A kit and method for detecting cyanobacteria

Abstract

The present application relates to a kind of kit and method for detecting ultramicro blue algae, the kit includes a pair of primers with sequence as SEQ ID NO:1 and 2 respectively.Compared with prior art, the present application gets rid of the dependence on professional taxonomist and large instrument equipment, and the routine water quality analyst can realize the quantification of this specific group of ultramicro blue algae by real-time fluorescent quantitative PCR;The operation method is simple and convenient, ordinary water quality testing personnel can master in a short time, and the efficiency is greatly improved;Strong specificity, specific primers designed based on rbcL gene can exclude the interference of diatom, green algae and other heterotrophic bacteria in water;Data is accurate and objective, and has strong comparability, and is expected to become a national standard detection method.Through the present application, the bottleneck of ultramicro blue algae quantitative detection depending on large instrument equipment such as flow cytometer can be broken through, and the detection efficiency of ultramicro blue algae in water is greatly improved, which provides an important technical means for water quality safety management and early warning.

Description

Technical Field

[0001] This invention belongs to the fields of microalgae molecular biology applications and water environment monitoring, specifically relating to a reagent kit and method for detecting ultramicroalgae. Background Technology

[0002] Ultramicroalgae are a group of photosynthetic autotrophic prokaryotes with extremely small particle sizes (0.2-2.0 μm), mainly composed of genera *Synechococcus*, *Prochlorococcus*, and *Cyanobium*, and are important primary producers in aquatic ecosystems. In freshwater and terrestrial waters, *Synechococcus* and *Cyanobium* often dominate. Despite their tiny size, ultramicroalgae can account for more than 50% of total phytoplankton biomass in some oligotrophic or even mesotrophic waters. Due to their extremely high specific surface area, ultramicroalgae are highly adaptable to their environment and can efficiently utilize low concentrations of nutrients, allowing them to maintain high population densities even in environments where large cyanobacteria cannot proliferate. In recent years, with the increasing global warming and eutrophication of water bodies, the abnormal proliferation and aggregation of ultramicroalgae in water bodies have become increasingly frequent, and their potential ecological and health hazards have gradually attracted widespread attention.

[0003] Existing studies have confirmed that some ultramicroscopic cyanobacteria possess the ability to produce microcystins, neurotoxins, and odor substances (such as 2-MIB), directly threatening drinking water safety and the quality of aquaculture. These tiny toxin-producing cells can penetrate conventional water treatment processes, causing toxins to accumulate in humans and drinking water sources. Ultramicroscopic cyanobacteria are extremely small in size but have a large specific surface area, exhibiting strong adaptability and expansion capabilities in extreme environments. They easily gain a competitive advantage in warmer surface waters, altering the phytoplankton community structure.

[0004] For water treatment plants that rely on surface water as their source, the frequent occurrence of ultrafine cyanobacteria poses a serious challenge to conventional water treatment processes. Due to their tiny size, traditional water treatment methods struggle to completely remove them, leading to the entry of ultrafine cyanobacteria cells and their metabolic products into the water supply system, threatening water safety. From the perspective of drinking water safety, the concealment of ultrafine cyanobacteria presents a significant challenge to water treatment monitoring. Traditional microscopic counting methods are insufficient for accurately counting and classifying these micrometer-sized algal cells, resulting in their long-term neglect in routine water quality monitoring. These tiny algal cells can penetrate the coagulation, sedimentation, and filtration barriers of water treatment plants, entering the treated water and increasing its biological risk. They may also form biofilms in the water transmission network and even produce biotoxic secondary metabolites.

