A probe composition, gene chip, kit and method for detecting common eukaryotic algae

Abstract

The present invention discloses a probe composition, a gene chip, a kit and a method for detecting common eukaryotic algae, wherein the probe composition of common eukaryotic algae includes the probe shown in SEQ ID No.1-SEQ ID No.115, and also discloses a gene chip, a kit and a detection method prepared by using the above probe composition. The present invention uses probes within the whole genome range and adopts a gene chip for hybridization. By interpreting the hybridization signal of the probe group, it can be determined whether 12 common eukaryotic algae exist, and the 12 common eukaryotic algae are detected. Compared with traditional detection methods, it has the advantage of high resolution, and the detection method is simple to operate, simple and convenient to process data, and high in accuracy. The detection method has important application value for long-term dynamic monitoring of water quality deterioration in water bodies, and provides strong support for guiding algae bloom control and water ecological restoration.

Description

Technical Field

[0001] The invention relates to a biochip and a microorganism detection method, and in particular to a probe composition, a gene chip, a kit and a method for detecting common eukaryotic algae. Background Art

[0002] Eukaryotic algae are a group of low-level autotrophic eukaryotic plants that can photosynthesize without differentiated roots, stems, and leaves. They appeared about 1.5 billion to 1.4 billion years ago. The forms include single cells, various groups, filaments, thallus, tubular bodies, etc. The size ranges from a few microns to several meters (kelp), and even hundreds of meters (macroalgae). The structure is simple and there is no obvious tissue differentiation. Eukaryotic algae can be divided into Chlorophyta, Charophyta, Euglenophyta, Bacillariophyta, etc. Among them, Chlorophyta and Bacillariophyta are two important algae groups. They are widely distributed in water bodies around the world and are sensitive to environmental changes and human activities.

[0003] Chlorophyta includes a large and diverse group of unicellular and multicellular eukaryotic algae that contain chlorophyll and steroids in their cells, enabling them to photosynthesize. These algae are widely distributed in different aquatic environments, including lakes, rivers, reservoirs, and oceans, through various lifestyles, such as planktonic, attached, and free-floating. Due to their different requirements for light and nutrients, Chlorophyta plays an important role in the ecosystem function of water bodies, affecting aquatic oxygen production, carbon cycle, and food supply chain for benthic organisms. In addition, the species diversity and distribution pattern of Chlorophyta are of great significance to the health of water bodies and the maintenance of biodiversity. For example, some green algae can form large-scale algal blooms, which not only affect the ecosystem, but may also pose a threat to the availability of water resources and human health.

[0004] The diatoms include the subphylum Bacillariophyta and the subphylum Chrysophyta. They contain siliceous shells in their cells and are widely distributed in water bodies. Their lifestyles include planktonic, benthic and epiphytic. Due to the high adaptability and diversity of their silicon-rich cell shells, diatoms can survive and reproduce widely in different water environments. In ecosystem functions, diatoms play an important role in maintaining the ecological balance and biodiversity of water bodies through photosynthesis and silicon cycling. Their biomass and community structure have a significant impact on water transparency, oxygen production and the food chain of benthic organisms. In addition, the growth of diatoms is closely related to environmental factors such as temperature, light and nutrients, which poses a challenge to the monitoring and management of water quality.

[0005] It is known that some eukaryotic algae have biological and environmental value: for example, Chlorella can absorb carbon dioxide and nutrients such as nitrogen and phosphorus in water through photosynthesis, and release oxygen, thereby reducing the concentration of organic and inorganic substances in water, reducing the accumulation of pollutants, increasing the dissolved oxygen content, and maintaining the acid-base balance of the water body. In this way, a clean, comfortable and healthy living environment can be created for aquatic animals. However, some algae blooms frequently break out, causing environmental pollution. Real-time monitoring and rapid identification of algae species that cause outbreaks are still an outstanding problem. Accurate identification of algae species is very important for assessing the hazards of algal blooms and formulating prevention and control measures.

