Method for detecting multi-drug resistance of ticks based on lamp technology

Abstract

The present application relates to the technical field of biological experiments, and discloses a tick multi-drug resistance detection method based on LAMP technology, comprising the following steps: parallel manufacturing of a spatially encoded solid-phase carrier module, sequence design and synthesis of a cis-tethered self-locking molecular recognition module, execution of a solid-phase anchoring assembly and conformational locking procedure, release of tick sample nucleic acids, construction of a heterogeneous mixed reaction system, thermodynamic control amplification and in-situ signal transduction, and discretization result decoding and interpretation. The present application constructs high-density cis-self-locking probes with thermodynamic locking functions on the surface of a solid-phase carrier, and uses a base competitive displacement mechanism between a target sequence and a probe terminal toe point region to strictly control the amplification reaction. Only a completely matched target sequence can open the stem-loop structure of the probe and expose a polymerase extension site, thereby blocking non-specific hybridization and error initiation. This design effectively overcomes the defect of high background interference in traditional isothermal amplification technology.

Description

Technical Field

[0001] This invention relates to the field of biological experimental technology, specifically to a method for detecting multidrug resistance in ticks based on LAMP technology. Background Technology

[0002] Ticks, as vectors for various zoonotic infectious diseases, carry pathogens that have developed resistance to specific antibiotics, making this a key challenge in public health prevention and control. To accurately guide clinical medication and block the spread of drug-resistant strains, nucleic acid-based molecular diagnostic technologies are widely used in screening for drug-resistant genes. Among these, loop-mediated isothermal amplification (LAMP) technology, with its advantages of not requiring complex temperature-controlled cycling equipment, extremely high amplification efficiency, and relatively relaxed requirements on sample purity, has gradually become an important technique for rapid on-site detection of pathogens and diagnosis in primary healthcare institutions.

[0003] In current applications, LAMP detection primarily relies on four or six primers designed for specific regions of the target gene. Under isothermal conditions, DNA polymerases with strand displacement activity drive the synthesis and displacement of DNA strands. This technique is typically performed in a liquid-phase system, or the primers are immobilized on a solid-phase support for solid-phase amplification.

[0004] However, traditional LAMP technology faces the problem of non-specific amplification in practical applications due to the lack of strict initiation control of the amplification mechanism. Because LAMP primers are long and highly concentrated, they are prone to self-folding or transient mismatch hybridization with non-target sequences during reaction preparation or under low-temperature conditions. Once these non-specific bindings are extended by the polymerase, they enter an exponential amplification cycle, generating a large amount of background noise. This inability to distinguish between target signals and background interference easily leads to false positives, making it difficult to meet the high standards of detection specificity and signal-to-noise ratio. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for detecting multidrug resistance in ticks based on LAMP technology, which solves the problems mentioned in the background section.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for detecting multidrug resistance in ticks based on LAMP technology, comprising the following steps: Step S1: Parallel fabrication of spatially encoded solid-phase carrier modules; using a flow-focusing microfluidic chip device, microsphere matrix units with a first average diameter are fabricated respectively. Microsphere matrix units with a second average diameter And microsphere matrix units with a third average diameter The microsphere matrix unit The microsphere matrix unit With the microsphere matrix unit During polymerization, chemically active anchoring groups are introduced through copolymerization. Step S2: Sequence design and synthesis of cis-tethered self-locking molecular recognition module; cis-tethered self-locking primers are designed and synthesized for tick multidrug resistance-related genes and internal reference genes; the cis-tethered self-locking primers contain 5' amino modification, locking domain, branching migration region and toe point domain; Step S3: Execution of solid-phase anchoring assembly and conformation locking procedure; the cis-tethered self-locking primer is modified with the 5'-terminal amino group and attached to the microsphere matrix unit. The microsphere matrix unit Or the chemically active anchoring groups on the surface of the microsphere matrix unit M3 undergo covalent coupling; after the coupling reaction is completed, an annealing locking procedure is performed, which causes the cis-tethered self-locking primer to fold spontaneously using the intramolecular proximity effect, forcing the locking domain to pair complementaryly with the amplification initiation domain to form a stem-loop structure, physically blocking the 3' extension site of the amplification initiation domain. Step S4: Release of nucleic acid from tick samples; The tick samples to be tested are mechanically ground and heat-treated in sodium hydroxide alkaline lysis buffer, and the pH value is adjusted using neutralization buffer to obtain a biological sample containing the target nucleic acid to be tested. Step S5: Construction of a heterogeneous mixed reaction system; mixing the microsphere matrix units loaded with the cis-tethered self-locking primers in a single reaction vessel. The microsphere matrix unit and the microsphere matrix unit Add a liquid phase reagent containing Bst2.0 WarmStart DNA polymerase, heme chloride, and a chromogenic substrate, as well as the biological sample prepared in step S4; Step S6: Thermodynamically controlled amplification and in situ signal transduction; the heterogeneous mixed reaction system is incubated in a constant temperature environment; if the biological sample contains mutant target nucleic acid, an interface restriction loop-mediated isothermal amplification reaction is triggered, and the generated amplification product combines with the heme chloride to form G4-DNAzyme, catalyzing the chromogenic substrate to generate a colored product; Step S7: Decoding and interpreting the discretization results; observing the microsphere matrix units that have settled at the bottom of the reaction vessel. The microsphere matrix unit With the microsphere matrix unit The color and particle size characteristics are used to determine the results based on the two-dimensional coding rules for particle size and color.

[0007] Preferably, the specific method of parallel manufacturing in step S1 includes: By independently adjusting the ratio parameter of the dispersed phase flow rate to the continuous phase flow rate of the flow-focusing microfluidic chip device, the microsphere matrix units are controlled respectively. The microsphere matrix unit and the microsphere matrix unit The particle size is used to achieve spatial coding based on physical dimensions; The prepared microsphere matrix unit The microsphere matrix unit and the microsphere matrix unit It is stored in anhydrous ethanol or isopropanol solvent to maintain the reactivity of the chemically active anchoring groups.

[0008] Preferably, the structural features of the cis-tethered self-locking primer in step S2 include: incorporation of a locked nucleic acid monomer into the locking domain of the cis-tethered self-locking primer; The specific site for recognizing single nucleotide polymorphisms is configured in the branch migration region of the cis-tethered self-locking primer, and the specific site spatially corresponds to the locked nucleic acid modification position of the locking domain, thereby constructing a thermodynamic energy barrier for wild-type target nucleic acids.

[0009] Preferably, the specific execution logic of the annealing locking procedure in S3 includes: The system covalently coupled with the cis-tethered self-locking primers was heated to 95 degrees Celsius and held for a preset time, and then slowly cooled to 25 degrees Celsius at a rate of 0.1 degrees Celsius per second. The slow cooling process forces the locking domain to pair complementaryly with the amplification initiation domain, forming the thermodynamically stable stem-loop structure.

[0010] Preferably, the release of nucleic acid from the tick sample in step S4 includes: The mechanical grinding process disrupts the tick's cuticle and cellular structure; the heat at 95 degrees Celsius promotes protein denaturation and the release of genomic DNA. After the neutralization buffer adjusts the pH of the lysis products to neutral, the unpurified crude lysis buffer is directly used as the biological sample in subsequent reactions.

[0011] Preferably, the concentration of the sodium hydroxide alkaline lysis solution in step S4 is 20 mmol / L to 50 mmol / L.

[0012] Preferably, the configuration requirements for the heterogeneous mixed reaction system in step S5 include: The single reaction vessel simultaneously contains 50 to 100 of the microsphere matrix units. 50 to 100 of the aforementioned microsphere matrix units and 50 to 100 of the aforementioned microsphere matrix units ; The volume of the biological sample added accounts for 5% to 20% of the total volume of the heterogeneous mixed reaction system.

[0013] Preferably, the thermodynamically controlled amplification mechanism in step S6 is as follows: When the mutant target nucleic acid is present in the biological sample, the mutant target nucleic acid opens the stem-loop structure of the cis-tethered self-locking primer through a toe-point mediated strand displacement reaction; When only wild-type target nucleic acid is present in the biological sample, the wild-type target nucleic acid has a base mismatch with the branch migration region, which cannot overcome the energy barrier caused by the modification of the locked nucleic acid monomer, and the chain substitution reaction is blocked, so the cis-tethered self-locking primer remains locked.

[0014] Preferably, the in-situ signal transduction mechanism in step S6 includes: After the interface-restricted loop-mediated isothermal amplification reaction is initiated, the amplification product forms a long-chain DNA containing G-quadruplexes inside the corresponding microsphere matrix unit. The heme chloride in the long-chain DNA capture reaction system is assembled into the G4-DNAzyme; The G4-DNAzyme catalyzes the redox reaction of the chromogenic substrate within the pores of the microsphere matrix unit, causing the corresponding microsphere matrix unit to exhibit a specific color.

[0015] Preferably, the two-dimensional encoding rules for particle size and color in step S7 include: The microsphere matrix unit The green indicator shows that the detection system is effective. The microsphere matrix unit A green color indicates that the biological sample has pyrethroid resistance. The microsphere matrix unit A green color indicates that the biological sample has organophosphate resistance; The microsphere matrix unit The microsphere matrix unit and the microsphere matrix unit All samples remain colorless and transparent, indicating that the biological samples are sensitive to the corresponding drugs.

[0016] This invention provides a method for detecting multidrug resistance in ticks based on LAMP technology. It has the following beneficial effects: 1. This invention constructs a high-density cis-locked probe with thermodynamic locking function on the surface of a solid-phase support. Utilizing a competitive base substitution mechanism between the target sequence and the probe's terminal toe region, it achieves strict control over the amplification reaction. Only a perfectly matched target sequence can open the probe's stem-loop structure and expose the polymerase extension site, thereby blocking non-specific hybridization and errors at the thermodynamic equilibrium level. This design effectively overcomes the high background interference of traditional isothermal amplification techniques and significantly improves the signal-to-noise ratio and specificity for detecting minute variations in tick multidrug resistance genes.

[0017] 2. This invention employs an interface-restricted amplification strategy, utilizing covalent anchoring technology to confine nucleic acid amplification products within the electric double layer of the microsphere surface, and relying on the volume repulsion effect between polymer chains to form an upright, ordered brush-like conformation. This physical spatial confinement mechanism effectively curbs the free diffusion of amplification products into the liquid phase, avoids cross-contamination and signal interference between products of different coding microspheres, and ensures the authenticity of data from each detection channel.

[0018] 3. This invention constructs an automated data processing logic that includes system drift correction by applying a discretization decoding and dynamic threshold determination method based on statistical principles. This method utilizes the measured signal of the internal standard reference to compensate for baseline fluctuations in the optical detection equipment in real time, and adaptively adjusts the positive determination criteria according to the distribution pattern of the negative background signal, effectively offsetting systematic errors caused by batch-to-batch reagent differences and changes in ambient temperature. Attached Figure Description

[0019] Figure 1 This is a flowchart of a method for detecting multidrug resistance in ticks based on LAMP technology. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example: Please see the appendix Figure 1 This invention provides a method for detecting multidrug resistance in ticks based on LAMP technology. The method comprises the core hardware and biochemical components of a multidrug resistance discretization detection system for ticks. The detection system consists of a spatially encoded solid-phase carrier module, a cis-tethered self-locking molecular recognition module, and an in-situ signal transduction and colorimetric module.

[0022] The spatially encoded solid-phase support module consists of an array of microspheres polymerized from polyethylene glycol diacrylate hydrogel. Each module comprises at least three groups of microsphere matrix units with non-overlapping particle size distributions. The microsphere matrix units are defined by their physical diameters. The detection channel is defined as a characteristic parameter. Microsphere matrix unit. Having the first average diameter Microsphere matrix unit This is used to establish a detection channel for pyrethroid resistance genes. Microsphere matrix unit. Having the second average diameter Microsphere matrix unit Used to establish a detection channel for organophosphate resistance genes. Microsphere matrix unit. Having a third average diameter Microsphere matrix unit This is used to establish a detection channel for tick internal reference genes. First average diameter. Second average diameter With the third average diameter There are physical size differences between them; The porous framework of the microsphere matrix unit is modified with a high density of chemically active anchoring groups. Chemically active anchoring groups Selected from N-hydroxysuccinimide ester group or carboxyl group. When chemically active anchoring group... When the group is a carboxyl group, the microsphere matrix unit also needs to undergo activation treatment with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (Sulfo-NHS) to form an intermediate active ester. Chemically active anchoring groups. Uniformly distributed within the three-dimensional network structure of the microsphere matrix units. Chemically active anchoring groups. The molar concentration of the substance must satisfy the condition shown in formula (1):

[0023] In formula (1), The molar concentration of the chemically active anchoring group. This represents the number of primer molecules designed and loaded onto a single microsphere matrix unit. Represents the volume of a single microsphere matrix unit. Represents Avogadro's constant. The volume of a single microsphere matrix unit. According to physical diameter The volume of the sphere is calculated according to the formula for sphere volume. Meeting the condition of formula (1) ensures that the microsphere matrix unit provides sufficient covalent bonding sites to achieve high-density primer loading; The cis-tethered self-locking molecular recognition module consists of chimeric primer molecules designed for specific single nucleotide polymorphism sites. These chimeric primer molecules are cis-tethered self-locking primers. The cis-tethered self-locking primers are chemically modified single-stranded oligonucleotide molecules, containing, sequentially from the 5' to 3' end, an anchoring domain, a locking domain, a toe domain, a signal domain, and an amplification initiation domain. The anchoring domain is formed by a C6-amino linker modified at the 5' end and is used to connect with chemically active anchoring groups. An amide condensation reaction occurs.

