Method for high-sensitivity detection of helicobacter pylori through Argonaute one-pot biosensing and application

Abstract

The invention belongs to the technical field of microbiological detection, and discloses an Argonaute-based biosensor for helicobacter pylori detection, a method and application, the biosensor comprises a TtAgo-LAMP one-pot reaction system, and every 25 [mu] L of the reaction system comprises TtAgo, guide DNAmix, Buffer I, dNTPs mix, a 10 * primer mix-vacA, BSA with a mass concentration of 2%, Bst DNA polymerase, a helicobacter pylori genome to be detected, a fluorescent probe report and ddH2O. The invention designs a biosensor which is based on an Argonaute system and is combined with an LAMP (Loop-Mediated Isothermal Amplification) assisted one-pot method detection sensing platform, so that signal transduction and amplification are realized. The method is used for detecting effective targets in helicobacter pylori and is used for rapidly and sensitively detecting and applying the helicobacter pylori.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and in particular to an Argonaute-based biosensor, method and application for Helicobacter pylori detection. Background Art

[0002] Helicobacter pylori (H. pylori) is a spiral-shaped, Gram-negative bacterium that is widely present in the human gastric mucosa. It is a key factor in the development of gastric cancer. The global infection rate of H. pylori is approximately 50% (18.9% to 87.7%). Epidemiological surveys in my country have shown that the H. pylori infection rate in China is approximately 40% to 90%, which means that approximately 700 to 800 million people in my country have been infected with H. pylori. According to the World Health Organization's International Agency for Research on Cancer (WHO-IARC), by 2040, the annual burden of gastric cancer will increase to approximately 1.8 million new cases and 1.3 million deaths.

[0003] The pathogenicity of Helicobacter pylori (H. pylori) is driven by multiple virulence factors, of which cytotoxin-associated gene A (cagA) and vacuolarging cytotoxin A (vacA) stand out as being directly associated with disease severity. These virulence factors mediate their pathogenic effects by directly interacting with gastric epithelial cells, triggering proinflammatory signaling cascades, disrupting mucosal integrity, and promoting carcinogenesis through dysregulation of host cell apoptotic and proliferative pathways. Notably, strain-specific differences in virulence factor expression contribute to diverse clinical outcomes in individuals infected with H. pylori. Although the vacA gene is ubiquitous in all H. pylori strains, only approximately 60% of clinical isolates express functional cagA cytotoxin, and approximately 50% of isolates display biologically active vacA toxin. This variability highlights the critical role of monitoring vacA expression patterns, as its ubiquitous genomic presence, combined with variable toxin activity, serves as a key biomarker for identifying high-risk infections and predicting disease progression.

[0004] Currently, H. pylori detection methods include both invasive and non-invasive methods. Invasive methods such as endoscopy and biopsy have numerous drawbacks, including being invasive, resource-intensive, and inconvenient, particularly for elderly or critically ill patients. Conventional urea breath tests can be used to detect H. pylori, but they suffer from limited specificity. Culture-based typing methods are often time-consuming and challenging, necessitating the development of rapid, non-invasive, and cost-effective molecular diagnostics for H. pylori genotyping using saliva or stool samples, which remains crucial for targeted eradication. Furthermore, genotypic analysis of H. pylori mutants for antibiotic resistance is crucial for selecting effective antibiotics for eradication therapy. The development of non-invasive tools for identifying infections with highly virulent H. pylori strains is needed to inform targeted prevention and eradication programs. Ideally, expanded point-of-care testing (POCT) and rapid DNA extraction, coupled with thermal cycler-free H. pylori genotyping and resistance testing using saliva or stool samples, would be the most suitable preventive approach.

[0005] Currently, molecular biological methods, such as polymerase chain reaction (PCR) and gene sequencing, are commonly used for H. pylori detection. However, these methods are time-consuming and require sophisticated instruments with thermal cycling, making them unsuitable for rapid point-of-care testing (POCT). With the development of isothermal amplification, various isothermal techniques have gradually gained attention, such as RCA, RPA, NASBA, HDA, and LAMP. LAMP, a novel nucleic acid amplification technique proposed by Notomi et al. in 2000, has been widely used in fields such as pathogen detection and food safety testing. LAMP can achieve specific and highly sensitive target gene amplification in a short period of time at a constant temperature (typically 60°C-65°C) and is independent of thermal cycling. Typically, four to six target-specific primers are required for amplification. Notably, LAMP exhibits high amplification efficiency for low concentrations of nucleic acid targets and can be detected using real-time or end-point fluorescence measurements, which undoubtedly improves detection sensitivity. However, LAMP also has some drawbacks. Due to the large number of primers, primer dimers are easily formed, and nonspecific amplification and false positives are highly likely to occur. Therefore, there is an urgent need to develop new assays and strategies to detect H. pylori.

[0006] Argonaute programmable nucleases are divided into eukaryotic Argonaute (eAgo) and prokaryotic Argonaute (pAgo). The most widely studied in the field of biosensing is prokaryotic Argonaute, such as PfAgo / TtAgo. Argonaute is a multi-converting enzyme that does not require the target to contain any specific protospacer adjacent motif (PAM). It has programmability, sequence specificity, high sensitivity and single-base resolution, making it an ideal tool for nucleic acid detection sensors. However, it is often necessary to add a pre-amplification step to improve sensitivity. Common isothermal amplification methods include LAMP / RPA. Several sensing strategies for nucleic acid detection based on the Argonaute system have been developed. Ago's multiple flipping activity and programming capabilities can develop a simultaneous detection platform for multiple genes.

[0007] Therefore, one or several new related detection kits, sensors, etc. are urgently needed.

[0008] In this work, a TtAgo-based biosensor was developed for rapid, accurate, portable, ultrasensitive, and visual detection of Helicobacter pylori. By integrating LAMP technology with the synergistic action of TtAgo nuclease, a novel single-tube closed nucleic acid detection system was constructed. This single-tube reaction design effectively eliminates the risk of contamination caused by aerosol diffusion of amplification products.

[0009] Through searching, no patent publication documents related to the patent application of the present invention have been found. Summary of the Invention

[0010] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide an Argonaute-based biosensor, method and application for Helicobacter pylori detection.

[0011] The technical solution adopted by the present invention to solve its technical problem is:

[0012] A one-pot detection platform integrating TtAgo cleavage and LAMP amplification realizes a method for rapid and sensitive detection of Helicobacter pylori. In the method, when target DNA is present, TtAgo can cleave the target DNA under the guidance of guide DNA. The generated secondary ssDNA acts as a new guide DNA to bind to TtAgo and guide it to specifically cleave a fluorescent probe. TtAgo cleavage is based on strict base complementary pairing, based on which the detection platform is designed. When Helicobacter pylori is present in the sample, the Helicobacter pylori genomic target is released after treatment with a rapid nucleic acid extraction reagent. LAMP primers specifically recognize the vacA target sequence and, through LAMP amplification, can obtain a vacA amplicon. TtAgo, under the guidance of the corresponding guide DNA, cleaves the vacA amplicon to obtain a secondary ssDNA as a new guide DNA to guide TtAgo to cleave the fluorescent probe. The entire process is carried out in a single reaction tube, realizing rapid and ultrasensitive detection of Helicobacter pylori.

[0013] An Argonaute-based biosensor for detecting Helicobacter pylori, the biosensor comprising a TtAgo-LAMP one-pot reaction system, wherein each 25 μL of the TtAgo-LAMP one-pot reaction system comprises:

[0014] 50 μM TtAgo 1 μL, 10 μM Guide DNA mix-vacA 2.5 μL, Buffer I 2.5 μL, dNTPs mix 2.5 μL, 10× primer mix-vacA 2.5 μL, 2% BSA 2.5 μL, 70 μM Bst DNA polymerase 1.5 μL, Helicobacter pylori genome to be detected 5 μL, 4 μM fluorescent probe vacA-reporter 2.5 μL, ddH2O 2.5 μL;

[0015] The Buffer I comprises: 200 mM Tris-HCl, pH 8.8, 100 mM KCl, 100 mM (NH4)2SO4, 100 mM MgSO4, 1% Tween-20; 200 mM betaine;

[0016] 10× primer mix-vacA gene amplification includes: 2 μM vacA-F3, 2 μM vacA-B3, 16 μM vacA-FIP, 16 μMvacA-BIP, 4 μM vacA-Loop F (i.e., LF), 4 μM vacA-Loop B (i.e., LB);

[0017] Guide DNA mix-vacA includes: 10μM vacA-Guide DNA 1, 10μM vacA-Guide DNA 2;

[0018] dNTPs mix includes: 10mM dATP, 10mM dTTP, 10mM dCTP, 10mM dGTP;

[0019] The gene sequences of vacA-F3, vacA-B3, vacA-FIP, vacA-BIP, vacA-Loop F (LF), vacA-Loop B (LB), vacA-Guide DNA 1, vacA-Guide DNA 2, and fluorescent probe vacA-reporter are SEQ ID No. 1-9, respectively.

[0020] Application of the above-mentioned biosensor in the preparation of detection of vacA target in Helicobacter pylori.

[0021] A method for rapid, automated detection of Helicobacter pylori using the integrated TtAgo cleavage and LAMP amplification technologies of a biosensor not intended for disease diagnosis or treatment, wherein when target DNA is present, TtAgo is able to cleave the target DNA under the guidance of a guide DNA, and the resulting secondary ssDNA can act as a new guide DNA to bind to TtAgo and guide it to specifically cleave a fluorescent probe. TtAgo cleavage is based on strict base complementary pairing, and a detection platform is designed based on this.

[0022] When Helicobacter pylori is present in a sample, treatment with a rapid nucleic acid extraction reagent releases the H. pylori genomic target. LAMP primers specifically recognize the vacA target sequence, generating vacA amplicons through LAMP amplification. TtAgo, guided by the corresponding guide DNA, cleaves the vacA amplicon, generating secondary ssDNA that serves as a new guide DNA to guide TtAgo cleavage of the fluorescent probe. This entire process enables rapid, ultrasensitive detection of H. pylori.

