A common algal bloom isothermal amplification liquid-phase nucleic acid chip and its application

Abstract

This invention relates to the field of nucleic acid detection technology, specifically disclosing a liquid-phase nucleic acid chip for isothermal amplification of common algal blooms and its application. The method includes: collecting water samples and filtering to enrich algal cells; lysing the algal cells using cell lysis buffer to extract DNA; adding the extracted DNA to the liquid-phase nucleic acid chip for isothermal amplification; detecting the Ct value using a qPCR instrument; and determining the species and cell density of the algal bloom based on a preset standard curve. This invention directly quantifies algal cell density using algal gene copy number, with significantly higher accuracy than microscopic counting, and does not rely on the experience of professional personnel. It can be widely applied in frontline water resource management work.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection technology, specifically relating to a liquid-phase nucleic acid chip for isothermal amplification of common algal blooms and its application. Background Technology

[0002] Under the multiple pressures of global climate change and eutrophication, the risk of algal blooms in lake and reservoir water sources is becoming increasingly severe. Increased algal cell numbers can severely damage freshwater ecosystems, leading to large-scale mortality of aquatic organisms. Furthermore, algal cells synthesize and analyze large amounts of algal organic matter. When high-algae raw water enters downstream water treatment plants, the increased algal cells clog filters. Algal organic matter affects the efficiency of coagulation and disinfection processes. Unremoved algal organic matter may undergo anaerobic fermentation in the water supply network, producing large amounts of sulfur-containing odorous compounds, leading to water quality deterioration and affecting the safety of urban residents' water supply.

[0003] Currently, manual microscopic observation and counting is a common method for classifying and counting algae in water blooms based on their morphological characteristics. It relies heavily on the experience and extensive knowledge of algal taxonomy of professionals, demanding extremely high levels of expertise from the observers. Water samples contain a rich diversity of algae, some with highly similar shapes, making differentiation extremely difficult, resulting in low accuracy and time-consuming processes. In recent years, some automated algae counting devices have been developed, incorporating algal databases. These devices automatically identify algal cell types by photographing algae images and utilizing big data methods, achieving semi-automated algae monitoring. However, this technology still relies on morphological characteristics for identification and classification, and the diversity of algal cell types varies across different regions. The completeness of the database significantly impacts the accuracy of the monitoring results. In conclusion, existing technologies for identifying algal blooms and quantifying their cell counts still have serious shortcomings.

[0004] Among the various published patent applications for algal bloom monitoring, for example, application number CN202310287882.3 discloses an algal monitoring method and system. This method involves photographing planktonic algae in a water sample and using a preset image vision algorithm to identify and count the algae in the captured images. The device automatically performs sampling, sedimentation collection, slide preparation, photography, and planktonic algal identification and counting, improving the standardization of algal detection. However, this method still cannot achieve high-precision monitoring of some specific algal blooms, only roughly identifying algal cell species based on algal morphology, resulting in low accuracy.

[0005] As living organisms, algal bloom cells possess highly specific genetic material, and their ribosomal genes exhibit significant differences among different algal species, making them ideal biomarkers for identifying various algal species. Therefore, developing a liquid-phase biochip based on isothermal amplification to accurately identify and quantify specific nucleic acid fragments in algal cells, thereby rapidly and accurately identifying common algal blooms and their cell densities, is urgently needed. This technology can be widely applied in the monitoring and management of algal blooms in lakes and reservoirs. Summary of the Invention

[0006] The purpose of this invention is to provide an isothermal amplification liquid-phase nucleic acid chip for common algal blooms and its applications, in order to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A liquid-phase nucleic acid chip for isothermal amplification of common algal blooms, the chip comprising a specific primer combination for detecting seven algal blooms, including: Microcystis, Crescentella, Syngonium, Euglena, Polydinium, Cryptophyta, and Phaeophyta.

[0009] The specific nucleic acid amplification primers for algal blooms are shown in the table below:

[0010]

[0011] Preferably, the reaction system of the chip includes:

[0012] Upstream primer: 0.2 μL;

[0013] Downstream primer: 0.2 μL;

[0014] SYBR Greenpre-mix: 10μL;

[0015] Sterile distilled water: 7.6 μL;

[0016] DNA template: 0.5 μL.

