A dual-mode nucleic acid detection method based on LAMP multi-primer collaborative labeling

Abstract

This application relates to the field of nucleic acid detection technology in molecular biology. The present invention discloses a dual-mode nucleic acid detection method based on LAMP multi-primer synergistic labeling, belonging to the field of molecular biology nucleic acid detection technology. This invention differentiates the inner and loop primers in the LAMP primer set by labeling: any one inner primer carries a first affinity molecule (such as biotin) or introduces biotin-coupled deoxyadenosine triphosphate / cytidine into the reaction system; the two loop primers carry a fluorescent group / quencher group and a second affinity molecule, respectively. During LAMP amplification under the action of Bst DNA polymerase, the chain substitution reaction affects the effect of the quencher group on the loop primer on the fluorescent group, causing the fluorescence signal to dissipate, thus achieving real-time monitoring of the amplification process; the final amplification product, because it simultaneously carries two affinity molecules, can be captured and colorimetrically developed by LFB, achieving visual detection. The purpose of this application is to solve the problem that existing LAMP detection methods fail to synergistically utilize their inherent multi-primer resources.

Description

Technical Field

[0001] This invention relates to the field of molecular biology nucleic acid detection technology, and in particular to a dual-mode nucleic acid detection method based on LAMP multi-primer synergistic labeling. Background Technology

[0002] Loop-mediated isothermal amplification (LAMP), first reported by Notomi et al. in 2000, is a widely used isothermal nucleic acid amplification method. This technique utilizes Bst DNA polymerase with strand displacement activity, and designs 4-6 specific primers targeting 6-8 regions of the target gene. Highly efficient amplification can be achieved under isothermal conditions (60-70℃), eliminating the need for a precision thermal cycler. LAMP technology features high amplification efficiency (achieving 10-1 nucleotides within 30-60 minutes). 9 -10¹ 0 With its advantages such as high nucleic acid amplification (multiple times), high specificity, and simple operation, it has been widely used in fields such as pathogen detection, food safety monitoring, and real-time on-site diagnosis.

[0003] With the continuous development of LAMP technology, researchers have developed various methods for interpreting amplified products. Early detection methods included turbidimetry to detect magnesium pyrophosphate precipitates generated during the reaction, and fluorescent dye methods such as SYBR Green and calcein. While these methods were simple to operate, they struggled to distinguish between specific amplifications and non-specific products, easily leading to false positives. To improve detection specificity, subsequent studies introduced labeling strategies: for example, introducing probes labeled with fluorescent groups or modifying loop primers (LF / LB) to form small fragments that can be cleaved by enzymes for real-time fluorescence detection; or labeling inner primers (FIP / BIP) or loop primers (LF / LB) with antigen tags, combined with a lateral flow biosensor (LBB) for visualization. Furthermore, some studies have combined LAMP with CRISPR / Cas systems to improve specificity, but these methods introduce additional enzyme and probe systems, increasing reaction complexity and cost. For example, Chinese patent CN119372346A discloses a loop-mediated isothermal amplification system assisted by a double-stranded probe for detecting Brucella. It designs amplification primers and detection double-stranded probes targeting the Brucella-specific gene bcsp31, integrating loop-mediated isothermal amplification (LAMP) with a foot-mediated strand substitution-based double-stranded probe (TP) to form a TP. The LAMP unit allows for real-time monitoring of amplification results using a PCR fluorescence instrument or visualization identification using a nanobiosensor. While achieving dual-mode detection, it still requires additional probe systems, resulting in complex structures and high costs.

[0004] However, existing LAMP detection methods mostly focus on single marker sites and single output patterns, failing to synergistically utilize the inherent multi-primer resources of LAMP. Specifically, few studies have integrated real-time monitoring of the amplification process with visualized confirmation of the final results by using differentiated markers for inner primers, loop primers, or even outer primers within the same reaction system. This results in significant room for improvement in terms of the dimension of detection information, result reliability, and scenario adaptability of existing methods.

[0005] Therefore, how to fully explore the potential of LAMP multi-primer and build a detection platform with higher integration, richer information dimensions, and wider application scenarios remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a dual-mode nucleic acid detection method based on LAMP multi-primer synergistic labeling, which solves the problem that existing LAMP detection methods fail to synergistically utilize the inherent multi-primer resources of LAMP.

