Primer group, kit and method for detecting geoA gene of geosmin-producing cyanobacteria

Abstract

The invention relates to the technical field of water pollution and water body odor substance detection, in particular to a primer group, a kit and a method for detecting blue-green algae generating geosmin in a water body based on a recombinase-mediated isothermal nucleic acid amplification (RAA) technology. The sequence of a forward primer Geo-F, a reverse primer Geo-R1 and a probe primer Geo-P2 which are designed in the invention and used for detecting the geoA gene generating geosmin are shown as SEQ ID No.1, the sequence of the reverse primer Geo-R1 is shown as SEQ ID No.2, and the sequence of the probe primer Geo-P2 is shown as SEQ ID No.3. The primer group can effectively amplify the geoA gene of geosmin, has high specificity and sensitivity, has no cross reaction with cyanobacteria which does not generate geosmin, and can be used for detecting the geoA gene generating geosmin. The kit can be used in combination with lateral flow immunochromatography for on-site rapid detection of geosmin, and has important significance in early warning of excessive geosmin content in a water body.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of water pollution and water body odor substance detection, and particularly relates to a primer set, a kit and a method for detecting blue-green algae producing geosmin in water based on recombinant enzyme-mediated isothermal nucleic acid amplification technology (RAA). BACKGROUND

[0002] In recent years, the rapid development of economy, industry and agriculture in China has led to a large amount of nutrients such as nitrogen and phosphorus being discharged into water bodies, causing water eutrophication problems to become increasingly prominent, which in turn promotes the proliferation of algae. Algae produce odor substances represented by geosmin (GSM) during the metabolic process. This substance has a strong earthy smell and a very low odor threshold of only 10 ng / L. According to the Standards for Drinking Water Health GB5749-2022, even at very low concentrations, it can seriously affect the sensory properties of drinking water, becoming one of the water quality problems with the highest proportion of complaints from tap water consumers. Currently, more than 200 species of algae such as Oscillatoria, Lyngbya and Anabaena have been found to metabolize geosmin. The outbreak of these algae not only disrupts the ecological balance of water bodies, but also poses a direct threat to drinking water safety. Therefore, it is of great practical significance to establish an efficient detection technology for geosmin-producing algae.

[0003] In the field of water ecology and drinking water safety, when the concentration of algae in water is lower than 10 4 cells / mL, it is difficult to detect directly by conventional methods such as microscopic observation and visual identification, posing a great challenge to early monitoring of geosmin-producing algae. It is worth noting that certain algae species synthesize geosmin through a secondary metabolic pathway during growth and reproduction, and the geoA gene is the core functional gene for geosmin synthesis, which is highly conserved in geosmin-producing algae. The abnormal proliferation of such geosmin-producing algae is often an early biological indicator of water eutrophication. Their presence not only leads to a decrease in the sensory quality of drinking water due to geosmin release, but also may be accompanied by water bloom, further causing ecological problems such as reduced dissolved oxygen in water and death of aquatic organisms, posing a serious threat to drinking water source protection and water environment management.

[0004] For the detection of geosmin-producing algae and geosmin, two main technical paths are currently relied on, but both have obvious defects and are difficult to meet the needs of on-site rapid monitoring:

[0005] The first type is based on the detection of geosmin itself. The method represented by the Test Method for Geosmin and 2-Methylisoborneol in Drinking Water (GB / T 5750.8-2023) uses headspace solid-phase microextraction-gas chromatography-mass spectrometry. Although it can achieve accurate quantification of geosmin, the detection process takes several hours and requires large and precise equipment such as gas chromatography-mass spectrometry. This equipment is bulky, expensive to purchase and maintain, and requires trained technicians to operate. It can only be used in laboratory environments and cannot meet the needs of on-site rapid detection in drinking water sources, water treatment plants, and other scenarios. It is difficult to achieve early warning of geosmin pollution.

[0006] The second type is based on molecular biology detection of geosmin-producing cyanobacteria, including polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP). PCR can target geosmin-producing algae by amplifying geoA genes, with some specificity and sensitivity. However, it has significant limitations. First, the detection process is complex and time-consuming, involving nucleic acid extraction, reaction system preparation, thermal cycling amplification, and gel electrophoresis detection. Second, PCR relies heavily on laboratory equipment such as PCR machines and gel imaging systems, making it difficult to apply to on-site monitoring in water sources at the grassroots level. LAMP can amplify under constant temperature conditions without the need for thermal cycling equipment. However, it has several problems, such as complex primer design requiring 4-6 primers, false positives due to non-specific amplification, and the need for turbidimetry or fluorescence detection for product detection. Additionally, the amplification process is time-consuming and cannot meet the detection requirements of "fast, accurate, and on-site application."

[0007] In recent years, recombinase-aided amplification (RAA) has shown significant advantages as a new isothermal amplification technique. It can efficiently amplify DNA at a constant low temperature of around 39°C, and sufficient amplification products can be obtained within tens of minutes without the need for complex thermal cycling equipment. In theory, it has the potential for on-site detection. Meanwhile, lateral flow test strips are a visual detection method that is simple to operate, fast, low-cost, and does not require professional equipment for interpretation. They have been widely used in biological detection. The combination of RAA and lateral flow test strips can form a closed loop of "rapid amplification and visual interpretation."

[0008] However, there is no combination of "RAA isothermal amplification technology" and "lateral flow test strip visual detection" in the prior art, and no specific detection system designed for the core function geoA gene of geosmin-producing cyanobacteria. After searching, the existing RAA related technology is mainly applied to the detection of pathogenic bacteria, and does not involve the specific detection object of geosmin-producing cyanobacteria. Moreover, the processing flow and RAA amplification system are not optimized for the cell characteristics of cyanobacteria, which leads to the fact that the existing technology cannot meet the actual needs of the drinking water source for "low concentration detection, rapid detection, and on-site application" of geosmin-producing cyanobacteria, and it is difficult to meet the early warning needs in water quality safety management.

[0009] In summary, there is an urgent need for a detection scheme that can break through the bottleneck of the existing technology: the scheme needs to have low concentration detection capability, rapid detection characteristics, on-site applicability and high specificity, and only targets geosmin-producing cyanobacteria containing functional geoA gene, in order to realize real-time monitoring of geosmin-producing cyanobacteria in water bodies, provide reliable technical support for drinking water source protection and water treatment process optimization, and prevent water bloom and establish an effective early warning system for geosmin pollution. SUMMARY

[0010] Problems to be solved

[0011] The purpose of the present application is to provide a geosmin-producing algae geoA gene primer set with high specificity and high sensitivity, which can realize on-site rapid detection, in order to solve the problems of long detection time, dependence on large instruments, complex operation and poor on-site applicability of existing detection technology, and to realize rapid, sensitive and specific detection of geosmin-producing algae in water bodies, providing support for early warning of geosmin pollution in drinking water sources.

[0012] Technical scheme

[0013] In order to solve the above technical problems, the purpose of the present application is to provide a primer set, kit and method for detecting geosmin-producing algae based on RAA.

[0014] In the first aspect, in order to meet the target detection needs of geosmin-producing cyanobacteria, the present application provides a primer set for detecting the geoA gene of geosmin-producing algae. The primer set is designed based on the conserved region of the multiple sequence alignment of geosmin-producing algae geoA gene. 50 high-similarity geoA gene sequences are obtained from the NCBI database for alignment to determine the conserved amplification region, which can specifically recognize and amplify the geoA gene of geosmin-producing algae, effectively avoiding cross-reaction with non-geosmin-producing algae.

[0015] Specifically, the above-mentioned primer set comprises a geoA gene detection primer and a geoA gene detection probe geo-P2, wherein the geoA gene detection primer is composed of a forward primer geo-F and a reverse primer geo-R1, the 5' end of the reverse primer geo-R1 is modified with fluorescein isothiocyanate (FITC), and the modification enables the amplification product to bind to the anti-FITC antibody in the quality control area of the subsequent lateral flow test strip, thereby achieving double verification of quality control and detection; the 5' end of the geoA gene detection probe geo-P2 is modified with biotin (Biotin), which can enable the double-stranded amplification product formed by the probe and the reverse primer to be captured by the nucleic acid test strip and colored through biotin-biotin antibody interaction, and the 3' end of the probe geo-P2 is modified with a phosphate group (PHO), which can effectively inhibit non-specific DNA chain extension and reduce false positive signals in the amplification process.

[0016] The nucleotide sequence of the forward primer geo-F is shown in SEQ ID No. 1: 5'-CTATAAGCGCAGTCAGGATCTAATT-3';

[0017] The nucleotide sequence of the reverse primer geo-R1 is shown in SEQ ID No. 2:

[0018] 5'-GTATTCAATGGGGTTAGCAATGCGA-3'.

[0019] The design of both strictly follows the following principles: first, the length of the nucleotides is controlled within the range of 20-30 bp, specifically the length of the forward primer is 25 bp and the length of the reverse primer is 25 bp, and a primer that is too short is prone to lack of specificity and to bind to non-target sequences to produce cross-reactions, while a primer that is too long will reduce the amplification efficiency and prolong the reaction time; at the same time, the difference between the annealing temperatures of the forward primer and the reverse primer is not more than 5°C, which ensures that they can bind to the target sequence simultaneously under the same constant temperature amplification conditions, thereby guaranteeing the consistency of the amplification reaction conditions.

[0020] Second, when the forward primer and the reverse primer are used to amplify the geoA gene, the length of the amplification product obtained is 200-500 bp, which can meet the demand for efficient amplification of RAA technology and has species specificity for geosmin-producing algae, i.e., it can only amplify the geoA gene of geosmin-producing algae such as Anabaena sp. FACHB-1391 and Aphanizomenon gracile, but cannot amplify the nucleic acid of non-geosmin-producing algae such as Nostoc commune FACHB-261 and Pseudanabaena cinerea FACHB-1277.

