Method for synchronously and quantitatively determining epithelial cells of Atlantic salmon, karenia mikimotoi and vibrio parahaemolyticus in seawater

Abstract

The invention relates to a method for synchronously and quantitatively determining Atlantic salmon epithelial cells, Karenia mikimotoi and Vibrio parahaemolyticus in seawater, and belongs to the technical field of detection. Respectively adding the extracted eDNA into a reaction tube of an LAMP reaction system filled with a targeted Atlantic salmon marker gene Cytb, a Karenia mikimotoi marker gene ITS2 and a Vibrio parahaemolyticus marker gene tlh, and then putting the reaction tube into a full-automatic electronic gene amplification analyzer to obtain slopes kf, ka and kb of a linear fitting equation corresponding to a kinetic curve exponential phase, and substituting the Atlantic salmon epithelial cells, the Karenia mikimotoi and the vibrio parahaemolyticus into formulas LogNf = 52.96 kf + 0.27, LogNa = 88.13 ka-0.54 and LogNb = 70.79 kb < 2 > + 35.67 kb + 0.70, and respectively calculating the numbers Nf, Na and Nb of the Atlantic salmon epithelial cells, the Karenia mikimotoi and the vibrio parahaemolyticus in the detected seawater sample. According to the method, the number of target animal cells, algae cells and microbial cells can be synchronously obtained within 2 hours.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of detection, and particularly relates to a method for synchronously and quantitatively determining Atlantic salmon epithelial cells, kathironella and Vibrio parahaemolyticus in seawater. BACKGROUND

[0002] In marine resource and environmental investigation, it is difficult for the prior art to obtain qualitative and quantitative information of animals, plants, algae and microorganisms within 2 hours, and high timeliness data cannot be provided for scientific research, environmental monitoring, fishery production and other activities. By analyzing the gene information carried in environmental DNA (eDNA), it is possible to provide qualitative and quantitative information of animals, plants, algae and microorganisms in seawater at the same time. In particular, the combination of isothermal gene amplification (LAMP) technology and eDNA technology has paved the way for on-site analysis. However, since the eDNA samples collected from seawater are often complex, so far there is no detection technology that can obtain the amount of complete cells of animals, algae and microorganisms in seawater eDNA on-site, synchronously, qualitatively and quantitatively on a marine investigation ship.

[0003] Atlantic salmon (Salmo salar) Salmo salarSalmon is a migratory fish with high economic and ecological value. Adults inhabit the North Atlantic Ocean, and its delicious flesh has made it a core species in global aquaculture. Wild populations play a crucial role in maintaining the balance of river and marine ecosystems. Traditional salmon resource surveys mainly rely on trawl fishing, acoustic detection, and manual counting. These methods have limitations such as high cost, significant disturbance to fish populations, low spatiotemporal resolution, and difficulty in covering complex habitats. eDNA-based technology monitors species by detecting shed material (mainly epithelial cells) in the water. Its advantages include non-invasiveness, high sensitivity, high efficiency, and applicability to large-scale surveys. Recent research progress shows that eDNA combined with gene amplification (such as qPCR and LAMP) can accurately identify fish marker genes (Ramey AM, McKeeman CM, Petrou EL, et al. Environmental DNA as a Tool for Better Understanding the Distribution, Abundance, And Health of Atlantic Salmon and Pacific Salmon. Fisheries, 2024, 49(4): 169-180). Li et al. (Li Y, Xue H, Fei Y, et al. A rapid and closed-tube method based on the dual-color fluorescence loop-mediated isothermal amplification for visual detection of Atlantic salmon) Salmo salar A method for identifying Atlantic salmon using dual-color fluorescence discrimination of LAMP amplification results has recently been developed (Food Chemistry, 2023, 405: 134975). However, this method can only qualitatively detect the presence of the target gene.