[0007] To address the aforementioned problems, the technical solution adopted in this invention is: a dual-mode nucleic acid detection method based on LAMP multi-primer co-labeling, comprising the following steps: Step 1: providing a set of LAMP primers, wherein the primer set includes at least one pair of outer primers F3 / B3, one pair of inner primers FIP / BIP, and one pair of circular primers LF / LB; Step 2: labeling the inner primers to carry a first affinity molecule; or directly introducing dNTPs modified with the free first affinity molecule into the reaction system; Step 3: labeling the circular primers such that one carries both a fluorescent group and a quenching group, wherein the fluorescence is quenched in the free state by the fluorescent group and the quenching group; the other carries a second affinity molecule, and the second affinity molecule is different from the first affinity molecule; Step 4: using the nucleic acid to be tested as a template, in Bst... LAMP amplification is performed under the action of DNA polymerase. During the amplification process, the fluorescence signal is monitored in real time. The effect of the chain displacement reaction on the quenching group is used to trigger the emission of the fluorescence signal of the fluorescent group. Step 5: After the amplification is completed, the amplification product is dropped into the sample well of the lateral flow nanobiosensor strip for development. Visual detection is achieved by capturing the first affinity molecule and the second affinity molecule on the test strip.

[0008] Furthermore, the first affinity molecule is biotin, and the second affinity molecule is either FITC / FAM or digoxin.

[0009] Furthermore, the fluorescent group is selected from any one of FAM, HEX, ROX, and Cy5. When the fluorescent group is selected from FAM or HEX, the quenching group is selected from BHQ1; when the fluorescent group is selected from ROX or Cy5, the quenching group is selected from BHQ2.

[0010] Furthermore, the LAMP amplification temperature is 60-70℃ and the time is 30-60 minutes.

[0011] Furthermore, the detection line of the lateral flow nanobiosensor strip is immobilized with an antibody against the second affinity molecule, and the quality control line is immobilized with nanoparticles that capture the first affinity molecule.

[0012] Furthermore, the fluorescent group and the quenching group on the ring primer are connected by a spacer base.

[0013] Furthermore, the nucleic acid to be tested is the IS6110 gene of Mycobacterium tuberculosis complex or the pol gene of human immunodeficiency virus.

[0014] Furthermore, the number of spacer bases between the fluorescent group and the quencher group on the ring primer is greater than 5.

[0015] Furthermore, the fluorescent group and the second affinity molecule are the same molecule, selected from FAM.

[0016] A reagent for implementing a dual-mode nucleic acid detection method based on LAMP multi-primer co-labeling, characterized in that it comprises the outer primer, labeled inner primer, labeled loop primer, Bst DNA polymerase, reaction buffer, Biotin-dATP / dCTP, and lateral flow nanobiosensor strip as described in claim 1.

[0017] The technical principles and beneficial effects of this solution are as follows: This proposal suggests a synergistic labeling strategy that fully leverages the advantages of LAMP multi-primer technology. By differentially labeling the loop primers and inner primers, a dual-mode detection platform is constructed, combining real-time fluorescence monitoring and lateral flow visualization capabilities. Specifically, the loop primers (LF / LB) are simultaneously labeled with a fluorescent / quencher pair and a biotin tag, while the inner primers (FIP / BIP) are labeled with an antigen tag (such as digoxigenin), allowing the two types of primers to perform their respective functions and provide complementary signals. The working mechanism of this strategy is characterized by dual synergy: firstly, based on the novel mechanism of "chain displacement-mediated fluorescence dissipation" between the fluorescent and quencher groups on the loop primers, real-time fluorescence monitoring of the amplification process is achieved; secondly, by utilizing the biotin on the loop primers and the antigen tag on the inner primers, the amplification final product simultaneously carries a dual-affinity label, which can be specifically captured by the lateral flow test strip, enabling terminal visualization of the amplification results. Thus, the same reaction system can output two independent signals in parallel: a real-time fluorescence kinetic curve and a lateral flow chromogenic band, forming cross-validation of results and improving the reliability of the detection. Compared with existing single-label or single-mode detection strategies, this strategy has the following advantages: (1) High information integration: Real-time fluorescence amplification curve (providing amplification kinetic information) and LFB colorimetric results (providing endpoint visualization confirmation) are obtained simultaneously in the same reaction system. The two signals verify each other, which significantly reduces the risk of false positives / false negatives.