[0006] The existing identification and classification methods of eukaryotic algae species are mainly based on traditional morphological detection. However, due to the simple external morphology and subtle differences in internal physiological structure and high phenotypic plasticity, some algae are difficult to distinguish and identify, which poses a huge challenge to the development of algal taxonomy. Traditional methods for detecting eukaryotic algae mainly include microscopic observation, biomass measurement and pigment analysis. Although traditional methods have accumulated a lot of experience and data in the classification and identification of eukaryotic algae, they also have obvious limitations. For example, the method of observation with a microscope is not only time-consuming and requires professional knowledge and operating experience, but also the eukaryotic algae in environmental samples are of various species, the proportion of some species is extremely low, and the accuracy is easily affected by subjective factors. Methods such as biomass measurement and pigment analysis can only give more general or indirect evaluation results.

[0007] With the development of molecular biology technology, polymerase chain reaction (PCR) and high-throughput sequencing (NGS) technologies are also often used in the detection and identification of eukaryotic algae. For example, 16S rRNA sequencing technology uses specific primers to target and amplify the conserved regions of 16S rRNA in the genomes of various species of eukaryotic algae. It can quantify environmental samples at the genus level, but cannot identify them to the species level. Other technical methods such as fluorescence in situ hybridization (FISH) and enzyme-linked immunosorbent assay (ELISA) are also unable to quickly and accurately identify various eukaryotic algae in environmental samples to the species level.

[0008] Gene chip technology is a high-throughput, high-sensitivity nucleic acid detection technology. Its basic principle is to fix a large number of specific nucleic acid probes on a tiny solid surface, and detect and analyze the genetic information in the sample by hybridizing with the target nucleic acid sequence in the sample. Gene chip technology can detect hundreds to thousands of genes simultaneously in a single experiment, and has shown great application potential in the detection of eukaryotic algae in aquatic bodies. Applying gene chip technology to the field of eukaryotic algae detection in aquatic bodies may provide us with a novel and efficient method to more comprehensively and rapidly understand the distribution and population dynamics of eukaryotic algae in water bodies. This application is expected to further promote the development of aquatic ecosystem monitoring technology and provide strong support for water quality evaluation, eutrophication warning and ecological environmental protection. Summary of the invention

[0009] In order to solve one of the above problems, the present invention provides a probe composition, a gene chip, a kit and a method for detecting common eukaryotic algae.

[0010] In order to achieve the above object, the present invention adopts the following technical means:

[0011] The first aspect of the present invention provides a probe composition for detecting common eukaryotic algae, the probe composition comprising a detection Chlorella vulgarisA probe group for detecting Scenedesmus sp. NREL 46B-D3, wherein 11 probe sequences in the probe group are shown as SEQ ID No.1 to SEQ ID No.11, a probe group for detecting Scenedesmus sp. NREL 46B-D3, wherein 8 probe sequences in the probe group are shown as SEQ ID No.12 to SEQ ID No.19, a probe group for detecting Amphora coffeaeformis, wherein 9 probe sequences in the probe group are shown as SEQ ID No.20 to SEQ ID No.28, a probe group for detecting Ankistrodesmus sp. CCAC 3332 B, wherein 10 probe sequences in the probe group are shown as SEQ ID No.29 to SEQ ID No.38, a probe group for detecting Coelastrummicroporum, wherein 10 probe sequences in the probe group are shown as SEQ ID No.39 to SEQ ID No.48, a probe group for detecting Pediastrum duplex, wherein 10 probe sequences in the probe group are shown as SEQ ID No.49 to SEQ IDNo.58, a probe group for detecting Tetraedron minutum, wherein 10 probe sequences in the probe group are shown as SEQ The probe group for detecting Nitzschia inconspicua is shown in SEQ ID No.59 to SEQ ID No.68, the 9 probe sequences in the probe group are shown in SEQ ID No.69 to SEQ ID No.77, the probe group for detecting Synedra sp. RCC2510, the 9 probe sequences in the probe group are shown in SEQ ID No.78 to SEQ ID No.86, the probe group for detecting Cyclotella atomus, the 10 probe sequences in the probe group are shown in SEQ ID No.87 to SEQ ID No.96, the probe group for detecting Achnanthes kuwaitensis, the 10 probe sequences in the probe group are shown in SEQ ID No.97 to SEQ ID No.106, the probe group for detecting Navicula sp. RCC3092, the 9 probe sequences in the probe group are shown in SEQ ID No.107 to SEQ ID No.115.