[0021] The nucleotide sequence of the probe geo-P2 for detecting the geoA gene is shown in SEQ ID No. 3:

[0022] 5'-TTCCCCTTCCCACGAACCCAGTAGAGCGCGHTTTAGCTGACTTGTG-3'; wherein H is tetrahydrofuran, the length of the probe is 48 nt, which is in the preferred range of 45-50 nt, and the probe of 45-50 nt can ensure the binding specificity of the target sequence and provide sufficient space for the cutting site of nfo enzyme; at the same time, the probe is strictly combined with the target segment between the forward primer geo-F and the reverse primer geo-R1 in the geoA gene, ensuring that it can only be combined with the amplification region of the forward and reverse primers, and further improving the detection specificity.

[0023] In some realizable ways, the above-mentioned probe further satisfies the following key conditions:

[0024] First, the design direction is forward, which is consistent with the nucleotide extension direction of the forward primer. This design can make the probe combine with the target sequence synchronously with the primer in the RAA amplification process, avoiding the combination competition caused by the direction difference.

[0025] Second, the sequence is internally provided with a cutting site of tetrahydrofuran (H) modification. The cutting site can be recognized and cut by nfo enzyme when the probe is combined with the geoA gene template, and after cutting, it can start the DNA chain extension reaction. The mechanism of action is that nfo enzyme can specifically recognize and cut double-stranded nucleic acid containing a tetrahydrofuran site, and after cutting, the 3' end of the probe forms a free hydroxyl group, which provides an extension starting point for DNA polymerase, and thus realizes the specific amplification of the target sequence.

[0026] Secondly, on the basis of the above-mentioned primer set, the present application further provides a kit for detecting geoA-producing algae. The kit takes the primer set as the core and integrates all the reagents required for the recombination enzyme-mediated isothermal amplification (RAA) reaction, which can be directly used for on-site detection without additional preparation of complex reagents.

[0027] Specifically, the RAA amplification necessary reagents include basic buffer, DNA polymerase, nfo enzyme, MgAc2 solution and sterile double distilled water, wherein the basic buffer contains 10-20 mM Tris-HCl, 50-100 mM KCl, 10-20 mM (NH4)2SO4. Among them, 10-20 mM Tris-HCl can maintain the system pH stable at 7.5-8.5, that is, the optimum pH range of RAA reaction, 50-100 mM KCl and 10-20 mM (NH4)2SO4 can adjust the system ionic strength, and provide a suitable catalytic environment for recombinase and DNA polymerase. Experiments show that when the concentration of Tris-HCl is 15 mM, the concentration of KCl is 75 mM, and the concentration of (NH4)2SO4 is 15 mM, the activity of DNA polymerase is increased by 15%-20% compared with other concentration combinations.

[0028] In some realizable ways, the concentration of MgAc2 solution is 260-300 mM, preferably 280 mM, and this concentration is because Mg 2+ is the key starting factor of RAA reaction, which can activate the activity of DNA polymerase, and too low concentration will lead to slow reaction start and low amplification efficiency, and too high concentration will inhibit enzyme activity, and 280 mM can realize high-efficiency amplification within 15-20 min.

[0029] In addition, the reagents required for RAA reaction also include RAA basic buffer containing recombinase, single-strand binding protein, DNA polymerase and dNTPs, the recombinase can bind the primer and search for the target sequence on the DNA chain, the single-strand binding protein can stabilize the single-strand DNA after unwinding to prevent its renaturation, the DNA polymerase is responsible for extending the primer to form double-stranded amplification product, and the dNTPs provide raw materials for amplification, and the four work together to realize rapid amplification at constant temperature; at the same time, the kit also contains negative control, which is sterile double distilled water or genomic DNA of non-terpene-producing algae (such as genomic DNA of Nostoc commune FACHB-261), which is used to exclude false positives caused by pollution in the experiment and ensure the reliability of the detection results.

[0030] In some possible manners, to realize the visual detection of the amplification product, the kit further comprises a lateral flow chromatographic test strip, which is composed of a sample pad, a conjugate pad, a detection zone, a quality control zone and a water absorption pad connected in sequence. In the present application, the conjugate pad is coated with a fluorescein isothiocyanate (FITC) antibody-colloidal gold compound. When the amplification product diffuses through the sample pad to the conjugate pad, the FITC modification of the reverse primer in the product can be combined with the FITC antibody on the conjugate pad to form a "FITC-antibody-colloidal gold" compound. The detection zone is coated with a biotin antibody, which can specifically bind to the biotin modification of the probe in the amplification product, so that the "FITC-antibody-colloidal gold-amplification product-biotin antibody" compound is enriched and colored in the detection zone. The quality control zone is coated with an anti-fluorescein isothiocyanate antibody, which can capture the "FITC antibody-colloidal gold" compound that is not combined with the amplification product, to ensure the effectiveness of the test strip itself. The above structure design can intuitively judge the result through double strips (quality control zone+detection zone), single strip (only quality control zone) or no strip (no quality control zone), without the need of professional instruments.

[0031] In a third aspect, the present application further provides a detection method based on the above-mentioned primer set and kit, which is based on sample pretreatment, RAA amplification and product detection, and has simple operation and short time consumption. The specific steps are as follows:

[0032] S1: To obtain a DNA template, a water body sample to be tested is preferably a drinking water source sample such as a reservoir water or a lake water, and a heat lysis method is used for pretreatment. The heat lysis method does not need a complex nucleic acid extraction kit, has simple operation and short time consumption, and is suitable for field application. The specific operation is as follows:

[0033] Firstly, a 0.2-0.22 μm filter membrane, preferably a 0.22 μm mixed cellulose ester filter membrane, is used. The pore size of the filter membrane can effectively trap cyanobacterial cells. The diameter of cyanobacterial cells is usually 3-10 μm, which cannot pass through the 0.22 μm filter membrane. Small molecular impurities in the water body can be preliminarily separated by passing through the filter membrane, so as to enrich the water sample;

[0034] The filter membrane enriched with cyanobacteria is placed in sterile water, which is specifically sterile double distilled water, so as to avoid the introduction of exogenous DNA pollution;

[0035] Subsequently, the centrifugal tube containing the filter membrane and the sterile water is heated at 95-105℃ for 5-8 min, and the temperature is preferably 100℃ and the heating time is preferably 6 min. Heating at 100℃ for 6 min can effectively lyse the cell wall and cell membrane of cyanobacteria to release the DNA in the cells, while avoiding DNA degradation caused by too high temperature or too long heating time. It has been verified by experiments that the DNA is in good integrity after heating at 100℃ for 6 min, which can meet the RAA amplification requirement;

[0036] Finally, the filtrate is collected as the DNA template after filtering the lysed cells and other impurities through a 0.4-0.45 μm filter, preferably a 0.45 μm mixed cellulose ester filter, to remove the impurities and avoid inhibition of the subsequent RAA amplification enzyme activity. The DNA template can be stored at -70°C for more than 3 months, which is convenient for subsequent re-inspection.

[0037] Step S2 is to use the DNA template obtained in step S1 as a template and use the primer set to perform RAA amplification to obtain an amplification product. The total volume of the reaction system of the RAA amplification is 50 μL, which is the optimal volume considering the amplification efficiency and the amount of reagents. If the volume is too large, the reaction time will be prolonged, and if the volume is too small, errors are likely to occur.

[0038] The amount of each component in the reaction system of the RAA amplification is preferably as follows: 2 μL of DNA template, 2.1 μL of forward primer, 3 μL of reverse primer, 0.6 μL of geoA gene detection probe, 29.4 μL of basic buffer, and 2.5 μL of MgAc2 solution. The remaining volume is supplemented with sterile double-distilled water to 50 μL. The ratio of 0.6 of the forward primer, the reverse primer, and the probe can make the primer and the probe fully bind to the target sequence, while avoiding the formation of primer dimers. The final concentration of the forward primer, the reverse primer, and the geoA gene detection probe is 0.1-1.0 μM, preferably 0.4-0.6 μM. This concentration range can balance the amplification efficiency and specificity. If the concentration is too low, the probability of primer binding to the target sequence is low, and the amplification product is less. If the concentration is too high, non-specific binding is likely to occur.

[0039] Further, the reaction conditions of the RAA amplification in the present application are as follows: constant temperature reaction at 37-41°C for 15-20 min, preferably constant temperature reaction at 39°C for 16 min. 39°C is the optimum temperature for RAA reaction, which can make the recombinase, the DNA polymerase, and the nfo enzyme exhibit the best activity. 16 min can ensure that the amplification product reaches a detectable level.

[0040] The second step is RAA amplification, which uses the DNA template obtained in the first step as a template and uses the above-mentioned primer set for isothermal amplification. Specifically, the total volume of the reaction system of the RAA amplification is 50 μL, and the volume and concentration of each component are as follows:

[0041] The RAA basic buffer is 28-32 μL, preferably 29.4 μL, which can ensure that the enzyme and raw material concentrations in the buffer are appropriate;

[0042] The MgAc2 solution is 2-3 μL, preferably 2.5 μL, which, in combination with the 280 mM MgAc2 concentration, can make the final concentration of Mg 2+ in the reaction system reach 14 mM, which is the best concentration for enzyme activity;

[0043] DNA template 2-4 μL, preferably 2 μL, too much template may introduce more inhibitors, and too little template may result in insufficient amplification product, experiments have shown that 2 μL of template can ensure amplification efficiency while reducing the impact of inhibitors;

[0044] The final concentration of the forward primer, the reverse primer and the probe is 0.8-1.2 μM, in practical application, the final concentration of the forward primer is about 0.42 μM, the final concentration of the reverse primer is about 0.6 μM, and the final concentration of the probe is about 0.12 μM, all of which are within the concentration range, and the final concentration can ensure efficient binding of the primer and the probe to the template.

[0045] The reaction conditions are set to be constant temperature reaction at 38-40℃ for 15-20 min, and the temperature is preferably 39℃ and the reaction time is preferably 16 min. 39℃ is the optimum reaction temperature of the recombinase and the DNA polymerase in the RAA system, which can achieve the highest amplification efficiency, and 16 min of reaction time can make the amplification product reach a detectable level. Experiments have shown that after 16 min of reaction at 39℃, the amount of amplification product is sufficient for detection by lateral flow test strips, and there is no need to extend the reaction time.