[0024] The in-situ signal transduction and chromogenic module includes a liquid-phase reagent system for performing isothermal amplification and chromogenic reactions. The liquid-phase reagent system includes a DNA polymerase with strand displacement activity, a free set of helper primers, heme chloride, and a chromogenic substrate. The DNA polymerase is selected from Bst2.0 WarmStart DNA polymerase. The free helper primer set includes outer primer F3, outer primer B3, loop primer LF, and loop primer LB. The chromogenic substrate is selected from 2,2'-azono-bis-(3-ethylbenzothiazoline-6-sulfonic acid), and heme chloride is used to bind to the G-quadruplex structure generated from the amplification product to form a deoxyribonuclease with peroxidase activity.

[0025] The spatially encoded solid-phase carrier module and the cis-tethered self-locking molecular recognition module are physically fixed together via covalent chemical bonding. The amino group in the anchoring domain at the 5' end of the cis-tethered self-locking primer undergoes a nucleophilic substitution reaction with a chemically active anchoring group (e.g., an activated ester) on the microsphere matrix unit backbone, forming a stable amide bond. This amide bond covalently binds the cis-tethered self-locking primer within the three-dimensional network of the microsphere matrix unit. Constrained by the covalent bond, the locking domain and amplification initiation domain of the cis-tethered self-locking primer are spatially confined to a very small intramolecular range of motion. This intramolecular proximity effect results in a significantly higher effective collision frequency between the locking domain and the amplification initiation domain than between adjacent primer molecules, thus preferentially promoting intramolecular hybridization rather than intermolecular hybridization during annealing.

[0026] A thermodynamic control logic based on the Gibbs free energy difference is established between the cis-tethered self-locking molecular recognition module and the externally input target nucleic acid. This control logic relies on a toe-mediated strand displacement reaction mechanism. When the target nucleic acid is present, it binds to the toe domain on the cis-tethered self-locking primer, forming a nucleation complex. Subsequently, the target nucleic acid attempts to displace the locking domain through a branching migration process. The direction of this reaction is determined by the change in net free energy of the system at the isothermal amplification reaction temperature. The determination is made, and its calculation is shown in formula (2):

[0027] In formula (2), Gibbs free energy represents the Gibbs free energy of the double-stranded complex formed by the target nucleic acid and the cis-tethered self-locking primer. This represents the Gibbs free energy by which a cis-tethered self-locking primer maintains its stem-loop structure. For drug-resistant target nucleic acids containing single nucleotide mutations, due to perfect base complementarity, The absolute value is greater than The absolute value of makes such that A negative net free energy change drives the chain displacement reaction to proceed spontaneously, forcing the locking domain to dissociate and release the 3' end of the amplification initiation domain. At this point, the cis-tethered self-locking molecular recognition module transitions to an open logic state. For wild-type target nucleic acids, due to base mismatches in the branching migration region, the free energy penalty caused by the mismatch makes... The absolute value is less than The absolute value of leads to A positive net free energy change hinders the chain displacement reaction, allowing the cis-tethered self-locking primer to maintain its closed conformation.

[0028] The cis-tethered self-locking molecular recognition module and the in-situ signal transduction and colorimetric module achieve functional coupling through a cascaded biochemical reaction. When the cis-tethered self-locking molecular recognition module is in the open state, the released amplification initiation domain recruits Bst2.0 WarmStart DNA polymerase to initiate a loop-mediated isothermal amplification reaction. The amplification reaction generates a long-chain DNA product containing numerous repeating units on the solid-phase surface of the microsphere matrix unit. Each repeating unit contains a G-quadruplex sequence motif derived from the replication extension of the signal domain. As the amplification reaction proceeds, a high concentration of G-quadruplex structures accumulates inside the microsphere matrix unit. The G-quadruplex structure specifically captures heme chloride molecules in the liquid reagent system and assembles them into a catalytically active G4-DNAzyme complex. The G4-DNAzyme complex catalyzes the infiltration of chromogenic substrates into the microsphere matrix unit in situ, converting the chromogenic substrates into colored products. Since the long-chain DNA products are still linked to the microsphere matrix unit backbone through anchoring domains, and the high molecular weight of the long-chain DNA products hinders their diffusion outward through the hydrogel pores, the chromogenic signal is strictly confined within the specific geometric boundaries of the microsphere matrix unit.

[0029] Based on the above system architecture and logical connection principle, the discretization detection method for multidrug resistance in ticks described in this invention is implemented through seven consecutive and logically related steps: Step S1 involves the parallel fabrication of the spatially encoded solid-phase carrier module. This is achieved using a flow-focusing microfluidic chip device, through independent adjustment of the dispersed phase flow rate. With continuous phase velocity The ratio parameter was used to prepare materials with a first average diameter. microsphere matrix unit Having a second average diameter microsphere matrix unit and having a third average diameter microsphere matrix unit Microsphere matrix unit , and During the polymerization process, chemically active anchoring groups with molar concentrations meeting the requirements of the aforementioned formula (1) are introduced through copolymerization. The hydrodynamic control and droplet generation processes for microfluidic chips are well-known technologies in this field and will not be elaborated upon here. The prepared microsphere matrix units are stored in anhydrous ethanol or isopropanol solvent to maintain the reactivity of the chemically active anchoring groups; Step S2 involves the sequence design and synthesis of the cis-tethered self-locking molecular recognition module. Cis-tethered self-locking primers were designed and synthesized for the pyrethroid resistance gene mutation sites, organophosphate resistance gene mutation sites, and conserved sequences of the tick internal reference gene. During the design process, specific sites recognizing single nucleotide polymorphisms were positioned within the branching migration region of the cis-tethered self-locking primers, ensuring that the specific sites spatially correspond to the locking nucleic acid modification positions of the locking domain. The synthesis of the cis-tethered self-locking primers involved 5' end amino modification, locking domain locking nucleic acid monomer incorporation, and toe domain sequence construction. Step S3 involves the execution of solid-phase anchoring assembly and conformation locking procedures, in which the cis-tethered self-locking primers synthesized in step S2 are added to the microsphere matrix unit. , or The cis-tethered self-locking primers are covalently coupled to the chemically active anchoring groups on the microsphere matrix unit backbone via the 5' amino group. After the coupling reaction, an annealing locking procedure is performed. The annealing locking procedure involves heating the system to 95°C and holding it for 5 minutes, followed by cooling to 25°C at a rate of 0.1°C per second. During the slow cooling process, the cis-tethered self-locking primers covalently attached to the surface of the microsphere matrix unit spontaneously fold using intramolecular proximity. The folding process forces the locking domain to pair complementaryly with the amplification initiation domain, forming a thermodynamically stable stem-loop structure, thereby physically blocking the 3' extension site of the amplification initiation domain; Step S4 involves the release of nucleic acid from the tick sample. The tick sample is mechanically ground in an alkaline sodium hydroxide lysis buffer at a concentration of 20 to 50 mmol / L. Mechanical grinding disrupts the cuticle and cellular structure of the tick. The lysis products are then heated to 95°C and maintained for 10 minutes. The heat further promotes protein denaturation and the release of genomic DNA. An equal volume of neutralization buffer is added to the lysis products to adjust the pH to neutral. The unpurified crude lysis buffer is then used directly as the biological sample containing the target nucleic acid.

[0030] Step S5 involves the construction of a heterogeneous mixed reaction system, in which 50 to 100 microsphere matrix units loaded with self-locking primers are mixed in a single reaction vessel. 50 to 100 microsphere matrix units and 50 to 100 microsphere matrix units Add a liquid phase reagent containing Bst2.0 WarmStart DNA polymerase, free helper primers, heme chloride, and chromogenic substrate to the reaction vessel. Finally, add the biological sample prepared in step S4 to the reaction vessel, with the volume of the biological sample accounting for 5% to 20% of the total volume.

[0031] Step S6 involves thermodynamically controlled amplification and in situ signal transduction. The heterogeneous mixed reaction system is incubated at a constant temperature of 63°C to 65°C for 45 to 60 minutes. During the isothermal incubation, if the biological sample contains a mutant target nucleic acid corresponding to the microsphere matrix unit, the mutant target nucleic acid opens the stem-loop structure of the cis-tethered self-locking primer through a toe-point mediated strand displacement reaction, triggering an interfacial restriction loop-mediated isothermal amplification reaction. The amplification product forms a long-chain DNA containing a G-quadruplex inside the microsphere matrix unit, and further binds to heme chloride to form a G4-DNAzyme. The G4-DNAzyme catalyzes the generation of a colored product from the chromogenic substrate within the pores of the microsphere matrix unit. If the biological sample contains only wild-type target nucleic acid, due to the base mismatch between the target nucleic acid and the branching migration region, the energy barrier caused by the locked nucleic acid modification cannot be overcome, the strand displacement reaction is inhibited, the cis-tethered self-locking primer remains locked, and the amplification and chromogenic reactions do not occur. Step S7 involves decoding and interpreting the discretized results. After the reaction, the color and particle size characteristics of the microsphere matrix units settling at the bottom of the reaction vessel are directly observed with the naked eye. The results are determined based on the two-dimensional encoding rules of particle size and color: microsphere matrix units... The green indicator detection system is effective; based on this, the microsphere matrix unit... The green indicator sample exhibits pyrethroid resistance; microsphere matrix unit The green indicator sample exhibits organophosphate resistance. The colorless and transparent microsphere matrix unit indicates that the sample is sensitive to the corresponding drug.

[0032] This embodiment details the specific implementation of the microfluidic fabrication process for the spatially encoded hydrogel microsphere matrix in step S1 of the overall process described in Embodiment 1.

[0033] The microfluidic fabrication process is carried out on a flow-focusing microfluidic chip fabricated using polydimethylsiloxane soft lithography. The flow-focusing microfluidic chip features a cross-shaped microchannel structure, where the channel width (WW) at the cross-junction is designed to be 50 to 100 micrometers, and the channel height (HH) is designed to be 50 to 80 micrometers. This geometric constraint ensures that the generated droplets are within the micrometer scale.

[0034] The manufacturing process is divided into dispersed phase fluid preparation step S1-1, continuous phase fluid preparation step S1-2, droplet shearing generation step S1-3, and photopolymerization curing step S1-4. In the dispersed phase fluid preparation step S1-1, an aqueous prepolymer solution for forming the hydrogel framework is prepared. The aqueous prepolymer solution contains polyethylene glycol diacrylate as the framework monomer, 2-hydroxy-2-methyl-1-phenyl-1-propanone as the photoinitiator, and N-hydroxysuccinimide acrylate as the functionalizing monomer. The number average molecular weight of polyethylene glycol diacrylate is selected as 700, and the volume percentage concentration of polyethylene glycol diacrylate in the aqueous prepolymer solution is set to 15% to 25%. The functionalizing monomer N-hydroxysuccinimide acrylate is dissolved in dimethyl sulfoxide to form a mother liquor, which is then added to the aqueous prepolymer solution to achieve a final concentration of N-hydroxysuccinimide acrylate of 5 mmol / L to 10 mmol / L. The mass percentage concentration of the photoinitiator is set to 1%. The solvent used is 10 mmol / L phosphate buffered saline (PBS, pH 7.4).

[0035] In continuous phase fluid preparation step S1-2, an oil phase fluid is prepared to provide shear force and an isolating environment. Fluorinated oil HFE-7500 is selected as the oil phase fluid. To maintain droplet stability and prevent merging, a perfluoropolyether surfactant with a mass percentage concentration of 2% is added to the oil phase fluid. The perfluoropolyether surfactant reduces the surface tension at the oil-water interface, promoting the formation of stable droplets.

