Primer group, kit and method for detecting mic gene of cyanobacteria capable of producing 2-methylisoborneol

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 cyanobacteria generating 2-methylisoborneol in a water body based on a recombinase-mediated isothermal nucleic acid amplification (RAA) technology. The primer group is designed based on a mic gene conserved region of the 2-methylisoborneol-producing cyanobacteria, and comprises a forward primer, a reverse primer and a specific probe; the 5'end of the reverse primer is modified with a fluorescent marker, the 5 'end of the probe is modified with biotin, the 3' end of the probe is modified with a phosphorylation group, and nuclease cleavage sites are contained in the probe. The primer group disclosed by the invention can effectively amplify the mic gene of the 2-methylisoborneol, has relatively high specificity and sensitivity, has no cross reaction with cyanobacteria which does not generate the 2-methylisoborneol, and can be combined with lateral flow immunochromatography to be used for on-site rapid detection of the 2-methylisoborneol; the method is of great significance in early warning of excessive content of 2-methylisoborneol in the water body.

Description

Technical Field

[0001] This invention relates to the field of water pollution and water odor detection technology, specifically to a primer set, kit, and method for detecting cyanobacteria that produce 2-methylisoborneol in water using recombinase-mediated isothermal nucleic acid amplification (RAA) technology. Background Technology

[0002] Algae are a group of ecologically important lower photosynthetic organisms. They exhibit diverse morphologies, with the smallest individuals measuring only 1-2 μm in diameter, while the largest, such as giant kelp, can reach lengths of over 60 meters. They generally possess photosynthetic pigments and chloroplasts, and metabolize autotrophically through photosynthesis. They exist in various forms, including unicellular, colonial, and filamentous forms. Under suitable environmental conditions such as eutrophication, algae readily exhibit exponential growth and reproduction, producing various secondary metabolites during their metabolism. Among these, odorous substances, represented by geosmin and 2-methylisoborneol (2-MIB), have a particularly significant impact on drinking water quality. 2-MIB, a typical algal odor-causing substance, has an extremely low odor threshold (only 10 ng / L), which can cause a noticeable musty smell in water, seriously damaging the sensory properties and safety of drinking water. 2-MIB has been isolated and identified from more than 200 species of algae, including Oscillatoria, Lycopodium, Lycopodium moniliforme, Lycopodium spp., and Anabaena. Odor problems caused by algal secondary metabolites have become a major technical challenge in the global drinking water treatment field, with 2-MIB-related complaints accounting for the highest proportion of complaints from tap water users.

[0003] Research on odor-causing pollutants in drinking water in my country started later than in developed countries such as Europe and the United States, but relevant standards have been gradually improved: The "15 Water Quality Indicators for Drinking Water" promulgated in 1956 only provided a qualitative description of the sensory standards for drinking water (colorless and transparent water, no off-odor or taste at 20℃ and 50℃); the "Hygienic Standard for Drinking Water" (GB5749-1985) in 1986 continued this qualitative provision without setting limits for odor-causing substances; the "Hygienic Standard for Drinking Water" (GB5749-2006) implemented in 2006, although still providing a qualitative description of "odor and taste", added a 2-MIB limit (10 ng / L) in Appendix A as a reference indicator. The "Hygienic Standard for Drinking Water" (GB5749-2022), implemented on April 1, 2023, further focuses on sensory indicators, explicitly specifying the 2-MIB limit requirement (10 ng / L) in the extended indicators, reflecting the country's emphasis on monitoring odor-causing substances in drinking water.

[0004] In the field of aquatic ecology and drinking water safety, cyanobacteria, as a type of photosynthetic prokaryotes, typically have cell diameters ranging from 3 to 10 μm. When the concentration of cyanobacteria in water is below 10... 4When the concentration is in cells / mL, it is difficult to detect directly using conventional methods such as microscopic observation and visual identification, posing a significant challenge to early monitoring. It is noteworthy that certain cyanobacteria species (such as Anabaena, Oscillatoria, Aphanizomenon, and Phormidium) synthesize 2-methylisoborneol (2-MIB) through secondary metabolic pathways during their growth and reproduction. This substance has an extremely low olfactory threshold of only 10 ng / L, and even at very low concentrations, it can impart a strong earthy odor to water, significantly impairing the sensory quality of drinking water and leading to a substantial increase in complaints from tap water users. More importantly, the abnormal proliferation of 2-MIB-producing cyanobacteria is often an early biological indicator of eutrophication. Its presence not only foreshadows potential water quality risks but may also be accompanied by algal blooms, further triggering ecological problems such as reduced dissolved oxygen and the death of aquatic organisms, posing a serious threat to the protection of drinking water sources and the management of the water environment.

[0005] Currently, the detection of 2-MIB-producing cyanobacteria and 2-MIB mainly relies on two technical approaches, but both have significant drawbacks:

[0006] The first category is detection techniques based on the 2-MIB compounds themselves, represented by the "Test Method for Odor Substances Geosmin and 2-Methylisoborneol in Drinking Water" (GB / T32470-2016). This method uses headspace solid-phase microextraction-gas chromatography-mass spectrometry (GC-MS). Although it can achieve accurate quantification of 2-MIB, the entire detection process takes more than 4 hours and requires large and sophisticated equipment such as GC-MS instruments. Such equipment is bulky, expensive, and requires professional technicians to operate and maintain. It can only be used in laboratory environments and cannot meet the needs of rapid on-site detection in drinking water sources, water treatment plants, and other scenarios, making it difficult to achieve early warning of 2-MIB pollution.

[0007] The second category is molecular biology detection technologies based on 2-MIB-producing cyanobacteria, including polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP). While PCR technology possesses a certain degree of specificity and sensitivity, it has significant limitations: firstly, its detection process is cumbersome, requiring multiple steps such as nucleic acid extraction, system preparation, thermal cycling amplification, and gel electrophoresis detection, resulting in a lengthy process; secondly, PCR technology is highly dependent on specialized laboratory equipment such as PCR instruments and gel imaging systems, and requires highly trained technicians, making it difficult to operate outside the laboratory environment and thus unsuitable for on-site monitoring of grassroots water sources. Although LAMP technology does not require thermal cycling equipment and can amplify under isothermal conditions, it suffers from complex primer design (requiring the simultaneous design of 4-6 primers), susceptibility to non-specific amplification leading to false positives, and reliance on turbidimeters or fluorescence detectors for product detection. Furthermore, amplification takes over 60 minutes, similarly failing to meet the detection requirements of "rapid, accurate, and applicable on-site."

[0008] In recent years, recombinase-mediated isothermal nucleic acid amplification technology (... RecombinaseAidedAmplification, RAA (Rapid Amplification As a novel isothermal amplification technology, it exhibits significant advantages: it can achieve efficient DNA amplification at a constant low temperature of around 39°C, obtaining sufficient amplification products within tens of minutes, and it does not require complex thermal cycling equipment, theoretically possessing the potential for on-site detection. RAA products can be detected by agarose gel electrophoresis. Meanwhile, lateral flow strips (LFS), as a visual detection method, have the advantages of simple operation, fast detection, low cost, and no need for professional instruments, and have been widely used in the field of pathogen detection.

[0009] However, current technologies lack a solution that combines RAA isothermal amplification technology with lateral flow test strip visualization detection, and designs a specific detection system targeting the key functional genes (mic genes, the core genes for 2-MIB synthesis, which are highly conserved in 2-MIB-producing cyanobacteria) of these algae. Existing RAA-related technologies are mostly applied to the detection of pathogens (such as Clostridium perfringens and Salmonella), not specifically targeting 2-MIB-producing cyanobacteria. Furthermore, they fail to optimize sample pretreatment and amplification systems for the cellular characteristics of cyanobacteria, thus failing to meet the practical needs of drinking water sources for rapid, sensitive, and specific detection of 2-MIB-producing cyanobacteria, and thus hindering early warning and process optimization decisions in water quality safety management.

[0010] In summary, there is an urgent need for a detection solution that can overcome the limitations of existing technologies: this solution must have the capability to detect low concentrations (below 10). 4This technology features several key characteristics: high cell / mL detection rate for 2-MIB-producing cyanobacteria, rapid detection (within 30 minutes), field applicability (no large instruments required, easy to operate), and high specificity (only for 2-MIB-producing cyanobacteria). These features enable real-time monitoring of 2-MIB-producing cyanobacteria in water bodies, providing reliable technical support for drinking water source protection and water treatment process optimization. Ultimately, this helps prevent algal blooms and establish an effective early warning system for 2-MIB pollution. Summary of the Invention

[0011] The problem to be solved

[0012] The purpose of this invention is to provide a primer set, kit, and method for detecting the mic gene of 2-methylisoborneol-producing cyanobacteria based on recombinase-mediated isothermal nucleic acid amplification (RAA) technology. This invention addresses the shortcomings of existing detection technologies, such as long detection time, reliance on large equipment, complex operation, and inability to perform rapid on-site detection. It enables rapid, sensitive, and specific detection of 2-methylisoborneol-producing cyanobacteria in water bodies, providing support for early warning of 2-MIB pollution in drinking water sources.