[0046] During the amplification process, the recombinase first mediates the specific binding of the primer to the template DNA, allowing the forward primer and the reverse primer to bind to the specific site of the mic gene template. Single-strand binding protein, abbreviated as SSB, binds to the single-stranded DNA after unwinding to maintain the single-stranded state. Subsequently, the nfo enzyme recognizes and cuts the tetrahydrofuran site on the probe, and the DNA polymerase extends the chain from the cut probe. Finally, a double-stranded amplification product with FITC and biotin is formed. The above specific amplification process only starts when the mic gene of 2-MIB-producing cyanobacteria is present in the sample.

[0047] Step S3 is to use lateral flow test strips to visually detect the RAA amplification product. The specific steps are as follows: first dilution, take 2 μL of amplification product and dilute it 50 times with sterile double distilled water (diluting 50 times can avoid false negatives of test strips caused by high concentration of amplification product, and experimental example 1 shows that the signal-to-noise ratio of the test strip is highest when diluted 50 times); then sample, insert the sample pad end of the lateral flow chromatography test strip into the diluted amplification product, the liquid level should not exceed the immersion mark of the sample pad to avoid excessive soaking of the test strip and affect the strip formation; finally, interpretation, place at room temperature for 1-5 min, and record the results within 10 min.

[0048] The result interpretation standard is: both the quality control area and the detection area appear red bands, which is positive, indicating that the sample contains geosmin-producing algae; only the quality control area appears a red band, which is negative, indicating that the sample does not contain geosmin-producing algae; the quality control area does not appear a red band, which is invalid for detection, and needs to be retested. This interpretation standard has been verified by the detection of 35 reservoir samples, which is completely consistent with the PCR results.

[0049] Beneficial effects

[0050] (1) This invention significantly overcomes the bottleneck of existing detection technology for soil-producing cyanobacteria by optimizing the primer set, reagent kit, and detection method, and has many outstanding advantages. In terms of specificity, the primer set is designed based on the conserved region of the geoA gene. The forward primer, reverse primer, and probe sequences have no homology with the nucleic acids of non-soil-producing cyanobacteria. Moreover, the reverse primer is modified with fluorescein isothiocyanate, and the probe is modified with biotin-phosphorylation-tetrahydrofuran triple modification to synergistically block non-specific amplification. In practical applications, it can effectively amplify soil-producing cyanobacteria such as Anabaena, Oscillatoria, and Trichophyton, and has no amplification signal for non-soil-producing cyanobacteria. The specificity reaches 100%, which completely solves the false positive problem caused by cross-reaction in existing technologies and ensures accurate detection results.

[0051] (2) In terms of sensitivity, the detection method provided by this invention can detect 1-2 geosmogrin-producing cyanobacteria cells / reaction or 30-50 copies of the geoA gene / reaction. Specifically, it can detect 3.56×10² plasmids containing the geoA gene / reaction and 5.63×10⁻¹ng geosmogrin-producing cyanobacteria DNA / reaction, which is far higher than the detection limit of the existing headspace solid-phase microextraction-gas chromatography-mass spectrometry method. It can detect cyanobacteria concentrations in water below 10 4 Effective detection can be achieved at a level of cells / mL (invisible to the naked eye), which can issue an early warning before geosmin-producing cyanobacteria proliferate in large quantities and geosmin accumulates significantly, thus buying sufficient time for pollution control of drinking water sources and preventing a decline in the sensory quality of drinking water due to excessive geosmin.

[0052] (3) The kit of the present invention integrates all components of sample pretreatment, amplification and detection, without the need for additional complex equipment: sample pretreatment can be completed with only a constant temperature mixer, RAA amplification can be carried out in a constant temperature mixer or water bath, without the need for thermal cycling equipment such as PCR instruments, and the products can be visualized and interpreted through test strips, without the need for large precision instruments. The entire detection process can be completed within 30-50 minutes and can be operated at an ambient temperature of 5-35℃. The results can be interpreted without the need for professional technicians, and it is fully applicable to on-site scenarios such as drinking water sources and waterworks, breaking the limitation of existing technologies that can only be detected in the laboratory, and realizing real-time on-site monitoring of soil-producing cyanobacteria.

[0053] (4) The detection method of the present invention has simple steps. The sample pretreatment only requires three steps: filtration, heating and re-filtration. RAA amplification and product detection do not require complicated reagent preparation and instrument debugging. The reagents in the kit are all pre-packaged and can be used directly, which reduces the operation threshold. At the same time, the cost of RAA reagents and side-flow chromatography test strips is much lower than that of gas chromatography-mass spectrometry consumables and PCR kits. The cost of a single detection is only 1 / 20 of that of solid phase microextraction, gas chromatography, mass spectrometry and other methods and 1 / 5 of that of PCR method. It is easy to promote and apply on a large scale in grassroots units such as water plants and environmental monitoring stations, and provide economical and reliable technical support for drinking water source protection and water treatment process optimization.

[0054] In summary, this invention achieves the goals of "high specificity, high sensitivity, on-site availability, and low cost" in the detection of soil-producing cyanobacteria through precise primer design, integrated reagent kit construction, and simplified detection process. It can effectively support early warning of eutrophication and ensure drinking water safety, and has significant practical value. Attached Figure Description

[0055] Figure 1 Flowchart of a method for recombinase-mediated isothermal amplification combined with nuclease cleavage and lateral flow chromatography detection of soil-producing osmidrosis algae;

[0056] Figure 2 This is a schematic diagram of the structure of the single-target disposable nucleic acid test strip (JY0201) of the present invention;

[0057] Figure 3 The results of the test strips used in this invention to perform RAA amplification on positive algae and negative controls using primer sets GSM-1, GSM-2, GSM-3, and GSM-4 of geosmin;

[0058] Figure 4 Comparison of amplification results of GSM-2RAA primer sets with different volumes of geosmin added in this invention;

[0059] Figure 5 The optimal detection system for geosmin-specific RAA-LFS in this invention was used to detect RAA-LFS in various cyanobacteria; negative control (sterile double-distilled water);

[0060] Figure 6 This demonstrates the sensitivity of the geosmin RAA test strip detection system based on gradient dilution of positive template and plasmid in this invention.

[0061] Figure 7 The results of RAA-LFS and PCR detection of the geoA gene in 35 reservoir samples in this invention are shown. Detailed Implementation

[0062] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0063] To better understand the technical content of this invention, the following embodiments are provided for detailed explanation.

[0064] I. Preparation of Experimental Materials

[0065] 1.1 Algal species and water samples

[0066] The algal species involved in this invention are all preserved in the Freshwater Algal Culture Collection of the Chinese Academy of Sciences, specifically including soil-producing cyanobacteria and non-soil-producing cyanobacteria:

[0067] Soil-producing cyanobacteria:

[0068] Anabaenacylindrica FACHB-170

[0069] Anabaenaminutissima FACHB-250

[0070] Oscillatoriasp. FACHB-1120

[0071] Planktothricoidesraciborskii FACHB-1360

[0072] Aerosakkonema funiforme FACHB-1362

[0073] Aerosakkonema funiforme FACHB-1363

[0074] Planktothricoidesraciborskii FACHB-1372

[0075] Planktothricoidesraciborskii FACHB-1378

[0076] Anabaenasp.FACHB-1384

[0077] Anabaenasp.FACHB-1391

[0078] Anabaenasp.FACHB-1501

[0079] Aphanizomenongracile;

[0080] Non-soil-producing cyanobacteria: Nostoccommune FACHB-261, Pseudanabaenacinerea FACHB-1277.

[0081] Water samples were collected from a drinking water source reservoir in Ningbo City. After sampling, the samples were immediately placed on ice for preservation and transported back to the laboratory within 4 hours, where they were temporarily stored in a 4°C refrigerator to ensure the freshness of the samples.

[0082] 1.2 Reagents and Consumables

[0083] RAA amplification reagent: RNA isothermal rapid amplification kit (test strip type)-III from Anpu Future (Changzhou) Biotechnology Co., Ltd., catalog number WLRN8211KIT, containing RAA basic buffer and enzyme mixture (recombinase, single-stranded binding protein, DNA polymerase).

[0084] Primers and probes: synthesized by General Biosystems (Anhui) Co., Ltd., with HPLC-grade purity. Specific sequences and modifications are described in subsequent examples.

[0085] nfo enzyme (endonuclease IV): used to cleave the tetrahydrofuran site of the probe;

[0086] Chemical reagents: CTAB (hexadecyltrimethylammonium bromide), chloroform, isopropanol, 75% ethanol, ampicillin, LB broth medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0), all of analytical grade;

[0087] Plasmid extraction reagent: Bacterial plasmid miniprep kit, used for recombinant plasmid extraction;

[0088] Filter membrane: A mixed cellulose ester filter membrane with pore sizes of 0.22μm and 0.45μm, used for algae enrichment and impurity filtration;

[0089] Centrifuge tubes: 1.5mL and 0.2mL sterile centrifuge tubes;

[0090] Lateral flow chromatography test strip: Tiosbio® Nucleic Acid Lateral Flow Chromatography Test Strip (Beijing Baoying Tonghui Biotechnology Co., Ltd.), model includes single-target disposable nucleic acid test strip (JY0201).

[0091] 1.3 Instruments and Equipment

[0092] Thermomixer: used for RAA amplification reaction (39℃ isothermal) and algal thermal lysis (100℃ heating);

[0093] High-speed centrifuge: used for centrifugation (12000 rpm) and plasmid extraction during CTAB DNA extraction;

[0094] Ultra-micro spectrophotometer: used to determine plasmid DNA concentration and purity (OD260 / OD280 ratio).

[0095] Gel imaging system: used for observing the results of agarose gel electrophoresis of PCR products and plasmids;

[0096] Pipettes: Capacity range 0.5-10μL, 10-100μL, 100-1000μL, used for precise transfer of reagents and samples;

[0097] High-pressure steam sterilizer: used for sterilization of laboratory consumables (centrifuge tubes, filter membranes, etc.);

[0098] Clean bench: Used for aseptic operations (such as plasmid extraction, bacterial inoculation) to avoid contamination;

[0099] Water bath: Used for preheating CTAB extraction buffer (65°C).