[0018] (2) Simple labeling strategy: No need for exogenous probes (such as molecular beacons, TaqMan probes) or additional enzyme systems (such as CRISPR / Cas), nor is it necessary to insert a small fragment containing an enzyme cleavage site into the 5' end of the primer (such as a loop primer) and introduce the corresponding restriction endonuclease (such as Nb. BsrDI). It can be achieved simply by chemically modifying the loop primer. Labeling is simple and flexible, has little impact on LAMP amplification efficiency, and has good system compatibility.

[0019] (3) Wide adaptability to different scenarios: It is suitable for both laboratory environments equipped with fluorescence detection equipment and on-site detection scenarios that only require constant temperature heating devices and LFB, thus meeting the detection needs of different application scenarios.

[0020] (4) Good universality: As long as a LAMP primer set can be designed, the labeling strategy of this invention can be used for detection, and is not limited to specific target nucleic acid sequences or pathogens.

[0021] (5) High specificity: Since LAMP technology itself relies on multiple primers to jointly recognize multiple regions of the target sequence, and the dual signal output of this invention further verifies that non-specific amplification is less likely to produce double positive results.

[0022] (6) The fluorescent group and the second affinity molecule can be the same molecule. When using FAM labeling, the FAM in the FAM-BHQ1 pair can function as both a fluorescent group, which is collected by the fluorescence detector, and as a second affinity molecule, i.e., an antigen tag, which is captured by the anti-FAM antibody on the LFB detection line, achieving dual-purpose labeling. In the field of LAMP detection, fluorescent groups and affinity molecules are usually considered to be completely different functional components, used for instrument detection and test strip detection, respectively. Combining the two into a single molecule reduces the number of labeling sites, lowers the difficulty and cost of synthesis, and avoids steric interference between multiple tags. It also solves the compatibility problem of labeling strategies: if the fluorescent group and the second affinity molecule are two different molecules, they need to be labeled at different positions on the same primer, increasing the difficulty and uncertainty of synthesis; while the dual role of FAM only requires one labeling site. Attached Figure Description

[0023] Figure 1 The results of the LAMP amplification temperature range test in this invention (amplification temperature is 62-69℃, DW is the distilled water control group). Figure 2Results of fluorescence emission testing of novel LAMP fluorescent labels (A: Gel electrophoresis image; B: Real-time turbidity monitoring image; C: Fluorescence signal detection image). Figure 3 Results of fluorescence effervescence assays using novel fluorescent primers with different loops for LAMP. Figure 4 Experimental results on the effect of different fluorescent labeling distances on fluorescence dispersion in novel LAMP systems; Figure 5 Results of tests on strand displacement-triggered fluorescence emission using Bst DNA polymerases from different sources; Figure 6 Results of a dual-mode detection experiment combining real-time fluorescence and nanobiosensing (A: Gel electrophoresis image; B: Real-time turbidity monitoring image; C: LFB detection result image; D: Real-time fluorescence detection result image). Figure 7 Sensitivity evaluation results of LAMP dual-mode detection (A: LFB detection results; B: Real-time fluorescence detection results). Figure 8 Specificity evaluation of LAMP dual-mode detection (A1-C1: LFB detection results; A2-C2: real-time fluorescence detection results). Figure 9 Validation of the universality of LAMP dual-mode detection (A: Gel electrophoresis image B: Real-time turbidity monitoring image C: LFB detection result image D: Real-time fluorescence detection result image). Detailed Implementation

[0024] The following detailed description illustrates the specific implementation methods: Example 1: (1) Materials, reagents and instruments involved in the embodiments of the present invention The main bacterial strains and nucleic acid samples are shown in Table 1, including the MTB reference strain (H37Rv), nontuberculous mycobacteria, and other common pathogens.

[0025] Table 1. Nucleic acids from different pathogens used in this invention

[0026] Note: The nucleic acids of the above pathogens were all obtained from the Guizhou Provincial Center for Disease Control and Prevention. Main reagents: Bst DNA polymerase (Beyotime Biotechnology Co., Ltd., China), Bst 2.0 DNA polymerase (Tianjin Huidexin Technology Development Co., Ltd., China), Bst 4.0 DNA polymerase (Tianjin Huidexin Technology Development Co., Ltd., China), matching reaction buffer, dNTPs (Beyotime Biotechnology Co., Ltd., China), MgSO4 (Beyotime Biotechnology Co., Ltd., China), Biotin-dCTP / Biotin-dATP (Tianjin Huidexin Technology Development Co., Ltd., China), betaine (SIGMA, USA), and nucleic acid extraction kit (Xi'an Tianlong Technology Co., Ltd., China).

