Rapid molecular detection method for vector organisms based on LAMP (loop-mediated isothermal amplification) technology

Abstract

The invention discloses an LAMP (loop-mediated isothermal amplification) technology-based rapid molecular detection method for vector organisms, belongs to the technical field of vector organism detection, and realizes comprehensive improvement of detection performance through multi-dimensional technology optimization. In a primer design stage, a multi-target primer group is constructed aiming at a conserved gene region, high-specificity recognition of a target sequence is ensured by combining bioinformatics verification, and the non-specific amplification risk is reduced from a molecular recognition source. According to the reaction system, key parameters are accurately optimized through a temperature gradient pre-experiment, real-time turbidity monitoring in the isothermal amplification process is matched, whole-process dynamic regulation and control from sample treatment to result output are achieved, and efficient amplification is ensured. By introducing the CRISPR-Cas12a system, nucleic acid amplification and visual detection are seamlessly connected, detection signals are amplified by utilizing the attached cutting activity of the CRISPR-Cas12a system, and results which can be distinguished by naked eyes are interpreted by matching with a lateral flow test strip without depending on complex detection equipment.

Description

Technical Field

[0001] This invention belongs to the field of vector-borne disease detection technology, specifically a rapid molecular detection method for vector-borne diseases based on LAMP technology. Background Technology

[0002] Vector-borne disease molecular detection is a modern detection method based on molecular biology techniques for the rapid and accurate identification and monitoring of vector organisms such as mosquitoes, flies, ticks, and fleas, and the pathogens they carry. This technology primarily utilizes polymerase chain reaction (PCR), quantitative real-time PCR (qPCR), loop-mediated isothermal amplification (LAMP), and gene sequencing to specifically amplify and analyze the DNA or RNA of vector organisms, thereby determining their species, genetic characteristics, and whether they carry pathogens such as Plasmodium, dengue virus, and Lyme disease spirochetes. Compared to traditional morphological identification methods, molecular detection offers higher sensitivity, specificity, and automation, making it suitable for epidemiological surveys, disease early warning, and the development of control strategies. With the development of high-throughput sequencing and artificial intelligence technologies, vector-borne disease molecular detection is evolving towards higher efficiency, portability, and intelligence, becoming a crucial technological support in the fields of public health and infectious disease control.

[0003] However, existing technologies suffer from several drawbacks, including insufficient specificity leading to non-specific amplification, unoptimized reaction conditions resulting in long amplification times and low efficiency, reliance on expensive detection equipment increasing costs and operational complexity, lack of internal control validation making it difficult to eliminate false negatives, unclear result interpretation standards relying on subjective judgment, and an imperfect quality control system with insufficient control settings leading to poor reliability and traceability of results. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid molecular detection method for vector-borne organisms based on LAMP technology in order to solve the problems mentioned above.

[0005] The technical solution adopted in this invention is as follows: A rapid molecular detection method for disease vectors based on LAMP technology, the method comprising the following steps:

[0006] S1: After collecting the target vector biological samples, mechanically grind them and add lysis buffer containing RNase inhibitors. The pretreated samples are immediately transferred to the DNA extraction step in S2 to avoid nucleic acid degradation.

[0007] S2: Sample DNA was extracted using a combination of magnetic bead extraction and thermal lysis. During the extraction process, the DNA purity was ensured to meet the sequence specificity requirements of the primers designed in S3 by adjusting the lysis temperature and the amount of magnetic beads. The extracted product was directly used in the preparation of the reaction system in S4.

[0008] S3: Design and synthesize LAMP primers. Design a multi-target primer set containing internal reference genes for conserved gene regions of vector organisms. The primer sequences must be verified by BLAST to ensure cross-reactivity with non-target organisms. The concentration of the synthesized primer powder must match the system concentration requirements of S4.

[0009] S4: Mix buffer, dNTPs, MgSO4, Bst DNA polymerase and primers synthesized in S3 in proportion, add DNA template extracted in S2 and immediately perform a temperature gradient pre-experiment to determine the optimal reaction temperature to ensure the amplification efficiency of S5.

[0010] S5: Place the reaction system configured in S4 in a 63℃ constant temperature device for 45 minutes. During the reaction, the amplification curve is dynamically observed through a real-time turbidity monitoring system. When the turbidity reaches the threshold, the detection program in S6 is automatically started.

[0011] S6: Add the Cas12a protein and specific crRNA complex to the amplification product. Use its collateral cleavage activity to cleave the fluorescently labeled reporter probe. The resulting fluorescent signal is developed by the lateral flow test strip. The color development result needs to be interpreted simultaneously with the internal control verification in S7.

