A probe combination, gene chip, kit and method for detecting Aphanizomenon bloom-aquae

Abstract

The present invention discloses a probe combination for detecting Aphanizomenon bloom-aquae, comprising a sequence as shown in SED ID No. 1-99. Also disclosed are a gene chip, a kit, and a detection method prepared using the probe combination. This method utilizes probes from across the entire genome and hybridizes on a gene chip. By interpreting the hybridization signals from the probe set, the presence of eight Aphanizomenon bloom-aquae species can be determined, enabling detection of all eight Aphanizomenon bloom-aquae species. Compared to traditional detection methods, this method offers the advantages of high resolution, simple operation, convenient data processing, and high accuracy. This detection method has significant application value for the long-term dynamic monitoring of water quality deterioration, providing strong support for guiding algal bloom control and aquatic ecological restoration.

Description

Technical Field

[0001] The present 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 bloom-aquae. Background Art

[0002] Aphanizomenon Aphanizomenon ) is a freshwater floating filamentous algae belonging to the Nostoc order of the Cyanobacteria phylum. Their most obvious feature is that the terminal cells of the filaments taper or become hair-like, extending into colorless cells. Aphanizomenon can form blooms in freshwater bodies, also known as cyanobacterial blooms, which have caused many environmental problems. In certain seasons of some lakes, Aphanizomenon blooms and Microcystis blooms will compete and alternate, forming a unique cyanobacterial bloom phenomenon. Some species of Aphanizomenon may produce toxins, such as Aphanizomenon blooms ( A. water-flower ), Aphanizomenon 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, species of Aphanizomenon include Aphanizomenon blooms-aquae, Aphanizomenon gracilis, and Aphanizomenon yissava, of which Aphanizomenon blooms-aquae is the most common. Aphanizomenon blooms-aquae is found worldwide, primarily in freshwater. Its morphological characteristics are characterized by straight or slightly curved filaments, either single or connected laterally in bundles. The cells in the middle of the filaments are short cylindrical, more or less square, and contain pseudovacuoles. The terminal cells become more or less tapering, elongating into colorless cells. The gelatinous sheath is indistinct. Heteromorphic cells are intercellular and vary in shape, including cylindrical, nearly spherical, and elliptical. Aphanizomenon blooms live in the water and can reproduce in large numbers. Under certain conditions, dense blooms can form, which can cause water quality problems and ecological impacts.

[0004] Currently, the identification and classification of Aphanizomenon primarily relies on traditional morphological analysis. However, due to the diverse and variable morphology of the genus Aphanizomenon and the lack of clear distinctions between genera like Anabaena, subtle differences in external morphology and internal physiological structures, as well as high phenotypic plasticity, are often difficult to distinguish and identify based on simple external morphology and internal physiological structures. This presents a significant challenge for the classification and identification of this genus. Conventional methods for detecting Aphanizomenon primarily include microscopic observation, biomass measurement, and pigment analysis. While these traditional methods have accumulated a wealth of experience and data in classification and identification, they also have significant limitations. For example, microscopic observation is time-consuming, requires specialized knowledge and operational experience, and is susceptible to subjective factors. Methods such as biomass measurement and pigment analysis can only provide general or indirect assessments, and their resolution is insufficient, making it impossible to specifically distinguish species or even strains, nor can they accurately quantify the presence of cyanobacteria. Molecular biological methods have also been applied to the study of Aphanizomenon, but research has tended to focus on the detection and identification of cyanobacterial toxins and toxin-producing genes. For example, high-performance liquid chromatography can be used to determine the composition and content of Aphanizomenon toxins, or polymerase chain reaction (PCR) can be used to amplify specific gene fragments of microcystins. When assessing the extent of water pollution, the quantitative and qualitative analysis of algae and the toxins they produce are equally important. As Aphanizomenon is the primary microalgae responsible for cyanobacterial blooms, developing faster and more accurate qualitative and quantitative methods is essential. 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:

