction temperature of PfAgo protein, and the Mg 2+ concentration respectively, and each group of experiments is repeated three times. And because the food detection matrix is relatively complex, the influence of common food additives on the system stability is also evaluated. As Figure 4 shown, the optimal protein concentration of PfAgo finally optimized by the present invention is 1.0 μM, the optimal concentration of guide DNA is 0.5 μM, the optimal reaction temperature is 93 °C, the optimal concentration of magnesium ions is 10 mM, the negative and positive can be distinguished after 5 min of reaction, and a relatively high fluorescence signal can be obtained after 15 min, that is, the shortest reaction time is 15 min, and food additives have little influence on the cleavage system, and the experimental system is stable.

[0170] Subsequently, the present invention further optimizes the key factors of the reverse fluorescence test strip. Based on the aggregation of AuNP@DNA on the test strip as an indicator, the present invention optimizes the concentrations of streptavidin (SA) on the test line and control line of the test strip, as well as the optimal addition amount of the probe AuNPs@DNA to improve the detection performance of the system. T line SA-biotin-T1 concentration: 10 μL of colloidal gold probe AuNPs@DNA and 10 μL of the PfAgo reaction system were dropped onto test strips with SA-biotin-T1 at different scribing concentrations (0.25, 0.50, 0.75, 1.00, 1.50, 2.00 mg / mL), and the final volume was made up to 50 μL with PBS buffer at pH 7.4, mixed well, incubated at 37 °C for 10 min, and the visualization image was recorded with a smartphone to screen out the optimal concentration of SA-biotin-T1. The C line SA-biotin-C1 concentration was optimized in the same way. Then, the scribing concentrations of the optimized SA-biotin-T1 and SA-biotin-C1 were fixed, different amounts of AuNPs@DNA (0, 2, 4, 6, 8, 10, 12 μL) and 10 μL of the PfAgo reaction system were added, and the final volume was made up to 50 μL with PBS buffer at pH 7.4, mixed well, and incubated at 37 °C for 10 min. Then the mixture was dropped onto the test strip loaded into the cartridge, and the visualization image was recorded with a smartphone to screen out the optimal volume of AuNPs@DNA. Moreover, the test strip is vulnerable to the environment (such as temperature, humidity, light, etc.), packaging (packaging damage or poor sealing), time (shelf life), production and other factors (contact with volatile, corrosive and other chemical substances), etc., which have an adverse impact on the performance of the test strip. Therefore, its stability was also verified. As Figure 5 shown, the optimal concentration of SA on the finally optimized test line is 1 mg / mL, the optimal concentration of SA on the control line is 3 mg / mL, and the optimal addition amount of the probe AuNPs@DNA is 10 μL. The results output by the prepared test strip still show obvious differences between positive and negative after 48 h, and the detection results are still accurate after the prepared test strip is stored in a dry and dark place for 120 days. Therefore, the detection performance of the test strip prepared by the present invention is stable.