[0012] S7: Use the internal control primers designed in S3 to perform a second amplification of the amplification product. Detect the internal control band by agarose gel electrophoresis. If the internal control is not detected, it is necessary to go back to S2 to re-extract DNA to ensure that false negatives caused by sample processing are eliminated.

[0013] S8: Combining the color intensity of the test strip in S6 and the internal reference verification results in S7, establish a three-level judgment standard: strong positive (double band on the test strip + positive internal reference), weak positive (weak band on the test strip + positive internal reference), and negative (no band or negative internal reference). The judgment results are automatically entered into the quality control system in S9.

[0014] S9: Verify the results of positive controls, negative controls, and blank controls throughout the entire process from S1 to S8. If the control results are abnormal, a system alarm will be triggered and possible sources of error will be indicated. After the verification is passed, a report containing sample information, test results, and confidence level will be generated.

[0015] In a preferred embodiment, in step S1, the collection and pretreatment of vector-borne organism samples requires selecting active adult or larval vectors, collecting them using sterile collection tubes, and immediately storing them at -20°C. For sample processing, a single individual is placed in a 2mL centrifuge tube, and 500μL of Tris-HCl lysis buffer containing 1% β-mercaptoethanol is added. The sample is homogenized using a tissue homogenizer at 3000rpm for 3 minutes in an ice bath. After centrifugation at 12000rpm for 5 minutes, the supernatant is collected. The entire process is controlled at 4°C to maintain nucleic acid stability. The processed sample must be transferred to step S2 within 15 minutes.

[0016] In a preferred embodiment, in step S2, rapid DNA extraction employs a magnetic bead method combined with thermal lysis. 200 μL of the supernatant obtained in step S1 is added to a centrifuge tube containing 50 μL of magnetic bead suspension, and the tube is placed in a 65°C metal bath for thermal lysis for 10 minutes, gently mixing every 2 minutes. After lysis, 400 μL of washing buffer is added, the tube is placed on a magnetic rack for adsorption for 2 minutes, and the supernatant is discarded. This washing step is repeated 3 times. Finally, 50 μL of TE elution buffer preheated to 60°C is added, the mixture is vortexed, and incubated at 60°C for 5 minutes. After separation on a magnetic rack, 40 μL of the elution buffer is used directly in step S4. The extracted product must have an A260 / A280 ratio within the range of 1.8-2.0 as determined by Nanodrop.

[0017] In a preferred embodiment, in step S3, LAMP primer design and synthesis target the conserved region of the 18S rRNA gene in vector organisms. A multi-target primer set containing four primers (F3, B3, LF, and LB) is designed using PrimerExplorer V5 software. The primer lengths are controlled at 20-22 nt for F3 / B3 and 32-35 nt for LF / LB, with GC content maintained between 45% and 55%. The designed primer sequences are subjected to homology comparison using the NCBI BLAST database to ensure homology with non-target organism sequences is less than 70%. The synthesized primer powder is dissolved in RNase-free water to prepare a 100 μM stock solution, aliquoted, and stored at -20°C. Before use, it is diluted to working concentrations of 0.2 μM for F3 / B3 and 1.6 μM for LF / LB to match the reaction system in S4.

[0018] In a preferred embodiment, in step S4, the optimized reaction system configuration uses a 25 μL reaction system, with the following added sequentially: 12.5 μL of 2×Isothermal Amplification Buffer, 2 μL of a 10 mM dNTP mixture, 3 μL of 25 mM MgSO4 solution, 1 μL of 8 U / μL Bst DNA polymerase fragment, 4 μL of the primer mixture prepared in step S3, 2 μL of DNA template extracted by L2, and finally, the volume is made up with nuclease-free water. Immediately after the system configuration is complete, a temperature gradient pre-experiment is performed, setting five gradients: 60℃, 61℃, 62℃, 63℃, and 64℃, with each gradient reacting for 40 minutes. The temperature with the highest amplification efficiency is determined by real-time turbidity monitoring and used as the reaction temperature for step S5.

[0019] In a preferred embodiment, in step S5, the isothermal amplification reaction involves placing the optimized reaction system from S4 into a real-time turbidity monitor, setting the reaction temperature to the optimal temperature determined in the pre-experiment, and maintaining the temperature for 45 minutes. The instrument records the optical density value at 650 nm every 2 minutes. When the increase in optical density value after three consecutive monitoring values ​​exceeds 10 times the baseline value, it is automatically determined to be a positive amplification, and the detection program in step S6 is immediately initiated. After the reaction is completed, the amplification product is temporarily stored in a 4°C refrigerator for subsequent verification.

