A rapid detection method for drug resistance of a vector organism

Abstract

The application discloses a kind of vector biological resistance rapid detection method.In the application, by mutant sequence hybridization recognition and step S4 acid environment synergistic trigger nanocage door opening, phenotype esterase activity and genotype mutation information are locked in the same color event.This double-locked linkage mechanism avoids the signal distortion caused by non-target esterase interference or metabolic fluctuations when single indicator detection is carried out, and the initiation of color reaction is strictly limited under the premise that the target exists, which fundamentally eliminates the basis for false positive and false negative occurrence.The method improves the accuracy and persuasiveness of the detection result, and reduces the misjudgment rate caused by sample individual difference or complex matrix.The closed-loop reaction formed completely eliminates the nucleic acid amplification and electrophoresis link.This not only saves the total detection time, but also saves the thermal cycling equipment and gel electrophoresis device, and the operation can be completed without supporting molecular biology laboratory in primary site.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically a rapid detection method for drug resistance in disease vectors. Background Technology

[0002] Disease vectors such as mosquitoes, flies, and cockroaches are the transmission agents for many infectious diseases, including malaria, dengue fever, and Japanese encephalitis. For a long time, chemical pesticides have been the primary means of controlling vector populations and blocking the spread of vector-borne infectious diseases. However, with the large-scale and continuous use of pesticides, the problem of pesticide resistance in disease vectors has become increasingly prominent, posing a major challenge to global public health control. Timely and accurate monitoring of pesticide resistance levels in disease vectors is crucial for guiding the scientific selection and rational rotation of pesticides and delaying the further development of resistance.

[0003] Currently, the detection of drug resistance in disease vectors is mainly divided into three categories of methods. The first category is the traditional bioassay method, including the adult mosquito contact tube method, larval immersion method, and spot method recommended by the World Health Organization.

[0004] The second category is biochemical detection methods, which mainly reflect the level of resistance indirectly by measuring the activity of enzymes related to drug resistance in the vector organism. Among them, esterase activity detection is a widely used method. Its principle is to use colorimetry to measure the activity of non-specific esterases or acetylcholinesterases in the sample. The higher the enzyme activity, the stronger the ability to metabolize organophosphates or pyrethroid insecticides, and the higher the level of resistance.

[0005] The third category is molecular detection methods, which primarily determine resistance by detecting mutation sites in genes related to drug resistance in vector-borne organisms. Several polymerase chain reaction-based molecular detection techniques have been reported and applied, such as PCR-restriction fragment length polymorphism (PCR), TaqMan probe assays, and multiplex SNaPshot assays. These methods directly read resistance information at the gene level and exhibit high sequence specificity.

[0006] However, no existing technology can integrate phenotypic enzyme activity information and genotypic mutation information into the same detection system, achieving mutual verification and coupled judgment between the two. When detecting a single indicator, the problem of misjudgment caused by metabolic fluctuations or non-target interference has not yet been effectively resolved. Summary of the Invention

[0007] The purpose of this invention is to provide a rapid detection method for drug resistance in vector-borne organisms in order to solve the problems mentioned above.

[0008] The technical solution adopted in this invention is as follows: A rapid detection method for drug resistance in disease vectors, characterized by comprising the following steps: S1: The whole vector organism or the midgut / head tissue after dissection is placed in lysis buffer, mechanically homogenized, and briefly centrifuged at low speed. The supernatant turbid suspension is used as the detection stock solution. This stock solution does not undergo nucleic acid extraction or protein purification, directly retaining all endogenous esterases and genomic DNA, serving as the common source of material for subsequent dual recognition.

[0009] S2: Mix the quantitative detection stock solution with magnetic microspheres immobilized with esterase substrate analog ligands. Incubate by rotation at room temperature to allow the microspheres to capture the active esterase in the suspension. After incubation, place the reaction tube on a magnetic rack. Once the microspheres have completely adhered to the wall, slowly aspirate the supernatant. This supernatant should not be discarded and must be retained separately. Wash the magnetic beads twice with hypotonic buffer to remove physically adsorbed proteins. Finally, resuspend the washed magnetic bead complex in a small amount of phosphate buffer, and add the previously retained supernatant back into the resuspended solution at a volume ratio to ensure that the reaction system contains both the magnetically bound esterase and free sample DNA.