[0013] Technical solution

[0014] To address the aforementioned technical problems, the present invention aims to provide a primer set, kit, and method for detecting 2-methylisoborneol-producing cyanobacteria based on RAA.

[0015] Firstly, this invention provides a primer set for detecting the mic gene of 2-methylisoborneol-producing cyanobacteria. The primer set is designed based on the conserved region sequence of the mic gene in 2-methylisoborneol-producing cyanobacteria. The mic gene is a key functional gene for the synthesis of 2-MIB in 2-MIB-producing cyanobacteria. Its conserved region sequence is highly consistent in 2-MIB-producing cyanobacteria, but shows significant differences in non-2-MIB-producing cyanobacteria. Designing the primer set based on this conserved region ensures detection specificity. Specifically, the primer set includes a forward primer, a reverse primer, and a specific probe, which work together to achieve specific amplification of the mic gene and subsequent detection.

[0016] The nucleotide sequence of the forward primer is shown in SEQ ID NO.1:

[0017] 5'-GTAGCGACCACCATCCTGCAA-3', this sequence is 21bp in length, which is within the preferred length range of 20-30bp. This length range can ensure the specificity of primer binding to template while reducing the probability of primers forming secondary structures themselves, thus avoiding affecting amplification efficiency;

[0018] The nucleotide sequence of the reverse primer is shown in SEQ ID NO.2:

[0019] 5'-AAGCCTGATGACCGCGTGATGG-3', this sequence is 22bp in length, which also meets the design requirement of 20-30bp, and the annealing temperature difference between the forward and reverse primers does not exceed 5℃, which can ensure that both can efficiently bind to the template under the same RAA amplification conditions, avoiding uneven amplification efficiency caused by differences in annealing temperature.

[0020] In this invention, the forward and reverse primers work together to specifically amplify a specific fragment of the mic gene from 2-MIB-producing cyanobacteria. The amplified product is 306 bp in length, falling within the preferred range of 200-500 bp. This length range satisfies the requirements for efficient RAA amplification (too long a fragment increases amplification time, while too short a fragment leads to non-specific amplification) and allows for convenient detection via electrophoresis or lateral flow test strips. Experimental verification (e.g., detection of various cyanobacteria such as *Anabaena cylindrica* and *Oscillatoria*) shows that the amplified product corresponds only to 2-MIB-producing algae, exhibiting no cross-reactivity and excellent species specificity.

[0021] To enable subsequent visualization detection of the lateral flow test strip, the 5' end of the reverse primer is modified with a fluorescent marker, specifically fluorescein isothiocyanate (FITC). FITC can specifically bind to the anti-FITC antibody on the control line of the lateral flow test strip, providing a basis for judging the validity of the test results.

[0022] The nucleotide sequence of the specific probe is shown in SEQ ID NO.3:

[0023] 5'-TACATTGTGGTCTCATTCGTCCAAATGCTHCGCATGGCTCCCGTC-3'.

[0024] In some feasible implementations, the probe of this invention satisfies several design requirements:

[0025] First, the design direction is forward, consistent with the nucleotide extension direction of the forward primer, which ensures that the probe and primer cooperate to bind to the template during amplification;

[0026] Secondly, the length is 48nt, which is within the preferred range of 45-50nt. This length can balance the binding specificity and binding efficiency of the probe and the template, avoiding insufficient specificity due to the length being too short or decreased binding efficiency due to the length being too long.

[0027] Third, the 5' end is modified with biotin, which can specifically bind to streptavidin on the detection line of the lateral flow test strip, enabling visualization of the amplification product;

[0028] Fourth, the 3' end is modified with a phosphorylation group, which can inhibit the non-specific extension of the probe under the action of DNA polymerase, avoid the probe itself acting as a primer to induce non-specific amplification, and reduce false positives;

[0029] Fifth, the sequence contains a nuclease cleavage site, corresponding to the "H" in the sequence. This "H" is a tetrahydrofuran modification site, a specific cleavage site of endonuclease IV (nfo enzyme). During RAA amplification, when the probe specifically binds to the mic gene template of 2-MIB-producing cyanobacteria, the nfo enzyme can recognize and cleave this tetrahydrofuran site. Subsequently, DNA polymerase initiates DNA chain extension starting from the cleaved probe, forming a double-stranded amplification product containing both FITC (from the reverse primer) and biotin (from the probe). This design further enhances the specificity of amplification, initiating effective amplification only when the mic gene template is present, significantly reducing the risk of false positives caused by non-specific amplification.

[0030] Secondly, in order to achieve the efficient application of the above primer set, the present invention further provides a kit for detecting 2-methylisoborneol produced by cyanobacteria. The kit contains the primer set described in any of the above-mentioned items, as well as the reagents required for recombinase-mediated isothermal amplification (RAA) reaction, which can be used directly for on-site detection without the need for additional preparation of complex reagents.

[0031] In some feasible methods, the reagents required for the RAA reaction specifically include RAA basal buffer, MgAc2 solution, and negative control. RAA basal buffer is the core reaction system for RAA amplification, containing recombinase, single-stranded binding protein (SSB), DNA polymerase, and dNTPs. The recombinase can be RecA protein, which mediates the specific binding of primers to template DNA, achieving DNA double-strand unwinding and primer binding without helicase. The single-stranded binding protein (SSB), preferably derived from *E. coli*, binds to unwound single-stranded DNA, preventing renaturation and maintaining the single-stranded state to facilitate primer binding and strand extension. The DNA polymerase can be a large fragment of BstDNA polymerase, which has 5 to 3 prime polymerase activity and strong strand displacement activity, allowing for efficient DNA strand extension under isothermal conditions. dNTPs, or deoxyribonucleoside triphosphates, include dATP, dTTP, dCTP, and dGTP, serving as raw materials for DNA synthesis. The synergistic effect of the above components ensures that RAA amplification is carried out efficiently under constant temperature conditions of 38-40℃.

[0032] In some feasible methods, the concentration of the MgAc2 solution is 260-300 mM, preferably 280 mM. 2+MgAc2 is an essential coenzyme for enzymes such as DNA polymerase and recombinase in the RAA system. Too low a concentration will lead to insufficient enzyme activity, thus affecting amplification efficiency; too high a concentration will inhibit enzyme activity and increase the risk of nonspecific amplification. Through testing the effect of different MgAc2 concentrations on amplification products, a 280 mM MgAc2 solution achieved the optimal RAA amplification efficiency.

[0033] The negative control is specifically sterile ultrapure water. Its function is to eliminate false positives caused by reagent contamination or operational contamination during the testing process, thereby ensuring the accuracy of the test results.

[0034] In some feasible methods, the primer concentrations in the kit are set as follows: the working concentrations of both the forward and reverse primers are 8-12 μM, and the working concentration of the probe is 8-12 μM, with a preferred concentration of 10 μM for all three. This concentration range ensures that the primers and probes have sufficient concentrations in the reaction system to bind to the template, while avoiding the formation of primer dimers or probe-primer dimers due to excessive concentrations. Through screening tests on the effect of different primer concentrations on amplification specificity, a working concentration of 10 μM can minimize the generation of non-specific products while ensuring amplification efficiency.

[0035] Thirdly, based on the aforementioned primer set and kit, this invention also provides a method for detecting 2-methylisoborneol produced by cyanobacteria. This method combines the high efficiency of RAA isothermal amplification with the convenience of lateral flow test strips, and specifically includes the following steps:

[0036] The first step is sample processing. The purpose of this step is to obtain DNA templates suitable for RAA amplification, which are pretreated using a thermal lysis method. Thermal lysis requires no complex nucleic acid extraction kits, is simple to operate, and is quick, making it suitable for on-site applications. The specific operation is as follows:

[0037] First, a 0.2-0.22μm filter membrane is used, preferably a 0.22μm mixed cellulose ester filter membrane. The pore size of this filter membrane can effectively trap cyanobacterial cells. The diameter of cyanobacterial cells is usually 3-10μm, which cannot pass through the 0.22μm filter membrane. Small molecule impurities in the water can be initially separated through the filter membrane, thereby enriching the water sample.

[0038] The filter membrane enriched with cyanobacteria is placed in sterile water, specifically sterile double-distilled water, to avoid introducing exogenous DNA contamination.