[0020] In a preferred embodiment, in step S6, a binary complex of Cas12a protein and specific crRNA is first constructed in advance. The spacer sequence of the crRNA must be completely complementary to the target region of the LAMP amplification product, enabling precise recognition of the target sequence through base pairing. This complex is in a quiescent state when it does not bind to the target sequence. Once it recognizes and binds to the specific amplification product generated in step S5, the nuclease domain of Cas12a will be activated and exhibit non-specific collateral cleavage activity.

[0021] A fluorescently labeled reporter probe, typically composed of a non-specific DNA sequence, is added to the reaction system, with a fluorophore and a quencher labeled at each end. When Cas12a is activated, it randomly cleaves the reporter probe, causing the fluorophore and quencher to separate, generating free fluorescent signal molecules. For visual detection, the reaction mixture is dropped onto the sample pad of a lateral flow test strip. The chromatography buffer inside the test strip carries the mixture towards the absorbent pad, passing the detection line and control line coated with an antibody against the fluorescent group.

[0022] Free fluorescent signal molecules are captured and enriched by the antibody on the test line, forming a visible colored band. Uncut, intact probes fail to develop color due to the presence of quenching groups. Simultaneously, the control line captures excess Cas12a-crRNA complexes or other internal control molecules in the system, ensuring the test strip chromatography process proceeds correctly. The colorimetric results must be interpreted within 10 minutes. The color intensity of the test line is positively correlated with the target concentration, while the absence of color in the control line indicates that the test strip is invalid and requires retesting.

[0023] This step, together with the internal control validation in S7, forms a rigorous result cross-checking mechanism. When a colored band appears on the S6 test strip, it is necessary to simultaneously confirm whether the amplification result of the internal control gene in S7 is positive. If the internal control is negative, it indicates an abnormality in the sample processing or amplification process, and the colorimetric result of S6 is deemed invalid. This two-step collaborative interpretation design effectively reduces the risk of false positives. Furthermore, through the high specificity of the CRISPR system and the convenience of the test strips, it achieves rapid conversion from molecular amplification to visual results. The entire process requires no complex instruments and is suitable for on-site testing scenarios.

[0024] In a preferred embodiment, in step S7, the internal reference gene verification uses the β-actin internal reference primer pair designed in step S3. 5 μL of the amplification product from step S5 is used for secondary amplification. The reaction system contains 10 μL of 2×PCR Mix, 0.5 μL each of forward and reverse primers, 5 μL of template, and water to a final volume of 20 μL. The amplification conditions are: 95℃ pre-denaturation for 3 minutes, 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, and 72℃ extension for 30 seconds, for a total of 35 cycles, with a final extension at 72℃ for 5 minutes. The amplification product is separated by 2% agarose gel electrophoresis. After electrophoresis at 100V for 30 minutes, the sample is observed under UV light. If a clear band appears at 200 bp, the internal reference is considered positive; otherwise, the DNA sample needs to be re-extracted from step S2.

[0025] In a preferred embodiment, in step S8, a three-level result determination standard is established for comprehensive result judgment. When the lateral flow test strip detection line shows a distinct purplish-red band and the control line is colored in step S6, it is determined to be strongly positive based on the positive result of the internal reference in step S7; when the detection line shows a light purplish-red band and the control line is colored, it is determined to be weakly positive based on the positive result of the internal reference; when the detection line is not colored or the control line is not colored, it is determined to be negative regardless of the internal reference result. The determination process must be completed within 10 minutes under natural light conditions, and the results are entered into the system using dedicated software, simultaneously recording the sample number, detection time, and operator information.

[0026] In a preferred embodiment, in step S9, the quality control and report generation includes three quality control groups: a positive control, a negative control, and a blank control. The positive control uses target plasmid DNA of known concentration, the negative control uses non-target biological DNA, and the blank control contains only reaction buffer. When the positive control fails to show the expected amplification or the negative control shows non-specific amplification, the system automatically triggers a red alarm and indicates possible sources of contamination or reagent failure. After all control results are verified, the system generates a test report containing sample collection information, the basis for determining the test results, and a confidence score. The report must be reviewed and signed by the laboratory director.

[0027] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0028] 1. This invention achieves a comprehensive improvement in detection performance through multi-dimensional technical optimization. During primer design, a multi-target primer set is constructed targeting conserved gene regions. Combined with bioinformatics validation, this ensures highly specific recognition of the target sequence, reducing the risk of non-specific amplification from the molecular recognition source. The reaction system undergoes precise optimization of key parameters through temperature gradient pre-experiments. Coupled with real-time turbidity monitoring during isothermal amplification, dynamic control of the entire process from sample processing to result output is achieved, ensuring efficient amplification. The introduction of the CRISPR-Cas12a system seamlessly integrates nucleic acid amplification with visual detection. Utilizing its attached cleavage activity to amplify the detection signal, and in conjunction with lateral flow test strips, results can be interpreted visually without relying on complex detection equipment, thus improving the overall accuracy of the detection process.