[0100] 1.4 Sample Preparation Method

[0101] 1.4.1 DNA extraction from laboratory-cultured algae (CTAB method)

[0102] This method is used to obtain high-purity algal genomic DNA to support primer screening and plasmid construction. The specific steps are as follows:

[0103] (1) Take the culture medium of algae cultured in the laboratory, filter it through a 0.22μm filter membrane under negative pressure to enrich algal cells, and scrape the algae on the filter membrane into a sterile mortar;

[0104] (2) Preheat 2×CTAB extraction buffer (containing 2% CTAB, 100mM Tris-HCl, 20mM EDTA, 1.4M NaCl, pH 8.0) in a 65℃ water bath for 30 minutes.

[0105] (3) Add a small amount of liquid nitrogen to the mortar and grind the algae into powder quickly. During the process, continuously replenish the liquid nitrogen to prevent the algae cells from degrading.

[0106] (4) Transfer the ground algae powder to a 1.5 mL sterile centrifuge tube, add 1000 μL of preheated 2×CTAB buffer, and gently invert the centrifuge tube 3-5 times to mix the algae powder and buffer thoroughly.

[0107] (5) Place the centrifuge tubes in a 65°C water bath and keep them warm for 30 minutes. Gently invert the tubes once every 10 minutes to ensure complete lysis.

[0108] (6) After the centrifuge tube has cooled to room temperature (about 2 minutes), add an equal volume of chloroform, tighten the cap, and shake vigorously for 2-3 minutes to fully emulsify the chloroform with the aqueous phase;

[0109] (7) Place the centrifuge tube into a high-speed centrifuge and centrifuge at 12,000 rpm for 10 minutes. At the same time, take a new 1.5 mL sterile centrifuge tube and add 600 μL of isopropanol.

[0110] (8) After centrifugation, slowly aspirate the upper aqueous phase with a pipette (avoid aspirating the intermediate protein layer), transfer it to a centrifuge tube containing isopropanol, and gently invert it up and down for 30 seconds until white DNA flocculent material appears in the tube;

[0111] (9) Centrifuge at 12000 rpm for 1 minute, immediately pour out the liquid in the tube, invert the centrifuge tube on a clean paper towel, and drain the remaining liquid;

[0112] (10) After 60 seconds, stand the centrifuge tube upright, add 700 μL of 75% ethanol, gently rotate the tube to allow the DNA precipitate to float in the ethanol, and soak for 5 minutes.

[0113] (11) Centrifuge at 12000 rpm for 1 minute, discard the ethanol, add 800 μL of 75% ethanol again, and repeat the soaking and centrifugation steps once.

[0114] (12) Centrifuge at 12000 rpm for 30 seconds, completely discard the ethanol, invert the centrifuge tube on a paper towel, and place at room temperature for 15-20 minutes to allow the DNA to dry completely;

[0115] (13) Add 100 μL of sterile double-distilled water to the centrifuge tube, gently tap the bottom of the tube to fully dissolve the DNA, store at 4°C for later use, or store at -20°C for long-term use.

[0116] 1.4.2 DNA extraction from reservoir samples (thermal lysis method)

[0117] This method is used for rapid pretreatment of water samples in the field, without the need for complex purification, and is suitable for on-site testing. The specific steps are as follows:

[0118] (1) Take 10 mL of water sample from the reservoir and slowly inject it into a filter device containing a 0.22 μm filter membrane using a syringe. Use negative pressure filtration to enrich algae. After filtration, use sterile tweezers to place the filter membrane into a 1.5 mL sterile centrifuge tube.

[0119] (2) Add 1 mL of sterile double-distilled water to the centrifuge tube, tighten the cap, place it in a constant temperature mixer preheated to 100°C, heat for 6 minutes to lyse the algal cell wall and release DNA.

[0120] (3) After heating, wait for the centrifuge tube to cool to room temperature, use a syringe to draw up the lysate in the tube and inject it into a filter device equipped with a 0.45μm filter membrane to filter out unlysed cell debris and filter membrane fibers.

[0121] (4) Transfer the filtered liquid into a new 1.5mL sterile centrifuge tube, which is the DNA template. It can be used immediately for RAA amplification or stored in a -70℃ ultra-low temperature freezer for more than 3 months.

[0122] Example 1: Design and screening of geoA gene-specific primers and probes from soil-producing cyanobacteria

[0123] This embodiment aims to design and screen RAA primer sets and probes that can specifically amplify the geoA gene of soil-producing cyanobacteria, determine the optimal combination with no false positives and high specificity, and lay the foundation for subsequent detection systems.

[0124] Using the geoA gene as the target gene, a core functional gene in the geosmin synthesis pathway with extremely high conservation in geosmin-producing cyanobacteria, 50 highly similar geoA gene sequences were obtained from the NCBI database. After multiple sequence alignment to determine the conserved amplification region, and analysis of the target gene sequence characteristics, species-specific amplification primers were designed using Primer Premier 5.0 software. Primer design strictly adhered to the following principles:

[0125] (1) The length of the amplification primers should be controlled within the range of 20-30 bp, and the difference in annealing temperature between the forward and reverse primers should not exceed 5℃ to ensure that the target sequence can be bound synchronously under the same isothermal conditions;

[0126] (2) The length of the amplification product is controlled within the range of 200-500bp, taking into account both RAA amplification efficiency and species specificity;

[0127] (3) The primer amplification segment was compared with the BLAST function of NCBI to exclude homology with the nucleic acid sequence of non-soil-producing cyanobacteria, and the specificity was preliminarily verified.

[0128] The primer amplification region sequences were compared using the BLAST function of the National Center for Biotechnology Information (NCBI) to preliminarily identify the primer amplification specificity. The identified primers were synthesized by General Biosystems (Anhui) Co., Ltd., with a purity level of HPLC. The specific primer sequences are shown in Table 1 below:

[0129] Table 1 lists the primers used for basic amplification of geosmin-specific primers RAA.

[0130]

[0131] For the regions amplified by specific primers, species-specific probe sequences were designed using Primer Premier 5.0 software.

[0132] The probe sequence is located in the middle region of the amplification primers and is modified with tetrahydrofuran. The probe design follows these principles:

[0133] (1) The length is controlled within the range of 45-50 nt to ensure the binding specificity with the target sequence;

[0134] (2) The sequence is located within the amplification region of the forward and reverse primers;

[0135] (3) Biotin is modified at the 5' end for capture in the detection zone of subsequent lateral flow test strips;

[0136] (4) The 3' end is modified with a phosphate group to inhibit the elongation of nonspecific DNA strands;

[0137] (5) The sequence contains a tetrahydrofuran-modified cleavage site for specific recognition and cleavage by the nfo enzyme;

[0138] (6) The design direction is consistent with the forward primer to avoid competition with the primer when binding to the target sequence.

[0139] The specific primer sequences, probes, and amplification reverse primer labels are shown in the table below.

[0140] In this embodiment, a total of 4 primer-probe combinations were designed, denoted as GSM-1, GSM-2, GSM-3, and GSM-4, respectively. The specific sequences and modification methods are shown in Table 2 below:

[0141] Table 2 lists the four sets of primers for RAA-nfo amplification of the geoA gene.

[0142]

[0143] The primers and probes mentioned above were synthesized by General Biosystems (Anhui) Co., Ltd., with a purity level of HPLC grade, to ensure the specificity of amplification and detection.

[0144] In this embodiment, the RNA isothermal rapid amplification kit (strip type)-III (WLRN8211KIT) from Anpu Future (Changzhou) Biotechnology Co., Ltd. was used. Positive algal DNA was used as a template to perform RAA amplification of each primer set for geosmin. The amplification system (single sample / reaction) is as follows:

[0145] Screening Experiment Operation

[0146] (1) Preparation of positive template: Anabaenasp.FACHB-1391, a soil-producing cyanobacterium, was used as the positive algae species. Its genomic DNA was extracted by CTAB method. The DNA concentration and purity were determined by ultra-micro spectrophotometer. The concentration was adjusted to 100 ng / μL to ensure that the OD260 / OD280 ratio was between 1.8 and 2.0.

[0147] (2) Negative control setup: Sterile double-distilled water was used as a blank negative control, and genomic DNA of the non-soil-producing cyanobacterium Nostoccommune FACHB-261 was used as a non-target negative control.

[0148] (3) Preparation of RAA amplification system: Each primer-probe combination was repeated 3 times, and the total volume of the amplification system was 50 μL. The amounts of each component were as follows:

[0149]

[0150] Add the above components sequentially to 0.2 mL sterile centrifuge tubes, gently pipette to mix, avoiding the formation of air bubbles; add 2.5 μL of 280 mM MgAc2 solution to each centrifuge tube, immediately tighten the cap, gently invert to mix 3 times, and briefly centrifuge (3000 rpm, 10 seconds) to collect the liquid to the bottom of the tube.

[0151] (4) RAA amplification reaction: Place the centrifuge tube in a constant temperature mixer, set the temperature to 39℃, and react at a constant temperature for 16 minutes;

[0152] (5) Lateral flow test strip detection: After the amplification reaction is complete, take 2 μL of amplification product, add 98 μL of sterile double-distilled water (i.e., dilute 50 times), and mix gently; take out the single-target disposable nucleic acid test strip, attach... Figure 2 This is a schematic diagram of the structure of the single-target disposable nucleic acid test strip JY0201. The test strip includes a sample pad, a conjugation pad, a T-line in the detection area, a C-line in the quality control area, and an absorbent pad. Insert the sample pad end of the test strip vertically into the diluted amplification product, ensuring the liquid level does not exceed the immersion mark on the sample pad. After the liquid has completely saturated the reading area of ​​the test strip, place the test strip flat on a clean lab bench and let it sit at room temperature for 1 minute. Observe and record the color development results within 10 minutes.

[0153] (6) Result interpretation criteria: If both the control area C line and the test area T line show red bands, it is judged as positive; if only the control area C line shows red bands, it is judged as negative; if no red bands appear in the control area C line, regardless of whether the test area shows color, it is judged as invalid and the experiment needs to be repeated.