[0012] In some embodiments, the detection targets of common eukaryotic algae include Chlorella vulgaris, Scenedesmus sp. NREL 46B-D3 、Amphora coffeaeformis、Ankistrodesmus sp. CCAC3332B 、Coelastrum microporum、Pediastrum duplex、Tetahedron minutum、Nitzschia inconspicua, Synedra sp. RCC2510 、Cyclotella atomus、Achnanthes kuwaitensis、 Navicula sp.There are probes for 12 detection targets such as RCC3092, and each detection target probe contains a probe group consisting of 8-10 specific detection probes.

[0013] In some specific embodiments of the present invention, the probe composition is a mixture of multiple probe groups; in some specific embodiments, the probe composition includes probe groups corresponding to the detection of 1, 2, 3...12 detection targets; in a preferred specific embodiment, the probe composition includes probes for all 12 detection targets, and contains all 115 probes corresponding to the detection probes for common eukaryotic algae, and can detect the above eukaryotic algae simultaneously. The probe is a specific probe that only binds to the target eukaryotic algae DNA sequence and does not bind to any other eukaryotic algae DNA sequence; the probe design step includes preliminary screening of specific probes, sensitivity screening of oligonucleotide fragments, and consistency screening of physical and chemical properties.

[0014] The present invention also provides the use of the probe composition described in the first aspect in preparing a gene chip or a kit for detecting common eukaryotic algae, wherein the detection targets of the common eukaryotic algae include Chlorella vulgaris, Scenedesmus sp. NREL 46B-D3 、Amphora coffeaeformis、Ankistrodesmus sp. CCAC3332B 、Coelastrum microporum、Pediastrum duplex、Tetahedron minutum、Nitzschia inconspicua, Synedra sp. RCC2510 、Cyclotella atomus、Achnanthes kuwaitensis、 Navicula sp. There are 12 detection targets such as RCC3092, and the detection target can include one, multiple or all of them.

[0015] The second aspect of the present invention provides a gene chip for detecting common eukaryotic algae, comprising the probe combination described above.

[0016] The third aspect of the present invention provides a kit for detecting common eukaryotic algae, wherein the kit comprises the probe composition described in the first aspect or the gene chip described in the first aspect.

[0017] In some embodiments, the kit further comprises a DNA extraction reagent for the sample to be tested, a DNA purification reagent, and a DNA fluorescent labeling reagent.

[0018] A fourth aspect of the present invention provides a method for detecting common eukaryotic algae, comprising the following steps:

[0019] (1) Filter the water sample to be tested, collect the filtered particles and complete DNA extraction;

[0020] (2) The extracted DNA is sheared and purified to obtain gDNA;

[0021] (3) Performing PCR on the gDNA obtained in step (2) using random primers and ATCG bases with fluorescent groups to obtain a gDNA sequence with fluorescent groups;

[0022] (4) performing a hybridization experiment on the fluorescently labeled gDNA obtained in (3) and the gene chip described above in a hybridization oven;

[0023] (5) reading the results of the hybridization experiment in (4) and determining the detection results of common eukaryotic algae based on the read probe signals;

[0024] Among them, the detection targets of common eukaryotic algae include Chlorella vulgaris、Scenedesmus sp. NREL 46B-D3 、Amphora coffeaeformis、Ankistrodesmus sp. CCAC 3332B 、Coelastrum microporum, Pediastrum duplex, Tetrahedron minutum, Nitzschia inconspicua, Synedra sp. RCC2510 ,Cyclotella atomus,Achnanthes kuwaitensis,Navicula sp. RCC3092.