[0171] Example 8, Sensitivity and Selectivity

[0172] The detection performance of the method of the present invention was analyzed. Based on the optimized PfAgo reaction system concentration, the LAMP amplification products of single hly or lde genes at different concentrations were cleaved. The composition of PfAgo fluorescence analysis (PfAgo-fluorescence, PfAgo-FL) in a 100 μL reaction volume included: 1.5 μM PfAgo, 0.5 μM Guide DNAs, 200 nM reporter gene (FAM-ssDNA-BHQ1), 10× reaction buffer (200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 100 mM MgSO4, 800 mM betaine, 1% Tween-20, pH 8.8), and 10 μL LAMP amplicon. Incubate at 95 °C for 15 min, and obtain the quantified fluorescence intensity of the incubated PfAgo system through a multifunctional microplate reader, or replace the reporter gene (FAM-ssDNA-BHQ1) in the reaction with Linker DNA 1 (the sequence is shown in SEQ ID NO.11) for PfAgo cleavage for PfAgo-reverse fluorescent enhancement lateral flow test strip (PfAgo-rLFTS) analysis. Take 10 μL of the incubated PfAgo system and add 10 μL of probe AuNPs@DNA 1 to 30 μL of PBS buffer (10 mM NaCl, 10 mM KCl, 25 mM Na2HPO4, 7.8 mM K2HPO4, pH = 7.4), bind at 37 °C for 10 min, and output the results through visual colorimetry and reverse fluorescence test strips, such as Figure 6 A-C. The results showed that the detection limit of the method of the present invention could reach 1 CFU / mL, and the dynamic detection range was 10 8 CFU / mL. To verify the selectivity of the proposed biosensor, genomic solutions containing Listeria monocytogenes, human cervical cancer cell line HeLa, methicillin-resistant Staphylococcus aureus, Salmonella typhimurium, Escherichia coli, Bacillus subtilis, Agrobacterium tumefaciens, Staphylococcus aureus, Pichia pastoris, Acetobacter, Lactobacillus, and Pseudomonas aeruginosa were added respectively to verify the selectivity of the detection platform. The results are as Figure 6 shown in D-F. The results showed that only Listeria monocytogenes had fluorescence output, there was no aggregation of AuNPs on the test line of the visual test strip, and there was fluorescence output of the R6G fluorophore on the reverse fluorescence test strip, further indicating that the detection specificity of this method was good.

[0173] Example 9. Reproducibility and repeatability

[0174] In addition, the repeatability and reproducibility of the biosensor of the present invention were verified to obtain the relative standard deviation (RSD) value, as Figure 7 shown. The RSD values of all calculated repeatability and reproducibility were less than 8%, indicating acceptable repeatability and reproducibility. Repeatability and reproducibility are key issues in the development of biosensors. Generally, it is recommended that the RSD value be less than 10% as a prerequisite for verifying the reliability of the developed biosensor.

[0175] Example 10, Method Comparison

[0176] In addition, 30 spiked samples (including 7 negative and 23 positive samples) were randomly selected for double-blind detection. First, the method of the present invention was used for detection, and then qPCR was used for parallel detection (TaqMan / SYBR Green). Fluorescence signal results were obtained on a qPCR instrument, fluorescence signals were obtained on an ELISA reader, visual results were obtained by visual inspection on a reverse fluorescence test strip, and reverse fluorescence results were obtained using a chemiluminescence / fluorescence imaging analysis system. As Figure 8 shown, the accuracy of the method of the present invention is equivalent to or better than that of qPCR. In the ROC curve results of the spiked sample detection, the area under the curve (AUC) based on the reverse fluorescence test strip for hly was 1.0, 0.944 for qPCR (TaqMan), and 0.956 for qPCR (SG). The radar chart also showed the same results. In addition, the Bland-Altman analysis well demonstrated the consistency between the method of the present invention and qPCR (as Figure 9 - 10 ). The analysis of the double-blind verification cohort results showed that both the fluorescence signal of Listeria monocytogenes and the reverse fluorescence enhanced test strip could effectively distinguish contaminated and uncontaminated samples (P < 0.0001). It was observed that the content of Listeria monocytogenes in the 23 contaminated samples was higher than that in the uncontaminated samples. The Real-time qPCR data also showed the expression difference of the Listeria monocytogenes genome between negative and contaminated samples (P < 0.0001); however, there was still a certain proportion of signal overlap between the two cohorts. In contrast, the multiple result output methods of the method of the present invention were mutually verified to ensure the accuracy of the detection. The specific cleavage of PfAgo also avoided false positive results caused by non-specific amplification of LAMP alone. The specificity, accuracy, and sensitivity of the method of the present invention were all 100%, which was better than the SYBR Green and TaqMan methods of qPCR. In this experiment, Listeria chromogenic agar plates and agarose gel electrophoresis were also used to detect Listeria monocytogenes, as Figure 11As shown. However, the traditional plate counting method is complex in operation, cumbersome in steps, has high requirements for personnel technology, and the accuracy of colony counting is easily affected by subjective judgment. The detection takes a long time. The culture conditions limit the detection of microbial species, the selectivity of the culture medium is limited, the detection sensitivity is low, and it cannot be used for on-site detection. The LAMP amplification method is simple in operation, can intuitively judge the presence of the target, and the cost is also low, but it cannot perform quantitative detection, takes a long time, and cannot judge and distinguish the amplification of single targets. Therefore, compared with other methods, the method of the present invention has good accuracy, specificity, and sensitivity, and can be used for portable on-site detection of foodborne pathogenic bacteria.