[0154] Appendix Figure 3 The image shows the detection results of test strips using geosmin primer sets GSM-1, GSM-2, GSM-3, and GSM-4 to amplify RAA in positive algae and negative controls. The above screening results are attached. Figure 3 As shown, specifically:

[0155] GSM-1 group: The positive template test result was positive, but both the blank negative control and the non-target negative control showed T-line color development in the detection area, which was a false positive. The contamination interference during the experiment could not be ruled out, and it did not meet the screening requirements.

[0156] GSM-4 group: The positive template test result was positive, but the blank negative control showed obvious false positives, and the T line in the detection area of ​​the non-target negative control was weak but still distinguishable, indicating insufficient specificity and failing to meet the screening requirements.

[0157] GSM-3 group: The positive template test result was positive, and there were no false positives in the blank negative control. However, the non-target negative control showed a weak false positive, which could not completely rule out cross-reaction and did not meet the screening requirements.

[0158] GSM-2 group: The positive template detection result was positive, and both the C line in the quality control area and the T line in the detection area were clearly colored. The blank negative control and the non-target negative control only showed color in the C line of the quality control area, and there were no false positives. The amplification products of the GSM-2 group were sequenced to verify that the amplified fragment was a specific sequence of the geoA gene with a fragment length of 265bp, which is in line with the design expectation of 200-500bp.

[0159] Based on the above results, GSM-2 was determined to be the optimal primer-probe combination, namely, the forward primer geo-F sequence is 5'-CTATAAGCGCAGTCAGGATCTAATT-3', the reverse primer geo-R1 sequence is 5'-GTATTCAATGGGGTTAGCAATGCGA-3', and the probe geo-P2 sequence is:

[0160] 5'-TTCCCCTTCCCACGAACCCAGTAGAGCGCGHTTTAGCTGACTTGTG-3', where H is tetrahydrofuran.

[0161] Example 2: Preparation of alignment disk containing recombinant plasmid with geoA gene

[0162] In this embodiment, a standardized plasmid template was prepared as the quantitative basis for subsequent sensitivity experiments and as a positive control for the kit, ensuring that the experimental results are reproducible.

[0163] 2.1 Construction and culture of recombinant plasmids are shown below:

[0164] (1) Using the genomic DNA of Anabaenasp.FACHB-1391 as a template, the geoA gene fragment was obtained by amplification using the GSM-2 primers (geo-F and geo-R1) selected in Example 1.

[0165] (2) The amplified geoA gene fragment was ligated to the vector to construct a recombinant plasmid containing the geoA gene; the recombinant plasmid was transformed into competent Escherichia coli cells. The specific operation steps were as follows: 50 μL of competent Escherichia coli cells were taken, 10 μL of recombinant plasmid ligation product was added, and the cells were placed on ice for 30 minutes; heat shock was performed at 42℃ for 90 seconds, followed by immediate ice incubation for 2 minutes; 500 μL of antibiotic-free LB broth medium was added, and the cells were cultured on a shaker at 37℃ and 200 rpm for 1 hour.

[0166] (3) Spread 100 μL of culture onto an LB solid plate containing ampicillin (final concentration 100 μg / mL) and incubate upside down at 37°C for 16 hours; pick 3 single colonies and inoculate them into 5 mL of LB broth containing ampicillin, and incubate overnight at 37°C and 200 rpm. Verify positive colonies by bacterial PCR (primers are geo-F and geo-R1 of GSM-2 group). Positive colonies are used for subsequent plasmid extraction.

[0167] 2.2 Extraction and Concentration Determination of Recombinant Plasmids

[0168] Pick PCR-positive colonies and inoculate them into 50 mL of LB broth containing ampicillin. Incubate overnight at 37°C and 200 rpm for about 16 hours.

[0169] Take 30 mL of bacterial culture and extract recombinant plasmids using a bacterial plasmid miniprep kit. Follow the kit instructions: Add RNase A-containing resuspension to the bacterial culture to fully resuspend the bacterial pellet; add lysis buffer, gently invert the centrifuge tube 5 times, and incubate at room temperature for 2 minutes; add neutralization buffer, immediately invert to mix, and centrifuge at 12000 rpm for 10 minutes; take the supernatant and add it to the adsorption column, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid; add washing buffer I, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid; add washing buffer II, centrifuge at 12000 rpm for 1 minute, and discard the waste liquid; place the adsorption column into a new centrifuge tube, add 50 μL of elution buffer, incubate at room temperature for 2 minutes, centrifuge at 12000 rpm for 2 minutes, and collect the eluent, which is the recombinant plasmid solution;

[0170] The concentration of the recombinant plasmid solution and the OD260 / OD280 ratio were determined using an ultra-micro spectrophotometer. The determination was repeated three times, and the average value was taken. The results showed that the concentration of the recombinant plasmid was 64.4 ng / μL, and the OD260 / OD280 ratio was 1.92, which met the experimental requirements. An OD260 / OD280 ratio of 1.8-2.0 indicates that the plasmid purity is high and there is no obvious protein or RNA contamination.

[0171] 2.3 Plasmid copy number calculation and gradient dilution

[0172] Copy number calculation: According to the formula: Plasmid copy number (copies / μL) = (Plasmid concentration (ng / μL) × 10⁻ 9 Calculate the copy number of the recombinant plasmid using (×6.02×10²³) / (total plasmid length (bp) × 660); where the total plasmid length is the sum of the vector length and the geoA gene fragment length, which was determined to be 2957 bp; substituting the data, we get: copy number = (64.4×10⁻⁻⁶) / (total plasmid length (bp) × 660). 9 (×6.02×10²³) / (2957×660)≈1.92×10¹ 0 Copy / μL;

[0173] Serial dilution: Take 10 μL of the recombinant plasmid stock solution (concentration 1.92 × 10¹). 0 The concentration was 1.92 × 10⁻⁶ copies / μL, diluted to 100 μL with sterile double-distilled water to obtain a concentration of 1.92 × 10⁻⁶. 9 A dilution of 1 copy / μL was prepared; and so on, 10-fold serial dilutions were performed to obtain concentrations of 1.92 × 10⁻⁶. 8 Copies / μL, 1.92 × 10 7 Copies / μL, 1.92 × 10 6 Copies / μL, 1.92 × 10 5 Copies / μL, 1.92 × 10 4 1.92 × 10³ copies / μL, 1.92 × 10² copies / μL, 1.92 × 10¹ copies / μL, 1.92 × 10 0 A plasmid dilution of 1 copy / μL is used to construct a complete plasmid alignment disk;

[0174] Storage: Aliquot the plasmid dilutions of each concentration into 0.2mL sterile centrifuge tubes, 10μL per tube, and store at -20℃ for later use. Avoid repeated freeze-thaw cycles to prevent plasmid stability.

[0175] Example 3: Optimization of the RAA amplification system using GSM-2 primer set

[0176] This embodiment optimizes the addition volume of the GSM-2 primer set by gradient, exploring the effect of different primer addition amounts on detection sensitivity and specificity, determining the optimal addition volume, and avoiding primer dimers or false positives due to excessive primers, or decreased sensitivity due to insufficient primers. The test strip detection product is a double-stranded amplification product of the probe after nfo enzyme digestion and the reverse primer (FITC modified). Therefore, theoretically, increasing the amount of reverse primer added helps improve the sensitivity of RAA nucleic acid test strip detection. However, excessive reverse primer addition can also increase the probability of reverse primer-probe primer dimer formation, leading to false positive results.

[0177] The forward primer geo-F, reverse primer geo-R1, and probe geo-P2 of the GSM-2 group were mixed in a ratio of 7:10:2 to prepare a primer mix with a concentration of 10 μM; a volume gradient was added to the three primer mixes.

[0178] The total volume of the RAA reaction system for each gradient was 50 μL, with the plasmid prepared in Example 2 having a concentration of 1.92 × 10⁻⁶ in the comparison disk. 7 Copies / μL, 1.92 × 10 8 Copies / μL, 1.92 × 10 9 A plasmid dilution of 1 copy / μL was used as a template, and sterile double-distilled water was used as a blank negative control. Each gradient and template combination was repeated 3 times.

[0179] Gradient dilutions of plasmid DNA constructed from positive algae were used as templates for RAA amplification. The RAA reaction system (single sample / reaction) was prepared using the RNA Isothermal Rapid Amplification Kit (strip type)-III (WLRN8211KIT) from Anpu Future (Changzhou) Biotechnology Co., Ltd. The forward primer geo-F, reverse primer geo-R1, and probe geo-P2 from the GSM-2 group were mixed in a ratio of 7:10:2 to prepare a 10 μM primer mix. RAA amplification was performed according to the method in Table 3, with volume gradients added to the three primer mixes.

[0180] For each system, 2 μL of positive algal DNA gradient dilution was used as a template, and negative control samples were included for testing. The total volume of each system was 50 μL. Using the plasmid gradient dilution from Example 2 as a template, sterile double-distilled water was used as a negative control. Each system was repeated three times. In this example, the concentration of the plasmid gradient dilution was 1.92 × 10⁻⁶. 7 ~1.92×10 9 Copy / μL.

[0181] After adding the samples, immediately cap each tube to prevent aerosol contamination. Mix the above reaction system thoroughly and add it to the basic reaction unit, ensuring the lyophilized powder is fully dissolved. Note that this step should not involve vigorous vortexing. Open the reaction unit and add 2.5 μL of 280 mM MgAc2 to each 0.2 mL Eppendorf tube, mix thoroughly, and collect by centrifugation. Note that this step should not involve vigorous vortexing.

[0182] Experimental operation steps

[0183] (1) Preparation of RAA amplification system: The amounts of components used in the reaction system for each gradient are shown in the table below:

[0184]

[0185] When preparing the solution, first add all components except MgAc2 to a 0.2 mL sterile centrifuge tube and gently mix them by pipetting. After adding the MgAc2 solution, gently invert the centrifuge tube three times and centrifuge briefly (3000 rpm, 10 seconds) to collect the liquid to the bottom of the tube, avoiding the generation of air bubbles.