[0023] Further, the following steps are included:

[0024] (1) When Helicobacter pylori is present in the sample, the Helicobacter pylori genome will be released after treatment with a rapid nucleic acid extraction reagent. The genome is added to the LAMP amplification system, and the qPCR program is set to 65°C for 40 minutes;

[0025] (2) After the reaction is completed, real-time fluorescence value measurement is performed through qPCR, and the analysis curve is automatically output to determine the test results.

[0026] Furthermore, the formula of the rapid nucleic acid extraction reagent is: 20mM Tris with a pH of 8.0, 25mM NaCl, 2.5mM EDTA, a volume concentration of 0.05% (wt / vol) SDS, and a volume concentration of 2% (wt / vol) PVP-40, that is, 2.42g of Tris, 14.6g of NaCl and 20g of PVP-40 are dissolved in 900mL of water, and then 5mL of 500mM EDTA with a pH of 8.0 and 5mL of SDS with a volume concentration of 10% wt / vol are added. Finally, the pH is adjusted to 8.0 with hydrochloric acid, and water is added to a final volume of 1L.

[0027] Furthermore, the specific steps are as follows:

[0028] (1) Design of rapid nucleic acid extraction device;

[0029] The device comprises an inner tube and an outer tube which are coaxially and tightly threadedly mounted. The inner tube is arranged above the outer tube, and the outer tube is made of soft plastic. A filter membrane is coaxially and tightly integrated at the bottom of the outer tube. The inner tube and the outer tube are coaxially sleeved to form two independent chambers. The inner tube contains a rapid nucleic acid extraction reagent, and the outer tube can contain a sample to be tested. The flow rate is controlled by a through hole with a 2 mm aperture. The bottom of the outer tube is coaxially and tightly connected to a protective cover which can be detachably provided. The filter membrane is also provided on the outer tube in the protective cover. An inner tube through hole is formed on the bottom of the inner tube, and the top of the outer tube is connected to the inner tube. The inner tube is connected to form a through hole in the outer tube. When the inner tube is rotated, the through hole in the inner tube and the through hole in the outer tube can be arranged vertically and connected, so that the chambers of the inner and outer tubes are closely connected. During use, the staggered design of the inner tube and the bottom disc can initially effectively block the connection between the extraction chamber and the reagent chamber. After the sample is loaded into the extraction chamber, the inner tube is rotated to align the orifices, and the nucleic acid extraction reagent can be quickly released from the reagent chamber. Combined with the up and down shaking operation, the genome can be efficiently extracted. At the same time, the filter membrane at the bottom of the outer tube can effectively remove impurities, completing the nucleic acid purification process.

[0030] (2) The sample to be tested is mixed with a rapid genome nucleic acid extraction reagent to release the Helicobacter pylori genome;

[0031] Mix the sample and the rapid nucleic acid extraction reagent in a 1:1 ratio in a rapid nucleic acid extraction device, i.e., add 500 μL of sample and then 500 μL of rapid nucleic acid extraction reagent, shake to mix, and let it stand for 30 seconds to 10 minutes;

[0032] (3) CTAB method was used to extract genomic DNA from samples;

[0033] (1) Grinding: Weigh 0.1 g of sample into a sterilized mortar and crush thoroughly on ice. Then add 1 mL of sterile water to thoroughly resuspend the feces. Centrifuge at 4°C for 10 min at 10,000 rpm and collect the supernatant.

[0034] (2) Hexadecyltrimethylammonium bromide (CTAB) method

[0035] To each 500 μL sample that had been pretreated, 30 μL of 10% SDS solution, 3 μL of 20 g / L proteinase K, and 4 μL of RNase A were added, mixed, and then placed in a 37°C water bath for 1 hour; 100 μL of 5 mol / L NaCl solution was added to each tube, mixed by inversion, and then 80 μL of CTAB / NaCl (a 10% CTAB, 0.7 mol / L NaCl aqueous solution) was added, mixed gently, and placed in a 65°C water bath for 10 minutes; an equal volume of phenol / chloride was added to the above mixed solution. Mix the mixture of phenol / chloroform and isopropanol, with the volume ratio of phenol:chloroform:isopropanol being 25:24:1. Centrifuge at 4°C for 10 min at 12000 rpm, take the supernatant, add 0.6 times the volume of isopropanol to the supernatant, and mix gently; centrifuge at 4°C for 10 min at 12000 rpm, discard the supernatant, wash the precipitate with 1 mL of pre-cooled 75% ethanol, centrifuge at 4°C for 5 min at 7500 rpm, discard the ethanol, place in a ventilated environment to allow the alcohol to evaporate, and then add 30 μL of sterile water to fully dissolve;

[0036] (4) Primer design

[0037] The specific gene vacA sequence in the Helicobacter pylori genome was selected to design LAMP primers for Helicobacter pylori detection; the specific gene sequence vacA in the vacA genome, i.e., GenBank accession number AP017632, was selected to design LAMP primers for detection; the gene sequences of the LAMP primers are SEQ ID No. 1-6 in order;

[0038] (5) Perform LAMP amplification on the extracted genome;

[0039]

[0040]

[0041] (6) Preparation of AuNPs and AuNPs@DNA;

[0042] 1) A method for preparing colloidal gold nanoparticles (AuNPs), comprising the following steps: adding 100 μL of 250 mM chloroauric acid trihydrate (HAuCl4·3H2O) to 100 mL of ultrapure water and heating the mixture to boiling under magnetic stirring; rapidly injecting 3 mL of 38.84 mM trisodium citrate until the solution turns a stable, transparent red and continuously stirring for 15 minutes; cooling the mixture to room temperature and concentrating the solution 5-fold by centrifugation at 12,000 rpm to obtain an AuNPs solution, which was stored at 4°C until further use;

[0043] 2) Preparation of gold nanoparticle-modified nucleic acid chain probes, AuNPs@DNA, comprising the following steps: mixing 5 μL of 100 μM thiol-modified nucleic acid chain vacA-SH-DNA with 2 μL of 0.5 M tris(2-chloroethyl) phosphate (TCEP) at pH 7.7, and incubating at room temperature in the dark for 2 h to activate the thiol groups; adding the activated vacA-SH-DNA to 200 μL of a high-concentration AuNPs solution, freezing the solution at −20°C for 2 h, and centrifuging at 12,000 rpm for 20 min after thawing; discarding the supernatant, resuspending and washing the solution with 200 μL of ultrapure water, and repeating the centrifugation twice to obtain the AuNPs@DNA probes, which were resuspended in 200 μL of ultrapure water and stored at 4°C until use;

[0044] 7) A method for preparing a test strip, comprising the following steps:

[0045] A PVC base plate, a sample pad, a conjugate pad, a NC membrane, and a water-absorbing pad are provided as the main components of the test strip; a test line, namely the T line, is prepared by incubating 4.4 mg / mL streptavidin SA with 100 μM biotinylated T probe vacA-T-biotin in 25 mM, pH 5.0 MEST buffer at 37°C for 2 h to obtain a 1 mg / mL SA-biotin-T complex; a continuous film scribing machine (HM8000, China) from Shanghai Jinbiao Biotechnology Co., Ltd. is used to precisely spray-print the complex on a preset position on the NC membrane at a line density of 1 μL / cm; a control line, namely the C line, is prepared by using the same method to prepare a 3 mg / mL SA-biotin-C complex, which is streaked onto the NC membrane at a rate of 1 μL / cm; a conjugate pad is treated by soaking it in a pH 5.0 buffer containing 5 mM polyvinylpyrrolidone (PVP-1000), 0.15 M sucrose, 0.45 mM BSA, and 2% Tween-20. 7.4% PBS buffer and dry overnight; assemble the test strips: fix the sample pad, treated conjugate pad, scribed NC membrane, and absorbent pad on the PVC base in this order, cut into 3.8 mm wide test strips, and place them in a plastic card; package and store: seal the test strips and desiccant in an aluminum foil bag for later use;

[0046] (8) Amplification and cleavage reactions are performed simultaneously to output fluorescent signals / colorimetric signals;

[0047] Combine the LAMP amplification system with the TtAgo reaction system and place the reaction tube in the qPCR [(QuantStudio TM 1Plus) from Shanghai Thermo Fisher Scientific Instrument Co., Ltd. (China). At 65°C, fluorescence signals were collected every 1 minute for a total of 40 minutes. Real-time fluorescence value measurements were performed, and analysis curves were automatically output to determine the test results. Alternatively, the reaction tube was placed in a 3D-printed portable visualization device we previously developed (patent application number 2023102268358 has been published) to obtain fluorescence image detection results. The vacA-reporter was replaced with a vacA-Linker chain. The reaction system and other reaction reagents remained unchanged and the tube was heated in a 65°C metal bath for 40 minutes. The reaction product was then incubated with a pre-prepared nanogold probe at room temperature for 10 minutes, dropped into a test strip, and the reaction results were observed.

[0048] A lateral flow assay was established to detect the results: when there is no target, vacA-GuideDNAs will not guide TtAgo to cut the vacA-Linker chain, and the test line, i.e., the T line, will capture the vacA-SH-DNA-Linker coupler that is complementary to its base, thereby displaying color on the T line. Excess vacA-SH-DNA will pair with the nucleic acid chain on the C line, causing the colloidal gold to aggregate on the C line for color development. In the presence of target, positive samples will cause TtAgo to cut the vacA-Linker chain, and only show a signal on the quality control line, i.e., the C line. Since the vacA-Linker chain is broken, it will not be recognized by the nucleic acid chain on the T line, so there is no signal on the test line (T line), which indicates the presence of the target sample.

[0049] When the vacA target is present, the fluorescence intensity of the positive group (i.e., with the target) increases compared with the fluorescence value of the negative control group (i.e., without the target), and it is green fluorescence under 302nm ultraviolet irradiation. An obvious signal appears on the quality control line of the test strip, which proves that the test object is a Helicobacter pylori-contaminated sample.

[0050] The one-pot reaction system of TtAgo-LAMP for each 25 μL is:

[0051] 50μM TtAgo 1μL, 10μM Guide DNA mix-vacA 2.5μL, Buffer I 2.5μL, dNTPsmix 2.5μL, 10× primer mix-vacA 2.5μL, 2% BSA 2.5μL, 70μM Bst DNA polymerase 1.5μL, genome to be detected 5μL, 4μM fluorescent probe vacA-reporter 2.5μL, ddH2O 2.5μL.