[0017] A method for detecting common algal blooms using the above-mentioned liquid-phase nucleic acid chip includes the following steps:

[0018] (1) Collect water samples and filter to enrich algal cells;

[0019] (2) Use cell lysis buffer to lyse algal cells and extract DNA;

[0020] (3) The extracted DNA is added to the liquid phase nucleic acid chip and isothermal amplification reaction is performed;

[0021] (4) The Ct value was detected by qPCR instrument, and the species and cell density of algae blooms were determined according to the preset standard curve.

[0022] Preferably, the cell lysis buffer contains: 15 g / L CTAB, 75 mM Tris-HCl, and 15 mM EDTA, and the lysis conditions are a metal bath treatment at 70–80°C for 15–20 minutes.

[0023] Preferably, the isothermal amplification reaction program is as follows: pre-denaturation at 94°C for 40 seconds, followed by 40 cycles of 94°C for 5 seconds and 60°C for 30 seconds.

[0024] Preferably, the standard curve is established through the following steps:

[0025] DNA from seven algal bloom species at known concentrations was serially diluted, and qPCR was performed to plot the relationship between Ct value and algal cell density.

[0026] Preferably, the water sample is natural surface water, and a 0.45μm filter membrane is used for filtration.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] (1) The liquid phase nucleic acid chip of the present invention is easy to use and has low cost.

[0029] (2) This invention can simultaneously monitor seven common algal blooms through isothermal amplification.

[0030] (3) The nucleic acid chip of the present invention utilizes the specificity of algal genes to accurately identify 7 common algal blooms, and has strong specificity.

[0031] (4) The present invention uses nucleic acid chips to monitor common algal blooms, which takes a short time (30-60 minutes).

[0032] (5) The present invention utilizes nucleic acid chips to determine the density of common algal blooms, with high sensitivity and a detection limit as low as 1-10 algal cells / mL.

[0033] (6) The nucleic acid chip of the present invention directly quantifies algal cell density by utilizing the copy number of algal genes. Its accuracy is significantly higher than that of microscopic counting, and it does not rely on the experience judgment of professionals. It can be widely used in front-line work of water resource management. Attached Figure Description

[0034] Figure 1 The standard curves of Ct values ​​and algal cell density for isothermal amplification of liquid-phase nucleic acid chips for the seven types of algal blooms in this invention are shown.

[0035] Figure 2This is a schematic diagram of a common algal bloom isothermal amplification liquid-phase nucleic acid chip structure according to the present invention;

[0036] Figure 3 This is a diagram showing the monitoring results of dominant algal blooms in lakes and reservoirs in Embodiment 2 of the present invention. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1:

[0039] Please see Figures 1-3 As shown, a liquid-phase nucleic acid chip for isothermal amplification of common algal blooms is illustrated. The chip includes a specific primer combination for detecting seven algal bloom species, namely: Microcystis, Crescentella, Syngonium, Euglena, Polydinium, Cryptophyta, and Phaeocystis.

[0040] The specific nucleic acid amplification primers for algal blooms are shown in the table below:

[0041]

[0042] In one embodiment of the present invention, the reaction system of the chip includes:

[0043] Upstream primer: 0.2 μL;

[0044] Downstream primer: 0.2 μL;

[0045] SYBRGreenpre-mix: 10μL;

[0046] Sterile distilled water: 7.6 μL;

[0047] DNA template: 0.5 μL.

[0048] A method for detecting common algal blooms using the above-mentioned liquid-phase nucleic acid chip includes the following steps:

[0049] Step 1: Take 300-500mL of natural surface water and use a 0.45µm filter membrane to obtain an enrichment of algal bloom cells;

[0050] Step 2: Add 50-100 μL of cell lysis buffer (15 g / L CTAB, 75 mM Tris-HCl, 15 mM EDTA) to the enrichment of algal bloom cells, treat in a metal bath at 70-80℃ for 15-20 min, centrifuge (12000 rpm, 5 min) to remove algal cell precipitate, and take 50-80 μL of supernatant to obtain nucleic acid (DNA) sample;

[0051] Step 3: Isothermal amplification of liquid-phase nucleic acid chips using algae (Microcystis, Crescentella, Syngonium, Euglena, Dinoflagellate, Cryptophyte, and Phaeophyte) was performed. The chips consisted of 8 wells (each well containing: 0.2 μL primer-F, 0.2 μL primer-R, 10 μL SYBR Green pre-mix, and 7.6 μL sterile distilled water). After adding 0.5 μL of the nucleic acid sample extracted in Step 2 to the liquid-phase chip, the chip was placed in a qPCR instrument. The reaction program was set to: pre-denaturation at 94°C for 40 s, followed by 40 cycles of 94°C, 5 s, 60°C, 30 s. The Ct values ​​of these 7 typical algal blooms were obtained.

