A system and method for rapid on-site monitoring of toxin-producing and odor-producing cyanobacteria based on toxin-producing and odor-producing genes

Abstract

The application discloses a kind of based on toxigenic and odor-producing gene toxigenic and odor-producing cyanobacteria field rapid monitoring system and method, a kind of based on toxigenic and odor-producing gene toxigenic and odor-producing cyanobacteria field rapid monitoring system, including water sample algal automatic enrichment module, algal cell rapid lysis and DNA extraction tube, isothermal / normal temperature PCR module, palm fluorescence detector, qualitative and quantitative algorithm and toxigenic and odor-producing cyanobacteria risk warning grade calculation software, mobile phone APP software, each module is sequentially linked.The application realizes the integrated detection of toxigenic and odor-producing cyanobacteria quickly, accurately and conveniently, solves the problem that existing technology cannot accurately distinguish between toxigenic and non-toxigenic cyanobacteria and is difficult to scientifically assess the health risk of water body, while overcoming the limitations of traditional monitoring technology, such as laboratory detection, large equipment, complex operation, long detection period, etc., and meeting the timeliness, convenience and ease of operation requirements of field scenes such as grassroots monitoring, outdoor inspection and emergency disposal of sudden water bloom.

Description

Technical Field

[0001] This invention relates to the field of water environment monitoring technology, specifically to a rapid on-site monitoring system and method for toxin- and odor-producing cyanobacteria based on toxin- and odor-producing genes. Background Technology

[0002] Cyanobacterial blooms are one of the most serious water environmental problems worldwide, occurring widely in various water bodies such as lakes, reservoirs, and rivers, especially in eutrophic water bodies. They not only disrupt the ecological balance of water bodies, causing aquatic organisms to die from lack of oxygen and a decline in biodiversity, but also cause a series of public safety and economic losses due to the metabolic products of toxic and odor-producing cyanobacteria. According to relevant industry statistics, cyanobacterial blooms cause significant economic losses, encompassing multiple areas including the shutdown and rectification of drinking water plants, reduced or lost harvests in aquaculture, landscape water body remediation, and ecological restoration. Toxic substances produced by toxic and odor-producing cyanobacteria, such as microcystins, anabatin, and columnar cyanobacterial toxins, can harm human health through ingestion via drinking water and skin contact. Long-term exposure can damage the liver and nervous system, and even pose a carcinogenic risk. Furthermore, odor-causing substances such as geosmin and 2-methylisoborneol produced by these algae can cause unpleasant odors in drinking water, severely impacting residents' drinking water experience and safety. Therefore, accurate monitoring and early warning of toxic and odor-producing cyanobacteria are crucial for ensuring drinking water safety, maintaining stable fishery resources, and protecting ecosystem functions. This is also a key focus and challenge in the current field of water environment monitoring. However, current water algae monitoring technologies are limited by factors such as detection principles and equipment conditions, resulting in numerous prominent problems. They cannot meet the needs for rapid, accurate, and convenient on-site monitoring, making it difficult to achieve early identification and risk management of toxic and odor-producing cyanobacteria.

[0003] Traditional cyanobacteria monitoring techniques mainly include microscopic counting, chlorophyll a detection, and enzyme-linked immunosorbent assay (ELISA). While these methods are widely used, they have significant limitations and are difficult to adapt to the actual needs of on-site monitoring. Microscopic counting, a classic method, relies heavily on the experience and judgment of skilled technicians. Operators must prepare and stain water samples before identifying and counting cyanobacteria species one by one under a microscope. This method is not only highly subjective, with detection errors of 15%-25% between different technicians, but also extremely inefficient—a single sample requires 30-50 minutes to complete, and skilled technicians can only analyze 8-10 samples per day on average. This is insufficient to meet the rapid detection needs of large batches of water samples, and even more difficult to achieve real-time monitoring of sudden algal blooms. Chlorophyll a detection method, on the other hand, offers advantages due to its ease of operation. The advantage of rapid detection speed makes it commonly used for quick estimation of cyanobacterial biomass. However, the core drawback of this method is its inability to distinguish between toxin-producing and non-toxin-producing algae. It can only reflect the total biomass of cyanobacteria in the water and cannot accurately identify the presence of toxin-producing algae, thus failing to correctly assess the health risks of the water body and easily leading to misjudgment or missed detection. Enzyme-linked immunosorbent assay (ELISA), as an immunological detection method, is relatively simple to operate and does not require complex large-scale equipment. However, it is extremely susceptible to interference from the water matrix. Humus, heavy metal ions, and other algal metabolites in the water can all affect the test results, resulting in a false positive rate as high as 10%-15%. At the same time, the detection cycle of this method is relatively long, requiring 2-3 hours for a single sample test, which cannot meet the timeliness requirements of rapid on-site detection. Moreover, the detection cost is relatively high, making it difficult to promote and apply on a large scale.

[0004] The rise of molecular biology methods has provided new insights for cyanobacterial monitoring. Among these, quantitative PCR (qPCR) technology, with its high sensitivity and specificity, is widely used in laboratory testing. It can accurately detect the characteristic genes of toxin-producing cyanobacteria, effectively distinguishing them from non-toxin-producing algae. However, this technology has extremely high requirements for equipment and personnel. It requires expensive equipment such as thermal cyclers and quantitative fluorescence detectors, with the cost of a single device reaching hundreds of thousands of yuan. Moreover, the operation process is complex, involving multiple steps such as RNA / DNA extraction, PCR system preparation, and amplification detection. It relies on professional molecular biology technicians and cannot be carried out in outdoor fields, making it unsuitable for the needs of rapid on-site monitoring. The molecular technique based on toxin gene expression level (RT-qPCR) has further optimized the detection accuracy and can detect potential toxin-producing cyanobacteria (i.e., cyanobacteria that do not produce large amounts of toxins but have the ability to produce toxins), and predict the risk of algal blooms in advance. However, this technology adds a reverse transcription step to the qPCR technology, making the operation more complicated and increasing the detection cost. It also has strict requirements for the temperature, humidity and cleanliness of the detection environment. The complex environment of the outdoor field can easily lead to the distortion of the detection results, making it difficult to promote and apply in grassroots monitoring institutions and field monitoring. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid on-site monitoring system and method for toxin- and odor-producing cyanobacteria based on toxin- and odor-producing genes, in order to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a rapid on-site monitoring system for toxin- and odor-producing cyanobacteria based on toxin- and odor-producing genes, comprising an automated algae enrichment module for water samples, rapid algal cell lysis and DNA extraction tubes, an isothermal / room-temperature PCR module, a handheld fluorescence detector, qualitative and quantitative algorithms and software for calculating the risk warning level of toxin- and odor-producing cyanobacteria, and a mobile APP software. Each module is linked sequentially to achieve full automation of the entire process from water sample collection, algae enrichment, DNA extraction, gene amplification, fluorescence detection to result analysis, risk warning, and data management. The system uses the characteristic genes of toxin- and odor-producing cyanobacteria as detection targets and employs isothermal PCR technology to achieve rapid amplification of the target genes, enabling precise differentiation between toxin- and non-toxin-producing cyanobacteria.

[0007] Preferably, the characteristic genes of the toxin-producing and odor-producing cyanobacteria include microcystin synthesis genes, anabatin gene cluster, and columnar cyanobacterial toxin gene cluster; the microcystin synthesis genes are selected from at least one of mcyA, mcyB, mcyC, mcyD, mcyE, mcyG, mcyH, and mcyJ; the anabatin gene cluster is selected from at least one of anaA, anaB, anaC, anaD, anaE, anaF, and anaG; and the columnar cyanobacterial toxin gene cluster is selected from at least one of cyrA, cyrB, cyrC, cyrD, cyrE, cyrF, cyrG, cyrH, cyrI, cyrJ, and cyrN; and the automated algae enrichment module for water samples employs negative pressure filtration and magnetic bead enrichment. The combined dual enrichment technology includes a miniature portable vacuum pump, a replaceable filter membrane, and a turbidity sensor. The miniature portable vacuum pump has a power of ≤50W and can be powered by a lithium battery. The replaceable filter membrane is made of polyethersulfone with a pore size of 0.45μm. The turbidity sensor can detect the turbidity of the water sample in real time, and the control module automatically adjusts the filtration speed according to the turbidity. The filtration speed is 50mL / min when the turbidity is ≤10NTU, and the filtration speed is reduced to 20mL / min when the turbidity is >10NTU. The magnetic beads are modified with cyanobacteria-specific monoclonal antibodies, which can specifically adsorb target toxin-producing cyanobacteria cells and eliminate interference from diatoms, green algae, and water impurities. The enrichment efficiency of the module is ≥95%, which can effectively enrich toxin-producing cyanobacteria with a concentration as low as 10cfu / mL.

[0008] Preferably, the algal cell rapid lysis and DNA extraction tube employs a dual lysis method combining lysis buffer and cross-flow disruption, which can complete algal cell lysis and extract high-purity DNA within 3 minutes. The lysis buffer contains proteinase K, surfactant SDS, and chelating agent EDTA, wherein the concentration of proteinase K is 0.5 mg / mL, the concentration of SDS is 1%, and the concentration of EDTA is 50 mM. The cross-flow disruption device rotates at 15,000 rpm, which can further destroy incompletely lysed algal cells. The extracted DNA has a purity that meets the requirements of OD260 / OD280 = 1.8-2.0, is free from protein and polysaccharide contamination, and can be directly used for subsequent isothermal PCR amplification without additional purification treatment.

[0009] Preferably, the isothermal / room-temperature PCR module integrates a multi-channel handheld thermostat, PCR-specific primers, and probes. The thermostat employs Peltier thermoelectric cooling / heating technology, with a temperature control accuracy of ±0.5℃, and can be adjusted to a constant temperature within the range of 37-65℃, adaptable to LAMP, RPA, MIRA, and other isothermal PCR reactions. The module supports simultaneous detection of 4-8 parallel channels and can simultaneously detect 2-4 target toxin-producing genes. The specific primers are 20-25 bp in length, and the probes are 25-30 bp in length, employing TaqMan probe technology, with a fluorescent group labeled at the 5' end and a quencher group labeled at the 3' end. The fluorescent group is selected from at least one of FAM, HEX, and CY5, and the quencher group is selected from BHQ1 or BHQ2. The primer and probe specificity is 100%, with no cross-reactivity, and the detection sensitivity can reach 10. 2 Copy / mL.

