Probe combination, gene chip, kit and method for detecting connezothrix aquaticum

Abstract

The invention discloses a probe combination for detecting connezothrix aquaticum, and the probe combination comprises sequences shown as SED ID NO.1-99. The invention also discloses a gene chip prepared by using the probe combination, a kit and a detection method. According to the present invention, the probes in the whole genome range are adopted, the gene chip is adopted to perform hybridization, the hybridization signal of the probe group is interpreted to determine whether the eight kinds of the hydronematoda aquae exist or not, the detection of the eight kinds of the hydronematoda aquae is achieved, and compared with the traditional detection method, the detection method has advantages of high resolution, simple operation, simple and convenient data processing, and low detection cost. And the detection method has important application value for long-term dynamic monitoring of water quality deterioration of the water body, and provides powerful support for guiding algal bloom control and water ecological restoration.

Description

Technical Field

[0001] The invention belongs to the technical field of gene chips and microorganisms, and particularly relates to a probe combination, a gene chip, a kit and a method for detecting Aphanizomenon flos-aquae. Background Art

[0002] Aphanizomenon Aphanizomenon ) is a freshwater floating filamentous algae belonging to the family Nostocaceae of the order Nostocales of the phylum Cyanobacteria. Their most obvious feature is that the terminal cells of the filaments taper or become hair-like, extending into colorless cells. Aphanizomenon can form algal blooms in freshwater bodies, also known as cyanobacterial blooms, which have caused many environmental problems. In certain seasons in some lakes, Aphanizomenon blooms and Microcystis blooms will compete and erupt alternately, forming a unique cyanobacterial bloom phenomenon. Some species of the genus Aphanizomenon may produce toxins, such as Aphanizomenon blooms ( A. water-flower )、Acanthosiphon gracilis( A. slender ) and Aphanizomenon elsa A. issatschenkoi ) can produce paralytic shellfish toxins, which harm aquatic animals and can endanger human health through the food chain.

[0003] In my country's freshwater bodies, the species of Aphanizomenon include Aphanizomenon bloom-water, Aphanizomenon tenuissima and Aphanizomenon yisha, among which Aphanizomenon bloom-water is the most common. Aphanizomenon bloom-water is widely distributed in the world, mainly in freshwater. Its morphological characteristics are mainly manifested in straight or slightly curved algae filaments, single or connected to the sides of the algae filaments in bundles. The cells in the middle of the algae filaments are short cylindrical, more or less square, with pseudovacuoles. The terminal cells become more or less tapered and elongated into colorless cells. The gelatin sheath is unclear. Heteromorphic cells grow intercellularly, in various shapes, cylindrical, nearly spherical, and elliptical. Aphanizomenon bloom-water has a planktonic life and can reproduce in large numbers. Under certain conditions, it can form dense water blooms, which may cause water quality problems and ecological impacts.

[0004] At present, the identification and classification methods of Aphanizomenon are mainly based on traditional morphological detection. However, due to the diverse and variable morphology of the genus Aphanizomenon and the lack of obvious differences between the genus and other cyanobacteria such as Anabaena, it is often difficult to distinguish and identify based on the simple external morphology and subtle differences in internal physiological structure and high phenotypic plasticity, which makes the classification and identification of this genus face great challenges. Conventional detection methods of Aphanizomenon mainly include microscopic observation, biomass determination and pigment analysis. Although traditional methods have accumulated a lot of experience and data in classification and identification, they also have obvious limitations. For example, the method of observation with a microscope is time-consuming, requires professional knowledge and operating experience, and its accuracy is easily affected by subjective factors. Methods such as biomass determination and pigment analysis can only give more general or indirect evaluation results, and the resolution of detection and identification is insufficient, and it is impossible to specifically distinguish species or even strains, and it is impossible to perform accurate quantification. Molecular biological methods have also been used to study Aphanizomenon, but the research direction is more inclined to the detection and identification of cyanobacterial toxins and toxin-producing genes. For example, the composition and content of Aphanizomenon toxins can be determined by high performance liquid chromatography, or a certain gene fragment of microcystin can be amplified by polymerase chain reaction (PCR). For the assessment of the degree of water pollution, the quantitative and qualitative analysis of algae and the toxins produced by algae are equally worthy of attention. Aphanizomenon is the main microalgae in the outbreak of cyanobacteria, so it is necessary to develop faster and more accurate qualitative and quantitative methods for it. Summary of the invention

[0005] In order to solve at least one of the above problems, the present invention provides a probe combination, a gene chip, a kit and a method for detecting Aphanizomenon flos-aquae.