[0039] The centrifuge tubes containing the filter membrane and sterile water were then heated at 95-105℃ for 5-7 minutes, preferably 100℃, for 6 minutes. Heating at 100℃ for 6 minutes effectively lyses the cyanobacterial cell wall and cell membrane, releasing intracellular DNA, while avoiding DNA degradation caused by excessively high temperatures or prolonged heating. Experiments have verified that after heating at 100℃ for 6 minutes, the DNA integrity is good and can meet the requirements for RAA amplification.

[0040] Finally, the solution was filtered using a 0.4-0.45 μm filter membrane, preferably a 0.45 μm mixed cellulose ester filter membrane. This process removed impurities such as lysed cell debris, preventing them from inhibiting the activity of the subsequent RAA amplification enzyme. The filtrate was then collected as a DNA template.

[0041] The second step is RAA amplification, using the DNA template obtained in the first step as a template, and isothermal amplification is performed using the primer set described above. Specifically, the total volume of the RAA amplification reaction system is 50 μL, and the volume and concentration of each component are as follows:

[0042] RAA basic buffer 28-32 μL, preferably 29.4 μL, this volume can ensure that the enzyme and raw material concentrations in the buffer are appropriate;

[0043] Adding 2-3 μL of MgAc2 solution, preferably 2.5 μL, combined with a MgAc2 concentration of 280 mM, can reduce the amount of Mg in the reaction system. 2+ The final concentration reached 14 mM, which is the optimal concentration for enzyme activity.

[0044] 2-4 μL of DNA template, preferably 2 μL. Too much template may introduce more inhibitors, while too little template may lead to insufficient amplification products. Experimental results show that 2 μL of template can reduce the impact of inhibitors while ensuring amplification efficiency.

[0045] The final concentrations of the forward primer, reverse primer, and probe are all 0.8-1.2 μM. In practical applications, the final concentrations of the forward primer are approximately 0.42 μM, the reverse primer is approximately 0.6 μM, and the probe is approximately 0.12 μM, all within this concentration range. This final concentration ensures that the primers and probes bind to the template efficiently.

[0046] The reaction conditions were set at a constant temperature of 38-40℃ for 15-20 minutes, with 39℃ being the preferred temperature and 16 minutes the preferred reaction time. 39℃ is the optimal reaction temperature for recombinase and DNA polymerase in the RAA system, achieving the highest amplification efficiency. The 16-minute reaction time allows the amplified products to reach detectable levels. Experimental tests showed that after 16 minutes of reaction at 39℃, the amount of amplified products was sufficient for detection by lateral flow test strips, eliminating the need to extend the reaction time.

[0047] During amplification, the recombinase first mediates the specific binding of the primers to the template DNA, enabling the forward and reverse primers to bind to specific sites on the mic gene template. Single-stranded binding protein (SSB) binds to the unwound single-stranded DNA to maintain its single-stranded state. Subsequently, the nfo enzyme recognizes and cleaves the tetrahydrofuran site on the probe. DNA polymerase then extends the strand starting from the cleaved probe, ultimately forming a double-stranded amplification product containing FITC and biotin. This specific amplification process is only initiated when the mic gene of a 2-MIB-producing cyanobacterium is present in the sample.

[0048] The third step is the detection of amplification products. Lateral flow test strips are used to visually detect the RAA amplification products, thereby determining whether cyanobacteria producing 2-methylisoborneol are present in the sample. The lateral flow test strips used include a control line (C line) and a test line (T line). Anti-FITC antibody is immobilized on the C line, and streptavidin is immobilized on the T line. The specific detection process is as follows:

[0049] Take 2 μL of LRAA amplification product and dilute it 50 times with sterile double-distilled water. This dilution operation can avoid the test strip from having a band tail or false negative due to excessive concentration of amplification product. Insert the conjugate pad end of the test strip into the diluted amplification product, and ensure that the liquid level does not exceed the upper limit mark of the sample pad immersion.

[0050] After the interpretation area is fully soaked, lay the test strip flat for 1 minute and record the test results within 10 minutes. The interpretation criteria are as follows: If both the C line and the T line are colored, the result is positive, indicating the presence of cyanobacteria that produce 2-methylisoborneol in the sample. At this time, FITC on the amplification product binds to the anti-FITC antibody on the C line, and biotin binds to streptavidin on the T line, forming two visible bands.

[0051] If the C line shows color but the T line does not, the result is negative, indicating that there is no cyanobacteria producing 2-methylisoborneol in the sample. In this case, only FITC in the amplified product or the free reverse primer FITC in the system binds to the antibody on the C line, and there is no specific binding on the T line. If the C line does not show color, regardless of whether the T line shows color, the result is invalid and the test strip needs to be replaced and tested again. The absence of a C line indicates that the test strip is faulty or there is an improper operation, such as insufficient sample volume or insufficient soaking time. In such cases, the test result is unreliable.

[0052] In some feasible embodiments, the detection method of the present invention achieves highly sensitive detection of 2-methylisoborneol-producing cyanobacteria through synergistic optimization of sample processing, RAA amplification, and amplification product detection, with a detection limit of no more than 50 2-methylisoborneol-producing cyanobacteria cells / reaction, or no more than 70 mic gene copies / reaction.

[0053] Achieving this detection limit relies on multi-stage technical support: in the sample processing stage, the enrichment effect of the filter membrane can detect low concentrations of cyanobacteria (such as those below 10 in water). 4 The lateral flow test strip effectively retains cyanobacteria (cells / mL), increasing the initial amount of DNA template for subsequent amplification. In the RAA amplification stage, the specific primer set and probe designed based on the conserved region of the mic gene, combined with the optimized reaction system and reaction conditions, can efficiently amplify the target gene fragment, generating sufficient amplification products even with extremely low template amounts. In the amplification product detection stage, the lateral flow test strip can accurately capture amplification products through the specific binding of FITC and anti-FITC antibodies, biotin and streptavidin, avoiding background signal interference and further improving detection sensitivity.

[0054] Experimental verification showed that, using *Anabaena fischeri* FACHB-1277 as the test subject, its cells were serially diluted to 4800 cells / reaction, 480 cells / reaction, 48 cells / reaction, and 4.8 cells / reaction. Using the method of this invention, only concentrations of 48 cells / reaction and above could detect a positive result. Simultaneously, using a recombinant plasmid containing the mic gene as a template, it was serially diluted to 6.82 × 10⁻⁶. 3 Copy / reaction, 6.82 × 10 2 Copy / reaction, 6.82 × 10 1 Copy / reaction, 6.82 × 10 0 The copy / reaction test results showed that a concentration of 68.2 copies / reaction or higher could detect a positive result, which fully demonstrates that the detection limit of the method of this invention fully meets the requirements of no more than 50 cells / reaction and no more than 70 copies / reaction. It can meet the needs of early monitoring of 2-MIB-producing cyanobacteria in water bodies. Even if the concentration of cyanobacteria is at a level that is difficult to observe by conventional methods, it can achieve accurate detection, providing a reliable technical guarantee for early warning of 2-MIB pollution.

[0055] Beneficial effects

[0056] This invention overcomes many shortcomings of existing 2-MIB detection technologies by designing specific primer sets, matching reagent kits, and detection methods, and has significant technical advantages, specifically reflected in the following aspects:

[0057] (1) Regarding detection specificity, the primer set of this invention is designed based on the conserved region of the mic gene of 2-MIB-producing cyanobacteria. The forward and reverse primers can specifically amplify a 306bp mic gene fragment. The specific probe, through the synergistic effect of the tetrahydrofuran modification site and the nfo enzyme, further limits the effective amplification to only when the mic gene template is present. Experimental verification (detection of 14 cyanobacteria including Anabaena, Anabaena, Auricularia auricula-judae, and Oscillatoria) shows that this primer set only produces positive amplification for 2-MIB-producing cyanobacteria (such as Anabaena pyrenoidosa FACHB-1277), and there is no cross-reaction with non-2-MIB-producing cyanobacteria. The specificity is significantly better than that of existing PCR technology, because some PCR primers may bind to homologous sequences of non-target cyanobacteria, leading to false positives. This ensures the accuracy of the detection results at the molecular level.

[0058] (2) Regarding detection sensitivity, the detection method of the present invention, through optimization of the RAA amplification system and sample processing steps, achieves a detection limit of no more than 50 2-methylisoborneol-producing cyanobacterial cells / reaction or no more than 70 mic gene copies / reaction. This sensitivity is significantly higher than that of the GB / T32470-2016 method, which has a detection limit of approximately 10 ng / L for 2-MIB. This corresponds to a cyanobacterial cell concentration far exceeding the cell detection limit of the present invention, enabling early detection even at low cyanobacterial cell concentrations, because below 10 ng / L... 4 The concentration of cells / mL is too small to be observed with a conventional microscope, allowing sufficient time for early warning of 2-MIB contamination and overcoming the shortcomings of existing technologies in providing early warning.

