Method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy

Abstract

This application relates to the technical field of spectral identification, and provides a method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy. The method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy includes: taking a standard sample of Thalassiosira weissflogii and a test solution of Thalassiosira weissflogii; subjecting the test solution of Thalassiosira weissflogii to elution treatment to obtain a test sample of Thalassiosira weissflogii; drying the standard sample of Thalassiosira weissflogii and the test sample of Thalassiosira weissflogii; measuring the Raman spectra of the standard sample of Thalassiosira weissflogii and the test sample of Thalassiosira weissflogii, and judging the nutritional status of Thalassiosira weissflogii in the test solution of Thalassiosira weissflogii based on the peak changes at 1003 cm-1, 1057 cm-1, 1470 cm-1, 1155 cm-1, 1515 cm-1 and 2930.67 cm-1. The method provided by this application can effectively predict the occurrence of red tides by comparing the spectral characteristics of the standard sample of Thalassiosira weissflogii and the test sample of Thalassiosira weissflogii to judge the nutritional status of microorganisms in the water to be tested, and then judge the water quality.

Description

Technical Field

[0001] This application belongs to the technical field of spectral identification, and particularly relates to a method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy. Background Art

[0002] Red tide is a harmful ecological phenomenon usually formed due to the explosive proliferation of red tide algae in water areas. When a red tide occurs, the water body can be red or black, which will cause hypoxia in the water body, death of organisms, and loss of fishery resources. Thalassiosira weissflogii is a common diatom in the genus Thalassiosira of the phylum Bacillariophyta and is also a typical red tide alga. Its size is usually about 20 μm, square in shape, and can be pure cultured in f / 2 medium in the laboratory.

[0003] It is very important to predict red tides by detecting the quality of water bodies. So far, there are mainly the following two methods for predicting red tides by monitoring the quality of water bodies: First, using on-site tracking and monitoring by ships. Monitoring personnel use instruments on the ship to observe and collect water samples in a timely manner for indoor analysis. By detecting the chlorophyll concentration, water temperature, etc. of the samples, the water body state is analyzed, and by comparing with the water body values before the occurrence of red tides, it is predicted whether a red tide will occur in this area. Second, deploying marine water quality detection buoys to monitor the changes in marine water quality in real time.

[0004] However, the first method requires a large amount of water body information collection, with high labor intensity for monitoring personnel, long time consumption, cumbersome experimental steps prone to errors, long cycle and slow response, and high requirements for the physical strength, energy and experience of experimental personnel, which cannot meet the needs of disaster reduction and prevention work. Although deploying buoys can reduce the experimental intensity, this method not only requires a large amount of manpower and material resources to deploy, recover buoys and analyze data, but also the buoys cannot work properly in extreme environments such as heavy rain, and are severely restricted by external conditions. Summary of the Invention

[0005] The purpose of this application is to provide a method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy, aiming to solve the problems that the existing methods for predicting red tides by detecting the quality of water bodies are complex, and the influencing factors of data results are complex, resulting in reduced reliability of the results.

[0006] To achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] This application provides a method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy in the first aspect, including:

[0008] Taking a standard sample of Thalassiosira weissflogii and a test solution of Thalassiosira weissflogii;

[0009] Elute the Thalassiosira weissflogii test solution to obtain a Thalassiosira weissflogii test sample; dry the Thalassiosira weissflogii standard sample and the Thalassiosira weissflogii test sample.

[0010] Measure the Raman spectra of the Thalassiosira weissflogii standard sample and the Thalassiosira weissflogii test sample, and judge the nutritional status of Thalassiosira weissflogii in the Thalassiosira weissflogii test solution by the peak changes at 1003 cm -1 、1057 cm -1 、1470 cm -1 、1155 cm -1 、1515 cm -1 and 2930.67 cm -1 .

[0011] Preferably, judging the nutritional status of Thalassiosira weissflogii in the Thalassiosira weissflogii test solution by the peak changes at 1003 cm -1 、1057 cm -1 、1470 cm -1 、1155 cm -1 、1515 cm -1 and 2930.67 cm -1 includes:

[0012] If, compared with the Thalassiosira weissflogii standard sample, in the Raman spectrum of the Thalassiosira weissflogii test sample, the intensities at 1003 cm -1 and 1057 cm -1 decrease, and at the same time, the intensities at 1155 cm -1 、1470 cm -1 、1515 cm -1 and 2930.67 cm -1 increase, then the Thalassiosira weissflogii test sample is in a state of oligotrophy.

[0013] Preferably, the decrease in the intensities at 1003 cm -1 and 1057 cm -1 includes: compared with the Thalassiosira weissflogii standard sample, the Raman spectrum of the Thalassiosira weissflogii test sample does not show peaks at 1003 cm -1 and 1057 cm -1 , or

[0014] compared with the Thalassiosira weissflogii standard sample, the peak intensities of the Raman spectrum of the Thalassiosira weissflogii test sample at 1003 cm -1 and 1057 cm -1 decrease by 50% or more.