[0006] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a probe combination for detecting Aphanizomenon flos-aquae, the probe combination comprising a probe for detecting Aphanizomenon water-flower The probe set of 2012 / KM1 / D3, the sequences are shown in SED ID NO.1 to 12; detection Aphanizomenon water-flower The probe set of FACHB-1040, the sequences are shown in SED ID NO. 13 to 25; detection Aphanizomenon water-flower The probe set for FACHB-1290, the sequences are shown in SED ID NOs. 26 to 37; Aphanizomenon water-flower The probe set of FACHB-1416, the sequences are shown in SED ID NOs. 38 to 50; detection Aphanizomenon water-flower The probe set for LD13, the sequences are shown in SED ID NOs. 51 to 62; detection Aphanizomenon water-flower The probe set for MDT14a, the sequences are shown in SED ID NOs. 63 to 74; detection Aphanizomenon water-flowerThe probe set of NIES-81, the sequences are shown in SED ID NOs. 75 to 87; detection Aphanizomenon water-flower The probe set of WA102, the sequences are shown in SED ID NOs. 88 to 99.

[0007] In some embodiments of the present invention, the Aphanizomenon flos-aquae is Aphanizomenon flos- water 2012 / KM1 / D3, Aphanizomenon-flowers-waters FACHB-1040, Aphanizomenon flos- water FACHB-1290, Aphanizomenon water-flower FACHB-1416, Aphanizomenon flos- water LD13, Aphanizomenon water-flower MDT14a, Aphanizomenon water-flower NIES-81 and Aphanizomenon water-flower At least one of WA102 is a target detection of Aphanizomenon, and the probe combination includes all probes in the probe group of the target detection of Aphanizomenon.

[0008] In some specific embodiments of the present invention, the probe combination is a mixture of multiple probe groups; in some specific embodiments, the probe combination includes probe groups corresponding to the detection of 1, 2, 3...8 detection targets; in a preferred specific embodiment, the probe combination includes probes for all 8 detection targets, and contains all 99 probes of the detection probes corresponding to the detection of Aphanizomenon bloom-aquae, and can detect the above-mentioned Aphanizomenon bloom-aquae simultaneously. The probe is a specific probe that only binds to the target Aphanizomenon bloom-aquae DNA sequence and does not bind to any other Aphanizomenon bloom-aquae DNA sequence; the probe design steps include preliminary screening of specific probes, sensitivity screening of oligonucleotide fragments, and consistency screening of physical and chemical properties.

[0009] The present invention also provides the use of the probe combination described in the first aspect in preparing a gene chip or a kit for detecting Aphanizomenon flos-aquae, wherein the detection targets of Aphanizomenon flos-aquae include Aphanizomenon flos- water 2012 / KM1 / D3, Aphanizomenon-flowers-waters FACHB-1040, Aphanizomenon flos- water FACHB-1290, Aphanizomenon water-flower FACHB-1416, Aphanizomenon flos- water LD13, Aphanizomenon water-flower MDT14a, Aphanizomenon water-flower NIES-81 and Aphanizomenon water-flowerThere are 8 detection targets such as WA102, and the detection targets may include one, multiple or all of them. The corresponding probe combination includes all probes in the probe group corresponding to the detection target.

[0010] For example, when the target detection is Aphanizomenon Aphanizomenon water-flower FACHB-1416, the probe combination includes detection Aphanizomenon water-flower The probe set of FACHB-1416, the sequences are all the probes shown in SED ID NOs. 38 to 50; when the target detection is Aphanizomenon Aphanizomenon water-flower 2012 / KM1 / D3, Aphanizomenon water-flower FACHB-1290, Aphanizomenon water-flower WA102, the probe combination includes detection Aphanizomenon water-flower 2012 / KM1 / D3, Aphanizomenon flos- water FACHB-1290 and Aphanizomenon water-flower All probes in the probe set of WA102 include sequences shown in SEDID NOs. 1 to 12, sequences shown in SED ID NOs. 26 to 37, and sequences shown in SED ID NOs. 88 to 99.

