Kit for detecting multiple drug-resistant genes of helicobacter pylori and quantitatively monitoring treatment based on paper sensor

Abstract

The invention relates to the technical field of helicobacter pylori multi-drug-resistant gene detection, and discloses a paper sensor-based helicobacter pylori multi-drug-resistant gene detection and treatment quantitative monitoring kit, which comprises an isothermal amplification module, which comprises a loop-mediated isothermal amplification reaction system and is used for carrying out isothermal amplification on 23S rRNA gene and rdxA gene of helicobacter pylori; the paper sensor module comprises a nitrocellulose membrane, the nitrocellulose membrane comprises a test area and a control area, a biotin labeled probe aiming at 23S rRNA gene and rdxA gene mutant amplification products is fixed in the test area, and a biotin labeled probe aiming at reference gene cgt amplification products is fixed in the control area; and the color development module is used for performing color development and quantitative analysis on the detection result of the paper sensor module. The kit disclosed by the invention can be used for detecting various helicobacter pylori multi-drug-resistant genes, can be used for quantitatively monitoring, and is rapid and convenient to monitor.

Description

Technical Field

[0001] This invention relates to the field of Helicobacter pylori multidrug resistance gene detection technology, and more specifically, to a kit for detecting and quantitatively monitoring Helicobacter pylori multidrug resistance genes based on paper sensors. Background Technology

[0002] Helicobacter pylori (Hp) is a Gram-negative bacterium that primarily lives in the human stomach and duodenum, and is spiral, S-shaped, or arc-shaped. Numerous studies have confirmed that Hp is closely related to various gastric diseases, including chronic gastritis, peptic ulcers, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma, and is one of the important pathogenic factors leading to the occurrence and development of these diseases.

[0003] Currently, the main treatment for Helicobacter pylori infection involves antibiotics combined with proton pump inhibitors and bismuth preparations. However, with the widespread use of antibiotics, drug resistance in Helicobacter pylori is becoming increasingly serious, leading to a continuous decline in the eradication rate of traditional empirical treatment regimens, with eradication rates in some areas even falling below 60%. The emergence of drug resistance not only increases treatment costs and prolongs the treatment cycle but may also lead to recurring symptoms, increasing patient suffering and posing a significant challenge to public health. For example, resistance rates to commonly used antibiotics such as clarithromycin, levofloxacin, and metronidazole are showing an upward trend in different regions, with clarithromycin resistance rates exceeding 30% in some areas and levofloxacin resistance rates approaching 50%.

[0004] Helicobacter pylori infection is a significant pathogenic factor for gastritis, peptic ulcers, and gastric cancer. Clarithromycin is one of the core drugs in first-line eradication regimens commonly used in clinical practice. Its antibacterial mechanism mainly relies on binding to the 23S rRNA in the 50S subunit of the Helicobacter pylori ribosome, inhibiting its protein synthesis. However, specific site mutations in the 23S rRNA gene can severely affect the binding efficiency of clarithromycin, leading to a significant increase in drug resistance. Studies have shown that mutations at bases 2142 and 2143 of the 23S rRNA gene (most commonly A2142G / C and A2143G) are the main mechanism causing clarithromycin resistance. These point mutations alter the conformation of the ribosomal active site, thereby preventing clarithromycin from binding and resulting in antibacterial inactivation. Because these mutation sites are highly concentrated and exist in the form of single-base variations, they are very suitable for precise identification and typing using single nucleotide detection strategies. In addition, metronidazole is another key drug for treating Helicobacter pylori. Its bactericidal mechanism relies on the reduction of Helicobacter pylori within the bacteria to toxic intermediate metabolites, which damage DNA. However, mutations in the rdxA gene (encoding an oxidoreductase) in Helicobacter pylori, especially missense mutations, premature termination mutations, or large deletions, significantly reduce its reductive metabolic activity, preventing metronidazole activation and ultimately leading to resistance. Among reported metronidazole-resistant strains, the rdxA gene exhibits a wide mutation spectrum, with Q50Stop (i.e., the 50th amino acid being replaced by a stop codon instead of glutamine) being a representative mutation type. This type of mutation can lead to complete loss of RdxA protein function and is an important molecular marker for metronidazole resistance. Unlike 23S rRNA, rdxA mutations typically involve loss of function; therefore, detection methods must consider both premature termination point mutations and structural changes such as fragment deletions.