[0015] Preferably, the 1155 cm -1, 1470 cm -1 , 1515 cm -1 and 2930.67 cm -1 The intensity at is enhanced, including: compared with the Thalassiosira weissflogii standard sample, the Raman spectrum of the Thalassiosira weissflogii sample to be tested has an increased peak intensity at 1155 cm -1 , 1470 cm -1 , 1515 cm -1 and 2930.67 cm -1 by 5 times or more.

[0016] Preferably, the elution treatment of the Thalassiosira weissflogii sample to be tested includes:

[0017] Centrifuging the Thalassiosira weissflogii sample to be tested, removing the supernatant, adding water to elute the collected precipitate, and then centrifuging again to obtain the Thalassiosira weissflogii sample to be tested.

[0018] Preferably, the centrifugation method is: centrifuging at 8000 - 10000 g for 3 - 5 min.

[0019] Preferably, the drying treatment includes natural air drying, air extraction drying or normal temperature pressure drying.

[0020] Preferably, the method for obtaining the Thalassiosira weissflogii sample to be tested is:

[0021] In-situ collecting a water sample from the water area to be detected, separating and purifying the Thalassiosira weissflogii in the water sample to obtain the Thalassiosira weissflogii sample to be tested.

[0022] The second aspect of the present application provides a detection system for identifying the nutritional status of Thalassiosira weissflogii, including:

[0023] A sample acquisition module for acquiring a Thalassiosira weissflogii standard sample and a Thalassiosira weissflogii sample to be tested;

[0024] A sample processing module for eluting the Thalassiosira weissflogii sample to be tested to obtain a Thalassiosira weissflogii sample to be tested; drying the Thalassiosira weissflogii standard sample and the Thalassiosira weissflogii sample to be tested;

[0025] A Raman spectrum detection and judgment module for measuring the Raman spectra of the Thalassiosira weissflogii standard sample and the Thalassiosira weissflogii sample to be tested, and through 1003 cm -1 , 1057 cm -1 , 1470 cm -1 , 1155 cm -1 , 1515 cm -1 and 2930.67 cm -1The peak change at [specific location] is used to determine the nutritional status of Thalassiosira weissflogii in the to-be-detected Thalassiosira weissflogii solution.

[0026] The third aspect of this application provides a terminal device, including a memory, a processor, and a computer program stored in the memory and executable on the processor. It is characterized in that when the processor executes the computer program, the method described in the first aspect is implemented.

[0027] The fourth aspect of this application provides a computer-readable storage medium. The computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the method described in the first aspect is implemented.

[0028] The method for identifying the nutritional status of Thalassiosira weissflogii provided by this application only collects the Raman spectrum of a single cell of Thalassiosira weissflogii, and compares the peak changes of the Thalassiosira weissflogii standard sample and the to-be-detected Thalassiosira weissflogii sample at 1003 cm -1 , 1057 cm -1 , 1470 cm -1 , 1155 cm -1 , 1515 cm -1 and 2930.67 cm -1 to determine the nutritional status of microorganisms in the to-be-detected water body, and further determine the quality of the water body, which can effectively predict the occurrence of red tides. By using this method to identify the nutritional status of Thalassiosira weissflogii and further predict red tides, the method is simple and reliable, can reduce the work intensity of testers, and uses high-precision and advanced instruments to make the experimental results not affected by external conditions such as the experience of operators and the dosage of reagents, reduce the values that need to be tested, make the response faster, and the results more accurate.

[0029] It can be understood that the beneficial effects of the above second aspect to the fourth aspect can refer to the relevant descriptions in the first aspect above, and will not be elaborated here. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0031] Figure 1 are the Raman spectra of three Thalassiosira weissflogii provided by the embodiments of this application;

[0032] Figure 2 is the intensity comparison diagram of the Raman spectra of three Thalassiosira weissflogii provided by the embodiments of this application at the lipid peak (1057 cm -1 );

[0033] Figure 3 It is a schematic diagram of a detection system provided by an embodiment of the present application for identifying the nutritional status of Thalassiosira weissflogii;

[0034] Figure 4 It is a schematic diagram of the structure of a terminal device provided by an embodiment of the present application. Detailed implementation manners

[0035] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application clearer and more understandable, the present application will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0036] It should be understood that in various embodiments of the present application, the magnitudes of the serial numbers of the above processes do not mean the order of execution. Some or all steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

[0037] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "the" and "said" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0038] The Raman spectrum of each cell is determined by its cell composition. Single-cell Raman spectroscopy is a rapid and non-destructive vibrational spectroscopy analysis method, which supports rapid determination at the molecular level of single cells and judges changes in cell structure. Specifically, the Raman spectroscopy recording system records the vibrations, rotations and other low-frequency modes of the inherent properties of molecules, which can represent the molecular or chemical fingerprint system of molecules. According to the Raman spectra of different cells, lasers can reveal the internal chemical information of single cells, and the superposition of all biochemical information constitutes the Raman spectrum of cells. Therefore, through the Raman spectra of cells, we can deeply understand their gene expression, biosynthesis of specific compounds, cell composition, characteristic structures, physiological states and metabolic characteristics. Utilizing this characteristic of Raman spectroscopy, the nutritional status of microorganisms in water can be monitored, and then the rapid identification of water quality can be achieved.