[0010] S3: Add the lyophilized and reconstituted DNA nanocage working solution to the above resuspension. The nanocage is pre-encapsulated with a high concentration of horseradish peroxidase, and its outer chain consists of two oligonucleotides, whose sequences are complementary to the upstream and downstream regions of the resistance mutation site, respectively. Then, quickly add the esterase-specific substrate solution, mix well, and transfer the entire solution to a clear microplate. At this point, if the sample DNA contains a resistance mutation sequence, it will progressively hybridize with the nanocage chain, but the threshold for opening the cage gate has not yet been reached.

[0011] S4: The microplate was placed in a thermostatic shaker and reacted in the dark at the set temperature. Simultaneously, an equal volume of a mixture of o-phenylenediamine and hydrogen peroxide was injected into each well as a start signal. After the reaction began, the esterase on the magnetic beads continuously hydrolyzed the substrate, releasing organic acids, causing a slow decrease in the pH of the local microenvironment within the pores. This acidic condition synergistically with the chain hybridization that occurred in S3, jointly inducing an allosteric change in the nanocage structure, fully opening the cage doors and releasing horseradish peroxidase. The free horseradish peroxidase then catalyzed the oxidation of o-phenylenediamine with hydrogen peroxide to produce an orange-yellow quinone compound. The color formation rate was positively correlated with the esterase acid production rate and the degree of mutagenic hybridization; a decrease in either significantly reduced the final color development slope.

[0012] S5: Starting from the injection of the chromogenic solution, at the end of the preset time window, use a microplate reader to read the absorbance value of each well at a wavelength of 450 nm. Subtract the background from the reading of the parallel control wells lacking only the nanocage to obtain the net absorbance difference. Substitute this difference into the absorbance-resistance index standard curve obtained in advance from multiple known resistance grades of strains, and calculate the resistance index value of the sample through linear interpolation.

[0013] S6: Compare the calculated resistance index with the critical threshold for distinguishing between sensitive and resistant individuals. If the index is greater than or equal to the threshold, the individual is classified as resistant; if it is lower than the threshold, the individual is classified as sensitive. The determination result is output immediately after the plate is read. The entire process does not involve any primer extension or thermal cycling, and all reactions are completed within the closed wells, eliminating the need to open the lids to transfer products and thus avoiding the risk of aerosol contamination from nucleic acid amplification.

[0014] In a preferred embodiment, in step S1, 500 μL of phosphate buffer is added to a single adult vector insect homogenizer. The buffer is adjusted to pH 7.4 and contains 0.01% Triton X-100 to aid cell disruption. The homogenate is manually ground at 800 rpm for 3 minutes under ice bath conditions until homogenized. The homogenate is transferred to a 1.5 mL centrifuge tube and centrifuged at 3000 × g for 5 minutes at 4°C to remove unbroken tissue fragments and cuticle precipitate. The supernatant milky white suspension is carefully aspirated as the detection stock solution. The pH of the stock solution is measured using a pH meter. If it deviates from pH 7.0–7.6, a small amount of dilute hydrochloric acid or dilute sodium hydroxide is added to adjust it back to ensure that the subsequent esterase capture and nucleic acid hybridization reactions are in the optimal acid-base environment. The detection stock solution must be used within 30 minutes. If a delay is required, it should be temporarily stored on ice for no more than 2 hours to prevent endogenous proteases from degrading the target esterase or nuclease from degrading free DNA.

[0015] In a preferred embodiment, in step S2, 100 μL of the detection stock solution is mixed with 20 μL of a magnetic microsphere suspension containing a surface-coupled esterase substrate analog ligand. The microspheres have a diameter of 200 nm and a concentration of approximately 10 mg / mL. The mixture is incubated at 400 rpm for 15 min in a 25°C constant-temperature shaker to allow the microspheres to fully capture the active esterase in the suspension. After incubation, the centrifuge tube is placed on a magnetic rack and allowed to stand for 2 min until the microspheres are completely adhered to the wall. The supernatant is then slowly aspirated with a pipette and transferred to a new centrifuge tube for safekeeping. This supernatant contains free genomic DNA released from the sample, which serves as the basis for subsequent gene recognition. The adherent magnetic beads are resuspended twice in 200 μL of washing buffer containing 0.05% Tween-20, with shaking for 1 min each time, followed by magnetic separation to discard the washing buffer, in order to remove physically adsorbed contaminants and unbound enzymes.