[0036] In droplet shearing generation steps S1-3, an aqueous prepolymer and an oil-phase fluid are injected into a flow-focusing microfluidic chip using a precision injection pump. The aqueous prepolymer, as the dispersed phase fluid, is injected through the central channel, while the oil-phase fluid, as the continuous phase fluid, is injected through the side channels. At the cross-junction, the continuous phase fluid applies fluid shear force and viscous stress to the dispersed phase fluid, forcing the dispersed phase fluid to break apart and form monodisperse droplets encapsulating the prepolymer components. The droplet generation process follows a capillary number (Ca) controlled droplet flow mechanism. The generated droplet diameter... With the dispersed phase flow rate and continuous phase velocity The ratio between them conforms to the scaling law shown in formula (3):

[0037] In formula (3), Represents the width of the microfluidic channel node. and This is an empirical constant determined by the geometry of the microfluidic chip and the viscosity of the fluid. Equation (3) shows that, in the channel size... Under the premise of constant conditions, by changing the dispersed phase flow rate With continuous phase velocity The ratio of [value] can precisely control the droplet diameter. This characteristic directly supports the parallel fabrication of the three microsphere matrix units with different particle sizes described in Example 1.

[0038] In photopolymerization curing steps S1-4, the generated droplets flow with the fluid into a serpentine curing channel downstream of the microfluidic chip. This serpentine curing channel is positioned below the irradiation area of ​​a 365 nm ultraviolet light source. Ultraviolet light penetrates the polydimethylsiloxane cover sheet and irradiates the flowing droplets. The photoinitiator HMPP absorbs the ultraviolet light energy to generate free radicals, initiating a free radical copolymerization reaction between polyethylene glycol diacrylate and N-hydroxysuccinimide acrylate. This copolymerization reaction transforms the liquid droplets into solid hydrogel microsphere matrix units. Because N-hydroxysuccinimide acrylate participates in the copolymerization reaction, the chemically active anchoring groups (NHS esters) are covalently fixed to the cross-linked network backbone, rather than through simple physical adsorption. The cured hydrogel microsphere matrix units are collected from the chip outlet and immediately cleaned with isopropanol to remove residual oil phase fluid and surfactants from the surface, ultimately obtaining a clean, space-encoded hydrogel microsphere matrix.

[0039] In the aforementioned microfluidic manufacturing process, the specific implementation steps of particle size encoding involve changing the driving parameters of a precision injection pump to independently and sequentially execute the microsphere matrix unit. Preparation steps S1-5, microsphere matrix unit Preparation steps S1-6 and microsphere matrix unit Preparation steps S1-7 are used to achieve this. Three independent preparation steps construct a coding system based on differences in physical diameter. The physical coding system uses microsphere matrix units of different diameters generated under different hydrodynamic conditions as independent reaction vessels and information carriers. Microsphere matrix unit In preparation steps S1-5, the volumetric flow rate of the continuous phase fluid is... Set to a fixed value of 30 microliters per minute to determine the volumetric flow rate of the dispersed phase fluid. The flow rate is set at 2 microliters per minute. Under these hydrodynamic conditions, the dispersed phase fluid is sheared and fractured by the continuous phase fluid, generating a first average diameter. The droplets are monodisperse droplets of 100 micrometers. These droplets enter a widened solidification channel with a width and height greater than 150 micrometers, where they recover their spherical shape under surface tension. They are then cured by ultraviolet light to obtain microsphere matrix units. Microsphere matrix unit As a dedicated vector for detecting pyrethroid resistance genes; Microsphere matrix unit In preparation steps S1-6, the volumetric flow rate of the continuous phase fluid is maintained. The volumetric flow rate of the dispersed phase fluid is 30 microliters per minute. The flow rate was adjusted to 5 μL / min. As the relative flux of the dispersed phase fluid increased, the droplet formation frequency decreased and the volume of a single droplet increased, resulting in a second average diameter. The droplets were monodisperse at 190 micrometers in size. After sufficient relaxation and solidification within a widened solidification channel with a width and height greater than 250 micrometers, the droplets were obtained as microsphere matrix units. Microsphere matrix unit As a dedicated carrier for the detection channel of organophosphorus resistance genes.

[0040] Microsphere matrix unit In preparation steps S1-7, the volumetric flow rate of the continuous phase fluid is maintained. The volumetric flow rate of the dispersed phase fluid is 30 microliters per minute. The flow rate is further increased to 10 microliters per minute. At this point, the fluid shearing effect is relatively weakened, and the dispersed phase fluid can fill more volume before breaking apart, generating a third average diameter. The droplets were monodisperse at 280 micrometers in size. After sufficient relaxation and solidification, the droplets solidified within a widened solidification channel with a width and height greater than 350 micrometers, yielding microsphere matrix units. Microsphere matrix unit This serves as a dedicated vector for the tick internal reference gene detection channel. To ensure the microsphere matrix unit... Microsphere matrix unit With microsphere matrix unit Accurate differentiation after mixing and detection is crucial, and particle size uniformity control during the preparation process is essential. Utilizing the stability of the laminar flow field in the microfluidic chip, the coefficient of variation (CV) of the microsphere matrix unit was controlled to be below 3%. The calculation of the coefficient of variation follows formula (4):

[0041] In formula (4), The standard deviation representing the particle size distribution of the matrix units of microspheres in the same batch. This represents the arithmetic mean of the particle size of the microsphere matrix units in the same batch. Meeting the condition of less than 3% in formula (4) ensures the microsphere matrix units... Particle size distribution range (97-103 micrometers), microsphere matrix unit The particle size distribution range (184-196 micrometers) and the microsphere matrix unit The particle size distribution range (271-289 micrometers) is statistically non-overlapping. This non-overlapping particle size distribution characteristic establishes a definite mapping relationship between physical size and biological target, so that the decoding of detection results does not rely on additional fluorescence spectroscopy analysis, but can be completed by channel identification based solely on the relative size observed by the microsphere matrix unit with portable optical magnification equipment.

[0042] Complete the microsphere matrix unit Microsphere matrix unit and microsphere matrix units After physical fabrication, the obtained microsphere matrix units are still dispersed in a continuous phase of fluorinated oil containing surfactants, and the chemical groups on the surface need to be in a chemically reactive state with coupling capabilities to adapt to subsequent biomolecule coupling. Therefore, surface activation and post-cleaning treatment steps S1-8 need to be performed. Step S1-8 is specifically divided into a phase transfer cleaning step S1-8-1 and a carboxyl in-situ activation step S1-8-2, depending on the type of functional monomer selected during the polymerization of the microsphere matrix units. Step S1-8-1, the cleaning of the phase transfer molecule, applies to the microsphere matrix units directly polymerized using acrylate-N-hydroxysuccinimide as the functional monomer in the aforementioned steps. Because the N-hydroxysuccinimide ester groups introduced through copolymerization have water-sensitive hydrolytic properties, a completely anhydrous solvent cleaning procedure must be performed to prevent the active sites from being deactivated. A fluorinated oil solution of 1H,1H,2H,2H-perfluoro-1-octanol (20% by volume) is added as a demulsifier to the oil phase suspension containing the microsphere matrix units. 1H,1H,2H,2H-perfluoro-1-octanol can disrupt the stable layer formed by the perfluoropolyether surfactant on the surface of the microsphere matrix units. The mixture is transferred to a centrifuge tube and centrifuged at 1000 rpm for 2 minutes. Because the density of the fluorinated oil is greater than that of the hydrogel, the microsphere matrix units aggregate in the upper layer, adsorbing the lower layer of fluorinated oil phase. The microsphere matrix units were then resuspended in anhydrous acetone or anhydrous ethanol, centrifuged again, and the supernatant was removed. This washing process was repeated 3 to 5 times to thoroughly remove fluorinated oil and surfactants. After washing, the microsphere matrix units were suspended in anhydrous ethanol and stored in a sealed, dry environment at 4°C to maintain the amine reactivity of the N-hydroxysuccinimide ester groups. The in-situ activator step S1-8-2 is applicable to microsphere matrix units with carboxyl groups generated by polymerization in the dispersed phase fluid preparation step S1-1 using acrylic acid instead of acrylate-N-hydroxysuccinimide ester as the functional monomer. In this case, the amount of acrylic acid added to the dispersed phase fluid must ensure that the molar concentration of carboxyl groups in the finally cured microsphere matrix unit meets the requirements of the aforementioned formula (1). After cleaning to remove the oil phase, the carboxyl groups on the surface of such microsphere matrix units need to be converted from a chemically inert state to a chemically active state. The microsphere matrix units with carboxyl groups are dispersed in 0.1 mol / L 2-(N-morpholine)ethanesulfonate.

[0043] Step S2-1 involves constructing the topology of a cis-tethered self-locking primer chimera. The cis-tethered self-locking primer is designed as a linear single-stranded oligonucleotide containing six functional domains, which are arranged sequentially from the 5' end to the 3' end as follows: anchoring domain ( ), flexible spacer domain ( ), Locking domain ( ), toe point domain ( ), signal domain ( ) and amplification initiation domain ( This topology, through the synergistic effect of its functional domains, enables solid-phase support anchoring, interfacial steric hindrance elimination, thermodynamic conformational locking, target-specific recognition, in-situ signal transduction, and amplification reaction initiation at the single-molecule level. Anchoring area ( Located at the 5' end of the cis-tethered self-locking primer, it is responsible for establishing the covalent link between the primer and the microsphere matrix unit. The anchoring domain is a C6 amino linker modified with a 5' nucleotide (-( - The C6 amino linker provides a primary amine group that can undergo a nucleophilic substitution reaction with the active ester groups on the surface of the microsphere matrix unit to generate a stable amide bond, thereby directionally and persistently immobilizing the primer at the solid-phase interface; Flexible spacer domain ( The flexible spacer connects the anchoring and locking regions, providing the necessary spatial freedom to overcome the steric hindrance and charge repulsion effects on the solid-phase support surface. The sequence of the flexible spacer is either a non-specific sequence of 5 to 10 thymine nucleotides (Poly-T) or a repeating hexaethylene glycol (HEG) non-nucleic acid linker arm. The flexible spacer does not participate in subsequent complementary pairing; its physical length ensures that the subsequent functional region extends beyond the electrical double layer and hydration layer of the microsphere surface, significantly increasing the collision probability between the primer functional region and target nucleic acid and enzyme molecules in the liquid phase. Locked domain Adjacent to the flexible spacer domain, its sequence is designed to be completely complementary to the 3' end sequence of the amplification initiation domain. The length of the locking domain is set to 10 to 15 nucleotides. To improve the melting temperature of the stem-loop structure and its ability to recognize single-base mismatches, 2 to 3 locked nucleic acid (LNA) monomers are introduced into the locking domain sequence. The LNA monomers restrict the ribose conformation through a 2'-O,4'-C methylene bridge, significantly enhancing the thermodynamic stability of the binding between the locking domain and the amplification initiation domain, and preventing non-specific amplification leakage in the absence of a target.

[0044] Toe point domain ( The toe domain, located between the lockout and signal domains, serves as the nucleation site for initiating strand substitution reactions. The sequence of the toe domain is designed to be completely complementary to conserved regions of adjacent mutation sites in the target nucleic acid sequence. The toe domain is 6 to 8 nucleotides in length and does not participate in internal complementary pairing within the primer. In the locked state, where the primer folds to form a stem-loop structure, the toe domain maintains an overhang conformation, allowing externally free target nucleic acids to preferentially bind via a driving force of reduced Gibbs free energy, thereby initiating the branching migration process. signal domain ( Located between the toe domain and the amplification initiation domain, the signal output module is constructed after the amplification product is generated. The signal domain consists of a guanine-rich nucleotide sequence, specifically containing a GGGT sequence motif with 2 to 4 tandem repeats. After the amplification reaction is complete, the sequence folds to form a G-quadruplex structure and specifically binds to heme chloride molecules in solution, assembling into a G4-DNAzyme with peroxidase activity, catalyzing the color development of the substrate. Amplification Initiation Domain Located at the 3' end of the cis-tethered self-locking primer is the initiation region for loop-mediated isothermal amplification. The amplification initiation domain contains the F1c and F2 regions. The F1c region sequence is complementary to the F1 region upstream of the target nucleic acid, and the F2 region sequence is identical to the F2 region downstream of the target nucleic acid. The 3'-terminal portion of the amplification initiation domain is complementary to the locking domain, resulting in the 3'-OH end being physically masked in the locked state, blocking DNA polymerase binding and elongation until the strand displacement reaction occurs.

