Fluorine content detection method based on fluoride riboswitch regulation transcription and application

Abstract

The invention relates to the technical field of fluorine content detection, and discloses a fluorine content detection method for fluoride riboswitch regulation and transcription and application. According to the present invention, with the biological element of the fluoride ribose switch, the fluorine ions in the sample to be detected can be captured and combined with high sensitivity and high specificity, and the fluorine ion concentration can be converted into the fluorescence signal based on the CRISPR technology so as to amplify the fluorine ion concentration signal based on the fluorescence signal. Due to the fact that the combination of the fluoride riboswitch and the transcription reaction are high in efficiency and stability, the detection method can achieve rapid and accurate detection of the fluorine content of the to-be-detected sample. The fluorine content detection method provided by the invention is a method integrating the advantages of low cost, high sensitivity, high specificity, simplicity and convenience in operation, reliable result and the like, and has application prospects in industrial detection and temporary detection scenes.

Description

Technical Field

[0001] This invention relates to the field of fluoride content detection technology, and in particular to a method and application for fluoride content detection based on transcriptional regulation by fluoride riboswitch. Background Technology

[0002] Antarctic krill (Euphausia superba) is one of the most abundant and successfully propagated single biological resources on Earth. Rich in nutrients, Antarctic krill contains not only protein, essential amino acids, and unsaturated fatty acids such as linoleic acid and linolenic acid, but also various minerals including calcium, potassium, magnesium, and strontium, making it a resource with immense development potential. For example, Antarctic krill oil, developed from krill, is rich in phospholipid-type polyunsaturated fatty acids and astaxanthin, exhibiting activities such as inhibiting liver steatosis, anti-allergy, antioxidant, lipid-lowering, blood sugar-lowering, and cardioprotective effects. However, due to the high fluoride content in Antarctic krill, excessively high fluoride levels in krill products can affect human health. Therefore, it is necessary to test the fluoride content in Antarctic krill to facilitate appropriate measures for controlling the fluoride content of Antarctic krill and its products.

[0003] For the detection of fluoride content, existing technologies mainly employ ion-selective electrode (ISE), ion chromatography (IC), and spectrophotometric colorimetry. However, each of these methods has its advantages and disadvantages when applied to the detection of fluoride content in Antarctic krill, and none are entirely suitable for the specific requirements of fluoride content detection in Antarctic krill. Among these, the fluoride ion-selective electrode method is the most commonly used method in laboratories, offering advantages such as simple and convenient operation and low cost. However, the detection results are highly sensitive to environmental conditions; temperature changes or stirring speeds can affect the experimental results. Ion chromatography offers advantages such as high detection sensitivity, good selectivity, and low detection limits, but its stringent sample pretreatment requirements and high detection costs make it unsuitable for routine testing in most laboratories. While the spectrophotometric colorimetric method using fluoride reagents is relatively simple to operate and process, the high levels of astaxanthin in Antarctic krill can interfere with absorbance readings, thus affecting the final experimental results and resulting in lower detection accuracy.

[0004] For example, in the prior art, Chinese patent application CN102967646A discloses a rapid detection method for fluoride ion content in Antarctic krill. This method utilizes a coaxial probe method to determine the dielectric properties of NaF deionized water solutions, plots and determines a standard curve of dielectric loss rate for NaF deionized water solutions at concentrations of 0.1 mg / ml-50 mg / ml under low-frequency conditions of 300 MHz-3000 MHz. Then, Antarctic krill powder is refluxed and heated for 1-4 hours to prepare a NaF deionized water solution sample. The fluoride content of the sample is detected based on the dielectric loss rate and the standard curve. However, the method provided by this patent requires 1-4 hours to prepare the sample, resulting in a relatively long total detection time. Furthermore, the dielectric constant in the solution is affected by various factors, including solute concentration and temperature. Temperature affects the thermal motion of solute molecules, thus affecting the degree of polarization. Therefore, the detection results for fluoride content are relatively unstable and have low data reliability under different temperatures and sample concentrations. Summary of the Invention

[0005] To address the technical problems of excessively long detection time and low detection accuracy in Antarctic krill, this invention provides a method and application for detecting fluoride content by regulating transcription through fluoride riboswitch.