[0029] 2. In this invention, a systematic quality control design ensures the reliability and practicality of the test results. The internal reference gene verification mechanism operates synchronously with the main detection process, effectively identifying problems such as sample degradation or extraction failure, and avoiding false negative results. A three-level judgment standard, combined with colorimetric intensity and internal reference verification results, provides accurate grading for target samples at different concentration gradients. The entire process quality control system simultaneously monitors positive, negative, and blank controls, automatically triggering alarms and indicating the source of error in abnormal situations, ensuring the traceability of test results. The entire detection process, from sample collection to report generation, achieves standardized operation, adapting to the needs of rapid on-site testing scenarios, providing efficient and reliable molecular diagnostic technology support for vector-borne disease monitoring and control, and improving detection efficiency. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the process principle of the present invention. Detailed Implementation

[0031] 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.

[0032] Example:

[0033] Reference Figure 1 A rapid molecular detection method for vector-borne diseases based on LAMP technology, comprising the following steps:

[0034] S1: After collecting the target vector biological samples, mechanically grind them and add lysis buffer containing RNase inhibitors. The pretreated samples are immediately transferred to the DNA extraction step in S2 to avoid nucleic acid degradation.

[0035] S2: Sample DNA was extracted using a combination of magnetic bead extraction and thermal lysis. During the extraction process, the DNA purity was ensured to meet the sequence specificity requirements of the primers designed in S3 by adjusting the lysis temperature and the amount of magnetic beads. The extracted product was directly used in the preparation of the reaction system in S4.

[0036] S3: Design and synthesize LAMP primers. Design a multi-target primer set containing internal reference genes for conserved gene regions of vector organisms. The primer sequences must be verified by BLAST to ensure cross-reactivity with non-target organisms. The concentration of the synthesized primer powder must match the system concentration requirements of S4.

[0037] S4: Mix buffer, dNTPs, MgSO4, Bst DNA polymerase and primers synthesized in S3 in proportion, add DNA template extracted in S2 and immediately perform a temperature gradient pre-experiment to determine the optimal reaction temperature to ensure the amplification efficiency of S5.

[0038] S5: Place the reaction system configured in S4 in a 63℃ constant temperature device for 45 minutes. During the reaction, the amplification curve is dynamically observed through a real-time turbidity monitoring system. When the turbidity reaches the threshold, the detection program in S6 is automatically started.

[0039] S6: Add the Cas12a protein and specific crRNA complex to the amplification product. Use its collateral cleavage activity to cleave the fluorescently labeled reporter probe. The resulting fluorescent signal is developed by the lateral flow test strip. The color development result needs to be interpreted simultaneously with the internal control verification in S7.

[0040] S7: Use the internal control primers designed in S3 to perform a second amplification of the amplification product. Detect the internal control band by agarose gel electrophoresis. If the internal control is not detected, it is necessary to go back to S2 to re-extract DNA to ensure that false negatives caused by sample processing are eliminated.

[0041] S8: Combining the color intensity of the test strip in S6 and the internal reference verification results in S7, establish a three-level judgment standard: strong positive (double band on the test strip + positive internal reference), weak positive (weak band on the test strip + positive internal reference), and negative (no band or negative internal reference). The judgment results are automatically entered into the quality control system in S9.

[0042] S9: Verify the results of positive controls, negative controls, and blank controls throughout the entire process from S1 to S8. If the control results are abnormal, a system alarm will be triggered and possible sources of error will be indicated. After the verification is passed, a report containing sample information, test results, and confidence level will be generated.

[0043] In step S1, the collection and pretreatment of vector-borne organism samples requires selecting active adult or larval vectors. After collection using sterile collection tubes, the samples should be immediately stored at -20°C. For sample processing, a single individual should be placed in a 2mL centrifuge tube, and 500μL of Tris-HCl lysis buffer containing 1% β-mercaptoethanol should be added. The sample should be homogenized using a tissue homogenizer at 3000rpm for 3 minutes in an ice bath. The homogenate should be centrifuged at 12000rpm for 5 minutes, and the supernatant should be collected. The entire process should be conducted at 4°C to maintain nucleic acid stability. The processed samples must be transferred to step S2 within 15 minutes.