[0011] The detection results of the designed Aphanizomenon were judged according to the fluorescence signal intensity of the strain-specific probe group and the proportion of the dominant fluorescent probe to all probes.

[0012] The second aspect of the present invention provides a gene chip for detecting Aphanizomenon flos-aquae, comprising the probe combination described in the first aspect.

[0013] The third aspect of the present invention provides a kit for detecting Aphanizomenon flos-aquae, wherein the kit comprises the probe combination described in the first aspect or the gene chip described in the second aspect.

[0014] In some embodiments of the present invention, the kit further comprises a DNA extraction reagent for the sample to be tested, a DNA purification reagent, and a DNA fluorescent labeling reagent.

[0015] A fourth aspect of the present invention provides a method for detecting Aphanizomenon flos-aquae, comprising the following steps: (1) Filter the water sample to be tested, collect the filtered particles and complete DNA extraction; the extracted DNA is sheared and purified to obtain gDNA; (2) diluting the gDNA obtained in step (1) to a fixed concentration, performing PCR using random primers and ATCG bases with fluorescent groups to obtain gDNA sequences with fluorescent groups; (3) performing a hybridization experiment on the fluorescently labeled gDNA obtained in (2) and the chip according to claim 3 in a hybridization oven; (4) Scanning and imaging the hybridization experiment results of (3), and judging the detection results of the designed Aphanizomenon according to the fluorescence signal intensity of the strain-specific probe group and the proportion of the dominant fluorescent probe to all probes; wherein the Aphanizomenon bloom-water algae includes Aphanizomenon water-flower 2012 / KM1 / D3, Aphanizomenon-flowers-waters FACHB-1040, Aphanizomenon water-flower FACHB-1290, Aphanizomenon water-flower FACHB-1416, Aphanizomenon water-flower LD13, Aphanizomenon water-flower MDT14a, Aphanizomenon flower-water NIES-81 and Aphanizomenon flos-aquae WA102.

[0016] In some embodiments of the present invention, the probe positivity threshold is 300, that is, when the fluorescence signal intensity is greater than 300, it indicates that the probe is detected; the strain probe group positivity rate threshold is 70%, that is, when more than 70% of the probes in the strain probe group are detected, it indicates that the strain is detected.

[0017] In some specific embodiments of the present invention, the DNA extraction, fragmentation and purification in step (1), the fluorescent labeling in step (2) and the hybridization in step (3) can be performed using conventional methods or kits in the art.

[0018] In some specific embodiments of the present invention, the hybridization experiment results in step (5) are read by scanning the chip using a laser scanner. The fluorescently labeled probe area will emit light of a specific wavelength, which will be captured by the scanner. Finally, the scanned image data will be converted into digital signals.

[0019] Further, the data signals were screened and the species detection of Aphanizomenon was determined: the microarray data was preprocessed to eliminate systematic differences between samples and filter out false positive detections. First, the raw signal intensity was corrected by subtracting the background intensity from the foreground intensity of the probe for background correction. Next, the probe signal intensity was normalized using the background correction data as the input matrix. The purpose was to adjust the overall signal intensity between different samples to ensure that they were comparable. In order to reduce false positives, the probe detection rate of each species also needs to be calculated. The probe detection rate is defined as the number of probes detected for a species divided by the number of all designed probes for that species. If the probe detection rate of a species is greater than the set ratio threshold, it is classified as positive to ensure that only true positive results are considered.

[0020] In the present invention, the sample to be tested may come from any water source, including but not limited to any flowing or non-flowing water source such as rivers, streams, creeks, seas, lakes, reservoirs, ponds, etc.

[0021] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: 1. At present, the identification and classification of Aphanizomenon still remains in the morphological detection method, but due to the diverse and changeable morphology of the genus Aphanizomenon and the lack of obvious differences between the genus and other cyanobacteria such as Anabaena, it is difficult to distinguish and identify it based on simple external morphology and internal physiological structure. The present invention establishes a specific probe detection based on the whole genome range for Aphanizomenon in water, filling the gap in related detection technology.

[0022] 2. The present invention utilizes gene chip technology to design species / strain-specific probes within the entire genome, which can simultaneously detect strains of Aphanizomenon flos-aquae in 8 water bodies, and has higher resolution and sensitivity than other PCR methods.