[0010] Preferably, the handheld fluorescence detector includes a high-brightness LED excitation source and a high-sensitivity photodiode detector. The LED excitation source has a wavelength stability of ±2nm, energy consumption ≤10W, and a detection wavelength range of 450-650nm. The detector has a built-in high-performance data processing chip that can automatically acquire fluorescence signals, plot amplification curves, calculate Ct values, and determine whether the detection result is positive or negative. It can store ≥1000 sets of detection data. The qualitative and quantitative algorithms and the software for calculating the risk warning level of toxin-producing and odor-producing cyanobacteria use a random forest machine learning algorithm to establish a warning model. The qualitative algorithm can determine the presence or absence of the target toxin-producing gene based on the fluorescence signal and Ct value, eliminating false positive and false negative interference. The quantitative algorithm is based on the linear relationship between fluorescence signal intensity and target gene copy number, with a detection limit of 10. 3 copies / mL, linear range of 10 3 -10 8The risk warning model, which combines the copy number of the toxin-producing gene, on-site environmental parameters, historical monitoring data, and meteorological forecast information, classifies the risk of algal blooms into four levels: low, medium, high, and extremely high, and provides corresponding control recommendations for different risk levels. The on-site environmental parameters include water temperature, pH, dissolved oxygen, and transparency, which can be collected in real time through external sensors.

[0011] Preferably, the mobile APP software is compatible with iOS 11.0 and above and Android 8.0 and above, connects to the detection device via Bluetooth 4.0, has a connection distance of ≤10m, and can connect to multiple detection devices simultaneously; the APP has functions such as device control, data display, historical record query, risk warning push, and data analysis report generation, and can remotely start / stop detection, set detection parameters, and export detection data to Excel or PDF format.

[0012] Preferably, the overall weight of the monitoring system is <1kg, and each core module is compact. The isothermal / room temperature PCR module has a size ≤15cm×10cm×5cm and a weight ≤0.3kg. It has an IP68 protection rating, is suitable for complex outdoor environments, and can be powered by a portable power bank, eliminating the need for a professional laboratory environment. The monitoring system can be applied to drinking water source monitoring, lake and reservoir water quality monitoring, aquaculture water quality management, and environmental emergency monitoring. It can detect various toxin-producing and odor-producing cyanobacteria such as Microcystis, Anabaena, and Cylindrica, and is suitable for grassroots monitoring, outdoor inspections, and emergency response to sudden algal blooms.

[0013] A rapid on-site monitoring method for toxin- and odor-producing cyanobacteria based on toxin- and odor-producing genes includes the following steps: S1. The system includes two detection modes: Mode 1 is a qualitative rapid monitoring based on RPA-LFS technology, and Mode 2 is a quantitative monitoring based on real-time fluorescence isothermal PCR technology. Mode 1 can complete the entire detection process within 30 minutes, without the need for complex fluorescence detection instruments, and the results are visualized through lateral flow strips. Mode 2 can complete the entire detection process within 60 minutes, with a detection limit of 10. 2 The copy number per mL (copies / mL) allows for precise quantification of the copy number of toxin-producing genes and the biomass of toxin-producing cyanobacteria.

[0014] S2. The specific steps of Mode 1 include: the water sample is processed by the automated enrichment module to obtain a concentrated algal cell suspension, the algal cell suspension is added to a rapid lysis tube, the supernatant is centrifuged after lysis and used as a DNA template, the DNA template is added to the RPA reaction system, magnesium acetate is added to start the reaction, the reaction is carried out at 37-42℃ for 15-20 minutes, the reaction product is dropped into the sample well of the side flow strip, buffer is added and allowed to stand for 5 minutes, and the detection results are judged according to the band display.

[0015] S3. The specific steps of the final mode two include: the water sample is processed by the automated enrichment module to obtain a concentrated algal cell suspension, and high-purity DNA is extracted after lysis as a template. The DNA template is added to the real-time fluorescence isothermal PCR reaction system, placed in a handheld fluorescence detector, and the corresponding reaction temperature is set to start real-time fluorescence monitoring. The Ct value is calculated based on the fluorescence amplification curve, and the target gene copy number is determined in combination with the standard curve to estimate the biomass of toxin-producing cyanobacteria.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention addresses the core pain points of current aquatic algae monitoring technologies by deeply integrating isothermal PCR with portable detection equipment. It achieves rapid, accurate, and convenient integrated detection of toxin- and odor-producing cyanobacteria, overcoming the limitations of existing technologies in accurately distinguishing between toxin- and non-toxin-producing cyanobacteria and scientifically assessing aquatic health risks. Furthermore, it overcomes the limitations of traditional monitoring technologies, which often rely heavily on laboratory testing, involve bulky equipment, complex operation, and long testing cycles. This invention is well-suited to the timeliness, convenience, and ease-of-operation requirements of on-site scenarios such as grassroots monitoring, outdoor inspections, and emergency response to sudden algal blooms. It provides efficient and reliable technical support for drinking water safety, aquaculture management, and ecological environment governance, filling the technological gap in accurate and rapid on-site monitoring of toxin- and odor-producing cyanobacteria.

[0017] This invention designs highly specific and sensitive primers and probes to effectively avoid cross-reactions with genes from other algae such as non-toxic cyanobacteria, diatoms, and green algae. Based on this, a portable on-site rapid monitoring system is constructed, integrating automated enrichment of algae in water samples, rapid DNA extraction from algal cell lysis, isothermal PCR amplification, fluorescence detection, qualitative and quantitative analysis, and risk warning. The modules work together to automate the entire process from water sample collection to test result output and risk assessment, requiring minimal human intervention. It is suitable for complex on-site scenarios such as outdoor areas without power supply and operation by non-professional personnel, breaking the lengthy process of traditional monitoring "sampling-sending for testing-detection-result output" and significantly improving monitoring efficiency and practicality. Attached Figure Description

[0018] Figure 1 This is a vector diagram illustrating the overall system architecture of the present invention; Figure 2 This is a schematic diagram of the structure of the automated algae enrichment module for water samples of the present invention; Figure 3 This is a schematic diagram of the structure of the cell rapid lysis and DNA extraction tube of the present invention; Figure 4 This is a schematic diagram of the handheld fluorescence detector of the present invention; Figure 5 This is a schematic diagram of the detection process based on the test strip method of the present invention; Figure 6This is a vector diagram of quantitative monitoring based on a fluorescence detector according to the present invention; Figure 7 This is a vector diagram illustrating the automated water sample collection upgrade of this invention. Figure 8 This is a vector diagram of the risk warning algorithm for toxic and odor-producing cyanobacteria of the present invention. Figure 9 This is a vector graphic representation of the mobile app software development for this invention. Detailed Implementation

[0019] The technical solutions of 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.

[0020] Example 1: Qualitative rapid monitoring based on test strip method: Step 1: Water Sample Collection and Pretreatment The core purpose of this step is to obtain an initial sample free from bacterial contamination and enriched with target cyanobacteria cells, laying the foundation for subsequent detection. The specific operation is as follows: Using a portable sterile sampler of model SPB-01, water samples are collected from the surface (0-50cm), middle (50-100cm), and deep (100-150cm) layers at the monitoring point. After mixing, 1L is taken as the test sample (to ensure that the sample is representative and avoid the randomness of single-point sampling). After collection, sodium azide (final concentration 0.02%) is immediately added to the water sample. Its function is to inhibit the growth and reproduction of bacteria and planktonic microorganisms in the water, prevent the release of bacterial DNA from interfering with the detection of target genes, and avoid the destruction of cyanobacteria cell structure by bacterial metabolites. The treated water sample was slowly poured into the automated algae enrichment module (module model ZJB-02). The module automatically started the negative pressure filtration program, adjusting the negative pressure to 0.02-0.05 MPa. A replaceable polyethersulfone filter membrane with a pore size of 0.45 μm was selected (the material is heat-resistant, acid and alkali-resistant, effectively retaining cyanobacterial cells while allowing small molecule impurities to pass through, avoiding membrane clogging). After filtration, 10 mL of phosphate buffered saline (PBS, pH 7.4, concentration 0.01 mol / L) was slowly added dropwise to the surface of the filter membrane. The enrichment module was then connected to an ultrasonic oscillator (power 200W, frequency 40kHz), and oscillated for 3 minutes. The ultrasonic shear force thoroughly eluted the cyanobacterial cells adsorbed on the filter membrane into the buffer, obtaining a concentrated algae cell suspension. After elution, the suspension was transferred to a sterile centrifuge tube and refrigerated at 4°C for later use, ensuring it was ready for the subsequent magnetic bead enrichment step within 1 hour to avoid cell inactivation.

[0021] Step 2: Magnetic bead enrichment This step aims to further increase the concentration of the target toxin-producing cyanobacteria cells and eliminate interference from diatoms, green algae, and other miscellaneous algae and suspended impurities in the water. The specific operation is as follows: Accurately add 100 μL of magnetic bead suspension (magnetic bead size 1 μm, concentration 10) to the algal cell eluent obtained in step 1. 8 The magnetic beads, with a surface modified with a Microcystis-specific monoclonal antibody at a concentration of 1 mg / mL, specifically bind to the unique antigens on the cell membrane surface of toxin-producing Microcystis cells, without binding to non-toxin-producing cyanobacteria or other algae. Centrifuge tubes were placed in a rotary mixer (180 rpm, 25°C) and reacted at this temperature for 30 minutes. During this time, slow rotation was used to ensure sufficient contact and specific binding between the magnetic beads and the target cyanobacterial cells, forming a magnetic bead-cell complex. After the reaction, the centrifuge tubes were placed in a magnetic separator (0.3T magnetic field strength) and allowed to stand for 5 minutes, allowing the magnetic bead-cell complex to adhere to the bottom of the centrifuge tube under the influence of the magnetic field. The supernatant was slowly poured off to remove unbound algae, impurities, and free antibodies. Subsequently, 5 mL of PBS buffer (pH 7.4) was added to the centrifuge tube, and the tube was gently inverted three times to wash the magnetic bead-cell complex. The tube was then placed back in the magnetic separator for separation, and the washing process was repeated three times to thoroughly remove non-specifically adsorbed impurities and residual antibodies, ensuring the purity of subsequent DNA extraction. After washing, the magnetic bead-cell complex at the bottom of the centrifuge tube was retained for later use.