[0045] After completing the chimeric topology construction of the cis-tethered self-locking primers described in step S2-1, in order to achieve high-resolution recognition of single nucleotide polymorphism sites—that is, to specifically detect mutant target sequences in the presence of a large amount of wild-type background sequence interference—it is necessary to further perform the competitive reaction thermodynamic design described in steps S2-2 to S2-4. The core of the competitive design strategy lies in utilizing the difference in Gibbs free energy between intramolecular hybridization and intermolecular hybridization to construct a thermodynamic control mechanism for single base mismatches.

[0046] Step S2-2 involves the spatial mapping and layout of single nucleotide polymorphism sites. When designing the sequences of cis-tethered self-locking primers, the amplification initiation domain must be included. The base position corresponding to the single nucleotide polymorphism site is designed in the adjacent to the toe domain ( The stem initiation region of the target generatrix, i.e., the very tip of the branching migration process (within 0 to 6 bases from the toe region). The amplification initiation domain sequence is designed to be perfectly complementary to the mutant target sequence, i.e., the amplification initiation domain carries the mutant base. Locking domain ( The sequence of the target nucleic acid is designed to be completely complementary to the amplification initiation domain. In this configuration, when an external mutant target nucleic acid invades and binds to the toe domain, it can successfully open the stem-loop structure through branching migration to form a perfectly paired double strand. However, when a wild-type target nucleic acid invades, it encounters a base mismatch at the initiation stage of branching migration, which leads to the obstruction of branching migration and reverse dissociation, preventing the opening of the stem-loop structure to release the 3'-OH terminus of the amplification initiation domain. Steps S2-3 involve sequence thermodynamic optimization based on the free energy criterion. To ensure that the chain substitution reaction occurs only in the presence of a mutant target, and is inhibited in the presence of a wild-type target, the stem-loop structure stability of the cis-tethered self-locking primer must be considered. The stability of the primer-target hybridization complex was quantitatively balanced, and the sequence length and GC content were fine-tuned according to the thermodynamic criterion shown in formula (5):

[0047]

[0048] In formula (5), The change in net Gibbs free energy representing the chain displacement reaction triggered by the mutant target. The change in net Gibbs free energy represents the substitution reaction triggered by the wild-type target. and These represent the standard Gibbs free energies for the formation of double-stranded complexes between the primer and the mutant target, and between the primer and the wild-type target, respectively. The stability of the stem-loop structure was improved by adjusting the length of the locking domain and the GC content. It falls within a specific energy threshold range. This energy threshold range ensures that the energy released by the mutant target binding is sufficient to overcome the energy barrier to open the stem-loop structure (i.e., the reaction proceeds spontaneously), while the wild-type target suffers from a Gibbs free energy penalty due to a single base mismatch. The energy released by its binding is insufficient to offset the energy required to open the stem-loop (i.e., the reaction is not spontaneous), thus achieving competitive inhibition of the wild-type sequence; Steps S2-4 involve using locked nucleic acid modification to enhance thermodynamic discriminability. To further expand the expression in formula (5)... and The difference between them, in the amplification initiation domain Or locking domain ( Locked nucleic acid (LNI) monomers are introduced at positions corresponding to single nucleotide polymorphism (SNP) sites. The rigid structure of LNI monomers restricts the conformational flexibility of the nucleic acid backbone, resulting in extremely high thermal stability when binding to the complementary strand, but also making them highly sensitive to mismatched bases. When a wild-type target attempts to bind to an amplification initiation domain containing LNI modification, the LNI forces the mismatched site to maintain a specific conformation, leading to significant thermodynamic instability and energy penalties. This design mitigates the effects of single-base mismatches... The value is increased from 1,000 to 3,000 kcal / mol for ordinary DNA-DNA hybridization to 4,000 to 8,000 kcal / mol, thereby significantly improving the specificity factor of detection without changing the sequence length.

[0049] Based on the competitive sequence design principles determined in steps S2-2 to S2-4, to ensure that the cis-tethered self-locking primers can maintain the expected conformational state in the temperature and ionic environment of the actual loop-mediated isothermal amplification reaction, further conformational thermodynamic verification and parameter correction as described in steps S2-5 to S2-7 are required. Conformational thermodynamic verification aims to eliminate metastable traps in sequence design through quantitative calculations, ensuring precise digital control of the lock-up / open state transitions. Steps S2-5 involve calculating the ground-state energy based on the nearest-neighbor thermodynamic model. The enthalpy change of the cis-tethered self-locking primer in its folded state is calculated using the nearest-neighbor model. With entropy change Because the primer sequences contain locked nucleic acid modified monomers, calculations must be performed using a specific set of thermodynamic parameters for the DNA / LNA mixed double strands. For pure DNA base pairs, the Santa Lucia unified parameter set is used; for DNA-LNA hybrid base pairs, the LNA nearest neighbor parameter set determined by McTigue or Tolstrup is used. The calculation process sets the standard reaction temperature to 60°C to 65°C, and the standard Gibbs free energy for the formation of the stem-loop structure by the cis-tethered self-locking primers is... Follow formula (6):

[0050] In formula (6), The absolute temperature (Kelvin) representing the reaction system; The total enthalpy change representing the formation of the stem-loop structure is derived from the sum of the contributions of conventional DNA-DNA stacking enthalpy change and LNA-induced conformational pre-organization enthalpy change. The total entropy change representing the formation of the stem-ring structure is obtained through formula (6). The value must be negative, and its absolute value must be sufficient to resist conformational dissociation caused by thermal fluctuations.

[0051] Steps S2-6 involve the correction of ionic strength for free energy and the calculation of equilibrium probability. Because the loop-mediated isothermal amplification reaction buffer contains a high concentration of magnesium ions (…),… ) and monovalent cations ( or High concentrations of cations shield the electrostatic repulsion between the phosphate backbones of nucleic acids, significantly improving the stability of the double-stranded structure. Therefore, the ion concentration parameters in the buffer solution (usually...) must be controlled. Substituting the concentration (6 mmol / L to 8 mmol / L) into the salt concentration correction algorithm, the results obtained in step S2-5 are... Make corrections. Based on the corrected free energy, calculate the probability that the cis-tethered self-locking primer is in a locked conformation in the target-free state. The calculation follows formula (7):

[0052] In formula (7), Represents the ideal gas constant. This represents the equilibrium constant between the closed conformation and the random coil conformation. To prevent nonspecific amplification, the design must meet the following requirements: at the reaction system preparation temperature (25 degrees Celsius), The value must be greater than 99.99%; at the reaction operating temperature (65 degrees Celsius), The value needs to be maintained above 98%. If the calculation result shows... If the set threshold is not reached, it indicates that the risk of leakage is too high, and it is necessary to return to step S2-3 to increase the length of the locking domain or increase the proportion of locked nucleic acid modification.

[0053] Steps S2-7 involve the screening and elimination of metastable secondary structures. Besides the expected stem-loop structure, long single-stranded DNA molecules are highly prone to folding into unexpected hairpin structures, inner loops, or dimers; these unexpected metastable structures are called kinetic traps. A polymorphic secondary structure prediction algorithm is used to traverse all possible secondary structure ensembles that the cis-tethered self-locking primer might form. The focus is on checking for self-folding regions within the amplification priming domain and whether the toe domain is accidentally involved in secondary structures. If the toe domain bases are found to participate in intramolecular hydrogen bonding with a pairing probability exceeding 5%, the design is deemed unacceptable. The toe domain must remain completely single-stranded free to ensure barrier-free binding of the target nucleic acid via collisions. For parasitic secondary structures formed within the amplification priming domain, if their Gibbs free energy is below -3 kcal / mol, a degenerate base substitution strategy is used to disrupt the complementarity of the parasitic secondary structure, ensuring that the cis-tethered self-locking primer exhibits only a single, designed stem-loop conformation in the locked state.

[0054] To securely load the designed and synthesized cis-tethered self-locking primers onto the coding microsphere matrix unit and ensure that the primers revert to the intended thermodynamically locked conformation, step S3 needs to be performed, including the following steps: Step S3-1 involves the directional covalent coupling of primer molecules on the microsphere surface. The microsphere matrix units activated by step S1-8-1 or step S1-8-2 are then... Microsphere matrix unit and microsphere matrix units Each sample was placed in a separate reaction vessel, and each vessel was filled with a solution of a cis-tethered self-locking primer. cis-tethered self-locking primers and cis-tethered self-locking primers Phosphate-buffered saline (PBS, pH 7.4) was used. The primer solution concentration was set at 5 μmol / L to 10 μmol / L, and the microsphere density in the reaction system was controlled at [value missing] / mL. One to The reaction was carried out with shaking at room temperature for 2 to 4 hours. During this process, the primary amine group on the C6 amino linker modified at the 5' end of the cis-tethered self-locking primer acted as a nucleophile, undergoing a nucleophilic substitution reaction with the N-hydroxysuccinimide ester group on the surface of the microsphere matrix unit, removing the N-hydroxysuccinimide molecule and forming a stable covalent amide bond. By controlling the reaction time and primer concentration, the primer loading density on the microsphere surface was controlled at approximately [value missing] per square micrometer. One to 10 Each molecule is used to ensure spatial accessibility for subsequent reactions; Step S3-2 involves the chemical blocking of residual active sites. After the covalent coupling reaction, to eliminate non-specific adsorption caused by unreacted active ester groups remaining on the surface of the microsphere matrix units, an ethanolamine solution (pH 8.0) with a final concentration of 50 mmol / L to 100 mmol / L is added to each reaction vessel. Ethanolamine molecules, utilizing their high diffusion rate, rapidly react with the remaining N-hydroxysuccinimide ester groups, converting the residual active sites into electrically neutral and hydrophilic hydroxyethylamide groups. The blocking reaction is carried out at room temperature for 30 to 60 minutes. After blocking, the microspheres are washed three times by centrifugation using a TE buffer containing 0.05% Tween-20 (10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH 8.0) to remove unbound primer molecules, detached N-hydroxysuccinimide, and excess ethanolamine.

[0055] Step S3-3 involves the thermodynamic annealing and conformational locking of the surface primers. Because cis-tethered self-locking primers may form metastable intramolecular entanglements or nonspecific adsorptions on the microsphere surface during chemical coupling, they must be reset to the stem-loop locked state with the lowest Gibbs free energy through a thermal annealing procedure. The cleaned coded microsphere probes are then... Encoded microsphere probes and coded microsphere probes Resuspended in solutions containing 100 mmol / L sodium chloride (NaCl) and 10 mmol / L magnesium chloride (MgCl₂), respectively. 2） The primers were placed in a high-salt renaturation buffer. The suspension was heated to 90°C and held for 5 minutes to fully open all secondary structures of the primer molecules. Subsequently, the temperature was slowly cooled to 25°C at a rate of 1°C per minute. During this slow cooling, the cis-tethered self-locking primers, according to the thermodynamic design described in Example 3, spontaneously folded to form a stable hairpin structure consisting of complementary pairing of the locking domain and the amplification priming domain, thus achieving conformational locking. The resulting encoded microsphere probe was stored in TE buffer at 4°C for later use.

[0056] After solid-phase covalent coupling and active site blocking are completed in steps S3-1 and S3-2, the cis-tethered self-locking primers, although chemically anchored to the microsphere surface, remain in a thermodynamically disordered or metastable folded state at the microscopic level. To activate the primers' self-locking function, the thermodynamic annealing and conformational locking procedures described in steps S3-3 to S3-4 need to be further executed. These procedures eliminate kinetic traps at the solid-phase interface through precise temperature control, forcing the primer molecules into the designed minimum free energy state.

[0057] Step S3-3 involves the construction and conformational dissociation of a high-salt renaturation environment. During chemical coupling, the cis-tethered self-locking primers on the microsphere surface readily form intermolecular entanglement or non-specific surface adsorption structures due to intermolecular electrostatic repulsion and steric hindrance. To correct folding errors, the coded microsphere probes washed in step S3-2 are... Encoded microsphere probes and coded microsphere probes The components were resuspended separately in a high-salt refolding buffer. The high-salt refolding buffer consisted of 100 mmol / L to 150 mmol / L sodium chloride (NaCl) and 10 mmol / L to 20 mmol / L magnesium chloride (MgCl₂). 2） And 10 mmol / L Tris-HCl (pH 8.0). High concentrations of monovalent cations in high-salt refolding buffer (… ) and divalent cations ( The Debye-Hückel shielding effect neutralizes the negative charge of the DNA phosphate backbone, significantly reducing the electrostatic repulsion barrier during primer folding and providing the necessary ionic environment for the subsequent formation of a tight stem-loop structure. The suspension is placed in a temperature-programmed instrument and heated to 90-95°C at a rate of 5°C per minute, then held at this temperature for 5-10 minutes. Under these high-temperature conditions, all hydrogen bonds and non-covalent interactions within or between molecules of the cis-tethered self-locking primer are disrupted, and the primer molecules transform into a fully extended single-stranded random coil state, thus completely eliminating non-specific molecular folding and entanglement.