[0016] In a preferred embodiment, in step S2, after washing, the magnetic bead complex is resuspended in 80 μL of phosphate buffer, and the previously stored 20 μL of supernatant is added back to the suspension to restore the final volume to 100 μL. At this point, the reaction system contains both the esterase captured by the magnetic beads and the free DNA of the sample, and both originate from the same single individual, ensuring a natural pairing of phenotypic and genotypic information.

[0017] In a preferred embodiment, in step S3, the preparation of the DNA nanocage requires the pre-design of three or more oligonucleotide chains in a Mg²⁺-containing environment. + Horseradish peroxidase (HRP) is self-assembled into a three-dimensional cage structure via programmed cooling annealing in a buffer solution. During assembly, three oligonucleotides are first hybridized to form a three-way branched double-stranded DNA, followed by three-dimensional self-assembly to ensure all complementary strands at the ends pair and close. The cage's edges are formed by 12 double-stranded B-DNA helices, and short thymine linkers form its corners. For encapsulation, the enzyme and pre-assembled half-cages are mixed in a 1:1 stoichiometric ratio and incubated. A bridging strand is then added to connect the half-cages into a complete cage. The outer locking chain consists of two oligonucleotides, their sequences complementary to the upstream and downstream regions of the resistance mutation site, respectively. Both ends of the chain have 8 bp sticky ends for connecting the cage. After assembly, unencapsulated enzyme and excess DNA strands are removed by agarose gel electrophoresis. The resulting nanocage has an internal cavity size of approximately 20 nm × 20 nm × 17 nm, sufficient to accommodate horseradish peroxidase molecules with a diameter of approximately 5 nm.

[0018] In a preferred embodiment, during step S3, the magnetic bead complex resuspension obtained in S2 is mixed with the DNA nanocage working solution at a volume ratio of 1:1 to control the final concentration of the nanocages at 100–200 nmol / L. Esterase substrate solution is added to a final concentration of 1–5 mmol / L, and after mixing, the mixture is transferred to a clear 96-well microplate, with 100 μL added to each well. The mixture is incubated at room temperature (25–30°C) for 5–10 min to allow progressive hybridization of the resistant mutant sequence in the sample DNA with the nanocage chains. At this stage, the hybridization has not yet reached the threshold for cage opening and requires subsequent acidic conditions to trigger it.

[0019] In a preferred embodiment, in step S4, when the colorimetric reaction is initiated, an equal volume of a mixed colorimetric solution of o-phenylenediamine and hydrogen peroxide is injected into each well, resulting in a final concentration of o-phenylenediamine of 0.5–1.0 mmol / L and a final concentration of hydrogen peroxide of 1–5 mmol / L. The microplate is placed in a constant-temperature shaker at 37°C to prevent light exposure, and the shaking speed is controlled at 300–500 r / min to ensure uniform mass transfer. After the reaction begins, the esterase on the magnetic beads continuously hydrolyzes the substrate, releasing organic acids, causing the local pH in the wells to gradually decrease from the initial pH 7.4 to the pH 6.0–6.5 range. This acidic environment, combined with the already occurred chain hybridization, creates a synergistic effect, jointly inducing an allosteric transition in the nanocage conformation. Once the cage doors are fully opened, the internally encapsulated horseradish peroxidase is released. The released horseradish peroxidase then catalyzes the oxidation of o-phenylenediamine with hydrogen peroxide to generate an orange-yellow quinone compound. The rate of color formation is positively correlated with the rate of acid production by the esterase and the degree of mutagenic hybridization.