[0025] In some specific embodiments of the present invention, the DNA extraction in step (1), the fragmentation and purification in step (2), the fluorescent labeling in step (3) and the hybridization in step (4) can be performed using conventional methods or kits in the art.

[0026] 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.

[0027] Further, the data signals are screened, and the detection of eukaryotic algae species is determined: the microarray data is preprocessed to eliminate systematic differences between samples and filter out false positive detections. First, the original signal intensity is corrected by subtracting the background intensity from the foreground intensity of the probe for background correction. Next, the probe signal intensity is normalized using the background correction data as the input matrix. The purpose is to adjust the overall signal intensity between different samples to ensure comparability between them. After normalization, undetected probes are identified and deleted. 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 and to reduce the false positive rate.

[0028] 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.

[0029] Beneficial Effects of the Invention

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] 1. Most of the existing molecular detection technologies for eukaryotic algae limit the detection target area to the 16S rRNA ribosomal gene. Since the target detection area is small, the resolution is low and it is impossible to identify the species and below. The present invention uses gene chip technology and species-specific probes in the whole genome to detect eukaryotic algae at the species level, making up for the defect of insufficient resolution of existing detection technologies.

[0032] 2. The present invention uses gene chip technology to simultaneously detect the presence of 12 common eukaryotic algae in water bodies. The detection method is simple to operate. 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 eukaryotic algae species detection method has important application value for the long-term dynamic monitoring of water quality deterioration, and can guide algal bloom control and water ecological restoration. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The following is a diagram showing the arrangement design of detection probes in a common eukaryotic algae detection gene chip of the present invention;

[0034] Figure 2 The results of the specificity experiment of the gene chip for detecting common eukaryotic algae in Example 3 of the present invention are shown;

[0035] Figure 3 The result display diagram of the application of common eukaryotic algae detection gene chip to detect common eukaryotic algae in three reservoirs in Shenzhen according to Example 4 of the present invention is shown, wherein the three water source reservoirs are: Shenzhen Reservoir (SZ), Sanzhoutian (SZT) and Tiegang Reservoir (TG). DETAILED DESCRIPTION

[0036] 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.

[0037] 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.

[0038] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.

[0039] Example 1 Design and screening of eukaryotic algae species-specific probes

[0040] The detection probe is an oligonucleotide fragment of a certain length that can bind to a specific target molecule and be detected by a certain detection method. The purpose of designing a specific detection probe is to find a probe that only binds to the DNA sequence of a given eukaryotic algae species and does not bind to the DNA sequence of any other species. The probe design method includes the following steps:

[0041] 1. Dataset collection:

[0042] In order to screen eukaryotic algae species-specific probes, the main data prepared include two parts, the target species genome sequence and the background genome sequence. Here we use the genomes of 12 eukaryotic algae as the target sequence, as shown in Table 1, and all eukaryotic algae genome sequences collected in the public database are set as the background sequence.

[0043] Table 1 Genomic information used to design eukaryotic species

[0044]

[0045] 2. Preliminary selection of species-specific probes:

[0046] All target eukaryotic algae genome sequences were broken into Kmer fragments of 50mer length, and a background sequence Kmer fragment hash library was established to record the frequency of occurrence of Kmer and the species information to which it belongs. Then a Kmer fragment hash library of the genome sequence was established. The target sequence Kmer library was compared and analyzed with the background sequence Kmer library, and the Kmers that only existed in the target sequence Kmer library but not in the background sequence Kmer library were selected as the alternative specific probe library for the species.