[0004] Karenia mikimotoi ( Karenia mikimotoi ☐ is a common, highly toxic red tide algae. When it proliferates in large numbers to form red tides, it produces hemolytic toxins and neurotoxins, leading to mass fish deaths and causing devastating damage to shellfish farming (Wang C, Xu Y, Gu H, et al. Potential geographical distribution of harmful algal blooms caused by the toxic dinoflagellate). Karenia mikimotoiIn the China Sea. Science of the Total Environment, 2024, 906: 167741). Detection and monitoring of *Karenella mikimotoi* mainly rely on microscopic counting, remote sensing, and flow cytometry. These methods have limitations such as high implementation difficulty, time-consuming and labor-intensive methods, and difficulty in achieving accurate quantification. Combining gene analysis and eDNA technology is expected to significantly improve the efficiency of detecting this algae (Jacobs-Palmer E, Gallego R, Cribari K, et al. Environmental DNAmetabarcoding for simultaneous monitoring and ecoLogical assessment of manyharmful algae. Frontiers in EcoLogy and Evolution, 2021, 9: 612107). LAMP technology provides the possibility for rapid, on-site detection of *Karenella mikimotoi* in water bodies (Han X, Zhao T, Yan T, et al. Rapid and sensitive detection of Karenia mikimotoi (by loop-mediated isothermal amplification combined with a lateral flow dipstick. Environmental Science and Pollution Research, 2022, 29: 24696-24703). However, there is currently no LAMP method for simultaneous qualitative and quantitative determination of Karenia mikimotoi at the survey site.

[0005] Vibrio parahaemolyticus ( Vibrio parahaemolyticus Silver nanoparticles are halophilic Gram-negative bacteria commonly found in estuaries and coastal waters. They are one of the main pathogens causing foodborne gastroenteritis in humans and are also opportunistic pathogens in aquatic organisms such as shrimp and crabs (Yang Q, Hou X, Lu F, et al. Evidently diverse effects of silvernanoparticles on Vibrio parahaemolyticusEnvironmental Science: Nano, 2025, 12(5): 2657-2666). Traditional methods for detecting V. parahaemolyticus in seawater and aquaculture water mainly rely on culture isolation and biochemical identification, which takes several days (Benjakul S, Sukkapat P, Palamae S, et al. Development of colorimetric multiplex loop-mediated isothermal amplification combined with lateral flow dipstick (mLAMP-LFD) assay for the identification of TDH toxin-producing Vibrio parahaemolyticus . Journal of Food Composition and Analysis, 2025, 137:106924). The combination of eDNA technology and LAMP technology is expected to provide a possibility for on-site and efficient detection of V. parahaemolyticus in seawater (Bass D, Christison K W, Stentiford G D, et al. Environmental DNA / RNA for pathogen and parasite detection, surveillance, and ecology. Trends in Parasitology, 2023, 39(4): 285-304). Patent CN201710421611.7 discloses a method for selectively detecting live V. parahaemolyticus in seawater, which includes steps of collecting total bacteria by membrane filtration, treating the collected sample with sodium azide, and determining the target gene by LAMP. Patent CN202410524010.9 discloses a method for detecting live V. parahaemolyticus in estuarine water, which works as follows: In estuarine water samples, there are generally intracellular DNA in living cells and extracellular DNA released from dead cells. In the case of V. parahaemolyticus, both intracellular DNA and extracellular DNA contain its marker gene tlh . When the estuarine water sample is directly added to the preloaded LAMP reaction system (which is used for specific analysis of the target gene of V. parahaemolyticus), the LAMP reaction system can be used to detect the presence of V. parahaemolyticus in the estuarine water sample. tlhAfter the sample (containing LAMP) is added to detection tube M, the tube opening is sealed and placed in a detection channel of the electronic gene amplification instrument's detection unit. The detection channel is heated to 42°C, and a square wave pulse signal with specific parameters is applied to the mixture in detection tube M through the instrument's functional component—a capacitively coupled non-contact conductivity electrode—causing the active bacterial cells to lyse and release intracellular DNA. After the square wave pulse is applied, the total DNA in the mixture includes proto-extracellular DNA and proto-intracellular DNA released from the lysed live cells. When the same estuarine water sample is added to detection tube N pre-filled with LAMP reaction mixture, the tube opening is sealed and placed in another detection channel of the electronic gene amplification instrument, and the temperature is also raised to 42°C, but a square wave pulse signal is not applied to the mixture in the detection tube through the capacitively coupled non-contact conductivity electrode. In this case, the live bacterial cells do not lyse, and the total DNA in the mixture only includes proto-extracellular DNA. The detection channel of the electronic gene amplification instrument is heated to 61°C, and... Bst Under the action of DNA polymerase, primers specifically amplify DNA in the reaction system using DNA as a template. tlh Gene fragments. An automated online monitoring system for the LAMP biochemical reaction process in detection tubes M and N, reporting the peak time of the rate curves. T m and T n . T m and T n Value and tlh Gene fragment quantity and cell quantity are inversely proportional; substituting them into the formula... T (s) = -260.0Log C The concentration of Vibrio parahaemolyticus in the estuary water sample was calculated as (CFU) + 2704.7, corresponding to the total DNA (extracellular DNA + intracellular DNA released from the lysis of viable Vibrio parahaemolyticus). C total (Unit: CFU / mL) and Vibrio parahaemolyticus concentration corresponding to extracellular DNA C background (Unit: CFU / mL). The concentration difference between the two ( C total - C background The concentration of live Vibrio parahaemolyticus in the sample can then be calculated. C net (Unit: CFU / mL). The entire time from sampling to obtaining the concentration of active Vibrio parahaemolyticus in estuarine water does not exceed 1 hour. However, studies have shown that this specific square wave pulse cannot disrupt animal, plant, and algal cells, and therefore cannot be applied to the development of techniques for simultaneously determining animal, plant, algal, and microbial samples.