[0020] In a preferred embodiment, in step S4, signal reading begins from the injection of the chromogenic solution. Within a 15–30 min time window after reaction initiation, the absorbance value of each well at 450 nm is read using a microplate reader. Background interference is subtracted from the readings of the parallel control wells lacking only the nanocages to obtain the net absorbance difference. If dynamic monitoring of the reaction process is required, readings can be taken every 2–3 min between 15 and 30 min, and the slope value of the linear interval is substituted into the standard curve. The entire chromogenic reaction is completed within the sealed wells; there is no need to open the caps to transfer the product. Subsequent determinations can be made based on the absorbance values ​​after the readings are completed.

[0021] In a preferred embodiment, in step S5, timing begins from the injection of the chromogenic solution. After 20 minutes of reaction, the microplate is placed in a microplate reader for dual-wavelength absorbance measurement using 450 nm as the dominant wavelength and 650 nm as the reference wavelength. The reference wavelength is used to correct for non-specific light scattering caused by the liquid meniscus and the plate material. Each well is read three times and the average value is taken. A control well containing only equal amounts of magnetic beads and nanocages but without esterase substrate is used as a background blank. The net absorbance difference is obtained by subtracting the background blank value from the absorbance values ​​of each sample well. This net difference is substituted into a pre-constructed resistance index standard curve. This standard curve is obtained by mixing the supernatant of homogenates from at least five concentration gradients of resistant strains and an equal amount of supernatant from homogenates of sensitive strains, and then measuring the results using the same procedure. The linear relationship between the absorbance difference and the resistance index is fitted using the least squares method, and the coefficient of determination must be higher than 0.98 for it to be usable. The calculated resistance index is dimensionless, and its value directly reflects the combined level of esterase activity intensity and mutant sequence abundance in the sample.

[0022] In a preferred embodiment, in step S6, before judgment, it is necessary to confirm that the linear range of the standard curve covers the net absorbance value of the sample to be tested. If it exceeds the range, the sample needs to be diluted with buffer and the S1 to S5 procedures repeated. The calculated resistance index is compared with a critical threshold of 1.00. The critical threshold is set based on the mean resistance index of a large number of sensitive strains plus three times the standard deviation. This threshold has been verified through field population pre-experiments and fixed in this method. An index greater than or equal to 1.00 is judged as a resistant individual, and an index less than 1.00 is judged as a sensitive individual. The judgment results are recorded in the well plate layout table and labeled with the corresponding individual number. The entire detection process, from homogenization to result reading, takes less than 45 minutes. The microplates and magnetic racks used are portable devices, suitable for rapid on-site screening at field sites or grassroots quarantine stations, without the need to return to the laboratory for subsequent confirmation.

[0023] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. In this invention, the mutation sequence hybridization identification in step S3 and the acidic environment in step S4 synergistically trigger the opening of the nanocage, locking phenotypic esterase activity and genotype mutation information into the same chromogenic event. This dual-locking linkage mechanism avoids signal distortion caused by non-target esterase interference or metabolic fluctuations during single-indicator detection. The initiation of the chromogenic reaction is strictly limited to the presence of the target, fundamentally eliminating the basis for false positives and false negatives. This method improves the accuracy and persuasiveness of the detection results and reduces the misjudgment rate caused by individual sample differences or matrix complexity.

[0024] 2. In this invention, the closed-loop reaction completely eliminates the nucleic acid amplification and electrophoresis steps. This not only saves the total detection time but also eliminates the need for thermal cycling equipment and gel electrophoresis devices, allowing the operation to be completed on-site without the need for a dedicated molecular biology laboratory. Simultaneously, the colorimetric reaction is carried out within the sealed wells without the need for opening the cap for transfer, reducing the risk of aerosol cross-contamination during operation and lowering the requirements for professional training of operators. This method improves detection efficiency and portability, while reducing hardware dependence and the risk of contamination. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the process principle of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Reference Figure 1 , Example 1: Validating the accuracy and repeatability of a method using known resistant and susceptible strains: Twenty Culex pipiens pallens resistant strains and 20 susceptible strains, both domesticated in the laboratory over a long period, were selected, with each strain serving as a separate test sample. The resistant strains underwent 30 consecutive generations of deltamethrin screening, exhibiting a resistance multiple of 45.6 times; the susceptible strains had never been exposed to any insecticides.