[0006] The specific technical solution of this invention is as follows: This invention provides a method for detecting fluoride content based on transcriptional regulation by a fluoride riboswitch, comprising the following steps: Step S1: Provide a detection reaction system, the detection reaction system comprising template DNA, Cas12a protein, double-stranded DNA, and single-stranded DNA fluorescent probe; The template DNA sequence contains a fluoride riboswitch sequence. When the fluoride riboswitch sequence binds to fluoride ions, crRNA is transcribed. When the fluoride riboswitch sequence does not bind to fluoride ions, crRNA is not transcribed. The sequence of the double-stranded DNA is complementary to the sequence of the crRNA, and is used to activate the Cas12a protein to cleave the single-stranded DNA fluorescent probe. The single-stranded DNA fluorescent probe is used to generate a fluorescent signal after being cleaved; Step S2: Add the sample to be tested into the detection reaction system, react to the endpoint, and obtain the fluoride ion content in the sample based on the intensity of the fluorescence signal.

[0007] This invention utilizes the biological element of a fluoride riboswitch to achieve highly sensitive and specific capture and binding of fluoride ions in the sample. Based on CRISPR technology, it converts fluoride ion concentration into a fluorescence signal, thereby amplifying the fluoride ion concentration signal. Due to the high efficiency and stability of the binding and transcription reactions of the fluoride riboswitch, this invention enables rapid and accurate detection of fluoride content in the sample.

[0008] As a preferred embodiment of the above-mentioned fluoride content detection method, the nucleotide sequence of the fluoride riboswitch sequence is shown in SEQ ID NO.2.

[0009] As a preferred embodiment of the above-mentioned fluoride content detection method, the nucleotide sequence of the template DNA is shown in SEQ ID NO.1.

[0010] As a preferred embodiment of the above-mentioned fluoride content detection method, the nucleotide sequence of the complementary sequence of the double-stranded DNA and crRNA is shown in SEQ ID NO.3.

[0011] As a preferred embodiment of the above-mentioned fluoride content detection method, the single-stranded DNA fluorescent probe comprises a DNA strand, a fluorescent group FAM labeled at the 5' end of the DNA strand, and a quencher group BHQ1 labeled at the 3' end of the DNA strand, wherein the nucleotide sequence of the DNA strand is TTATT.

[0012] As a preferred embodiment of the above-mentioned fluoride content detection method, the detection reaction system comprises the following components: template DNA 1~5 nM, NTP 0.1~1 mM, cell-free extract volume percentage of 5%~25%, Cas12a protein 0.1~0.5 μM, double-stranded DNA 0.5~2 nM, and single-stranded DNA fluorescent probe 0.5~1 nM.

[0013] As a preferred embodiment of the above-mentioned fluoride content detection method, in step S2, the reaction time to the endpoint is 20-60 minutes.

[0014] As a preferred method for detecting fluoride content, the fluoride ion content in the sample to be tested is obtained based on the intensity of the fluorescence signal, including the following steps: Sodium fluoride standard samples of different concentrations were added to the detection reaction system, and the fluorescence signal intensity of the reaction endpoint corresponding to the sodium fluoride standard samples of different concentrations was collected. A standard curve of fluorescence signal intensity-sodium fluoride concentration was plotted. Based on the fluorescence signal intensity of the sample to be tested and the standard curve of fluorescence signal intensity-sodium fluoride concentration, the sodium fluoride concentration data corresponding to the fluorescence signal intensity of the sample to be tested was obtained.

[0015] As a preferred method for detecting the fluoride content, the preparation method of the sample to be tested is as follows: drying and pulverizing Antarctic krill, adding hydrochloric acid to extract the fluoride from the Antarctic krill powder, obtaining an extract, which is then diluted or not diluted to obtain the sample to be tested.

[0016] Based on the above, the present invention provides an application of a detection method in detecting fluoride content in Antarctic krill.