[0023] 3. The present invention has the advantage of simple operation. Compared with the detection method based on sequencing, it omits the steps of PCR and sequencing of the target area, and the data processing is simpler and more convenient. This high-throughput, high-resolution and convenient detection method has important application value for the long-term dynamic monitoring of water quality deterioration caused by Aphanizomenon, and can guide algal bloom control and water ecological restoration. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic diagram of a gene chip pattern according to Example 1 of the present invention is shown; Figure 2 Two groups of specific experimental test results in Example 3 of the present invention are shown; Figure 3 The figure shows the detection results of water samples from 5 wild environments in Example 4 of the present invention. DETAILED DESCRIPTION

[0025] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0027] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0028] Example 1 Screening of specific probes for Aphanizomenon flos-aquae and preparation of chip 1. Dataset collection: In order to screen specific probes for Aphanizomenon bloom-aquae strains, the main data prepared include two parts, the target strain genome sequence and the background genome sequence. Here we use the genomes of 8 species of Aphanizomenon bloom-aquae as the target sequence, and all other cyanobacterial genome sequences collected in the public database are set as the background sequence. The genome information used to design the Aphanizomenon bloom-aquae strain is shown in Table 1.

[0029] Table 1 Genomic information used to design Aphanizomenon flos-aquae strains

[0030] 2. Preliminary selection of specific probes for Aphanizomenon flos-aquae strains The target Aphanizomenon genome sequence is completely broken into Kmer fragments of 50mer length, and a background sequence Kmer fragment hash library is established to record the frequency of occurrence of Kmer and the information of the strain to which it belongs. Then a Kmer fragment hash library of the genome sequence is established. The target sequence Kmer library is compared and analyzed with the background sequence Kmer library, and the Kmer that only exists in the target sequence Kmer library but not in the background sequence Kmer library is selected as the alternative specific probe library of the strain.

[0031] 3. Elimination of potential non-specific binding probes from candidate specific probes Studies have shown that if the probe has more than 20 consecutive base matches with the non-target sequence, potential non-specific binding may occur. Using the NCBI Blast program, the candidate specific probes were aligned with the background Kmer library. Based on the alignment results, the candidate specific probes that had more than 20 consecutive base matches with the background Kmer were removed.

[0032] 4. Screening of probe physical and chemical properties The strain-specific probes obtained after the previous step are unique in the genome of the strain and do not match more than 20 consecutive bases with the genomes of other species. The remaining specific probes are then screened for physical and chemical properties. The main conditions include: (1) The free energy of nucleic acid between the probe sequence and the target sequence (unit: kcal / mol). If the free energy of nucleic acid is less than -30, the probe sequence is removed. (2)If the continuous identical bases of the probe appear 5 times, then the complexity of this probe is too low, and this probe sequence is removed; (3)Finally screen the strain-specific probes according to the melting temperature Tm value (65 < Tm < 95) and GC content (0.2 < GC content < 0.8).

[0033] Finally, 99 strain-specific probes of Aphanizomenon flos-aquae distributed throughout the genome were screened out, and the specific probes are shown as SED ID NO.1 - SED ID NO.99. The information of the strain-specific probes of Aphanizomenon flos-aquae is shown in Table 2.

[0034] Table 2 Strain-specific probes of Aphanizomenon flos-aquae

[0035] 5. Chip preparation Adopt the conventional chip preparation method to synthesize the above-mentioned specific probes through a commercial company and integrate them into a gene chip, and this gene chip has the function of specifically detecting Aphanizomenon. The style of the gene chip is as Figure 1 shown.

[0036] Example 2 Method for rapidly detecting Aphanizomenon in water by using the chip of the present invention 1. Collection of water algae microorganisms Preparation before sampling: (1)Select a suitable ultrafiltration membrane filter with a pore size of 0.22 - 0.45 microns.

[0037] (2)Ensure that the filtration equipment, including the filter holder and pipeline, is clean and disinfected.

[0038] (3)Prepare sterile containers, tubes and collection bottles for sampling.

[0039] Sampling collection: (1)Determine the sampling location according to the research purpose and relevant water sources.

[0040] (2)Determine the volume of water sample to be collected according to the analysis requirements and microbial load.

[0041] (3)Start the water flow and adjust the filtration equipment to a controlled flow rate, usually 100 - 500 ml / min.

[0042] (4)Pass the water sample through the ultrafiltration membrane filter to ensure that all the water samples are filtered.