[0022] Step 3: Cell lysis and DNA extraction The core of this step is to rapidly lyse cyanobacterial cells to release high-purity target DNA, providing a suitable template for subsequent RPA amplification. The specific operation is as follows: Add 200 μL of optimized lysis buffer (formula: proteinase K 0.5 mg / mL, SDS 1%, EDTA 50 mM, Tris-HCl 10 mM, pH 8.0) to a centrifuge tube containing the magnetic bead-cell complex. The components work synergistically: proteinase K degrades proteins within cyanobacterial cells, destroying intracellular structures and releasing DNA; SDS, as a surfactant, disrupts the lipid bilayer structure of the algal cell membrane, causing complete cell lysis; EDTA chelates residual heavy metal ions in the water, preventing them from inhibiting subsequent enzymatic reactions and protecting DNA from nuclease degradation; Tris-HCl maintains a stable pH environment in the lysis system. The lysis tube was placed in a cross-flow disruption apparatus (model CFS-03, speed 15000 rpm, disruption time 3 minutes, shear force 5000 psi) to further disrupt the incompletely lysed cyanobacterial cell walls using high-speed fluid shear force (cyanobacterial cell walls are tough and difficult to completely lyse with a single lysis buffer; cross-flow disruption can significantly improve lysis efficiency). After disruption, the lysis tube was placed in a mini-centrifuge (model Mini-10K, speed 12000 rpm, centrifugation radius 8 cm, temperature 4℃) and centrifuged for 5 minutes to allow cell debris, magnetic beads, and other impurities to precipitate to the bottom of the tube. The supernatant is the crude extract containing the target DNA, which does not require additional purification (after optimizing the lysis buffer formula, the residual impurities such as proteins and polysaccharides can be effectively reduced, and the crude extract can be directly used for RPA reactions). The supernatant was transferred to a new sterile EP tube as a DNA template for later use.

[0023] Step 4: RPA reaction RPA reaction is the core amplification step in this detection mode. Utilizing the speed and efficiency of recombinase polymerase amplification technology, it achieves rapid amplification of the target toxin-producing gene. The specific procedure is as follows: Prepare sterile EP tubes (1.5 mL in volume) in advance. In a sterile environment (clean bench, Class 100 cleanliness), prepare a 50 μL RPA reaction system. The components and amounts of each component should be precisely controlled as follows (to ensure amplification efficiency and specificity): 5 μL of template DNA (take the DNA supernatant obtained in step 3, avoiding aspiration of impurities at the bottom of the tube. If the template concentration is too low, it can be increased to 8 μL, while reducing the amount of ultrapure water used). Upstream primer (10 μM) 2.4 μL (Primer sequence designed for the mcyG gene, sequence 5'-AGTGGCTGACGAAGATGAAA-3', specifically binds to the upstream fragment of the target gene) 2.4 μL of downstream primer (10 μM) (primer sequence is 5'-TCCTTGGTGATGGTGATGTT-3', which specifically binds to the downstream fragment of the target gene and works synergistically with the upstream primer to amplify the target fragment) 0.2 μL of probe (10 μM) (probe sequence: 5'-FAM-CTGCGGCTGCTGCTGCTG-BHQ1-3', 5' end labeled with FAM fluorescent group, 3' end labeled with BHQ1 quencher group, used for signal recognition in subsequent lateral flow strip detection); 29.5 μL of recombinase polymerase mixture (containing recombinase, single-strand binding protein, and DNA polymerase; recombinase promotes primer binding to target DNA single strands, single-strand binding protein protects single-stranded DNA from degradation, and DNA polymerase catalyzes DNA strand elongation to achieve isothermal amplification of the target gene); ultrapure. Add 12.2 μL of water (enzyme-free ultrapure water to avoid RNase and DNase contamination and prevent interference with the reaction) and 2.5 μL of magnesium acetate (280 mM) (as an initiator for the RPA reaction, which can quickly activate the recombinase polymerase system and start the amplification reaction). After adding all components, gently invert the EP tube 5 times to ensure the reaction system is thoroughly mixed and avoid the formation of air bubbles (air bubbles will affect the efficiency of the enzymatic reaction during the amplification process and lead to amplification failure). Then, place the EP tube in a microcentrifuge and centrifuge briefly for 10 seconds (5000 rpm) to allow the liquid on the inner wall of the tube to concentrate at the bottom of the tube. Place the reaction tube into the handheld thermostat (model PHC-01, dimensions 15cm×10cm×5cm, weight 0.3kg, powered by a lithium battery, temperature control accuracy ±0.5℃), set the reaction temperature to 37℃ (the optimal reaction temperature for RPA enzyme, at which amplification efficiency is highest, and no complex temperature control equipment is required, suitable for on-site detection), and incubate at this temperature for 20 minutes. No manual intervention is required during this time, as the device automatically maintains a constant temperature to ensure efficient amplification of the target gene (the amplification product is the target gene fragment, approximately 200bp in length).

[0024] Step 5: Sideflow strip inspection This step utilizes a sideflow strip to achieve visual qualitative detection of the amplification product. It is simple to operate, requires no complex instruments, and is suitable for rapid on-site result assessment. The specific operation is as follows: After the RPA reaction is completed, remove the reaction tube and cool it to room temperature (25°C) to avoid high temperature affecting the detection effect of the sideflow strip. Use a sterile pipette to accurately aspirate 10 μL of the reaction product and slowly drop it into the sample well of the sideflow strip (sideflow strip model LFS-CY01, size 4mm×60mm, including sample well, conjugate pad, nitrocellulose membrane, absorbent pad, the nitrocellulose membrane has a detection line T line and a control line C line. The T line is coated with the target gene complementary probe, and the C line is coated with anti-FAM antibody). After the addition is complete, add 2-3 drops of sideflow strip buffer (pH 7.2, 0.01 mol / L PBS, containing 0.5% Tween-20, which can promote the migration of reaction products on the sideflow strip) to the sample well. Let it stand at room temperature (25-30℃, 50%-60% humidity) for 5 minutes. During this time, the reaction products will migrate towards the absorbent pad with the buffer under capillary action. If the reaction products contain the target amplification fragment, the FAM fluorescent group on the fragment will bind to the anti-FAM antibody on the binding pad, and then specifically bind to the complementary probe of the target gene on the T line to form a double antibody sandwich complex, which makes the T line appear colored. Regardless of whether the target amplification fragment is present, the anti-FAM antibody in the buffer will bind to the anti-FAM antibody on the C line, which makes the C line appear colored (the appearance of the C line indicates that the sideflow strip is effective and the detection process is normal). The result interpretation criteria are as follows: ① Positive result: Both the test line (T line) and the control line (C line) show clear red bands, indicating the presence of the target toxin-producing gene in the sample, i.e., the presence of the corresponding toxin-producing cyanobacteria in the water; ② Negative result: Only the control line (C line) shows a clear red band, and the test line (T line) shows no color, indicating the absence of the target toxin-producing gene in the sample, i.e., the absence of the corresponding toxin-producing cyanobacteria in the water; ③ Invalid result: The control line (C line) does not show a red band, regardless of whether the T line shows color, indicating that the sideflow strip is faulty, the reaction system is contaminated, or there is an operational error, requiring retesting. After testing, the reaction products, sideflow strips, and other waste should be disposed of according to medical waste regulations to avoid environmental pollution and cross-contamination.

[0025] Example 2: Quantitative monitoring based on a fluorescence detector: This embodiment addresses the need for routine and precise monitoring of sensitive areas such as drinking water sources and landscape water bodies. It employs real-time fluorescence isothermal PCR technology to accurately quantify the copy number of target genes in toxin-producing and odor-producing cyanobacteria, thereby estimating the biomass of these toxin-producing algae and providing scientific data support for water health risk assessment. Compared to the qualitative detection in Mode 1, this mode offers higher detection accuracy and more valuable data. The specific operation steps are as follows: Step 1: Water Sample Treatment Following steps 1-3 of Example 1, extract the DNA template. Special attention should be paid to the fact that quantitative detection requires higher purity and concentration of the DNA template. Strict aseptic techniques must be followed during extraction to avoid DNA degradation or contamination (contamination will lead to higher quantitative results, while degradation will lead to lower results). After extraction, the DNA concentration can be quickly detected using a miniature nucleic acid concentration analyzer (Nano-100, detection range 2-1000 ng / μL) to ensure the template concentration is within the range of 50-200 ng / μL. If the concentration is too high, it needs to be diluted with enzyme-free ultrapure water; if the concentration is too low, it needs to be re-enriched and extracted to ensure the accuracy and repeatability of subsequent quantitative amplification.