[0017] Compared with the prior art, the present invention has the following technical effects: (1) This invention utilizes the biological element of a fluoride riboswitch to achieve high sensitivity and specificity in capturing and binding fluoride ions in the sample to be tested. Based on CRISPR technology, it realizes the conversion of fluoride ion concentration into a fluorescence signal, thereby amplifying the fluoride ion concentration signal based on the fluorescence signal. Since the binding and transcription reactions of the fluoride riboswitch are highly efficient and stable, the detection method of this invention can achieve rapid and accurate detection of the fluoride content in the sample to be tested.

[0018] (2) The fluoride content detection method provided by the present invention is based on the transcription reaction of cell-free extract, without the need to add commercial RNA transcriptase, which is cheaper and has the advantage of low detection cost.

[0019] (3) The fluorine content detection method provided by the present invention is a method that integrates the advantages of low cost, high sensitivity, high specificity, simple operation and reliable results, and has application prospects in industrial detection and temporary detection scenarios. Attached Figure Description

[0020] Figure 1 The figure shows the results of the optimization experiment for DNA template concentration in this invention; Figure 2 This is a graph showing the results of the screening test for the amount of cell-free extract added in this invention; Figure 3 This is a standard curve of fluorescence intensity versus sodium fluoride concentration (negative logarithm) according to the present invention. Figure 4 This is a graph showing the interference results of different ions on the detection of fluoride ions in this invention. Detailed Implementation

[0021] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0022] This embodiment provides a method for detecting fluoride content based on fluoride riboswitch-regulated transcription, including the following steps: Step S1: Provide a detection reaction system, the detection reaction system comprising template DNA, Cas12a protein, double-stranded DNA, and single-stranded DNA fluorescent probe; The template DNA sequence contains a fluoride riboswitch sequence. When the fluoride riboswitch sequence binds to fluoride ions, crRNA is transcribed. When the fluoride riboswitch sequence does not bind to fluoride ions, crRNA is not transcribed. The sequence of the double-stranded DNA is complementary to the sequence of the crRNA, and is used to activate the Cas12a protein to cleave the single-stranded DNA fluorescent probe. The single-stranded DNA fluorescent probe is used to generate a fluorescent signal after being cleaved; Step S2: Add the sample to be tested into the detection reaction system, react to the endpoint, and obtain the fluoride ion content in the sample based on the intensity of the fluorescence signal.

[0023] The method described in this embodiment utilizes the biological element of a fluoride riboswitch to achieve high sensitivity and specificity in capturing and binding fluoride ions in the sample. Based on CRISPR technology, it converts fluoride ion concentration into a fluorescence signal, thereby amplifying the fluoride ion concentration signal. Because the binding and transcription reactions of the fluoride riboswitch are highly efficient and stable, this detection method can achieve rapid and accurate detection of the fluoride content in the sample.

[0024] The nucleotide sequence of the template DNA is shown in SEQ ID NO.1, the nucleotide sequence of the fluoride riboswitch sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the double-stranded DNA is shown in SEQ ID NO.3 (crRNA complementary strand). The single-stranded DNA fluorescent probe is FAM-TTATT-BHQ1, comprising a DNA strand, a fluorescent group FAM labeled at the 5' end of the DNA strand, and a quencher group BHQ1 labeled at the 3' end of the DNA strand, wherein the nucleotide sequence of the DNA strand is TTATT.

[0025] The two DNA sequences of the double-stranded DNA are shown below: 5'-AAGTTTACCTTATTAGGTACTGTTATCGTC-3'; 5'-GACGATAACAGTACCTAATAAGGTAAACTT-3'.

[0026] The RNA sequence of the crRNA is shown below: 5'-UAAUUUCUACUAAGUGUAGAUCCUUAUUAGGUACUGUUAUC-3'.

[0027] In one embodiment, the detection reaction system comprises the following components: template DNA 1-5 nM, NTP 0.1-1 mM, cell-free extract volume percentage of 5%-25%, Cas12a protein 0.1-0.5 μM, double-stranded DNA 0.5-2 nM, and single-stranded DNA fluorescent probe 0.5-1 nM.

[0028] Template DNA, double-stranded DNA, and single-stranded DNA fluorescent probes are obtained through whole-genome synthesis technology. Cell-free extracts are derived from cell-free extracts of E. coli, and other raw materials are sourced from commercially available sources.