[0007] The first aspect of the present invention provides a probe combination for detecting Aphanizomenon flos-aquae, wherein the probe combination comprises a probe for detecting Aphanizomenon water-flower The probe set for 2012 / KM1 / D3, the sequences are shown in SED ID NO. 1 to 12; Aphanizomenon water-flower The probe set for FACHB-1040, the sequences are shown in SED ID NOs. 13 to 25; 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 for FACHB-1416, the sequences of which are shown in SED ID NOs. 38 to 50; Aphanizomenon water-flower The probe set for LD13, the sequences are shown in SED ID NOs. 51 to 62; Aphanizomenon water-flower The probe set for MDT14a, the sequences are shown in SED ID NOs. 63 to 74; Aphanizomenon water-flower The probe set for NIES-81, the sequences are shown in SED ID NOs. 75 to 87; Aphanizomenon water-flower The probe set of WA102, the sequences are shown in SED ID NOs. 88 to 99.

[0008] 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.

[0009] In some embodiments of the present invention, the probe combination is a mixture of multiple probe sets. In some embodiments, the probe combination includes probe sets corresponding to detection of one, two, three, or eight detection targets. In a preferred embodiment, the probe combination includes probes for all eight detection targets and contains all 99 probes corresponding to detection of Aphanizomenon bloom-aquae, enabling simultaneous detection of each of the aforementioned Aphanizomenon bloom-aquae. These probes are specific probes that bind only to the target Aphanizomenon bloom-aquae DNA sequence and not to any other Aphanizomenon bloom-aquae DNA sequence. Probe design steps include preliminary screening for specific probes, sensitivity screening of oligonucleotide fragments, and screening for consistency in physical and chemical properties.

[0010] The present invention also provides the use of the probe combination of the first aspect in preparing a gene chip or a kit for detecting Aphanizomenon blooms-aquae, wherein the detection targets of Aphanizomenon blooms-aquae include Aphanizomenon flos- water 2012 / KM1 / D3, Aphanizomenonflos-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. The detection targets may include one, multiple or all of them, and the corresponding probe combination includes all probes in the probe group corresponding to the detection target.

[0011] 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 of all probes shown in SED ID NO. 38 to 50; when the target 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 the sequences shown by SEDID NOs. 1 to 12, the sequences shown by SED ID NOs. 26 to 37, and the sequences shown by SED ID NOs. 88 to 99.

[0012] The detection results of the designed Aphanizomenon were judged based on the fluorescence signal intensity of the strain-specific probe group and the proportion of the dominant fluorescent probe to all probes.

[0013] 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.

[0014] A third aspect of the present invention provides a kit for detecting Aphanizomenon bloom-aquae, the kit comprising the probe combination described in the first aspect or the gene chip described in the second aspect.

[0015] 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.

[0016] A fourth aspect of the present invention provides a method for detecting Aphanizomenon bloom-aquae, comprising the following steps:

[0017] (1) The water sample to be tested is filtered, the particulate matter obtained by filtration is collected and DNA is extracted; the extracted DNA is sheared and purified to obtain gDNA;

[0018] (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;

[0019] (3) performing a hybridization experiment on the fluorescently labeled gDNA obtained in (2) and the gene chip described in the second aspect in a hybridization oven;

[0020] (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 blooms include 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.

[0021] 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.

[0022] 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.

[0023] 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 emits light of a specific wavelength, which is captured by the scanner. Finally, the image data obtained by the scan is converted into a digital signal.

[0024] Furthermore, 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 comparability between them. In order to reduce false positives, the probe detection rate of each species also needed 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.

[0025] 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.

[0026] Beneficial effects of the present invention

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] Currently, the identification and classification of Aphanizomenon remains reliant on morphological detection methods. However, due to the diverse and variable morphology of the genus Aphanizomenon and the lack of clear distinctions between it and other cyanobacteria, such as Anabaena, differentiation and identification based on simple external morphology and internal physiological structures is difficult. This invention establishes a genome-wide, specific probe-based detection method for Aphanizomenon in water, filling a gap in related detection technology.

[0029] 2. The present invention utilizes gene chip technology to design genome-wide species / strain-specific probes, which can simultaneously detect Aphanizomenon bloom-aquae strains in eight water bodies, with higher resolution and sensitivity than other PCR methods.