[0005] Therefore, providing a simple, rapid, accurate, and low-cost method and kit that can simultaneously monitor Helicobacter pylori multidrug resistance genes and conduct quantitative monitoring of treatment is of significant clinical and social value for improving the diagnosis and treatment of Helicobacter pylori infection. Summary of the Invention

[0006] In view of this, the present invention proposes a kit for the detection and quantitative monitoring of Helicobacter pylori multidrug resistance genes based on paper sensors, aiming to solve the technical problem that current technologies cannot rapidly, accurately and quantitatively detect Helicobacter pylori multidrug resistance genes.

[0007] This invention provides a kit for detecting Helicobacter pylori multidrug resistance genes and quantitatively monitoring treatment based on a paper sensor, comprising:

[0008] The isothermal amplification module includes a loop-mediated isothermal amplification reaction system for isothermal amplification of the 23S rRNA gene and rdxA gene of Helicobacter pylori.

[0009] A paper sensor module includes a nitrocellulose membrane, which has a test area and a control area. The test area is immobilized with biotin-labeled probes targeting mutant amplification products of the 23S rRNA gene and the rdxA gene, and the control area is immobilized with biotin-labeled probes targeting amplification products of the internal reference gene cgt.

[0010] The color development module is used to perform color development and quantitative analysis on the detection results of the paper sensor module.

[0011] Preferably, the primer set for the 23S rRNA gene A2143G mutation site includes outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB, wherein the sequences of outer primers F3 and B3 are shown in SEQ ID NO:1 and SEQ ID NO:2; the sequences of inner primers FIP and BIP are shown in SEQ ID NO:3 and SEQ ID NO:4; and the sequences of loop primers LF and LB are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0012] Preferably, the primer set for the rdxA gene Q50Stop mutation site includes outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB, wherein the sequences of outer primers F3 and B3 are shown in SEQ ID NO:7 and SEQ ID NO:8; the sequences of inner primers FIP and BIP are shown in SEQ ID NO:9 and SEQ ID NO:10; and the sequences of loop primers LF and LB are shown in SEQ ID NO:11 and SEQ ID NO:12.

[0013] Preferably, the primer set for the internal reference gene cgt includes outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB, wherein the sequences of outer primers F3 and B3 are shown in SEQ ID NO:13 and SEQ ID NO:14; the sequences of inner primers FIP and BIP are shown in SEQ ID NO:15 and SEQ ID NO:16; and the sequences of loop primers LF and LB are shown in SEQ ID NO:17 and SEQ ID NO:18.

[0014] Preferably, the biotin-labeled probes in the test region include probes targeting the A2143G mutation site of the 23S rRNA gene, probes targeting the A2142G / C mutation site of the 23S rRNA gene, and probes targeting the Q50Stop mutation site of the rdxA gene, with sequences as shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; the biotin-labeled probes in the control region include probes targeting the internal reference gene cgt, with a sequence as shown in SEQ ID NO:22.

[0015] Preferably, the loop-mediated isothermal amplification reaction system includes ThermoPol buffer and Mg2+. 2+ Betaine, dNTPs, Bst DNA polymerase, and template DNA.

[0016] Preferably, the nitrocellulose membrane of the paper sensor module is further provided with a sample pad and a conjugate pad, and the conjugate pad contains a labeled particle probe, which is a colloidal gold nanoparticle conjugated with an anti-FAM antibody.

[0017] Preferably, the colorimetric module can be a portable test strip reader, image analysis software, or a mobile phone image acquisition system.

[0018] Preferably, the method of using the kit includes the following steps:

[0019] Sample processing: Collect clinical samples and extract Helicobacter pylori genomic DNA;

[0020] Isothermal amplification: Genomic DNA is added to the loop-mediated isothermal amplification reaction system;

[0021] Detection: The amplification product of the isothermal amplification is added to the sample loading area of ​​the paper sensor module. Under capillary action, the amplification product hybridizes with the biotin-labeled probe and then binds to the colloidal gold nanoparticles on the binding pad, forming colored bands in the test area and control area.