[0027] The main instruments used in the experiments of this invention are: fully automated nucleic acid extractor (GeneRotex96, Xi'an Tianlong Technology Co., Ltd., China), real-time quantitative PCR instrument (GENRIER 96 E, Xi'an Tianlong Technology Co., Ltd., China), gel imaging system (GelDoc Go, Bio-Rad, USA), and real-time turbidity gene detection system (LA-500, Rongyan Biotechnology (China) Co., Ltd.).

[0028] (2) Fabrication of lateral flow nanobiosensors (LFB) Construction of Lateral Flow Nanobiosensor Strips: This invention allows for the design of 1-2 target gene capture regions for single and dual target gene detection. The lateral flow nanobiosensor (LFB) structure used is designed as follows: a plastic-bonded backplate, on which a sample pad, conjugation pad, nitrocellulose membrane, absorbent pad, and panel are sequentially mounted; streptavidin-modified nanoparticles (SA-G) embedded in the conjugation pad can bind to biotin for color development; the reaction area can be configured with detection line 1 (TL1) and / or detection line 2 (TL2), and a control line (CL), and anti-fluorescein antibody (anti-FAM / anti-FITC), digoxigenin antibody (anti-Dig), and bovine serum albumin conjugated to biotin are sequentially embedded in the corresponding reaction areas as capture agents; the developing solvent is phosphate-buffered saline (PBS). During detection, 1-2 μL of amplification product is added to the sample pad of the test strip, and the result is read after standing for 1-5 minutes.

[0029] (3) LAMP primer design and multifunctional markers LAMP reaction primers were designed using an online website based on Primer Premier 5.0. A total of six primers were constructed, comprising three primer pairs, each typically 15-25 nucleotides in length. These consisted of outer primers F3 / B3, inner primers FIP / BIP, and loop primers LF / LB. Functional primers were labeled according to the product detection mode, such as fluorescence detection or lateral flow nanobiosensor strip detection. Primer synthesis and labeling were performed by Sangon Biotech (Shanghai) Co., Ltd., and the primers were purified by HPLC. The primer sequences and labeling positions used in this invention are shown in Table 2.

[0030] Table 2 Primer sequences and modifications

[0031] Note: MTB-IS6110: is the IS6110 target gene of Mycobacterium tuberculosis; HIV-pol: is the pol target gene of HIV.

[0032] FAM: 6-Carboxyfluorescein; CY5: cyanine 5; BHQ1: Quencher group 1; BHQ2: Quencher group 2; Biotin: Biotin. mer: Monomeric unit; nt: Nucleitide.

[0033] Preferred labeling scheme description: Fluorescent groups can be selected from FAM, HEX, ROX, Cy5, etc., and quenching groups can be selected from BHQ1, BHQ2, etc.

[0034] The first affinity molecule is preferably biotin, and the second affinity molecule is preferably fluorescein isothiocyanate (FITC) / 5-carboxyfluorescein (FAM) or digoxigenin.

[0035] The first affinity marker is placed after the fifth base at the 3' end of the inner primer (FIP / BIP) to reduce the impact of 3' end steric hindrance on the efficiency of synthetic chain elongation. This spatially-based reduction of non-specific polymerization has a relatively small impact.

[0036] The second affinity molecular marker is on one of the loop primers (LF / LB). After the reaction is complete, the amplicon will be integrated with the Biotin-FITC / FAM / Dig tag, which can be captured and colored by a specific antibody in the LFB reaction region, enabling visual detection.

[0037] The fluorescent group and the quenching group are labeled on the loop primer (LF / LB), usually spaced more than 10 linker bases apart, to prevent the distance from being too close and affecting the fluorescence emission intensity.

[0038] The labeling of fluorescent and quenching groups on the circular primers (LF / LB) is highly flexible, unlike Taq Man probes, which are usually labeled at both ends and cannot be flexibly arranged.