[0028] S1 Procedure: Place a single adult mosquito in a pre-cooled glass homogenizer, add 500 μL of pH 7.4 phosphate buffer containing 0.01% Triton X-100, and manually homogenize at 800 r / min for 3 min under ice bath conditions until homogenized. Transfer the homogenate to a 1.5 mL centrifuge tube, centrifuge at 3000×g for 5 min at 4 °C to remove debris, and use the supernatant as the test solution. Measure and adjust the pH to the range of 7.0–7.6.

[0029] S2 Procedure: Mix 100 μL of the test stock solution with 20 μL of magnetic bead suspension (magnetic bead diameter 200 nm, concentration 10 mg / mL). Incubate at 25 °C and 400 r / min for 15 min with shaking. After incubation, perform magnetic separation for 2 min, collect the supernatant, and wash the magnetic beads twice with 200 μL of washing buffer, shaking for 1 min each time, followed by magnetic separation and discarding the solution. After washing, resuspend the magnetic beads in 80 μL of phosphate buffer and add 20 μL of supernatant to bring the final volume to 100 μL.

[0030] S3 procedure: Add 50 μL of 100–200 nmol / L DNA nanocage working solution to the resuspension, add esterase substrate to a final concentration of 2 mmol / L, mix well, transfer to a 96-well microplate, 100 μL per well, and incubate at 25 °C for 8 min.

[0031] S4 procedure: Inject an equal volume of o-phenylenediamine and hydrogen peroxide mixture into each well to achieve final concentrations of 0.8 mmol / L and 3 mmol / L, respectively, and react at 37 °C and 400 r / min in the dark for 20 min.

[0032] S5 Procedure: Measure absorbance at a wavelength of 450 nm, using 650 nm as the reference wavelength. Take three readings per well and average the results. Subtract the background blank to obtain the net absorbance difference. The standard curve is fitted by measuring the absorbance of a mixture of five strains with resistance indices of 0.5, 1.0, 2.0, 3.0, and 4.0. The linear equation is A = 0.285 × RI + 0.020, with a coefficient of determination R² = 0.973. Substitute the net absorbance of the samples into this equation to calculate the resistance index.

[0033] S6 operation: Resistance index ≥1.00 is judged as resistant, <1.00 is judged as sensitive.

[0034] The test results are as follows:

[0035] The resistance index of resistant strains ranged from 2.05 to 2.63, while that of sensitive strains ranged from 0.38 to 0.66. The two sets of data were completely separated, and the decision threshold of 1.00 was precisely in the middle of the two distribution ranges. The standard deviation of the resistant strains was 0.15, and that of the sensitive strains was 0.08. The difference in standard deviation stemmed from the different scales of the absolute values ​​of the signals rather than differences in repeatability—the resistant strains had a higher signal-to-noise ratio when the mean absorbance was 0.687, while the photon count fluctuations of the sensitive strains were relatively amplified when the signal was close to the detection lower limit. The coefficients of variation for both strains were within the acceptable range for similar colorimetric methods. These results indicate that this method can accurately distinguish between known resistant and sensitive strains.

[0036] Example 2: Simultaneous detection of resistance levels using field-collected samples and comparison with traditional bioassay methods: Fifty adult houseflies were randomly captured in an agricultural pest infestation area. Each fly was individually placed in a centrifuge tube, labeled, and transported to the on-site laboratory in an ice bath. Each sample underwent both this method and traditional bioassay using a membrane-based method. For the membrane-based method, filter paper was treated with a 5% deltamethrin acetone solution. After the acetone evaporated, the filter paper was placed in the filter paper for 1 hour of contact. The fly was then transferred to a clean container with sugar water. Mortality was assessed after 24 hours; a mortality rate below 80% was considered indicative of resistance.