[0044] In step S2, rapid DNA extraction employs a magnetic bead method combined with thermal lysis. 200 μL of the supernatant obtained in step S1 is added to a centrifuge tube containing 50 μL of magnetic bead suspension. The tube is then placed in a 65°C metal bath for thermal lysis for 10 minutes, gently mixing every 2 minutes. After lysis, 400 μL of washing buffer is added, and the tube is placed on a magnetic rack for adsorption for 2 minutes. The supernatant is then discarded. This washing step is repeated three times. Finally, 50 μL of TE elution buffer preheated to 60°C is added, and the mixture is vortexed and incubated at 60°C for 5 minutes. After separation on a magnetic rack, 40 μL of the elution buffer is used directly in step S4. The extracted product must have an A260 / A280 ratio within the range of 1.8-2.0 as determined by Nanodrop.

[0045] In step S3, LAMP primer design and synthesis target the conserved region of the 18S rRNA gene in vector organisms. A multi-target primer set containing four primers (F3, B3, LF, and LB) is designed using PrimerExplorer V5 software. Primer lengths are controlled at 20-22 nt for F3 / B3 and 32-35 nt for LF / LB, with GC content maintained between 45% and 55%. The designed primer sequences are then compared for homology using the NCBI BLAST database to ensure homology with non-target organism sequences is less than 70%. The synthesized primer powder is dissolved in RNase-free water to prepare a 100 μM stock solution, aliquoted, and stored at -20°C. Before use, it is diluted to working concentrations of 0.2 μM for F3 / B3 and 1.6 μM for LF / LB to match the reaction system in step S4.

[0046] In step S4, the optimized reaction system was configured using a 25 μL reaction volume. The following were added sequentially: 12.5 μL of 2×Isothermal Amplification Buffer, 2 μL of a 10 mM dNTP mixture, 3 μL of 25 mM MgSO4 solution, 1 μL of 8 U / μL Bst DNA polymerase fragment, 4 μL of the primer mixture prepared in step S3, and 2 μL of the DNA template extracted in step S2. The volume was then brought to the final volume with nuclease-free water. Immediately after the system was configured, a temperature gradient experiment was conducted, setting five gradients: 60℃, 61℃, 62℃, 63℃, and 64℃. Each gradient was reacted for 40 minutes. The temperature with the highest amplification efficiency was determined by real-time turbidity monitoring and used as the reaction temperature for step S5.

[0047] In step S5, the isothermal amplification reaction involves placing the optimized reaction system from S4 into a real-time turbidity monitor. The reaction temperature is set to the optimal temperature determined in the pre-experiment, and the reaction is carried out at this temperature for 45 minutes. The instrument records the optical density value at 650 nm every 2 minutes. When the increase in optical density value after three consecutive monitoring values ​​exceeds 10 times the baseline value, the amplification is automatically determined to be positive, and the detection program in step S6 is immediately initiated. After the reaction is completed, the amplification product is temporarily stored at 4°C for subsequent verification.

[0048] In step S6, a binary complex of Cas12a protein and specific crRNA must first be constructed. The spacer sequence of the crRNA must be completely complementary to the target region of the LAMP amplification product, enabling precise recognition of the target sequence through base pairing. This complex is in a quiescent state when it does not bind to the target sequence. Once it recognizes and binds to the specific amplification product generated in step S5, the nuclease domain of Cas12a will be activated and exhibit non-specific collateral cleavage activity.

[0049] A fluorescently labeled reporter probe, typically composed of a non-specific DNA sequence, is added to the reaction system, with a fluorophore and a quencher labeled at each end. When Cas12a is activated, it randomly cleaves the reporter probe, causing the fluorophore and quencher to separate, generating free fluorescent signal molecules. For visual detection, the reaction mixture is dropped onto the sample pad of a lateral flow test strip. The chromatography buffer inside the test strip carries the mixture towards the absorbent pad, passing the detection line and control line coated with an antibody against the fluorescent group.

[0050] Free fluorescent signal molecules are captured and enriched by the antibody on the test line, forming a visible colored band. Uncut, intact probes fail to develop color due to the presence of quenching groups. Simultaneously, the control line captures excess Cas12a-crRNA complexes or other internal control molecules in the system, ensuring the test strip chromatography process proceeds correctly. The colorimetric results must be interpreted within 10 minutes. The color intensity of the test line is positively correlated with the target concentration, while the absence of color in the control line indicates that the test strip is invalid and requires retesting.

[0051] This step, together with the internal control validation in S7, forms a rigorous result cross-checking mechanism. When a colored band appears on the S6 test strip, it is necessary to simultaneously confirm whether the amplification result of the internal control gene in S7 is positive. If the internal control is negative, it indicates an abnormality in the sample processing or amplification process, and the colorimetric result of S6 is deemed invalid. This two-step collaborative interpretation design effectively reduces the risk of false positives. Furthermore, through the high specificity of the CRISPR system and the convenience of the test strips, it achieves rapid conversion from molecular amplification to visual results. The entire process requires no complex instruments and is suitable for on-site testing scenarios.