[0039] The affinity molecule (antigen) tag and fluorescent group on the loop primer can be the same, such as the FAM tag, which can be used as both a fluorescent group and an antigen tag.

[0040] (4) Establishment of the LAMP reaction system LAMP reaction system (total volume 25 μL): 2.5 μL Isothermal buffer × 10 (200mM Tris-HCl, 100mM KCl, 100mM (NH4)2SO4, 20mM MgSO4, 1% Triton X-100) or 12.5 μL Isothermal buffer × 2, 1-1.5 μL MgSO4 (final concentration 6-8mM), 1.4 μL dNTPs (final concentration 1.4 mM each), betaine (optional, final concentration 0.6-1.2 M), 1.1-1.4 μL mixed primers (containing 1.6 μM FIP / BIP, 0.4-0.8 μM LF / LB, 0.2-0.4 μM F3 / B3), 1-5 μL template DNA, and ddH2O to a final volume of 25 μL.

[0041] LAMP amplification reaction conditions: reaction temperature 62-69℃, isothermal for 30-60 minutes. Experimental results show... Figure 1 As shown, 62-69℃ corresponds to real-time turbidity detection during LAMP-IS6110 amplification; DW represents distilled water, serving as the corresponding template-free blank control. Figure 1 It can be seen that the LAMP reaction can be effectively amplified within the reaction temperature range of 62-69℃.

[0042] LAMP amplification reaction conditions: Reaction temperature 62-69℃, isothermal for 30-60 minutes. Experimental results show (e.g.) Figure 1 LAMP reactions can be effectively amplified within a reaction temperature range of 62-69℃.

[0043] Example 2: Experiment to verify the chain substitution-triggered fluorescence dissipation mechanism To verify that the generation of the fluorescence signal in this invention does indeed depend on the chain substitution reaction, the following verification experiment was designed: Verification 1: Fluorescence Emission Test of Novel LAMP Fluorescent Labels Real-time fluorescence monitoring was performed using a real-time fluorescence PCR instrument or a portable fluorescence detection device, collecting fluorescence signals every 15-30 seconds during amplification. This was achieved using IS6110-LF fluorescent and quencher-labeled fluorescent polymerases. (SEQ ID NO: 7) LAMP reactions were performed using primers that replaced the IS6110-LF primers, with unlabeled LAMP-IS6110 primers used as a control, and a corresponding template-free control group was established. Experimental results are as follows: Figure 2 As shown, a represents unlabeled LAMP-IS6110 amplification, and a-DW represents the corresponding template-free amplification control; b represents amplification containing IS6110-LF. Labeled LAMP-IS6110 amplification, b-DW is the corresponding template-free amplification control; DW represents distilled water. Figure 2 It can be seen that gel electrophoresis ( Figure 2 A) and real-time turbidity meter ( Figure 2 B) Both can detect LAMP amplification products from labeled and unlabeled groups; however, only IS6110-LF labeled with fluorescent and quenching groups can detect these products. Fluorescent signals can only be stably detected by a qPCR instrument in the presence of primers. As the reaction proceeds, the fluorescence intensity accumulates and then plateaus, exhibiting a typical "S-shaped" curve. Figure 2 C).

[0044] Verification 2: Fluorescence Emission Test of Novel Fluorescent Primers with Different Loops for LAMP By labeling different circular primers IS6110-LF (SEQ ID NO: 7) and SEQ ID NO: 9 (IS6110-LB) The universality of the circular primer markers was tested. Experimental results are as follows: Figure 3 As shown, where LF Indicates that it contains IS6110-LF Labeled LAMP-IS6110 amplification, LF -DW corresponds to the template-free amplification control; LB This indicates that it contains IS6110-LB. Labeled LAMP-IS6110 amplification, LF -DW represents the template-free amplification control; DW indicates distilled water. Figure 3 It can be seen that, regardless of the marking IS6110-LF The group is still IS6110-LB Stable fluorescence signals were detected in all LAMP-IS6110 amplifications of the labeled groups.