[0037] The procedure is the same as in Example 1, except that in S1, a whole housefly homogenate was used, and in S4, the colorimetric reaction time was adjusted to 25 min to accommodate the low esterase expression level of this species. All other parameters remained unchanged. The standard curve was refitted using the housefly homogenate matrix. The curve equation used in this experiment was A = 0.302 × RI + 0.015, with a coefficient of determination R² = 0.968. The detection results are shown in the table below:

[0038] The test results are as follows: The resistance rate detected in 50 field samples was 32%, close to the historical resistance monitoring data of 31% in the local area. The highest index of 0.96 in the sensitive group and the lowest index of 1.03 in the resistant group were located on either side of the judgment threshold of 1.00 with a very small gap, indicating that some of the samples collected this time are in the transitional stage of resistance succession. This continuous distribution characteristic is consistent with the true genetic structure of the wild population. Eight samples with an index in the range of 0.85–1.15 were selected from the 50 samples for three parallel replicate determinations. The coefficient of variation of the resistance index was 4.2%–7.1%, with good intra-batch repeatability. Four samples had a mortality rate of 76%–78% in the drug film method. The drug film method was initially determined to be resistant. This method provided a resistance determination consistent with subsequent retests, avoiding the time cost of repeated determinations near the critical value in traditional methods.

[0039] The total time required for this method is less than 45 minutes. It does not require feeding the insects to the next generation or waiting 24 hours for mortality statistics, while the drug film method requires at least 28 hours from preparation to final result reading. The results of the two methods are completely consistent.

[0040] Example 3: Using artificially constructed interference samples to verify methods against non-target interference: Three sets of artificial interference samples were designed, with 10 replicates in each set, all using the supernatant of homogenate from the sensitive strain *Culex pipiens pallens* as the base matrix. The first set consisted of a pure homogenate from the sensitive strain without any added interfering substances. The second set contained porcine pancreatic lipase at a final concentration of 2 U / mL added to the homogenate to simulate interference from non-target esterase activity. The third set contained acetic acid at a final concentration of 5 mmol / L added to the homogenate to simulate interference from an acidic environment not derived from esterases. All three sets of samples were simultaneously tested using this method and a traditional single-index colorimetric method measuring only esterase activity.

[0041] The single-index colorimetric method uses the same o-phenylenediamine and hydrogen peroxide colorimetric system as S4, but omits the nanocage step. An equal volume of colorimetric solution is added directly to the supernatant of the sensitive strain homogenate, and the absorbance at 450 nm is read under the same reaction conditions. The positive threshold is set at 0.55, which is the mean signal of the pure sensitive strain using the single-index method (0.34) plus three times the standard deviation (0.07), rounded down, representing the upper limit of the signal in normal sensitive individuals. The standard curve for this method is the same as in Example 1. All three groups of samples are substituted into A = 0.285 × RI + 0.020 to calculate the resistance index. The net absorbance of the pure sensitive group (0.168) corresponds to an index of 0.52, the net absorbance of the porcine pancreatic lipase group (0.182) corresponds to an index of 0.57, and the net absorbance of the acetic acid group (0.177) corresponds to an index of 0.55. All three are below 1.00.

[0042] The test results are shown in the table below:

[0043] After adding porcine pancreatic lipase, the resistance index of this method only slightly increased from 0.52 to 0.57, still far below the critical threshold of 1.00. This is because porcine pancreatic lipase has a low affinity for the esterase substrate of this method, and the proton flux produced is insufficient to open the cage door to the release threshold. This phenomenon precisely reflects the nonlinear characteristics of the nanocage acidity response curve—slow acid production cannot accumulate to below pH 6.0 in a short time, and the cage door remains closed. After adding acetic acid, the index of this method was 0.55. Although an acidic environment was achieved, the nanocage chains did not hybridize with the resistant mutant DNA, and the single condition did not meet the opening requirement, so the signal did not increase. In contrast, the single-index colorimetric method showed that the signal value of the porcine pancreatic lipase group rose to 0.87, exceeding the 0.55 threshold and causing false positives. This is because the single-index method directly measures the total esterase activity and cannot distinguish the contributions of target esterases and non-target esterases. The signal value of the acetic acid group surged to 1.92. The mechanism is that a large number of protons directly changed the pH environment of the colorimetric system. The orange-yellow quinone product generated by the oxidation of o-phenylenediamine by peroxide showed a significantly increased molar extinction coefficient under acidic conditions, meaning that the absorbance reading corresponding to the same product concentration was higher. The single-index method is completely ineffective against this type of non-enzymatic acid-base interference. The above results fully demonstrate that the dual synergistic gating mechanism of the nanocage in this method can effectively shield against the dual interference of non-target esterases and acid-base changes from non-enzymatic sources.