[0058] Steps S3-4 involve slow cooling renaturation and conformational locking. After high-temperature denaturation, a programmed cooling process is initiated to induce the cis-tethered self-locking primers to self-fold according to the thermodynamic path designed in Example 3. The cooling program is set to slowly reduce the system temperature from 95°C to 25°C at a linear rate of 0.5°C to 1.0°C per minute. During the slow cooling process, as the system temperature gradually approaches the melting temperature of the complementary double strands of the locking domain and the amplification initiation domain... Intramolecular hydrogen bonds preferentially form between the locking domain and the amplification initiation domain. Since intramolecular hybridization is kinetically superior to intermolecular hybridization, and the locking domain sequence described in step S2 is completely complementary to the 3' end of the amplification initiation domain, the cis-tethered self-locking primer will spontaneously fold into a stable stem-loop structure. In this stem-loop structure, the 3' hydroxyl end of the amplification initiation domain is physically masked by the locking domain and forms a double-stranded hybridization state, thereby achieving spatial blocking of DNA polymerase extension activity. This physical process is the specific implementation of conformational locking. Through slow cooling, the primer molecules can reach thermodynamic equilibrium, avoiding falling into metastable traps of local minima, ensuring that most surface primers are ultimately in the locked conformation with the lowest Gibbs free energy. After annealing, the encoded microsphere probe is collected by centrifugation at 4°C and resuspended in a high-salt preservation buffer (10 mmol / L Tris-HCl, 150 mmol / L NaCl, 5 mmol / L MgCl2, 0.05% Proclin 300, pH 8.0). The preservation buffer retains a certain ionic strength and magnesium ions to maintain the charge shielding environment required for the primer stem-loop structure and prevent spontaneous dissociation of the stem-loop structure due to a sudden drop in ionic strength. The resulting product is a cis-locked coding microsphere probe with zero background characteristics.

[0059] After the thermodynamic annealing process is completed in steps S3-3 and S3-4, the cis-tethered self-locking primers establish the expected stem-loop conformation on the surface of the microspheres. At this point, steps S3-5 to S3-6 need to be further explained, involving the verification of the molecular mechanism of self-locking formation and the quantitative evaluation of the locking efficiency.

[0060] Steps S3-5 involve the stabilization of the self-locking structure driven by the cis-tethering effect. At the microsphere solid-phase interface, the folding of the cis-tethered self-locking primers follows the intramolecular reaction kinetics priority principle. Due to the anchoring domain ( This confines the primers within a specific spatial volume, creating a locking domain. With amplification initiation domain ( Local effective concentration between ) The concentration is significantly higher than the average concentration of free molecules in the solution. According to polymer solution theory, the local effective concentration of two complementary segments connected by a flexible spacer domain can reach the order of 1 to 10 mmol / L. The entropy effect generated by this high local effective concentration makes the formation of intramolecular hairpin structures far superior to the formation of intermolecular dimers in terms of Gibbs free energy. The specific self-locking formation criterion follows formula (8):

[0061] In formula (8), The change in free energy represents the formation of intramolecular self-locking. This represents the change in free energy for the formation of intermolecular dimers. To account for the conformational entropy loss caused by the formation of ring structures, This refers to the translational entropy loss caused by intermolecular bonding. Because cis-tethering significantly reduces entropy loss ( ), making This results in a more negative value, which thermodynamically drives the primers to form a single-molecule self-locking conformation on the solid surface with near 100% probability, rather than an intermolecular cross-linked network. This mechanism ensures that the probes on each microsphere matrix unit operate independently without interference. Steps S3-6 involve verifying the structural integrity of the self-locking conformation. To verify whether the annealing procedure described in step S3-4 successfully induced the formation of the self-locking structure, thermodynamic stability analysis of the prepared coded microsphere probe is required. Due to the light scattering interference of the microspheres and the short double-chain characteristics of the stem-ring structure, the conventional SYBR Greenl dye method is insufficiently sensitive. Therefore, differential scanning calorimetry (DSC) or ultraviolet absorbance-based microcalorimetric melting analysis is used. An appropriate amount of the coded microsphere probe suspension is placed in a detection cell, and the temperature is linearly increased from 40°C to 95°C at a rate of 1°C per minute. The change in heat capacity (Cp) or absorbance is monitored to obtain the melting transition curve. A qualified cis-self-locking coded microsphere probe should exhibit a single endothermic peak or absorbance transition point at the designed stem-ring melting temperature of 75°C to 80°C. If multiple transitions occur or the transition temperature is significantly lower than the design value, it indicates the presence of non-specific folding or incomplete self-locking formation.

[0062] Steps S3-7 involve polymerase extension blocking assays and background leakage rate calculations. The core function of the locking mechanism is to physically block the extension of the DNA polymerase to the 3'-OH end of the amplification initiation domain. A blank control reaction system is constructed containing only BstDNA polymerase, dNTPs (deoxyribonucleoside triphosphates), and reaction buffer, but without the target nucleic acid. Encoded microsphere probes are added to the system, and a simulated amplification reaction is performed at a constant temperature of 65°C for 60 minutes, with fluorescence signals monitored (a trace amount of SYBR Green I or Evagreen dye is added to indicate the amplification products). The locking efficiency of the locking structure (… Quantitative calculations are performed using formula (9):

[0063] In formula (9), The background fluorescence growth rate (i.e., leakage rate) representing the encoded microsphere probe under target-free conditions. The fluorescence growth rate (i.e., maximum elongation rate) represents the rate at which an excess (more than 1 μmol per liter) of single-stranded DNA completely complementary to the locking domain sequence (unlocking strand) is introduced to forcibly open the stem-loop structure. To meet the requirements of high-sensitivity detection, the locking efficiency of the prepared encoded microsphere probe is... The accuracy must be greater than 99.5%. This indicator confirms that after self-locking, the 3'-OH end of the amplification initiation domain is effectively buried deep inside the double helix structure and cannot be reached by the active site of DNA polymerase, thus achieving a conformational lock state with zero background.

[0064] After completing the preparation and validation of the encoded microsphere probes, step S4 needs to be performed to address the biological characteristics of tick samples, which have a hard chitinous exoskeleton on their epidermis and contain large amounts of PCR inhibitors (such as heme and polysaccharides) in their body fluids. Step S4 utilizes a synergistic mechanism of physical mechanical shearing and chemical denaturation and dissolution to disrupt the tissue integrity of the tick and the cell wall structure of the pathogen, releasing the nucleic acids encapsulated within the organism into the liquid phase system. This includes the following steps: Step S4-1 involves mechanical homogenization based on high-frequency oscillation. Collected tick samples (whole ticks or partial tissue) are placed in 2 mL reinforced centrifuge tubes containing pre-cooled lysis buffer. The reinforced centrifuge tubes are pre-loaded with 200-300 mg of a mixture of 1.4 mm diameter zirconia beads and 2.8 mm diameter stainless steel beads at a 3:1 mass ratio. This multi-scale abrasive media design utilizes the high kinetic energy of the large-diameter stainless steel beads to break the tick's hard chitinous exoskeleton, while simultaneously using the concentrated shear force of the small-diameter zirconia beads to break up soft tissue and cellular structures. The reinforced centrifuge tubes are placed in a high-throughput tissue homogenizer, with the oscillation frequency set to 50-60 Hz and the runtime to 60-90 seconds. Under the shear and impact forces generated by the high-frequency reciprocating motion, the tick tissue is rapidly pulverized into a homogenous suspension, achieving initial exposure of the pathogen.

[0065] Step S4-2 involves constructing a potent chemical lysis environment. To further disrupt the cell membrane, cell wall, or capsid structure of pathogens (bacteria, rickettsiae, viruses) and inhibit the activity of released nucleases, a chemical lysis buffer is used in conjunction with the mechanical grinding step. The lysis buffer consists of a high concentration of ionizing salts, a nonionic surfactant, and a reducing agent. The specific formulation is: 4 mol / L to 6 mol / L guanidine isothiocyanate (GuSCN), 50 mmol / L Tris-HCl (pH 7.5), 25 mmol / L EDTA, 296 to 496 (v / v) Triton X-100, and 1% (v / v) β-mercaptoethanol. Guanidine isothiocyanate, as a strong lysis buffer, can disrupt the hydration layer of proteins, leading to protein denaturation and dissolution, while effectively inactivating endogenous RNase and DNase; Triton X-100 disrupts the lipid bilayer; β-mercaptoethanol assists in the degradation of chitin-binding proteins and mucins by reducing disulfide bonds. The amount of lysis buffer used ( Based on the wet weight of the tick sample ( The adjustment is made dynamically, following formula (10):

[0066] In formula (10), The liquid-to-solid ratio coefficient is set to 10 μL per milligram. The minimum volume required to maintain the grinding fluid environment was set at 200 μL. The amount of lysis buffer determined by formula (10) ensured that while maintaining a high-concentration lysis environment, sufficient fluid medium was provided to disperse the heat generated by grinding and prevent nucleic acid thermal degradation. Step S4-3 involves enzymatic digestion and heat shock nucleic acid release. To thoroughly remove histones and nucleocapsid proteins tightly bound to nucleic acids, 20 μL of proteinase K solution with a final concentration of 20 mg / mL is added to the homogenized homogenate. The mixture is placed in a constant-temperature metal bath and incubated at 55°C for 10 to 15 minutes. During this period, proteinase K non-specifically degrades various proteins in the sample, reduces the viscosity of the homogenate, and releases protein-encapsulated nucleic acids. Subsequently, the temperature of the metal bath is rapidly increased to 95 to 98°C and maintained for 5 minutes. This high-temperature treatment has a dual function: on the one hand, it completely inactivates proteinase K and residual pathogens, ensuring biosafety; on the other hand, it uses heat to destroy the thick-walled structure of difficult-to-lyse Gram-positive bacteria or fungi and unfold the secondary structure of nucleic acids. After treatment, the lysate is centrifuged at 12,000 rpm for 3 minutes, and the supernatant is collected as the test sample containing pathogen nucleic acids. Because the lysis buffer contains high concentrations of guanidine isothiocyanate and EDTA, these components strongly inhibit subsequent enzymatic amplification reactions. Therefore, the supernatant cannot be used directly for amplification. Instead, it must be purified by microsphere hybridization capture and washing as described in step S5 before it can be used in the amplification and detection process.

[0067] To ensure that the lysis products obtained from step S4-3 can efficiently participate in the hybridization and capture of the encoded microspheres described in subsequent step S5, the thermodynamic state of the lysis products must be maintained and the chemical environment adjusted. Because the high concentration of guanidine isothiocyanate introduced in step S4-2 has extremely strong denaturing ability, direct contact with the encoded microspheres without adjustment may result in the probe failing to form a stable double-stranded structure or causing the surface modification of the microspheres to detach. Therefore, steps S4-4 to S4-5, namely the thermal denaturation maintenance and chemical neutralization adjustment steps, need to be performed.