[0039] Red tides are usually formed due to the explosive proliferation of red tide algae in water areas. Red tide algae usually have the ability of over-compensatory growth. Red tide algae growing under oligotrophic conditions will experience explosive proliferation due to their over-compensatory growth ability when encountering a large amount of nutrient supplementation, thus triggering red tides. By culturing Thalassiosira weissflogii under nutrient deficiency conditions and analyzing the changes in the physiological and biochemical composition of Thalassiosira weissflogii cells under different nutrient states in combination with Raman spectroscopy technology, and comparing their Raman spectra, it is finally found that the Raman spectrum of Thalassiosira weissflogii under the oligotrophic state is significantly different from its Raman spectrum under the normal nutrient state, which indicates that the nutrient supply status in water can be retrieved based on the Raman spectrum. In view of this, the embodiments of the present application can retrieve the state of the water body and then predict the possibility of red tide algae outbreak and the occurrence of red tides by quickly and non-destructively detecting the nutrient state (metabolic activity) of red tide algae in the water body with high precision.

[0040] The first aspect of the embodiments of the present application provides a method for identifying the nutrient state of Thalassiosira weissflogii using Raman spectroscopy, including:

[0041] S01. Take a standard sample of Thalassiosira weissflogii and a test solution of Thalassiosira weissflogii;

[0042] S02. Elute the test solution of Thalassiosira weissflogii to obtain a test sample of Thalassiosira weissflogii; dry the standard sample of Thalassiosira weissflogii and the test sample of Thalassiosira weissflogii;

[0043] S03. Measure the Raman spectra of the standard sample of Thalassiosira weissflogii and the test sample of Thalassiosira weissflogii, and judge the nutrient state of Thalassiosira weissflogii in the test solution of Thalassiosira weissflogii by the peak changes at 1003 cm -1 、1057 cm -1 、1470 cm -1 、1155 cm -1 、1515 cm -1 and 2930.67 cm -1 .

[0044] The method for identifying the nutrient state of Thalassiosira weissflogii provided by the embodiments of the present application only collects the Raman spectrum of a single cell of Thalassiosira weissflogii, and compares the standard sample of Thalassiosira weissflogii and the test sample of Thalassiosira weissflogii at 1003 cm -1 、1057 cm -1 、1470 cm -1 、1155 cm -1 、1515 cm -1 and 2930.67 cm -1The peak change at [location] is used to judge the nutritional status of microorganisms in the water body to be detected, and then to judge the water quality, which can effectively predict the occurrence of red tides. By using this method to identify the nutritional status of *Thalassiosira weissflogii* and then predict red tides, the method is simple and reliable, which can reduce the work intensity of testers, and the experimental results are not affected by external conditions such as the experience of operators and the dosage of reagents by using high-precision instruments, reduce the values to be tested, make the response faster and the results more accurate.

[0045] Specifically, in the above step S01, the standard sample of *Thalassiosira weissflogii* can be obtained by purchasing from a market biotechnology company, and there is no strict requirement in the embodiments of the present application. In some embodiments, the standard sample of *Thalassiosira weissflogii* is obtained by pure culture of *Thalassiosira weissflogii* in F / 2 medium.

[0046] The liquid sample to be tested of *Thalassiosira weissflogii* is a liquid sample containing *Thalassiosira weissflogii* in the water area to be detected. In some embodiments, the method for obtaining the liquid sample to be tested of *Thalassiosira weissflogii* is: in-situ collecting the water sample in the water area to be detected, separating and purifying *Thalassiosira weissflogii* in the water sample to obtain the liquid sample to be tested of *Thalassiosira weissflogii*. This method can in-situ extract *Thalassiosira weissflogii* in the water area to be detected and maintain its characteristics in the water area to be tested, making the test results accurate and reliable. It should be noted that in the embodiments of the present application, the *Thalassiosira weissflogii* obtained by separation and purification is not recultured to avoid changes in the nutritional status and metabolic components of *Thalassiosira weissflogii* during the culture process, resulting in changes in its Raman spectral characteristics and affecting the reliability of the Raman spectral comparison results between the standard sample of *Thalassiosira weissflogii* and the sample to be tested of *Thalassiosira weissflogii*.

[0047] In the embodiments of the present application, there is no strict requirement for separating and purifying *Thalassiosira weissflogii* in the water sample, and it only needs to extract *Thalassiosira weissflogii* in the water sample.