[0177] Among them, the nucleic acid sequences used in qPCR are as follows:

[0178] hly-qPCR-F: The nucleic acid sequence is as shown in SEQ ID NO.15;

[0179] hly-qPCR-R: The nucleic acid sequence is as shown in SEQ ID NO.16;

[0180] hly-TaqMan-probe: The nucleic acid sequence is as shown in SEQ ID NO.17;

[0181] Example 11. Application of the sensor in detecting Listeria monocytogenes

[0182] The potential of the method of the present invention to detect Listeria monocytogenes contamination in daily foods (such as milk, beef, and shrimp) was studied. As Figure 12 shown, by adding Listeria monocytogenes cultures at different concentrations, the detection limits of milk, beef, and shrimp reached 10 0 CFU / mL, which was consistent with the sensitivity detection results. All kinds of spiked samples could be significantly detected within 45 minutes. The method of the present invention was comparable or better for most Listeria monocytogenes tests.

[0183] Based on the operability of the test strip, the present invention also verified the ability of the method of the present invention for dual-gene detection, and detected the hly gene and lde gene of Listeria monocytogenes, as Figure 13When neither of the two targets is present in the system, the PfAgo-FL output method exhibits low fluorescence, and the test strips T1 and T2 lines of the PfAgo-rLFTS output method show visible red changes to the naked eye. At the same time, the R6G fluorescence on T1 and T2 lines is quenched, and there is aggregation of colloidal gold on the C line to form a red band; when only a single target is present in the system, there is an increase in single fluorescence, and one of the T1 and T2 lines of the reverse fluorescence lateral flow test strip shows visible red changes to the naked eye. At the same time, the R6G fluorescence corresponding to the red change on T1 and T2 lines is quenched, and there is still aggregation of colloidal gold on the C line to form a red band; when both targets are present in a system simultaneously, both fluorescences increase, and the T1 and T2 lines of the reverse fluorescence lateral flow test strip do not show visible red changes to the naked eye. At the same time, there is obvious R6G fluorescence on T1 and T2 lines, and there is aggregation of colloidal gold on the C line to form a red band. The excellent performance of the method of the present invention for simultaneous dual-target detection can well distinguish the excellent detection performance of the target genes contained in Listeria monocytogenes.

[0184] In summary, the method proposed by the present invention is comparable or even superior in performance indicators such as sensitivity, dynamic range, and detection time compared with the methods reported in most literatures. These detailed data strongly confirm that the biosensing strategy of the present invention has good applicability in detecting Listeria monocytogenes in real samples, and also demonstrates that the method of the present invention has great potential in food detection and early diagnosis of Listeria monocytogenes infection, and is expected to bring new breakthroughs and applications to related fields.

[0185] Sequences used in the present invention:

[0186]

[0187] The related technologies for detecting Listeria monocytogenes in recent years were sorted out and compared with the sensor of the present invention. As shown in Table 1, the output mode of the present invention is a multimodal output mode, including fluorescence, colorimetric signals of reverse fluorescence enhanced test strips, and reverse fluorescence signals. It can be used for POCT detection through a visual test strip without large-scale instrument equipment; in addition, the detection limit of the present invention is 1 CFU / mL, which is comparable or even superior to the detection limits of existing technologies; on the other hand, the present invention is applicable to the screening of hly and lde targets in all Listeria monocytogenes. In summary, the detection sensor of the present invention is superior to existing detection technologies in terms of sensitivity, detection method, and detection range, and can be used for POCT detection at the same time.