[0043] (5)Collect the filtrate containing smaller particles and molecules into a sterile container for subsequent analysis or treatment.

[0044] (6)Disconnect the filter holder and carefully transfer the microbial cells retained on the filter membrane to a sterile collection bottle or tube.

[0045] Sample processing: (1) Flush the filter holder and tubing with sterile water or buffer to recover any remaining microbial cells in the system.

[0046] (2) Transfer the retained microbial cells on the filter membrane to a sterile container or tube containing a preservation solution (such as sterile PBS) to prevent cell degradation.

[0047] (3) Ensure that the filter membrane is completely immersed in the preservation solution to maintain the viability of microbial cells during transportation and storage.

[0048] (4) Label each container or tube with a unique identifier that includes the sampling location, date, and other relevant information.

[0049] Transportation and storage: (1) Place the collected microbial samples in an insulated cooler or ice packs for transportation to maintain the appropriate temperature.

[0050] (2) Minimize exposure to extreme temperatures, sunlight, and other environmental factors that may affect the viability or composition of microorganisms.

[0051] (3) Transport samples to the laboratory as quickly as possible, preferably within 24-48 hours, to avoid changes in the microbial flora.

[0052] (4) Once at the laboratory, store the sample at a temperature appropriate for the analysis, such as refrigeration or freezing, to maintain the viability of the microorganisms.

[0053] Sample preparation: (1) Use sterile sampling tubes or containers to collect environmental samples.

[0054] (2) If necessary, homogenize solid samples using a sterile mortar and pestle or other appropriate method.

[0055] (3) Alternatively, samples can be snap-frozen in liquid nitrogen and stored at -80°C until further processing.

[0056] 2. DNA Extraction (1) Wear sterile gloves and ensure the operating area is clean.

[0057] (2) Depending on the sample size and manufacturer's instructions, add an appropriate amount of lysis buffer to the sample tube to ensure that the sample is completely suspended.

[0058] (3) Add proteinase K and SDS to the sample tube at the recommended concentrations. Mix gently by inverting the sample tube several times.

[0059] (4) Incubate the sample tube at an appropriate temperature (usually 55-65°C) for a certain period of time (e.g., 1-2 hours) to allow the cells to undergo enzymatic hydrolysis and protein digestion.

[0060] (5) Perform a phenol:chloroform:isoamyl alcohol extraction by adding an equal volume of a mixture of phenol:chloroform:isoamyl alcohol to the sample tube. Mix thoroughly by inverting the sample tube several times.

[0061] (6) Centrifuge the sample tube at an appropriate speed and time (e.g., 12,000 rpm, 10 min) to separate the aqueous phase (containing DNA) from the organic phase.

[0062] (7) Carefully transfer the aqueous phase to a new tube, avoiding disturbing the interface or the organic phase.

[0063] (8) Perform a chloroform:isoamyl alcohol extraction by adding an equal volume of the chloroform:isoamyl alcohol mixture to a new tube. Mix thoroughly by inverting the sample tube several times.

[0064] (9) Centrifuge the tube at an appropriate speed and time to separate the aqueous phase.

[0065] (10) Transfer the aqueous phase to a new tube, leaving behind any interfacial contaminants.

[0066] (11) Precipitate the DNA by adding an equal volume of isopropanol. Mix gently by inverting the tube.

[0067] (12) Incubate at -20°C or -80°C for at least 1 hour to precipitate the DNA.

[0068] (13) Centrifuge the tube at an appropriate speed and time to precipitate the DNA.

[0069] (14) Discard the supernatant and wash the DNA pellet with 70% ethanol to remove residual contaminants.

[0070] (15) Air-dry the DNA pellet or gently blow dry any residual ethanol using nitrogen gas.

[0071] (16) Dissolve the DNA pellet in sterile water or buffer. Mix gently to ensure complete dissolution.

[0072] (17) Optionally, measure the concentration and purity of DNA using a DNA quantification kit.