[0048] In some embodiments, in the step of separating and purifying *Thalassiosira weissflogii* in the water sample, the water sample is centrifuged at 1000 - 3000 RPM. Among them, although too high a rotation speed can separate microalgae, high rotation speed is likely to damage algal cells and is not easy to culture, while too low a rotation speed may not be able to separate algal cells well. It should be understood that other separation methods can also be applicable to separating *Thalassiosira weissflogii* in the water sample in the embodiments of the present application. Generally, before using other separation methods, the centrifugation separation method can also be used to remove some impurities, and then corresponding separation is carried out. In some embodiments, in a sterile environment, repeated centrifugation separation with sterile water can achieve the purification effect, and at least 20 times of repeated centrifugation is required to achieve the purification effect.

[0049] In some embodiments, scribing separation is used to isolate and purify *Thalassiosira weissflogii* in a water sample. Exemplarily, an inoculation loop is used to dip into the microalgae suspension and then scribed on an agar plate. After the sample is cultured in an artificial climate chamber for a period of time (generally at least two weeks), microalgae are picked from a single microalgae colony and placed in a clean and sterilized liquid medium with the same composition for culture. In some embodiments, pure *Thalassiosira weissflogii* can be directly used for scale-up culture.

[0050] In some embodiments, dilution separation is used to isolate and purify *Thalassiosira weissflogii* in a water sample. The principle is as follows: A microalgae suspension with a specific concentration is continuously diluted with a culture medium or sterilized distilled water until there is only one microalgae in each droplet. Specifically, the diluted microalgae suspension is dropped on a clean glass slide and observed under a microscope. If there is only one algal cell in each droplet in the field of view, it is transferred into a pre-prepared culture solution for culture. Alternatively, the diluted microalgae suspension can be directly added to a 96-well plate at one droplet per well for culture, and then the wells with only single microalgae propagation (only one type of microalgae in the same micro-well) are picked for scale-up culture.

[0051] In some embodiments, micropipette separation is used to isolate and purify *Thalassiosira weissflogii* in a water sample. The principle is as follows: Before the experiment, the micropipette or micro glass spotting tube is softened at the flame of an alcohol lamp, and then quickly removed from the flame and quickly stretched. It is evenly clamped at an appropriate aperture with medical pliers to obtain a smooth micropipette tip, and the aperture of the micropipette tip should be at least more than twice the size of the algal cells to be aspirated. When aspirating algal cells, it is necessary to be slow. Due to the fluid shear force, too fast aspiration speed will cause damage to the algal cells, and the micropipette tip should not be too large, otherwise it will be difficult to aspirate a single target microalgae cell. After the capillary tube is made, it is connected to a 2 μl, 5 μl, 10 μl or 100 μl pipette tip, and finally connected to a pipettor. When using micropipette separation, an ordinary optical microscope can be used. Since its stage is relatively low, the operation is relatively convenient, especially for the problem of hand shaking encountered by beginners. When aspirating cells by the micropipette method, multiple separations are often required. Regarding capillary action, the capillary tip needs to be placed in sterilized distilled water first to wet the capillary to reduce capillary action, and then the wetted capillary is used to aspirate a single microalgae cell in the sample solution. If more than one microalgae cell is aspirated, the aspirated liquid is blown into sterilized and clean distilled water and the operation is repeated. After repeating many times, single pure algal cells can be obtained.

[0052] In some embodiments, the Thalassiosira weissflogii in water samples is isolated and purified by the 96-well plate method. The principle is as follows: The water sample to be separated is diluted until each drop contains approximately one microalgae cell, and then the diluted algal solution is added drop by drop to a 96-well plate containing a separation medium, about 250 μl per well. The medium can be liquid or solid. After the obtained sample is enriched and cultured in an artificial incubator with preset culture conditions for a period of time, pure microalgae are collected.

[0053] In the above step S02, eluting the test solution of Thalassiosira weissflogii can remove impurities in the test solution, thereby removing background interference in Raman spectroscopy measurement and improving the accuracy of detection. Further, the eluted Thalassiosira weissflogii is enriched, increasing the content of Thalassiosira weissflogii in the test sample, so that the test sample contains Thalassiosira weissflogii.

[0054] In some embodiments, when performing Raman spectroscopy measurement on the test sample of Thalassiosira weissflogii, 10 - 30 cells are randomly selected to measure their Raman spectra and the average value is taken to improve the accuracy. In this case, eluting and enriching the test solution of Thalassiosira weissflogii can increase the concentration of Thalassiosira weissflogii in the test sample, avoid repeated sampling, and thus improve the measurement efficiency of Raman spectroscopy.