[0188] Table 1 Comparison of methods for detecting LM with existing technologies

[0189]

[0190] Although embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes, and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the content disclosed in the embodiments.

Claims

1. A method for dual, rapid, and portable detection of *Listeria monocytogenes* by integrating PfAgo cleavage and LAMP amplification with fluorescence and reverse fluorescence-enhanced lateral flow technology, characterized in that: The method is based on the Argonaute binding isothermal amplification detection technology, integrating the signal amplification system of PfAgo cleavage and LAMP isothermal amplification, and then combining with the reverse fluorescence lateral flow technology to sensitively, selectively and accurately detect Listeria monocytogenes on-site in a short time.

2. The method according to claim 1, characterized in that: When the target DNA is present, PfAgo can cleave the target DNA under the guidance of the guiding DNA, and the generated secondary ssDNA can bind to the empty-PfAgo as a new guiding DNA and guide its specific cleavage of the fluorescent probe. The cleavage of PfAgo is based on strict base complementary pairing, and a dual detection platform is designed based on this. When only pathogenic and ciprofloxacin-resistant genes are present in the system, hly amplicons and lde amplicons can be obtained after LAMP amplification. PfAgo cleaves the hly amplicon and lde amplicon respectively under the guidance of the corresponding guiding DNA to obtain secondary ssDNA 1 and ssDNA2, which serve as new guiding DNAs to guide PfAgo to cleave fluorescent probe 1 (double-labeled with 5’FAM and 3’BHQ1, complementary to ssDNA 1) and fluorescent probe 2 (double-labeled with 5’ROX and 3’BHQ2, complementary to ssDNA2) respectively. When only pathogenic Listeria monocytogenes is present in the system, only hly amplicons can be obtained after LAMP amplification. After PfAgo cleavage under the guidance of the corresponding guiding DNA, the obtained secondary ssDNA 1 guides PfAgo to cleave fluorescent probe 1. When ciprofloxacin-resistant Listeria monocytogenes is present in the system, only lde amplicons can be obtained after LAMP amplification. After PfAgo cleavage under the guidance of the corresponding guiding DNA, the obtained secondary ssDNA2 guides PfAgo to cleave fluorescent probe 2. When none of the above three bacteria are present in the system, there are no amplicons and no fluorescent probes are cleaved, and no fluorescence is generated. Similarly, in the reverse fluorescence lateral flow test strip, when pathogenic and ciprofloxacin-resistant genes are present in the system, L1 and L2 reporter molecules replace FAM-ssDNA-BHQ1 and ROX-ssDNA-BHQ2 for PfAgo cleavage; rhodamine 6G (R6G)-labeled streptavidin cannot form T1-Biotin-SA / R6G and T2-Biotin-SA / R6G on T1 and T2 lines to specifically bind to the probe-labeled AuNPs, namely AuNPs@DNA 1) and AuNPs@DNA 2, respectively, with the L1 and L2 reporter molecules. There is no visible red change on T1 and T2. At the same time, there is obvious R6G fluorescence on T1 and T2 lines, and red aggregation on the C line; when only pathogenic Listeria monocytogenes is present in the system, only the hly amplicon can be obtained after LAMP amplification. In the reverse fluorescence lateral flow test strip, the L2 reporter molecule is not cleaved, while the L1 reporter molecule is cleaved. A specific binding of AuNPs@DNA / L2 / T2-Biotin-SA / R6G will form on the T2 line, while this binding will not form on the T1 line. A visible red change will form on the T2 line. At the same time, the R6G fluorescence on the T1 line is quenched, and red aggregation occurs on the C line; when only ciprofloxacin-resistant Listeria monocytogenes is present in the system, only the lde amplicon can be obtained after LAMP amplification. In the reverse fluorescence lateral flow test strip, a visible red change will form on the T1 line. At the same time, the R6G fluorescence on the T2 line is quenched, and red aggregation occurs on the C line; when none of the above three bacteria are present in the system, there is no amplicon. In the reverse fluorescence lateral flow test strip, there is no visible red change on T1 and T2. At the same time, the R6G fluorescence on T1 and T2 lines is quenched, and red aggregation occurs on the C line. This method outputs fluorescence signals through an enzyme-labeled instrument and visual signals through a reverse fluorescence lateral flow test strip. This method can achieve highly sensitive detection of Listeria monocytogenes based on the R6G fluorescence intensity signal, and this signal is negatively correlated with the generation of visible AuNPs signals on the strip, providing a possibility for rapid and sensitive on-site detection.