[0022] Results analysis: The presence and extent of drug resistance gene mutations can be determined by visual observation or colorimetric analysis.

[0023] Preferably, the isothermal amplification time is 20 to 30 minutes.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The kit of the present invention can simultaneously detect drug resistance gene mutations of Helicobacter pylori to clarithromycin and metronidazole, including the A2143G and A2142G / C mutation sites of the 23S rRNA gene and the Q50Stop mutation site of the rdxA gene, thus realizing the simultaneous detection of multiple drug resistance genes.

[0026] (2) This invention employs loop-mediated isothermal amplification (LAMP) technology, which allows DNA amplification at a constant temperature without the need for a thermal cycler. The detection time is short, and the entire detection process can be completed within one hour. The paper sensor module design eliminates the need for complex instruments and professional technicians; simply adding the sample to the sample loading area allows for visual observation or analysis using simple equipment, making it suitable for on-site use. Furthermore, LAMP technology boasts high amplification efficiency, enabling the amplification of target DNA by 10^ ... 9 The combination of biotin-avidin high-affinity capture system improves detection sensitivity, enabling the detection of trace amounts of drug resistance gene mutations.

[0027] (3) Specific primers and probes were designed for different drug resistance gene mutation sites. They can only be perfectly paired and bound when there are mutated bases in the target sequence, avoiding interference from wild-type sequences and ensuring the specificity of detection. The colorimetric module can be used to perform quantitative or semi-quantitative analysis of the detection results, establish the relationship between color intensity and target DNA concentration, realize the graded assessment of Helicobacter pylori drug resistance mutation load, and provide a more accurate basis for clinical treatment.

[0028] (4) The reagent kit structure of this invention achieves physical isolation between amplification and detection, reducing the risk of contamination. The "L"-shaped folding design of the test strip vertically separates the amplification reaction area (vertical) and the detection area (horizontal) during the amplification stage, avoiding cross-contamination caused by premature contact of amplification products with the detection area; product migration occurs after unfolding during the detection stage, ensuring detection accuracy. The "isothermal air heater" of the long strip reaction frame stably provides a constant temperature of 60-65℃, meeting the strict temperature requirements of the LAMP reaction (at which Bst DNA polymerase activity is highest), avoiding a decrease in amplification efficiency or non-specific amplification caused by temperature fluctuations, and improving detection sensitivity. The foldable hinge allows the test strip to quickly switch between "amplification mode" (L-shaped, inserted into the reaction frame) and "detection mode" (unfolded, lateral migration) without transferring samples or changing equipment; the exposed detection end design makes the results more directly observable, which can be read by the naked eye, a portable reader, or a mobile phone system, without the need for complex instruments, making it suitable for primary healthcare institutions or rapid on-site testing. The control line (C line) of the test strip must develop color in any valid test; failure to develop color indicates that the test strip is invalid. By combining the detection of the internal reference gene cgt, the comparison between the C line and the T line can eliminate operational errors and further improve the accuracy of quantitative monitoring. Attached Figure Description

[0029] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0030] Figure 1 This is a schematic diagram of the target amplification L-shaped test strip of the present invention;

[0031] Figure 2 A schematic diagram of a target detection test strip and a long strip reaction frame;

[0032] Among them, 1-sample pad, 2-binding pad, 3-nitrocellulose membrane, 4-absorbent pad, 5-target detection area, 6-foldable hinge, 7-L-shaped groove, 8-isothermal air heater. Detailed Implementation

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0034] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] This invention provides a kit for detecting Helicobacter pylori multidrug resistance genes and quantitatively monitoring treatment based on a paper sensor, comprising:

[0039] The isothermal amplification module includes a loop-mediated isothermal amplification reaction system for isothermal amplification of the 23S rRNA gene and rdxA gene of Helicobacter pylori.

[0040] A paper sensor module includes a nitrocellulose membrane, which has a test area and a control area. The test area is immobilized with biotin-labeled probes targeting mutant amplification products of the 23S rRNA gene and the rdxA gene, and the control area is immobilized with biotin-labeled probes targeting amplification products of the internal reference gene cgt.