[0005] Traditional microscopic counting methods are limited by the resolution of optical microscopes (approximately 0.2 μm), making it difficult to classify ultramicroscopic cyanobacteria with uniform morphological characteristics and easily confusing them with heterotrophic bacteria. Furthermore, it is difficult to distinguish closely related groups with significant physiological differences (such as Synechococcus and dichromatic algae) based solely on morphological characteristics. While flow cytometry can utilize the autofluorescence of phycoerythrin and phycocyanin for rapid counting, it still has limitations in differentiating ecotypes and elucidating genetic diversity. Molecular biology techniques, particularly real-time quantitative PCR and high-throughput sequencing, have made it possible to accurately identify and quantify ultramicroscopic cyanobacteria. These techniques can directly capture specific genetic markers from environmental DNA, thus overcoming the challenge of the invisibility of ultramicroscopic algae.

[0006] While amplicon sequencing based on the 16S rRNA gene can reflect the overall structure of the community, the high conservation of the 16S rRNA gene makes it difficult to achieve accurate quantification of ultramicroalgae.

[0007] Currently, there are no reported molecular detection methods for ultramicroscopic cyanobacteria. Therefore, it is essential to develop a simple, rapid, highly sensitive, and highly specific quantitative detection method for ultramicroscopic cyanobacteria. Summary of the Invention

[0008] This invention investigates and analyzes the rbcL gene within ultramicroscopic cyanobacteria cells, screening out conserved sequences located in the core region of the gene, and designing specific primers based on these sequences. These primers, designed based on functional genes, can specifically amplify the sequence of ultramicroscopic cyanobacteria, eliminating interference from diatoms, green algae, and other heterotrophic bacteria in the water.

[0009] Based on the above research, the present invention provides a kit for detecting ultramicroalgae, comprising a pair of primers with sequences as shown in SEQ ID NO:1 and 2, respectively.

[0010] In one specific implementation, the kit is a conventional PCR kit or a real-time quantitative PCR kit.

[0011] In one specific implementation, the ultramicro cyanobacteria are selected from the genus Synechococcus (… Synechococcus ), Prochlorococcus ( Prochlorococcus ) and the genus *Dictyophora* ( Cyanobium One or more combinations of ).

[0012] The present invention also provides a method for detecting ultramicroalgae in water samples, including the step of amplifying the water sample to be tested using a pair of primers with sequences as shown in SEQ ID NO:1 and 2, respectively. When a fragment of 202 bp is amplified, it is determined that the water sample to be tested contains ultramicroalgae.

[0013] In one specific implementation, the ultramicro cyanobacteria are selected from the genus Synechococcus (… Synechococcus ), Prochlorococcus ( Prochlorococcus ) and the genus *Dictyophora* ( Cyanobium One or more combinations of ).

[0014] In one specific implementation, the method includes the following steps: S1: The water sample to be tested is filtered through a membrane; S2: Using the filtered genomic DNA of the water sample to be tested as a template, PCR amplification was performed using the primers described above; S3: Make a judgment based on the amplification results.

[0015] In one specific implementation, in S1, the pore size of the filter membrane used for filtration is 0.22 μm.

[0016] In one specific implementation, the method is PCR, in which the PCR amplification product is subjected to electrophoresis, and the presence or absence of a specific amplification band at 202 bp is used to determine the amplification.

[0017] In one specific implementation, the method is real-time quantitative PCR, and S2 includes the following steps: S21: Extract genomic DNA from the water sample to be tested as a template, perform PCR amplification using the primers, and record the Ct value after the reaction is complete; S2: A standard curve was prepared using plasmid standards containing the target fragment of the rbcL gene from ultramicroalgae; S23: Substitute the Ct value of the water sample to be tested into the standard curve to calculate the copy number of the target fragment of the rbcL gene; S24: Estimate the cell density of ultramicroalgae in the water sample to be tested based on the copy number.

[0018] In one specific implementation, the amplification program in the CR amplification reaction in S21 is as follows: pre-denaturation at 95°C for 300 s, followed by 40 amplification cycles, with each cycle consisting of 15 s at 95°C, 30 s at 60°C, and 30 s at 72°C.

[0019] Compared with the prior art, the present invention has the following advantages and effects: 1) It eliminates the dependence on professional taxonomic technicians and large instruments such as optical microscopes and flow cytometers. Conventional water quality analysts can use real-time quantitative PCR to quantify the specific group of ultramicro algae.