[0052] In step S7, the internal control gene verification uses the β-actin internal control primer pair designed in step S3. 5 μL of the amplification product from step S5 is used for secondary amplification. The reaction system contains 10 μL of 2×PCR Mix, 0.5 μL each of forward and reverse primers, 5 μL of template, and water to a final volume of 20 μL. The amplification conditions are: 95℃ pre-denaturation for 3 minutes, 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, and 72℃ extension for 30 seconds, for a total of 35 cycles, with a final extension at 72℃ for 5 minutes. The amplification products are separated by 2% agarose gel electrophoresis. After electrophoresis at 100V for 30 minutes, the samples are observed under UV light. If a clear band appears at 200 bp, the internal control is considered positive; otherwise, the DNA sample needs to be re-extracted from step S2.

[0053] In step S8, a three-tiered result determination standard is established. When the lateral flow test strip in step S6 shows a distinct purplish-red band and the control line is colored, it is determined to be strongly positive based on the positive result of the internal reference in step S7. When the test line shows a light purplish-red band and the control line is colored, it is determined to be weakly positive based on the positive result of the internal reference. When the test line is not colored or the control line is not colored, it is determined to be negative regardless of the internal reference result. The determination process must be completed within 10 minutes under natural light conditions. The results are entered into the system using dedicated software, and the sample number, testing time, and operator information are recorded simultaneously.

[0054] In step S9, the quality control and report generation process sets up three quality control groups: positive control, negative control, and blank control. The positive control uses target plasmid DNA at a known concentration, the negative control uses non-target biological DNA, and the blank control contains only reaction buffer. When the positive control fails to show the expected amplification or the negative control shows non-specific amplification, the system automatically triggers a red alarm and indicates possible sources of contamination or reagent failure. After all control results are verified, the system generates a test report containing sample collection information, the basis for result determination, and a confidence score. The report must be reviewed and signed by the laboratory director.

[0055] From the above, we can conclude that:

[0056] In this invention, a comprehensive improvement in detection performance is achieved through multi-dimensional technical optimization. During the primer design stage, a multi-target primer set is constructed targeting conserved gene regions. Combined with bioinformatics validation, this ensures highly specific recognition of the target sequence, reducing the risk of non-specific amplification from the molecular recognition source. The reaction system undergoes precise optimization of key parameters through temperature gradient pre-experiments. Coupled with real-time turbidity monitoring during isothermal amplification, dynamic control of the entire process from sample processing to result output is achieved, ensuring efficient amplification. The introduction of the CRISPR-Cas12a system seamlessly integrates nucleic acid amplification with visual detection. Utilizing its attached cleavage activity to amplify the detection signal, and in conjunction with lateral flow test strips, results can be interpreted visually without relying on complex detection equipment.

[0057] In this invention, a systematic quality control design ensures the reliability and practicality of the test results. The internal reference gene validation mechanism operates synchronously with the main detection process, effectively identifying problems such as sample degradation or extraction failure, avoiding false negative results. A three-tiered judgment standard, combining colorimetric intensity and internal reference validation results, provides precise grading for target samples at different concentration gradients. A comprehensive quality control system simultaneously monitors positive, negative, and blank controls, automatically triggering alarms and indicating the source of error in abnormal situations, ensuring the traceability of test results. The entire testing process, from sample collection to report generation, is standardized, adapting to the needs of rapid on-site testing scenarios and providing efficient and reliable molecular diagnostic technology support for vector-borne disease monitoring and control.

[0058] 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.