[0006] The combination of eDNA technology and LAMP technology provides the possibility of quickly obtaining qualitative and quantitative information of animals, plants, algae and microorganisms on the sea survey site. However, not all ships used for marine resource and environment investigation are professional research vessels, and even if they are professional research vessels, the smaller models cannot carry large and valuable instruments and equipment. Therefore, so far, the international community still needs to develop a universal and efficient marine biological investigation technology applicable to various laboratories and investigation ships. In addition, when data from eDNA is used to infer and calculate the qualitative and quantitative information of marine organisms in seawater, the genetic information from intact cells (usually living cells) is often more valuable and can truly and directly reflect the timely status of the marine ecological environment. Therefore, it is required to detect without interference from free DNA (extracellular DNA) in seawater. SUMMARY

[0007] The present application provides a method for simultaneously and quantitatively determining Atlantic salmon epithelial cells, K. micronesiaca and V. parahaemolyticus in seawater, which can simultaneously qualitatively and quantitatively determine animal cells, algal cells and microbial cells within 2 hours.

[0008] The present application is realized by the following technical scheme: a method for simultaneously determining the number of Atlantic salmon epithelial cells, K. micronesiaca and V. parahaemolyticus in seawater, which is not for the purpose of disease treatment and prevention, extracts eDNA, adds the extracted eDNA into reaction tubes containing LAMP reaction systems targeting Atlantic salmon marker genes Cytb、 K. micronesiaca marker genes ITS2 and V. parahaemolyticus marker genes tlh , then places the reaction tubes into a full-automatic electronic gene amplification analyzer to determine the LAMP biochemical reaction kinetics curves of the Atlantic salmon marker genes Cytb , K. micronesiaca marker genes ITS2 and V. parahaemolyticus marker genes tlh in real time online, reads the slopes of the linear fitting equations corresponding to the exponential phase of the kinetics curves k f , k a and k b , and substitutes them into the formula Log N f (cells / L) = 52.96 k f (mV / s) + 0.27, Log N a (cells / L) = 88.13 k a (mV / s) - 0.54 and LogN b ( cells / L) = 70.79 k b 2 ( mV / s) + 35.67 k b ( mV / s) + 0.70, respectively, the number of Atlantic salmon epithelial cells, Mithraculum karen and Vibrio parahaemolyticus intact cells in the measured seawater sample is calculated N f 、 N a and N b .