[0041] The color development module is used to perform color development and quantitative analysis on the detection results of the paper sensor module.

[0042] In this invention, the test area (T line) is also immobilized with avidin molecules, such as streptavidin, which can capture biotin-labeled probe-product complexes with high affinity; the control area (C line) is immobilized with secondary antibodies or other control molecules targeting the reporter label, used to verify the effective operation of the test strip.

[0043] The paper sensor described in this invention utilizes the high affinity of biotin-avidin, enabling the target product to be enriched on the test line by the capture probe even at very low concentrations, thereby improving detection sensitivity.

[0044] In this invention, the primer set for the 23S rRNA gene A2143G mutation site includes outer primers F3 and B3, inner primers FIP and BIP, and circular primers LF and LB. The sequences of outer primers F3 and B3 are shown in SEQ ID NO:1 and SEQ ID NO:2; the sequences of inner primers FIP and BIP are shown in SEQ ID NO:3 and SEQ ID NO:4; and the sequences of circular primers LF and LB are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0045] Specifically, SEQ ID NO:1 is: 5'-GAAGTAAGGGCTTTGGCTCT-3';

[0046] Specifically, SEQ ID NO:2 is: 5'-ATGGAACTTCTACGCTTCCA-3';

[0047] Specifically, SEQ ID NO:3 is: 5'-GCTCTTATGGAACTTCTACGCTTCCA-GAAGTAAGGGCTTCGGCTCT-3';

[0048] Specifically, SEQ ID NO:4 is: 5'-TATGGAACTTCTACGCTTCCAGAAGTA AGGGCTCTGGCTCT-3';

[0049] Specifically, SEQ ID NO:5 is: 5'-GGCTCTTATGGAACTTCTAC-3';

[0050] Specifically, SEQ ID NO:6 is: 5'-TCTACGCTTCCAGAAGTAA-3';

[0051] Specifically, SEQ ID NO:7 is: 5'-AACGCCATGGCTGCGTG-3';

[0052] Specifically, SEQ ID NO:8 is: 5'-GTAAACGTACGTAGCCAT-3';

[0053] Specifically, SEQ ID NO:9 is: 5'-GTAAACGTACGTAGCCAT-AACGCCATG GCTACGTGTT-3';

[0054] Specifically, SEQ ID NO:10 is: 5'-GTAAACGTACGTAGCCAT-AACGCCAT GGCTGCGTGTT-3';

[0055] Specifically, SEQ ID NO:11 is: 5'-CATGGCTACGTGTTGTAA-3';

[0056] Specifically, SEQ ID NO:12 is: 5'-CATGGCTGCGTGTTGTAA-3';

[0057] Specifically, SEQ ID NO:13 is: 5'-GTGCCTTATATTGGCTCT-3'

[0058] Specifically, SEQ ID NO:14 is: 5'-AAGAAGCCAAGATGGTG-3'

[0059] Specifically, SEQ ID NO:15 is: 5'-AAGAAGCCAAGATGGTG-GTGCCTTATATTGGCTCTTT-3'

[0060] Specifically, SEQ ID NO:16 is: 5'-AAGAAGCCAAGATGGTG-GTGCCTTATATTGGCTCTTT-3'

[0061] Specifically, SEQ ID NO:17 is: 5'-CTTTCCGTGCCTTATATT-3'

[0062] Specifically, SEQ ID NO:18 is: 5'-CTTTCCGTGCCTTATATT-3'

[0063] Specifically, SEQ ID NO:19 is: 5'-Biotin-GAAGTAAGGGCTTCGGCTCTTATGGA-3';

[0064] Specifically, SEQ ID NO:20 is: 5'-Biotin-TGAAGTAAGGGCTCTGGCTCTTATGGA-3'

[0065] Specifically, SEQ ID NO:21 is: 5'-Biotin-AACGCCATGGCTACGTGTTGTAA-3'

[0066] Specifically, SEQ ID NO:22 is: 5'-Biotin-GTGCCTTATATTGGCTCTTTCC-3'

[0067] In this invention, the primer set for the Q50Stop mutation site of the rdxA gene includes outer primers F3 and B3, inner primers FIP and BIP, and circular primers LF and LB. The sequences of outer primers F3 and B3 are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively; the sequences of inner primers FIP and BIP are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively; and the sequences of circular primers LF and LB are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively.