[0059] The foregoing description enables those skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A rapid molecular detection method for vector-borne organisms based on LAMP technology, characterized in that: The method includes the following steps: S1: After collecting the target vector biological samples, mechanically grind them and add lysis buffer containing RNase inhibitors. The pretreated samples are immediately transferred to the DNA extraction step in S2 to avoid nucleic acid degradation. S2: Sample DNA was extracted using magnetic bead method combined with thermal lysis technology. During the extraction process, the DNA purity was ensured to meet the sequence specificity requirements of the primer design in S3 by adjusting the lysis temperature and the amount of magnetic beads. The extracted product was directly used in the preparation of the reaction system in S4. S3: Design and synthesize LAMP primers. Design a multi-target primer set containing internal reference genes for conserved gene regions of vector organisms. The primer sequences need to be verified by BLAST for cross-reactivity with non-target organisms. The concentration of the synthesized primer powder must match the system concentration requirements of S4. S4: Mix buffer, dNTPs, MgSO4, Bst DNA polymerase and primers synthesized in S3 in proportion, add DNA template extracted in S2 and immediately perform temperature gradient pre-experiment to determine the optimal reaction temperature to ensure the amplification efficiency of S5. S5: Place the reaction system configured in S4 in a 63℃ constant temperature device for 45 minutes. During the reaction, the amplification curve is dynamically observed through a real-time turbidity monitoring system. When the turbidity reaches the threshold, the detection program in S6 is automatically started. S6: Add the Cas12a protein and specific crRNA complex to the amplification product, and use its collateral cleavage activity to cleave the fluorescently labeled reporter probe. The resulting fluorescent signal is developed by the lateral flow test strip. The color development result needs to be interpreted simultaneously with the internal control verification in S7. S7: Use the internal control primers designed in S3 to perform a second amplification of the amplification product. Detect the internal control band by agarose gel electrophoresis. If the internal control is not detected, it is necessary to go back to S2 to re-extract DNA to ensure that false negatives caused by sample processing are eliminated. S8: Combining the color intensity of the test strips in S6 and the internal reference verification results in S7, establish a three-level judgment standard: strong positive; weak positive; negative; and the judgment results are automatically entered into the quality control system in S9. S9: Verify the results of positive controls, negative controls, and blank controls throughout the entire process from S1 to S8. If the control results are abnormal, a system alarm will be triggered and possible sources of error will be indicated. After the verification is passed, a report containing sample information, test results, and confidence level will be generated.

2. The rapid molecular detection method for vector-borne diseases based on LAMP technology as described in claim 1, characterized in that: In step S1, the collection and pretreatment of vector organism samples requires selecting active adult or larval vector organisms. After collection using sterile collection tubes, the samples should be immediately stored at -20°C. For sample processing, a single individual should be placed in a 2mL centrifuge tube, and 500μL of Tris-HCl lysis buffer containing 1% β-mercaptoethanol should be added. The sample should be homogenized for 3 minutes at 3000rpm using a tissue homogenizer under ice bath conditions. After centrifugation at 12000rpm for 5 minutes, the supernatant should be collected. The entire process should be carried out at 4°C to maintain nucleic acid stability. The processed samples should be transferred to step S2 within 15 minutes.

3. The rapid molecular detection method for vector-borne pathogens based on LAMP technology as described in claim 1, characterized in that: In step S2, rapid DNA extraction employs a magnetic bead method combined with thermal lysis. 200 μL of the supernatant obtained in step S1 is added to a centrifuge tube containing 50 μL of magnetic bead suspension. The tube is then placed in a 65°C metal bath for thermal lysis for 10 minutes, gently mixing every 2 minutes. After lysis, 400 μL of washing buffer is added, and the tube is placed on a magnetic rack for adsorption for 2 minutes. The supernatant is then discarded. This washing step is repeated three times. Finally, 50 μL of TE elution buffer preheated to 60°C is added. After vortexing and incubation at 60°C for 5 minutes, 40 μL of the elution buffer is used directly in step S4 after magnetic rack separation. The extracted product must have an A260 / A280 ratio within the range of 1.8-2.0 as determined by Nanodrop.

4. The rapid molecular detection method for vector-borne organisms based on LAMP technology as described in claim 1, characterized in that: In step S3, LAMP primer design and synthesis target the conserved region of the 18S rRNA gene in vector organisms. A multi-target primer set containing four primers (F3, B3, LF, and LB) is designed using PrimerExplorer V5 software. The primer lengths are controlled at 20-22 nt for F3 / B3 and 32-35 nt for LF / LB, with GC content maintained between 45% and 55%. The designed primer sequences need to be compared for homology using the NCBI BLAST database to ensure that the homology with non-target organism sequences is less than 70%. The synthesized primer powder is dissolved in RNase-free water to prepare a 100 μM stock solution, aliquoted, and stored at -20°C. Before use, it is diluted to working concentrations of 0.2 μM for F3 / B3 and 1.6 μM for LF / LB to match the reaction system in S4.

5. The rapid molecular detection method for vector-borne organisms based on LAMP technology as described in claim 1, characterized in that: In step S4, the optimized reaction system configuration uses a 25 μL reaction system, with the following added sequentially: 12.5 μL of 2×Isothermal Amplification Buffer, 2 μL of 10 mM dNTP mixture, 3 μL of 25 mM MgSO4 solution, 1 μL of 8 U / μL Bst DNA polymerase fragment, 4 μL of primer mixture prepared in step S3, and 2 μL of DNA template extracted in step S2. Finally, the volume is made up with nuclease-free water. Immediately after the system configuration is completed, a temperature gradient pre-experiment is performed, setting five gradients: 60℃, 61℃, 62℃, 63℃, and 64℃. Each gradient is reacted for 40 minutes. The temperature with the highest amplification efficiency is determined by real-time turbidity monitoring and used as the reaction temperature for step S5.