[0009] Further, the preparation method of the eDNA is: Step one, the eDNA in seawater is collected on the filter membrane by suction filtration method, the filter membrane is taken out from the suction filtration device, and then is put into the detection tube preloaded with DNase I Buffer, and is incubated in 36℃ water bath with shaking, so that the extracellular DNA attached to the filter membrane is removed by enzymatic hydrolysis; Step two, 10% of triton X-100 by volume is added to the detection tube treated in step one, and the liquid in the detection tube is boiled and kept boiling for 4 min by continuously heating in water bath; Step three, after the treatment in step two, the detection tube is taken out and quickly cooled to obtain a lysis mixture, and the mixture is centrifuged to obtain a supernatant containing dissolved DNA.

[0010] Further, the LAMP reaction system comprises 20 mmol / L Tris-HCl, 10 mmol / L KCl, 10 mmol / L (NH4)2SO4, 6 mmol / L MgSO4, 0.1% Tween 20, 1.4 mmol / L dNTPs, 0.4 mol / L betaine, 8 U / µL DNA polymerase and primers.

[0011] Further, the primer sequence is as shown in SEQ ID NO. 1-16.

[0012] Further, the reaction parameters of the full-automatic electronic gene amplification analyzer are as follows: working temperature 58 ℃, excitation voltage 16 V, excitation frequency 2.0 MHz, signal acquisition period 1 s, and acquisition time length 30 min.

[0013] Compared with the prior art, the method of the present application can simultaneously obtain the number of animal (Atlantic salmon), algal (Mithraculum karen) and microbial (Vibrio parahaemolyticus) cells in seawater through simple operation, and ensures the number of intact cells, and the data is not disturbed by the extracellular DNA in the environment.

[0014] This invention establishes highly specific and accurate quantitative algorithm models for three significantly different organisms: aquatic animals (Atlantic salmon), algae (Karenella mikimotoi), and bacteria (Vibrio parahaemolyticus). Through experimental verification, the quantitative relationship between the slope of the exponential phase of the LAMP reaction kinetic curve and the logarithmic value of the cell number was determined for the first time: a linear model for Atlantic salmon and Karenella mikimotoi, and a polynomial model for Vibrio parahaemolyticus.

[0015] These algorithms not only achieve direct conversion from LAMP reaction kinetic signals to the number of intact cells, but also have extremely high coefficients of determination (R² > 0.99), ensuring the reliability of quantitative results. Compared with existing technologies that can usually only achieve qualitative or rough quantitative analysis of a single type of organism, the algorithm system of this invention breaks through the technical barriers caused by differences in biological categories, and can simultaneously output the precise cell concentration of three types of targets within 2 hours, providing a new, efficient, and integrated on-site quantitative monitoring method for marine ecological environment and aquaculture water. This invention, through system optimization, amplifies the LAMP reaction of three different species using the same physicochemical parameters, realizing the simultaneous analysis of three cross-species cell categories; (4) It does not involve large and expensive instruments and equipment, and can be analyzed on-site on marine survey vessels, eliminating the time and cost of sample preservation, turnover, and transportation. The total cycle does not exceed 2 hours, providing high-time data. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the measurement process; Figure 2 To target Atlantic salmon epithelial cell marker genes Cytb LAMP reaction kinetics curves; F1, F2, F3, F4, F5, and F6 correspond to cell concentrations of 1×10⁻⁶. 1 cells / L, 1×10 2 cells / L, 1×10 3 cells / L, 1×10 4 cells / L, 1×10 5 cells / L and 1×10 6 The test tube contains cells / L; Pp and Pn correspond to the positive and negative controls, respectively. Figure 3 To target the marker gene of Karenia mikimotoi ITS2 LAMP reaction kinetics curves; A1, A2, A3, A4, A5, and A6 correspond to cell concentrations of 1 × 10⁻⁶. 1 cells / L, 1×10 2 cells / L, 1×10 31 x 10 4 1 x 10 5 1 x 10 6 1 x 10 1 1 x 10 2 1 x 10 3 1 x 10 4 1 x 10 5 1 x 10 6 1 x 10 Pp and Pn correspond to positive control and negative control, respectively. Figure 4 LAMP reaction kinetics curve targeting Vibrio parahaemolyticus marker gene tlh B1, B2, B3, B4, B5 and B6 correspond to detection tubes with cell concentration of 1 x 10 1 1 x 10 2 1 x 10 3 1 x 10 4 1 x 10 5 1 x 10 6 1 x 10 Pp and Pn correspond to positive control and negative control, respectively.