[0068] In this invention, the primer set for the internal reference gene cgt includes outer primers F3 and B3, inner primers FIP and BIP, and circular primers LF and LB. The sequences of outer primers F3 and B3 are shown in SEQ ID NO:13 and SEQ ID NO:14; the sequences of inner primers FIP and BIP are shown in SEQ ID NO:15 and SEQ ID NO:16; and the sequences of circular primers LF and LB are shown in SEQ ID NO:17 and SEQ ID NO:18.

[0069] In this invention, the biotin-labeled probes in the test region include probes targeting the A2143G mutation site of the 23S rRNA gene, probes targeting the A2142G / C mutation site of the 23S rRNA gene, and probes targeting the Q50Stop mutation site of the rdxA gene, with sequences shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; the biotin-labeled probes in the control region include probes targeting the internal reference gene cgt, with a sequence shown in SEQ ID NO:22.

[0070] In this invention, the loop-mediated isothermal amplification reaction system includes ThermoPol buffer and Mg2+. 2+ Betaine, dNTPs, Bst DNA polymerase, and template DNA.

[0071] In this invention, the nitrocellulose membrane of the paper sensor module is further provided with a sample pad and a conjugate pad, and the conjugate pad is dry-embedded with a labeled particle probe, which is a colloidal gold nanoparticle conjugated with an anti-FAM antibody.

[0072] In this invention, the color development module can be a portable test strip reader, image analysis software, or a mobile phone image acquisition system.

[0073] In this invention, the method of using the reagent kit includes the following steps:

[0074] Sample processing: Collect clinical samples and extract Helicobacter pylori genomic DNA;

[0075] Isothermal amplification: Genomic DNA is added to the loop-mediated isothermal amplification reaction system;

[0076] Detection: The amplification product of the isothermal amplification is added to the sample loading area of ​​the paper sensor module. Under capillary action, the amplification product hybridizes with the biotin-labeled probe and then binds to the colloidal gold nanoparticles on the binding pad, forming colored bands in the test area and control area.

[0077] Results analysis: The presence and extent of drug resistance gene mutations can be determined by visual observation or colorimetric analysis.

[0078] In the sample processing described in this invention, the sample collection can be carried out using techniques well known to those skilled in the art, namely, gastric mucosal biopsy tissue or non-invasive fecal samples. In the embodiments of this invention, gastric mucosal biopsy tissue is preferred as the sample.

[0079] The working principle of the kit described in this invention is as follows: After the solution containing the amplification product and probe is added to the sample application area of ​​the test strip, the liquid flows laterally along the test strip under capillary action, passing sequentially through the binding pad and the detection area. When the FAM tag carried on the amplification product encounters the binding pad, it is captured and bound by the colloidal gold-labeled anti-FAM antibody, forming a "gold-labeled-product" complex. Continuing to flow to the test area, the biotin probe in the complex is captured by avidin immobilized on the membrane, and the complex accumulates and immobilizes at the T-line. Due to the attached colloidal gold particles, the test line gradually shows a red band, forming a visible signal. Conversely, if there is no mutated amplification product in the sample, the biotin probe will not effectively bind to the DNA, the gold-labeled probe cannot remain in the test area, and the T-line will not show a color change. When the test strip further flows to the control area, the remaining colloidal gold label (regardless of whether it binds to the product) will be captured by the control line (e.g., through binding with an anti-antibody), showing a color check line to confirm effective test strip operation and sufficient sample flow. The entire lateral flow detection process is generally completed within 5 to 10 minutes. When a target mutation is present, the T line will show a red band; if no target is present, only the C line will show color, indicating a negative result.

[0080] The reagent kit structure of this invention is as follows: to achieve amplification and detection line folding isolation: filter paper containing isothermal amplification reagent is placed at the bottom layer, forming an "L"-shaped folded structure with the laminar flow membrane. After adding the pretreated sample, the absorbent paper containing the isothermal reaction system is mixed with the filter paper reaction system. Amplification is performed for 20-30 minutes in an isothermal thermal environment of about 62°C. Then the "L" shape is straightened, and the reaction product flows to the target detection area at one end and is recognized by the target.