[0059] (3) In terms of detection efficiency and on-site applicability, the detection method of the present invention takes only about 30 minutes in total, which is much shorter than the GB / T32470-2016 method (which takes several hours) and the PCR method; and no large precision instruments are required. Only a constant temperature mixer and a lateral flow test strip are needed to complete the detection. The constant temperature mixer is small in size and highly portable. The test strip detection does not require professional operating skills. Ordinary staff can complete the detection after simple training. It can be directly applied on-site in drinking water sources, waterworks and other places, which solves the core problem of existing technologies relying on laboratories and being unable to detect on-site.

[0060] (4) In terms of application value, the method of the present invention has been verified by actual samples. The RAA-LFS detection results are completely consistent with the PCR detection results, and the accuracy has been fully confirmed. This method can be used for daily monitoring of drinking water sources to achieve early detection of 2-MIB-producing cyanobacteria and early warning of 2-MIB pollution; it can also be used for the optimization of water treatment processes in waterworks, adjusting algae removal and deodorization process parameters according to the detection results; at the same time, it is of great significance in the prevention and control of algal blooms, as it can monitor changes in cyanobacterial communities through regular detection, providing data support for the formulation of algal bloom prevention and control measures, and ultimately ensuring the safety of drinking water quality, with significant practical value and social benefits.

[0061] In summary, this invention comprehensively improves existing 2-MIB detection technology in terms of specificity, sensitivity, efficiency, and applicability by designing a mic gene-specific primer set, constructing a kit containing this primer set, and establishing a RAA-lateral flow test strip detection method. It creatively realizes rapid, sensitive, and specific on-site detection of 2-methylisoborneol-producing cyanobacteria, filling the gap in on-site detection technology in this field. Attached Figure Description

[0062] Figure 1 Flowchart of a recombinase-mediated isothermal amplification combined with nuclease cleavage and lateral flow chromatography (RAA-nfo-LFS) method for detecting 2-methylisoborneol-producing cyanobacteria;

[0063] Figure 2 This is a schematic diagram of the structure of a single-target disposable nucleic acid test strip (JY0201);

[0064] Figure 3 The results of RAA amplification tests on positive algae and negative controls using primer sets MIB-1, MIB-2, MIB-3, and MIB-4 of 2-methylisoborneol are shown.

[0065] Figure 4 The results show the sensitivity comparison of RAA amplification of samples with graded dilutions of plasmid using the methyl isoborneol primer sets MIB-2 and MIB-3.

[0066] Figure 5 Comparison of MIB-3RAA amplification results with different volumes of 2-methylisoborneol primer sets;

[0067] Figure 6 The optimal detection system for methyl isochonol-specific RAA-LFS was used for the detection of RAA-LFS in various cyanobacteria, with a negative control (sterile double-distilled water).

[0068] Figure 7 To assess the sensitivity of the 2-methylisoborneol RAA test strip detection system based on gradient dilution of positive template plasmids;

[0069] Figure 8 Results of mic gene RAA-LFS and PCR detection in 35 reservoir samples. Detailed Implementation

[0070] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0071] To better understand the technical content of this invention, the following embodiments are provided for detailed explanation.

[0072] Unless otherwise stated, the terminology used in this invention generally has the meanings commonly understood by those skilled in the art. Various processes and methods not described in detail in the embodiments are conventional methods known in the art.

[0073] Unless otherwise specified, the experimental methods described in the following examples are conventional. Unless otherwise specified, the experimental materials, consumables, and reagents used in the following examples are all commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0074] The algal sample sources involved in this invention are:

[0075] The water samples containing algae producing 2-methylisoborneol involved in this invention were collected in Ningbo City. After collection, the samples were placed on ice and quickly returned to the laboratory for storage at 4°C.

[0076] The algal samples involved in this invention are all preserved in the Freshwater Algae Culture Collection of the Chinese Academy of Sciences.

[0077] The experimental materials used in this embodiment and comparative example need to be prepared in advance to ensure the reproducibility of the experiment: In terms of algal species, all cyanobacterial samples were preserved in the Freshwater Algae Culture Collection of the Chinese Academy of Sciences, including 14 strains of 2-MIB-producing cyanobacteria (Anabaenacylindrica FACHB-170, Oscillatoria sp. FACHB-1120, Pseudanabaena cinerea FACHB-1277, etc.) and non-2-MIB-producing cyanobacteria (Nostoccommune FACHB-261, Aphanizomenon gracile, etc.). The reservoir water samples were collected in Ningbo City, and after sampling, they were preserved in an ice bath and quickly sent back to the laboratory for refrigeration at 4°C.

[0078] For reagents, RAA amplification was performed using the RNA isothermal rapid amplification kit (strip type)-III (catalog number WLRN8211KIT, containing RAA basic buffer and enzyme mixture) from Anpu Future (Changzhou) Biotechnology Co., Ltd. Lateral flow test strips were single-target disposable nucleic acid detection test strips (model JY0201) from Beijing Baoying Tonghui Biotechnology Co., Ltd. Primers and probes were synthesized by General Biosystems (Anhui) Co., Ltd. (HPLC grade purity). MgAc2 (analytical grade) was purchased from Sinopharm Group, and nfo enzyme (nuclease IV) was purchased from NEB (catalog number M0299S). For instruments, the isothermal mixer was a MK2000-1A from Hangzhou Aosheng Instrument Co., Ltd., 1.5mL and 0.2mL sterile centrifuge tubes were purchased from Axygen, and the gel imaging system was a Bio-Rad ChemiDoc XRS+.

[0079] In the RAA-specific primer screening experiment, the following algae were used as experimental subjects. Algal DNA was extracted as amplification template, and an equal volume of ultrapure water was used as a negative control. Specifically, these included:

[0080] Anabaenacylindrica FACHB-170

[0081] Anabaenaminutissima FACHB-250

[0082] NostoccommuneFACHB-261,

[0083] Oscillatoriasp. FACHB-1120

[0084] Pseudanabaenacinerea FACHB-1277

[0085] Planktothricoidesraciborskii FACHB-1360

[0086] Aerosakkonema funiforme FACHB-1362

[0087] Aerosakkonema funiforme FACHB-1363

[0088] Planktothricoidesraciborskii FACHB-1372

[0089] Planktothricoidesraciborskii FACHB-1378

[0090] Anabaenasp.FACHB-1384, Anabaenasp.FACHB-1391

[0091] Anabaenasp. FACHB-1501, Aphanizomenongracile.

[0092] The sample preparation method involved in this invention is as follows:

[0093] Laboratory methods for extracting DNA from cultured algae:

[0094] (1) Enrichment of algae through a 0.22µm filter membrane;

[0095] (2) Preheat 2×CTAB (Cetyltrimethylammonium bromide) extraction buffer in a 65°C water bath;

[0096] (3) Take a small amount of experimental material (about 300 mg) and place it in a mortar. Grind it into powder with liquid nitrogen.

[0097] (4) Add 1000 μL of 2×CTAB extraction buffer and stir gently;

[0098] (5) Pour the ground liquid into 1.5ml sterile centrifuge tubes, filling the tubes to about two-thirds full.

[0099] (6) Place it in a water bath or constant temperature box at 65℃, shake it gently every 10 minutes, and take it out after 30 minutes;

[0100] (7) After cooling for 2 minutes, add chloroform until the tube is full, and shake vigorously for 2-3 minutes to mix the two evenly;

[0101] (8) Centrifuge at 12,000 rpm for 10 min in a centrifuge. At the same time, add 600 μL of isopropanol to another new sterile centrifuge tube.

[0102] (9) After centrifuging at 12000 rpm for 1 min, gently aspirate the supernatant with a pipette and transfer it into a centrifuge tube containing isopropanol. Gently shake the centrifuge tube up and down for 30 sec to fully mix the isopropanol with the water layer until DNA flocculent matter is visible.

[0103] (10) After centrifuging at 12000 rpm for 1 min, immediately discard the liquid, being careful not to pour out the white DNA precipitate. Invert the centrifuge tube onto a spread paper towel.

[0104] (11) After 60 seconds, stand the centrifuge tube upright, add 700 μL of 75% ethanol, gently rotate, and flick the tip of the tube with your finger to make the DNA clumps at the bottom of the tube float in the liquid.