[0030] 3. The present invention has the advantage of simple operation. Compared with sequencing-based detection methods, it omits the steps of PCR and sequencing of the target area, and 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

[0031] Figure 1 A schematic diagram of the gene chip format of Example 1 of the present invention is shown;

[0032] Figure 2 The figures show the results of two groups of specific experimental tests in Example 3 of the present invention;

[0033] Figure 3 The figure shows the detection results of water samples from 5 wild environments in Example 4 of the present invention. DETAILED DESCRIPTION

[0034] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0036] The technical solution of the present application will be further described in detail below in conjunction with specific implementation methods.

[0037] Example 1 Screening of specific probes for Aphanizomenon flos-aquae and preparation of chip

[0038] 1. Dataset collection:

[0039] To screen for probes specific for Aphanizomenon blooms-aquae strains, the data required primarily consisted of two parts: the target strain genome sequence and the background genome sequence. Here, the genomes of eight Aphanizomenon blooms-aquae strains were used as target sequences, while the genome sequences of all other cyanobacteria collected from public databases were used as the background sequence. The genomic information used to design Aphanizomenon blooms-aquae strains is shown in Table 1.

[0040] Table 1 Genomic information used to design Aphanizomenon flos-aquae strains

[0041]

[0042] 2. Preliminary selection of specific probes for Aphanizomenon blooms-aquae

[0043] The target Aphanizomenon genome sequence was completely broken into 50-mer Kmer fragments. A hash library of the background sequence Kmer fragments was created, recording the Kmer occurrence frequency and strain information. A hash library of the genome sequence Kmer fragments was then created. The target sequence Kmer library was compared with the background sequence Kmer library, and Kmers that were present only in the target sequence Kmer library but not in the background sequence Kmer library were selected as candidate specific probe libraries for the strain.

[0044] 3. Elimination of potential non-specific binding probes from candidate specific probes

[0045] Studies have shown that potential nonspecific binding may occur if a probe has more than 20 consecutive base matches with a non-target sequence. Using the NCBI Blast program, candidate specific probes were aligned with a background Kmer library. Based on the alignment results, candidate specific probes with more than 20 consecutive base matches with background Kmers were removed.

[0046] 4. Screening of probe physical and chemical properties

[0047] The strain-specific probes obtained after the previous step. These probe sequences uniquely exist in the genome of this strain and have no more than 20 consecutive base matches with the genomes of other species. Subsequently, physical and chemical property screening was performed on the remaining specific probes. The main conditions include:

[0048] (1) The nucleic acid free energy (Free energy, unit: kcal / mol) of the probe sequence and the target sequence. If the nucleic acid free energy is less than -30, then this probe sequence is removed;

[0049] (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;

[0050] (3) Final screening of the strain-specific probes was carried out according to the melting temperature Tm value (65 < Tm < 95) and GC content (0.2 < GC content < 0.8).

[0051] Finally, 99 strain-specific probes of Aphanizomenon flos-aquae distributed throughout the genome were obtained. 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.

[0052] Table 2 Strain-specific probes of Aphanizomenon flos-aquae

[0053]

[0054] 5. Chip preparation

[0055] Using a conventional chip preparation method, the above-mentioned specific probes were synthesized by a commercial company and integrated into a gene chip, and this gene chip has the function of specifically detecting Aphanizomenon flos-aquae. The style of the gene chip is as Figure 1 shown.

[0056] Example 2 Method for rapidly detecting Aphanizomenon flos-aquae in water by using the chip of the present invention

[0057] 1. Collection of water body algal microorganisms

[0058] Preparation before sampling:

[0059] (1) Select a suitable ultrafiltration membrane filter with a pore size of 0.22 - 0.45 micrometers. <00001​​​​​​​​​(1) Determine the sampling location based on the research purpose and relevant water sources.

[0064] (2) Determine the volume of water sample to be collected based on the analysis requirements and microbial load.

[0065] (3) Start the water flow and adjust the filter equipment to a controlled flow rate, typically 100-500 ml / min.

[0066] (4) Pass the water sample through an ultrafiltration membrane filter to ensure that all the water sample is filtered.

[0067] (5) Collect the filtrate containing smaller particles and molecules into a sterile container for subsequent analysis or processing.