3. The method according to claim 1, characterized in that: Including the following steps: The reverse fluorescence enhanced test strip consists of five main components, including a PVC backplane, a sample pad, a conjugate pad, an NC membrane, and an absorbent pad; for the T and C lines, different concentrations of SA, Biotin-modified DNA strands T1 and C1 are incubated with the fluorescent dye R6G in buffer MEST at 37 °C for 2 h, and then drawn onto the NC membrane at a rate of 1 μL / cm; the conjugate pad needs to be soaked in a specific buffer and then dried overnight; finally, the sample pad, the treated conjugate pad, the drawn NC membrane, and the absorbent pad are assembled on the backing card, cut into test strips 3.8 mm wide, and placed in a plastic card to obtain the reverse fluorescence enhanced test strip; First, add the designed SH-DNA to TCEP and activate it in a metal heating block at 37°C for 2 h. Then, add the colloidal gold solution with a particle size of 20 nm synthesized by reducing chloroauric acid with trisodium citrate. After pipetting and mixing evenly, place it in a -20°C refrigerator and freeze for 2 h. After thawing at room temperature, centrifuge at 12,000 rpm for 20 min, discard the supernatant, resuspend and wash twice with ultrapure water. The red precipitate is the AuNPs@DNA probe. Resuspend it in ultrapure water again and store it at 4°C; Inoculate Listeria monocytogenes resistant to ciprofloxacin in TSB medium and culture it in a shaker at 37°C for 24 h. Extract the genome of Listeria monocytogenes using a rapid extraction reagent. Add the genome to the LAMP dual-target amplification system and incubate at 65°C for 15 min. Add the obtained amplification product to the PfAgo system for cleavage reaction and incubate at 95°C for 15 min. Incubate the incubated PfAgo system with the probe AuNPs@DNA at 37°C for 10 min; Collect the fluorescence signal of the reaction solution after PfAgo cleavage using a microplate reader, or drop the mixed solution of the PfAgo system and the probe AuNPs@DNA incubated well on a plastic card equipped with a reverse fluorescence enhancement test strip, and observe the aggregation change of colloidal gold on the test strip with the naked eye, and observe the fluorescence quenching of the fluorescent group R6G on the test strip under ultraviolet light irradiation. To distinguish positive samples from negative samples.