[0068] Step S4-4 involves maintaining the denatured state of nucleic acids and eliminating secondary structures. Although high-temperature treatment was performed in step S4-3, during solution cooling, long-chain genomic DNA molecules are highly susceptible to intramolecular folding or random intermolecular renaturation, forming complex secondary structures that can obscure the target sequence region. To ensure that the released tick pathogen nucleic acid exists as fully extended single-stranded DNA (ssDNA), the supernatant obtained from centrifugation in step S4-3 needs to be transferred to an incubator preheated to 95 degrees Celsius and maintained for 2 to 3 minutes. This operation aims to thermodynamically disrupt the hydrogen bonds and base stacking forces within the nucleic acid molecules, ensuring full exposure of the target sequence, unraveling the nucleic acid secondary structure, and reducing steric hindrance effects on subsequent hybridization capture. Steps S4-5 involve the chemical neutralization of the lysis environment and the construction of hybridization conditions. To transform the strongly denatured lysis buffer into a suitable buffer environment for nucleic acid hybridization, 1.5 to 2 times the volume of hybridization neutralization buffer needs to be rapidly added to the supernatant maintained at 95°C. The hybridization neutralization buffer consists of: 2.0 mol / L to 3.0 mol / L sodium chloride, 200 mmol / L sodium citrate, 0.196 mmol / L sodium dodecyl sarcosinate, and a Tris-HCl buffer system at pH 7.0. The mechanism of action of the hybridization neutralization buffer is based on the extremely high concentration of sodium ions (… It significantly increases the ionic strength of the solution, neutralizes the negative charge of the DNA phosphate backbone, and counteracts the destructive effect of ionizing salts on double-strand stability. Simultaneously, through dilution, the final concentration of guanidine isothiocyanate is reduced to a hybridization-promoting range of 1.5 mol / L to 2.0 mol / L. Within this concentration range, the strong denaturing ability of guanidine isothiocyanate is partially inhibited by the high-salt environment, instead manifesting as a reduction in the probability of non-specific mismatches during nucleic acid annealing and an increase in hybridization specificity. The final concentrations of each component in the mixed system (…) Follow the dilution formula (11):

[0069] In formula (11), and These represent the concentration and volume of a specific component in the lysis supernatant, respectively. and These represent the concentration and added volume of the corresponding component in the hybridization neutralization buffer, respectively. After treatment in steps S4-5, the chemical environment of the mixture has been neutralized and adjusted to a state conducive to DNA-specific binding, and it can be immediately used for the hybridization and capture reaction with the cis-locked coding microsphere probe described in step S5.

[0070] Steps S4-6 involve the direct delivery of the lysis mixture and the construction of a solid-phase enrichment environment. Specifically, the hybridization neutralization mixture prepared in step S4-5 is directly aspirated and transferred in its entirety or in large volumes (50 μL to 200 μL) as the injection solution into hybridization reaction wells or microfluidic chip reaction chambers pre-loaded with high-density cis-locked coding microsphere probes. During this process, to ensure that the complex biological matrix in the injection solution (including denatured tick protein fragments, heme residues, and high concentrations of guanidine isothiocyanate) does not interfere with the molecular recognition process on the microsphere surface, and to maximize nucleic acid capture efficiency, the injection operation must follow the principles of volume exclusion and kinetic enrichment. Unlike conventional PCR systems, which require strict control of the injection volume (usually less than 10% of the reaction volume), this step allows the injection solution to serve as the main medium for the hybridization reaction. Specifically, the preservation solution containing the coding microsphere probes is centrifuged to remove the supernatant, or the microspheres are pre-dried and fixed at the bottom of the reaction wells, and then the injection solution treated in step S4-5 is added. At this point, the liquid environment in the reaction system is entirely composed of the injection solution, and the microspheres are directly suspended in the lysis-neutralization mixture containing the target nucleic acid. This injection method is optimized according to the following fluid transport formula (12):

[0071] In formula (12), This represents the effective concentration of microspheres in the reaction system. This is the volume of the injected liquid. The dynamic viscosity of the pyrolysis fluid. Let be the diffusion rate constant of the nucleic acid molecule. This is the preset hybridization time. By maintaining a high microsphere concentration ( ), compensated for the viscosity of the pyrolysis fluid ( The effect of ) on the diffusion rate, ensuring that within a finite time ( Within the microsphere, the vast majority of target nucleic acids can effectively collide with and bind to the probes on the surface of the microsphere.

[0072] After completing purification-free sample injection in steps S4-6, the reaction system proceeds to step S5, the construction and hybridization capture stage of the solid-liquid mixed reaction system. In this stage, the cis-locked encoded microsphere probes prepared in steps S1 to S3 serve as the solid-phase component, performing the core target recognition and enrichment functions, including the following steps: Step S5-1 involves the construction and standardization of a multiplex detection solid-phase microsphere library. To achieve simultaneous detection of multiple tick-borne pathogens, the solid-phase component is not a single type of microsphere, but rather a suspension array composed of multiple encoded microsphere probes targeting different pathogens in a specific ratio. Specifically, it constructs specific microsphere subpopulations targeting Lyme disease spirochetes, Rickettsiae, Anaplasma, and Babesia. Each microsphere subpopulation is distinguished by the encoding sequence described in step S1 and modified with cis-locking primers targeting conserved sequences of specific pathogens. In the mixed reaction system, the number density of each microsphere subpopulation (…) The total surface area of ​​the solid phase components must be kept consistent to ensure the quantitative parallelism of the detection signal. ) and injection volume ( The relationship between them follows formula (13):

[0073] In formula (13), Representing the The number of microspheres corresponding to each pathogen Let be the radius of the microsphere. The lowest detection limit concentration for the target pathogen. The binding constant between the probe and the target. The probe coverage factor on the surface of the microspheres is denoted by formula (13). The amount of microspheres added, determined by formula (13), ensures that the solid-phase surface provides sufficient binding sites to capture low-abundance target nucleic acids under the large-volume injection conditions described in steps S4-6.

[0074] Step S5-2 involves target-induced conformational rearrangement and stem-loop unlocking. When the lysis-neutralization mixture containing tick pathogen nucleic acid is fully contacted with the solid-phase microsphere library, a specific molecular recognition event occurs after incubation at a hybridization temperature of 60°C to 65°C for 10 to 20 minutes. At this point, the cis-locked coding microsphere probe is in the stem-loop locked state described in step S3, and the 3'-OH end of the amplification initiation domain is masked. When the target single-stranded DNA that is completely complementary to the locking domain and the amplification initiation domain is present in the solution, the target sequence invades the stem-loop structure through a foothold-mediated strand substitution mechanism. Due to the thermodynamic stability of the target-probe hybridization complex ( The stability of the structure is significantly higher than that of the self-locking stem-ring structure. The probe undergoes a conformational inversion, forcibly opening the double strands at the stem to form a rigid double-helix hybrid. This process releases the previously locked 3'-OH terminus, exposing it to the liquid phase. This step achieves a conformational switching function, meaning the solid-phase probe only converts from a locked conformation to an open conformation in the presence of a specific target. Its conversion efficiency ( This can be described by formula (14):

[0075] In formula (14), The concentration of probes that have bound to the target and opened the stem-loop. The formula, representing the total probe concentration on the surface of the microspheres, reveals that the self-locking probes respond only to perfectly matched target sequences, while remaining locked to non-specific sequences with mismatches, thus providing extremely high specificity recognition capabilities.

[0076] Step S5-3 involves solid-phase washing and inhibitor removal. After hybridization capture, the target nucleic acid is tightly bound to the microsphere surface via hydrogen bonds, while high concentrations of guanidine isothiocyanate, heme, tick tissue fragments, and non-specifically adsorbed nucleic acids remaining in the lysis buffer are still suspended in the liquid phase or loosely attached to the microsphere surface. To construct an amplification compatibility buffer environment suitable for enzymatic amplification, a physical washing procedure is required. For superparamagnetic microspheres, an external magnetic field is applied to adsorb the microspheres onto the reaction tube wall, and the supernatant is removed; for non-magnetic microspheres, the microspheres are centrifuged at 3000 rpm to settle, and the supernatant is removed; for the microfluidic chip implementation, a washing buffer is introduced by controlling the fluid valve to flush the microsphere trapping area in a laminar flow manner. Subsequently, a washing buffer containing 10 mmol / L Tris-HCl (pH 8.0) and 0.05% Tween-20 is added to the microsphere precipitate, the microspheres are resuspended, and separated again. The washing steps are repeated 2 to 3 times. In step S5-3, solid-phase microspheres are transferred from the complex lysis matrix to a clean buffer system, and the concentration of residual inhibitors is diluted to below 10⁻⁶ / 10⁻⁶ of the original concentration. In other words, the physical separability of the solid-phase carrier replaces the column chromatography step in traditional nucleic acid extraction, solving the technical problem that the high-concentration lysis buffer cannot be directly amplified in step S4.

[0077] Steps S5-4 involve the preparation and functionalization of a universal isothermal amplification premix. Unlike traditional PCR reaction systems that require the preparation of specific primer mixtures for each pathogen, this embodiment employs a solid-phase specific-liquid-phase universal separation design strategy. The universal isothermal amplification premix contains the ionic environment, substrate, and non-anchored primers required to maintain BstDNA polymerase activity. Specifically, the components are: 20 mmol / L Tris-HCl (pH 8.8), 10 mmol / L ammonium sulfate ((NH4)2SO4), 50 mmol / L potassium chloride (KCl), and 8 mmol / L magnesium sulfate (MgSO4). 4） And 1.4 mmol / L of deoxyribonucleoside triphosphates (dNTPs). Among them, magnesium sulfate provides magnesium ions ( The concentration was optimized to 8 mmol / L, slightly higher than that of conventional PCR systems, designed to act as a cofactor for DNA polymerase, promoting strand displacement activity and stabilizing the formation of magnesium pyrophosphate precipitate. In addition, 1 mol / L of betaine was added to the universal isothermal amplification premix. Betaine, as an isothermal amplification enhancer, can lower the melting temperature of DNA double strands in GC-rich regions, eliminating secondary structure blockage during amplification. Regarding primer configuration, the premix contains a pool of free reverse primers paired with the solid-phase probe. This pool contains specific reverse primers designed for each target pathogen, all mixed at equimolar concentrations. These free primers diffuse freely in the liquid phase, binding to the extended template strands on the solid-phase microspheres, thereby forming a complete amplification cycle at the solid-liquid interface. Step S5-5 involves resuspending the reaction system and adjusting the enzyme-substrate ratio. The microsphere precipitate washed in step S5-3 is completely resuspended in the universal isothermal amplification premix prepared in step S5-4, and Bst2.0 DNA polymerase (large fragment) with strand displacement activity is added. To ensure efficient amplification of the target anchored on the solid-phase surface, while avoiding non-specific amplification background caused by excessive enzyme, the polymerase activity units in the liquid phase are... The dosage needs to be precisely matched according to the amount of solid microspheres added. The amount of polymerase used follows the enzyme kinetic formula (15):

[0078] In formula (15), The total number of microspheres in the reaction system. The effective surface area of ​​a single microsphere (unit: square micrometers). The saturated enzyme activity constant required per unit solid phase surface area (set to 1.5 × 10⁻⁶). -5 U / μm 2 ), This is the total volume of the liquid phase. The minimum enzyme concentration required to maintain the activity of the liquid substrate was set at 0.32 U / μL. The amount of enzyme added, determined by formula (15), ensured the high-density amplification requirement on the solid surface while maintaining the enzymatic stability of the liquid environment.

[0079] Steps S5-6 involve liquid-phase background suppression and reaction initiation preparation. Although the microspheres have been washed, a background suppression mechanism needs to be introduced into the liquid-phase environment to further reduce potential non-specific amplification (such as false positive signals from primer dimer formation). Single-stranded binding protein (SSB) is added to the resuspended reaction system at a final concentration of 0.1 μmol / L. SSB specifically binds to free single-stranded primers in the liquid phase, preventing non-specific annealing or self-initiation at low temperatures. At reaction temperatures above 60°C, SSB undergoes thermal inactivation or detachment, releasing primers to participate in normal specific amplification. This hot-start mechanism, combined with a conformational switch at the solid-phase level, constructs a chemical-physical dual-specificity protection system. After completing the above steps, the solid-liquid phase mixed reaction system is constructed. The system contains both specifically encoded microspheres anchored to the target nucleic acid and a universal liquid medium filled with amplification substrate and polymerase, and is in a state of readiness to enter the isothermal amplification and real-time detection stage described in step S6.