[0068] (6) Disconnect the filter holder and carefully transfer the microbial cells retained on the filter membrane into a sterile collection bottle or tube.

[0069] Sample processing:

[0070] (1) Flush the filter holder and tubing with sterile water or buffer to recover any remaining microbial cells in the system.

[0071] (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.

[0072] (3) Ensure that the filter membrane is completely immersed in the preservation solution to maintain the viability of microbial cells during transportation and storage.

[0073] (4) Label each container or tube with a unique identifier that includes the sampling location, date, and other relevant information.

[0074] Transportation and storage:

[0075] (1) Place the collected microbial samples in an insulated cooler or ice pack for transportation to maintain the appropriate temperature.

[0076] (2) Minimize exposure to extreme temperatures, sunlight, and other environmental factors that may affect the viability or composition of microorganisms.

[0077] (3) Transport samples to the laboratory as quickly as possible, preferably within 24–48 hours, to avoid changes in the microbial community.

[0078] (4) Upon arrival at the laboratory, store the sample at the temperature required for analysis, such as refrigeration or freezing, to maintain the viability of the microorganisms.

[0079] Sample preparation:

[0080] (1) Use sterile sampling tubes or containers to collect environmental samples.

[0081] (2) If necessary, homogenize solid samples using a sterile mortar and pestle or other appropriate method.

[0082] (3) Alternatively, samples can be snap-frozen in liquid nitrogen and stored at -80°C until further processing.

[0083] 2. DNA Extraction

[0084] (1) Wear sterile gloves and ensure the operating area is clean.

[0085] (2) Add an appropriate amount of lysis buffer to the sample tube according to the sample size and manufacturer's instructions to ensure that the sample is completely suspended.

[0086] (3) Add proteinase K and SDS to the sample tube at the recommended concentrations. Mix gently by inverting the sample tube several times.

[0087] (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.

[0088] (5) Perform a phenol:chloroform:isoamyl alcohol extraction by adding equal volumes of a phenol:chloroform:isoamyl alcohol mixture to the sample tube. Mix thoroughly by inverting the sample tube several times.

[0089] (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.

[0090] (7) Carefully transfer the aqueous phase to a new tube, avoiding disturbing the interface or the organic phase.

[0091] (8) Perform a chloroform:isoamyl alcohol extraction by adding an equal volume of chloroform:isoamyl alcohol mixture to a new tube. Mix thoroughly by inverting the sample tube several times.

[0092] (9) Centrifuge the tube at an appropriate speed and time to separate the aqueous phase.

[0093] (10) Transfer the aqueous phase to a new tube, leaving behind any interfacial contaminants.

[0094] (11) Precipitate the DNA by adding an equal volume of isopropanol. Mix gently by inverting the tube.

[0095] (12) Incubate at -20°C or -80°C for at least 1 hour to precipitate the DNA.

[0096] (13) Centrifuge the tube at an appropriate speed and time to precipitate the DNA.

[0097] (14) Discard the supernatant and wash the DNA pellet with 70% ethanol to remove residual contaminants.

[0098] (15) Air-dry the DNA pellet or gently blow dry any remaining ethanol using nitrogen gas.

[0099] (16) Dissolve the DNA pellet in sterile water or buffer. Mix gently to ensure complete dissolution.

[0100] (17) Optionally, measure the concentration and purity of DNA using a DNA quantification kit.

[0101] 3. DNA magnetic bead purification

[0102] The main steps of DNA purification are:

[0103] (1) Equilibrate OnePure MagBeads at room temperature for 30 minutes and vortex thoroughly to ensure there is no obvious precipitation of magnetic beads;

[0104] (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 it stand at room temperature for 5 minutes;

[0105] (3) Place the PCR tube / eight-tube strip on a magnetic rack, wait for the solution in the tube to clear, and discard the supernatant;

[0106] (4) Add 200 μL of 80% freshly prepared ethanol to the PCR tube / eight-tube strip, let it stand for 30 seconds, then discard the supernatant and repeat the steps until the supernatant is removed relatively cleanly;

[0107] (5) Place the PCR tube / eight-tube strip on a magnetic rack and let it stand at room temperature for 1 to 2 minutes until the magnetic beads are dry and cracked, 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;

[0108] (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 thoroughly, then quickly collect the liquid from the tube wall and place at room temperature for 3 minutes.