[0055] In some embodiments, the treatment of eluting the test solution of Thalassiosira weissflogii includes: centrifuging the test solution of Thalassiosira weissflogii, removing the supernatant, adding water to wash the collected precipitate, and then centrifuging again to obtain the test sample of Thalassiosira weissflogii. Through the first centrifugation treatment, that is, centrifuging the test solution of Thalassiosira weissflogii, the Thalassiosira weissflogii in the test solution can be enriched and some impurities can be removed; washing with water removes impurities in Thalassiosira weissflogii, thereby removing background interference in Raman spectroscopy measurement and improving the accuracy of detection; further, centrifuging again can enrich Thalassiosira weissflogii.

[0056] In some embodiments, the method of centrifugation treatment is: centrifuging at 8000 - 10000 g for 3 - 5 min. In this case, both the elution and enrichment of Thalassiosira weissflogii can be achieved, and the influence of centrifugation conditions on Thalassiosira weissflogii can be avoided, and the rupture of Thalassiosira weissflogii cells caused by too high centrifugation conditions can be avoided.

[0057] When performing Raman spectroscopy measurement on Thalassiosira weissflogii, in order to avoid cell movement during Raman spectroscopy measurement, which is not only not conducive to observation but also leads to the spectral data collected not being the actual test data, affecting the accuracy of the measurement result. In the embodiments of the present application, by drying the standard sample of Thalassiosira weissflogii and the test sample of Thalassiosira weissflogii, the cells are fixed when performing Raman spectroscopy measurement on Thalassiosira weissflogii, thereby improving the reliability of the detection result.

[0058] In some embodiments, the drying treatment includes natural air drying, air extraction drying or normal temperature pressure drying. These drying methods will not damage the cells of *Thalassiosira weissflogii*, thus improving the detection reliability of *Thalassiosira weissflogii*. Among them, normal temperature refers to a temperature of 5 to 45 °C. Under this condition, the higher the temperature, the more conducive to drying.

[0059] Exemplarily, the above step S02 includes: centrifuging 1 ml of the *Thalassiosira weissflogii* liquid to be detected at 10,000 g for 3 min, and discarding the supernatant; after eluting with water, centrifuging again at 10,000 g for 3 min, discarding the supernatant, and finally leaving about 10 - 20 μL of the sample as the *Thalassiosira weissflogii* sample to be detected. Take 2 μL of the *Thalassiosira weissflogii* sample to be detected and drop it onto a special slide for measuring Raman spectra, and let it dry naturally.

[0060] In the above step S03, the Raman spectra of the *Thalassiosira weissflogii* standard sample and the *Thalassiosira weissflogii* sample to be detected can be measured on a Raman spectrometer by the Raman spectrum measurement method. In some embodiments, the Raman spectrum measurement of the *Thalassiosira weissflogii* standard sample may not be carried out synchronously with the Raman spectrum measurement of the *Thalassiosira weissflogii* sample to be detected. The Raman spectrum measurement of the *Thalassiosira weissflogii* standard sample can be carried out before the measurement of the *Thalassiosira weissflogii* sample to be detected, and the Raman spectrum of the *Thalassiosira weissflogii* standard sample can be saved. When comparing the spectral characteristics with the Raman spectrum of the *Thalassiosira weissflogii* sample to be detected later, the Raman spectrum of the *Thalassiosira weissflogii* standard sample can be directly called.

[0061] In the embodiments of the present application, the Raman spectra of the *Thalassiosira weissflogii* standard sample and the *Thalassiosira weissflogii* sample to be detected are compared, and the peak changes at 1003 cm -1 、1057 cm -1 、1470 cm -1 、1155 cm -1 、1515 cm -1 and 2930.67 cm -1 are analyzed to judge the nutritional status of *Thalassiosira weissflogii* in the *Thalassiosira weissflogii* liquid to be detected.

[0062] In some embodiments, by the peak changes at 1003 cm -1 、1057 cm -1 、1470 cm -1 、1155 cm -1 、1515 cm -1 and 2930.67 cm -1 to judge the nutritional status of *Thalassiosira weissflogii* in the *Thalassiosira weissflogii* liquid to be detected, including:

[0063] If in the Raman spectrum of the *Thalassiosira weissflogii* sample to be detected compared with the *Thalassiosira weissflogii* standard sample, at 1003 cm-1 and at 1057 cm -1 the intensity decreases, and at the same time, at 1155 cm -1 and 1470 cm -1 and 1515 cm -1 and 2930.67 cm -1 the intensity increases, then the sample of Thalassiosira weissflogii to be tested is in an oligotrophic state.

[0064] In some embodiments, the intensity decreases at 1003 cm -1 and 1057 cm -1 including: compared with the standard sample of Thalassiosira weissflogii, the Raman spectrum of the sample of Thalassiosira weissflogii to be tested does not show peaks at 1003 cm -1 and 1057 cm -1 In some embodiments, compared with the standard sample of Thalassiosira weissflogii, the Raman spectrum of the sample of Thalassiosira weissflogii to be tested has a peak intensity reduction of 50% or more at 1003 cm -1 and 1057 cm -1 or more.