[0029] The cell-free extract is used to replace commercially available RNA transcriptase to catalyze the transcription reaction. The cell-free extract can be derived from prokaryotic cell extracts, such as *Escherichia coli* and *Bacillus subtilis*, or from eukaryotic cells, such as *Saccharomyces cerevisiae*, or from plant cells, such as wheat germ. In this embodiment of the invention, a cell-free extract derived from *E. coli* is used, and the preparation method is as follows: *E. coli* BL21 (OD21) in the mid-log phase is selected. 600 =0.7), the bacterial cells were collected by centrifugation at 4℃ and 5000 g for 10 minutes, and the cell pellet was washed three times with pre-cooled S30 Buffer to remove residual culture medium.

[0030] In one embodiment, in step S2, the reaction time to the endpoint is 20-60 minutes.

[0031] In one embodiment, the fluoride ion content in the sample to be tested is obtained based on the intensity of the fluorescence signal, including the following steps: Sodium fluoride standard samples of different concentrations were added to the detection reaction system, and the fluorescence signal intensity of the reaction endpoint corresponding to the sodium fluoride standard samples of different concentrations was collected. A standard curve of fluorescence signal intensity-sodium fluoride concentration was plotted. Based on the fluorescence signal intensity of the sample to be tested and the standard curve of fluorescence signal intensity-sodium fluoride concentration, the sodium fluoride concentration data corresponding to the fluorescence signal intensity of the sample to be tested was obtained.

[0032] In one embodiment, the method for preparing the sample to be tested is as follows: drying and pulverizing Antarctic krill, adding hydrochloric acid to extract fluoride from the Antarctic krill powder to obtain an extract, which is then diluted or not diluted to obtain the sample to be tested.

[0033] To make the present invention clearer, the following embodiments are provided.

[0034] Example 1 A method for detecting fluoride content in Antarctic krill includes the following steps: (1) Preparation of a combined detection reaction system of transcription reaction system and CRISPR-Cas12a fluorescence detection system: Template DNA 5 nM, NTP 0.2 mM, cell-free extract volume ratio 3 μL, 5X T7 buffer 4 μL, double-stranded DNA 1 nM, single-stranded DNA fluorescent probe 0.8 nM and Cas12a protein 0.1 μM, and the total volume was made up to 20 μL with sterile water.

[0035] The nucleotide sequence of the template DNA is shown in SEQ ID NO.1, the nucleotide sequence of one strand of the double-stranded DNA is shown in SEQ ID NO.3, and the single-stranded DNA fluorescent probe is FAM-TTATT-BHQ1.

[0036] (2) To determine the optimal concentration of template DNA, the template DNA in the composite detection reaction system of step (1) was replaced with concentrations of 0.1 nM, 0.5 nM, 1 nM, and 5 nM, respectively, to prepare five composite detection reaction systems with a total volume of 20 μL. 0.5 μL of 4 mM sodium fluoride solution was added to each composite detection reaction system to bring the final sodium fluoride concentration to 0.1 mM before the reaction. The reaction was carried out at 37°C for 30 minutes, and the fluorescence intensity of the system after the reaction was detected. The results are shown in […]. Figure 1 .

[0037] Next, five 20 μL composite reaction detection systems were prepared with template DNA concentrations of 0.1 nM, 0.5 nM, 1 nM, and 5 nM, respectively. 0.5 μL of 8 mM sodium fluoride solution was added to each composite reaction system to bring the final sodium fluoride concentration to 0.2 mM. The reaction was carried out at 37°C for 30 minutes, and the fluorescence intensity of the system was measured after the reaction. The results are shown in [Figure number missing]. Figure 1 .

[0038] Next, five 20 μL composite reaction detection systems were prepared with template DNA concentrations of 0.1 nM, 0.5 nM, 1 nM, and 5 nM. 0.5 μL of 20 mM sodium fluoride solution was added to each composite reaction system to bring the final sodium fluoride concentration to 0.5 mM. The reaction was carried out at 37°C for 30 minutes, and the fluorescence intensity of the system was measured after the reaction. The results are shown in [Figure number missing]. Figure 1 . Depend on Figure 1 The results showed that the fluorescence change value caused by different concentrations of sodium fluoride reached its maximum when the template DNA was 5 nM. Therefore, a DNA template concentration of 5 nM was selected for subsequent detection of sodium fluoride concentration. (3) To determine the optimal concentration of the cell-free extract, the cell-free extract in the composite detection reaction system of step (1) was replaced with concentrations of 1 μL, 3 μL, and 5 μL, respectively, to prepare three composite reaction detection systems with a total volume of 20 μL. 0.5 μL of 8 mM sodium fluoride solution was added to each composite detection reaction system to achieve a sodium fluoride concentration of 0.2 mM. The system was then incubated at 37°C. The fluorescence intensity of the reaction system was detected using fluorescence kinetics. The results are shown in […]. Figure 2 .