[0081] In this invention, the isothermal amplification time is preferably 20 to 30 minutes.

[0082] Example 1

[0083] (1) Sample collection: Gastric mucosal biopsy tissue, approximately 2-3 mm, was collected from the patient and immediately placed in MH liquid culture medium for low-temperature transport and storage; DNA was extracted using a commercially available genomic DNA extraction kit according to the following steps:

[0084] Gastric mucosal biopsy tissue was placed in lysis buffer, proteinase K was added, and incubated overnight at 37°C; centrifuged at 12000 rpm for 10 minutes, and the supernatant was collected; the supernatant was added to a centrifuge column, centrifuged at 12000 rpm for 1 minute, and the filtrate was discarded; wash buffer was added, centrifuged at 12000 rpm for 1 minute, and the filtrate was discarded; the washing step was repeated once; centrifuged at 12000 rpm for 2 minutes to remove residual wash buffer; the centrifuge column was placed in a new centrifuge tube, nucleic acid preservation solution was added, and the mixture was incubated at room temperature for 5 minutes; centrifuged at 12000 rpm for 1 minute, and the genomic DNA solution was collected and stored at -20°C for later use;

[0085] (2) Synthesize primer sets targeting the A2143G mutation site, A2142G / C mutation site, Q50Stop mutation site of rdxA gene, and internal reference gene cgt; design LAMP primers and biotin-labeled probes based on Helicobacter pylori gene mutation sites. The primers include outer primer F3 / B3, inner primer FIP / BIP, and optional LF / LB, and are dissolved in TE buffer at a concentration of 10 μmol / L.

[0086] (3) Prepare the reaction mixture, specifically including: ThermoPol buffer (10×): 2.5 μL; inner primer FIP (40 μmol / L): 1.0 μL; inner primer BIP (40 μmol / L): 1.0 μL; outer primer F3 (10 μmol / L): 1.0 μL; outer primer B3 (10 μmol / L): 1.0 μL; loop primer LF (20 μmol / L): 0.5 μL; loop primer LB (20 μmol / L): 0.5 μL; avidin-labeled dNTPs (10 mmol / L): 3.0 μL; betaine (5 mol / L): 4.0 μL; MgSO4 (100 mmol / L): 1 μL; Bst enzyme (8 U / μL): 1 μL; template DNA: 2.5 μL; ddH2O: 6.0 μL, with a total volume of 25 μL;

[0087] (4) Isothermal amplification: The reaction mixture was placed in a constant temperature heating device at 63°C and amplified for 20 minutes;

[0088] (5) To prepare a paper sensor, cut the nitrocellulose membrane to a suitable size, dilute the biotin-labeled probe with 0.05 mol / L carbonate buffer (pH 9.6) to a concentration of 1 μg / mL, and spot the probe onto the test area and control area of ​​the nitrocellulose membrane using a spotting instrument. Dry at 37°C overnight. Spray the colloidal gold nanoparticle solution bound to anti-FAM antibody onto the glass fiber membrane and dry at 37°C overnight to make a conjugate pad. Then, attach the sample pad, conjugate pad, nitrocellulose membrane and absorbent pad to the base plate in sequence to make a paper sensor.

[0089] (6) Mix the amplification product with the pre-prepared flow buffer at a ratio of 1:1 and add it to the sample loading area of ​​the paper sensor. Under capillary action, the liquid flows laterally along the test paper, passing through the conjugate pad and the detection area in sequence. After 5-10 minutes, observe the color development of the test area and the control area.

[0090] By visually observing the color bands in the test and control areas, if a red band appears in both the test and control areas, it indicates the presence of the corresponding drug resistance gene mutation in the sample; if no red band appears in the test area but a red band appears in the control area, it indicates the absence of the corresponding drug resistance gene mutation in the sample; if no red band appears in the control area, the testing process is invalid and needs to be repeated.

[0091] Subsequently, a portable test strip reader or mobile phone image acquisition system can be used to obtain the color intensity of the test area and control area. The gray value or RGB value is calculated by image analysis software. According to the pre-established standard curve, the color intensity is converted into the concentration of target DNA, thereby realizing the quantitative assessment of Helicobacter pylori drug resistance mutation load.