[0047] The initial screening of probes should also consider the following factors:

[0048] GC content: The GC content of the designed probes should be optimized, and it is necessary to ensure that the overall GC content of all designed probes tends to be consistent to guarantee the stability of hybridization. In addition, it is also necessary to consider whether the GC content of the target species is too high or too low, and select an appropriate GC content range accordingly.

[0049] Tm value: The Tm value of the designed probes should be set according to the experimental conditions, and it is necessary to ensure that the overall Tm value of all designed probes tends to be consistent to guarantee the stability of hybridization. Appropriate GC content and Tm value are helpful to improve the success rate of hybridization experiments.

[0050] Secondary structure: The probes should avoid containing palindromic sequences that can form stable secondary structures, otherwise it may affect the hybridization efficiency with the target sequence.

[0051] Specificity: The designed probes should specifically bind only to the target sequence and not to any other non-target DNA sequences.

[0052] 3. Elimination of potentially non-specific binding probes among alternative specific probes:

[0053] Some studies have pointed out that if a probe has more than 20 consecutive base matches with a non-target sequence, potential non-specific binding may occur. Using the Blast program of NCBI, align the sequences of alternative specific probes with the background Kmer library. According to the alignment results, eliminate the probes among the alternative specific probes that have more than 20 consecutive base matches with the background Kmer.

[0054] 4. Screening of probe physicochemical properties:

[0055] The species-specific probes obtained after the above step are unique to the genome of this species and have no more than 20 consecutive base matches with the genomes of other species. Subsequently, physicochemical property screening is carried out on the remaining specific probes, and the main conditions include:

[0056] (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 remove this probe sequence;

[0057] (2) If the same base appears continuously 5 times in the probe, then the complexity of this probe is too low, and remove this probe sequence;

[0058] (3) Finally screen the species-specific probes according to the melting temperature Tm value (65℃ < Tm < 95℃) and GC content (0.2 < GC content < 0.8). And select 8 to 11 probes respectively.

[0059] Finally, 115 eukaryotic algae species-specific probes distributed throughout the genome were screened. The probe sequences are shown in SEQ ID No. 1-SEQ ID No. 115. The probe sets, probe numbers, and specific sequence information corresponding to different eukaryotic algae species are shown in Table 2.

[0060] Table 2 Probe information of different eukaryotic algae species

[0061]

[0062] 5. Preparation of gene chips

[0063] The conventional gene chip preparation method is adopted to integrate the above-mentioned specific probes into a gene chip, and the gene chip has the eukaryotic algae-specific detection function.

[0064] The other types of probes in the gene chip are common built-in probes in general DNA chips and do not affect the detection content of this patent. The schematic diagram of the arrangement design of the detection probes in the gene chip is shown in FIG. Figure 1 shown.

[0065] Example 2 Method for rapidly detecting eukaryotic algae in water using the gene chip of the present invention

[0066] 1. Collection of eukaryotic algae microorganisms in water

[0067] Preparation before sampling:

[0068] (1) Select a suitable ultrafiltration membrane filter with a pore size of 0.22-0.45 μm.

[0069] (2) Ensure that the filtration equipment, including the filter holder and pipes, are clean and disinfected.

[0070] (3) Prepare sterile containers, tubes, and collection bottles for sampling.

[0071] Sampling collection:

[0072] (1) Determine the sampling location based on the research purpose and relevant water sources.

[0073] (2) Determine the volume of water samples to be collected based on the analysis requirements and microbial load.

[0074] (3) Start the water flow and adjust the filtration equipment to a controlled flow rate, typically 100-500 ml / min.

[0075] (4) Pass the water sample through an ultrafiltration membrane filter to ensure that all the water sample is filtered.

[0076] (5) Collect the filtrate containing smaller particles and molecules into a sterile container for subsequent analysis or processing.

[0077] (6) Disconnect the filter holder and carefully transfer the microbial cells retained on the filter membrane into a sterile collection bottle or tube.

[0078] Sample processing:

[0079] (1) Flush the filter holder and tubing with sterile water or buffer to recover any remaining microbial cells in the system.