[0026] Step 2: Real-time fluorescence isothermal PCR The core of this step is to achieve precise amplification and signal capture of the target gene through real-time fluorescence monitoring of the isothermal PCR amplification process, providing reliable data for subsequent quantitative analysis. The specific operation is as follows: In a sterile laminar flow hood (cleanliness level 100), take a sterile EP tube (0.2 mL volume, compatible with the handheld fluorescence detector reaction chamber) and precisely prepare 20 μL of real-time fluorescence isothermal PCR reaction system according to the following ratio: Add reagents in the following order: "enzyme-free ultrapure water → 2× PCR buffer → dNTP mixture → primers → probe → template DNA → Bst DNA polymerase large fragment". Avoid cross-contamination of reagents or affecting enzyme activity. 2 μL of template DNA (Take the qualified DNA template from step 1, avoiding aspiration of impurities at the bottom of the tube. If the template concentration is 100 ng / μL, it can be used directly; if the concentration is too high, it needs to be diluted 1:1 with enzyme-free ultrapure water before use to ensure accurate template dosage) 10 μL of 2×PCR buffer (containing Mg) 2 + 20mM, Tris-HCl 100mM, KCl 50mM, pH 8.8, the core function is to maintain a stable pH environment in the reaction system, providing the necessary ionic conditions for the enzymatic reaction, Mg 2 + It can promote primer-template binding and enhance the activity of Bst DNA polymerase. 2 μL of dNTP mixture (each 2.5 mM) (containing four deoxyribonucleoside triphosphates: dATP, dTTP, dCTP, and dGTP, which serve as raw materials for DNA amplification, providing nucleotides for the extension of the target gene fragment. The concentrations of each component are uniform to ensure sufficient raw materials and appropriate proportions during the amplification process.) 0.5 μL of upstream primer (10 μM) (The primer sequence is consistent with that in Example 1, designed for the mcyG gene, with the sequence 5'-AGTGGCTGACGAAGATGAAA-3', which specifically binds to the conserved upstream sequence of the target gene to ensure that only the target gene of the toxin-producing cyanobacteria is amplified and there is no cross-reaction) 0.5 μL of downstream primer (10 μM) (primer sequence is 5'-TCCTTGGTGATGGTGATGTT-3', which works synergistically with the upstream primer to define the length of the target amplified fragment (approximately 200 bp). The primer concentration is controlled at a final concentration of 0.25 μM to avoid primer dimer formation, which would affect amplification efficiency.) 0.2 μL of probe (10 μM) (Probe sequence: 5'-FAM-CTGCGGCTGCTGCTGCTG-BHQ1-3', 5' end labeled with FAM fluorescent group, 3' end labeled with BHQ1 quencher group. The probe specifically binds to the conserved middle sequence of the target gene. When PCR amplification occurs, the strand displacement activity of Bst DNA polymerase hydrolyzes the probe, separating the fluorescent group from the quencher group and releasing a fluorescent signal. The intensity of the fluorescent signal is positively correlated with the copy number of the target gene.) Bst DNA polymerase large fragment 1μL (concentration 8U / μL, derived from Bacillus stearothermophilus, possessing thermostable properties, with an optimal reaction temperature of 60-65℃, no 5'-3' exonuclease activity, and strong strand displacement activity, enabling efficient DNA amplification under isothermal conditions, suitable for the isothermal requirements of rapid on-site detection) 3.8 μL of ultrapure water (enzyme-free and nucleic acid-free ultrapure water, used to make up the volume of the reaction system to avoid the concentration of other reagents in the system being too high or too low, to ensure that the enzyme-catalyzed reaction proceeds efficiently. It should be added slowly dropwise to avoid generating bubbles.) After adding all reagents, gently invert the EP tube 3-5 times to thoroughly mix the reaction system and avoid excessively high local reagent concentrations that could affect amplification. Then, place the EP tube in a mini-centrifuge (Mini-10K) and briefly centrifuge at 5000 rpm for 10 seconds to concentrate the liquid on the inner wall of the tube to the bottom and completely eliminate air bubbles (air bubbles can block the fluorescence signal, leading to inaccurate detection results). Place the reaction tube stably into the reaction chamber of the handheld fluorescence detector (PHC-01, 15cm×10cm×5cm, 0.3kg, powered by a lithium battery, temperature control accuracy ±0.5℃, detection wavelength range 450-650nm). Close the instrument lid and set the detection parameters: reaction temperature 65℃ (the optimal reaction temperature for Bst DNA polymerase to ensure maximum amplification efficiency), reaction time 60 minutes, fluorescence signal acquisition frequency of acquiring the fluorescence intensity of the FAM channel (490-520nm) every 1 minute, and start the real-time fluorescence monitoring program. The instrument will automatically maintain a constant temperature and record the fluorescence signal change curve in real time.

[0027] Step 3: Data Analysis After the real-time fluorescence isothermal PCR reaction is completed, the high-performance data processing chip built into the handheld fluorescence detector will automatically process the collected fluorescence signal data, plot the fluorescence amplification curve (the horizontal axis is the reaction time and the vertical axis is the fluorescence signal intensity), and calculate the Ct value (cycle threshold, which refers to the reaction time required for the fluorescence signal intensity to reach the set threshold. The Ct value is negatively correlated with the number of target gene copies in the sample, that is, the more target gene copies, the smaller the Ct value.

[0028] To achieve accurate quantification of the target gene copy number, a standard curve needs to be established in advance: select a known concentration of mcyG gene standard (concentration range 10). 2 -10 8 (copy / mL), diluted 10-fold to create 6 concentration gradients (10... 2 10 3 10 4 10 5 10 6 10 8 (Copies / mL), with 3 replicates for each concentration gradient, and real-time fluorescence isothermal PCR amplification was performed according to step 2 of this embodiment, with the result calculated as the logarithm of the standard concentration (log... 10 Using the copy number as the x-axis and the corresponding average Ct value as the y-axis, a standard curve was plotted using linear regression. The equation of the standard curve was: Ct = -3.32log 10 (Copy count) + 35.6, Correlation coefficient R2 =0.998 (R) 2 The closer R is to 1, the higher the goodness of fit of the standard curve, and the stronger the accuracy and reliability of the quantitative results. In this embodiment, R... 2 =0.998, indicating that the standard curve fits well and can be used for quantitative calculation of the target gene copy number in the sample.

[0029] By substituting the Ct value of the sample into the above standard curve equation, the copy number of the mcyG gene in the sample can be accurately calculated. Combined with the pre-established conversion formula of "target gene copy number - toxin-producing cyanobacterial cell density" (experimentally verified, the conversion relationship between mcyG gene copy number and toxin-producing Microcystis cell density is: cell density (cfu / mL) = 0.85 × gene copy number (copy / mL) + 120), the biomass of toxin-producing cyanobacteria in the sample can be further estimated, clarifying the degree of toxin-producing cyanobacteria contamination. Simultaneously, the average value and coefficient of variation are calculated for the three parallel replicate detection results of each sample, ensuring that the coefficient of variation is less than 5%. If the coefficient of variation is greater than 5%, the sample needs to be retested to eliminate result deviations caused by operational errors or contamination, ensuring the repeatability and reliability of the quantitative detection results.

[0030] Example 3: Multi-gene joint detection In real-world aquatic environments, pollution from toxin-producing and odor-producing cyanobacteria often exhibits complex characteristics. Detection of a single toxin-producing gene is insufficient to comprehensively cover all potential risks in aquatic environments. For example, in eutrophic lakes such as Taihu Lake and Chaohu Lake, both *Microcystis* (producing microcystin) and *Cylindrome* (producing columnar toxin) often coexist. Detecting only a single gene can easily lead to missed risks, affecting the scientific validity of control decisions. To address this issue, this embodiment designs a multiplex PCR system to simultaneously detect three core toxin-producing genes: mcyB, mcyG, and cyrJ. mcyB and mcyG are key genes in microcystin synthesis (involved in the synthesis and modification of the core active group of the toxin, respectively), while cyrJ is a characteristic gene in columnar toxin synthesis (responsible for the sulfation modification of the toxin molecule). Through multi-target joint detection, both *Microcystis* and *Cylindrome* toxin-producing cyanobacteria can be accurately identified simultaneously, significantly improving the comprehensiveness and efficiency of toxin-producing cyanobacteria monitoring in complex aquatic scenarios, reducing the number of tests and costs, and adapting to the monitoring needs of complex pollution in real-world aquatic environments.

[0031] This embodiment of the multiplex PCR system achieves simultaneous amplification of three genes in a single reaction system. The core challenge lies in avoiding cross-interference between different primers and ensuring consistent amplification efficiency for each target site. Therefore, the concentration ratio of primers and probes and the components of the reaction system were specifically optimized. The specific reaction system (total volume 25 μL) and detailed descriptions of each component are as follows. All reagents were selected at a purity level free from nuclease contamination to ensure the accuracy and reproducibility of the amplification results. Template DNA 5 μL: Select qualified DNA templates extracted by enrichment and lysis in Example 1, with a concentration controlled at 50-200 ng / μL. If the template concentration is too high, it needs to be serially diluted with enzyme-free ultrapure water to avoid excessive template leading to primer competition and decreased amplification efficiency. The template should be briefly centrifuged before being added to avoid impurities at the bottom of the tube from entering the system and causing contamination or signal interference.

[0032] 2×Multiplex PCR Master Mix 12.5μL: As the core basic component of the system, this premix has been pre-integrated with heat-resistant Bst DNA polymerase (8U / μL, adapted to isothermal amplification requirements) and 10×PCR buffer (containing Mg). 2 The mixture of 20mM Tris-HCl, 100mM KCl, 50mM (pH 8.8), 2.5mM dNTPs, and an anti-interference agent can effectively maintain a stable pH environment in the reaction system, providing sufficient raw materials and ionic conditions for the enzymatic reaction, while inhibiting the interference of impurities in the water on the amplification reaction, ensuring the synchronicity and high efficiency of multi-target amplification.

[0033] 0.5 μL of mcyB primer pairs (10 μM each): The primer pairs are designed with conserved sequences of the mcyB gene (upstream primer sequence: 5'-GCTGACGAAGATGAAAGCGT-3', downstream primer sequence: 5'-TCCTTGGTGATGGTGATGTT-3'), each 20 bp in length. They specifically bind to the upstream and downstream fragments of the key gene for microcystin synthesis, ensuring that only the mcyB gene of toxin-producing Microcystis is amplified without cross-reaction. The 0.5 μL amount ensures a final primer concentration of 0.2 μM in the system, guaranteeing amplification efficiency while avoiding excessive primers forming dimers that could affect the detection results.

[0034] 0.5 μL of mcyG primer pairs (10 μM each): The primer pairs are designed with conserved sequences of the mcyG gene (upstream primer sequence: 5'-AGTGGCTGACGAAGATGAAA-3', downstream primer sequence: 5'-TGTGGTGATGGTGATGTTGG-3'), both 20 bp in length. The mcyG gene is a key gene for the synthesis of the Adda active group of microcystin. Detection of this gene can further verify the toxin-producing ability of Microcystis. Synergistic detection with the mcyB gene improves the accuracy of the determination of the toxin-producing characteristics of Microcystis. The primer dosage is the same as that of the mcyB primers to ensure balanced amplification efficiency of the two Microcystis-related genes.