[0065] In some embodiments, the intensity increases at 1155 cm -1 and 1470 cm -1 and 1515 cm -1 and 2930.67 cm -1 including: compared with the standard sample of Thalassiosira weissflogii, the Raman spectrum of the sample of Thalassiosira weissflogii to be tested has a peak intensity increase of 5 times or more at 1155 cm -1 and 1470 cm -1 and 1515 cm -1 and 2930.67 cm -1 or more.

[0066] Through the above two types of peak changes, it can be determined that the sample of Thalassiosira weissflogii to be tested is in an oligotrophic state. Under such conditions, the red tide algae growing under oligotrophic conditions will have explosive proliferation due to the overcompensation growth ability when encountering a large amount of nutrient element supplementation, thus triggering a red tide, and thus predicting the occurrence of a red tide.

[0067] The following is described in conjunction with specific embodiments.

[0068] A method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy, including:

[0069] In the laboratory, a eutrophic Thalassiosira weissflogii standard sample, an oligotrophic Thalassiosira weissflogii, and Thalassiosira weissflogii cultured under silicon deficiency were cultured in artificial seawater; the three types of Thalassiosira weissflogii in the exponential growth phase were subjected to elution treatment to obtain a Thalassiosira weissflogii standard sample, an oligotrophic Thalassiosira weissflogii sample, and a Thalassiosira weissflogii sample cultured under silicon deficiency; the Thalassiosira weissflogii standard sample, the oligotrophic Thalassiosira weissflogii sample, and the Thalassiosira weissflogii sample cultured under silicon deficiency were dried.

[0070] The Raman spectra of the Thalassiosira weissflogii standard sample, the oligotrophic Thalassiosira weissflogii sample, and the Thalassiosira weissflogii sample cultured under silicon deficiency were measured, and the peak changes at 1003 cm -1 , 1057 cm -1 , 1470 cm -1 , 1155 cm -1 , 1515 cm -1 and 2930.67 cm -1 were used to judge the nutritional status of Thalassiosira weissflogii in the test solution of Thalassiosira weissflogii. There are obvious differences in the Raman spectra of Thalassiosira weissflogii grown under oligotrophic and normal conditions (eutrophic).

[0071] Figure 1 are the Raman spectra of the three types of Thalassiosira weissflogii (test conditions: Raman spectra of Thalassiosira weissflogii obtained under a 50× microscope objective, 532 nm laser, 5 mw laser power, 5 s measurement time, and 30 s accumulation times; the abscissa represents the wave number of bond vibration, and the ordinate represents the Raman peak intensity). As shown in the figure, the lipid peak (1057 cm -1 ) of the diatom cells in the silicon-deficient group is significantly higher than that of the Thalassiosira weissflogii standard sample (eutrophic group), which indicates that the lack of only a single nutritional condition can also cause changes in its Raman spectrum, and this change makes the Raman spectrum under this condition significantly different from the spectrum of Thalassiosira weissflogii grown under normal nutritional conditions. It can be seen from Figure 1 that: compared with the Thalassiosira weissflogii standard sample, the Raman peaks of the Thalassiosira weissflogii under nutritional stress are significantly weakened at 1003 cm -1 and 1057 cm -1 , while the intensities at 1155 cm -1 , 1470 cm -1 , 1515 cm -1 and 2930.67 cm -1 are significantly increased.

[0072] Figure 2 is the intensity comparison diagram of the Raman spectra of the three types of Thalassiosira weissflogii at the lipid peak (1057 cm -1 ). It can be seen from Figure 2It can be seen that there are very obvious differences in the lipid peak intensities of Thalassiosira weissflogii in three growth states, indicating that the Raman spectrum of Thalassiosira weissflogii can detect changes in the lipid content of algae, directly reflect its vegetative growth state, and thus invert the water environment, judge the nutritional environment of other microorganisms, and take timely measures to prevent the explosive proliferation of microorganisms, eliminating the possibility of red tide occurrence from the source.

[0073] The lipid peak (1057 cm -1 ) of diatom cells in the silicon-deficient group is significantly higher than that of the Thalassiosira weissflogii standard sample (eutrophic group), which shows that: First, the Raman spectrum is very sensitive, and even a single change in nutritional conditions will be directly reflected in the spectrum; Second, any change in conditions will cause the algal spectrum under that condition to deviate from the spectrum of the normal nutritional state, indicating that silicon deficiency is conducive to diatoms producing lipids, which is consistent with the results measured by chemical methods before. However, single-cell Raman spectroscopy can detect changes in the inclusions of diatom cells more quickly, providing a basis for monitoring the cell state of red tides represented by diatoms and predicting the risk of red tide occurrence.