[0080] (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.

[0081] (3) Ensure that the filter membrane is completely immersed in the preservation solution to maintain the viability of microbial cells during transportation and storage.

[0082] (4) Label each container or tube with a unique identifier that includes the sampling location, date, and other relevant information.

[0083] Transportation and storage:

[0084] (1) Place the collected microbial samples in an insulated cooler or ice packs for transportation to maintain the appropriate temperature.

[0085] (2) Minimize exposure to extreme temperatures, sunlight, and other environmental factors that may affect the viability or composition of microorganisms.

[0086] (3) Transport samples to the laboratory as quickly as possible, preferably within 24-48 hours, to avoid changes in the microbial flora.

[0087] (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.

[0088] Sample preparation:

[0089] (1) Use sterile sampling tubes or containers to collect environmental samples.

[0090] (2) If necessary, homogenize solid samples using a sterile mortar and pestle or other appropriate method.

[0091] (3) Alternatively, samples can be snap-frozen in liquid nitrogen and stored at -80°C until further processing.

[0092] 2. DNA Extraction

[0093] (1) Wear sterile gloves and ensure the operating area is clean.

[0094] (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.

[0095] (3) Add proteinase K and SDS to the sample tube at the recommended concentrations. Mix gently by inverting the sample tube several times.

[0096] (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.

[0097] (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.

[0098] (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.

[0099] (7) Carefully transfer the aqueous phase to a new tube, avoiding disturbing the interface or the organic phase.

[0100] (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.

[0101] (9) Centrifuge at an appropriate speed and time to separate the aqueous phase.

[0102] (10) Transfer the aqueous phase to a new tube, leaving behind any interfacial contaminants.

[0103] (11) Precipitate the DNA by adding an equal volume of isopropanol. Mix gently by inverting the tube.

[0104] (12) Incubate at -20°C or -80°C for at least 1 hour to precipitate the DNA.

[0105] (13) Centrifuge the tube at an appropriate speed and time to precipitate the DNA.

[0106] (14) Discard the supernatant and wash the DNA pellet with 70% ethanol to remove residual contaminants.

[0107] (15) Air-dry the DNA pellet or gently blow dry any residual ethanol using nitrogen gas.

[0108] (16) Dissolve the DNA pellet in sterile water or buffer. Mix gently to ensure complete dissolution.

[0109] (17) Optionally, measure the concentration and purity of DNA using a DNA quantification kit.

[0110] 3. DNA magnetic bead purification

[0111] The main steps of DNA purification are:

[0112] (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;

[0113] (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;

[0114] (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;

[0115] (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.

[0116] (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;

[0117] (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.

[0118] (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.

[0119] 3. DNA fluorescent labeling

[0120] This example uses the Agilent SureTag Complete DNA Labeling Kit, which includes the following steps:

[0121] (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℃;

[0122] (2) When the time is reached, immediately place the sample on ice to cool;

[0123] (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;

[0124] (4) Use a pipette to blow or vortex to mix, then quickly centrifuge to collect the liquid on the tube wall and remove bubbles;

[0125] (5) Place the reaction system on a PCR instrument, set the heated cover temperature to 105°C, and run the following program:

[0126] 37℃ for 4 hours, 95℃ for 3 minutes, hold at 4℃.

[0127] 4. Purification of fluorescently labeled DNA products

[0128] This example uses the Agilent Oligo aCGH / ChIP-on-chip Hybridization Kit, which includes the following steps:

[0129] (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;

[0130] (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;

[0131] (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;

[0132] (4) Use Nanodrop one to measure the total nucleic acid concentration and the corresponding dye labeling concentration;

[0133] (5) Use a concentrator to dry the sample to a volume of 10 μL.