[0020] 2) The operation method is simple and convenient. Ordinary water quality testers can master it in a short time, and more samples can be tested per unit time, which greatly improves efficiency.

[0021] 3) High specificity: Specific primers designed based on the rbcL gene can eliminate interference from diatoms, green algae and other heterotrophic bacteria in the water.

[0022] 4) The data is accurate, objective, and highly comparable, and is expected to become a national standard testing method.

[0023] This invention overcomes the bottleneck of relying on large instruments and equipment such as flow cytometers for quantitative detection of ultrafine cyanobacteria, greatly improving the detection efficiency of ultrafine cyanobacteria in water bodies and providing an important technical means for water quality safety management and early warning. Attached Figure Description

[0024] Figure 1 This represents the specificity detection results of primer pairs for different algal strains in the laboratory. Lane M is the marker (DL2000), lanes 1-6 are for Microcystis, Slender Dichromatic Algae, Rashomon, Soft-Fingered Algae, Chlorella, and Syngonium, respectively, and lane 7 is the negative control.

[0025] Figure 2 The relative abundance of species at the genus level after amplification of environmental samples using primer pairs.

[0026] Figure 3 The standard curve is shown (the vertical axis represents the Ct value, and the horizontal axis represents the gene copy number log10). Detailed Implementation

[0027] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0028] 1. Primer design The rbcL gene encodes the large subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase, a key functional gene for carbon fixation in algae. Ultramicroscopic cyanobacteria (such as Synechococcus) typically carry Form IA RubisCO, while most freshwater cyanobacteria and green algae carry Form IB. This genetic difference provides a molecular basis for designing specific primers for ultramicroscopic cyanobacteria. Compared to 16S rRNA genes, the copy number of the rbcL gene shows a better correlation with cell biomass and photosynthetic activity, more accurately reflecting the biomass of ultramicroscopic cyanobacteria in aquatic bodies.

[0029] The rbcL gene sequences of green algae, diatoms, ultramicroscopic cyanobacteria, and other cyanobacteria were compared to select conserved gene sequence fragments. These conserved gene sequence fragments were then compared with rbcL gene sequences from other algae to further confirm conserved and specific gene sequence fragments. Primers were then designed for the specific sequence, and their specificity was verified. Specific primers were designed for the rbcL gene of ultramicroscopic cyanobacteria in this experiment, targeting a fragment size of 202 bp. Primer information is shown in Table 1.

[0030] Table 1 Primer Sequences 2. Specificity detection of different algal strains by primer pairs in the laboratory The above primers were used to target Microcystis (Microcystis jirovecii) respectively. Microcystis ), delicate bicolor algae ( Cyanobium gracile ), Rabdosia rubescens ( Raphidiopsis raciborskii ), fine silk algae ( Aphanizomenon gracile Chlorella ( Chlorella ) and needle algae ( Synedra PCR amplification was performed, followed by agarose gel electrophoresis to verify the specificity of the primers. Except for *Slender Bicolor Algae*, all other algal species were obtained from the freshwater algae strain bank of the Wuhan Institute of Hydrobiology, Chinese Academy of Sciences.

[0031] DNA was extracted from these six algal species using standard methods. PCR amplification was performed on each of the six algal strains using designed primers. The PCR reaction volume was 20 μL, containing 1 μL of DNA template, 0.4 μL of forward primer (10 μmol / L), 0.4 μL of reverse primer (10 μmol / L), 10 μL of 2×Taq Plus MasterMix (Dye), and ddH2O to bring the volume to 20 μL. The PCR program was as follows: 94℃ pre-denaturation for 3 minutes, 94℃ denaturation for 15 seconds, 59℃ annealing for 15 seconds, 72℃ extension for 10 seconds, for a total of 34 cycles, with a final extension at 72℃ for 3 minutes. After the PCR reaction, 8 μL of the amplified product was added to each well of a 1% agarose gel and electrophoresed at 100V for 20 minutes. The electrophoresis results were then captured using a gel imaging system.