[0109] (7) Place the PCR tube / eight-tube strip on a magnetic rack, wait for the solution in the tube to clarify, and transfer 42 μL of the supernatant to a new EP tube for labeling in the next step.

[0110] 3. DNA fluorescent labeling

[0111] This example uses the Agilent SureTag Complete DNA Labeling Kit, which includes the following steps:

[0112] (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℃;

[0113] (2) When the time is up, immediately place the sample on ice to cool;

[0114] (3) After the sample was centrifuged, the following reagents were added directly: 1 μL sterile water, 5.5 μL 5× Reaction buffer, 2.75 μL 10× dNTP mix, 0.25 μL Cyanine 3-dUTP, and 0.5 μL Exo(-)Klenow, for a total of 27.5 μL;

[0115] (4) Use a pipette to blow or vortex to mix, then quickly centrifuge to collect the liquid on the tube wall to remove bubbles;

[0116] (5) Place the reaction system on a PCR instrument, set the heated lid temperature to 105°C, and run the following program:

[0117] 37℃ for 4 hours, 95℃ for 3 minutes, hold at 4℃.

[0118] 4. Purification of fluorescently labeled DNA products

[0119] This example uses the Agilent Oligo aCGH / ChIP-on-chip Hybridization Kit, which includes the following steps:

[0120] (1) Centrifuge the labeled product to confirm column sorting. Add 125 μL of 1× TE to the sample three times each time to rinse the labeled tube and transfer it to the purification column. Centrifuge at 14,000 × g for 10 min.

[0121] (2) Discard the filtrate, place the collection tube back into the collection column, add 480 μL of 1×TE (pH 8.0) to the collection column, cover the lid, and centrifuge at 14,000 × g for 10 min;

[0122] (3) Remove the collection column and invert it into a new 2 mL centrifuge tube. Mark the tube accordingly and centrifuge at 1000 × g for 1 min. The purified sample (samples from the same hybridization area, purified and recovered in the same collection tube) is obtained (volume is approximately 40-64 μL). Transfer the purified product into a PCR tube.

[0123] (4) Determine the total nucleic acid concentration and the corresponding dye labeling concentration using Nanodrop one;

[0124] (5) Use a concentrator to dry the sample to a volume of 10 μL.

[0125] 5. Hybridization of target sample fluorescent DNA with Aphanizomenon flos-aquae microarray

[0126] This example uses the Agilent Oligo aCGH / ChIP-on-chip Hybridization Kit, which includes the following steps:

[0127] (1) Configuring a hybrid system

[0128] The chip hybridization system is shown in Table 3. After preparation, 45 μL of hybridization system was added to the above sample concentrated to 10 μL and mixed by pipetting. After a short spin, the reaction system was placed on a PCR instrument with the heated lid set to 105°C and the following program was run: 98°C for 3 min, 37°C for 30 min, and 37°C hold.

[0129] Table 3 Chip hybridization system

[0130]

[0131] (2) Hybridization

[0132] 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.

[0133] b. Then, pipette 47 μL of the 37°C sample from the previous step into the center of the rubber ring on the gasket, avoiding bubbles. Place the chip upside down on the gasket.

[0134] c. Then close the chamber lid and tighten the knob;

[0135] d. Place each assembled device into the incubator rotation rack. Take a balanced chamber and rotate the hybridization chamber vertically to wet the slide and assess the mobility of the air bubble.

[0136] e. Set the hybridization rotator speed to 20 rpm and hybridize at 67°C for 22 hours.

[0137] (3) Chip cleaning

[0138] After hybridization, remove the chip from the microarray and place it in Wash Solution 1 (Agilent kit reagent) at room temperature, shaking and washing at 250 rpm for 5 minutes at room temperature. Then, use Wash Solution 2 (Agilent kit reagent) at 200 rpm at 39°C for 1 minute. Finally, remove the liquid from the chip surface and scan within 4 hours.

[0139] 6. Fluorescence result scanning and signal analysis

[0140] (1) Import the microarray data file into the microarray data preprocessing software.