[0052] Furthermore, the components of Buffer I are: 200mM Tris-HCl, pH 8.8, 100mM KCl, 100mM (NH4)2SO4, 100mM MgSO4, 1% Tween-20; 200mM betaine;

[0053] 10× primer mix-vacA gene amplification includes: 2 μM vacA-F3, 2 μM vacA-B3, 16 μM vacA-FIP, 16 μMvacA-BIP, vacA-Loop F (i.e., LF), vacA-Loop B (i.e., LB);

[0054] Guide DNA mix-vacA includes: 10μM vacA-Guide DNA 1, 10μM vacA-Guide DNA 2;

[0055] dNTPs mix includes: 10mM dATP, 10mM dTTP, 10mM dCTP, 10mM dGTP;

[0056] The gene sequences of vacA-F3, vacA-B3, vacA-FIP, vacA-BIP, vacA-Loop F (LF), vacA-Loop B (LB), vacA-Guide DNA 1, vacA-Guide DNA 2, and fluorescent probe vacA-reporter are SEQ ID No. 1-9, respectively.

[0057] Furthermore, the minimum detection limit of the method is 1 CFU / mL; the detection time of the method from nucleic acid extraction, LAMP amplification, TtAgo cleavage and signal output only takes 40 minutes.

[0058] Application of the above method in preparing a vacA target and / or a kit for detecting Helicobacter pylori.

[0059] Application of the above method in preparing the vacA gene for detecting Helicobacter pylori.

[0060] The advantages and positive effects achieved by the present invention are:

[0061] 1. This invention designs a biosensor based on the Argonaute system combined with a LAMP amplification-assisted one-pot detection sensing platform to achieve signal transduction and amplification. It is used to detect effective targets in Helicobacter pylori and is used for rapid and ultrasensitive Helicobacter pylori detection and application.

[0062] 2. The present invention is based on the Argonaute system combined with isothermal amplification technology. By combining TtAgo with LAMP, it provides a rapid, visual and portable bioassay method based on the Argonaute system for ultra-sensitive one-pot detection and application of Helicobacter pylori. The TtAgo protein used is a thermophilic protein with thermal stability, which avoids reactions at room temperature during the sample addition process and reduces experimental errors.

[0063] 3. The method established by the present invention has a short detection time, requiring only 40 minutes from nucleic acid extraction, LAMP amplification, TtAgo cleavage and signal output, which is superior to most existing detection technologies.

[0064] 4. The one-pot design in the method of the present invention avoids cross contamination to a great extent.

[0065] 5. The sensor of the present invention is sensitive, with a minimum detection limit of 1 CFU / mL, which meets the prescribed threshold. Furthermore, the biosensor based on the Argonaute system has been validated for detection of clinical samples.

[0066] 6. The present invention has designed a rapid nucleic acid extraction device that can extract Helicobacter pylori genomic DNA within 30 seconds, greatly simplifying the nucleic acid extraction process and shortening the time. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 A schematic diagram of an experiment principle in the present invention; wherein, A: is the experimental process and principle of the present invention; B: is a schematic diagram of a rapid nucleic acid extraction device; C: is a schematic diagram of a test strip;

[0068] Figure 2 This is a diagram showing the specificity verification results of TtAgo cleavage of LAMP products in the range of 35-95°C in the present invention;

[0069] Figure 3This is a feasibility diagram explored in the present invention; A: The DNA sequences used in the present invention include the target gene, guide DNA, and reporter gene; B: Fluorescence spectrum of each sample (excitation wavelength is 484nm, emission wavelength range is between 500nm and 650nm, and the fluorescence reading during detection is selected at 529nm); C: Fluorescence image of each sample captured using a homemade 3D-printed visualizer; D: Real-time fluorescence intensity at different time points and fluorescence intensity at the end of 40 minutes; E: Test strip results for each sample;

[0070] Figure 4 Figure 1 shows the optimized LAMP-TtAgo one-pot platform for capturing real-time fluorescence curves using fluorescence quantitative PCR and fluorescence images captured by a 3D printing visualizer. A: Bst concentration; B: TtAgo concentration; C: guide DNA concentration; D: dNTP mixture addition amount; E: primer mixture addition amount; F: reporter gene concentration.

[0071] Figure 5 Figure 1 shows the optimized buffer composition of the present invention; A: Real-time fluorescence curves of the LAMP-TtAgo one-pot method using different buffer reactions (Ctrl: no buffer added; 1: Buffer 1; 2: TtAgo Buffer; 3: NEBuffer; 4: NEBuffer 2.1; 5: TRBuffer; 6: NEBuffer 3.1); B: Fluorescence value heat map analysis of the LAMP-TtAgo one-pot method using different buffer reactions; C: Different concentrations of Mg 2+ Effects on TtAgo cleavage activity (Ctrl: 0 mM Mg 2+ );D: different concentrations of Mg 2+ Heat map analysis of TtAgo cleavage activity (Ctrl: 0 mM Mg 2+ ); E: Effect of different concentrations of betaine on TtAgo cleavage activity (Ctrl: no buffer added); F: Heat map analysis of different concentrations of betaine on TtAgo cleavage activity (Ctrl: no buffer added);

[0072] Figure 6 Figures showing the sensitivity and specificity of the LAMP-TtAgo one-pot assay of the present invention; A: Real-time fluorescence curves of H. pylori at different concentrations at different time points and real-time fluorescence intensity at the end of 40 minutes; B: Fluorescence images of H. pylori samples at different concentrations captured using a homemade 3D-printed visualizer; C: Test strip results of H. pylori samples at different concentrations; (DF) Selectivity of the platform for detecting H. pylori over other common pathogens.

[0073] Figure 7 These are the test diagrams of real samples in the present invention; wherein, A: the real-time fluorescence intensity curves, endpoint fluorescence value measurements, fluorescence images collected by a 3D printing visualization instrument, and test strip test results of the method of the present invention for detecting different concentrations of Helicobacter pylori in pork; B: the real-time fluorescence intensity curves, endpoint fluorescence value measurements, fluorescence images collected by a 3D printing visualization instrument, and test strip test results of the method of the present invention for detecting different concentrations of Helicobacter pylori in mouse feces; C: the real-time fluorescence intensity curves, endpoint fluorescence value measurements, fluorescence images collected by a 3D printing visualization instrument, and test strip test results of the method of the present invention for detecting different concentrations of Helicobacter pylori in saliva; D: the real-time fluorescence intensity curves, endpoint fluorescence value measurements, fluorescence images collected by a 3D printing visualization instrument, and test strip test results of the method of the present invention for detecting different concentrations of Helicobacter pylori in milk;

[0074] Figure 8 Figure 1 is a comparison chart of the methods of the present invention; wherein, A: heat map analysis comparison of the method of the present invention, qPCR (SG), qPCR (TaqMan), and LAMP (qPCR); B: visualization of the method of the present invention and test strip results; C: ROC curve analysis of the six methods; D E: Bland-Altman plots of the method of the present invention and qPCR; FG: correlation between the method of the present invention and qPCR; H: radar chart of detection accuracy, sensitivity, and specificity analysis of the method of the present invention and some other methods; I: repeatability and reproducibility chart of the present invention. DETAILED DESCRIPTION

[0075] The present invention will be further described below with reference to the following examples. The following examples are descriptive rather than restrictive, and the scope of protection of the present invention cannot be limited by the following examples.

[0076] The various experimental operations involved in the specific embodiments are all routine techniques in the field. For parts not specifically annotated in this document, ordinary technicians in this field can refer to various commonly used reference books, scientific literature or related instructions, manuals, etc. before the filing date of this invention to implement them.

[0077] An Argonaute-based biosensor for detecting Helicobacter pylori, the biosensor comprising a TtAgo-LAMP one-pot reaction system, wherein each 25 μL of the TtAgo-LAMP one-pot reaction system comprises:

[0078] 50 μM TtAgo 1 μL, 10 μM Guide DNA mix-vacA 2.5 μL, Buffer I 2.5 μL, dNTPs mix 2.5 μL, 10× primer mix-vacA 2.5 μL, 2% BSA 2.5 μL, 70 μM Bst DNA polymerase 1.5 μL, Helicobacter pylori genome to be detected 5 μL, 4 μM fluorescent probe vacA-reporter 2.5 μL, ddH2O 2.5 μL;

[0079] The Buffer I comprises: 200 mM Tris-HCl, pH 8.8, 100 mM KCl, 100 mM (NH4)2SO4, 100 mM MgSO4, 1% Tween-20; 200 mM betaine;

[0080] 10× primer mix-vacA gene amplification includes: 2 μM vacA-F3, 2 μM vacA-B3, 16 μM vacA-FIP, 16 μMvacA-BIP, 4 μM vacA-Loop F (i.e., LF), 4 μM vacA-Loop B (i.e., LB);

[0081] Guide DNA mix-vacA includes: 10μM vacA-Guide DNA 1, 10μM vacA-Guide DNA 2;

[0082] dNTPs mix includes: 10mM dATP, 10mM dTTP, 10mM dCTP, 10mM dGTP;

[0083] The gene sequences of vacA-F3, vacA-B3, vacA-FIP, vacA-BIP, vacA-Loop F (LF), vacA-Loop B (LB), vacA-Guide DNA 1, vacA-Guide DNA 2, and fluorescent probe vacA-reporter are SEQ ID No. 1-9, respectively.

[0084] Application of the above-mentioned biosensor in the preparation of detection of vacA target in Helicobacter pylori.

[0085] A method for rapid, automated detection of Helicobacter pylori using the integrated TtAgo cleavage and LAMP amplification technologies of a biosensor not intended for disease diagnosis or treatment, wherein when target DNA is present, TtAgo is able to cleave the target DNA under the guidance of a guide DNA, and the resulting secondary ssDNA can act as a new guide DNA to bind to TtAgo and guide it to specifically cleave a fluorescent probe. TtAgo cleavage is based on strict base complementary pairing, and a detection platform is designed based on this.

[0086] When Helicobacter pylori is present in a sample, treatment with a rapid nucleic acid extraction reagent releases the H. pylori genomic target. LAMP primers specifically recognize the vacA target sequence, generating vacA amplicons through LAMP amplification. TtAgo, guided by the corresponding guide DNA, cleaves the vacA amplicon, generating secondary ssDNA that serves as a new guide DNA to guide TtAgo cleavage of the fluorescent probe. This entire process enables rapid, ultrasensitive detection of H. pylori.