[0032] The results are as follows Figure 1 As shown, the primers only amplified DNA samples from *Dichroa stenoptera*, with an amplified band of 202 bp. No amplification was observed in DNA samples from other algal strains besides *Dichroa stenoptera*, indicating that the primers are specific to *Dichroa stenoptera*.

[0033] 3. Specific detection of primer pairs in environmental samples The specificity of the primers for environmental samples was verified using DNA samples from the filter membrane of raw water from a water plant in Xinxiang section of the South-to-North Water Diversion Project in August. The designed ultramicroalgae-specific primers were used to amplify the DNA from the filter membrane of the raw water from the water plant in August. The PCR reaction system was 50 μL, containing 2 μL of DNA template, 1 μL of PCR forward primer (10 μmol / L), 1 μL of PCR reverse primer (10 μmol / L), 25 μL of 2×Taq Plus MasterMix (Dye), and ddH2O to bring the system to 50 μL. The PCR amplification program was: 94℃ pre-denaturation for 3 minutes, 94℃ denaturation for 15 seconds, 59℃ annealing for 15 seconds, 72℃ extension for 10 seconds, for a total of 34 cycles, with a final extension at 72℃ for 3 minutes. The PCR products were separated using 1% TBE agarose gel electrophoresis, and the target band of 202 bp was excised under gel imaging. The excised gel fragment was recovered using a gel extraction kit, and the recovered DNA was sequenced. Meanwhile, high-throughput sequencing was performed on the raw water filter membrane of the water plant in August. The sequencing results were compared with the sequencing results after gel recovery to determine the specificity of the designed primers and their actual coverage of field samples.

[0034] Sequencing results show that ( Figure 2 The genus with the highest relative abundance in the amplified products was Synechococcus (…). Synechococcus ), accounting for about 50%; followed by the genus *Dichromatic* (…). Cyanobium The proportion of these algae is approximately 49%. This primer can specifically detect ultramicroscopic cyanobacteria from complex environmental samples, unaffected by interference from other species, with a detection rate exceeding 99%. Therefore, the primers designed in this study can specifically detect ultramicroscopic cyanobacteria in environmental samples.

[0035] 4. Quantitative detection of samples The target DNA fragment from *Slender Two-Colored Algae* was amplified using the primers described above. The PCR reaction system and gel recovery procedure were the same as in Example 1. The recovered target gene fragment was used for gene cloning.

[0036] The cloning vector used was the 5-min TA / Blunt-Zero Cloning Kit. The total ligation reaction volume was 5 μL: 4 μL PCR product and 1 μL vector (5×TA / Blunt-Zero Cloning Mix), reacted at room temperature for 5 minutes. The ligation product was transformed into Fast-T1 chemocompetent cells, plated on LB agar plates containing ampicillin, and incubated overnight for 12 hours. Single colonies were picked for PCR detection to screen for positive clones.

[0037] Positive clone bacterial cultures were used for plasmid extraction, and plasmids were extracted using a plasmid miniprep kit. The concentration of plasmid DNA was measured using micro-UV spectrophotometry, and the plasmid concentration was converted to copy number based on the molecular weight of the recombinant plasmid. The conversion formula is as follows: Copy number (copies / μL) = plasmid concentration (ng / μL) × 6.02 × 10 23 / [ length (bp) × 660 × 10 9 ] Dilute plasmid DNA to 10. 4 ~10 9 A concentration gradient of copies / μL was used to obtain quantitative standards. qPCR was used for quantitative analysis of the standards. The qPCR reaction system was 10 μL: 5 μL Mix, 0.2 μL each of forward and reverse primers (10 μmol / L), 1 μL of the DNA to be tested, and ddH2O to bring the reaction system to 10 μL. The reaction program was: 95℃ pre-denaturation for 300 s, 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 40 cycles. Each sample was prepared in triplicate, and a template-free negative control was included in each experiment. After the reaction, a standard curve was plotted with the logarithm of the initial template amount of the standards on the x-axis and the cycle threshold (Ct) on the y-axis.