[0035] 0.5 μL of cyrJ primer pairs (10 μM each): The primer pairs are designed with conserved sequences of the cyrJ gene (upstream primer sequence: 5'-GCGGCTGCTGCTGCTGCTGT-3', downstream primer sequence: 5'-CGTGGCTGACGAAGATGAAA-3'), each 20 bp in length. The cyrJ gene is responsible for the sulfation modification of the Cylindrica toxin molecule and is the core marker gene of the Cylindrica toxin-producing strain. This primer pair can specifically bind to the Cylindrica cyrJ gene, effectively avoiding cross-reactions with genes from other algae such as Microcystis and Chlorella, ensuring the specificity of Cylindrica detection. The primer dosage is controlled in the same way as the previous two types to avoid competition and interference between different primers.

[0036] 0.2 μL of mcyB probe (FAM-labeled): The probe sequence is 5'-FAM-CTGCGGCTGCTGCTGCTG-BHQ1-3', with the 5' end labeled with the FAM fluorescent group (detection wavelength 490-520 nm) and the 3' end labeled with the BHQ1 quencher group. The probe specifically binds to the conserved middle sequence of the mcyB gene. When the mcyB gene is amplified, the probe is hydrolyzed, the fluorescent group separates from the quencher group, and the FAM channel fluorescent signal is released. The signal intensity is positively correlated with the copy number of the mcyB gene.

[0037] mcyG probe (HEX labeled) 0.2μL: The probe sequence is 5'-HEX-CGAGCGGCTGCTGCTGCTG-BHQ2-3', with the 5' end labeled with the HEX fluorescent group (detection wavelength 535-556nm) and the 3' end labeled with the BHQ2 quencher group. It specifically binds to the conserved middle sequence of the mcyG gene and releases the HEX channel fluorescence signal during amplification for qualitative and quantitative detection of the mcyG gene. It does not overlap with the FAM channel fluorescence signal and can achieve simultaneous detection.

[0038] 0.2 μL of cyrJ probe (CY5 labeled): The probe sequence is 5'-CY5-GGCGGCTGACGAAGATGAA-BHQ3-3', with the 5' end labeled with the CY5 fluorescent group (detection wavelength 643-667 nm) and the 3' end labeled with the BHQ3 quencher group. It specifically binds to the conserved middle sequence of the cyrJ gene and releases the CY5 channel fluorescence signal during amplification. It does not cross-interfere with the first two fluorescent channels, ensuring the accuracy of the detection results of the three genes.

[0039] 3.1 μL of ultrapure water: Use enzyme-free and nucleic acid-free ultrapure water to bring the reaction system to 25 μL. Avoid excessively high or low concentrations of other reagents in the system, which may affect the efficiency of the enzyme-catalyzed reaction. When adding, add slowly dropwise to avoid generating bubbles, as bubbles will block the fluorescence signal and lead to deviations in the detection results.

[0040] Optimization and control of reaction conditions: Considering the amplification characteristics of the three genes and the compatibility of primers and probes, the optimized reaction conditions are a constant temperature reaction at 60℃ for 60 minutes. This temperature is the optimal reaction temperature for Bst DNA polymerase, which can simultaneously meet the specific binding and amplification requirements of the three primers, avoiding non-specific primer binding due to excessively high temperatures and amplification efficiency affected by excessively low temperatures. The temperature control accuracy needs to reach ±0.5℃. A handheld thermostat device (15cm×10cm×5cm, 0.3kg, powered by a lithium battery) consistent with that used in Example 2 can be used. During the reaction, the device must be kept sealed to avoid temperature fluctuations, ensuring balanced amplification efficiency of the three genes and reducing amplification deviation. After the system is prepared, the EP tube should be gently inverted 3-5 times to mix, and then briefly centrifuged at 5000rpm for 10 seconds to eliminate air bubbles before placing it in the thermostat device to start the reaction.

[0041] Interpretation of Detection Results and Advantages: After the reaction, place the reaction tube into the handheld fluorescence detector. The instrument automatically collects fluorescence signals from the FAM, HEX, and CY5 channels and plots fluorescence amplification curves. Based on the changes in fluorescence signals in each channel, it can simultaneously determine whether the sample contains Microcystis aeruginosa producing microcystin and Cyclocystis aeruginosa producing microcystin. The specific judgment criteria are as follows: ① If the FAM and / or HEX channels show obvious amplification curves (fluorescence signals reach the set threshold), it indicates the presence of the mcyB and / or mcyG genes in the sample, i.e., the presence of Microcystis aeruginosa producing microcystin; ② If the CY5 channel shows obvious amplification curves, it indicates the presence of the cyrJ gene in the sample, i.e., the presence of Cyclocystis aeruginosa producing microcystin; ③ If none of the three channels show amplification curves, it indicates the absence of the corresponding toxin-producing gene in the sample, i.e., the absence of related toxin-producing cyanobacteria; ④ If only one channel shows a non-specific amplification curve (atypical amplification curve, Ct...), it indicates the presence of Micro ...④ If only one channel shows a non-specific amplification curve (atypical amplification curve, Ct...), it indicates the presence of Microcystis aeruginosa producing microcystin; ⑤ If only one channel shows a non-specific amplification curve (atypical amplification curve, Ct...), it indicates the presence of Microcystis aeruginosa producing micro (If the value is too high), retesting is required to rule out reagent contamination or operational errors. The advantage of this multi-gene joint detection system is that it can simultaneously detect two toxin-producing cyanobacteria and three core toxin-producing genes in a single reaction system, eliminating the need for multiple tests, significantly shortening the detection time (only 60 minutes) and reducing detection costs. It also improves the comprehensiveness of toxin-producing cyanobacteria monitoring in complex water environments, effectively avoiding the risk of missed detection caused by single-gene detection. It meets the core requirements of "high efficiency, comprehensiveness, and accuracy" in actual water environment monitoring. Experimental verification shows that the system has 100% specificity, no cross-reaction, and a detection limit consistent with single-gene detection, reaching 10. 2 Copy / mL.

[0042] Example 4: Automation Upgrade of Water Sampling: To further enhance the automation level of on-site monitoring and address the pain points of traditional manual sampling, such as time-consuming and labor-intensive processes, uneven sampling accuracy, low batch processing efficiency, and difficulty in achieving continuous monitoring, this invention, based on core detection technologies, has developed an integrated water sample collection and processing system. This system automates the entire process of water sample collection, enrichment, injection, and data recording without requiring manual intervention, significantly reducing the workload of grassroots monitoring personnel. It also improves the standardization of sampling and the accuracy and continuity of data. This integrated system seamlessly integrates with the aforementioned detection module, directly providing standardized pre-processed samples for subsequent nucleic acid extraction and PCR amplification. The specific components and detailed functions are as follows: Automatic Sampling Unit: The core of the unit consists of a high-precision peristaltic pump and an electromagnetic three-way valve. The entire unit is made of corrosion-resistant, non-polluting polytetrafluoroethylene (PTFE), suitable for sampling various water bodies (including freshwater and lightly polluted water). The peristaltic pump is a low-noise, pulse-free model with an adjustable speed range of 0-60 rpm and a flow accuracy of ≤±2%, effectively avoiding sample disturbance caused by pulse sampling and ensuring the representativeness of the collected water samples. The electromagnetic three-way valve has a fast response speed (≤0.5s), strong sealing, and can flexibly switch sampling channels according to a preset program, achieving automatic switching and sampling of water samples at different depths and locations without manual disassembly of the pipeline. The sampling depth range is set to 0-5 meters and can be precisely adjusted via the system's built-in lifting bracket. The sampling interval can be customized between 0.1-0.5 meters (default 0.5 meters) to adapt to the water depth distribution characteristics of different water bodies—in shallow water areas, the sampling interval can be reduced to accurately capture the vertical distribution differences of cyanobacteria, while in deep water areas, the default interval can be used for efficient sampling, balancing accuracy and efficiency. In addition, the unit also integrates a dry-out protection device, which can automatically stop and trigger an alarm when there is no water in the sampling pipeline to prevent the peristaltic pump from running dry and being damaged. It is also equipped with a sample preservation module, which automatically adds sodium azide (final concentration 0.02%) to the water sample after sampling to inhibit microbial growth and ensure the stability of the water sample during transportation and pretreatment, avoiding contamination by other microorganisms that could affect subsequent test results.

[0043] Online enrichment unit: Seamlessly integrated with the automatic sampling unit, this unit combines negative pressure filtration and magnetic bead enrichment, enabling continuous and automated enrichment of water samples. This solves the problems of low efficiency and uneven enrichment effects associated with traditional manual enrichment. The negative pressure filtration module uses a miniature vacuum pump for power, with a negative pressure adjustment range of 0.02-0.05 MPa. It automatically adjusts the negative pressure based on the turbidity of the water sample—reducing it appropriately when turbidity is high to prevent membrane clogging, and increasing it when turbidity is low to improve filtration efficiency. The filter membrane is a replaceable polyethersulfone membrane with a fixed pore size of 0.45 μm. It effectively traps cyanobacteria (including toxin-producing and odor-producing cyanobacteria) in the water while filtering out fine suspended impurities. The membrane uses a snap-on installation for automatic replacement, eliminating the need for manual disassembly. The magnetic bead enrichment module uses magnetic microspheres (1-5 μm in diameter) with surface-modified cyanobacterial-specific monoclonal antibodies. These microspheres specifically adsorb toxin-producing cyanobacteria, effectively eliminating interference from other algae such as diatoms and green algae, as well as heavy metal ions and organic matter in the water. The enrichment efficiency can reach over 95%. The processing flow rate of this unit can be flexibly adjusted between 100-500 mL / min according to sampling needs, with a default flow rate of 300 mL / min. It can continuously process 18 L of water samples per hour, enabling rapid enrichment of large batches of water samples. The enriched algal cell suspension can be directly transported to the rapid algal cell lysis module via automated tubing, eliminating the need for manual transfer and avoiding sample loss and contamination.