[0105] (12) After centrifuging at 12000 rpm for 1 min, pour out the liquid, add 800 μL of 75% ethanol, gently rotate, and flick the tube tip with your finger to make the DNA clumps at the bottom of the tube float in the liquid.

[0106] (13) After centrifuging at 12000 rpm for 30 seconds, immediately discard the liquid and invert the centrifuge tube onto a spread paper towel; after a few minutes, straighten the centrifuge tube and dry the DNA.

[0107] (14) Add 100 μL of sterile double-distilled water to dissolve the DNA.

[0108] The method for DNA extraction from reservoir samples (thermal lysis method) is as follows:

[0109] (1) 10 ml of field sample was passed through a 0.22 µm filter membrane to enrich algae, and the filter membrane was placed in a 1.5 ml sterile centrifuge tube;

[0110] (2) Add 1 ml of sterile double-distilled water and heat it in a preheated constant temperature mixer at 100°C for 6 min;

[0111] (3) Use a syringe to draw up a portion of the liquid and filter out the impurities through a 0.45µm filter membrane;

[0112] (4) Transfer the filtered liquid into a new 1.5 ml sterile centrifuge tube as a DNA sample.

[0113] Tiosbio® nucleic acid lateral chromatography test strips are sourced from Beijing Baoying Tonghui Biotechnology Co., Ltd. To evaluate whether the RAA-LFS system used can be applied to the rapid detection of 2-methylisoborneol-producing algae, *Pseudanabaenacinerea* FACHB-1277, cultured in the laboratory, was used as the test subject, with other algae lacking the mic gene serving as negative controls. 1 ml of culture medium containing algae was heated at 100°C for 6 min using a constant temperature mixer. After filtering out impurities through a 0.45 µm filter, the corresponding algal sample pyrolysis buffer was obtained. 2 μL of the algal sample pyrolysis buffer was used as the amplification template for RAA. The algal pyrolysis buffer can be tested immediately or stored at -70°C for more than 3 months.

[0114] Example 1: This example aims to design and screen primers and probes specific to the mic gene of 2-MIB-producing cyanobacteria, in order to determine the optimal primer set that meets the requirements, clarify the sequence, modification method and screening criteria of primers and probes, and lay the foundation for subsequent detection.

[0115] Before the experiment, key materials need to be prepared: the target gene is the mic gene of 2-MIB-producing cyanobacteria, referring to conserved sequences such as GenBank accession numbers HQ897231 and KC174682; the algal species is Pseudanabaena cinerea FACHB-1277, preserved in the Freshwater Algae Culture Collection of the Chinese Academy of Sciences, which is a 2-MIB-producing positive algal species; sterile double-distilled water is used as a negative control.

[0116] Through extensive sequence analysis, our team targeted the mic gene, a key gene for 2-MIB synthesis, which exhibits over 98% conservation in 2-MIB-producing cyanobacteria. Species-specific amplification primers were designed using PrimerPremier 5.0 software. The primer design followed these principles:

[0117] (1) Primer length 20~30bp, annealing temperature difference between forward and reverse primers not exceeding 5℃, and as consistent or similar as possible;

[0118] (2) The amplified product is 200~500bp in length and has species specificity.

[0119] 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:

[0120] Table 1 - Primers for RAA basic amplification using methylisoborneol-specific primers.

[0121]

[0122] For the amplified region of specific primers, species-specific probe sequences were designed using PrimerPremier 5.0 software. The probe sequences were located in the middle region of the amplification primers and modified with tetrahydrofuran (THF). The probe design followed the following principles: ① The design direction was forward, i.e., the same direction as the forward primer; ② The length was 45–50 nt; ③ The 3' end was phosphorylated to inhibit DNA chain elongation; ④ A cleavage site, containing an NFO enzyme-specific cleavage site, was added to the sequence, modified with THF and labeled "H". This site is cleaved by NFO enzyme when the probe binds to the template DNA, initiating DNA chain elongation; ⑤ The 5' end was modified with biotin, so that the double-stranded amplification product of the probe and the FITC-modified reverse primer could be detected by nucleic acid test strips.

[0123] 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 sequences and modification methods of the four primer-probe sets are shown in the table below.

[0124] Table 2: Specific sequences and modification methods of the four primer-probe sets (MIB-1 to MIB-4)

[0125]

[0126] Using the RNA isothermal rapid amplification kit (strip type)-III (WLRN8211KIT) from Anpu Future (Changzhou) Biotechnology Co., Ltd., RAA amplification was performed with positive algal DNA as a template using various primer sets of 2-methylisoborneol. The amplification system (single sample / reaction) is as follows:

[0127]

[0128] The screening experiment used DNA from *Anabaena fischeri* FACHB-1277 as a positive template, and extracted it using the CTAB method: 300 mg of algal cells were ground in liquid nitrogen, 1 mL of 2×CTAB buffer preheated to 65℃ was added, and the mixture was incubated at 65℃ for 30 min. After chloroform extraction, the DNA was precipitated with isopropanol, washed twice with 75% ethanol, and dissolved in sterile double-distilled water. Sterile double-distilled water served as a negative control. RAA amplification was performed using four sets of primer-probe combinations.

[0129] The RAA amplification system should be prepared according to the following standard:

[0130] The total volume was 50 μL, including 29.4 μL of RAA basal buffer, 2.1 μL of forward primer (10 μM), 3 μL of reverse primer (10 μM), 0.6 μL of probe (10 μM), and 2 μL of DNA template. The remaining volume was made up with sterile double-distilled water. After mixing the system, 2.5 μL of 280 mg Ac2 solution was added, and the mixture was placed in a 0.2 mL centrifuge tube and incubated at 39 °C for 16 min.

[0131] After amplification, take 2 μL of the amplification product and dilute it 50 times with sterile double-distilled water, as shown in the attached diagram. Figure 2 - A schematic diagram of the single-target disposable nucleic acid test strip (JY0201). This diagram shows that the test strip contains a control line (C line) and a test line (T line). The C line is immobilized with anti-FITC antibody, and the T line is immobilized with streptavidin. During testing, immerse the test strip's conjugate pad end in the diluent, ensuring the liquid level does not exceed the maximum immersion limit. After laying it flat for 1 minute, interpret the results within 10 minutes. The interpretation criteria are: both the C line and the T line showing color indicates a positive result; only the C line showing color indicates a negative result; and no color showing on the C line indicates an invalid result.

[0132] The screening results are attached. Figure 3 As shown: The negative controls MIB-1 and MIB-4 produced false positives (T-line color development), indicating contamination and interference, and therefore could not be used as primers for detecting the mic gene. However, the RAA amplification products of MIB-2 and MIB-3 resulted in positive results on the test strip, and no false positives were observed in the negative controls; therefore, they could be used as primers for detecting the mic gene. Therefore, the 2-methylisoborneol primer set MIB-2 and MIB-3 were initially selected as the detection primers. The amplification products were cloned into the T-vector, and sequencing confirmed them as the target sequence. (Attached...) Figure 3 The results are from test strips used to amplify RAA in positive algae and negative controls using 2-methylisoborneol primer sets MIB-1 to MIB-4.

[0133] Upon testing, although MIB-2 could detect positive templates, subsequent verification showed that its sensitivity was low; only the MIB-3 group showed positive results for all positive templates, and the negative control had no false positives. Moreover, the amplification product was verified by cloning and sequencing to be a mic gene-specific fragment with a length of 306bp, which is within the preferred range of 200~500bp. Therefore, MIB-3 was determined to be the final primer set.

[0134] Example 2: To support subsequent primer set sensitivity screening experiments and provide a standardized template for sensitivity verification of the detection method, this example prepared a plasmid alignment disk containing the mic gene of cyanobacteria producing 2-methylisoborneol (2-MIB), clarifying its construction, culture, extraction and dilution process to ensure the reproducibility and accuracy of subsequent experiments.

[0135] The core materials used in the experiment are as follows: a recombinant plasmid containing the mic gene, constructed from the amplification products of the forward primer MIB-F (sequence 5'-GTAGCGACCACCATCCTGCAA-3') and the reverse primer MIB-R2 (sequence 5'-AAGCCTGATGACCGCGTGATGG-3'). Specifically, the 306bp mic gene fragment amplified by this primer pair was cloned into the pMD19-T vector, transformed into competent E. coli cells, and positive colonies containing the recombinant plasmid were screened. LB broth medium was prepared according to the standard formula (10g / L tryptone, 5g / L yeast extract, 10g / L NaCl, pH 7.0), with the addition of ampicillin antibiotic (final concentration 100μg / mL) for selective culture of positive E. coli. Plasmid extraction was performed using a bacterial plasmid miniprep kit to purify the recombinant plasmid. Concentration determination was performed using an ultra-micro spectrophotometer to detect the concentration and purity of the plasmid DNA.