[0038] The regression equation for the obtained standard curve is: y = -3.425 x + 42.07 ( Figure 3 The Ct value showed a high correlation with the log10 of the copy number of *Slender Dichroic Algae* (R0). 2 =0.9944). The amplification efficiency of qPCR (E=95.9%) is greater than 90%, indicating that qPCR using this primer can effectively detect the presence of ultramicroalgae.

[0039] Water samples from the test environment were collected and filtered through a 0.22 μm microporous membrane. Total genomic DNA was extracted and purified from the filtered algal samples. The extracted and purified DNA was then amplified and quantified using quantitative real-time PCR (qPCR). The Ct value of the sample was recorded after the reaction. Substituting the Ct value of the sample into the standard curve formula yielded the copy number of the rbcL gene in the ultramicroscopic cyanobacteria. Since the rbcL gene is a single-copy gene, the copy number can be considered equivalent to the cell number, thus determining the ultramicroscopic cyanobacteria cell density in the sample.

[0040] Using the above method, the copy number of the rbcL gene of ultramicroalgae in water samples can be obtained in a relatively short time.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A reagent kit for detecting ultramicroscopic cyanobacteria, characterized in that, It includes a pair of primers with sequences as shown in SEQ ID NO:1 and 2, respectively.

2. The reagent kit according to claim 1, characterized in that, This can be a standard PCR kit or a real-time quantitative PCR kit.

3. The reagent kit according to claim 1, characterized in that, The ultramicro cyanobacteria are selected from the genus Synechococcus ( Synechococcus ), Prochlorococcus ( Prochlorococcus ) and the genus *Dictyophora* ( Cyanobium One or more combinations of ).

4. A method for detecting ultrafine cyanobacteria in water samples, characterized in that, The method includes the step of amplifying the water sample to be tested using a pair of primers with sequences as shown in SEQ ID NO:1 and 2, respectively. When a fragment of 202 bp is amplified, it is determined that the water sample to be tested contains ultramicro algae.

5. The method according to claim 4, characterized in that, The ultramicro cyanobacteria are selected from the genus Synechococcus ( Synechococcus ), Prochlorococcus ( Prochlorococcus ) and the genus *Dictyophora* ( Cyanobium One or more combinations of ).

6. The method according to claim 4, characterized in that, Includes the following steps: S1: The water sample to be tested is filtered through a membrane; S2: Using the filtered genomic DNA of the water sample to be tested as a template, PCR amplification was performed using the primers described above; S3: Make a judgment based on the amplification results.

7. The method according to claim 6, characterized in that, In S1, the pore size of the filter membrane used for filtration is 0.22 μm.

8. The method according to claim 6, characterized in that, The method is PCR, in which the PCR amplification product is subjected to electrophoresis, and the presence of a specific amplification band at 202 bp is used to determine the amplification.

9. The method according to claim 6, characterized in that, The method is real-time quantitative PCR, and S2 includes the following steps: S21: Extract genomic DNA from the water sample to be tested as a template, perform PCR amplification using the primers, and record the Ct value after the reaction is complete; S2: A standard curve was prepared using plasmid standards containing the target fragment of the rbcL gene from ultramicroalgae; S23: Substitute the Ct value of the water sample to be tested into the standard curve to calculate the copy number of the target fragment of the rbcL gene; S24: Estimate the cell density of ultramicroalgae in the water sample to be tested based on the copy number.

10. The method according to claim 9, characterized in that, The amplification program in the CR amplification reaction of S21 is as follows: pre-denaturation at 95℃ for 300 s, followed by 40 amplification cycles, with each cycle consisting of 15 s at 95℃, 30 s at 60℃, and 30 s at 72℃.

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