[0087] Further, the following steps are included:

[0088] (1) When Helicobacter pylori is present in the sample, the Helicobacter pylori genome will be released after treatment with a rapid nucleic acid extraction reagent. The genome is added to the LAMP amplification system, and the qPCR program is set to 65°C for 40 minutes;

[0089] (2) After the reaction is completed, real-time fluorescence value measurement is performed through qPCR, and the analysis curve is automatically output to determine the test results.

[0090] Furthermore, the formula of the rapid nucleic acid extraction reagent is: 20mM Tris with a pH of 8.0, 25mM NaCl, 2.5mM EDTA, a volume concentration of 0.05% (wt / vol) SDS, and a volume concentration of 2% (wt / vol) PVP-40, that is, 2.42g of Tris, 14.6g of NaCl and 20g of PVP-40 are dissolved in 900mL of water, and then 5mL of 500mM EDTA with a pH of 8.0 and 5mL of SDS with a volume concentration of 10% wt / vol are added. Finally, the pH is adjusted to 8.0 with hydrochloric acid, and water is added to a final volume of 1L.

[0091] Furthermore, the specific steps are as follows:

[0092] (1) Design of rapid nucleic acid extraction device;

[0093] The device comprises an inner tube and an outer tube which are coaxially and tightly threadedly mounted. The inner tube is arranged above the outer tube, and the outer tube is made of soft plastic. A filter membrane is coaxially and tightly integrated at the bottom of the outer tube. The inner tube and the outer tube are coaxially sleeved to form two independent chambers. The inner tube contains a rapid nucleic acid extraction reagent, and the outer tube can contain a sample to be tested. The flow rate is controlled by a through hole with a 2 mm aperture. The bottom of the outer tube is coaxially and tightly connected to a protective cover which can be detachably provided. The filter membrane is also provided on the outer tube in the protective cover. An inner tube through hole is formed on the bottom of the inner tube, and the top of the outer tube is connected to the inner tube. The inner tube is connected to form a through hole in the outer tube. When the inner tube is rotated, the through hole in the inner tube and the through hole in the outer tube can be arranged vertically and connected, so that the chambers of the inner and outer tubes are closely connected. During use, the staggered design of the inner tube and the bottom disc can initially effectively block the connection between the extraction chamber and the reagent chamber. After the sample is loaded into the extraction chamber, the inner tube is rotated to align the orifices, and the nucleic acid extraction reagent can be quickly released from the reagent chamber. Combined with the up and down shaking operation, the genome can be efficiently extracted. At the same time, the filter membrane at the bottom of the outer tube can effectively remove impurities, completing the nucleic acid purification process.

[0094] (2) The sample to be tested is mixed with a rapid genome nucleic acid extraction reagent to release the Helicobacter pylori genome;

[0095] Mix the sample and the rapid nucleic acid extraction reagent in a 1:1 ratio in a rapid nucleic acid extraction device, i.e., add 500 μL of sample and then 500 μL of rapid nucleic acid extraction reagent, shake to mix, and let it stand for 30 seconds to 10 minutes;

[0096] (3) CTAB method was used to extract genomic DNA from samples;

[0097] (1) Grinding: Weigh 0.1 g of sample into a sterilized mortar and crush thoroughly on ice. Then add 1 mL of sterile water to thoroughly resuspend the feces. Centrifuge at 4°C for 10 min at 10,000 rpm and collect the supernatant.

[0098] (2) Hexadecyltrimethylammonium bromide (CTAB) method

[0099] To each 500 μL sample that had been pretreated, 30 μL of 10% SDS solution, 3 μL of 20 g / L proteinase K, and 4 μL of RNase A were added, mixed, and then placed in a 37°C water bath for 1 hour; 100 μL of 5 mol / L NaCl solution was added to each tube, mixed by inversion, and then 80 μL of CTAB / NaCl (a 10% CTAB, 0.7 mol / L NaCl aqueous solution) was added, mixed gently, and placed in a 65°C water bath for 10 minutes; an equal volume of phenol / chloride was added to the above mixed solution. Mix the mixture of phenol / chloroform and isopropanol, with the volume ratio of phenol:chloroform:isopropanol being 25:24:1. Centrifuge at 4°C for 10 min at 12000 rpm, take the supernatant, add 0.6 times the volume of isopropanol to the supernatant, and mix gently; centrifuge at 4°C for 10 min at 12000 rpm, discard the supernatant, wash the precipitate with 1 mL of pre-cooled 75% ethanol, centrifuge at 4°C for 5 min at 7500 rpm, discard the ethanol, place in a ventilated environment to allow the alcohol to evaporate, and then add 30 μL of sterile water to fully dissolve;

[0100] (4) Primer design

[0101] The specific gene vacA sequence in the Helicobacter pylori genome was selected to design LAMP primers for Helicobacter pylori detection; the specific gene sequence vacA in the vacA genome, i.e., GenBank accession number AP017632, was selected to design LAMP primers for detection; the gene sequences of the LAMP primers are SEQ ID No. 1-6 in order;

[0102] (5) Perform LAMP amplification on the extracted genome;

[0103]

[0104] (6) Preparation of AuNPs and AuNPs@DNA;

[0105] 1) A method for preparing colloidal gold nanoparticles (AuNPs), comprising the following steps: adding 100 μL of 250 mM chloroauric acid trihydrate (HAuCl4·3H2O) to 100 mL of ultrapure water and heating the mixture to boiling under magnetic stirring; rapidly injecting 3 mL of 38.84 mM trisodium citrate until the solution turns a stable, transparent red and continuously stirring for 15 minutes; cooling the mixture to room temperature and concentrating the solution 5-fold by centrifugation at 12,000 rpm to obtain an AuNPs solution, which was stored at 4°C until further use;

[0106] 2) Preparation of gold nanoparticle-modified nucleic acid chain probes, AuNPs@DNA, comprising the following steps: mixing 5 μL of 100 μM thiol-modified nucleic acid chain vacA-SH-DNA with 2 μL of 0.5 M tris(2-chloroethyl) phosphate (TCEP) at pH 7.7, and incubating at room temperature in the dark for 2 h to activate the thiol groups; adding the activated vacA-SH-DNA to 200 μL of a high-concentration AuNPs solution, freezing the solution at −20°C for 2 h, and centrifuging at 12,000 rpm for 20 min after thawing; discarding the supernatant, resuspending and washing the solution with 200 μL of ultrapure water, and repeating the centrifugation twice to obtain the AuNPs@DNA probes, which were resuspended in 200 μL of ultrapure water and stored at 4°C until use;

[0107] 7) A method for preparing a test strip, comprising the following steps:

[0108] A PVC base plate, a sample pad, a conjugate pad, a NC membrane, and a water-absorbing pad are provided as the main components of the test strip; a test line, namely the T line, is prepared by incubating 4.4 mg / mL streptavidin SA with 100 μM biotinylated T probe vacA-T-biotin in 25 mM, pH 5.0 MEST buffer at 37°C for 2 h to obtain a 1 mg / mL SA-biotin-T complex; a continuous film scribing machine (HM8000, China) from Shanghai Jinbiao Biotechnology Co., Ltd. is used to precisely spray-print the complex on a preset position on the NC membrane at a line density of 1 μL / cm; a control line, namely the C line, is prepared by using the same method to prepare a 3 mg / mL SA-biotin-C complex, which is streaked onto the NC membrane at a rate of 1 μL / cm; a conjugate pad is treated by soaking it in a pH 5.0 buffer containing 5 mM polyvinylpyrrolidone (PVP-1000), 0.15 M sucrose, 0.45 mM BSA, and 2% Tween-20. 7.4% PBS buffer and dry overnight; assemble the test strips: fix the sample pad, treated conjugate pad, scribed NC membrane, and absorbent pad on the PVC base in this order, cut into 3.8 mm wide test strips, and place them in a plastic card; package and store: seal the test strips and desiccant in an aluminum foil bag for later use;

[0109] (8) Amplification and cleavage reactions are performed simultaneously to output fluorescent signals / colorimetric signals;

[0110] Combine the LAMP amplification system with the TtAgo reaction system and place the reaction tube in the qPCR [(QuantStudio TM1Plus) from Shanghai Thermo Fisher Scientific Instrument Co., Ltd. (China). At 65°C, fluorescence signals were collected every 1 minute for a total of 40 minutes. Real-time fluorescence value measurements were performed, and analysis curves were automatically output to determine the test results. Alternatively, the reaction tube was placed in a 3D-printed portable visualization device we previously developed (patent application number 2023102268358 has been published) to obtain fluorescence image detection results. The vacA-reporter was replaced with a vacA-Linker chain. The reaction system and other reaction reagents remained unchanged and the tube was heated in a 65°C metal bath for 40 minutes. The reaction product was then incubated with a pre-prepared nanogold probe at room temperature for 10 minutes, dropped into a test strip, and the reaction results were observed.

[0111] A lateral flow assay was established to detect the results: when there is no target, vacA-GuideDNAs will not guide TtAgo to cut the vacA-Linker chain, and the test line, i.e., the T line, will capture the vacA-SH-DNA-Linker coupler that is complementary to its base, thereby displaying color on the T line. Excess vacA-SH-DNA will pair with the nucleic acid chain on the C line, causing the colloidal gold to aggregate on the C line for color development. In the presence of target, positive samples will cause TtAgo to cut the vacA-Linker chain, and only show a signal on the quality control line, i.e., the C line. Since the vacA-Linker chain is broken, it will not be recognized by the nucleic acid chain on the T line, so there is no signal on the test line (T line), which indicates the presence of the target sample.

[0112] When the vacA target is present, the fluorescence intensity of the positive group (i.e., with the target) increases compared with the fluorescence value of the negative control group (i.e., without the target), and it is green fluorescence under 302nm ultraviolet irradiation. An obvious signal appears on the quality control line of the test strip, which proves that the test object is a Helicobacter pylori-contaminated sample.