[0136] The experimental procedure is as follows: First, a single colony of positive E. coli containing the mic gene recombinant plasmid was picked and inoculated into 5 mL of LB broth medium containing ampicillin. The culture was then placed in a constant temperature shaker (200 r / min) at 37℃ overnight to ensure sufficient bacterial proliferation. After the culture was completed, 3 mL of bacterial solution was taken and the bacterial plasmid miniprep kit was prepared according to the instructions: the bacterial pellet was resuspended in a resuspension containing RNase A, the cells were lysed in lysis buffer, and the lysis system was neutralized in a neutralizing buffer. After centrifugation at 12000 rpm for 10 min, the supernatant was collected and the plasmid DNA was purified by the adsorption column of the kit. Finally, the plasmid solution was eluted with elution buffer and collected. To ensure the reliability of the concentration and purity data, the concentration of the extracted plasmid DNA was determined using an ultra-micro spectrophotometer. The operation was repeated 3 times, and the average value was taken as the final concentration.

[0137] The copy number was calculated based on the plasmid concentration, and the plasmid was diluted to 3.41 × 10⁻⁶. 10 Copy / μL, aliquoted and stored at -20℃ for later use; this is the stock solution of the recombinant plasmid after value determination. For subsequent experiments, dilute the stock solution with sterile double-distilled water to 3.41 × 10⁻⁶. 3 Copy / μL, then perform 10-fold serial dilutions to obtain 3.41×10⁻⁶ copies / μL. 3 Copies / μL, 3.41 × 10 2 Copies / μL, 3.41 × 10 1 Copy / μL and 3.41×10 0 A plasmid dilution of 1 copy / μL was used to construct a complete sample alignment disk, which served as a standard template for subsequent RAA amplification.

[0138] The plasmid alignment disk prepared in this embodiment can ensure the accuracy of template concentration in subsequent primer set sensitivity screening experiments, avoid screening errors caused by template differences, and provide quantitative basis for the sensitivity verification of the detection method, ensuring the comparability and reliability of subsequent experimental results.

[0139] Example 3: Based on the plasmid gradient dilution samples prepared above, in order to compare the detection sensitivity of the RAA-lateral flow chromatography test strips (LFS) with the two sets of primers MIB-2 and MIB-3, the optimal primer set was screened to provide a better reagent combination for subsequent experiments and actual detection.

[0140] The materials used in the experiment are as follows: the plasmid gradient dilution buffer was prepared in Example 2 at a concentration of 3.41 × 10⁻⁶. 3 ~3.41×10 0 Copy / μL series diluents;

[0141] The sequences and modification methods of the two primer-probe combinations (MIB-2 and MIB-3) are as follows:

[0142] MIB-2 group: forward primer MIB-F-, with the sequence 5'-GTAGCGACCACCATCCTGCAA-3', unmodified; reverse primer MIB-R1, with the sequence 5'-ACTACGGGATCGGCGATGTC-3', 5' end FITC modified.

[0143] Probe MIB-P2, its sequence:

[0144] 5'-CATACATGGTGGTCTCATTCGTCCAAATGHTACGCATGGCTCCCG-3', where the 5' end is biotinylated and the 3' end is phosphorylated.

[0145] MIB-3 group: forward primer MIB-F, with the sequence 5'-GTAGCGACCACCATCCTGCAA-3', unmodified; reverse primer MIB-R2, with the sequence 5'-AAGCCTGATGACCGCGTGATGG-3', 5' end FITC modified.

[0146] Probe MIB-P1, its sequence:

[0147] 5'-TACATTGTGGTCTCATTCGTCCAAATGCTHCGCATGGCTCCCGTC-3', 5' end biotin modification, 3' end phosphorylation modification.

[0148] RAA amplification reagents include a basic buffer and an enzyme mixture; the lateral flow chromatography test strip contains a control line (C line) and a detection line (T line), with the C line immobilized with anti-FITC antibody and the T line immobilized with streptavidin; the experimental instrument is a thermostat mixer for temperature control of RAA amplification.

[0149] The experimental method is as follows: MIB-2 and MIB-3 were used as primer sets, and the above four plasmid dilutions were used as templates. Each set was repeated three times, with sterile double-distilled water as a negative control. The total volume of the RAA amplification system was 50 μL, specifically composed of: 29.4 μL of basal buffer, 5.7 μL of the corresponding primer set (10 μM), 2 μL of plasmid template, and 2.5 μL of 280 mg Ac2 solution. The remaining volume was made up with sterile double-distilled water. The corresponding primer sets included the following two groups:

[0150] The MIB-2 kit consisted of 2.1 μL of forward primer, 3 μL of reverse primer, and 0.6 μL of probe.

[0151] The MIB-3 group consisted of 2.1 μL of forward primer, 3 μL of reverse primer, and 0.6 μL of probe.

[0152] After mixing the above system, place it in a constant temperature mixer and react at 39℃ for 16 min. After the reaction, take 2 μL of the amplification product and dilute it 50 times with sterile double-distilled water. Insert the conjugation pad end of the lateral flow chromatography test strip into the diluent, ensuring the liquid level does not exceed the upper limit mark of the sample pad on the test strip. After the readout area is completely wetted, lay the test strip flat for 1 min. Observe and record the results within 10 min. Both the control line and the test line show color, indicating a positive result; only the control line shows color, indicating a negative result; no color from the control line indicates an invalid result. The detection sensitivity of the two sets of primers is determined by the lowest template concentration for a positive result.

[0153] Appendix Figure 4 This section presents a comparison of the sensitivity of 2-methylisoborneol primer sets MIB-2 and MIB-3 for RAA amplification of serially diluted plasmid samples. The experimental results are attached. Figure 4 As shown: MIB-3 primer pair 3.41 × 10 1 Plasmid dilutions at concentrations of 3.41 × 10⁻⁶ copies / μL (approximately 68.4 copies / reaction based on 2 μL template) and higher all showed positive results, with clear detection lines. 0 No positive result was detected at a concentration of 3.41 × 10⁻⁶ copies / μL; the MIB-2 primer group could only detect 3.41 × 10⁻⁶. 3 Plasmid dilutions at concentrations of copies / μL (approximately 6840 copies / reaction based on 2μL template) and above, 3.41 × 10⁻⁶ 2 No positive results were detected at concentrations of copies / μL and below. Comparison between the two groups showed that the sensitivity of the MIB-3 group was significantly higher than that of the MIB-2 group, with a sensitivity increase of approximately 100-fold. Furthermore, no false positives were observed in the negative control of the MIB-3 group, demonstrating good specificity.

[0154] Based on the previous primer set specificity screening results, this embodiment further verifies the advantage of the MIB-3 group in terms of sensitivity, and finally determines MIB-3 as the optimal primer set, providing a reliable reagent basis for the establishment of subsequent detection methods and practical applications.

[0155] Example 4: To further improve the detection performance of the MIB-3 primer set for cyanobacteria producing 2-methylisoborneol (2-MIB), this example optimizes the addition volume of the primer set in the RAA-nfo-LFS system. Theoretically, the amount of reverse primer added can affect the detection sensitivity, but excessive amounts can easily lead to primer dimers and false positives. Therefore, the optimal addition volume needs to be determined through gradient experiments.

[0156] The materials used in the experiment were as follows: the positive template was a gradient dilution of plasmid containing the mic gene (constructed from the gene of a 2-MIB-producing cyanobacterium); the RAA amplification reagent was the RNA isothermal rapid amplification kit (strip type)-III (catalog number WLRN8211KIT) from Anpu Future (Changzhou) Biotechnology Co., Ltd., containing basic buffer and enzyme mixture (containing nfo enzyme); the MIB-3 primer set was prepared as a primer mix (concentration 10μM) with a ratio of forward primer: probe: reverse primer = 7:2:10; the MgAc2 solution concentration was 280mM; the experimental container was a 0.2mL eppendorf tube; the detection lateral flow chromatography test strip contained a control line (C line) and a detection line (T line), the C line was fixed with anti-FITC antibody, and the T line was fixed with streptavidin; the instruments were an isothermal mixer and a centrifuge.