[0134] 5. Hybridization of target sample fluorescent DNA with eukaryotic algae chip

[0135] This example uses the Agilent Oligo aCGH / ChIP-on-chip Hybridization Kit, which includes the following steps:

[0136] (1) After preparing the hybridization system according to Table 1, add 45 μL of the hybridization system to the sample concentrated to 10 μL and mix well with a pipette. After centrifugation, place the reaction system on a PCR instrument, set the temperature of the heated cover to 105°C, and run the following program: 98°C for 3 min, 37°C for 30 min, and 37°C hold;

[0137] Table 3 Chip hybridization system

[0138]

[0139] (2) Hybridization:

[0140] 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;

[0141] b. Then pipette 47 μL of the sample at 37℃ in the previous step into the middle of the rubber ring on the gasket to avoid bubbles, and then turn the chip upside down on the gasket; c. Then cover the chamber lid and tighten the knob;

[0142] d. Place each assembled device into the incubator rotating rack, take a balanced chamber, rotate the hybridization chamber vertically to wet the slide, and evaluate the fluidity of the bubble;

[0143] e. Set the hybridization rotator speed to 20 rpm and hybridize at 67°C for 22 hours.

[0144] (3) Chip cleaning After hybridization, remove the chip at room temperature and place it in wash solution 1 (reagent from the Agilent kit) at 250 rpm for 5 minutes at room temperature. Then use wash solution 2 (reagent from the Agilent kit) at 200 rpm for 1 minute at 39°C. Finally, remove the liquid on the chip surface and scan it within 4 hours.

[0145] 6. Fluorescence result scanning and signal analysis

[0146] (1) Import the microarray data file into the microarray data preprocessing software.

[0147] (2) Verify the integrity of the data file and ensure that the file format is compatible with the preprocessing software.

[0148] (3) Perform quality control checks to assess the overall quality of the data and identify potential problems such as outliers and artifacts, such as setting an outlier detection threshold of ±3 standard deviations;

[0149] (4) Removing or marking low-quality or unreliable probes or spots from the data, such as setting the probe removal to probes with signal intensity <100;

[0150] (5) Apply background correction methods to remove non-specific hybridization signals, such as setting background correction algorithms RMA, mas5, and model-based background correction parameters;

[0151] (6) Perform data normalization to adjust for systematic variations between arrays, such as quantile normalization and loess normalization;

[0152] (7) Export the preprocessed microarray data in an appropriate format for subsequent analysis, such as exporting CSV or Excel files.

[0153] Example 3 Detection specificity test of chip for eukaryotic algae

[0154] Specific detection means that the presence of the target species 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 mixed algae liquid of eukaryotic algae 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.

[0155] The experiment included two designs. Experiment 1 only included Micropore Coelastrum A eukaryotic algae. Experiment 2 included Boat sp. . RCC3092 and Tetrahedron minutum Two eukaryotic algae (see Figure 2 ).

[0156] The test results show that in Experiment 1, only Micropore Coelastrum The probes in the probe set have higher fluorescence signals, while the signals of other probe sets without added eukaryotic algae are relatively low, and overall lower than our probe positive threshold.

[0157] In Experiment 2, we added Boat sp. . RCC3092 and Tetrahedron minutum The results showed that the two eukaryotic algae were also successfully detected. The probes in their probe sets all had high fluorescence signals, while the probe signals of the other ten eukaryotic algae probe sets that were not added were very weak and did not pass the positive detection threshold ( Figure 2 ).

[0158] Conclusion: It is confirmed that the method of the present invention has a reliable detection effect at the eukaryotic algae species level.

[0159] Example 4 Detection and Analysis of Eukaryotic Algae in Water Samples from Three Reservoirs in Shenzhen

[0160] Three drinking water source reservoirs in Shenzhen, Guangdong Province were selected, and the eukaryotic algae detection chip of the present invention was used to investigate and analyze the eukaryotic algae in the reservoirs according to the method of Example 2. The three reservoirs were Shenzhen Reservoir (SZ), Sanzhoutian (SZT) and Tiegang Reservoir (TG), and samples were taken from three locations of each reservoir, namely the water inlet (RSK), the reservoir (KZ), and the water intake (QSK).