[0045] Verification 3: The effect of different fluorescent labeling distances on fluorescence egress in novel LAMP models The effect of labeling distance on fluorescence efflux was tested by adjusting the distance between the bases of the fluorescent group and the quencher group. The experimental results are as follows: Figure 4 As shown, among which, by Figure 4 It can be seen that LF With LF They are IS6110-LF With IS6110-LF The LAMP-IS6110 amplification curves involved; DW represents distilled water, which is LF. -DW and LF -DW corresponds to a template-free blank control. When the linker arms of the fluorescent group and the quencher group are close together (using SEQ ID NO: 8IS6110-LF) When participating in the LAMP reaction, its fluorescence emission intensity is weak (i.e., the quenching group has a strong quenching effect on the fluorescent group during chain substitution); when the distance between the fluorescent group and the base linker of the quenching group is large (using SEQ ID NO: 7 IS6110-LF) It participates in the LAMP reaction, and its fluorescence emission intensity is high (that is, the quenching effect of the quenching group on the fluorescent group is weakened during the chain substitution process), which is more conducive to the interpretation of the results.

[0046] Verification 4: Test of strand displacement triggering-fluorescence emission by Bst DNA polymerases from different sources LAMP amplification was tested using Bst DNA polymerases from different sources (including Bst DNA polymerase, Bst 2.0 DNA polymerase, and Bst 4.0 DNA polymerase) (using SEQ ID NO: 7 IS6110-LF). The universality of chain substitution-triggered fluorescence emission detection (involving the reaction) is demonstrated. Experimental results are as follows: Figure 5 As shown in the figure, Bst, Bst 2.0, and Bst 4.0 are the corresponding DNA polymerase amplification curves; DW represents distilled water, which is the template-free blank control for the corresponding amplification. As can be seen from the figure, LAMP amplification under Bst DNA polymerase, Bst 2.0 DNA polymerase, and Bst 4.0 DNA polymerase can all achieve strand displacement triggering-fluorescence emission detection.

[0047] The above results confirm the effectiveness of the chain displacement triggering-fluorescence dissipation mechanism of the present invention.

[0048] Explanation of the preferred chain substitution-triggered fluorescence dissipation mechanism: When amplifying on a real-time fluorescence PCR instrument, select the corresponding fluorescence acquisition channel (such as FAM / Cy5 channel) according to the labeled fluorescent molecule. Program settings: incubate at an isothermal temperature (such as any temperature between 62-69℃) for 5-10 minutes, continuously acquire fluorescence signals for 35-45 seconds every 15-25 seconds, and cycle for 35-50 times (the reaction time corresponding to one cycle can be set to 1 minute).

[0049] In the verification of the chain substitution-fluorescence dissipation mechanism, fluorescence signals were only detected when fluorescently labeled loop primers (LF / LB) participated in the amplification reaction. The role of loop primers (LF / LB) is to accelerate the LAMP reaction process (i.e., the efficiency of chain substitution), so its fluorescence generation mechanism is chain substitution triggering.

[0050] The circular primers, as novel fluorescent probes for LAMP isothermal amplification labeled with fluorescent and quenching groups, exhibit luminescence by influencing the darking effect of the quenching group on the fluorescent group through chain substitution. This results in fluorescence dissipation during the amplification reaction, which can be stably detected by instruments such as fluorescent PCR instruments, unlike the fluorescence release of Taq man probes in qPCR.

[0051] like Figure 4 As shown, when labeling circular primers (LF / LB) with fluorescent and quenching groups, the distance between the base linkers affects the intensity of fluorescence emission. It is recommended that the base linkers be greater than or equal to 15 bases for easier result interpretation.

[0052] Bst DNA polymerases from different sources (including Bst DNA polymerase, Bst 2.0 DNA polymerase, and Bst 4.0 DNA polymerase) can all trigger the strand substitution-fluorescence dissipation mechanism of LAMP amplification, regardless of the enzyme source, and only related to whether the LAMP reaction is initiated.