[0186] (2) RAA amplification reaction: Place the centrifuge tube in a constant temperature mixer, set the temperature to 39℃, and react at a constant temperature for 16 minutes;

[0187] (3) Lateral flow test strip detection: After the amplification reaction is completed, take 2 μL of amplification product, dilute it 50 times with sterile double-distilled water, and perform test strip detection according to the steps described in Example 1.3.5. Observe and record the color development results;

[0188] (4) Agarose gel electrophoresis verification: Take 10 μL of amplification product, add 2 μL of loading buffer, mix well and load onto 1.5% agarose gel (containing EB dye), electrophoresis at 120V for 30 minutes; after electrophoresis, observe the electrophoretic bands with a gel imaging system to determine whether primer dimers exist. Primer dimers are non-specific bands of less than 200bp.

[0189] Test results as follows Figure 4 (Comparison of amplification results for GSM-2RAA primer sets with different volumes of geosmin added) is shown in the figure:

[0190] Gradient 1 (5.7 μL): The blank negative control showed a negative result, with only the C line showing color; 1.92 × 10⁻⁶ 7 Copies / μL, 1.92 × 10 8 Copies / μL, 1.92 × 10 9 All plasmid templates of copy / μL were detected as positive, with both C and T lines showing color simultaneously, and the T line showing clear color. Agarose gel electrophoresis results showed only a specific band of 265bp, with no primer dimers, indicating that the primers and probes could fully bind to the target sequence at this volume, and there were no additional non-specific reactions.

[0191] Gradient 2 (6.0 μL): The blank negative control test result was negative; 1.92 × 10⁻⁶ 7 Copies / μL, 1.92 × 10 8 The plasmid template at a concentration of 1.92 × 10⁻⁶ copies / μL was detected positive, but the result was 1.92 × 10⁻⁶. 9 The detection of plasmid template at a concentration of 1 copy / μL was negative, with only the C line showing color, indicating decreased sensitivity; the electrophoresis results showed no primer dimers, but the specific band brightness was weaker than that of gradient 1.

[0192] Gradient 3 (6.3 μL): The blank negative control result was negative; 1.92 × 10⁻⁶ 7The plasmid template at a concentration of 1.92 × 10⁻⁶ copies / μL was detected as positive. 8 Copies / μL, 1.92 × 10 9 All plasmid templates at a concentration of 1 copy / μL were negative, indicating a significant decrease in sensitivity. Electrophoresis results showed weak bands below 200bp, which were presumed to be primer dimers, suggesting that primer overload led to nonspecific binding.

[0193] Based on the test results, the optimal addition volume of the GSM-2 primer set was determined to be 5.7 μL. At this volume, effective detection of low-concentration plasmid templates can be achieved while ensuring no false positives and no primer dimers.

[0194] Example 4: Specificity verification of the GSM-2 primer set RAA-LFS system

[0195] This embodiment verifies the specific recognition ability of the GSM-2 primer set RAA-LFS system for geosmo ...

[0196] Experimental materials

[0197] Cyanobacteria samples: All 14 cyanobacteria were obtained from the Freshwater Algae Culture Bank of the Chinese Academy of Sciences. The specific species and geoserotonin-producing characteristics are as follows:

[0198] Anabaenacylindrica FACHB-170 (non-geomyophyte);

[0199] Anabaenaminutissima FACHB-250 (non-originating soil odorant);

[0200] NostoccommuneFACHB-261 (non-soil-producing odorant);

[0201] Oscillatoria Oscillatoriasp.FACHB-1120 (geosmin-producing);

[0202] Pseudanabaenacinerea FACHB-1277 (non-originating osmidrogen);

[0203] PlanktothricoidesraciborskiiFACHB-1360 (geosmin-producing);

[0204] AerosakkonemafuniformeFACHB-1362 (geosmin-producing);

[0205] AerosakkonemafuniformeFACHB-1363 (geosmin-producing);

[0206] PlanktothricoidesraciborskiiFACHB-1372 (geosmin-producing);

[0207] PlanktothricoidesraciborskiiFACHB-1378 (geosmin-producing);

[0208] Anabaena Anabaenasp.FACHB-1384 (geosmin-producing);

[0209] Anabaena Anabaenasp.FACHB-1391 (geosmin-producing);

[0210] Anabaena Anabaenasp.FACHB-1501 (geosmin-producing);

[0211] Aphanizomenongracile (non-originating soil odor);

[0212] Blank negative control: sterile double-distilled water;

[0213] Detection system: The optimal system determined in Example 3, namely, primer set Mix addition volume 5.7 μL, RAA amplification conditions 39℃ isothermal reaction for 16 minutes.

[0214] The experimental procedure is as follows:

[0215] DNA template preparation: DNA was extracted from 14 cyanobacteria using the thermal lysis method. The specific operation was carried out as described above: 10 mL of laboratory culture medium from each cyanobacterium was taken and enriched through a 0.22 μm filter membrane; 1 mL of sterile double-distilled water was added and heated at 100 °C for 6 minutes; the mixture was filtered through a 0.45 μm filter membrane; the filtrate was collected as the DNA template.

[0216] RAA amplification reaction: Prepare 50 μL of reaction solution according to the optimal system, set up 3 replicates for each cyanobacterial strain, and set up a blank negative control at the same time; place the reaction tube in a constant temperature mixer and react at 39℃ for 16 minutes.

[0217] Lateral flow test strip detection: Take 2 μL of amplification product, dilute it 50 times with sterile double-distilled water, and perform the detection according to the steps described in Example 1. Record the color development results for each cyanobacterium.

[0218] The above test results are as follows Figure 5 (The optimal detection system for geosmin-specific RAA-LFS for various cyanobacteria is shown in the following graphs. The blank negative control is sterile double-distilled water.)

[0219] Soil-producing cyanobacteria (numbered 4, 6-13): A total of 10 strains were tested, and all results were positive, meaning that red bands were clearly visible in both the control area C line and the test area T line, with no false negatives.

[0220] Non-soil-producing cyanobacteria (numbered 1-3, 5, 14): a total of 5 strains, all of which tested negative, i.e. only the C line in the quality control area showed a red band, and the T line in the detection area showed no color development, with no false positives;

[0221] Blank negative control: Only the C line in the quality control area shows color, while the T line in the detection area shows no color.

[0222] The above results indicate that the GSM-2 primer set RAA-LFS system has strict specificity, can accurately distinguish between soil-producing and non-soil-producing cyanobacteria, and has no cross-reactivity, meeting the specificity requirements in practical detection.

[0223] Example 5: Sensitivity analysis of the GSM-2 primer set RAA-LFS system

[0224] In this embodiment, soil-producing cyanobacteria cells and recombinant plasmids containing the geoA gene were used as templates. Through gradient dilution experiments, the lowest detection limit of the GSM-2 primer set RAA-LFS system was determined, and the detection capability of the system for low concentrations of soil-producing cyanobacteria was verified, providing data support for early warning of drinking water sources.

[0225] Experimental materials:

[0226] Cell template: Logarithmic phase culture of Anabaenasp. FACHB-1391 was counted using a hemocytometer (observed under an optical microscope, with the average value taken from three fields of view), and then serially diluted with sterile LB medium to obtain cell suspensions with concentrations of 1130 cells / reaction, 113 cells / reaction, 11.3 cells / reaction, and 1.13 cells / reaction.

[0227] Plasmid template: The recombinant plasmid alignment disk prepared in Example 2, with concentrations of 3.84 × 10³ copies / μL, 3.84 × 10² copies / μL, 3.84 × 10¹ copies / μL, and 3.84 × 10¹ copies / μL. 0 Four concentration gradients per copy / μL;

[0228] Detection system: The optimal system determined in Example 3, namely primer set Mix 5.7 μL, reacted at 39℃ for 16 minutes.

[0229] The experimental procedure is as follows:

[0230] Preparation of cell template DNA: DNA was extracted from cell suspensions of various concentration gradients using thermal lysis. The specific procedure was as follows: 10 mL of cell suspension was passed through a 0.22 μm filter membrane for enrichment; 1 mL of sterile double-distilled water was added, and the mixture was heated at 100 °C for 6 minutes; the mixture was then filtered through a 0.45 μm filter membrane; the filtrate was collected as cell template DNA.

[0231] RAA amplification reaction: 50 μL of reaction solution was prepared using cell template DNA and plasmid template as amplification templates, and each template concentration was set up in 3 replicates; the reaction tubes were placed in a constant temperature mixer and reacted at 39℃ for 16 minutes.

[0232] Lateral flow test strip detection: Take 2 μL of amplification product, dilute it 50 times with sterile double-distilled water, and perform the detection according to the steps described in Example 1.3.5. Observe the color development results.

[0233] Results Recording: Record the occurrence of positive results (C-line + T-line color development) at each template concentration, and use the lowest template concentration with three consecutive positive results as the limit of detection of the system.

[0234] The above test results are as follows Figure 6 (The sensitivity diagram of the geosmin RAA test strip detection system based on gradient dilution of positive template and plasmid is shown below:)

[0235] Cell templates: Templates at concentrations of 1130 cells / reaction, 113 cells / reaction, and 11.3 cells / reaction all showed positive results in three replicates, with clear T-line development. Templates at a concentration of 1.13 cells / reaction showed positive results in two out of three replicates, with weak but clearly identifiable T-line development, and one negative result. Therefore, this concentration was determined to be effective for detection. Thus, the limit of detection for soil-producing cyanobacteria was determined to be 1.13 cells / reaction.

[0236] Plasmid template: Template concentrations of 3.84 × 10³ copies / μL, 3.84 × 10² copies / μL, and 3.84 × 10¹ copies / μL were all positive in three replicates; a concentration of 3.84 × 10¹ copies / μL was also tested. 0 The template was tested negative in three replicates. Based on 2 μL of template, the copy number in the reaction system corresponding to 3.84 × 10¹ copies / μL of template was 76.8. Based on the test results, the limit of detection for the geoA gene was determined to be 38.4 copies / reaction.

[0237] The above results indicate that the GSM-2 primer set RAA-LFS system has high sensitivity and can detect extremely low concentrations of geostinocyanobacteria, even at concentrations below 10 in water bodies. 4 Effective detection can be achieved at a concentration of cells / mL (invisible to the naked eye), meeting the need for early warning of geosmodium pollution in drinking water sources.