[0113] The one-pot reaction system of TtAgo-LAMP for each 25 μL is:

[0114] 50μM TtAgo 1μL, 10μM Guide DNA mix-vacA 2.5μL, Buffer I 2.5μL, dNTPsmix 2.5μL, 10× primer mix-vacA 2.5μL, 2% BSA 2.5μL, 70μM Bst DNA polymerase 1.5μL, genome to be detected 5μL, 4μM fluorescent probe vacA-reporter 2.5μL, ddH2O 2.5μL.

[0115] Furthermore, the components of Buffer I are: 200mM Tris-HCl, pH 8.8, 100mM KCl, 100mM (NH4)2SO4, 100mM MgSO4, 1% Tween-20; 200mM betaine;

[0116] 10× primer mix-vacA gene amplification includes: 2 μM vacA-F3, 2 μM vacA-B3, 16 μM vacA-FIP, 16 μMvacA-BIP, vacA-Loop F (i.e., LF), vacA-Loop B (i.e., LB);

[0117] Guide DNA mix-vacA includes: 10μM vacA-Guide DNA 1, 10μM vacA-Guide DNA 2;

[0118] dNTPs mix includes: 10mM dATP, 10mM dTTP, 10mM dCTP, 10mM dGTP;

[0119] The gene sequences of vacA-F3, vacA-B3, vacA-FIP, vacA-BIP, vacA-Loop F (LF), vacA-Loop B (LB), vacA-Guide DNA 1, vacA-Guide DNA 2, and fluorescent probe vacA-reporter are SEQ ID No. 1-9, respectively.

[0120] Furthermore, the minimum detection limit of the method is 1 CFU / mL; the detection time of the method from nucleic acid extraction, LAMP amplification, TtAgo cleavage and signal output only takes 40 minutes.

[0121] Application of the above method in preparing a vacA target and / or a kit for detecting Helicobacter pylori.

[0122] Application of the above method in preparing the vacA gene for detecting Helicobacter pylori.

[0123] Specifically, the relevant preparation and detection are as follows:

[0124] Example 1 Design of a rapid nucleic acid extraction device

[0125] The present invention designs a dual-chamber rapid nucleic acid extraction device based on low-viscosity polypropylene material. Figure 1As shown in B, the device includes an inner tube and an outer tube that are coaxially and tightly threaded together. The inner tube is arranged above the outer tube. The outer tube (capacity is 1mL) is made of soft plastic. The bottom of the outer tube is coaxially and tightly integrated with a filter membrane. The inner tube and the outer tube are coaxially sleeved to form two independent chambers. The inner tube contains a rapid nucleic acid extraction reagent, and the outer tube can contain a sample to be tested. The flow rate is controlled by a rotating disk assembly with a 2mm aperture at the bottom. The bottom of the outer tube is coaxially and tightly connected to a protective cover, and the filter membrane is also arranged on the outer tube inside the protective cover. An inner tube through hole is connected on the bottom of the inner tube, and an outer tube through hole is connected on the top of the outer tube. When the inner tube is rotated, the inner tube through hole and the outer tube through hole can be connected and arranged vertically, so that the chambers of the inner and outer tubes are tightly connected. Initially, the staggered design of the inner tube and the bottom disc can effectively block the connection between the extraction chamber and the reagent chamber. After loading the sample into the extraction chamber, the inner tube is rotated to align the orifices, rapidly releasing the nucleic acid extraction reagent from the chamber. Combined with an up-and-down shaking motion, this allows for efficient genome extraction. Simultaneously, the filter membrane at the bottom of the outer tube effectively removes impurities, completing the nucleic acid purification process. This integrated design significantly simplifies the traditional nucleic acid extraction process through mechanical innovation, providing a reliable sample pretreatment solution for rapid molecular testing.

[0126] Example 2 Rapid Extraction of Helicobacter pylori Genome

[0127] The present invention uses a formulated rapid nucleic acid extraction reagent to quickly extract the Helicobacter pylori genome from bacterial liquid, spiked samples, and saliva samples in clinical samples. The lysis buffer used for rapid nucleic acid extraction is pre-placed in an inner tube, and the circular hole of the inner tube is parallel to the small circular plate to ensure that the liquid cannot flow downward. Then, by simply rotating, the two circular holes will overlap. In this way, the lysis buffer and the sample to be tested are mixed together, and by shaking up and down, the genomic DNA in the sample to be tested can be quickly extracted. Then, the tube wall is squeezed to allow the liquid to pass through the filter membrane below to filter out impurities and allow the genome to drip into the reaction tube. The whole process will be completed within 30 seconds. Take 500 μL of the sample containing Helicobacter pylori and add it to the rapid nucleic acid extraction device, then add 500 μL of lysis solution, i.e., rapid 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), shake to mix, and let it stand for 30 seconds to 10 minutes.

[0128] The rapid nucleic acid extraction reagent was prepared by dissolving 2.42 g of Tris, 14.6 g of NaCl, and 20 g of PVP-40 in 900 mL of water. 5 mL of 500 mM EDTA at pH 8.0 and 5 mL of 10% wt / vol SDS were then added. The pH was adjusted to 8.0 with hydrochloric acid, and water was added to a final volume of 1 L.

[0129] In the present invention, the genome of clinical stool samples was extracted by the CTAB method, and the specific steps are as follows:

[0130] (1) Grinding: Weigh 0.1 g of feces into a sterilized mortar and crush thoroughly on ice. Then add 1 mL of sterile water and resuspend the feces thoroughly. Centrifuge at 4°C for 10 min at 10,000 rpm and collect the supernatant.

[0131] (2) Hexadecyltrimethylammonium bromide (CTAB method)

[0132] To each 500 μL pretreated sample, add 30 μL of 10% SDS, 3 μL of 20 g / L Proteinase K, and 4 μL of RNase A, mix thoroughly, and incubate in a 37°C water bath for 1 hour. Add 100 μL of 5 mol / L NaCl to each tube and mix thoroughly by inversion. Then, add 80 μL of 10% CTAB / NaCl (0.7 mol / L NaCl) and gently mix. Place in a 65°C water bath for 10 minutes. Add an equal volume of phenol / chloroform / isopropanol (25:24:1) to the above mixture, mix thoroughly, and centrifuge at 4°C for 10 minutes at 12,000 rpm. Remove the supernatant, add 0.6 times the volume of isopropanol to the supernatant, and gently mix. Centrifuge at 4°C for 10 min at 12000 rpm, discard the supernatant, wash the precipitate with 1 mL of pre-cooled 75% ethanol, centrifuge at 4°C for 5 min at 7500 rpm, discard the ethanol, place in a ventilated environment to allow the alcohol to evaporate, and then add 30 μL of sterile water to fully dissolve it.

[0133] Example 3 Primer design and LAMP amplification

[0134] exist LAMP Primer Design Tool version 1.3.0 website was used to design LAMP primers for detection based on the specific gene vacA sequence in the vacA genome (GenBank accession number AP017632) of the Helicobacter pylori genome. The gene sequences of the LAMP primers are SEQ ID No. 1-6.

[0135] The mixed LAMP amplification system is placed at 65°C (such as in a metal bath) and incubated for 30 minutes to obtain the LAMP amplification product. The specific LAMP amplification system is as follows:

[0136] Element Volume (μL) 10× primer mix-vacA 2.5 dNTPs mix (10 mM) 2.5 BufferI 2.5 2% BSA 2.5 BstDNA polymerase 0.5 Genome 5 <![CDATA[ddH2O]]> 9.5 Total volume 25

[0137] Example 4 Preparation of AuNPs and AuNPs@DNA

[0138] A colloidal gold solution with a particle size of 20 nm was synthesized by reducing chloroauric acid with trisodium citrate. 100 μL of 250 mM HAuC14·3H2O was added to 100 mL of ultrapure water and heated to boiling under constant magnetic stirring. Subsequently, 3 mL of 38.84 mM trisodium citrate was rapidly injected into the solution. The solution changed color from colorless to a stable, transparent red, and the color remained unchanged after 15 minutes of continuous stirring, indicating the successful synthesis of AuNPs. Finally, the resulting AuNP solution was cooled to room temperature and concentrated fivefold by centrifugation at 12,000 rpm to obtain the AuNP solution, which was stored at 4°C for further use.

[0139] The preparation of AuNPs-modified nucleic acid probes (AuNPs@DNA) is achieved through the formation of gold-sulfur (Au-S) covalent bonds. First, 5 μL of a thiol-modified nucleic acid (vacA-SH-DNA, 100 μM) was mixed with 2 μL of tris(2-chloroethyl) phosphate (TCEP, 0.5 M, pH 7.7). The mixture was incubated at room temperature in the dark for 2 h to activate the thiol groups. The activated vacA-SH-DNA was then added to 200 μL of the AuNPs solution and frozen at -20°C for 2 h. After thawing at room temperature, the solution was centrifuged at 12,000 rpm for 20 min. After centrifugation, the supernatant was discarded, resuspended in 200 μL of ultrapure water, and centrifuged at 12,000 rpm for 20 min. This was repeated twice. The red precipitate, representing the AuNPs@DNA probe, was resuspended in 200 μL of ultrapure water and stored at 4°C for subsequent experiments.

[0140] Example 5. Preparation of test strips

[0141] The test strip consists of five main components: a PVC base, a sample pad, a conjugate pad, a NC membrane, and an absorbent pad. For the test line (T line), SA (4.4 mg / mL) and vacA-T-biotin (100 μM) were added to a solution in MEST buffer (25 mM, pH 5.0). After incubation at 37°C for 2 hours, SA-biotin-T (1 mg / mL) was prepared and streaked onto the NC membrane at a rate of 1 μL / cm. For the C line, SA-biotin-C (3 mg / mL) was prepared using the same method and streaked onto the NC membrane at a rate of 1 μL / cm. The conjugate pad was soaked in PBS buffer (pH 7.4) supplemented with 5 mM PVP-1000, 0.15 M sucrose, 0.45 mM BSA, and 2% Tween-20, and then dried overnight. Finally, the sample pad, treated conjugate pad, scribed NC membrane, and absorbent pad were assembled in that order on a backing card, cut into 3.8 mm wide strips, and placed in a plastic card. All strips were sealed in aluminum foil bags with desiccant for subsequent experiments.