[0044] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the term "comprising" or any other variations thereof is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0045] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rapid detection method for drug resistance in disease vectors, characterized in that: Includes the following steps: S1: The whole or partial tissue of a single disease vector is placed in the lysis buffer for mechanical homogenization, followed by brief low-speed centrifugation to remove fragments, and the supernatant turbid suspension is used as the test solution. S2: Mix the quantitative detection stock solution with magnetic microspheres with esterase substrate analog ligands immobilized on the surface, and incubate at room temperature by rotation to allow the microspheres to capture the active esterase in the suspension; after incubation, magnetically separate and carefully aspirate the supernatant, but the supernatant needs to be retained separately. Wash the magnetic beads twice with hypotonic buffer to remove impurities and proteins. Resuspend the washed magnetic bead complex in a small amount of phosphate buffer and add back the retained supernatant so that the system contains both magnetically bound esterase and free sample DNA. S3: Add the lyophilized and reconstituted DNA nanocage working solution to the resuspension. The nanocage is pre-encapsulated with horseradish peroxidase inside, and the outer lock sequence is complementary to the upstream and downstream regions of the resistance mutation site. After adding the esterase-specific substrate solution, transfer it into a microplate. S4: Place the microplate in a constant temperature shaker to avoid light and inject a mixed colorimetric solution of o-phenylenediamine and hydrogen peroxide to initiate the reaction; the esterase hydrolyzes the substrate to release organic acids, causing a decrease in local pH. This acidic condition, in conjunction with chain hybridization, induces an allosteric transformation of the nanocage, fully opening the cage doors to release horseradish peroxidase, which catalyzes the generation of an orange-yellow product. S5: Start timing from the injection of the chromogenic solution, and read the absorbance at 450nm using an ELISA reader at the preset time endpoint. Subtract the background from the control wells lacking nanocages to obtain the net difference value. Substitute the net difference value into the pre-established absorbance-resistance index standard curve, and calculate the resistance index value of the sample through linear interpolation. S6: Compare the resistance index with the critical thresholds for susceptibility and resistance. Individuals with a resistance index greater than or equal to the threshold are classified as resistant, while those with a resistance index less than the threshold are classified as susceptible. The results are output immediately.

2. The rapid detection method for drug resistance in vector-borne organisms according to claim 1, characterized in that: In step S1, 500 μL of phosphate buffer is added to a single adult vector insect homogenizer. The pH of the buffer is adjusted to 7.4 and contains 0.01% Triton X-100 to aid cell disruption. The homogenate is manually ground at 800 r / min for 3 min under ice bath conditions until homogenized. The homogenate is transferred to a 1.5 mL centrifuge tube and centrifuged at 3000 × g for 5 min at 4°C to remove unbroken tissue fragments and cuticle precipitate. The upper milky white suspension is carefully aspirated as the test stock solution.

3. The rapid detection method for drug resistance in vector-borne organisms according to claim 1, characterized in that: In step S2, 100 μL of the detection stock solution is mixed with 20 μL of a magnetic microsphere suspension with a surface-coupled esterase substrate analog ligand. The microspheres have a diameter of 200 nm and a concentration of approximately 10 mg / mL. The mixture is then incubated in a 25°C constant temperature shaker at 400 r / min for 15 min to allow the microspheres to fully capture the active esterase in the suspension.

4. The rapid detection method for drug resistance in disease vectors according to claim 1, characterized in that: In step S2, after washing, the magnetic bead complex is resuspended in 80 μL of phosphate buffer, and the previously stored 20 μL of supernatant is added back to the suspension to restore the final volume to 100 μL. At this point, the reaction system contains both the esterase captured by the magnetic beads and the free DNA of the sample, and both originate from the same single individual.