[0039] Depend on Figure 2 The results showed that, compared with 1 μL of cell-free extract, the fluorescence intensity induced by sodium fluoride was significantly enhanced when 3 μL and 5 μL of cell-free extract were added. Moreover, the fluorescence intensity induced by 3 μL and 5 μL of cell-free extract was not significantly different. Therefore, the volume of cell-free extract added was selected as 3 μL for subsequent testing.

[0040] (4) Through the above experiments, the following composition and ratio of the composite detection reaction system were determined: Template DNA 5 nM, NTP 0.2 mM, cell-free extract volume ratio 3 μL, 5X T7 buffer 4 μL, double-stranded DNA 1 nM, single-stranded DNA fluorescent probe 0.8 nM and Cas12a protein 0.1 μM, and the total volume was made up to 20 μL with sterile water.

[0041] Sodium fluoride solution was added to the composite detection reaction system in this step to achieve final concentrations of 0.006, 0.012, 0.060, 0.12, 0.24, 0.48, 0.96, and 0.12 mM, respectively. The reaction was incubated at 37°C for 30 minutes, and then terminated by heating in a metal bath at 90°C for 1 minute. The fluorescence signal was detected using an ELISA reader. The assay was performed in triplicate, and a standard curve of fluorescence intensity versus sodium fluoride concentration (negative logarithm) was plotted. The linear relationship of the fitted curve was: Y = -789589.59881X + 2586382.92029 (R² = 0.99), with a linear range of 0.006~0.12 mM. The limit of detection (LOD) was calculated to be 0.001 mM.

[0042] The standard curve of fluorescence intensity versus sodium fluoride concentration (negative logarithm) is shown below. Figure 3 As shown. By Figure 3 It can be seen that there is a good linear relationship between fluorescence intensity and sodium fluoride concentration.

[0043] (5) Detection of fluoride content in Antarctic krill samples: Antarctic krill samples were separated into shells and meat, which were dried separately in an oven at 70°C, pulverized using a grinder, passed through a 40-mesh sieve, and stored in a desiccator.

[0044] Weigh 1.00 g of shrimp shell and shrimp meat samples that have passed through a 40-mesh sieve, place them in a 15 mL centrifuge tube, and add 10 mL of hydrochloric acid. Seal and extract for 1 h, gently shaking occasionally to promote complete extraction. After centrifugation at 5000 g for 10 min, take the supernatant, filter it through a 0.45 μm filter membrane, add 25 mL of distilled water to the filtrate, adjust the pH to 7.0 with NaOH, transfer it to a 50 mL volumetric flask, make up to the mark with ultrapure water, add 5 μL to the composite detection reaction system obtained in step (4), incubate at 37℃ for 30 min, and then heat in a 90℃ metal bath for 1 min to terminate the reaction. Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the fluorescence signal, perform three parallel measurements, and calculate the fluoride ion content in the shrimp shell and shrimp meat samples to be tested as 1995.82 mg / kg and 180.51 mg / kg, respectively, based on the working curve obtained in step (4).

[0045] (6) Verification of the method specificity of this embodiment To investigate the interference of common ions, a composite reaction detection system with a total volume of 20 μL was prepared, consisting of 10 steps (4). Fluoride ions or other ions with a concentration of 200 μmol / L were added to each system. These other ions included cations and anions, such as chlorobromoiodide ions, carbonate ions, sulfate ions, phosphate ions, hypophosphite ions, nitrate ions, and acetic anhydride ions. The fluorescence intensity of each system was measured after the reaction. The fluorescence intensity of the systems with added ions was compared with that with those with added fluoride ions to verify the interference effect of other ions on fluoride ions. Sterile water was used as the negative control group. The results are as follows: Figure 4 As shown.