6. The rapid molecular detection method for vector-borne organisms based on LAMP technology as described in claim 1, characterized in that: In step S5, the isothermal amplification reaction involves placing the optimized reaction system from S4 into a real-time turbidity monitor, setting the reaction temperature to the optimal temperature determined in the pre-experiment, and incubating at the same temperature for 45 minutes. The instrument records the optical density value at 650 nm every 2 minutes. When the increase in optical density value from three consecutive monitoring values ​​exceeds 10 times the baseline value, it is automatically determined to be a positive amplification, and the detection program in step S6 is immediately started. After the reaction is completed, the amplification product is temporarily stored in a 4°C refrigerator for subsequent verification.

7. The rapid molecular detection method for vector-borne organisms based on LAMP technology as described in claim 1, characterized in that: In step S6, a binary complex of Cas12a protein and specific crRNA needs to be constructed in advance. The spacer sequence of the crRNA must be completely complementary to the target region of the LAMP amplification product. Accurate recognition of the target sequence is achieved through base pairing. This complex is in a quiescent state when it does not bind to the target sequence. Once it recognizes and binds to the specific amplification product generated in step S5, the nuclease domain of Cas12a will be activated and exhibit non-specific collateral cleavage activity. A fluorescently labeled reporter probe is added to the reaction system. This probe is usually composed of a non-specific DNA sequence with a fluorescent group and a quencher group labeled at both ends, respectively. When Cas12a is activated, it randomly cleaves the reporter probe in the system, causing the fluorescent group and the quencher group to separate, generating free fluorescent signal molecules. To achieve visual detection, the reaction mixture is dropped onto the sample pad of the lateral flow test strip. The chromatography buffer inside the test strip will carry the mixture towards the absorbent pad, passing through the detection line and control line coated with anti-fluorescent antibody along the way. Free fluorescent signal molecules are captured and enriched by the antibody on the detection line, forming a visible colored band. Intact probes that have not been cleaved cannot develop color due to the presence of quenching groups. At the same time, the control line captures excess Cas12a-crRNA complex or other internal control molecules in the system to ensure that the test strip chromatography process is normal. The color development results must be interpreted within 10 minutes. The color intensity of the detection line is positively correlated with the target concentration. If the control line does not develop color, it indicates that the test strip is invalid and needs to be tested again.

8. The rapid molecular detection method for vector-borne organisms based on LAMP technology as described in claim 1, characterized in that: In step S7, the internal reference gene verification uses the β-actin internal reference primer pair designed in step S3. 5 μL of the amplification product from step S5 is used for secondary amplification. The reaction system contains 10 μL of 2×PCR Mix, 0.5 μL each of forward and reverse primers, 5 μL of template, and water to a final volume of 20 μL. The amplification conditions are: 95℃ pre-denaturation for 3 minutes, 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, and 72℃ extension for 30 seconds, for a total of 35 cycles, with a final extension at 72℃ for 5 minutes. The amplification product is separated by 2% agarose gel electrophoresis. After electrophoresis at 100V for 30 minutes, the sample is observed under UV light. If a clear band appears at 200 bp, the internal reference is considered positive; otherwise, the DNA sample needs to be re-extracted from step S2.

9. The rapid molecular detection method for vector-borne organisms based on LAMP technology as described in claim 1, characterized in that: In step S8, a three-level result judgment standard is established for comprehensive result judgment. When the lateral flow test strip detection line in step S6 shows a clear purple-red band and the quality control line is colored, it is judged as strongly positive in combination with the positive result of the internal reference in S7; when the detection line shows a light purple-red band and the quality control line is colored, it is judged as weakly positive in combination with the positive result of the internal reference; when the detection line is not colored or the quality control line is not colored, it is judged as negative regardless of the result of the internal reference. The judgment process must be completed within 10 minutes under natural light conditions, and the results are entered into the system through dedicated software, and the sample number, detection time and operator information are recorded simultaneously.

10. The rapid molecular detection method for vector-borne organisms based on LAMP technology as described in claim 1, characterized in that: In step S9, the quality control and report generation sets up three quality control groups: positive control, negative control, and blank control. The positive control uses target plasmid DNA of known concentration, the negative control uses non-target biological DNA, and the blank control contains only reaction buffer. The system generates a test report that includes sample collection information, the basis for judging the test results, and a confidence score. The report must be reviewed and signed by the laboratory director.

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