[0017] Figure 5 LAMP biochemical reaction kinetics curve targeting Atlantic salmon marker gene Cytb , Karenia mikimotoi marker gene ITS2 and Vibrio parahaemolyticus marker gene tlh ; Ta, Tb and Tf correspond to detection tubes targeting Karenia mikimotoi, Vibrio parahaemolyticus and Atlantic salmon epithelial cells, respectively; Na, Nb and Nf are positive controls for the three, respectively; Nc is negative control. DETAILED DESCRIPTION

[0018] The content of the present application is further explained by the following examples, but the protection scope of the present application is not limited in any form by the examples.

[0019] Example 1: Quantitative working curve of Atlantic salmon epithelial cells Step one, prepare the dispersion solution of Atlantic salmon epithelial single cells.

[0020] The aim of this protocol is to efficiently and gently dissociate the epidermal epithelial tissue of Atlantic salmon into single intact epithelial cells. The first step is Dispase II pretreatment. Dispase II is a neutral protease that specifically hydrolyzes the junction proteins (e.g., collagen type IV, fibronectin) between the cells and the basement membrane, thus completely stripping the whole epithelial tissue from the underlying dermal tissue with minimal damage to the cells themselves. The second step is Accutase digestion. Accutase is a mild dissociation enzyme cocktail containing protease and collagenase activity. It further hydrolyzes the junctions between epithelial cells (e.g., E-cadherin), dispersing the stripped epithelial tissue into single cells, with lower toxicity than traditional trypsin, better ensuring cell integrity and activity.

[0021] Instruments and consumables used: Biosafety cabinet, Thermo Fisher Scientific Herasafe KS12, provides a sterile operating environment, all operations are carried out in it.

[0022] Tabletop refrigerated centrifuge, Eppendorf Centrifuge 5424 R, used for 4°C low-temperature centrifugation to protect cell activity.

[0023] Handheld centrifuge, FUP 3MG handheld centrifuge, Qingdao Fute Technology Co., Ltd., field room temperature centrifugation.

[0024] Constant temperature oscillation metal bath, Dalong HCM100-Pro, accurate temperature control.

[0025] Inverted phase contrast microscope, Olympus CKX53, used to observe the degree of tissue digestion and cell morphology.

[0026] Automatic cell counter, Countess 3 FL Auto Cell Counter (Thermo Fisher), used for cell counting.

[0027] Precise electronic balance, ML503 (Mettler-Toledo Instruments Co., Ltd.), accurate weighing.

[0028] Electronic gene amplification analyzer, ER32, eDAQ Company, Australia, used to determine the LAMP amplification kinetics curve.

[0029] Magnetic stirrer, SL-FB-2, Shanghai Sile Instrument Co., Ltd., used for preparing buffer solution, etc.

[0030] pH meter, Mettler Toledo SevenExcellence, pH measurement.

[0031] Low temperature refrigerator, Haier DW-86L578, for storing reagents and materials.

[0032] Filter membrane, 0.22 μm and 0.45 μm, Merck-Millipore.

[0033] Disposable test tube, 5 mm, NORELL, for LAMP amplification container.

[0034] Surgical instruments, including sterile dissecting scissors, dissecting forceps, scalpel handle, blade, etc., Xinhua Medical Instrument Co., Ltd.

[0035] Culture dish, 90 mm, Dongguan Han Ning Biological Technology Co., Ltd.

[0036] Cell strainer, Falcon 70 μm Nylon Cell Strainer (Corning), for removing cell clumps and debris.

[0037] Centrifuge tube, 1.5 mL, 15 mL and 50 mL, Falcon Conical Centrifuge Tubes (Corning).

[0038] Pipette and pipette tips, Eppendorf Research plus series and matching pipette tips.

[0039] Dispase II, Sigma-Aldrich, D4693-1G (≥ 5.0 U / mg), dissolved in PBS, prepared into a working solution of 2.4 U / mL, filtered with a 0.22 μm filter membrane to remove bacteria, stored at -20℃ after aliquoting.