[0052] Step 4: Select 1L of common blooming algae such as *Microcystis*, *Ceratophyllum*, *Nephrolepis*, *Euglena*, *Polydinium*, *Cryptocybe*, and *Phaeodactylum*, and extract DNA using a column-based bacterial total DNA extraction kit after 10-fold serial dilution. Use these 10-fold serial dilutions as templates for qPCR reactions. Perform qPCR experiments on the standard DNA serially diluted samples using specific nucleic acid primers for these common blooming algae. The reaction mixture includes: 0.2 μL primer-F, 0.2 μL primer-F, 10 μL SYBR Green pre-mix, 2 μL DNA template, and 7.6 μL sterile distilled water. The reaction procedure is the same as in Step 3: Plot a standard curve of Ct values ​​versus algal cell density for isothermal amplification of the algal nucleic acid microarray.

[0053] Step 5: Using the CT values ​​obtained in Step 3, and based on the standard curve obtained in Step 4, calculate the cell density of a specific algal bloom.

[0054] Example 2:

[0055] like Figure 3 As shown in the figure, Longhu Lake, a key water source in Fujian Province, was selected as the site for the application of this technology. A six-month quantitative monitoring of the dominant algal blooms in the lake was conducted using an algal isothermal amplification liquid-phase nucleic acid chip. It was found that *Crescentia spp.* and *Microcystis spp.* were the dominant algal species. *Crescentia spp.* had a cell count as high as 50,000 cells / L, while *Microcystis spp.* had a cell count as high as only 350 cells / L, far lower than *Crescentia spp.*, indicating a mild to moderate algal bloom. This method demonstrates high specificity, high sensitivity, and rapid detection.

[0056] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A liquid-phase nucleic acid chip for isothermal amplification of common algal blooms, characterized in that, The chip includes a specific primer combination for detecting seven types of algal blooms, namely: Microcystis, Crescentella, Syngonium, Euglena, Dinoflagellate, Cryptophyte, and Phaeophyte.

2. The isothermal amplification liquid-phase nucleic acid chip for common algal blooms according to claim 1, characterized in that, The reaction system of the chip includes: Upstream primer: 0.2 μL; Downstream primer: 0.2 μL; SYBR Greenpre-mix: 10μL; Sterile distilled water: 7.6 μL; DNA template: 0.5 μL.

3. A method for detecting common algal blooms using the liquid-phase nucleic acid chip described in claim 1, characterized in that, Includes the following steps: (1) Collect water samples and filter to enrich algal cells; (2) Use cell lysis buffer to lyse algal cells and extract DNA; (3) The extracted DNA is added to the liquid phase nucleic acid chip and isothermal amplification reaction is performed; (4) The Ct value was detected by qPCR instrument, and the species and cell density of algae blooms were determined according to the preset standard curve.

4. The application method of a liquid-phase nucleic acid chip for isothermal amplification of common algal blooms according to claim 3, characterized in that, The cell lysis buffer contains 15 g / L CTAB, 75 mM Tris-HCl, and 15 mM EDTA, and the lysis conditions are a metal bath treatment at 70–80°C for 15–20 minutes.

5. The application method of a liquid-phase nucleic acid chip for isothermal amplification of common algal blooms according to claim 3, characterized in that, The isothermal amplification reaction program is as follows: pre-denaturation at 94°C for 40 seconds, followed by 40 cycles of 94°C for 5 seconds and 60°C for 30 seconds.

6. The application method of a liquid-phase nucleic acid chip for isothermal amplification of common algal blooms according to claim 3, characterized in that, The standard curve is established through the following steps: DNA from seven algal bloom species at known concentrations was serially diluted, and qPCR was performed to plot the relationship between Ct value and algal cell density.

7. The application method of a liquid-phase nucleic acid chip for isothermal amplification of common algal blooms according to claim 3, characterized in that: The water sample was a natural surface water body, and a 0.45μm filter membrane was used for filtration.

Images