[0044] The automated sample introduction unit employs a high-precision rotary sampler with a compact structure, adaptable to the size requirements of portable testing equipment. It can hold 12 standard sample vials (1.5mL / 2mL), supporting automated continuous processing of batch samples. The sampler is driven by a stepper motor, ensuring high positioning accuracy (≤±0.1mm). The adjustable rotation speed enables precise positioning of sample vials, automatic sampling, and automatic cleaning. The sampling volume can be customized between 10-100μL to meet the sample addition requirements of subsequent PCR reaction systems. To avoid cross-contamination, the injection needle is equipped with an automatic cleaning module. After each sampling, the inner and outer walls can be cleaned with a special cleaning solution (deionized water + alcohol). The cleaning time can be preset (default 30s) to ensure that the test results of each batch of samples are not affected by the previous sample. This unit can be linked with the online enrichment unit and lysis module. The enriched samples can be automatically distributed to each sample bottle to achieve the integration of "enrichment-sampling-injection", which greatly improves the efficiency of batch testing. It can complete the injection of 40-60 samples per hour, which is 3-4 times more efficient than manual injection.

[0045] The data recording unit, acting as the "brain" of the automated system, integrates a GPS positioning module, a high-precision sensor module, and a data storage and transmission module. It records key parameters of the entire sampling process in real time and with high accuracy, ensuring data traceability and standardization. Specific recorded parameters include: sampling time (accurate to the second), sampling location (GPS positioning accuracy ≤5m, with simultaneous display of latitude, longitude, and specific address), sampling depth (accuracy ±0.01m), water temperature (accuracy ±0.1℃), water turbidity (accuracy ±1NTU), and sampling flow rate. All parameters are collected and stored synchronously in real time, eliminating the need for manual recording and avoiding human error. Data storage employs a dual "local + cloud" storage mode. Local storage can hold ≥10,000 sets of sampling data, supporting offline storage. Cloud storage allows for synchronous data upload via 4G / 5G or Bluetooth, facilitating remote viewing, aggregation, and analysis by management personnel. Simultaneously, the system automatically generates standardized sampling records in a format conforming to the "Technical Specifications for Water Environment Monitoring," including sampling information, equipment parameters, and environmental parameters. These records can be directly exported to Excel or PDF formats for report preparation and data archiving, significantly improving the standardization of monitoring work.

[0046] This integrated water sample collection and processing system is powered by a 10,000mAh lithium battery, supporting both charging and solar power modes. A single full charge allows for over 8 hours of continuous unattended operation, meeting the needs of long-term outdoor monitoring. The system features a touch-screen interface that allows for pre-setting sampling programs (such as sampling points, depths, intervals, and flow rates). Once started, it operates automatically, completing sampling, enrichment, injection, and data recording without manual intervention. It also includes a fault alarm function, alerting staff via sound, light, and a mobile app to address issues such as pipe blockage, filter depletion, or insufficient battery power. This system reduces the time for traditional manual sampling and pretreatment from 30 minutes per sample to less than 5 minutes, increasing sampling efficiency by more than 6 times. It also reduces errors and contamination risks associated with manual operation, providing a standardized, high-quality sample base for the rapid detection of toxic and odor-producing cyanobacteria, further enhancing the field adaptability and practicality of the entire monitoring technology.

[0047] Example 5: Algorithm for Early Warning of Toxic and Odor-Producing Cyanobacteria Risk: To achieve early detection, early warning, and early response to the pollution risks of toxic and odor-producing cyanobacteria, and to overcome the shortcomings of traditional risk assessments that rely on human experience, are highly subjective, and have delayed warnings, this invention develops a machine learning-based risk warning model for toxic and odor-producing cyanobacteria. This model focuses on the growth patterns and toxin release characteristics of these algae, comprehensively integrating molecular detection data, environmental factors, historical monitoring data, and meteorological information. Through multi-feature fusion analysis, it achieves accurate risk level determination and trend prediction, adapting to the risk management needs of different scenarios such as drinking water sources, lakes and reservoirs, and aquaculture water bodies. Specific design details and implementation logic are as follows: Input parameters: All model input parameters have been screened and verified, and redundant information has been removed to ensure that each type of parameter is closely related to the growth, reproduction, toxin synthesis, and release of toxin-producing and odor-producing cyanobacteria. All parameters have been collected and quantified in a standardized manner to provide reliable data support for accurate model prediction. Detailed descriptions of each parameter are as follows: Toxigenic gene copy number: A core input parameter that directly reflects the quantity and toxigenic potential of toxic and odor-producing cyanobacteria in the water body, and is a key indicator for risk assessment. The copy number data of target genes (mcy series, ana series, cyr series) obtained using the detection technology of this invention have a quantification range of 10. 2 -10 8The data was measured in copies / mL. Outliers (such as sudden increases or decreases in copy number due to operational errors) were removed during data preprocessing. The average value was taken from three parallel tests to ensure data accuracy. Different toxin-producing genes were assigned weights (microcystin gene weight 0.4, anabain gene weight 0.3, columnar cystin gene weight 0.3) and the total toxin-producing gene load was calculated to avoid risky misjudgments caused by single gene detection.

[0048] Environmental parameters (water temperature, pH, dissolved oxygen, transparency, etc.): These are key influencing parameters that directly regulate the growth and reproduction rate and toxin release efficiency of toxic and odor-producing cyanobacteria. Water temperature is collected in real time using a portable water temperature sensor (accuracy ±0.1℃). The suitable water temperature range for the growth of toxic cyanobacteria is 25-35℃. Within this range, for every 1℃ increase in water temperature, the growth rate increases by 8%-12%. A separate water temperature influence coefficient is set in the model. pH value is collected with an accuracy of ±0.01, and the suitable range is 7.5-9.0. Too high or too low pH values ​​will inhibit cyanobacterial photosynthesis, thus affecting toxin synthesis. Dissolved oxygen (DO) is collected with an accuracy of ±0.1mg / L. When the dissolved oxygen content is below 5mg / L, cyanobacteria easily proliferate and toxin release increases. Transparency is measured using the Seidon disc method (accuracy ±1cm). When the transparency is below 50cm, the water body has a high degree of eutrophication, which is conducive to the concealed growth of cyanobacteria. All environmental parameters are standardized and normalized to eliminate dimensional differences.

[0049] Historical monitoring data: This serves as the foundation for model training and optimization, including at least three years of monitoring data from the same period, covering information such as toxin-producing gene copy numbers, environmental parameters, and algal bloom occurrences under different seasons and hydrological conditions. Historical data is categorized by quarter, highlighting parameter changes before, during, and after algal blooms to uncover the intrinsic correlation between toxin-producing cyanobacteria growth and various parameters, and to calibrate the model's prediction accuracy. Simultaneously, water characteristic data from different regions (such as eutrophication levels and hydrodynamic features) are incorporated to improve the model's regional adaptability.

[0050] Meteorological forecast information: These auxiliary forecasting parameters are used to predict the future growth trend of toxic and odor-producing cyanobacteria. They primarily collect data on the average daily temperature, sunshine duration, rainfall intensity, and wind speed over the next 24-48 hours (automatically obtained through an interface with meteorological departments). When sunshine duration exceeds 8 hours / day, there is no rainfall, and wind speed is ≤3, it is conducive to cyanobacteria photosynthesis and uplift, increasing the risk level. When rainfall intensity exceeds 20mm / 24h, it dilutes the concentration of cyanobacteria in the water, reducing short-term risk. Meteorological parameters are weighted (sunlight weight 0.3, rainfall weight 0.2, temperature weight 0.3, wind speed weight 0.2) and used in trend prediction calculations.

[0051] Algorithm Model: Considering the risk assessment requirements for toxic and odor-producing cyanobacteria, a random forest algorithm was selected to construct the early warning model. This algorithm has advantages such as resistance to overfitting, handling multiple feature variables, high prediction accuracy, and strong interpretability, making it suitable for risk assessment scenarios involving multi-parameter fusion. The specific design and implementation process is as follows: Model Training: The training dataset consists of monitoring data from eutrophic lakes such as Taihu Lake, Chaohu Lake, and Dianchi Lake, as well as 10 drinking water sources in my country, totaling over 10,000 valid samples. These samples cover different seasons, pollution levels, and water body types. The samples are divided into a training set (70%) and a test set (30%) in a 7:3 ratio. The training set is used for model parameter optimization, while the test set is used to verify model accuracy. During training, a grid search method is used to optimize parameters such as the number of decision trees (set to 100-200 trees, optimal 150 trees), decision tree depth (maximum depth 10-15 layers, optimal 12 layers), and feature sampling ratio to reduce the risk of overfitting. Simultaneously, cross-validation (5-fold cross-validation) is introduced to ensure model stability under different scenarios.

[0052] Risk Level Classification: The model outputs four risk levels (1-4), determined by a combination of factors including the toxin-producing gene copy number threshold, environmental suitability, and historical algal bloom probability. Each level corresponds to a clear quantitative standard and risk description, facilitating rapid assessment and control measures by on-site personnel. The specific classification is as follows: Level 1 (Low Risk): Total toxin-producing gene copy number <10 3 / mL, environmental conditions are unsuitable for the growth of toxic cyanobacteria (water temperature <20℃ or >35℃, pH <7.0 or >9.5, dissolved oxygen >8mg / L), there are no obvious records of algal blooms in the same period in history, and the weather forecast indicates no suitable growth conditions in the next 24-48 hours; at this level, the number of toxic cyanobacteria in the water is extremely small, there is no risk of toxin accumulation, no special control is required, only the frequency of routine inspections needs to be maintained.

[0053] Level 2 (Medium Risk): Total toxin-producing gene copy number 10 3 -10 4 / mL, environmental conditions are average (water temperature 20-25℃ or 30-35℃, pH 7.0-7.5 or 9.0-9.5, dissolved oxygen 5-8mg / L), historical algal bloom probability <20%, and weather forecast indicates no obvious suitable growth conditions in the next 24-48 hours; at this level, toxin-producing cyanobacteria are in a slow growth stage, with extremely low toxin release and a slight risk of accumulation, requiring increased inspection frequency (from once a week to twice a week) and real-time monitoring of parameter changes.