[0092] A schematic diagram of the test strip described in this invention is shown below. Figure 1 and Figure 2 As shown, where, Figure 1 This is a schematic diagram of a target amplification L-type test strip. Fold the sample end of the test strip backward 90° to form an "L" shape, so that the amplification reaction area (vertical) and the detection area (horizontal) are vertically aligned, making it easy to insert the folded end into the isothermal amplification reaction cell, while keeping the detection end facing upward for reading. Figure 2 This is a schematic diagram of a target detection test strip and a long strip reaction frame. The upper part shows a foldable "L" shaped position marked with a dotted line at the junction of the NC membrane and the absorbent pad, which can accommodate different operating modes before and after detection. The lower part is a schematic diagram of the long strip reaction frame, showing only the core structure. The isothermal air heater provides constant temperature amplification at 60–65℃, and the L-shaped groove is a slot / reaction cell into which the folded test strip can be inserted, ensuring that the amplification end is in the constant temperature zone, while the detection end is exposed for easy observation.

[0093] The reagent kit structure of this invention achieves physical isolation between amplification and detection, reducing the risk of contamination. The "L"-shaped folding design of the test strip vertically separates the amplification reaction area (vertical) and the detection area (lateral) during the amplification stage, avoiding cross-contamination caused by premature contact of amplification products with the detection area. Product migration occurs only after unfolding during the detection stage, ensuring detection accuracy. The "isothermal air heater" of the elongated reaction frame stably provides a constant temperature of 60-65°C, meeting the stringent temperature requirements of the LAMP reaction (at which BstDNA polymerase activity is highest), avoiding a decrease in amplification efficiency or non-specific amplification caused by temperature fluctuations, and improving detection sensitivity. The foldable hinge allows the test strip to quickly switch between "amplification mode" (L-shaped, inserted into the reaction frame) and "detection mode" (unfolded, lateral migration) without transferring samples or changing equipment. The exposed detection end design makes result observation more direct, and can be read by the naked eye, a portable reader, or a mobile phone system, eliminating the need for complex instruments and making it suitable for primary healthcare institutions or rapid on-site testing. The control line (C line) of the test strip must develop color in any valid test; failure to develop color indicates that the test strip is invalid. By combining the detection of the internal reference gene cgt, the comparison between the C line and the T line can eliminate operational errors and further improve the accuracy of quantitative monitoring.

Claims

1. A kit for detecting Helicobacter pylori multidrug resistance genes and quantitatively monitoring treatment based on paper sensors, characterized in that, include: The isothermal amplification module includes a loop-mediated isothermal amplification reaction system for isothermal amplification of the 23S rRNA gene and rdxA gene of Helicobacter pylori. A paper sensor module includes a nitrocellulose membrane, which has a test area and a control area. The test area is immobilized with biotin-labeled probes targeting mutant amplification products of the 23S rRNA gene and the rdxA gene, and the control area is immobilized with biotin-labeled probes targeting amplification products of the internal reference gene cgt. The biotin-labeled probes targeting amplification products of the internal reference gene cgt are used to improve the accuracy of quantitative monitoring. The color development module is used to perform color development and quantitative analysis on the detection results of the paper sensor module.

2. The kit for detecting and quantitatively monitoring Helicobacter pylori multidrug resistance genes and treatment based on a paper sensor according to claim 1, characterized in that, The primer set for the A2143G mutation site of the 23S rRNA gene includes outer primers F3 and B3, inner primers FIP and BIP, and circular primers LF and LB. The sequences of outer primers F3 and B3 are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; the sequences of inner primers FIP and BIP are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively; and the sequences of circular primers LF and LB are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

3. The kit for detecting and quantitatively monitoring Helicobacter pylori multidrug resistance genes and treatment based on a paper sensor according to claim 1, characterized in that, The primer set for the Q50Stop mutation site of the rdxA gene includes outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB. The sequences of outer primers F3 and B3 are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively; the sequences of inner primers FIP and BIP are shown in SEQ ID NO:9 and SEQ ID NO:10, respectively; and the sequences of loop primers LF and LB are shown in SEQ ID NO:11 and SEQ ID NO:12, respectively.