[0053] Example 3: Establishment of a dual-mode detection system combining real-time fluorescence and nanobiosensing Based on the chain substitution-triggered fluorescence escape mechanism of LAMP described above, we further explored the dimensions of information output. By integrating molecularly affinity markers (antigen tags), we constructed multifunctional LAMP primers to achieve dual-mode output detection of real-time fluorescence and lateral flow nanobiosensing (LFB). As shown in Table 2, by replacing the unlabeled LAMP-IS6110 primers with the corresponding IS6110-LF primers... The multifunctional primer sets IS6110-LB# (SEQ ID NO: 7), IS6110-LB# (SEQ ID NO: 10), and IS6110-BIP# (SEQ ID NO: 11), with the unlabeled LAMP-IS6110 primer set as a control and a corresponding template-free blank control, were used to test the detection capability of real-time fluorescence and LFB dual-mode output. Experimental results are as follows: Figure 6 As shown, a represents unlabeled LAMP-IS6110 amplification, and a-DW is the corresponding template-free amplification control; b represents LAMP-IS6110 amplification with dual-mode labeling, and b-DW is the corresponding template-free amplification control; DW represents distilled water; CL is the control line for LFB detection, and TL is the detection line for LFB detection (indicating a positive reaction). Figure 6 It can be seen that gel electrophoresis ( Figure 6 A) and real-time turbidity meter ( Figure 6 Both B) can detect LAMP amplification products from the dual-mode labeled group and the unlabeled group; however, the dual-mode labeled group can be detected by another LFB ( Figure 6 C) and qPCR instrument stably detected ( Figure 6 D).

[0054] Example 4: Sensitivity Evaluation of LAMP Dual-Mode Detection Based on the amplification efficiency of LMAP, to obtain a more accurate detection limit, the target DNA (such as MTBH37Rv genomic DNA) was serially diluted 5-fold at the pg level and 2-fold at the fg level using template serial dilution (concentration range: 100 pg / reaction, 20 pg / reaction, 4 pg / reaction, 800 fg / reaction, 400 fg / reaction, 200 fg / reaction, 100 fg / reaction). LAMP-MTB-IS6110 amplification was performed using the reaction system of Example 3 and the multifunctional primers of Example 5, and detection was performed using dual-mode output. Experimental results are as follows: Figure 7 As shown, A represents LFB detection, B represents real-time fluorescence detection, DW indicates distilled water as a template-free blank control, CL represents the quality control line for LFB detection, and TL represents the detection line for LFB detection (indicating a positive reaction).

[0055] Depend on Figure 7 It can be seen that when the template concentration is ≥400 fg / reaction, it can be induced by LFB ( Figure 7 A) Stable detection by qPCR instrument ( Figure 7 B).

[0056] The method of this invention has a detection limit (LoD) of 400 fg / reaction for MTB genomic DNA, which is approximately equivalent to 3.3 copies / μL.

[0057] Example 5: Specificity evaluation of LAMP dual-mode detection The test strains are shown in Table 1. MTB nucleic acid was used as a positive control, and DW was used as a template-free negative control to test the specificity of LAMP-MTB-IS6110. The test results are as follows: Figure 8As shown, A1-C1 represent LFB detection, and A2-C2 represent real-time fluorescence detection; P is the positive control for MTB nucleic acid, and DW indicates distilled water as a template-free blank control; CL is the control line for LFB detection, and TL is the detection line for LFB detection (indicating a positive reaction). Figure 8 It can be seen that only MTB H37Rv produced a positive fluorescence amplification curve and LFB colorimetric band, while all non-target strains showed no cross-reaction, exhibiting a negative flat fluorescence curve and LFB results with only the CL line.

[0058] Example 6: Sample Validation The method of this invention was used to test two groups of samples, including 30 test samples (from the 2022-2024 Chinese Center for Disease Control and Prevention tuberculosis molecular assessment samples) and 40 clinical sputum samples (from the 2023 monitoring samples of the Guizhou Provincial Center for Disease Control and Prevention). Consistency was compared using Xpert MTB / RIF as the reference standard. The test results are shown in Table 3 below: Table 3. Detection results of different sample groups of GeneXpert MTB / RIF and dual-mode LAMP-MTB-IS6110

[0059] Explanation of the target's applicable scope: It should be noted that when IS6110 is used as the target, this method is effective at least for Mycobacterium tuberculosis complex strains containing detectable copies of IS6110. Some MTBC strains (such as certain Mycobacterium bovis) have extremely low or absent IS6110 copies; for the detection of such strains, other targets of this invention (such as IS1081) or other methods in the art can be used. The scope of protection of this invention is not limited by the applicability to the specific strains described above.