[0141] (2) Verify the integrity of the data file and ensure that the file format is compatible with the preprocessing software.

[0142] (3) Perform quality control checks to assess the overall quality of the data and identify potential problems such as outliers and artifacts.

[0143] (4) Remove or mark low-quality or unreliable probes or spots from the data; for example, remove probes with signal intensity <100.

[0144] (5) Apply background correction methods to remove non-specific hybridization signals, such as RMA, mas5, and model-based background correction.

[0145] (6) Perform data normalization to adjust the systematic variation between arrays, such as quantile normalization and loess normalization.

[0146] (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.

[0147] Example 3 Detection specificity test of the chip for Aphanizomenon

[0148] Specific detection refers to the ability to specifically detect the presence of a target strain in a water sample. To test the specific detection performance of our probe and chip design, we used the method described in this paper to detect specific bacterial cultures within the detection range of Aphanizomenon. We compared the consistency of the test results with the experimental combination design and analyzed the false positive and false negative rates of the test results. The experiment set the probe positivity threshold at 300, meaning a fluorescence signal intensity greater than 300 indicates detection by the probe. The strain probe group positivity threshold was set at 70%, meaning a probe detection rate of 70% or higher indicates detection of the strain.

[0149] In this example, two groups of specific experiments were designed:

[0150] Specificity experiment 1: only contains Aphanizomenon flos-aquae FACHB-1416 A body of water containing Aphanizomenon flos-aquae.

[0151] Specificity experiment 2: only contains Aphanizomenon flos-aquae MDT14a A body of water containing Aphanizomenon flos-aquae.

[0152] Test results such as Figure 2 shown.

[0153] 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 the probe group has 11 probes 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 detection of strains, 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.

[0154] In Experiment 2, we only added Aphanizomenon flos-aquae MDT14a The results showed that all probe fluorescence signal values ​​for the target strain probe set exceeded 300, indicating that all probes were detected, with a probe detection rate of 100%, indicating that the strain was successfully detected. However, the probe signals of the other seven unadded Aphanizomenon probe sets were all below 300, failing to pass the probe set positivity threshold. Furthermore, the false positive and false negative rates for this experiment were both 0.

[0155] The results confirmed that the method of the present invention has a reliable detection effect at the level of Aphanizomenon strains.

[0156] Example 4 Detection and Analysis of Aphanizomenon Blooms in Water Samples from Wild Environments

[0157] Five water samples from the wild environment were selected: sample_1 to sample_5. The Aphanizomenon bloom-aquae detection chip of the present invention was used to investigate and analyze the Aphanizomenon bloom-aquae in the samples according to the method of Example 2.

[0158] The survey and analysis results are as follows Figure 3 shown.

[0159] 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 species 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 species of Aphanizomenon bloom-aquae.

[0160] The results confirmed that the present invention can successfully detect the strain types of Aphanizomenon blooms-aquae in water bodies in practical applications. When these eight strains of Aphanizomenon blooms-aquae exist simultaneously in a certain water body, they can be detected simultaneously using the DNA chip of the present invention.

[0161] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the present application.

Claims

1. A probe combination for detecting Aphanizomenon bloom-aquae, characterized by: The probe combination includes detection Aphanizomenon flos-aquae The probe set for 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; 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 for FACHB-1416, the sequences of which are shown in SED ID NOs. 38 to 50; Aphanizomenon flos-aquae The probe set for 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; Aphanizomenon flos-aquae The probe set for NIES-81, the sequences are shown in SED ID NOs. 75 to 87; 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 according to claim 1 in preparing a gene chip or a kit for detecting Aphanizomenon bloom-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 targets Aphanizomenon.

3. A gene chip for detecting Aphanizomenon bloom-aquae, characterized by: The method comprises the probe combination according to claim 1.

4. A kit for detecting Aphanizomenon bloom-aquae, characterized by: The kit comprises the probe combination according to claim 1 or the gene chip according to claim 3.

5. The 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 bloom-aquae, characterized in that: The steps include: (1) The water sample to be tested is filtered, the particulate matter obtained by filtration is collected and DNA is extracted; 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 blooms include 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, When more than 70% of the probes in the probe set of the strain were detected, it was indicated that the strain was detected.

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