4. The method according to claim 1, characterized in that: The specific steps are as follows: (1) Mix the sample to be tested with the genomic rapid extraction reagent to release the genome of Listeria monocytogenes; Mix the sample and the rapid extraction reagent at a ratio of 1:1, that is, add 100 μL of the sample and then add 100 μL of the rapid extraction reagent. The mixed solution of the sample and the rapid extraction reagent is shaken and mixed evenly and left standing for 30 s; Among them, the formula of the rapid extraction reagent is: 20 mM Tris with pH = 8.0, 25 mM NaCl, 2.5 mM EDTA, SDS with a mass concentration of 0.05%, PVP-40 with a mass concentration of 2%, and the solvent is water; The preparation steps for every 1 L of the rapid extraction reagent are: dissolve 2.42 g of Tris, 14.6 g of NaCl and 20 g of PVP-40 in 900 mL of water. Then add 5 mL of EDTA with pH 8.0 and 500 mM and 5 mL of SDS with a mass concentration of 10%, and finally adjust the pH to 8.0 with hydrochloric acid and add water to a final volume of 1 L; (2) Primer design; According to the whole genome sequence of the target strain of Listeria monocytogenes, select the specific genes hly and lde sequences in the sequence to design LAMP primers for the detection of Listeria monocytogenes; (3) Perform LAMP amplification on the extracted genome; Place the mixed LAMP amplification system in a 65°C constant temperature metal bath and incubate for 15 min to obtain the LAMP amplification product; (4) Preparation of AuNPs Synthesize colloidal gold solution with a particle size of 20 nm by reducing chloroauric acid with trisodium citrate. The specific steps are as follows: Add 250 mM HAuCl4·3H2O solution to ultrapure water and heat it to boiling under constant magnetic stirring; Subsequently, quickly inject 38.84 mM trisodium citrate solution into the solution; After the solution color changes from colorless to stable transparent red, continue stirring for 15 min with the color remaining unchanged, indicating the successful synthesis of AuNPs; Finally, stir the obtained AuNPs solution at room temperature for 15 min, cool it to room temperature, and centrifuge and concentrate it 5 times at 12,000 rpm to obtain the AuNPs solution, which is stored at 4 °C for further use; Among them, the ratio of HAuCl4·3H2O solution: ultrapure water: trisodium citrate solution in μL: mL: mL is 100:100:3; (5) Preparation of AuNPs@DNA probe The preparation process of the nucleic acid strand probe AuNPs@DNA modified with AuNPs is achieved through the formation of gold-sulfur (Au-S) covalent bonds: First, mix 100 μM nucleic acid strand SH-DNA solution containing thiol modification with 0.5 M tris(2-carboxyethyl)phosphine (TCEP) solution at pH 7.7; Incubate in the dark at room temperature for 2 h to activate the thiol group; Add the activated SH-DNA strand to the AuNPs solution and freeze it at -20 °C for 2 h; After thawing at room temperature, centrifuge at 12,000 rpm for 20 min; After centrifugation, discard the supernatant, add ultrapure water to resuspend and wash, centrifuge at 12,000 rpm for 20 min, discard the supernatant, and repeat 2 times. The red precipitate is the AuNPs@DNA probe, which is resuspended in 200 μL of ultrapure water and stored at 4 °C for subsequent experiments; Among them, the volume ratio of nucleic acid strand SH-DNA solution: (2-carboxyethyl)phosphine TCEP solution: AuNPs solution: ultrapure water is 5:2:200:200; (6) Preparation of reverse fluorescence-enhanced test strip The reverse fluorescence enhanced test strip consists of five main components, including a PVC bottom plate, a sample pad, a conjugate pad, an NC membrane, and an absorbent pad; for the T line, first dissolve rhodamine 6G (R6G) in PBS buffer at pH 7.4 to prepare a 100 mM solution. According to the molar ratio of R6G, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) of 1:5:5, slowly add EDC and NHS to the R6G solution while stirring, and continue stirring for 30 minutes to 1 hour to activate R6G and form an active intermediate capable of binding to streptavidin (SA); weigh SA and dissolve it in MEST buffer to prepare a 10 mg / mL solution; slowly drip the activated R6G solution into the SA solution while stirring to ensure sufficient contact; control the molar ratio of R6G to SA to be 5:1, and continue stirring and reacting at room temperature for 2 hours to allow the reaction of groups such as amino groups on R6G and SA molecules; then, add 100 μM of T1-Biotin or 100 μM of T2-Biotin to the mixed solution, with the molar ratio of SA to Biotin being 1:5, and react at room temperature for 30 min. Dilute the obtained R6G-SA-Biotin-T1 or R6G-SA-Biotin-T2 solution with a 10 kD microporous ultrafiltration membrane by centrifuging at 12,000 rpm for 40 min at 4 °C, dilute it with MEST solution to 1 mg / mL, and draw it on the NC membrane at a rate of 1 μL / cm; for the C line, use the same method to prepare an SA-Biotin-C1 / C2 solution with a final concentration of SA of 3 mg / mL and draw it on the NC membrane at a rate of 1 μL / cm; the conjugate pad needs to be soaked in PBS buffer at pH 7.4 containing 5 mM PVP-1000, 0.15 M sucrose, 0.45 mM BSA, and 2% (mass concentration) Tween-20, and then dried overnight; finally, assemble the sample pad, the treated conjugate pad, the drawn NC membrane, and the absorbent pad on the backing card, cut it into test strips 3.8 mm wide, and place them in a plastic card; all the test strips are sealed in an aluminum foil bag with a desiccant for subsequent experiments; Among them, the composition of the PBS buffer required for the reaction is: 10 mM NaCl, 10 mM KCl, 25 mM Na2HPO4, 7.8 mM K2HPO4, the solvent is water, and pH = 7.4; The composition of the MEST buffer is: 25 mM 2-(N-morpholino)ethanesulfonic acid (MES), 0.05% (mass concentration) of Tween-20, and the solvent is water; (7) Add the amplified product to the PfAgo system for cleavage reaction; Combine the LAMP amplification product obtained in step (3) with the PfAgo reaction system, incubate at 95 °C for 15 min, measure the fluorescence value by a microplate reader after the reaction ends, or incubate the reaction solution with AuNPs@DNA probe at 37 °C for 10 min and then drop it on the reverse fluorescence enhanced test strip to output the result; When the hly and lde targets are present, compared with the fluorescence value of the negative control group without targets, the fluorescence of the positive group with targets increases. Accordingly, the Linker-DNA is cleaved and cannot hybridize with the AuNPs@DNA probe and the T line, and thus cannot aggregate on the T line. At the same time, there is no aggregation of AuNPs on the T1 and T2 lines, and the R6G fluorescence is not quenched, proving that the bacterium is Listeria monocytogenes with pathogenicity and ciprofloxacin resistance.