4. The kit for detecting and quantitatively monitoring Helicobacter pylori multidrug resistance genes and treatment based on a paper sensor according to claim 1, characterized in that, The primer set for the internal reference gene cgt includes outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB. The sequences of outer primers F3 and B3 are shown in SEQ ID NO:13 and SEQ ID NO:14; the sequences of inner primers FIP and BIP are shown in SEQ ID NO:15 and SEQ ID NO:16; and the sequences of loop primers LF and LB are shown in SEQ ID NO:17 and SEQ ID NO:

18.

5. The kit for detecting and quantitatively monitoring Helicobacter pylori multidrug resistance genes and treatment based on a paper sensor according to claim 1, characterized in that, The biotin-labeled probes in the test region include probes targeting the A2143G mutation site of the 23S rRNA gene, probes targeting the A2142G / C mutation site of the 23S rRNA gene, and probes targeting the Q50Stop mutation site of the rdxA gene, with sequences shown in SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; the biotin-labeled probes in the control region include probes targeting the internal reference gene cgt, with a sequence shown in SEQ ID NO:

22.

6. The kit for detecting and quantitatively monitoring Helicobacter pylori multidrug resistance genes and treatment based on a paper sensor according to claim 1, characterized in that, The loop-mediated isothermal amplification reaction system includes ThermoPol buffer and Mg 2+ Betaine, dNTPs, Bst DNA polymerase, and template DNA.

7. The kit for detecting and quantitatively monitoring Helicobacter pylori multidrug resistance genes and treatment based on a paper sensor according to claim 1, characterized in that, The nitrocellulose membrane of the paper sensor module is further provided with a sample pad and a conjugate pad. The conjugate pad contains a labeled particle probe, which is a colloidal gold nanoparticle conjugated with an anti-FAM antibody.

8. The kit for detecting and quantitatively monitoring Helicobacter pylori multidrug resistance genes and treatment based on a paper sensor according to claim 1, characterized in that, The colorimetric module can be a portable test strip reader, image analysis software, or a mobile phone image acquisition system.

9. The kit for detecting and quantitatively monitoring Helicobacter pylori multidrug resistance genes based on a paper sensor as described in any one of claims 1 to 8, characterized in that, The structure of the kit includes: Sample pad: Located at the beginning of the test strip, it is used to hold the sample to be tested. It has good water absorption and sample diffusion properties, and can quickly absorb the sample and promote the sample to flow to the subsequent areas. Binding pad: It is coated with a binding antibody that is double-labeled with colloidal gold and biotin; Nitrocellulose membrane: It has a test line and a control line. The test line indicates a positive result; the control line is used to verify the validity of the test strip. Regardless of whether Helicobacter pylori is present in the sample, the control line should show a band. If it does not appear, it indicates that the test strip is invalid. Absorbent pad: Located at the end of the test strip, it quickly absorbs the liquid after passing through the test line and control line through capillary action, maintaining the continuous flow of the sample on the test strip and ensuring the smooth progress of the testing process; L-shaped groove: used to hold sample diluent, which is used to dilute the collected original sample to achieve a concentration range suitable for test strip detection; the L-shaped groove contains an isothermal amplification reaction cell.

10. The kit for detecting and quantitatively monitoring Helicobacter pylori multidrug resistance genes and treatment based on a paper sensor according to claim 1, characterized in that, The method of using the kit includes the following steps: Sample processing: Collect clinical samples and extract Helicobacter pylori genomic DNA; Isothermal amplification: Genomic DNA is added to the loop-mediated isothermal amplification reaction system; Detection: The amplification product of the isothermal amplification is added to the sample loading area of ​​the paper sensor module. Under capillary action, the amplification product hybridizes with the biotin-labeled probe and then binds to the colloidal gold nanoparticles on the binding pad, forming colored bands in the test area and control area. Results analysis: The presence and extent of drug resistance gene mutations are determined by visual observation or colorimetric analysis using a visual or colorimetric module. The isothermal amplification is performed at 60-65℃ for 20-30 minutes.

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