[0073] 3. DNA magnetic bead purification The main steps of DNA purification are: (1) Equilibrate OnePure MagBeads at room temperature for 30 minutes and vortex to mix thoroughly to ensure that there is no obvious precipitation of magnetic beads; (2) Add 50-100 μL of the DNA sample to be purified by magnetic beads to the PCR tube / eight-tube strip, then add an equal volume of OnePure MagBeads, vortex to mix, centrifuge to collect the liquid on the tube wall, and let stand at room temperature for 5 minutes; (3) Place the PCR tube / eight-tube strip on a magnetic rack, wait for the solution in the tube to become clear, and discard the supernatant; (4) Add 200 μL of 80% freshly prepared ethanol to the PCR tube / eight-tube strip, let stand for 30 seconds, then discard the supernatant. Repeat the steps until the supernatant is removed cleanly. (5) Place the PCR tube / eight-tube strip on a magnetic rack and leave it at room temperature for 1 to 2 minutes until the magnetic beads are dry or place the tube with the lid open on a 45°C metal bath until there is no water on the surface of the magnetic beads and no ethanol residue at the bottom of the tube. (6) Remove the PCR tube / eight-tube strip from the magnetic stand and add 43 μL of sterile water to resuspend the magnetic beads. Vortex or pipette to mix well, then quickly collect the liquid on the tube wall and leave at room temperature for 3 minutes. (7) Place the PCR tube / eight-tube strip on a magnetic rack, wait for the solution in the tube to become clear, and transfer 42 μL of the supernatant to a new EP tube for labeling in the next step.

[0074] 3. DNA fluorescent labeling This example uses the Agilent SureTag Complete DNA Labeling Kit, which includes the following steps: (1) Take 250 ng of gDNA purified from magnetic beads and make up the volume to 14.75 μL with sterile water, then add 2.75 μL of random primer, mix well, and perform the following denaturation reaction: 98℃ for 10 min, heated cover at 105℃; (2) When the time is reached, immediately place the sample on ice to cool; (3) After centrifuging the above sample, add the following reagents directly: 1 μL sterile water, 5.5 μL 5× Reaction buffer, 2.75 μL 10× dNTP mix, 0.25 μL Cyanine 3-dUTP, 0.5 μL Exo(-)Klenow, a total of 27.5 μL; (4) Use a pipette to blow or vortex to mix, then quickly centrifuge to collect the liquid on the tube wall and remove bubbles; (5) Place the reaction system on a PCR instrument, set the heated cover temperature to 105°C, and run the following program: 37℃ for 4 hours, 95℃ for 3 minutes, hold at 4℃.

[0075] 4. Purification of fluorescently labeled DNA products This example uses the Agilent Oligo aCGH / ChIP-on-chip Hybridization Kit, which includes the following steps: (1) Centrifuge the labeled product to confirm the column sorting, add 125 μL 1× TE and mix with the sample three times each time, rinse the labeled tube and transfer it to the purification column. Centrifuge at 14000×g for 10 min; (2) Discard the filtrate, put the collection tube back into the collection column, add 480 μL 1×TE (pH 8.0) to the collection column, cover the lid, and centrifuge at 14,000×g for 10 min; (3) Remove the collection column and invert it in a new 2 mL centrifuge tube, mark the tube accordingly, and centrifuge at 1000 × g for 1 min. The purified sample (the sample in the same hybridization area is purified and recovered in the same collection tube) is obtained (the volume is approximately between 40-64 μL). Transfer the purified product to a PCR tube; (4) Use Nanodrop one to measure the total nucleic acid concentration and the corresponding dye labeling concentration; (5) Use a concentrator to dry the sample to a volume of 10 μL.

[0076] 5. Hybridization of fluorescent DNA of target sample with Aphanizomenon flos-aquae microarray This example uses the Agilent Oligo aCGH / ChIP-on-chip Hybridization Kit, which includes the following steps: (1) Configuration of hybridization system The chip hybridization system is shown in Table 3. After preparation, 45 μL of the hybridization system was added to the sample concentrated to 10 μL and mixed with a gun. After centrifugation, the reaction system was placed on a PCR instrument, the temperature of the hot cover was set to 105°C, and the following program was run: 98°C for 3 min, 37°C for 30 min, and 37°C hold; Table 3 Chip hybridization system

[0077] (2) Hybridization a. First, place a clean gasket into the Agilent chamber with the gasket label facing up and align it with the rectangular part of the chamber bottom, making sure the gasket is flush with the chamber base; b. Then pipette 47 μL of the sample at 37°C in the previous step into the middle of the rubber ring on the gasket, avoiding bubbles, and place the chip upside down on the gasket; c. Then close the chamber lid and tighten the knob; d. Load each assembled device into the incubator rotation rack, take a balanced chamber, rotate the hybridization chamber vertically to wet the slide, and evaluate the mobility of the bubble.