[0142] Example 6 Feasibility Verification Experiment

[0143] To demonstrate the ability of the present invention to detect Helicobacter pylori, the present invention first verified the feasibility of the proposed LAMP-TtAgo one-pot reaction platform. The LAMP amplification temperature was 60-65°C. Within the physiological activity temperature range of TtAgo, we verified the ability to perform primary and secondary cleavage of the LAMP amplicon in two steps within the temperature range of 35 to 95°C. Figure 2 It can be seen that the specificity of TtAgo cutting LAMP products is the best at 65°C, and the positive control and negative control can be clearly distinguished. The uniform temperature provides a basis for the constant temperature reaction of TtAgo and LAMP one-pot method. On this basis, the present invention has developed a biosensor strategy for detecting Helicobacter pylori by LAMP-TtAgo one-pot reaction. First, the present invention established a LAMP-TtAgo one-pot biosensor strategy for detecting the vacA gene target of Helicobacter pylori. The 16nt short ssDNA produced by TtAgo primary cleavage, the two guide DNA sequences designed for the vacA gene amplification fragment, and the matching reporter DNA sequence, such as Figure 3 As shown in A. The feasibility of biosensors for Helicobacter pylori detection. Figure 3As shown in B, when Bst enzyme, dNTP, vacA-GuideDNA, TtAgo protein, target DNAs and primer are present in the system at the same time, a clear fluorescence absorption peak is generated at an excitation wavelength of 484nm and an emission wavelength of 529nm. In addition, no significant increase in fluorescence signal is observed when any component is missing, further verifying the feasibility of the experiment. Only when all components are present, the sample fluorescence image taken using the homemade 3D printing visualizer will show clear green fluorescence ( Figure 3 C). Then, we used a real-time quantitative PCR system to monitor the fluorescence intensity of different samples every 1 minute. The fluorescence signal was recorded at 65°C for 60 minutes, and the average of three experiments was taken. Only the positive samples showed a high fluorescence intensity, and the real-time fluorescence curve reached a plateau after 40 minutes (e.g. Figure 3 D real-time fluorescence curve). And at 40 min, compare the fluorescence intensity of different samples, such as Figure 3 The fluorescence intensity analysis of the endpoint of D shows that the fluorescence intensity of the positive sample is clearly different from that of other samples, which is consistent with the results reflected by the real-time fluorescence curve. The test strip established by the present invention is designed based on the principle of nucleic acid base pairing. Figure 3 E shows the experimental results of a test strip analysis. In the absence of Bst, dNTPs, vacA-Guide DNA, TtAgo, target DNA, and primer, vacA-Guide DNAs do not guide TtAgo to cleave the vacA-Linker chain. Instead, the test line (T line) captures the vacA-SH-DNA-Linker conjugate, which is complementary to its base, resulting in color development on the T line. Excess vacA-SH-DNA pairs with the nucleic acid chain on the C line, causing colloidal gold to accumulate on the C line for color development. In the presence of target, a positive sample causes TtAgo to cleave the vacA-Linker chain, resulting in a signal only on the control line (C line). Since the vacA-Linker chain is broken, it is not recognized by the nucleic acid chain on the T line, resulting in no signal on the test line (T line), indicating the presence of the target sample.

[0144] Example 7 Optimization of reaction conditions

[0145] In this study, we used fluorescence intensity as a metric to investigate the various variables that influence the one-pot LAMP-TtAgo method. These parameters included the dosages of the key components (Bst DNA polymerase, TtAgo, vacA-guide DNA, dNTPs, primers, and the vacA-reporter gene). Fluorescence signals were collected using a real-time quantitative PCR system, and the average of three tests was calculated. Two key principles should be considered during this optimization process: (I) ensuring that the fluorescence curve enters the exponential amplification phase more quickly; and (II) ensuring that the fluorescence curve has a high fluorescence intensity.

[0146] Different concentrations of Bst DNA polymerase (0.0, 1.4, 2.8, 4.2, 5.6, and 7.0 μM), TtAgo (0.0, 0.5, 1.0, 1.5, 2.0, and 2.5 μM), vacA-guide DNA (0.0, 0.5, 1.0, 1.5, 2.0, and 2.5 μM), dNTPs (0.0, 1.0, 1.5, 2.0, 2.5, and 3.0 μL), primer (0.0, 1.0, 1.5, 2.0, and 3.0 μL), and the vacA-reporter gene (0.0, 0.1, 0.2, 0.4, 0.6, and 0.8 μM) were combined in a one-pot LAMP-TtAgo assay system. The reaction was performed at 65°C for 40 minutes, and fluorescence was measured every 1 minute in three replicates. Optimal conditions were defined by a faster and higher fluorescence peak. Fluorescence images of the above samples were taken using a 3D printing visualizer, and the optimal conditions screened out were consistent with the fluorescence curve results. Figure 4 As shown in (AF), the optimal system for LAMP-TtAgo single-tube reaction is: 4.2μM Bst DNA polymerase, 2.0μM TtAgo, 1.0μM vacA-Guide DNA, 2.5μL dNTP, 2.5μL Primer, and 0.4μM reporter gene vacA-reporter.

[0147] In a one-pot reaction, the buffer is the core component that maintains the stability and functionality of the reaction system. Its role not only includes regulating the physical and chemical properties of the reaction environment, but also directly affects enzyme activity, nucleic acid amplification efficiency and detection specificity. The present invention systematically evaluated the effect of the reaction buffer composition on the performance of the LAMP-TtAgo one-pot detection platform. Six buffer systems were used for one-pot reaction optimization, and the reaction process was dynamically monitored by real-time quantitative PCR. Figure 5As shown in A, only Buffer I and TRBuffer can achieve effective exponential amplification and show significantly higher fluorescence signal intensity. Among them, Buffer I shows the best reaction kinetics, entering the exponential amplification phase at 10 minutes and reaching the plateau phase at around 30 minutes. Compared with TRBuffer, the reaction efficiency is higher. In order to verify the effect of buffer on detection specificity, we set up a buffer-free control (Ctrl) in the above system and evaluated the system by calculating the positive / negative signal-to-noise ratio (P / N ratio) at different time points. Figure 5 As shown in B, the P / N ratios of Buffer I and TRBuffer were significantly higher than those of other buffers, and were consistent with the real-time amplification curve ( Figure 5 A) Highly consistent, indicating that the optimized buffer system can effectively balance reaction speed and signal specificity.

[0148] As a key cofactor for Bst DNA polymerase and TtAgo endonuclease, Mg 2+ In order to optimize the metal ion environment of the reaction system, the present invention systematically evaluated the 0-12mM concentration gradient of Mg 2+ Effects on the efficiency of fluorescence quantitative PCR amplification ( Figure 5 C) Experimental data show that when Mg 2+ When the concentration increased from 2 mM to 10 mM, the real-time fluorescence intensity showed a dose-dependent enhancement, which indicated that within this concentration range, Mg 2+ It can effectively promote the synergistic effect of LAMP amplification and TtAgo cleavage. 2+ When the concentration of Mg was increased to 12 mM, although the amplification reached its peak at 20 min, the final fluorescence intensity of the system decreased significantly compared with the 10 mM group. 2+ This accelerates the strand displacement activity of Bst DNA polymerase, resulting in a shortened LAMP amplification cycle, which in turn prompts TtAgo to prematurely cut the newly generated double-stranded DNA product, resulting in insufficient accumulation of detectable fluorescent signals. Time kinetic analysis confirmed this mechanism (e.g. Figure 5 D). Although the 12 mM group entered the plateau phase earlier than the 10 mM group, based on the comprehensive evaluation of the reaction kinetic parameters and the integrity of the final product, 10 mM was finally determined to be the optimal Mg 2+ The concentration not only ensures efficient amplification, but also maintains moderate regulation of TtAgo cleavage, establishing a standardized reaction system for subsequent experiments.

[0149] The stability of enzymes is a key factor in the nucleic acid detection process. In this invention, we focused on the two proteins, Bst and TtAgo, to ensure their stability in a one-pot reaction system. Since betaine has the function of maintaining enzyme activity, the present invention selected different concentrations of betaine (0, 20, 40, 60, 80, 100 mM) and studied the results by real-time fluorescence monitoring. Among them, 20 mM betaine performed the best (such as Figure 5 E). In addition, each concentration of betaine was compared with a negative control (no buffer), as shown in FIG. Figure 5 As shown in Figure F, the fluorescence intensity of betaine was detected in a one-pot reaction ranging from 0 to 100 mM. However, compared with the negative control, 20 mM betaine showed the highest signal-to-noise ratio at 30 min, indicating that it stabilized the enzymatic reaction while minimizing background interference.

[0150] Example 8 Sensitivity and Selectivity

[0151] In order to make LAMP-TtAgo one-pot detection faster and more accurate, the Helicobacter pylori genome was extracted based on the optimized LAMP-TtAgo one-pot system and added to the system to trigger the LAMP-TtAgo one-pot reaction, outputting a fluorescent signal. The reaction time was 40 minutes and the detection limit could reach 1 CFU / mL (such as Figure 6 After the reaction is completed, the sample is placed in the 3D printed portable visual detection device we developed previously (application number 2023102268358), as shown in FIG. Figure 6 As shown in B, the fluorescence image shows that the detection 0 -10 6 There is obvious green fluorescence in the reaction tube with CFU / mL, while there is no obvious fluorescence in the negative control reaction tube. The sample is added to the test strip, and the test strip shows detection 10 0 -10 6 There is an obvious signal on the C line of CFU / mL ( Figure 6 C) The above proves that the detection system reacts normally in qPCR and outputs fluorescent signals, with a detection limit of 1 CFU / mL. Then, genomic solutions containing Staphylococcus aureus, Escherichia coli, Hela, human colon adenocarcinoma cell HT29, Bacillus subtilis, Lactobacillus, Salmonella, Pichia pastoris, Rhizobium, and Helicobacter pylori were added to verify the selectivity of the detection platform. Figure 6 As shown in DF, only Helicobacter pylori has fluorescence output, which further supports the good specificity of the one-pot system detection.