[0157] The experimental procedure is as follows: First, set up three primer mixes with volume gradients: 5.7 μL for the original system, 6.0 μL for adjustment system 1, and 6.3 μL for adjustment system 2. The total volume of each RAA reaction system is 50 μL, specifically composed of: 29.4 μL of basal buffer, the corresponding volume of primer mix (10 μM), and 2 μL of DNA template (a gradient dilution of positive algal DNA). The remaining volume is made up with sterile double-distilled water. After adding the sample, immediately cap the tube to avoid aerosol contamination. Gently mix the reaction system and add it to the basic reaction unit in the kit, ensuring the lyophilized powder is fully dissolved. Do not use a vortex mixer for vigorous shaking in this step to prevent damage to enzyme activity. Open the reaction unit and add 2.5 μL of 280 mg Ac2 solution to each 0.2 mL Eppendorf tube. Gently mix and centrifuge to collect the liquid. Again, do not use vigorous shaking. Place the Eppendorf tube in a constant temperature mixer and incubate at 39°C for 16 min. After the reaction, transfer the amplification product to a new Eppendorf tube and label it. Take 2 μL of the amplification product and dilute it 50 times with sterile double-distilled water. Insert the conjugation pad end of the lateral flow chromatography test strip into the diluent, ensuring the liquid level does not exceed the upper limit mark of the sample pad. After the reading area is completely wetted, lay it flat for 1 min and observe the color development results within 10 min. At the same time, set a negative control sample (using sterile double-distilled water as a template) to exclude false positives.

[0158] Table 3 - Adjustment of reverse primer addition amount in the RAA amplification system for methyl isoborneol specific detection primer set

[0159]

[0160] Appendix Figure 5 The amplification results of MIB-3RAA primer sets with different volumes of 2-methylisoborneol were compared. The experimental results are attached. Figure 5As shown: When the primer set MIB-3 was added in a volume of 5.7 μL, the negative control was negative, and the plasmid 10 7 times, 10 8 times, 10 9 All serially diluted plasmids tested positive; at a volume of 6.0 μL, the negative control remained negative, and all three plasmid dilutions showed positive results, with 10 being particularly positive. 9 The detection bands of the 10-fold dilution were brighter, indicating better sensitivity; although the negative control was negative at a volume of 6.3 μL, plasmid 10 9 The 1-fold dilution test was negative, and weak primer dimer activity was observed. Considering both sensitivity and specificity, the optimal addition volume for the MIB-3 primer set was determined to be 6.0 μL. This parameter provides a basis for the standardization of subsequent detection systems.

[0161] Example 5: After determining the optimal parameters of the MIB-3 primer set RAA-nfo-LFS system, this example verifies the system's specific recognition ability for 2-MIB-producing cyanobacteria by detecting multiple different cyanobacteria, ensuring that it can accurately distinguish between 2-MIB producers and non-producers.

[0162] The cyanobacteria samples used in the experiment were all from the Freshwater Algae Bank of the Chinese Academy of Sciences, specifically including 14 cyanobacteria strains:

[0163] Anabaenacylindrica FACHB-170

[0164] Anabaenaminutissima FACHB-250

[0165] NostoccommuneFACHB-261,

[0166] Oscillatoriasp. FACHB-1120

[0167] Pseudanabaenacinerea FACHB-1277

[0168] Planktothricoidesraciborskii FACHB-1360

[0169] Aerosakkonema funiforme FACHB-1362

[0170] Aerosakkonema funiforme FACHB-1363

[0171] Planktothricoidesraciborskii FACHB-1372

[0172] Planktothricoidesraciborskii FACHB-1378

[0173] Anabaenasp.FACHB-1384, Anabaenasp.FACHB-1391

[0174] Anabaenasp. FACHB-1501, Aphanizomenongracile.

[0175] The RAA-LFS detection system uses the optimal parameters determined above, with 6.0 μL of MIB-3 primer set added and reaction at 39℃ for 16 min; the negative control is sterile double-distilled water.

[0176] The experimental procedure is as follows: Cultures of the above 14 cyanobacteria were taken, and DNA templates were prepared by thermal lysis. 10 mL of algal solution was enriched through a 0.22 μm filter membrane, 1 mL of sterile double-distilled water was added, and the mixture was heated at 100 °C for 6 min. The solution was then filtered through a 0.45 μm filter membrane and the filtrate was collected. Each DNA template was used as an amplification template, and the reaction solution was prepared according to the optimal RAA-LFS system. After reacting at 39 °C for 16 min, 2 μL of the amplification product was diluted 50 times and tested with a lateral flow test strip. The color development results of each sample were observed and recorded. Simultaneous color development of both C line and T line indicated a positive result, while color development of only C line indicated a negative result.

[0177] Appendix Figure 6 The optimal detection system for 2-methylisoborneol-specific RAA-LFS was used for the detection of RAA-LFS in various cyanobacteria; negative control (sterile double-distilled water). The experimental results are as follows: Figure 6 As shown:

[0178] Ten 2-MIB-producing cyanobacteria, namely *Oscillatoria* FACHB-1120, *Pseudo-Anabaena* FACHB-1277, *Pseudo-Floating Fellows* FACHB-1360, *Aerophytic Fellows* FACHB-1362, *Aerophytic Fellows* FACHB-1363, *Pseudo-Floating Fellows* FACHB-1372, *Pseudo-Floating Fellows* FACHB-1378, *Anabaena* FACHB-1384, *Anabaena* FACHB-1391, and *Anabaena* FACHB-1501, were all positive (both C and T lines were visible). Four non-2-MIB-producing cyanobacteria, namely *Anabaena* FACHB-170, *Anabaena* FACHB-250, *Auricularia* FACHB-261, and *Aphanizomenongracile*, as well as the negative control, were all negative (only the C line was visible). The results demonstrate that the RAA-LFS system of the MIB-3 primer set has strict specificity, can accurately distinguish between 2-MIB-producing and non-2-MIB-producing cyanobacteria, and has no cross-reactivity.

[0179] Example 6: In this example, cyanobacterial cells and plasmids containing the mic gene were used as templates, and the detection limit of the MIB-3 primer set RAA-LFS system was determined by gradient dilution experiments to verify its ability to detect low concentrations of 2-MIB-producing cyanobacteria.

[0180] The materials used in the experiment were as follows: The cell template was the genomic solution of *Pseudanabaenacinerea* FACHB-1277, which was serially diluted to 4800 cells / reaction, 480 cells / reaction, 48 cells / reaction, and 4.8 cells / reaction after hemocytometer counting; the plasmid template was the recombinant plasmid corresponding to the amplified fragment of the MIB-3 primer set, which was serially diluted to 6.82 × 10³ copies / μL, 6.82 × 10² copies / μL, 6.82 × 10¹ copies / μL, and 6.82 × 10¹ copies / μL after concentration determination and copy number calculation. 0 Copy / μL; The RAA-LFS system uses the optimal parameters determined above; The lateral flow chromatography test strips and the isothermal mixer are the same as in the previous examples.

[0181] The experimental procedure is as follows: Cell templates were prepared by thermal lysis, and plasmid templates were prepared directly using dilution buffer; each gradient template was used as an amplification template, and 50 μL of reaction solution was prepared according to the optimal RAA-LFS system, and the reaction was carried out at 39℃ for 16 min; after the reaction, 2 μL of amplification product was taken and diluted 50 times with sterile double-distilled water, and detected by lateral flow test strips. Each gradient was set up in 3 replicates, and the lowest template concentration with positive results was recorded, which is the detection limit.

[0182] Appendix Figure 7The sensitivity of the 2-methylisoborneol RAA test strip detection system based on a gradient dilution of the positive template plasmid was assessed. The experimental results are as follows: Figure 7 As shown: In the cell template, 4800 cells / reaction, 480 cells / reaction, and 48 cells / reaction were all positive, with both C-line and T-line showing color development; 4.8 cells / reaction was negative, confirming the detection limit as 48 2-MIB-producing cyanobacterial cells / reaction; in the plasmid template, 6.82 × 10⁻⁶ cells / reaction... 3 Copy / μL, 6.82×10 2 Copy / μL, 6.82×10 1 All copies / μL were detected as positive, 6.82×10 0 The copy / μL test was negative, and the calculated detection limit for the mic gene copy was 68.2 copies / reaction. This result demonstrates that the RAA-LFS system using the MIB-3 primer set has high sensitivity and can detect low concentrations of 2-MIB-producing cyanobacteria.

[0183] Example 7: To verify the practical application value of the MIB-3 primer set RAA-LFS system, this example uses reservoir water samples as the detection object. By comparing with conventional PCR methods, the accuracy and reliability of its detection results are verified.

[0184] The materials used in the experiment were as follows: 35 water samples were collected from a drinking water source reservoir. After sampling, the samples were stored in an ice bath and transported back to the laboratory within 4 hours. The water sample pretreatment adopted the thermal lysis method (10 mL of water sample was enriched through a 0.22 μm filter membrane, 1 mL of sterile double-distilled water was added, heated at 100℃ for 6 min, filtered through a 0.45 μm filter membrane, and the filtrate was collected as a DNA template). The optimal parameters for the RAA-LFS system were determined in the above examples.