[0161] Test results such as Figure 3 shown.

[0162] The results show that the most abundant eukaryotic algae types were found in Shenzhen Reservoir (SZ). The second most abundant eukaryotic algae species were found in Tiegang Reservoir (TG). This example proves that the present invention can successfully detect the eukaryotic algae types contained in water bodies in practical applications.

[0163] 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 composition for detecting common eukaryotic algae, characterized in that: The probe composition includes a detection Chlorella vulgaris The probe set, the sequences of which are shown in SEQ ID No.1 to SEQ ID No.11, detects Scenedesmus sp. The probe set of NREL 46B-D3, the sequences of which are shown in SEQ ID No.12 to SEQ ID No.19, detects Amphora coffeaeformis The probe set, whose sequences are shown in SEQ ID No.20 to SEQ ID No.28, detects Ankistrodesmus sp. The probe set of CCAC 3332 B, the sequences of which are shown in SEQ ID No. 29 to SEQ ID No. 38, detects Coelastrum microporum The probe set, whose sequences are shown in SEQ ID No.39 to SEQ ID No.48, detects Pediastrum duplex The probe set, whose sequences are shown in SEQ ID No.49 to SEQ ID No.58, detects Tetraedron Minutum The probe set, whose sequences are shown in SEQ ID No.59 to SEQ ID No.68, detects Nitzschia inconspicua The probe set, whose sequences are shown in SEQ ID No.69 to SEQ ID No.77, detects Synedra sp. The probe set of RCC2510, the sequences of which are shown in SEQ ID No.78 to SEQ ID No.86, detects Cyclotella atomus The probe set, whose sequences are shown in SEQ ID No.87 to SEQ ID No.96, detects Achnanthes kuwaitensis The probe set, the sequences of which are shown in SEQ ID No.97 to SEQ ID No.106, detects Navicula sp. The probe set for RCC3092 has sequences shown in SEQ ID No.107 to SEQ ID No.

115.

2. Use of the probe composition according to claim 1 in preparing a gene chip or a kit for detecting common eukaryotic algae, characterized in that: The detection targets of the common eukaryotic algae include Chlorella vulgaris, Scenedesmussp. NREL 46B-D3, Amphora coffeaeformis , Ankistrodesmus sp. CCAC 3332 B Coelastrum microporum, Pediastrum duplex, Tetraedron minutum, Nitzschia inconspicua, Synedra sp. RCC2510, Cyclotella atomus, Achnanthes kuwaitensis, Navicula sp. RCC3092.

3. A gene chip for detecting common eukaryotic algae, characterized in that: The invention comprises the probe composition according to claim 1.

4. A kit for detecting common eukaryotic algae, characterized in that: The kit comprises the probe composition according to claim 1 or the gene chip according to claim 3.

5. A kit for detecting common eukaryotic algae 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 common eukaryotic algae, characterized in that: The steps include: (1) Filter the water sample to be tested, collect the filtered particles and complete DNA extraction; (2) The extracted DNA is sheared and purified to obtain gDNA; (3) Performing PCR on the gDNA obtained in step (2) using random primers and ATCG bases with fluorescent groups to obtain a gDNA sequence with fluorescent groups; (4) performing a hybridization experiment on the fluorescently labeled gDNA obtained in (3) and the gene chip according to claim 3 in a hybridization oven; (5) reading the hybridization experiment results of (4) and determining the eukaryotic algae detection results based on the read probe signals; Among them, the detection targets of common eukaryotic algae include Chlorella vulgaris, Scenedesmus sp. NREL46B-D3, Amphora coffeaeformis , Ankistrodesmus sp. CCAC 3332 B, Coelastrum microporum, Pediastrum duplex, Tetraedron minutum, Nitzschia inconspicua, Synedra sp. RCC2510, Cyclotella atomus, Achnanthes kuwaitensis, Navicula sp. RCC3092.

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