[0060] Example 6: Universality Validation of LAMP Dual-Mode Detection To verify that the labeling strategy of this invention is not limited to a specific target, the HIV-pol gene was selected for parallel validation. LAMP primers were redesigned for the above target (as shown in Table 2), and detection was performed using the same labeling strategy (inner primer labeled with biotin, and loop primer labeled with fluorescence, quenching, and second affinity molecules). Detection results are as follows: Figure 9 As shown: Gel electrophoresis ( Figure 9 A) and real-time turbidity meter ( Figure 9 Both B) can detect the amplification of LAMP products from the dual-mode labeled group and the unlabeled group; however, the dual-mode labeled group can be detected by another LFB ( Figure 9 C) and qPCR instrument stably detected ( Figure 9 (D) further confirms the universality and reliability of the labeling strategy of the present invention.

[0061] Explanation of dual-mode LAMP multifunctional primer markers: To further verify the universality of the dual-mode detection, an additional fluorescent group (FAM) and quencher group (BHQ1) pair were selected, and an affinity molecule marker (antigen tag) was integrated. The FAM tag can be used as both a fluorescent group and a second affinity molecule (antigen).

[0062] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics in the solutions is not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A dual-mode nucleic acid detection method based on LAMP multi-primer co-labeling, characterized in that: Includes the following steps: Step 1: Provide a set of LAMP primers, which shall contain at least one pair of outer primers F3 / B3, one pair of inner primers FIP / BIP, and one pair of circular primers LF / LB; Step 2: Label the inner primer to carry the first affinity molecule; or directly introduce free first affinity molecule-modified dNTPs into the reaction system; Step 3: Label the loop primers so that one of them carries both a fluorescent group and a quenching group, wherein the fluorescence is quenched in the free state by the fluorescent group and the quenching group; the other one carries a second affinity molecule, and the second affinity molecule is different from the first affinity molecule; Step 4: Using the nucleic acid to be tested as a template, LAMP amplification is performed under the action of Bst DNA polymerase. During the amplification process, the fluorescence signal is monitored in real time. The fluorescence signal is triggered by the effect of the strand displacement reaction on the quenching group. Step 5: After amplification, the amplification product is dropped into the sample well of the lateral flow nanobiosensor strip for development. Visual detection is achieved through the capture of the first affinity molecule and the second affinity molecule on the test strip.

2. The dual-mode nucleic acid detection method based on LAMP multi-primer co-labeling according to claim 1, characterized in that: The first affinity molecule is biotin, and the second affinity molecule is any one of FITC, FAM, or digoxigenin.

3. The dual-mode nucleic acid detection method based on LAMP multi-primer co-labeling according to claim 1, characterized in that: The fluorescent group is any one of FAM, HEX, ROX, and Cy5. When the fluorescent group is FAM or HEX, the quenching group is selected from BHQ1; when the fluorescent group is selected from ROX or Cy5, the quenching group is selected from BHQ2.

4. The dual-mode nucleic acid detection method based on LAMP multi-primer co-labeling according to claim 1, characterized in that: The LAMP amplification temperature is 60-70℃ and the time is 30-60 minutes.

5. The dual-mode nucleic acid detection method based on LAMP multi-primer co-labeling according to claim 1, characterized in that: The detection line of the lateral flow nanobiosensor strip is immobilized with an antibody against the second affinity molecule, and the quality control line is immobilized with nanoparticles that capture the first affinity molecule.

6. The dual-mode nucleic acid detection method based on LAMP multi-primer co-labeling according to claim 1, characterized in that: The fluorescent group and the quenching group on the circular primer are connected by a spacer base.

7. The dual-mode nucleic acid detection method based on LAMP multi-primer co-labeling according to claim 1, characterized in that: The nucleic acid to be tested is the IS6110 gene of Mycobacterium tuberculosis complex or the pol gene of human immunodeficiency virus.

8. The dual-mode nucleic acid detection method based on LAMP multi-primer co-labeling according to claim 6, characterized in that: The number of spacer bases between the fluorescent group and the quencher group on the circular primer is greater than 5.

9. The dual-mode nucleic acid detection method based on LAMP multi-primer co-labeling according to claim 1, characterized in that: The fluorescent group and the second affinity molecule are the same molecule, namely FAM.

10. A reagent for implementing the method according to any one of claims 1-9, characterized in that: It comprises the outer primer, labeled inner primer, labeled loop primer, Bst DNA polymerase, reaction buffer, Biotin-dATP / dCTP, and lateral flow nanobiosensing strip as described in claim 1.

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