[0054] Level 3 (High Risk): Total toxin-producing gene copy number 10 4 -10 5 / mL, environmental conditions are suitable for the growth of toxic cyanobacteria (water temperature 25-30℃, pH 7.5-9.0, dissolved oxygen 5-6mg / L), the historical probability of algal blooms is 20%-50% for the same period, and the weather forecast indicates suitable growth conditions in the next 24-48 hours (sunlight ≥8 hours / day, wind force ≤3). Under this level, the growth rate of toxic cyanobacteria accelerates, they begin to accumulate in large quantities, and the release of toxins increases, indicating a precursor to an algal bloom. It is necessary to initiate enhanced monitoring (test once a day), investigate the source of eutrophication in the water body, and prepare for emergency response.

[0055] Level 4 (Extremely High Risk): Total toxin-producing gene copy number > 10 5 / mL, environmental conditions are highly suitable for the growth of toxic cyanobacteria (water temperature 28-32℃, pH 7.8-8.5, dissolved oxygen 5-5.5mg / L), the probability of algal blooms during the same period in history is >50%, and the weather forecast indicates that the conditions will remain suitable for growth for the next 24-48 hours; at this level, toxic cyanobacteria have accumulated in large quantities, and there is a high possibility of a severe algal bloom, with the toxin release far exceeding the safety threshold, which will threaten drinking water safety, fishery production and the ecological environment, and an emergency response plan must be activated immediately.

[0056] Early warning rules: The early warning rules are dynamically adjusted based on the risk level output by the model and combined with real-time monitoring data to ensure the timeliness, relevance, and operability of early warning information, and to achieve closed-loop management of "early warning-response-feedback". The specific rules are as follows: Triggering condition: When the copy number of any toxin-producing gene is detected to be ≥10 3 If the number of toxin-producing gene copies increases by 0.5 mL (i.e., reaches the medium risk level or above), an early warning will be triggered immediately to avoid missed reports. If the number of toxin-producing gene copies increases for three consecutive tests, an early warning will be triggered even if the medium risk threshold has not been reached, enabling early intervention.

[0057] Early warning tiered push notification: Different levels of early warning information are sent according to the risk level to meet the needs of different control entities: Level 1 (low risk) is only recorded in the background of the mobile APP and no notification is pushed; Level 2 (medium risk) pushes a general notification to the mobile APP of on-site inspection personnel; Level 3 (high risk) pushes an emergency notification to inspection personnel and regional environmental protection control personnel, and is simultaneously displayed on the display screen at the monitoring point; Level 4 (extremely high risk) pushes a red alert to all relevant control personnel and emergency response teams, and simultaneously coordinates with local environmental protection and water affairs departments to ensure that relevant personnel are informed as soon as possible.

[0058] Trend Forecasting: Combining historical monitoring data patterns, real-time environmental parameters, and weather forecasts for the next 24-48 hours, the model calculates the trend of risk level changes to predict whether the risk level will escalate or degrade. If the risk level is predicted to escalate (e.g., from level 2 to level 3), a trend warning is sent out 6-12 hours in advance to allow time for response; if the risk level is predicted to degrade (e.g., from level 3 to level 2), a mitigation prompt is sent out to guide the gradual adjustment of control measures.

[0059] Control Recommendations: Targeted control recommendations are provided for different risk levels and application scenarios to ensure the effective implementation of early warning information: Low risk level (Level 1): Maintain routine inspections, regularly clean up debris in the water, and control the input of external nutrients; Medium risk level (Level 2): ​​Increase testing frequency, investigate surrounding pollution sources (such as domestic sewage and aquaculture wastewater discharge), and appropriately apply algae control agents (low concentration, no secondary pollution); High risk level (Level 3): Initiate enhanced algae control measures, combining physical dredging with chemical algae control, and strengthen the protection of drinking water source intakes; Extremely high risk level (Level 4): Immediately suspend drinking water intake, fully initiate emergency dredging, apply highly effective algae control agents, and simultaneously monitor the toxin content in the water. Normal water intake can only be resumed once the risk level drops below Level 2.

[0060] Model Validation: Validated by on-site monitoring data from Taihu Lake and Chaohu Lake, the prediction accuracy of this early warning model reaches over 95%, the risk level judgment error is ≤1 level, and the early warning response time is ≤5 minutes. Compared with traditional manual experience assessment, the early warning lead time is increased by 24-48 hours, which can effectively provide scientific and accurate decision support for the control of toxic and odorous cyanobacteria pollution.

[0061] Example 6: Mobile App Software Development To achieve networked and intelligent management of the monitoring process and address the pain points of scattered on-site monitoring data and inconvenient remote control, a comprehensive, user-friendly, and highly adaptable mobile app has been developed, tailored to the actual operational needs of grassroots monitoring and control personnel. This app serves as an auxiliary control and data management terminal for testing equipment, deeply integrating with portable testing equipment and risk warning algorithms to achieve closed-loop management of the entire process from detection and control to data archiving and risk management. It is compatible with both iOS (iOS 11.0 and above) and Android (Android 8.0 and above) mobile operating systems, supporting smartphones, tablets, and other mobile devices. Its core functional modules and detailed implementation scheme are as follows: A fully functional mobile app has been developed, with main functional modules including: Device connectivity module: Utilizes Bluetooth 4.0 Low Energy technology for wireless connection with portable testing equipment, balancing connection stability and power consumption control. The effective connection distance is ≤10m, and it can connect to multiple testing devices simultaneously (up to 8 devices), adapting to multi-point synchronous monitoring scenarios. The connection process employs automatic search and one-click pairing, eliminating the need for complex operations. After successful pairing, device information is automatically saved, allowing automatic connection to frequently used devices upon next app launch, reducing repetitive operations. Once connected, the operating status of each device can be monitored in real time, including key parameters such as battery level, testing progress, signal strength, filter membrane usage, and reaction temperature. If any abnormalities occur, such as insufficient battery, signal interruption, or filter membrane depletion, a local alert will be immediately triggered, facilitating timely troubleshooting and ensuring continuous testing. Simultaneously, it supports remote viewing and synchronization of device calibration parameters, ensuring consistent testing accuracy across all linked devices.

[0062] The detection control module enables remote control of the detection process, breaking the time and space limitations of on-site operation. It is suitable for scenarios such as unattended operation and remote inspection. Staff can remotely start or stop detection tasks via the APP and flexibly set various detection parameters according to detection needs, including isothermal PCR reaction temperature (adjustable from 37-65℃ with an accuracy of ±0.5℃), reaction time (customizable from 10-60 minutes), detection mode (qualitative / quantitative detection), fluorescence detection wavelength, etc. After parameter settings, they are automatically synchronized to the detection equipment without the need for manual adjustment on-site. During the detection process, the APP displays the detection progress in real time, including water sample enrichment progress, algal cell lysis progress, PCR amplification progress, fluorescence signal acquisition progress, etc., presented intuitively as a percentage. It also indicates the remaining time for each stage, which helps staff to reasonably arrange subsequent work. If it is necessary to terminate the detection urgently, it can be done with one click through the APP. The equipment will automatically stop the reaction and save the current detection data to avoid waste of samples and reagents.

[0063] The data display module employs a visual design, synchronously displaying various data transmitted from the testing equipment in real time. This ensures clear, intuitive, and easy-to-interpret test results. Core display content includes: target toxin-producing gene type (clearly labeled as mcy series, ana series, cyr series, etc.), gene copy number (precisely displaying numerical values ​​and units), test result determination (positive / negative, with the determination criteria indicated), and risk warning level (synchronously displaying risk levels 1-4 and corresponding descriptions). Data display supports multiple chart formats, including fluorescence amplification curves, gene copy number trend graphs, and risk level bar charts. Display modes can be switched as needed, and curves can be zoomed in and out to view details, facilitating staff analysis of the data's rationality. The interface design follows a simple and efficient principle, highlighting core data and expanding auxiliary data as needed. It supports a night mode to adapt to complex scenarios such as strong outdoor light and nighttime monitoring, ensuring staff can clearly view data in various environments.

[0064] Historical Data Module: Features powerful data storage and retrieval capabilities, employing a dual "local + cloud" storage mode. Local storage can hold ≥10,000 sets of test data, while cloud storage has no upper limit, ensuring data integrity and traceability. The query function supports precise multi-condition searches, allowing for queries based on test time (accurate to the second), test location (filtered by region and latitude / longitude), test type (qualitative / quantitative), gene type, risk level, and other conditions to quickly locate target data. Batch export of test data is supported in Excel or PDF formats. Excel format can be used for subsequent data statistical analysis, while PDF format can be directly used for test report preparation. Exported files are compatible with mainstream office software, requiring no additional format conversion. Furthermore, it supports batch deletion and highlighting of historical data, facilitating data classification and management by staff. Data retention periods can be set as needed, and expired data is automatically cleaned up, saving storage space.

[0065] Map Display Module: Integrates dual map interfaces from Gaode Maps and Baidu Maps, supporting offline map downloads and adapting to outdoor scenarios without network access. The location of each monitoring point is accurately marked on the map, using different colored icons to distinguish the risk level of each point (low risk green, medium risk yellow, high risk orange, and extremely high risk red). Key information such as the point name and monitoring time is indicated next to the icon, intuitively presenting the pollution distribution of toxic and odor-producing cyanobacteria in the area. Clicking on any point allows viewing detailed monitoring information, including monitoring data, risk level, environmental parameters, and historical monitoring records. Point navigation is supported, automatically planning the optimal route from the current location to the monitoring point, facilitating rapid on-site inspection and maintenance by staff. Simultaneously, manual addition and editing of monitoring points are supported, along with labeling of point types (drinking water sources, lakes, aquaculture water bodies, etc.), enabling personalized management of monitoring points.

[0066] The early warning push module is deeply integrated with risk warning algorithms to achieve accurate and tiered notifications of early warning information, ensuring that staff are promptly aware of risks and can respond quickly. Users can set early warning thresholds according to their permissions and customize early warning conditions based on gene type and risk level. When the test results reach the early warning threshold, the app automatically pushes early warning information via sound alerts, vibration alerts, mobile system notifications, and SMS (optional). Users can set the priority and frequency of reminders to avoid missing critical early warning information. The early warning information clearly indicates the warning location, risk level, test data, reason for the warning, and preliminary handling suggestions, facilitating staff to quickly grasp the situation and take countermeasures. Simultaneously, it supports batch viewing of early warning information, marking as read, and querying historical early warnings. It can also statistically analyze the number of early warnings and the distribution of early warning levels over a certain period, providing data support for regional pollution control.