[0080] Steps S5-7 involve controlling the spatial distribution and suspension stability of solid microspheres in the liquid medium. To ensure that each coded microsphere can function as an independent reaction and detection unit, and to avoid increased flow cytometry overlap and local substrate competition effects caused by microsphere aggregation, it is essential to ensure that the microspheres are monodisperse in the reaction system. The specific implementation involves placing the constructed reaction system in an ultrasonic water bath at a frequency of 40 kHz and a power of 50 W for 10 to 15 seconds, followed by intense vortex oscillation for 5 seconds. In the reaction system described in step S5, the number density of microspheres (… The mean free path between microspheres is determined by both the enzyme concentration and the liquid phase viscosity. To ensure that the local enzyme and substrate concentrations on the microsphere surface are not disturbed by the sink effect of neighboring microspheres during rapid amplification, the mean free path between microspheres must satisfy the spatial constraint condition set by formula (16):

[0081] In formula (16), To encode the diameter of the microspheres, The total number of microspheres in the system. The total volume of the reaction system is... The diffusion coefficient of the amplified substrate (dNTPs) is given by... Characteristic reaction time, The safety factor is set to 3 to 5. Formula (16) clarifies the physical boundary of the independent reaction microregion, that is, by controlling the concentration of microspheres and the volume of the system, it ensures that there is a sufficient liquid volume around each microsphere to provide the substrate reserve required for amplification, and avoids substrate depletion kinetic inhibition caused by excessive local density of microspheres; Steps S5-8 involve the chemical potential energy and local concentration effect at the solid-liquid interface. Although the reaction system is macroscopically homogeneous, it exhibits a significant core-shell structure at the microscopic scale. The high-density cis-locking probes modified on the surface of the solid microspheres form a polyelectrolyte brush layer with a high negative charge density due to the presence of the phosphate backbone. The polyelectrolyte brush layer enriches positively charged magnesium ions from the liquid phase through the Donnan effect. A high ionic strength reaction interface was constructed on the surface of the microspheres, consisting of DNA polymerases with some positively charged domains. Within this interface layer, the local effective concentration of key reaction cofactors ( The concentration was significantly higher than that in the liquid phase bulk ( This interface enrichment effect follows the Boltzmann distribution law described in formula (17):

[0082] In formula (17), The number of charges of the reactant ions. It is Faraday's constant. The zeta potential on the surface of the microspheres (typically -30 mV to -50 mV), Let be the ideal gas constant. The absolute temperature is used. Through the state construction in steps S5-8, the electrostatic attraction of the microsphere surface reduces the mass transfer resistance of reactants to the solid-phase surface, resulting in a higher amplification reaction rate on the solid-phase surface compared to the pure liquid-phase system. At this point, the entire solid-liquid mixed reaction system is in a reaction-ready state, requiring only a temperature signal to trigger the exponential amplification phase.

[0083] After completing the construction and state control of the reaction system in step S5, the system is placed in an isothermal amplification instrument, and the reaction temperature is set to 63°C to 65°C. The toe-point exchange control and interface-restricted amplification reaction described in step S6 is initiated. As the core signal conversion link of the entire detection process, step S6 utilizes the cis-locked structure designed in step S3 to construct a kinetic control mechanism based on toe-point exchange at the molecular level, realizing highly specific recognition and amplification initiation of the target sequence, including the following steps: Step S6-1 involves the thermodynamic control and specificity screening of target recognition. In the initial stage of the reaction, the high-density cis-locked probes on the surface of the solid microspheres are in a metastable stem-loop locked state, and their amplification initiation domains are blocked by complementary blocking domains through intramolecular hybridization. When the target nucleic acid of the tick pathogen is present in the liquid phase, the target sequence first binds to the single-stranded protrusion at the bottom of the probe stem-loop structure, i.e., the pre-defined toe region. This binding process provides the initial nucleation site. Subsequently, through a branching migration process of one-dimensional random walk, the target sequence gradually replaces the blocking domain in the probe. Only when the target sequence and the recognition domain of the probe are completely complementary can the thermodynamic stability of the newly formed probe-target double-stranded structure be sufficient to overcome the energy barrier required to open the stem-loop structure. The process follows the Gibbs free energy change formula (18):

[0084] In formula (18), This represents the net change in reaction free energy. This is due to the release of free energy during the formation of the probe-target double strand. The free energy consumed to open the stem-ring structure. This is a penalty for the initial entropy of the bimolecular reaction. If a single base mismatch exists in the target sequence (especially a mismatch located in a branching migration region), it will result in... A significant increase (absolute value decrease) makes The energy difference is close to or greater than zero, thus blocking the chain substitution process. This recognition mechanism based on energy difference constitutes thermodynamic selectivity control, effectively preventing erroneous amplification caused by non-specific sequences; Step S6-2 involves 3'-terminal release and polymerase-mediated extension initiation. Once the target sequence successfully completes the strand replacement process, the stem-loop structure of the probe is completely unblocked, exposing the previously masked 3'-hydroxyl terminus (3'-OH) of the amplification initiation domain to the liquid environment. At this point, the Bst2.0 DNA polymerase, free within the electric double layer on the microsphere surface, recognizes the 3'-OH terminus at the double-strand-single-strand junction and takes up dNTPs substrates from the liquid phase, extending the target sequence 5' to 3'. Notably, since the 5' end of the probe is chemically anchored to the microsphere surface, the extension product is essentially a direct extension of the probe sequence, forming a newly formed complementary strand covalently bonded to the solid surface. This process transforms the previously free target recognition event into a permanent chemical information record on the solid surface.

[0085] Step S6-3 involves the recycling of the substitution products and signal cascade amplification. To achieve high-sensitivity detection, step S6 also introduces a cyclic substitution mechanism based on secondary toe points. After the first round of extension, the free reverse primer in the liquid phase binds to the end of the nascent chain and performs reverse extension. When the reverse extension reaches the original anchoring point of the probe, the strong chain substitution activity of the polymerase strips the previously bound target chain from the probe. The released target chain, as a cyclic triggering factor, can diffuse again to the adjacent microsphere surface or the adjacent probe on the same microsphere surface, bind to the new toe point region, and initiate the next round of chain substitution and extension reaction. This target recycling mechanism enables a single target molecule to trigger conformational changes and amplification of hundreds or thousands of probes on the microsphere surface, and its kinetic rate equation is shown in Equation (19):

[0086] In formula (19), The amplification rate on the surface of the microspheres. The surface density of the extended probe, For target concentration, Let be the apparent catalytic constant of the system. It is the Michaelis constant. The surface density of the locked-state probe is the key factor. Through this mechanism, the reaction system achieves the conversion from linear hybridization signal to exponential amplification signal.

[0087] Step S6-4 involves the annealing of the reverse primer and the synthesis of the complementary strand. As the first extension reaction proceeds, the nascent DNA strand formed on the solid-phase surface contains a sequence region completely complementary to the free reverse primer in the liquid phase. Under isothermal conditions of 63°C to 65°C, the free reverse primer impacts the microsphere surface via Brownian motion and undergoes specific annealing with the 3' end of the nascent strand on the solid phase. Subsequently, Bst DNA polymerase catalyzes the extension of the reverse primer using the nascent strand on the solid phase as a template. This process synthesizes a second strand (i.e., the antisense strand) complementary to the original probe sequence, thus forming a double-stranded DNA structure on the microsphere surface. Due to the strong strand displacement activity of Bst polymerase, the double-stranded structure does not dissociate when the extension reaches the 5' anchoring end of the probe, but maintains a stable hybridization state. At this point, the probe density on the microsphere surface ( ) on reverse initiation efficiency ( The relationship between the two plays a decisive role and follows formula (20):

[0088] In formula (20), This is the primer binding rate constant. This represents the concentration of the free reverse primer in the liquid phase. is the steric hindrance coefficient. Equation (20) reveals the existence of a steric hindrance effect in the high-density probe cluster on the microsphere surface. To overcome this effect, this embodiment introduces long-chain flexible spacer arms in the probe design, increasing the accessibility of surface reaction sites; Steps S6-5 involve the specific introduction and optical readout of signal molecules. To distinguish amplified signals on different coding microspheres in a multiplex detection system and avoid interference from non-specific background in the liquid phase, this embodiment employs a fluorescently labeled nucleotide incorporation method or a universal fluorescent primer method. In the premixed solution described in step S5-4, dUTP or dCTP with fluorescent groups is incorporated at a ratio of 1:10 to 1:20. As the polymerase extension reaction on the microsphere surface proceeds, the fluorescently labeled nucleotide is taken up by the enzyme as a substrate and covalently integrated into the newly synthesized DNA strand. Because the amplification reaction occurs only on the surface of microspheres that have captured specific targets, the fluorescence signal is strictly confined to the specific coding region of the microsphere. Its fluorescence intensity (… ) and the length of double-stranded DNA synthesized on the surface ( ) and the quantity of amplified products ( The relationship is directly proportional to the relationship shown in formula (21):

[0089] In formula (21), The incorporation frequency of fluorescent nucleotides, The quantum yield of the fluorescent group is given. Compared to non-specific intercalation dyes, this method labels only the newly synthesized solid-phase product, significantly improving the signal-to-noise ratio.

[0090] Steps S6-6 involve the formation of an interface-restricted amplification field and crosstalk suppression. As the amplification cycle progresses, the amount of double-stranded DNA product on the microsphere surface increases exponentially. Unlike traditional liquid-phase PCR, the amplification product in this embodiment is strictly confined within the electric double layer of the microsphere surface, forming a surface amplification layer with a thickness of approximately 100 nm to 500 nm. The restriction effect mainly stems from the covalent chemical anchoring of the probe's 5' end to the microsphere surface, preventing the extended DNA chains from detaching from the microsphere and entering the liquid phase. Simultaneously, the electrostatic repulsion generated by the high negative charge density of the polyelectrolyte layer on the microsphere surface promotes the outward extension of the rigid DNA double strands, preventing product collapse and entanglement on the microsphere surface and maintaining the continuous accessibility of the polymerase. This physical-chemical dual mechanism effectively prevents leakage of the amplification product into the liquid phase, resulting in a high surface restriction rate (…). Defined by formula (22):

[0091] In formula (22), To determine the amount of product bound to the surface of the microspheres, This represents the amount of leakage products free in the solution. By maintaining an extremely high surface confinement rate, each microsphere is ensured to function as an independent optical signal point, making it possible to simultaneously detect multiple pathogens using different coded microspheres within the same reaction tube.

[0092] Steps S6-7 involve the conformational entropy constraint and spatial distribution of the amplification products on the microsphere surface. Because the 5' ends of the high-density cis-locked probes are firmly anchored to the microsphere surface by stable covalent bonds (such as amide or thioether bonds), as the amplification reaction proceeds, although the newly synthesized DNA double strands continuously extend in length, one end remains fixed. According to polymer physics theory, such single-end-fixed polymer chains exhibit a specific spatial conformation in good solvents. In the high ionic strength amplification buffer of this embodiment, there is a strong volume repulsion reaction between the high-density DNA strands on the surface. This effect forces the DNA double strands to extend in an orientation perpendicular to the microsphere surface, forming a highly ordered molecular array. For micrometer-scale coding microspheres with diameters much larger than the DNA radius of gyration, the average extension height of the amplification products (… ) and grafting density ( The relationship between them approximately follows the Alexander-de Genner scaling theory, as shown in formula (23):

[0093] In formula (23), This represents the number of base pairs in the amplification product. The effective monomer length of a single nucleotide is approximately 0.34 nanometers. , where represents the surface grafting density of the deep needle. Equation (23) shows that the physical thickness of the surface signal layer can be precisely controlled by adjusting the probe density and amplification length. This vertically oriented conformation not only maximizes the exposure area of ​​the fluorescent group and enhances the detection sensitivity, but also provides a low-damping mass transfer channel for the polymerase, avoiding reaction stagnation caused by surface congestion; Steps S6-8 involve the kinetic suppression of product detachment and the stability of signal localization. During prolonged high-temperature amplification, non-covalently bound molecules are prone to thermal dissociation. To ensure absolute signal localization, this embodiment relies on the extremely high bond energy of covalent chemical bonds (greater than 300 kJ / mol) to counteract the dissociation forces generated by thermal motion. In contrast, any non-specifically adsorbed nucleic acid fragments on the microsphere surface (adsorbed solely by van der Waals forces or electrostatics, with bond energies less than 20 kJ / mol) will rapidly detach under washing or reaction conditions at 65°C. Therefore, the fluorescence signal ultimately retained on the microsphere surface originates only from the product specifically initiated and covalently extended. Product retention rate ( The change of ) over time follows a modified form of the first-order kinetic decay equation, as shown in equation (24):

[0094] In formula (24), Let be the thermal dissociation rate constant of the covalent bond at the reaction temperature. For the reaction and washing time. Equation (24) proves the robustness of the signal from a kinetic perspective. This extreme physical stability ensures that even during violent fluid scouring or long-term reaction processes, the microspheres carrying specific codes and the amplification signals carried on their surfaces always maintain a strict one-to-one correspondence, eliminating the risk of false positives caused by product diffusion in traditional liquid-phase amplification.