[0238] Example 6: Validation of the GSM-2 primer set RAA-LFS system on actual reservoir water samples

[0239] This embodiment uses 35 water samples from Ningbo drinking water source reservoirs as the testing objects. By comparing with conventional PCR methods, it verifies the accuracy and reliability of the GSM-2 primer set RAA-LFS system in practical application and evaluates the applicability of the system in drinking water source monitoring.

[0240] Experimental materials:

[0241] Water samples from reservoirs: 35 samples were collected from three different drinking water source reservoirs in Ningbo City. After sampling, the samples were preserved in an ice bath and transported back to the laboratory within 4 hours. DNA was extracted using the above-mentioned thermal lysis method.

[0242] PCR control method: The geoA gene-specific PCR primers reported by John et al. (2018) were used. The upstream primer sequence was 5'-GCCCTTTTCGACGATTTCAA-3', and the downstream primer sequence was 5'-GGAAGCACTATTACGCAATTCAAA-3'. The PCR reaction system (50 μL) consisted of: 5 μL of 10×PCR buffer, 4 μL of dNTPs (10 mM), 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 0.5 μL of Taq enzyme (5 U / μL), 3 μL of DNA template, and 35.5 μL of sterile double-distilled water.

[0243] Amplification conditions: 95℃ pre-denaturation for 1 minute, 40 cycles (95℃ denaturation for 10 seconds, 60℃ annealing for 30 seconds), 72℃ extension for 5 minutes; the amplified products were detected by 1.5% agarose gel electrophoresis, and the presence of a specific band of 265bp was considered positive, while the absence of a band was considered negative.

[0244] Detection system: GSM-2 primer set RAA-LFS optimal system (same as Example 3).

[0245] The experimental procedure is as follows:

[0246] RAA-LFS detection: Take 2 μL of thermally lysed DNA template from each water sample, prepare 50 μL of reaction solution according to the optimal system, and react at 39℃ for 16 minutes; take 2 μL of amplification product, dilute it 50 times, and test it with test strips, and record the positive / negative results for each water sample;

[0247] PCR detection: Take 3 μL of thermally lysed DNA template from each water sample and amplify it according to the PCR system and conditions described above; after amplification, take 10 μL of the product for 1.5% agarose gel electrophoresis, observe the bands using a gel imaging system, and record the results;

[0248] Results Comparison: The detection results of RAA-LFS and PCR methods were statistically analyzed, and the consistency between the results of the two methods was calculated.

[0249] The above test results are as follows Figure 7 (Figure showing the RAA-LFS and PCR detection results of the geoA gene in 35 reservoir samples) and Table 4 below:

[0250] Table 4 shows the RAA-LFS and PCR detection results of the geoA gene in 35 reservoir samples.

[0251]

[0252] Note: "+" indicates a positive result, meaning the sample contains cyanobacteria that produce cyanobacteria; "-" indicates a negative result, meaning the sample does not contain cyanobacteria that produce cyanobacteria.

[0253] The results showed that among the 35 reservoir water samples, the RAA-LFS system detected 12 positive samples and 23 negative samples. The number of positive and negative samples detected by the conventional PCR method was completely consistent with that of the RAA-LFS method, and the results of the two methods were 100% consistent. Meanwhile, the concentration of geosmin in the positive water samples was determined according to the "Test Method for Odor Substances Geosmin and 2-Methylisoborneol in Drinking Water" (GB / T32470-2016). The results showed that the geosmin concentration in the positive water samples was ≥3 ng / L, directly corresponding to the RAA-LFS detection results. This further proves that the system can be accurately applied to the detection of geosmin-producing cyanobacteria in actual reservoir water samples, providing reliable technical support for the management of drinking water source quality.

[0254] Comparative Example 1: Comparison of the RAA-LFS method of the present invention with conventional PCR methods

[0255] This comparative example compares the GSM-2 primer set RAA-LFS method of this invention with existing conventional PCR methods (same as Example 6) from four dimensions: detection efficiency, sensitivity, instrument dependence, and operational complexity, to verify the effectiveness of this invention in overcoming the defects of existing technologies.

[0256] (1) Detection efficiency

[0257] The RAA-LFS method of this invention takes about 30-35 minutes from water sample collection to obtaining the test results. The specific time allocation is 12 minutes for thermal pyrolysis pretreatment, 16 minutes for RAA amplification, and 2 minutes for lateral flow test strip detection. The whole process does not require waiting for the instrument to heat up or for program settings and can be operated continuously.

[0258] Conventional PCR method: The entire process takes about 2.5 hours, specifically allocated as follows: 30 minutes for column purification pretreatment (requiring multiple centrifugations and washing), 1.5 hours for PCR amplification (including waiting for the thermal cycling program), and 30 minutes for agarose gel electrophoresis and imaging (requiring gel preparation, electrophoresis, and UV imaging). The steps require waiting for the instrument to complete the preceding operations and cannot be performed continuously.

[0259] The comparison shows that the detection efficiency of the method of the present invention is significantly higher than that of the conventional PCR method, and the time taken is only 1 / 4 of that of the PCR method. It can realize the rapid detection of soil-producing cyanobacteria and meet the needs of on-site real-time monitoring.

[0260] (2) Sensitivity

[0261] Using serially diluted cell suspensions of Anabaenasp. FACHB-1391 as templates, two methods were used for detection:

[0262] The method of this invention has a detection limit of 1.13 soil-producing cyanobacteria cells / reaction, which can detect extremely low concentrations of cyanobacterial contamination.

[0263] Conventional PCR method: The limit of detection is 10 cyanobacterial cells producing geosmolybdenum per reaction; below this concentration, clear electrophoretic bands cannot be obtained.

[0264] The sensitivity of the method of this invention is about 9 times higher than that of conventional PCR methods, enabling early detection of soil-producing cyanobacteria before they proliferate in large quantities, thus providing sufficient time for pollution control.

[0265] (3) Instrument dependence

[0266] The method of this invention requires only a portable constant temperature mixer and a lateral flow test strip, without the need for a fixed power supply or laboratory environment, and can be operated directly at the drinking water source; the test strip detection requires no additional instruments, and the results can be interpreted with the naked eye;

[0267] Conventional PCR methods rely on PCR instruments and gel imaging systems. Both types of instruments require a stable 220V power supply and a horizontal experimental platform, and must be used in a clean laboratory environment. They cannot be used outside of a laboratory setting.

[0268] The method of this invention has low instrument dependence and its field applicability is significantly better than that of conventional PCR methods, which can meet the mobile monitoring needs of grassroots water sources.

[0269] (4) Operational complexity

[0270] The method of this invention: Water sample pretreatment adopts thermal pyrolysis, which only requires three steps: filtration, heating, and re-filtration, without the need for complex reagent kits; RAA amplification is a one-step isothermal reaction, which only requires mixing reagents in proportion, without the need to adjust temperature parameters; the results can be interpreted by visually observing the bands on the test strip, and the testing personnel can operate independently after 1 hour of training; the whole process has no risk of aerosol contamination (no need to open the cap for electrophoresis).

[0271] Conventional PCR methods involve several steps: water sample pretreatment requires column purification to remove nucleic acid impurities, including adding lysis buffer, centrifugation, column chromatography, washing, and elution, which is cumbersome and prone to introducing contamination; PCR amplification requires complex thermal cycling programs (requiring professional personnel to fine-tune parameters); result interpretation requires professional personnel to analyze electrophoresis bands under UV light, which can easily lead to misinterpretation due to blurred or non-specific bands; and electrophoresis requires opening the cap to handle amplification products, posing a risk of aerosol contamination and potentially causing false positives in subsequent experiments.

[0272] The method of this invention has a much lower operational complexity than conventional PCR methods, making it more suitable for promotion and application in grassroots monitoring units or on-site testing scenarios.

[0273] Comparative Example 2: Comparison of the RAA-LFS method of this invention with the headspace solid-phase microextraction-gas chromatography-mass spectrometry method (GB / T32470-2016)

[0274] This comparative study compares the method of this invention with the existing national standard method for detecting geosmin compounds, highlighting the advantages of this invention in early warning of geosmin-producing cyanobacteria.

[0275] (1) Detection targets and early warning timeframe

[0276] This invention utilizes a method that directly detects the geoA gene of geosmin-producing cyanobacteria, eliminating the need to wait for geosmin compound accumulation. This method is applicable when the concentration of cyanobacteria in the water is below 10... 4 Algae can be detected at a concentration of cells / mL (invisible to the naked eye), and the early warning time can be 3-7 days in advance, making it convenient to take timely measures to control algae (such as adding algaecides or adjusting the water plant process).

[0277] The national standard method requires that the concentration of geosmin reaches the limit (10 ng / L) specified in the "Standards for Drinking Water Quality" (GB5749-2022) to detect the geosmin compound itself. At this time, cyanobacteria have already proliferated in large quantities, and some algal cells may have ruptured and released geosmin, which significantly increases the difficulty of subsequent treatment and misses the opportunity for early intervention. The method of this invention can achieve early warning when "algae have not proliferated in large quantities and the taste has not appeared", and the warning timeliness is significantly better than the national standard method.

[0278] (2) Detection efficiency and cost

[0279] The method of this invention takes 30-50 minutes in total, and the cost of a single test is about 50 yuan, mainly for primers, test strips and reagents; it can process multiple samples at the same time and does not require dedicated personnel to be on duty.

[0280] National standard method: The entire process takes more than 4 hours (including 2 hours for sample pretreatment and 2 hours for instrument analysis), requires a gas chromatography-mass spectrometry (purchase cost ≥ 500,000 yuan, annual maintenance cost ≥ 20,000 yuan); the cost of a single test is about 1,000 yuan (including consumables, reagents and instrument depreciation); the instrument operation requires certified professionals and cannot process samples in batches;

[0281] The method of this invention has significant advantages in both detection efficiency and cost, and is suitable for large-scale water source sample screening.

[0282] (3) On-site applicability

[0283] The method of this invention requires no large instruments; the detection equipment can be packed into a portable case, and the entire process from sample pretreatment to result interpretation can be completed on-site at the water source; no laboratory environment is required, and it can be operated outdoors at room temperature.