[0040] Accutase®, Innovative Cell TechnoLogies, AT104 ready-to-use solution, stored at 4℃.

[0041] Phosphate buffered saline (PBS), Acmec, 1X, pH 7.4.

[0042] Fetal bovine serum (FBS), Macklin, used for terminating digestion, inhibitors in serum can effectively inactivate protease.

[0043] DMEM / F-12 medium, Gibco, 11330032, used for resuspending cells.

[0044] DNase I and DNase I Buffer, Acmec, used for removing extracellular DNA.

[0045] 75% ethanol, for bench and instrument sterilization.

[0046] Procedure 1. Turn on the UV light of the biosafety cabinet for 30 min. Turn off the UV light and turn on the fan. Wipe the bench and all the reagent bottles and instruments to be used with 75% ethanol.

[0047] 2. Preheat the water bath to 37°C. Precool the centrifuge to 4°C.

[0048] 3. Take PBS and medium from the 4°C refrigerator. Take one tube of Dispase II working solution from the -20°C refrigerator and place it on ice to thaw slowly. Take Accutase and FBS from the 4°C refrigerator and pre-warm them in a 37°C water bath for about 5 min. After they are completely thawed / warmed, wipe the outer wall with 75% ethanol and place them in the biosafety cabinet.

[0049] 4. Prepare complete medium in the biosafety cabinet and store it at 4°C.

[0050] 5. Prepare the termination solution: add 6 mL of complete medium (FBS final concentration >10% to effectively terminate) into a 15 mL centrifuge tube.

[0051] 6. Take a fresh sample of Atlantic salmon skin and place it in a 90 mm petri dish containing pre-cooled PBS.

[0052] 7. Carefully remove subcutaneous fat and connective tissue with a sterile set of dissecting forceps and scissors.

[0053] 8. Transfer the cleaned fish skin to another petri dish containing fresh pre-cooled PBS and gently shake to wash. Repeat this step 2-3 times until the PBS is clear and blood cells and mucus are removed.

[0054] 9. Transfer the washed fish skin to a new sterile petri dish with the dermis facing down.

[0055] 10. Cut the fish skin into small pieces of about 0.5 cm x 0.5 cm with sterile scalpel blades. The operation should be done quickly to avoid tissue drying.

[0056] 11. Dispase II digestion (4°C overnight): Using sterile forceps, transfer each cut skin piece into a 50 mL centrifuge tube; add enough pre-cooled Dispase II working solution (2.4 U / mL) to the tube to ensure complete submersion of all tissue pieces (about 10-15 mL); cap the tube and place it on a shaker or platform in the 4°C refrigerator for slow shaking (~50 rpm) or static digestion for 16-18 h (overnight). Principle: This step aims to enzymatically digest the basement membrane at the dermal-epidermal junction. The low temperature and long incubation time ensure complete and gentle enzymatic digestion, avoiding damage to the epithelial cells.

[0057] 12. Epidermal tissue isolation: The next day, remove the centrifuge tube from the 4°C and place it in the safety cabinet. Using a wide-bore sterile pipette or forceps, gently transfer the skin pieces into another Petri dish containing pre-cooled PBS; using two fine dissecting forceps, one to hold the dermal layer and the other to gently peel the upper smooth, translucent epidermal tissue from the dermis, collect it into a new 15 mL centrifuge tube. Add 10 mL of pre-cooled PBS and wash the epidermal tissue by gently inverting the tube 3-5 times. Centrifuge at 200 x g for 5 min at 4°C, carefully discard the supernatant and remove residual Dispase II.

[0058] 13. Accutase digestion (37°C, 30-45 min): Add 3-5 mL of pre-warmed Accutase® solution to the pelleted epidermal tissue. Gently pipette the tissue pieces several times with a pipette to ensure complete contact with the enzyme solution. Cap the tube tightly and place it in a 37°C water bath for 30-45 min. Every 10 min, remove it and gently pipette (avoiding air bubbles) 10-15 times with a 1 mL pipette in the safety cabinet to aid mechanical dispersion.