5. The method according to claim 4, characterized in that: In step (3), the total volume of the LAMP amplification system is 25 μL per reaction, and the specific components are as follows: 2.5 μL of BufferⅠ, 2.5 μL of 10 μM dNTPs mix, 2.5 μL of 10× primer mix-hly, 2.5 μL of 2% BSA, 0.5 μL of Bst DNA polymerase, 10 μL of the genomic DNA to be detected, and 4.5 μL of ddH2O; Among them, the components of BufferⅠ are: 200 mM Tris-HCl, pH 8.8, 500 mM KCl, 80 mM (NH4)2SO4, 20 mM MgSO4, 1% (w / v) Tween-20, 600 mM betaine, and the solvent is water; The 10× primer mix-hly gene amplification includes: 2 μM F3, 2 μM B3, 16 μM FIP, 16 μM BIP, 4 μM Loop F, and 4 μM Loop B; The dNTPs mix includes: 10 mM dATP, 10 mM dTTP, 10 mM dCTP, 10 mM dGTP, and 10 mM dUTP.

6. The method according to claim 4, characterized in that: The PfAgo cleavage reaction system is as follows:

7. The method according to claim 4, wherein: In step (7), the PfAgo reaction system is: 1.0 μM PfAgo, 0.5 μM hly gene guide DNAs, 0.5 μM lde gene guide DNAs, 10 μL of Target, 0.2 μM fluorescent probe 1 / 0.2 μM Linker 1, ddH2O, and reaction buffer; Among them, the hly and lde gene Guide DNAs (Guide mix) include 3 corresponding guide DNAs, namely guide DNA 1, guide DNA 2, and guide DNA 3. The total concentration of the three guide DNAs is 10 μM, and the molar concentration ratio of the three guide DNAs is 1:1:1; The composition of the reaction buffer is: 200 mM Tris-HCl, 100 mM (NH4)2SO4, 100 mM KCl, 100 mM MgSO4, 800 mM betaine, 1% (w / v) Tween-20, pH = 8.8, and the solvent is water.