[0078] e. Set the hybridization rotator speed to 20 rpm and hybridize at 67°C for 22 hours.

[0079] (3) Chip cleaning After hybridization, remove the chip at room temperature, place it in wash solution 1 (reagent of the Agilent kit), set it to 250 rpm, and shake it for 5 minutes at room temperature; then use wash solution 2 (reagent of the Agilent kit) at 200 rpm, shake it for 1 minute at 39°C, finally remove the liquid on the surface of the chip and scan it within 4 hours.

[0080] 6. Fluorescence result scanning and signal analysis (1) Import the microarray data file into the microarray data preprocessing software.

[0081] (2) Verify the integrity of the data file and ensure that the file format is compatible with the preprocessing software.

[0082] (3) Perform quality control checks to assess the overall quality of the data and identify potential problems, such as outliers and artifacts.

[0083] (4) Remove or mark low-quality or unreliable probes or spots from the data; for example, remove probes with signal intensity < 100.

[0084] (5) Apply background correction methods to remove non-specific hybridization signals, such as RMA, mas5, and model-based background correction.

[0085] (6) Perform data normalization to adjust for systematic variations between arrays, such as quantile normalization and loess normalization.

[0086] (7) Export the preprocessed microarray data in an appropriate format, such as CSV or Excel, for subsequent analysis and calculation of the probe detection rate for each species.

[0087] Example 3 Detection specificity test of the chip for Aphanizomenon Specific detection means that the presence of the target strain in the water sample can be detected specifically. In order to test the specific detection effect of the probe and chip we designed, we used the method of the present invention to detect the specific bacterial liquid of Aphanizomenon within the detection range, compared the consistency between the test results and the experimental selected combination design, and analyzed the false positive and false negative ratios of the test results. The experiment set the probe positive threshold to 300, that is, when the fluorescence signal intensity is greater than 300, it means that the probe is detected; the strain probe group positive rate threshold is 70%, that is, when the strain probe detection rate is above 70%, it means that the strain is detected.

[0088] Two groups of specific experiments were designed in this example: Specificity experiment 1: only contains Aphanizomenon flos-aquae FACHB-1416 A body of water containing Aphanizomenon flos-aquae.

[0089] Specificity experiment 2: only contains Aphanizomenon flos-aquae MDT14a A body of water containing Aphanizomenon flos-aquae.

[0090] Test results such as Figure 2 shown.

[0091] In experiment 1, the strain Aphanizomenon flos-aquae FACHB-1416 Among the 12 probes in the probe group, the fluorescence signal intensity of 11 probes is greater than the probe positive threshold of 300, indicating that 11 probes in the probe group are detected, and the fluorescence signal intensity of the other probe is lower than 300, indicating that the probe is not detected. Therefore, the positive rate of the probe group is 91.7%, which exceeds the probe group positive rate threshold of 70% for judging the strain detection, indicating that the strain Aphanizomenon flos-aquae FACHB-1416 The probe group signals of the other strains that were not added were relatively low, lower than our probe positive threshold and probe group positive rate threshold.

[0092] In Experiment 2, we only added Aphanizomenon flos-aquae MDT14a The results showed that the fluorescence signal values ​​of all probes in the probe set of the target strain exceeded 300, that is, all probes were detected, and the probe detection rate was 100%, indicating that the strain was successfully detected, while the probe signals of the other 7 unadded Aphanizomenon probe sets were all below 300, and none of them passed the probe set positive rate threshold. In addition, the false positive and false negative rates of this experiment were both 0.

[0093] The results confirmed that the method of the present invention has a reliable detection effect at the level of Aphanizomenon strains.

[0094] Example 4 Detection and analysis of Aphanizomenon flos-aquae in water samples from the wild environment Five water samples from the wild environment were selected: sample_1-sample_5, and the Aphanizomenon bloom-water detection chip of the present invention was used to investigate and analyze the Aphanizomenon bloom-water in the samples according to the method of Example 2.

[0095] The survey and analysis results are as follows Figure 3 shown.