[0059] 14. Stop digestion and initial filtration: After the incubation period, the solution should appear cloudy and viscous to the naked eye. Pipette an equal volume of pre- aliquoted stop solution (complete medium with FBS) into the tube and mix gently to stop the Accutase digestion reaction. Repeat the gentle pipetting of the mixture at least 20-30 times with a 5 mL pipette until no large tissue clumps are visible.

[0060] 15. Filter the cell suspension through a pre-wetted 70 μm nylon cell strainer into a new 50 mL centrifuge tube. Rinse the original digestion tube and strainer with 5-10 mL of complete medium to recover all cells.

[0061] 16. Centrifugal washing: Centrifuge the filtered cell suspension at 200 x g for 8 min at 4°C. Carefully discard the supernatant, taking care not to disturb the cell pellet at the bottom. Resuspend the cell pellet with 10 mL of pre-chilled complete medium, and centrifuge again at 200 x g for 5 min at 4°C. Repeat this washing step once to completely remove residual enzymes and cell debris.

[0062] 17. Cell resuspension: Add an appropriate amount (e.g., 1-2 mL) of pre-chilled complete medium to the cell pellet according to the expected cell yield, and gently resuspend the cells thoroughly with a pipette to obtain a single-cell epithelial cell suspension.

[0063] 18. Cell density determination: Place the cell suspension on ice, and take 10 μL to a Countess cell counting chamber. Insert the counting chamber into the instrument, and the total cell concentration (cells / L) is automatically calculated and displayed.

[0064] 19. Dilute the cell suspension to 1 x 10 6 cells / L, 1 x 10 5 cells / L, 1 x 10 4 cells / L, 1 x 10 3 cells / L, 1 x 10 2 cells / L, and 1 x 10 1 cells / L, respectively, for standby use.

[0065] Step two, removal of extracellular DNA (free DNA) from the Atlantic salmon epithelial single-cell suspension.

[0066] Transfer 1 mL of each concentration gradient of the cell suspension into a glass tube (10 mm inner diameter) pre-loaded with 10 mL of DNase I Buffer. The DNase I Buffer consists of 40 mM Tris-HCl, 6 mM MgCl2, 2 mM CaCl2, and 6 U / mL of the nuclease DNase I. Insert the glass tube into the heating hole of a metal bath, and incubate at 36°C for 10 min with vortexing for 5 s every 2 min. Remove the extracellular DNA (free DNA) attached to the filter membrane by enzymatic digestion.

[0067] Step three, release the intracellular DNA by boiling method. Add 1 mL 10% triton X-100 into each glass tube, continue to heat until the liquid in the glass tube boils, and keep boiling for 4 min. During this process, the DNA in the Atlantic salmon epithelial cells will be released and the DNA binding proteins will be denatured to separate from the DNA under the combined action of triton X-100 (a “mild” ionic surfactant that helps to dissolve the cell membrane and nuclear membrane by destroying the lipid bilayer of the cell membrane) and high temperature of 100 ℃. At the same time, the nuclease DNase I is denatured and inactivated during the heating process to avoid damaging the DNA released by cell lysis.

[0068] Step four, prepare the supernatant containing soluble DNA. After step three, take out each glass tube and place it in an ice water mixture to rapidly cool down to obtain the lysis mixture. Then use a pipette to take 2 mL of the lysis mixture and evenly divide it into two 1.5 mL centrifuge tubes, and place them in a handheld centrifuge at 2000 x g for 2 min to precipitate the solid particles, cell debris, denatured proteins, etc. to the bottom of the centrifuge tube, ensuring effective precipitation without loss or damage to the DNA, to obtain supernatant containing a series of concentrations of dissolved DNA to provide templates for the LAMP reaction below.

[0069] Step five, prepare the LAMP biochemical system targeting the Atlantic salmon marker gene Cytb . Prepare 6 portions of LAMP biochemical reaction mixture with a total volume of 90 μL, each containing 20 mmol / L Tris-HCl, 10 mmol / L KCl, 10 mmol / L (NH4)2SO4, 6 mmol / L MgSO4, 0.1% Tween 20, 1.4 mmol / L dNTPs, 0.4 mol / L betaine, 8 U / µL Bst 2.0 WarmStart DNA polymerase and primers. Primer concentration: 1.6 μmol / L FIP and BIP, 0.8 μmol / L LB and LF, 0.2 μmol / L F3 and B3. The sequences of each primer are shown in Table 1.