8. The method according to claim 4, characterized in that: When pathogenic and ciprofloxacin-resistant genes exist in the system, hly amplicons and lde amplicons can be obtained through LAMP amplification. PfAgo cuts the hly amplicon and lde amplicon respectively under the guidance of the corresponding guide DNA to obtain secondary ssDNA 1 and ssDNA 2, which serve as new guide DNAs to guide PfAgo to cut the fluorescent probe 1 (FAM-ssDNA-BHQ1, complementary to ssDNA 1) and fluorescent probe 2 (ROX-ssDNA-BHQ2, complementary to ssDNA 2). In the reverse fluorescence lateral flow test strip, L1 and L2 reporter molecules replace FAM-ssDNA-BHQ1 and ROX-ssDNA-BHQ2 for PfAgo cleavage; SA labeled with R6G cannot form T1-Biotin-SA / R6G and T2-Biotin-SA / R6G on T1 and T2 lines to specifically bind to the probe-labeled AuNPs (AuNPs@DNA1) and AuNPs (AuNPs@DNA2) of L1 and L2 reporter molecules respectively, and no visible red change occurs on T1 and T2. At the same time, there is obvious R6G fluorescence on T1 and T2 lines, and red aggregation on the C line; when pathogenic Listeria monocytogenes exists in the system, only hly amplicons can be obtained through LAMP amplification. After PfAgo cleavage under the guidance of the corresponding guide DNA, the secondary ssDNA 1 is obtained to guide PfAgo to cut the fluorescent probe 1. In the reverse fluorescence lateral flow test strip, the L1 reporter molecule is cleaved, while the L2 reporter molecule is not cleaved. The T2 line will form a specific binding of AuNPs@DNA2 / L2 / T2-Biotin-SA / R6G, while the T1 line will not form this binding. A visible red change occurs on T2. At the same time, there is obvious R6G fluorescence on the T1 line, the R6G fluorescence on the T2 line is quenched, and red aggregation occurs on the C line; when ciprofloxacin-resistant Listeria monocytogenes exists in the system, only lde amplicons can be obtained through LAMP amplification. After PfAgo cleavage under the guidance of the corresponding guide DNA, the secondary ssDNA 2 is obtained to guide PfAgo to cut the fluorescent probe 2. In the reverse fluorescence lateral flow test strip, L2 is cleaved while L1 is not cleaved. The T1 line will form a specific binding of AuNPs@DNA1 / L1 / T1-Biotin-SA / R6G, while the T2 line will not form this binding. A visible red change occurs on T1. At the same time, the R6G fluorescence on the T1 line is quenched, there is obvious R6G fluorescence on the T2 line, and red aggregation occurs on the C line; when none of the above three bacteria exist in the system, there are no amplicons and no fluorescent probes are cleaved. In the reverse fluorescence lateral flow test strip, neither the L1 nor the L2 reporter molecule is cleaved.The R6G-labeled SA can form T1-Biotin-SA / R6G and T2-Biotin-SA / R6G on the T1 and T2 lines respectively, which specifically bind to the probe-labeled AuNPs, namely AuNPs@DNA1 and AuNPs@DNA2, with the L1 and L2 reporter molecules, resulting in a visible red change for T1 and T2; meanwhile, the R6G fluorescence on the T1 and T2 lines is quenched, and there is a red aggregation on the C line.

9. The method according to any one of claims 1 to 8, characterized in that: The lowest detection limit of the method is 1 CFU / mL; the detection time of the method from nucleic acid extraction, LAMP amplification, PfAgo cleavage, and signal output only takes 45 min.

10. Use of the method according to any one of claims 1 to 9 in the preparation of a medicament and / or kit for detecting Listeria monocytogenes.

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