[0074] As Figure 3 shown, the second aspect of the embodiment of the present application provides a detection system 3 for identifying the nutritional state of Thalassiosira weissflogii, including:

[0075] A sample acquisition module 31 for acquiring a Thalassiosira weissflogii standard sample and a Thalassiosira weissflogii sample to be detected;

[0076] A sample processing module 32 for eluting the Thalassiosira weissflogii sample to be detected to obtain a Thalassiosira weissflogii sample to be detected; drying the Thalassiosira weissflogii standard sample and the Thalassiosira weissflogii sample to be detected;

[0077] A Raman spectrum detection and judgment module 33 for measuring the Raman spectra of the Thalassiosira weissflogii standard sample and the Thalassiosira weissflogii sample to be detected, and judging the nutritional state of Thalassiosira weissflogii in the Thalassiosira weissflogii sample to be detected through the peak value changes at 1003 cm -1 、1057 cm -1 、1470 cm -1 、1155 cm -1 、1515 cm -1 and 2930.67 cm -1 。

[0078] In some embodiments, the Raman spectrum detection and judgment module 33 includes a detection unit and a judgment unit. Among them, the detection unit is used to measure the Raman spectra of the Thalassiosira weissflogii standard sample and / or the Thalassiosira weissflogii sample to be detected, and the judgment unit is used to judge the two according to the Raman spectra of the Thalassiosira weissflogii standard sample and the Thalassiosira weissflogii sample to be detected at 1003 cm -1 、1057 cm -1, 1470 cm -1 , 1155 cm -1 , 1515 cm -1 and 2930.67 cm -1 Analyze and judge the peak changes at these positions.

[0079] As Figure 4 shown, the third aspect of the embodiment of the present application provides a terminal device 400, including a memory 420, a processor 410, and a computer program 421 stored in the memory 420 and executable on the processor. When the processor executes the computer program, the method of the first aspect is implemented.

[0080] Exemplarily, the computer program 421 can be divided into one or more modules / units. One or more modules / units are stored in the memory 420 and executed by the processor 410 to complete the present application. One or more modules / units can be a series of computer program instruction segments capable of completing specific functions, and these instruction segments can be used to describe the execution process of the computer program 421 in the server 400. For example, the computer program 421 can be divided into a sample acquisition module, a sample processing module, and a Raman spectrum detection and judgment module. The specific functions of each module are as follows:

[0081] The sample acquisition module is used to acquire a Thalassiosira weissflogii standard sample and a Thalassiosira weissflogii sample to be detected;

[0082] The sample processing module is used to elute the Thalassiosira weissflogii sample to be detected to obtain a Thalassiosira weissflogii sample to be detected; dry the Thalassiosira weissflogii standard sample and the Thalassiosira weissflogii sample to be detected;

[0083] The Raman spectrum detection and judgment module is used to measure the Raman spectra of the Thalassiosira weissflogii standard sample and the Thalassiosira weissflogii sample to be detected, and judge the nutritional status of Thalassiosira weissflogii in the Thalassiosira weissflogii sample to be detected through the peak changes at 1003 cm -1 , 1057 cm -1 , 1470 cm -1 , 1155 cm -1 , 1515 cm -1 and 2930.67 cm -1 at these positions.

[0084] The server 400 may include, but is not limited to, a processor 410 and a memory 420. Those skilled in the art can understand that Figure 2 this is only an example of the server 400 and does not constitute a limitation on the server 400. It may include more or fewer components than shown in the figure, or combine certain components, or different components. For example, the server 400 may further include input / output devices, network access devices, buses, etc.

[0085] The processor 410 may be a Central Processing Unit (CPU), or may also be other general-purpose processors, Digital Signal Processors (DSPs), Application Specific Integrated Circuits (ASICs), Field-Programmable Gate Arrays (FPGAs), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. The general-purpose processor may be a microprocessor or the processor may also be any conventional processor, etc.

[0086] The memory 420 may be an internal storage unit of the server 400, such as the hard disk or memory of the server 400. The memory 420 may also be an external storage device of the server 400, such as a plug-in hard disk equipped on the server 400, a Smart Media Card (SMC), a Secure Digital (SD) card, a Flash Card, and so on. Further, the memory 420 may also include both the internal storage unit of the server 800 and the external storage device. The memory 420 is used to store the computer program 421 and other programs and data required by the server 400. The memory 420 may also be used to temporarily store the data that has been output or is to be output.

[0087] A fourth aspect of the embodiments of the present application provides a computer-readable storage medium storing a computer program, and when the computer program is executed by a processor, the method as described in the first aspect is implemented.

[0088] The embodiments of the present application also provide a computer program product, and when the computer program product runs on a terminal device, the terminal device is enabled to perform the automated biosynthesis of the foregoing method embodiments.