[0095] Based on the formation of solid-phase amplification products in the aforementioned embodiments, step S7: in-situ signal accumulation and discretization result decoding, regarding cascaded color development, aims to convert the amount of nucleic acid amplification at the microscale into a macroscopically detectable optical signal through a cascaded amplification mechanism, including the following steps: Step S7-1 involves the capture of primary signal molecules and the assembly of cascade complexes. To overcome the sensitivity limit of direct fluorescent labeling and support femtomolar detection limits, this step employs a biotin-streptavidin-phycoerythrin cascade amplification system. In the amplification process described in step S6, if biotinylated nucleotides are used instead of directly fluorescently labeled nucleotides, the double-stranded DNA product formed on the solid-phase surface carries a high density of biotin sites. In step S7-1, a streptavidin-phycoerythrin conjugate at a concentration of 2 to 5 micrograms per milliliter is added to the reaction system. Streptavidin, acting as a primary adapter, rapidly binds to the surface of the solid-phase amplification product due to its extremely high affinity for biotin. The binding process follows the kinetics described by the Langmuir adsorption isotherm, and its surface coverage (… Defined by formula (25):

[0096] In formula (25), In order to incorporate the equilibrium constant, This represents the bulk concentration of the coupling compound in the liquid phase. This is the spatial steric hindrance correction factor. The introduction of this technology clarifies the technical characteristics of steric hindrance regulation, demonstrating that by optimizing the hydrodynamic radius of the conjugate in a high-density amplification cluster, the macromolecule phycoerythrin can penetrate deep into the DNA brush layer, thereby maximizing the occupancy of signal sites.

[0097] Step S7-2 involves orthogonal spectral separation and high quantum yield signal enhancement. The phycoerythrin bound to the microsphere surface is a protein with an extremely high extinction coefficient (…). ) and a light-gathering complex of quantum yield. Unlike conventional organic dyes, PE has a broad excitation spectrum and a narrow emission spectrum, exhibiting a large effective Stokes shift. In the multiple detection system, this embodiment utilizes the principle of spectral orthogonality to strictly isolate the reporting signal from the encoded signal inside the microsphere in the wavelength domain. When excited by laser, the PE molecules on the surface of the microsphere emit strong fluorescence independently, without energy transfer (FRET) or crosstalk with the internal encoded dye. This ensures that the signals of identification and content quantification do not interfere with each other on the same microsphere, and their combined signal-to-noise ratio (SNR) is described by formula (26):

[0098] In formula (26), The fluorescence intensity of PE For system electronic and optical noise, Intensity. Through step S7-2, the physical separation of the detection signal and the encoded signal is achieved, which greatly reduces background noise.

[0099] Step S7-3 involves the discretization, decoding, and quantitative conversion of the optical signals. The microsphere array, after cascaded color development, is scanned one by one in a flow cytometer or fluorescence imaging system. The detection system simultaneously acquires the coded fluorescence signals of the microspheres. With the reported fluorescence signal ( The decoding logic module first determines the... Each microsphere is categorized into a specific pathogen detection channel (e.g., based on its coordinate position in a pre-defined two-dimensional color space) according to its location. or Subsequently, for each type of microsphere, the surface properties were statistically analyzed. Intensity. To eliminate signal fluctuations caused by differences in microsphere size, normalized fluorescence intensity is introduced as the final quantitative indicator. The calculation logic is shown in formula (27):

[0100] In formula (27), This represents the average non-specific adsorption fluorescence value of the blank control microspheres from the same batch. The effective scattering cross section of the microsphere, The calibration coefficient is the instrument response. Through the processing of formula (27), the continuous analog light signal is converted into discrete, comparable digital values. When the average NFI value of a certain type of microsphere exceeds the set positive threshold, the presence of the corresponding pathogen nucleic acid is determined. This processing flow realizes the conversion from analog light intensity to digital diagnostic results.

[0101] Step S7-4 involves the construction and dynamic calibration of the decoding criterion parameters. The discretized decoding logic is not based on fixed static values, but is generated in real time based on the statistical distribution characteristics of the negative control and internal standard microspheres in the current reaction batch. The system first uses the normal distribution quasi-Difference method to process the background fluorescence data of the negative microsphere population and extracts the background mean ( ) and background standard deviation ( Based on the statistical confidence interval theory, a specific microsphere is coded with a channel ( ), calculate the judgment threshold To ensure the specificity of the results and compensate for instrument fluctuations, the threshold calculation follows formula (28):

[0102] In formula (28), This is the confidence coefficient (values ​​from 3 to 5, corresponding to a confidence level of 99.7% or higher). The system drift correction factor is calculated using the following formula: ,in This represents the measured average fluorescence intensity of the current batch of internal standard microspheres. The factory calibration strength of the internal standard microspheres. Formula (28) can automatically correct baseline drift caused by optical system aging, temperature fluctuations, or minor optical path offsets; Step S7-5 involves the discretization and result output of the signal. The data processing module reads the output of each type of microsphere from step S7-3. The value is then compared with the threshold generated in step S7-4. The system performs multi-parameter logical operations to convert continuous values... The numerical value is converted into a discrete detection state. The mapping logic is shown in formula (29):

[0103] In formula (29), The coefficient of variation is the fluorescence intensity of similar microspheres. The upper limit of the dispersion tolerance is set at 15%. To determine the number of microspheres effectively collected, This is the minimum statistical sample size. Formula (29) introduces a multidimensional quality control mechanism: only when the signal strength is higher than the threshold and the signal distribution is uniform (low... A positive result is only determined when the sample size is sufficient. If the sample size is insufficient, an invalid status code is output, indicating that a retest is required. This mechanism effectively eliminates abnormal data caused by microsphere aggregation, bubble interference, or flow path blockage, ensuring the reliability of the final output results. Finally, the processed data is reconstructed into a structured diagnostic report, which clearly records the name of the pathogen (from...). Decoding and detection values The process involves determining the result (positive / negative / invalid) and the quality control status. This process completes the conversion from optical signal acquisition to clinical auxiliary diagnostic information.

[0104] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for detecting multidrug resistance in ticks based on LAMP technology, characterized in that, Includes the following steps: Step S1: Parallel fabrication of spatially encoded solid-phase carrier modules; using a flow-focusing microfluidic chip device, microsphere matrix units with a first average diameter are fabricated respectively. Microsphere matrix units with a second average diameter And microsphere matrix units with a third average diameter The microsphere matrix unit The microsphere matrix unit With the microsphere matrix unit During polymerization, chemically active anchoring groups are introduced through copolymerization. Step S2: Sequence design and synthesis of cis-tethered self-locking molecular recognition module; design and synthesize corresponding cis-tethered self-locking primers for tick multidrug resistance-related genes and internal reference genes respectively; The cis-tethered self-locking primers include a 5' amino-terminal modification, a locking domain, a branching migration region, and a toe domain. Step S3: Execution of solid-phase anchoring assembly and conformation locking procedure; the cis-tethered self-locking primer is modified with the 5'-terminal amino group and attached to the microsphere matrix unit. The microsphere matrix unit Or the chemically active anchoring groups on the surface of the microsphere matrix unit M3 undergo covalent coupling; after the coupling reaction is completed, an annealing locking procedure is performed, which causes the cis-tethered self-locking primer to fold spontaneously using the intramolecular proximity effect, forcing the locking domain to pair complementaryly with the amplification initiation domain to form a stem-loop structure, physically blocking the 3' extension site of the amplification initiation domain. Step S4: Release of nucleic acid from tick samples; The tick samples to be tested are mechanically ground and heat-treated in sodium hydroxide alkaline lysis buffer, and the pH value is adjusted using neutralization buffer to obtain a biological sample containing the target nucleic acid to be tested. Step S5: Construction of a heterogeneous mixed reaction system; mixing the microsphere matrix units loaded with the cis-tethered self-locking primers in a single reaction vessel. The microsphere matrix unit and the microsphere matrix unit Add a liquid phase reagent containing Bst2.0 WarmStart DNA polymerase, heme chloride, and a chromogenic substrate, as well as the biological sample prepared in step S4; Step S6: Thermodynamically controlled amplification and in situ signal transduction; the heterogeneous mixed reaction system is incubated in a constant temperature environment; if the biological sample contains mutant target nucleic acid, an interface restriction loop-mediated isothermal amplification reaction is triggered, and the generated amplification product combines with the heme chloride to form G4-DNAzyme, catalyzing the chromogenic substrate to generate a colored product; Step S7: Decoding and interpreting the discretization results; observing the microsphere matrix units that have settled at the bottom of the reaction vessel. The microsphere matrix unit With the microsphere matrix unit The color and particle size characteristics are used to determine the results based on the two-dimensional coding rules for particle size and color.

2. The method for detecting multidrug resistance in ticks based on LAMP technology according to claim 1, characterized in that, The specific methods of parallel manufacturing in step S1 include: By independently adjusting the ratio parameter of the dispersed phase flow rate to the continuous phase flow rate of the flow-focusing microfluidic chip device, the microsphere matrix units are controlled respectively. The microsphere matrix unit and the microsphere matrix unit The particle size is used to achieve spatial coding based on physical dimensions; The prepared microsphere matrix unit The microsphere matrix unit and the microsphere matrix unit It is stored in anhydrous ethanol or isopropanol solvent to maintain the reactivity of the chemically active anchoring groups.

3. The method for detecting multidrug resistance in ticks based on LAMP technology according to claim 1, characterized in that, The structural features of the cis-tethered self-locking primer in step S2 include: modification of the locking domain of the cis-tethered self-locking primer by incorporating a locked nucleic acid monomer; The specific site for recognizing single nucleotide polymorphisms is configured in the branch migration region of the cis-tethered self-locking primer, and the specific site spatially corresponds to the locked nucleic acid modification position of the locking domain, thereby constructing a thermodynamic energy barrier for wild-type target nucleic acids.

4. The method for detecting multidrug resistance in ticks based on LAMP technology according to claim 1, characterized in that, The specific execution logic of the annealing locking procedure in S3 includes: The system covalently coupled with the cis-tethered self-locking primers was heated to 95 degrees Celsius and held for a preset time, and then slowly cooled to 25 degrees Celsius at a rate of 0.1 degrees Celsius per second. The slow cooling process forces the locking domain to pair complementaryly with the amplification initiation domain, forming the thermodynamically stable stem-loop structure.

5. The method for detecting multidrug resistance in ticks based on LAMP technology according to claim 1, characterized in that, The release process of the nucleic acid from the tick sample in step S4 includes: The mechanical grinding process disrupts the tick's cuticle and cellular structure; the heat at 95 degrees Celsius promotes protein denaturation and the release of genomic DNA. After the neutralization buffer adjusts the pH of the lysis products to neutral, the unpurified crude lysis buffer is directly used as the biological sample in subsequent reactions.

6. The method for detecting multidrug resistance in ticks based on LAMP technology according to claim 5, characterized in that, The concentration of the sodium hydroxide alkaline lysis solution in step S4 is 20 mmol / L to 50 mmol / L.

7. The method for detecting multidrug resistance in ticks based on LAMP technology according to claim 1, characterized in that, The configuration requirements for the heterogeneous mixed reaction system in step S5 include: The single reaction vessel simultaneously contains 50 to 100 of the microsphere matrix units. 50 to 100 of the aforementioned microsphere matrix units and 50 to 100 of the aforementioned microsphere matrix units ; The volume of the biological sample added accounts for 5% to 20% of the total volume of the heterogeneous mixed reaction system.

8. The method for detecting multidrug resistance in ticks based on LAMP technology according to claim 1, characterized in that, The thermodynamically controlled amplification mechanism in step S6 is as follows: When the mutant target nucleic acid is present in the biological sample, the mutant target nucleic acid opens the stem-loop structure of the cis-tethered self-locking primer through a toe-point mediated strand displacement reaction; When only wild-type target nucleic acid is present in the biological sample, the wild-type target nucleic acid has a base mismatch with the branch migration region, which cannot overcome the energy barrier caused by the modification of the locked nucleic acid monomer, and the chain substitution reaction is blocked, so the cis-tethered self-locking primer remains locked.

9. The method for detecting multidrug resistance in ticks based on LAMP technology according to claim 1, characterized in that, The mechanism of in-situ signal transduction in step S6 includes: After the interface-restricted loop-mediated isothermal amplification reaction is initiated, the amplification product forms a long-chain DNA containing G-quadruplexes inside the corresponding microsphere matrix unit. The heme chloride in the long-chain DNA capture reaction system is assembled into the G4-DNAzyme; The G4-DNAzyme catalyzes the redox reaction of the chromogenic substrate within the pores of the microsphere matrix unit, causing the corresponding microsphere matrix unit to exhibit a specific color.

10. The method for detecting multidrug resistance in ticks based on LAMP technology according to claim 9, characterized in that, The two-dimensional encoding rules for particle size and color in step S7 include: The microsphere matrix unit The green indicator shows that the detection system is effective. The microsphere matrix unit A green color indicates that the biological sample has pyrethroid resistance. The microsphere matrix unit A green color indicates that the biological sample has organophosphate resistance; The microsphere matrix unit The microsphere matrix unit and the microsphere matrix unit All samples remain colorless and transparent, indicating that the biological samples are sensitive to the corresponding drugs.

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