[0185] The control method was conventional PCR, and the primers used were those reported by Suruzzaman et al. (Suruzzaman, M., et al., Evaluation of the MIB-producing potential based on real-time qPCR in drinking water reservoirs., Beijing, (2022)). The PCR reaction system was 50 μL, containing 5 μL 10× PCR buffer, 4 μL dNTPs, 1 μL forward and reverse primers, 0.5 μL Taq enzyme, and 3 μL DNA template. The amplification conditions were: 94℃ pre-denaturation for 5 min, 35 cycles (94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, and 72℃ final extension for 10 min). The products were detected by 1.5% agarose gel electrophoresis.

[0186] The experimental procedure is as follows: using the thermal lysis buffers of 35 reservoir water samples as templates, RAA-LFS detection and conventional PCR detection were performed respectively; the RAA-LFS detection was performed according to the optimal system and procedure, and positive / negative results were recorded; after the PCR detection was completed, gel imaging was used to observe whether specific bands appeared to determine the positive / negative results; the consistency of the detection results of the two methods was compared to verify the accuracy of the RAA-LFS system.

[0187] Appendix Figure 8 The results of mic gene RAA-LFS and PCR detection are shown for 35 reservoir samples. The above experimental results are as follows: Figure 8 The corresponding test results are shown in the table: Of the 35 reservoir water samples, the RAA-LFS system detected 16 positive samples and 19 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 showed a 100% agreement. This result demonstrates that the RAA-LFS system using the MIB-3 primer set can be accurately applied to the detection of 2-MIB-producing cyanobacteria in actual reservoir water samples, providing reliable technical support for 2-MIB pollution monitoring and early warning in drinking water sources.

[0188] Table 4-35 Reservoir Samples: RAA-LFS and PCR Detection Results of the Microgenic Gene.

[0189] Sample number RAA-LFS PCR 1 - - 2 - - 3 - - 4 - - 5 - - 6 - - 7 - - 8 - - 9 - - 10 + + 11 - - 12 - - 13 + + 14 + + 15 + + 16 - - 17 + + 18 + + 19 + + 20 + + 21 + + 22 + + 23 - - 24 - - 25 - - 26 - - 27 + + 28 + + 29 - - 30 + + 31 - - 32 - - 33 + + 34 - - 35 + +

[0190] In summary, this embodiment has screened out a primer set capable of detecting 2-methylisoborneol-producing algae through a series of verifications. This primer set can effectively amplify the 2-methylisoborneol target gene, has high specificity and sensitivity, and shows no cross-reactivity with other non-2-methylisoborneol-producing algae. It can be used for rapid identification of 2-methylisoborneol and is of great significance for effective early warning of odors in reservoir water.

[0191] Comparative Example 1: Comparison with existing conventional PCR methods

[0192] To highlight the technical advantages of this detection method, this comparative example compares the RAA-nfo-LFS method with existing conventional PCR methods (referring to the method reported by Suruzzaman et al. in 2022) from four dimensions: detection efficiency, sensitivity, instrument dependence, and operational complexity, to verify the effectiveness of this method in overcoming the shortcomings of existing technologies.

[0193] Comparison of detection efficiency: Using 35 reservoir water samples as the subjects, the total time from sample collection to obtaining results was approximately 30 minutes, including 12 minutes of thermal lysis pretreatment, 16 minutes of RAA amplification, and 2 minutes of test strip detection. The conventional PCR method requires 2.5 hours, including 30 minutes of column purification of nucleic acid, 1.5 hours of PCR amplification, and 30 minutes of electrophoresis and imaging. The time taken by this comparative method is only 1 / 5 of that of the PCR method, which significantly improves the detection efficiency.

[0194] Sensitivity comparison: Using serially diluted cells of *Anabaena gracilis* as templates, the detection limit of this method is 48 cells / reaction, while the detection limit of the conventional PCR method is 100 cells / reaction. This method improves sensitivity by about 2 times and can detect lower concentrations of 2-MIB-producing cyanobacteria, meeting the needs of early warning.

[0195] Instrument dependency comparison: This method only requires a portable constant temperature mixer and lateral flow test strips, and does not require a fixed laboratory environment; conventional PCR methods rely on PCR instruments and gel imaging systems, and cannot be used on-site at water sources, thus limiting their applicability.

[0196] Comparison of operational complexity: This method uses thermal lysis to prepare templates, eliminating the need for nucleic acid purification. RAA amplification is a one-step isothermal reaction, and results can be interpreted by visually observing the test strip. Grassroots testing personnel can operate independently after one hour of training. Conventional PCR methods require column purification to remove nucleic acid impurities, which is cumbersome and prone to aerosol contamination. The amplification process requires multiple thermal cycles, and result interpretation requires professional personnel to analyze electrophoresis bands under ultraviolet light. This method has a high operational threshold and is prone to misinterpretation.

[0197] In summary, the method of this invention effectively overcomes the shortcomings of existing PCR methods, such as "long time consumption, low sensitivity, dependence on professional instruments, and complex operation", and is more suitable for rapid on-site detection and early warning of 2-MIB cyanobacteria produced in drinking water sources.

[0198] 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 mic gene of cyanobacteria producing 2-methylisoborneol, characterized in that, The primer set is designed based on the conserved region of the mic gene of 2-methylisoborneol-producing cyanobacteria and includes a forward primer, a reverse primer, and a specific probe. The 5' end of the reverse primer is modified with a fluorescent marker, and the 5' end of the probe is modified with biotin, the 3' end is modified with a phosphorylation group, and the probe contains a nuclease cleavage site.

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

3. The primer set according to claim 1 or 2, characterized in that, The nucleotide sequence of the reverse primer is shown in SEQ ID NO.2: 5'-AAGCCTGATGACCGCGTGATGG-3'; The fluorescent marker is FITC, which is modified at the 5' end of the reverse primer.

4. The primer set according to any one of claims 1-3, characterized in that, The nucleotide sequence of the probe is shown in SEQ ID NO. 3: 5'-TACATTGTGGTCTCATTCGTCCAAATGCTHCGCATGGCTCCCGTC-3'; The nuclease cleavage site is a tetrahydrofuran-modified site, corresponding to "H" in the sequence.

5. The primer set according to claim 4, 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 nuclease cleavage site is a specific cleavage site of the nfo enzyme, i.e., endonuclease IV. When the probe binds to the mic gene template of algae producing 2-methylisoborneol, it can be recognized and cleaved by the nfo enzyme, thereby initiating DNA chain extension.

6. 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) The product length of the amplified algal 2-methylisoborneol mic gene by the forward and reverse primers is 200~500bp, and the amplified product corresponds only to algae that produce 2-methylisoborneol, and has species specificity.

7. A kit for detecting cyanobacteria that produce 2-methylisoborneol, 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 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.

9. The reagent kit according to claim 7 or 8, characterized in that, The concentration of the MgAc2 solution is 260-300 mM; in the primer set, the working concentrations of the forward and reverse primers are 8-12 μM, and the working concentration of the probe is 8-12 μM.

10. A method for detecting cyanobacteria that produce 2-methylisoborneol, characterized in that, Includes the following steps: (1) Sample processing: Water samples containing cyanobacteria were pretreated to obtain DNA templates; (2) RAA amplification: Using the DNA template obtained in step (1) as a template, RAA isothermal amplification is performed using the primer set described in any one of claims 1-4; (3) Detection of amplified products: The RAA amplified products were visually detected using lateral flow test strips to determine whether there were cyanobacteria that produced 2-methylisocamphexol.

11. The method according to claim 10, characterized in that, In step (1), the pretreatment is a thermal decomposition 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 or 11, characterized in that, In step (2), the reaction system for RAA isothermal amplification is 50 μL, including: 28-32 μL RAA basic buffer, 2-3 μL MgAc2 solution, 2-4 μL DNA template, and the final concentration of each primer and probe is 0.1-1.0 μM. The remaining volume is made up to 50 μL with ddH2O. The reaction conditions are: 38-40℃ constant temperature reaction for 15-20 min.

13. The method according to any one of claims 10-12, characterized in that, In step (3), the lateral flow test strip includes a control line (C line) and a detection line (T line); the interpretation criteria for the visual detection are: If both the C and T lines show color, the result is positive, indicating the presence of cyanobacteria that produce 2-methylisoborneol. If line C is colored and line T is not colored, then the result is negative; If line C does not show color, the result is invalid regardless of whether line T shows color.

14. The method according to any one of claims 10-13, characterized in that, The detection limit of the method is no more than 50 2-methylisoborneol-producing cyanobacterial cells / reaction, or no more than 70 mic gene copies / reaction.

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