[0089] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy, comprising: taking a standard sample of Thalassiosira weissflogii and a test solution of Thalassiosira weissflogii; eluting and enriching the test solution of Thalassiosira weissflogii to obtain a test sample of Thalassiosira weissflogii; drying the standard sample of Thalassiosira weissflogii and the test sample of Thalassiosira weissflogii; Measure the Raman spectra of the Thalassiosira weissflogii standard sample and the Thalassiosira weissflogii sample to be tested, and judge the nutritional status of Thalassiosira weissflogii in the Thalassiosira weissflogii sample to be tested by the peak changes at 1003 cm -1 , 1057 cm -1 , 1470 cm -1 , 1155 cm -1 , 1515 cm -1 and 2930.67 cm -1 . The peak changes at 1003 cm -1 , 1057 cm -1 , 1470 cm -1 , 1155 cm -1 , 1515 cm -1 and 2930.67 cm -1 are used to judge the nutritional status of *Thalassiosira weissflogii* in the *Thalassiosira weissflogii* test solution to be detected, including: If, in the Raman spectrum of the Thalassiosira weissflogii sample to be tested, compared with the Thalassiosira weissflogii standard sample, the intensities at 1003 cm -1 and 1057 cm -1 decrease, and at the same time, the intensities at 1155 cm -1 1470 cm -1 1515 cm -1 and 2930.67 cm -1 increase, then the Thalassiosira weissflogii sample to be tested is in an oligotrophic state; The elution treatment of the test solution of Thalassiosira weissflogii includes: centrifuging the test solution of Thalassiosira weissflogii, removing the supernatant, adding water to elute the collected precipitate, and then centrifuging again to obtain the test sample of Thalassiosira weissflogii.

2. The method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy according to claim 1, characterized in that, The intensity reduction at 1003 cm -1 and 1057 cm -1 is as follows: compared with the Thalassiosira weissflogii standard sample, the Raman spectrum of the Thalassiosira weissflogii sample to be tested does not show peaks at 1003 cm -1 and 1057 cm -1 or Compared with the Thalassiosira weissflogii standard sample, the Raman spectrum of the Thalassiosira weissflogii sample to be tested has a peak intensity reduction of more than 50% at 1003 cm -1 and 1057 cm -1 .

3. The method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy according to claim 1, characterized in that, The 1155 cm -1 , 1470 cm -1 , 1515 cm -1 and 2930.67 cm -1 show enhanced intensities, including: compared with the Thalassiosira weissflogii standard sample, the Raman spectra of the Thalassiosira weissflogii sample to be tested have peak intensities at 1155 cm -1 , 1470 cm -1 , 1515 cm -1 and 2930.67 cm -1 that increase by more than 5 times.

4. The method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy according to any one of claims 1 to 3, characterized in that, The centrifugation method is: centrifuging at 8000 - 10000 g for 3 - 5 minutes.

5. The method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy according to any one of claims 1 to 3, characterized in that, The drying treatment includes natural air drying, suction drying or normal temperature pressure drying.

6. The method for identifying the nutritional status of Thalassiosira weissflogii using Raman spectroscopy according to any one of claims 1 to 3, characterized in that, The method for obtaining the test solution of Thalassiosira weissflogii is: in-situ collecting a water sample in the water area to be detected, separating and purifying Thalassiosira weissflogii in the water sample to obtain a test solution of Thalassiosira weissflogii.

7. A detection system for identifying the nutritional status of Thalassiosira weissflogii, characterized in that, comprising: a sample acquisition module for acquiring a standard sample of Thalassiosira weissflogii and a test solution of Thalassiosira weissflogii; a sample processing module for eluting and enriching the test solution of Thalassiosira weissflogii to obtain a test sample of Thalassiosira weissflogii; drying the standard sample of Thalassiosira weissflogii and the test sample of Thalassiosira weissflogii; Raman spectroscopy detection and judgment module, used to measure the Raman spectra of the Thalassiosira weissflogii standard sample and the Thalassiosira weissflogii sample to be detected, and judge the nutritional status of Thalassiosira weissflogii in the Thalassiosira weissflogii sample to be detected by the peak changes at 1003 cm -1 , 1057 cm -1 , 1470 cm -1 , 1155 cm -1 , 1515 cm -1 and 2930.67 cm -1 ; Among them, the peak changes at 1003 cm -1 , 1057 cm -1 , 1470 cm -1 , 1155 cm -1 , 1515 cm -1 and 2930.67 cm -1 are used to judge the nutritional status of Thalassiosira weissflogii in the test solution of Thalassiosira weissflogii, including: if in the Raman spectrum of the test sample of Thalassiosira weissflogii, compared with the standard sample of Thalassiosira weissflogii, the intensities at 1003 cm -1 and 1057 cm -1 decrease, and at the same time, the intensities at 1155 cm -1 , 1470 cm -1 , 1515 cm -1 and 2930.67 cm -1 increase, then the test sample of Thalassiosira weissflogii is in a state of oligotrophy; The elution treatment of the test solution of Thalassiosira weissflogii includes: centrifuging the test solution of Thalassiosira weissflogii, removing the supernatant, adding water to elute the collected precipitate, and then centrifuging again to obtain the test sample of Thalassiosira weissflogii.

8. A terminal device, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that, when the processor executes the computer program, the method according to any one of claims 1 to 6 is implemented.

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