[0096] The results showed that the most strains of Aphanizomenon bloom-aquae were detected in sample_2, followed by sample_3, while sample_4 had the least number of Aphanizomenon bloom-aquae. Overall, the detection results of the five samples showed that the probe designed in the present invention could detect all eight common Aphanizomenon bloom-aquae.

[0097] The results confirmed that the present invention can successfully detect the strain types of Aphanizomenon bloom-aquae contained in water bodies in practical applications. When these eight strains of Aphanizomenon bloom-aquae exist simultaneously in a certain water body, they can be detected simultaneously by using the DNA chip of the present invention.

[0098] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A probe combination for detecting Aphanizomenon flos-aquae, characterized in that: The probe combination includes detection Aphanizomenon flos-aquae The probe set of 2012 / KM1 / D3, the sequences are shown in SED ID NOs. 1 to 12; Detection Aphanizomenon flos-aquae The probe set for FACHB-1040, the sequences are shown in SED ID NOs. 13 to 25; detection Aphanizomenon flos-aquae The probe set for FACHB-1290, the sequences are shown in SED ID NOs. 26 to 37; Aphanizomenon flos-aquae The probe set of FACHB-1416, the sequences are shown in SED ID NOs. 38 to 50; detection Aphanizomenon flos-aquae The probe set of LD13, the sequences are shown in SED ID NOs. 51 to 62; Detection Aphanizomenon flos-aquae The probe set for MDT14a, the sequences are shown in SED ID NOs. 63 to 74; detection Aphanizomenon flos-aquae The probe set of NIES-81, the sequences are shown in SED ID NOs. 75 to 87; detection Aphanizomenon flos-aquae The probe set of WA102, the sequences are shown in SED ID NOs. 88 to 99.

2. Use of the probe combination in preparing a gene chip or a kit for detecting Aphanizomenon flos-aquae, characterized in that: The algae blooms Aphanizomenon flos-aquae 2012 / KM1 / D3, Aphanizomenonflos- aquae FACHB-1040, Aphanizomenon flos-aquae FACHB-1290, Aphanizomenon flos-aquae FACHB-1416, Aphanizomenon flos-aquae LD13, Aphanizomenon flos-aquae MDT14a, Aphanizomenon flos-aquae NIES-81 and Aphanizomenon flos-aquae At least one of WA102 is a target detection of Aphanizomenon, and the probe combination includes all probes in the probe group of the target detection of Aphanizomenon.

3. A gene chip for detecting Aphanizomenon flos-aquae, characterized in that: The probe combination according to claim 1.

4. A kit for detecting Aphanizomenon flos-aquae, characterized in that: The kit comprises the probe combination according to claim 2 or the gene chip according to claim 3.

5. A kit for detecting Aphanizomenon bloom-aquae according to claim 4, characterized in that: The kit also includes a DNA extraction reagent for the sample to be tested, a DNA purification reagent and a DNA fluorescent labeling reagent.

6. A method for detecting Aphanizomenon flos-aquae, characterized in that: The steps include: (1) Filter the water sample to be tested, collect the filtered particles and complete DNA extraction; the extracted DNA is sheared and purified to obtain gDNA; (2) diluting the gDNA obtained in step (1) to a fixed concentration, performing PCR using random primers and ATCG bases with fluorescent groups to obtain gDNA sequences with fluorescent groups; (3) performing a hybridization experiment on the fluorescently labeled gDNA obtained in (2) and the chip according to claim 3 in a hybridization oven; (4) Scanning and imaging the hybridization experiment results of (3), and judging the detection results of the designed Aphanizomenon according to the fluorescence signal intensity of the strain-specific probe group and the proportion of the dominant fluorescent probe to all probes; wherein the Aphanizomenon bloom-water algae includes Aphanizomenon flos-aquae 2012 / KM1 / D3, Aphanizomenonflos-aquae FACHB-1040, Aphanizomenon flos-aquae FACHB-1290, Aphanizomenon flos-aquae FACHB-1416, Aphanizomenon flos-aquae LD13, Aphanizomenon flos-aquae MDT14a, Aphanizomenon flos-aquae NIES-81 and Aphanizomenon flos-aquae WA102.

7. The method according to claim 6, characterized in that: The method for determining the detection result is that when the detected probe fluorescence signal intensity is greater than the probe positive threshold of 300, it indicates that the probe is detected; when more than 70% of the probes in the probe group of the strain are detected, it indicates that the strain is detected.

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