[0152] Example 9 Application of the sensor in detecting Helicobacter pylori

[0153] In order to demonstrate the feasibility of using the one-pot platform to detect Helicobacter pylori in real samples, we used some real samples to test the performance and capability of the TtAgo-based detection method. We selected daily foods (such as milk, pork), saliva, and mouse feces, and added different concentrations of Helicobacter pylori to them. After the genome was extracted with a rapid nucleic acid extraction reagent, it was added to the one-pot reaction system, and the reaction products were analyzed by fluorescence, visualization, and test strip methods, such as Figure 7 As shown in A. The fluorescence method system was used to detect different concentrations of Helicobacter pylori in pork, with the concentration of Helicobacter pylori as the horizontal axis and the fluorescence intensity as the vertical axis. The fluorescence intensity of the pork samples containing Helicobacter pylori increased significantly, and the detection limit LOD was 1 CFU / mL, which was consistent with the sensitivity test results mentioned above. The visualization method system was used to detect different concentrations of Helicobacter pylori in pork, and fluorescence images were collected by a portable detection device. Positive samples had obvious green fluorescence, and negative samples did not capture fluorescence images. Similarly, we used a test strip system to detect Helicobacter pylori in pork samples. In samples containing Helicobacter pylori, the test strip only showed color on the C line, while negative samples showed color on both the C line and the T line, indicating that the test results of the test strip method were consistent with the results of the fluorescence method. The detection methods for mouse feces, saliva, and milk were consistent with those for pork ( Figure 7 The portable fluorescence detection device detected H. pylori at various concentrations in real samples, producing a bright green fluorescent signal. The test strips only produced a red signal along the C line for H. pylori samples of varying concentrations. The detection results were consistent across the three different methods. These data demonstrate that our constructed LAMP-TtAgo detection platform is reliable and capable of detecting bacteria in a variety of real samples.

[0154] In summary, the performance of the proposed method in terms of sensitivity, dynamic range, and detection time is comparable to or better than most reported methods. These data clearly demonstrate the applicability of the proposed biosensing strategy for real-world H. pylori detection. Furthermore, the detection of H. pylori in food samples, feces, and saliva samples demonstrates the potential of the proposed method for food detection and early diagnosis of H. pylori infection.

[0155] Example 10 Method Comparison

[0156] This study adopted a double-blind experimental design and used a single-tube method to detect and analyze clinical samples. The experimental samples included three biological samples: gastric mucosa (sample number = 22, 5 negative cases / 17 positive cases), saliva (sample number = 25, 18 negative cases / 7 positive cases) and feces (sample number = 25, 18 negative cases / 7 positive cases). For loop-mediated isothermal amplification technology (LAMP), we systematically compared three detection methods: 1) Real-time quantitative LAMP method based on SYBR Green fluorescence detection (LAMP-qPCR), which obtains fluorescence signals through real-time monitoring of qPCR instruments; 2) Colorimetric LAMP method (LAMP colorimetric), which uses the color change of phenol red indicator with pH value for naked eye interpretation. 3) Agarose gel electrophoresis (LAMP-AGGE), which detects amplified product bands by traditional electrophoresis technology. We first compared the method of the present invention with the qPCR method and the LAMP-qPCR method, such as Figure 8 As shown in Figure A, the diagnostic accuracy of the method of the present invention is comparable to that of the SYBR Green method and TaqMan probe method of qPCR. It is worth noting that, except for this method, the other three detection methods all showed varying degrees of false positive or false negative phenomena. As for the LAMP-TtAgo one-pot method, Figure 8 As shown in B, the results of the visualization method and the test paper method were consistent with those of the fluorescence method.

[0157] Receiver operating characteristic curve (ROC) analysis showed that the method of the present invention showed excellent diagnostic effect in clinical sample detection, with an area under the curve (AUC) of 1.0, which was significantly better than qPCR (SG) method (0.97), qPCR (TaqMan) (0.98), LAMP-qPCR (0.85), LAMP-AGGE (0.82) and LAMP colorimetric (0.88) ( Figure 8 C). Bland-Altman consistency analysis further verified the high consistency between the present invention and the two qPCR methods ( Figure 8 DE). The analysis results showed that all data points were within the 95% confidence interval, and the differences were concentrated near the zero baseline, indicating that the detection system has excellent analytical performance.

[0158] We further verified the quantitative consistency of the present invention with two standard qPCR methods (SYBR Green and TaqMan methods). Figure 8As shown in Figure 5, the linear correlation analysis between the present invention and the two qPCR methods showed that all r and ρ values ​​were distributed in the range of 0.8-1.0, confirming that there was a significant and highly positive correlation between the two sets of quantitative data. This study constructed a four-quadrant classification scatter plot based on the threshold (cut-off value) of the increase in fluorescence signal. This visualization strategy effectively achieved intuitive analysis of detection performance parameters and visual positioning of classification errors. In terms of diagnostic effect indicators, the specificity, accuracy, and sensitivity of the present invention were all 100%, and all parameters were superior to other detection methods ( Figure 8 H). In terms of method validation, this study evaluated the stability of the system through repeatability and reproducibility experiments according to the biosensor development standards. The results showed that the relative standard deviation (RSD) of the two experiments was less than 8% ( Figure 8 I), meeting the quality control requirement of an RSD threshold of less than 10%, confirming the excellent repeatability and reproducibility of the present invention. In summary, the present invention offers significant advantages in rapidity (single-tube closed-tube testing), accuracy (AUC = 1.0), and stability (RSD < 8%), providing an efficient and reliable technical solution for clinical pathogen detection.

[0159] The sequences used in the present invention are:

[0160]

[0161] Buffer components used in the present invention:

[0162]

[0163] Comparison of TtAgo-LAMP one-pot detection with existing technologies:

[0164] Recent technologies for detecting Helicobacter pylori have been reviewed and compared with the sensor of the present invention. As shown in Table 1, the present sensor uses fluorescence as its output, but can be used for point-of-care (POCT) testing through visualization of fluorescence, eliminating the need for large-scale instrumentation. Furthermore, the detection limit of the present invention is 1 CFU / mL, comparable to or even better than that of existing technologies. Furthermore, the present invention is suitable for screening vacA targets in all H. pylori species. In summary, the detection sensor of the present invention surpasses existing technologies in terms of sensitivity, detection method, and detection range, and is also suitable for POCT testing.

[0165] Table 1 Comparison of methods for detecting Helicobacter pylori with existing technologies

[0166]

[0167] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand 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 contents disclosed in the embodiments.

Claims

1. An Argonaute-based biosensor for Helicobacter pylori detection, characterized in that: The biosensor includes a TtAgo-LAMP one-pot reaction system. Each 25 μL of the TtAgo-LAMP one-pot reaction system is: 50 μM TtAgo 1 μL, 10 μM Guide DNA mix-vacA 2.5 μL, Buffer I 2.5 μL, dNTPs mix 2.5 μL, 10× primer mix-vacA 2.5 μL, 2% BSA 2.5 μL, 70 μM Bst DNA polymerase 1.5 μL, Helicobacter pylori genome to be detected 5 μL, 4 μM fluorescent probe vacA-reporter 2.5 μL, ddH2O 2.5 μL; The Buffer I comprises: 200 mM Tris-HCl, pH 8.8, 100 mM KCl, 100 mM (NH4)2SO4, 100 mM MgSO4, 1% Tween-20; 200 mM betaine; 10× primer mix-vacA gene amplification includes: 2 μM vacA-F3, 2 μM vacA-B3, 16 μM vacA-FIP, 16 μMvacA-BIP, 4 μM vacA-Loop F (i.e., LF), 4 μM vacA-Loop B (i.e., LB); Guide DNAmix-vacA includes: 10μM vacA-Guide DNA 1, 10μM vacA-Guide DNA 2; dNTPs mix includes: 10mM dATP, 10mM dTTP, 10mM dCTP, 10mM dGTP; The gene sequences of vacA-F3, vacA-B3, vacA-FIP, vacA-BIP, vacA-Loop F (LF), vacA-Loop B (LB), vacA-Guide DNA 1, vacA-Guide DNA 2, and fluorescent probe vacA-reporter are SEQ ID No. 1-9, respectively.

2. Use of the biosensor according to claim 1 in preparing a vacA target for detecting Helicobacter pylori.

3. A method for rapid and automated detection of Helicobacter pylori using the biosensor of claim 1 that integrates TtAgo cleavage and LAMP amplification technology for purposes other than diagnosis and treatment of a disease, characterized in that: In the method, when target DNA is present, TtAgo can cut the target DNA under the guidance of the guide DNA. The generated secondary ssDNA can act as a new guide DNA to bind to TtAgo and guide it to specifically cut the fluorescent probe. The cutting of TtAgo is based on strict base complementary pairing, based on which the detection platform is designed; When Helicobacter pylori is present in a sample, treatment with a rapid nucleic acid extraction reagent releases the H. pylori genomic target. LAMP primers specifically recognize the vacA target sequence, generating vacA amplicons through LAMP amplification. TtAgo, guided by the corresponding guide DNA, cleaves the vacA amplicon, generating secondary ssDNA that serves as a new guide DNA to guide TtAgo cleavage of the fluorescent probe. This entire process enables rapid, ultrasensitive detection of H. pylori.

4. The method according to claim 3, wherein: The steps include: (1) When Helicobacter pylori is present in the sample, the Helicobacter pylori genome will be released after treatment with a rapid nucleic acid extraction reagent. The genome is added to the LAMP amplification system, and the qPCR program is set to 65°C for 40 minutes; (2) After the reaction is completed, real-time fluorescence value measurement is performed through qPCR, and the analysis curve is automatically output to determine the test results.

5. The method according to claim 4, characterized in that: The formula of the rapid nucleic acid extraction reagent is: 20mM Tris (pH=8.0), 25mM NaCl, 2.5mM EDTA, 0.05% (wt / vol) SDS, and 2% (wt / vol) PVP-40. That is, 2.42g of Tris, 14.6g of NaCl, and 20g of PVP-40 are dissolved in 900mL of water, followed by the addition of 5mL of 500mM EDTA (pH 8.0) and 5mL of 10% wt / vol SDS. Finally, the pH is adjusted to 8.0 with hydrochloric acid, and water is added to a final volume of 1L.