[0046] Figure 4 The results show that the addition of different types of cations and anions has little interference with the detection system, indicating that the method of the present invention has excellent specificity for detecting fluoride ions.

[0047] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

[0049] Although specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, without departing from the direction of the invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting fluoride content based on transcriptional regulation by a fluoride riboswitch, characterized in that: Includes the following steps: Step S1: Provide a detection reaction system, the detection reaction system comprising template DNA, Cas12a protein, double-stranded DNA, and single-stranded DNA fluorescent probe; The template DNA sequence contains a fluoride riboswitch sequence. When the fluoride riboswitch sequence binds to fluoride ions, crRNA is transcribed. When the fluoride riboswitch sequence does not bind to fluoride ions, crRNA is not transcribed. The sequence of the double-stranded DNA is complementary to the sequence of the crRNA, and is used to activate the Cas12a protein to cleave the single-stranded DNA fluorescent probe. The single-stranded DNA fluorescent probe is used to generate a fluorescent signal after being cleaved; Step S2: Add the sample to be tested into the detection reaction system, react to the endpoint, and obtain the fluoride ion content in the sample based on the intensity of the fluorescence signal.

2. The method for detecting fluoride content based on transcriptional regulation by fluoride riboswitch as described in claim 1, characterized in that: The nucleotide sequence of the fluoride riboswitch sequence is shown in SEQ ID NO.

2.

3. The method for detecting fluoride content based on transcription regulated by fluoride riboswitch as described in claim 1 or 2, characterized in that: The nucleotide sequence of the template DNA is shown in SEQ ID NO.

1.

4. The method for detecting fluoride content based on transcriptional regulation by fluoride riboswitch as described in claim 1, characterized in that: The nucleotide sequence of the complementary sequence of the double-stranded DNA and crRNA is shown in SEQ ID NO.

3.

5. The method for detecting fluoride content based on transcriptional regulation by fluoride riboswitch as described in claim 1, characterized in that: The single-stranded DNA fluorescent probe includes a DNA strand, a fluorescent group FAM labeled at the 5' end of the DNA strand, and a quencher group BHQ1 labeled at the 3' end of the DNA strand, wherein the nucleotide sequence of the DNA strand is TTATT.

6. The method for detecting fluoride content based on transcriptional regulation by fluoride riboswitch as described in claim 1, characterized in that: The detection reaction system comprises the following components: template DNA 1-5 nM, NTP 0.1-1 mM, cell-free extract volume percentage of 5%-25%, Cas12a protein 0.1-0.5 μM, double-stranded DNA 0.5-2 nM, and single-stranded DNA fluorescent probe 0.5-1 nM.

7. The method for detecting fluoride content based on transcriptional regulation by fluoride riboswitch as described in claim 1, characterized in that: In step S2, the reaction time to the endpoint is 20-60 minutes.

8. The method for detecting fluoride content based on transcriptional regulation by fluoride riboswitch as described in claim 1, characterized in that: The fluoride ion content in the sample is obtained based on the intensity of the fluorescence signal, including the following steps: Sodium fluoride standard samples of different concentrations were added to the detection reaction system, and the fluorescence signal intensity of the reaction endpoint corresponding to the sodium fluoride standard samples of different concentrations was collected. A standard curve of fluorescence signal intensity-sodium fluoride concentration was plotted. Based on the fluorescence signal intensity of the sample to be tested and the standard curve of fluorescence signal intensity-sodium fluoride concentration, the sodium fluoride concentration data corresponding to the fluorescence signal intensity of the sample to be tested was obtained.

9. The method for detecting fluoride content based on transcriptional regulation by fluoride riboswitch as described in claim 1, characterized in that: The preparation method of the sample to be tested is as follows: Antarctic krill is dried and crushed, hydrochloric acid is added to extract the fluoride in the Antarctic krill powder to obtain an extract, which is then diluted or not diluted to obtain the sample to be tested.

10. The application of the detection method according to any one of claims 1 to 9 in the detection of fluoride content in Antarctic krill.

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