5. The rapid detection method for drug resistance in vector-borne organisms according to claim 1, characterized in that: In step S3, the preparation of DNA nanocages requires the pre-design of at least three oligonucleotide chains in a Mg²⁺-containing environment. + In the buffer solution, the three-dimensional cage structure is formed by self-assembly through programmed cooling and annealing; during assembly, three oligonucleotides are first assembled in two dimensions through hybridization to form a three-way branched double-stranded DNA, and then the complementary strands at the ends are completely paired and closed through three-dimensional self-assembly. The cage is formed by 12 double-stranded B-DNA helices forming the edge and short-stranded thymine linkers forming the cage corners. When encapsulating horseradish peroxidase, the enzyme is mixed with the pre-assembled half-cage in a 1:1 stoichiometric ratio and incubated. Then, bridging strands are added to connect the half-cages into a complete cage. The external lock is designed as two oligonucleotides, whose sequences are complementary to the upstream and downstream regions of the resistance mutation site, respectively. The lock has 8 bp sticky ends at both ends for connecting the cage. After assembly, unencapsulated enzymes and excess DNA strands are removed by agarose gel electrophoresis.

6. The rapid detection method for drug resistance in vector-borne organisms according to claim 1, characterized in that: In step S3, during the reaction operation, the magnetic bead complex resuspension obtained in S2 is mixed with the DNA nanocage working solution at a volume ratio of 1:1 to control the final concentration of the nanocage at 100–200 nmol / L; esterase substrate solution is added to a final concentration of 1–5 mmol / L, and after mixing, the whole mixture is transferred to a transparent 96-well microplate, with 100 μL added to each well.

7. The rapid detection method for drug resistance in disease vectors according to claim 1, characterized in that: In step S4, when the colorimetric reaction is started, an equal volume of a mixed colorimetric solution of o-phenylenediamine and hydrogen peroxide is injected into each well, so that the final concentration of o-phenylenediamine is 0.5–1.0 mmol / L and the final concentration of hydrogen peroxide is 1–5 mmol / L; the microplate is placed in a constant temperature shaker at 37°C to avoid light, and the shaking speed is controlled at 300–500 r / min to ensure uniform mass transfer.

8. The rapid detection method for drug resistance in vector-borne organisms according to claim 1, characterized in that: In step S4, signal reading begins from the injection of the chromogenic solution. Within a time window of 15–30 minutes after the reaction starts, the absorbance value of each well at a wavelength of 450 nm is read using an ELISA reader. The background interference is subtracted from the readings of the parallel control wells lacking only the nanocages to obtain the net absorbance difference. If dynamic monitoring of the reaction process is required, readings can be taken every 2–3 minutes between 15 and 30 minutes, and the slope value of the linear interval is substituted into the standard curve. The entire chromogenic reaction is completed in the closed wells without opening the caps to transfer the product. After reading, subsequent judgments can be made based on the absorbance values.

9. The rapid detection method for drug resistance in vector-borne organisms according to claim 1, characterized in that: In step S5, timing begins from the injection of the chromogenic solution. When the reaction reaches 20 minutes, the microplate is placed in an ELISA reader for dual-wavelength absorbance measurement using 450 nm as the main wavelength and 650 nm as the reference wavelength. The reference wavelength is used to correct for non-specific light scattering caused by the liquid meniscus and the plate material. Each well is read three times and the average value is taken. The control well containing only an equal amount of magnetic beads and nanocages but without esterase substrate is used as the background blank. The net absorbance difference is obtained by subtracting the background blank value from the absorbance value of each sample well. This net difference is substituted into the pre-constructed antibiotic resistance index standard curve, and the linear relationship between the absorbance difference and the antibiotic resistance index is fitted using the least squares method. The coefficient of determination must be higher than 0.

98.

10. The rapid detection method for drug resistance in vector-borne organisms according to claim 1, characterized in that: In step S6, before making a determination, it is necessary to confirm that the linear range of the standard curve covers the net absorbance value of the sample to be tested. If it exceeds the range, the sample needs to be diluted with buffer and the S1 to S5 procedures need to be repeated. The calculated resistance index is compared with the critical threshold of 1.

00. The critical threshold is set based on the mean resistance index of a large number of sensitive strains plus three times the standard deviation. This threshold has been verified by field population pre-experiments and fixed in this method. When the index is greater than or equal to 1.00, it is determined to be a resistant individual, and when the index is less than 1.00, it is determined to be a sensitive individual. The determination results are recorded in the well plate layout table and the corresponding individual number is marked.

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