6. The method according to claim 3, wherein: The specific steps are as follows: (1) Design of rapid nucleic acid extraction device; The device comprises an inner tube and an outer tube which are coaxially and tightly threadedly mounted. The inner tube is arranged above the outer tube, and the outer tube is made of soft plastic. A filter membrane is coaxially and tightly integrated at the bottom of the outer tube. The inner tube and the outer tube are coaxially sleeved to form two independent chambers. The inner tube contains a rapid nucleic acid extraction reagent, and the outer tube can contain a sample to be tested. The flow rate is controlled by a through hole with a 2 mm aperture. The bottom of the outer tube is coaxially and tightly connected to a protective cover which can be detachably provided. The filter membrane is also provided on the outer tube in the protective cover. An inner tube through hole is formed on the bottom of the inner tube, and the top of the outer tube is connected to the inner tube. The inner tube is connected to form a through hole in the outer tube. When the inner tube is rotated, the through hole in the inner tube and the through hole in the outer tube can be arranged vertically and connected, so that the chambers of the inner and outer tubes are closely connected. During use, the staggered design of the inner tube and the bottom disc can initially effectively block the connection between the extraction chamber and the reagent chamber. After the sample is loaded into the extraction chamber, the inner tube is rotated to align the orifices, and the nucleic acid extraction reagent can be quickly released from the reagent chamber. Combined with the up and down shaking operation, the genome can be efficiently extracted. At the same time, the filter membrane at the bottom of the outer tube can effectively remove impurities, completing the nucleic acid purification process. (2) The sample to be tested is mixed with a rapid genome nucleic acid extraction reagent to release the Helicobacter pylori genome; Mix the sample and the rapid nucleic acid extraction reagent in a 1:1 ratio in a rapid nucleic acid extraction device, i.e., add 500 μL of sample and then 500 μL of rapid nucleic acid extraction reagent, shake to mix, and let it stand for 30 seconds to 10 minutes; (3) CTAB method was used to extract genomic DNA from samples; (1) Grinding: Weigh 0.1 g of sample into a sterilized mortar and crush thoroughly on ice. Then add 1 mL of sterile water to thoroughly resuspend the feces. Centrifuge at 4°C for 10 min at 10,000 rpm and collect the supernatant. (2) Hexadecyltrimethylammonium bromide (CTAB) method To each 500 μL sample that had been pretreated, 30 μL of 10% SDS solution, 3 μL of 20 g / L proteinase K, and 4 μL of RNase A were added, mixed, and then placed in a 37°C water bath for 1 hour; 100 μL of 5 mol / L NaCl solution was added to each tube, mixed by inversion, and then 80 μL of CTAB / NaCl (a 10% CTAB, 0.7 mol / L NaCl aqueous solution) was added, mixed gently, and placed in a 65°C water bath for 10 minutes; an equal volume of phenol / chloride was added to the above mixed solution. Mix the mixture of phenol / chloroform and isopropanol, with the volume ratio of phenol:chloroform:isopropanol being 25:24:

1. Centrifuge at 4°C for 10 min at 12000 rpm, take the supernatant, add 0.6 times the volume of isopropanol to the supernatant, and mix gently; centrifuge at 4°C for 10 min at 12000 rpm, discard the supernatant, wash the precipitate with 1 mL of pre-cooled 75% ethanol, centrifuge at 4°C for 5 min at 7500 rpm, discard the ethanol, place in a ventilated environment to allow the alcohol to evaporate, and then add 30 μL of sterile water to fully dissolve; (4) Primer design The specific gene vacA sequence in the Helicobacter pylori genome was selected to design LAMP primers for Helicobacter pylori detection; the specific gene sequence vacA in the vacA genome, i.e., GenBank accession number AP017632, was selected to design LAMP primers for detection; the gene sequences of the LAMP primers are SEQ ID No. 1-6 in order; (5) Perform LAMP amplification on the extracted genome; (6) Preparation of AuNPs and AuNPs@DNA; 1) A method for preparing colloidal gold nanoparticles (AuNPs), comprising the following steps: adding 100 μL of 250 mM chloroauric acid trihydrate (HAuCl4·3H2O) to 100 mL of ultrapure water and heating the mixture to boiling under magnetic stirring; rapidly injecting 3 mL of 38.84 mM trisodium citrate until the solution turns a stable, transparent red and continuously stirring for 15 minutes; cooling the mixture to room temperature and concentrating the solution 5-fold by centrifugation at 12,000 rpm to obtain an AuNPs solution, which was stored at 4°C until further use; 2) Preparation of gold nanoparticle-modified nucleic acid chain probes, AuNPs@DNA, comprising the following steps: mixing 5 μL of 100 μM thiol-modified nucleic acid chain vacA-SH-DNA with 2 μL of 0.5 M tris(2-chloroethyl) phosphate (TCEP) at pH 7.7, and incubating at room temperature in the dark for 2 h to activate the thiol groups; adding the activated vacA-SH-DNA to 200 μL of a high-concentration AuNPs solution, freezing the solution at −20°C for 2 h, and centrifuging at 12,000 rpm for 20 min after thawing; discarding the supernatant, resuspending and washing the solution with 200 μL of ultrapure water, and repeating the centrifugation twice to obtain the AuNPs@DNA probes, which were resuspended in 200 μL of ultrapure water and stored at 4°C until use; 7) A method for preparing a test strip, comprising the following steps: A PVC base plate, a sample pad, a conjugate pad, a NC membrane, and a water-absorbing pad are provided as the main components of the test strip; a test line, namely the T line, is prepared by incubating 4.4 mg / mL streptavidin SA with 100 μM biotinylated T probe vacA-T-biotin in 25 mM, pH 5.0 MEST buffer at 37°C for 2 h to obtain a 1 mg / mL SA-biotin-T complex; a continuous film scribing machine (HM8000, China) from Shanghai Jinbiao Biotechnology Co., Ltd. is used to precisely spray-print the complex on a preset position on the NC membrane at a line density of 1 μL / cm; a control line, namely the C line, is prepared by using the same method to prepare a 3 mg / mL SA-biotin-C complex, which is streaked onto the NC membrane at a rate of 1 μL / cm; a conjugate pad is treated by soaking it in a pH 5.0 buffer containing 5 mM polyvinylpyrrolidone (PVP-1000), 0.15 M sucrose, 0.45 mM BSA, and 2% Tween-20. 7.4% PBS buffer and dry overnight; assemble the test strips: fix the sample pad, treated conjugate pad, scribed NC membrane, and absorbent pad on the PVC base in this order, cut into 3.8 mm wide test strips, and place them in a plastic card; package and store: seal the test strips and desiccant in an aluminum foil bag for later use; (8) Amplification and cleavage reactions are performed simultaneously to output fluorescent signals / colorimetric signals; Combine the LAMP amplification system with the TtAgo reaction system, place the reaction tube in a qPCR system, collect fluorescence signals every 1 minute for a total of 40 minutes at 65°C, perform real-time fluorescence value measurement, automatically output analysis curves, and determine the test results. Alternatively, place the reaction tube in a 3D-printed portable visualization device to obtain fluorescence image detection results. Replace the vacA-reporter with a vacA-linker chain, keep the reaction system and other reaction reagents unchanged, and heat in a 65°C metal bath for 40 minutes. Then, incubate the reaction product with a pre-prepared nanogold probe at room temperature for 10 minutes, drip it onto a test strip, and observe the reaction results. A lateral flow assay was established to detect the results: when there is no target, vacA-Guide DNAs will not guide TtAgo to cut the vacA-Linker chain, and the test line, i.e., the T line, will capture the vacA-SH-DNA-Linker coupler that is complementary to its base, thereby developing color on the T line. Excess vacA-SH-DNA will pair with the nucleic acid chain on the C line, causing the colloidal gold to accumulate on the C line for color development. In the presence of target, positive samples will cause TtAgo to cut the vacA-Linker chain, and a signal will only be displayed on the quality control line, i.e., the C line. Since the vacA-Linker chain is broken, it will not be recognized by the nucleic acid chain on the T line, so there is no signal on the test line (T line), indicating the presence of the target sample. When the vacA target is present, the fluorescence intensity of the positive group (i.e., with the target) increases compared with the fluorescence value of the negative control group (i.e., without the target), and it is green fluorescence under 302nm ultraviolet irradiation. An obvious signal appears on the quality control line of the test strip, which proves that the test object is a Helicobacter pylori-contaminated sample. The one-pot reaction system of TtAgo-LAMP for each 25 μL is: 50μM TtAgo 1μL, 10μM Guide DNAmix-vacA 2.5μL, Buffer I 2.5μL, dNTPs mix 2.5μL, 10× primer mix-vacA 2.5μL, 2% BSA 2.5μL, 70μM Bst DNA polymerase 1.5μL, genome to be detected 5μL, 4μM fluorescent probe vacA-reporter 2.5μL, ddH2O 2.5μL.

7. The method according to claim 8, characterized in that: The components of Buffer I are: 200mM Tris-HCl, pH 8.8, 100mM KCl, 100mM (NH4)2SO4, 100mM MgSO4, 1% Tween-20; 200mM betaine; 10× primer mix-vacA gene amplification includes: 2 μM vacA-F3, 2 μM vacA-B3, 16 μM vacA-FIP, 16 μMvacA-BIP, vacA-Loop F (i.e., LF), vacA-Loop B (i.e., LB); Guide DNA mix-vacA includes: 10μM vacA-Guide DNA 1, 10μM vacA-Guide DNA 2; dNTPs mix includes: 10mM dATP, 10mM dTTP, 10mM dCTP, 10mM dGTP; The gene sequences of vacA-F3, vacA-B3, vacA-FIP, vacA-BIP, vacA-Loop F (LF), vacA-Loop B (LB), vacA-Guide DNA 1, vacA-Guide DNA 2, and fluorescent probe vacA-reporter are SEQ ID No. 1-9, respectively.

8. The method according to any one of claims 3 to 7, characterized in that: The minimum detection limit of the method is 1 CFU / mL; the detection time of the method, including nucleic acid extraction, LAMP amplification, TtAgo cleavage and signal output, only takes 40 minutes.

9. Use of the method according to any one of claims 3 to 8 in preparing a target and / or a kit for detecting vacA in Helicobacter pylori.

10. Use of the method according to any one of claims 3 to 8 in preparing the vacA gene for detecting Helicobacter pylori.

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