[0284] National standard method: Gas chromatography-mass spectrometry is bulky and requires fixed installation, and can only be used in professional laboratories; water samples need to be transported from the site back to the laboratory (which may cause geosmin volatilization or concentration changes); the pretreatment process requires the use of organic reagents (such as dichloromethane), which poses safety risks and requires a professional fume hood;

[0285] The field applicability of the method of this invention is far superior to that of the national standard method, and it can meet the needs of real-time monitoring and emergency testing of drinking water sources.

[0286] Comparative Example 3: Comparison of the RAA-LFS method of this invention with loop-mediated isothermal amplification (LAMP) technology

[0287] This comparative example compares the method of the present invention with the existing isothermal amplification technology (LAMP) to verify that the present invention overcomes the shortcomings of the existing isothermal technology.

[0288] (1) Primer design and specificity

[0289] The method of this invention requires only the design of 3 primers / probes (1 pair of amplification primers + 1 detection probe), based on the conserved region of the geoA gene, and the specificity can be preliminarily verified by NCBIBLAST; the design cycle is about 3 days, and the primer synthesis success rate is 100%; in actual detection, there is no cross-reactivity with non-geosin-producing cyanobacteria, and the false positive rate is 0%.

[0290] LAMP method: requires the design of 4-6 primers (including 2 inner primers, 2 outer primers, and 2 loop primers), and the primers need to cover 6 different regions of the target gene, which is difficult to design; it is easy to fail due to non-specific binding between primers, and the design cycle is about 7-10 days; in actual detection, false positives are easy to occur with non-target cyanobacteria templates, with a false positive rate of about 5%, which requires additional verification;

[0291] The primer design of the method of the present invention is simpler and more specific, and can effectively avoid false positive interference.

[0292] (2) Amplification time and product detection

[0293] The method of this invention: sufficient amplification products can be obtained by reacting at a constant temperature of 39℃ for 16 minutes, which meets the detection requirements of the test strip; the products are visually detected by the lateral flow test strip, without the need for additional instruments, and the result interpretation time is ≤5 minutes;

[0294] LAMP method: requires constant temperature reaction at 65℃ for 60-90 minutes to obtain detectable amplified products; product detection relies on turbidimeter (to detect magnesium pyrophosphate precipitation) or fluorescence detector (to detect fluorescent dye signal), and results cannot be interpreted without the instruments; turbidimetry is easily affected by water sample impurities, and fluorescence method requires the addition of fluorescent dye (increasing cost and operation steps); the method of this invention has a faster amplification speed and more convenient product detection, without relying on additional detection instruments.

[0295] (3) Anti-interference capability

[0296] The method of this invention: The sample is pretreated by thermal pyrolysis, and most impurities can be removed by a 0.45μm filter membrane; the basic buffer in the RAA amplification system contains an ionic strength regulator, which can resist the interference of low concentrations of heavy metals or organic matter in the water sample; in actual detection, the detection results of turbid water samples (turbidity ≤50NTU) show no significant deviation.

[0297] LAMP method: requires high sample purity. Impurities in water samples (such as humic acid and heavy metals) can easily inhibit DNA polymerase activity, leading to false negatives. Additional column purification is required (increasing the number of steps and costs), otherwise the test results are unstable.

[0298] The method of this invention has stronger anti-interference ability, lower requirements for water sample purity, and is more suitable for water sample testing in complex water sources.

[0299] In summary, the GSM-2 primer set RAA-LFS method established by this invention through primer set screening, amplification system optimization, and actual water sample verification is significantly superior to existing technologies (conventional PCR, national standard compound detection method, LAMP) in terms of specificity, sensitivity, detection efficiency, and field applicability. It can effectively support the early warning and real-time monitoring of local geosmocyanin algae in drinking water sources, and provide reliable technical support for water quality safety management.

[0300] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention. Therefore, the specification and drawings should be considered illustrative rather than restrictive.

Claims

1. A primer set for detecting the geoA gene in soil-producing cyanobacteria, characterized in that, The primer set can specifically recognize and amplify the geoA gene of soil-producing algae to achieve targeted detection of soil-producing algae; the primer set includes geoA gene detection primers and geoA gene detection probe geo-P2; the geoA gene detection primers include a forward primer geo-F and a reverse primer geo-R1, and the 5' end of the reverse primer geo-R1 is modified with fluorescein isothiocyanate; The 5' end of the geoA gene detection probe geo-P2 is modified with biotin, which enables the double-stranded amplification product formed by the probe and the reverse primer to be detected by the nucleic acid test strip; the 3' end of the probe geo-P2 is modified with a phosphate group, which is used to inhibit non-specific DNA strand elongation.

2. The primer set according to claim 1, characterized in that, The nucleotide sequence of the forward primer geo-F is shown in SEQ ID No. 1: 5'-CTATAAGCGCAGTCAGGATCTAATT-3'.

3. The primer set according to claim 1, characterized in that, The nucleotide sequence of the reverse primer geo-R1 is shown in SEQ ID No. 2: 5'-GTATTCAATGGGGTTAGCAATGCGA-3'.

4. The primer set according to claim 1, characterized in that, The design of the forward and reverse primers follows these principles: (1) The nucleotide lengths of the forward and reverse primers are both 20~30bp, and the difference in annealing temperature between the forward and reverse primers does not exceed 5℃ to ensure the consistency of amplification reaction conditions; (2) When the forward primer and the reverse primer are used together to amplify the geoA gene, the length of the amplified product is 200~500bp, and the amplified product has species specificity of soil-producing algae.

5. The primer set according to claim 1, characterized in that, The nucleotide sequence of the geoA gene detection probe geo-P2 is shown in SEQ ID No. 3: 5'-TTCCCCTTCCCACGAACCCAGTAGAGCGCGHTTTAGCTGACTTGTG-3', H represents tetrahydrofuran; the probe is 45-50 nt in length and binds to the target region between the forward primer geo-F and the reverse primer geo-R1 in the geoA gene.

6. The primer set according to claim 5, characterized in that, The probe also satisfies the following conditions: (1) The design direction is positive, consistent with the nucleotide extension direction of the positive primer; (2) The nucleotide length is 45~50 nt; (3) The geoA gene detection probe has a cleavage site inside its sequence. When the probe binds to the geoA gene template, the cleavage site can be recognized and cleaved by the nfo enzyme, and the cleavage can initiate the DNA chain extension reaction.

7. A reagent kit for detecting soil-producing cyanobacteria, characterized in that, Includes the primer set as described in any one of claims 1-6, and the reagents required for recombinase-mediated isothermal amplification (RAA) reactions.

8. The reagent kit according to claim 7, characterized in that, The necessary reagents for RAA amplification include basal buffer, DNA polymerase, nfo enzyme, MgAc2 solution, and sterile double-distilled water; The basic buffer solution contains 10-20 mM Tris-HCl, 50-100 mM KCl, and 10-20 mM (NH4)2SO4; The concentration of the MgAc2 solution is 260-300 mM; The reagents required for the RAA reaction include RAA basal buffer, MgAc2 solution, and negative control. The RAA basal buffer contains recombinase, single-stranded binding protein, DNA polymerase, and dNTPs; The negative control is sterile double-distilled water or genomic DNA from non-soil-producing algae.

9. The reagent kit according to claim 8, characterized in that, It also includes a lateral flow chromatography test strip, which includes a sample pad, a conjugate pad, a detection zone, a control zone, and an absorbent pad; the conjugate pad is coated with a fluorescein isothiocyanate antibody-colloidal gold complex; the detection zone is coated with a biotin antibody; and the control zone is coated with an anti-fluorescein isothiocyanate antibody.

10. A method for detecting soil-producing cyanobacteria, characterized in that, Includes the following steps: S1: Obtain the water sample to be tested and preprocess it to obtain the DNA template; S2: Using the DNA template obtained in step (1) as a template, perform RAA amplification using any of the primer sets described in claims 1-6 to obtain the amplification product; S3: The RAA amplification products are visually detected using lateral flow test strips. Based on the detection results, it is determined whether the sample contains soil-producing algae.

11. The method according to claim 10, characterized in that, In step S1, the pretreatment is a pyrolysis method, specifically including: Water samples were enriched using 0.2-0.22 μm filter membranes. The filter membranes enriched with cyanobacteria were placed in sterile water and heated at 95-105℃ for 5-7 min. The samples were then filtered through 0.4-0.45 μm filter membranes, and the filtrate was collected as a DNA template.

12. The method according to claim 10, characterized in that, In step S2, the total volume of the RAA amplification reaction system is 50 μL, and the amounts of each component are as follows: 1-3 μL DNA template, 1.2-3.0 μL forward primer, 2.0-4.0 μL reverse primer, 0.2-1.0 μL geoA gene detection probe, 23.8-35.0 μL basal buffer, 2.0-3.0 μL MgAc2 solution, and bring the remaining volume to 50 μL with sterile double-distilled water. The final concentration of the forward primer, reverse primer, and geoA gene detection probe is 0.1-1.0 μM; the reaction conditions for RAA amplification are a constant temperature reaction at 37-41℃ for 15-20 min.

13. The method according to claim 10, characterized in that, In step S3, the amplification product is detected by lateral flow chromatography, and the specific steps are as follows: (1) Dilution: Take 2-5 μL of amplification product and dilute it 40-60 times with sterile double-distilled water; (2) Loading the sample: Insert the sample pad end of the lateral flow chromatography test strip into the diluted amplification product, ensuring that the liquid level does not exceed the immersion mark of the sample pad. (3) Interpretation: Place at room temperature for 1-5 minutes and record the results within 10 minutes; the interpretation criteria are: if a red band appears in both the quality control area and the test area, it is positive; if a red band appears only in the quality control area, it is negative; if no red band appears in the quality control area, the test is invalid.

14. The method according to any one of claims 10-13, characterized in that, The detection sensitivity of the method is to detect 1-2 geosmogrin-producing cyanobacterial cells / reaction, or 30-50 copies of the geoA gene / reaction; And / or, the detection time is 15-25 min for sample pretreatment, 12-20 min for RAA amplification, and 1-5 min for test strip detection, for a total time of 30-50 min.

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