[0067] The system settings module provides comprehensive parameter settings and account management functions, adapting to the needs of different users and ensuring stable and convenient APP operation. Device parameter settings allow for simultaneous adjustment of various parameters of the testing equipment, including fluorescence detection sensitivity, reaction temperature calibration, and sampling flow rate settings. Parameter backup and recovery are supported to prevent parameter loss due to accidental operation. User information settings allow users to modify account passwords, bind mobile phone numbers, and complete personal information. Multi-user management mode is supported, allowing different operation permissions to be set based on user identity (inspection personnel, control personnel, administrators). For example, inspection personnel can only view and upload test data, while administrators can modify parameters, manage all user accounts, and export all historical data, ensuring data security and operational standardization. Furthermore, the module supports automatic APP version updates, language switching (Chinese / English), and log viewing, facilitating timely access to the latest features and troubleshooting of operational faults, thus improving the APP's user experience.

[0068] 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 rapid on-site monitoring system for toxin- and odor-producing cyanobacteria based on toxin- and odor-producing genes, characterized in that: The system includes an automated algae enrichment module for water samples, rapid algal cell lysis and DNA extraction tubes, an isothermal / room temperature PCR module, a handheld fluorescence detector, qualitative and quantitative algorithms, software for calculating the risk warning level of toxin-producing and odor-producing cyanobacteria, and a mobile APP. These modules work together to automate the entire process from water sample collection, algae enrichment, DNA extraction, gene amplification, fluorescence detection, result analysis, risk warning, and data management. The system uses the characteristic genes of toxin-producing and odor-producing cyanobacteria as detection targets and employs isothermal PCR technology to rapidly amplify the target genes, accurately distinguishing between toxin-producing and non-toxin-producing cyanobacteria.

2. The rapid on-site monitoring system for toxin- and odor-producing cyanobacteria based on toxin- and odor-producing genes according to claim 1, characterized in that: The characteristic genes of the toxin-producing and odor-producing cyanobacteria include microcystin synthesis genes, anabatin gene cluster, and columnar cyanobacterial toxin gene cluster; the microcystin synthesis gene is selected from at least one of mcyA, mcyB, mcyC, mcyD, mcyE, mcyG, mcyH, and mcyJ; the anabatin gene cluster is selected from at least one of anaA, anaB, anaC, anaD, anaE, anaF, and anaG; the columnar cyanobacterial toxin gene cluster is selected from at least one of cyrA, cyrB, cyrC, cyrD, cyrE, cyrF, cyrG, cyrH, cyrI, cyrJ, and cyrN; and the automated algae enrichment module for water samples combines negative pressure filtration with magnetic bead enrichment. The combined dual enrichment technology includes a miniature portable vacuum pump, a replaceable filter membrane, and a turbidity sensor. The miniature portable vacuum pump has a power of ≤50W and can be powered by a lithium battery. The replaceable filter membrane is made of polyethersulfone with a pore size of 0.45μm. The turbidity sensor can detect the turbidity of the water sample in real time, and the control module automatically adjusts the filtration speed according to the turbidity. The filtration speed is 50mL / min when the turbidity is ≤10NTU, and the filtration speed is reduced to 20mL / min when the turbidity is >10NTU. The magnetic beads are modified with cyanobacteria-specific monoclonal antibodies, which can specifically adsorb target toxin-producing cyanobacteria cells and eliminate interference from diatoms, green algae, and water impurities. The enrichment efficiency of the module is ≥95%, which can effectively enrich toxin-producing cyanobacteria with a concentration as low as 10cfu / mL.

3. The rapid on-site monitoring system for toxin- and odor-producing cyanobacteria based on toxin- and odor-producing genes according to claim 1, characterized in that: The algal cell rapid lysis and DNA extraction tube employs a dual lysis method combining lysis buffer and cross-flow disruption, enabling algal cell lysis and high-purity DNA extraction within 3 minutes. The lysis buffer contains proteinase K, surfactant SDS, and chelating agent EDTA, with proteinase K concentration of 0.5 mg / mL, SDS concentration of 1%, and EDTA concentration of 50 mM. The cross-flow disruption device operates at 15,000 rpm, further disrupting incompletely lysed algal cells. The extracted DNA purity meets the requirements of OD260 / OD280 = 1.8-2.0, is free from protein and polysaccharide contamination, and can be directly used for subsequent isothermal PCR amplification without additional purification.

4. The rapid on-site monitoring system for toxin- and odor-producing cyanobacteria based on toxin- and odor-producing genes according to claim 1, characterized in that: The isothermal / room-temperature PCR module integrates a multi-channel handheld thermostat, PCR-specific primers, and probes. The thermostat utilizes Peltier thermoelectric cooling / heating technology, with a temperature control accuracy of ±0.5℃, and can be adjusted to a constant temperature within the range of 37-65℃, adaptable to LAMP, RPA, MIRA, and other isothermal PCR reactions. The module supports simultaneous detection of 4-8 parallel channels, and can simultaneously detect 2-4 target toxin-producing genes. The specific primers are 20-25 bp in length, and the probes are 25-30 bp in length, employing TaqMan probe technology, with a fluorescent group labeled at the 5' end and a quencher group labeled at the 3' end. The fluorescent group is selected from at least one of FAM, HEX, and CY5, and the quencher group is selected from BHQ1 or BHQ2. The primer and probe specificity is 100%, with no cross-reactivity, and the detection sensitivity can reach 10. 2 Copy / mL.

5. The rapid on-site monitoring system for toxin- and odor-producing cyanobacteria based on toxin- and odor-producing genes according to claim 1, characterized in that: The handheld fluorescence detector includes a high-brightness LED excitation source and a high-sensitivity photodiode detector. The LED excitation source has a wavelength stability of ±2nm, energy consumption ≤10W, and a detection wavelength range of 450-650nm. The detector has a built-in high-performance data processing chip that can automatically acquire fluorescence signals, plot amplification curves, calculate Ct values, and determine whether the detection result is positive or negative. It can store ≥1000 sets of detection data. The qualitative and quantitative algorithms and the software for calculating the risk warning level of toxin-producing and odor-producing cyanobacteria use a random forest machine learning algorithm to establish a warning model. The qualitative algorithm can determine the presence or absence of the target toxin-producing gene based on the fluorescence signal and Ct value, eliminating false positives and false negatives. The quantitative algorithm is based on the linear relationship between fluorescence signal intensity and target gene copy number, with a detection limit of 10. 3 copies / mL, linear range of 10 3 -10 8 The risk warning model, which combines the copy number of the toxin-producing gene, on-site environmental parameters, historical monitoring data, and meteorological forecast information, classifies the risk of algal blooms into four levels: low, medium, high, and extremely high, and provides corresponding control recommendations for different risk levels. The on-site environmental parameters include water temperature, pH, dissolved oxygen, and transparency, which can be collected in real time through external sensors.

6. The rapid on-site monitoring system for toxin- and odor-producing cyanobacteria based on toxin- and odor-producing genes according to claim 1, characterized in that: The mobile app is compatible with iOS 11.0 and above and Android 8.0 and above. It connects to the detection device via Bluetooth 4.0 with a connection distance of ≤10m and can connect to multiple detection devices simultaneously. The app has functions such as device control, data display, historical record query, risk warning push, and data analysis report generation. It can remotely start / stop detection, set detection parameters, and export detection data to Excel or PDF format.

7. The rapid on-site monitoring system for toxin- and odor-producing cyanobacteria based on toxin- and odor-producing genes according to claim 1, characterized in that: The monitoring system weighs less than 1 kg, and each core module is compact. The isothermal / room temperature PCR module has dimensions of ≤15cm×10cm×5cm and a weight of ≤0.3kg. It has an IP68 protection rating, is suitable for complex outdoor environments, and can be powered by a portable power bank, eliminating the need for a professional laboratory environment. The monitoring system can be applied to drinking water source monitoring, lake and reservoir water quality monitoring, aquaculture water quality management, and environmental emergency monitoring. It can detect various toxin-producing and odor-producing cyanobacteria such as Microcystis, Anabaena, and Cylindrica, and is suitable for grassroots monitoring, outdoor inspections, and emergency response to sudden algal blooms.

8. A rapid on-site monitoring method for toxin- and odor-producing cyanobacteria based on toxin- and odor-producing genes, as described in claims 1-7, characterized in that: Includes the following steps: S1. The system includes two detection modes: Mode 1 is a qualitative rapid monitoring based on RPA-LFS technology, and Mode 2 is a quantitative monitoring based on real-time fluorescence isothermal PCR technology. Mode 1 can complete the entire detection process within 30 minutes, without the need for complex fluorescence detection instruments, and the results are visualized through lateral flow strips. Mode 2 can complete the entire detection process within 60 minutes, with a detection limit of 10. 2 The copy number / mL can accurately quantify the copy number of toxin-producing genes and the biomass of toxin-producing cyanobacteria. S2. The specific steps of Mode 1 include: the water sample is processed by the automated enrichment module to obtain a concentrated algal cell suspension, the algal cell suspension is added to a rapid lysis tube, the supernatant is centrifuged after lysis and used as a DNA template, the DNA template is added to the RPA reaction system, magnesium acetate is added to start the reaction, the reaction is carried out at 37-42℃ for 15-20 minutes, the reaction product is dropped into the sample well of the side flow strip, buffer is added and allowed to stand for 5 minutes, and the detection results are judged according to the band display. S3. The specific steps of the final mode two include: the water sample is processed by the automated enrichment module to obtain a concentrated algal cell suspension, and high-purity DNA is extracted after lysis as a template. The DNA template is added to the real-time fluorescence isothermal PCR reaction system, placed in a handheld fluorescence detector, and the corresponding reaction temperature is set to start real-time fluorescence monitoring. The Ct value is calculated based on the fluorescence amplification curve, and the target gene copy number is determined in combination with the standard curve to estimate the biomass of toxin-producing cyanobacteria.

Images