[0070] Table 1 LAMP primer base sequences targeting Cytb the gene .

[0071] 2, each of the above reaction mixtures is placed in a detection tube, then 10 μL of the supernatant obtained in step four is added to each of the six detection tubes using a microsyringe, and the detection tube is sealed with a rubber plug. The detection tubes are labeled as F1, F2, F3, F4, F5 and F6, respectively, corresponding to 1 x 10 11 x 10 2 1 x 10 3 1 x 10 4 1 x 10 5 1 x 10 6 1 x 10 p 1 x 10 n .

[0072] Step six, prepare the electronic gene amplification analyzer. Open the working interface of the special software "EA-Staz" on the computer controlling the analyzer, and set the parameters of the electronic gene amplification analyzer as follows: working temperature 58 ℃, excitation voltage 16V, excitation frequency 2.0 MHz, signal acquisition period 1 s, and acquisition duration 30 min. Turn on the temperature rise, and it will rise to the preset temperature in about 15 min.

[0073] Step seven, determine the LAMP reaction kinetics curve. Put each test tube prepared in step six (including negative and positive controls) into a detection channel of the electronic gene amplification analyzer which has been warmed to 58 ℃, and click the "Start" key on the working interface of the special software "EA-Staz" to start recording and displaying the LAMP reaction kinetics curve in each test tube in real time.

[0074] Step eight, judge whether the biochemical reaction system is normal. The determination results are shown in Figure 2 . The positive control is a front parabolic curve, and the negative control is an approximate horizontal line, indicating that the LAMP amplification is normal, and the biochemical system is not contaminated by nucleic acids.

[0075] Step nine, analyze the data and determine the quantitative working curve 1. Click the "Integrated Area" key on the working interface of the special software "EA-Staz" to read out the integral areas S F1 , S F2 , S F3 , S F4 , S F5 and S F6219.37 V·s, 1044.99 V·s, 817.22 V·s, 625.10 V·s, 406.41 V·s and 219.37 V·s, respectively. The integral area S (V·s) and its corresponding cell concentration of Atlantic salmon epithelial cell dispersion (cells / L) and the Log value of the concentration were analyzed by using linear, polynomial and logarithmic models, respectively. The results are shown in Table 4 and Table 5, respectively. C Table 4 Relationship between integral area S and cell concentration of cell dispersion C ; Table 5 Relationship between integral area S and Log value of cell concentration of cell dispersion C .

[0076] 2、In the working interface of the special software "EA-Staz", click the "Apparent Conductivity ΔMax" key to read the maximum apparent conductivity change values AC F1 , AC F2 , AC F3 , AC F4 , AC F5 and AC F6 of F1, F2, F3, F4, F5 and F6 corresponding LAMP reaction kinetics curves, which are 491.92 mV, 415.80 mV, 325.74 mV, 249.68 mV, 155.45 mV and 84.90 mV, respectively. The maximum apparent conductivity change value AC (mV) and its corresponding cell concentration of Atlantic salmon epithelial cell dispersion (cells / L) and the Log value of the concentration were analyzed by using linear, polynomial and logarithmic models, respectively. The results are shown in Table 6 and Table 7, respectively. C Table 6 Relationship between maximum apparent conductivity change value AC and cell concentration of cell dispersion C ; Table 7 Relationship between maximum apparent conductivity change value AC and Log value of cell concentration of cell dispersion C .

[0077] 3、In the working interface of the special software "EA-Staz", click the "First-order Derivative" key to read the linear fitting equation slope of the exponential phase of each LAMP reaction kinetics curve corresponding to F1, F2, F3, F4, F5 and F6​​​​​​k F1 , k F2 , k F3 , k F4 , k F5 and k F6 They are 0.02 mV / s, 0.03 mV / s, 0.05 mV / s, 0.07 mV / s, 0.09 mV / s and 0.11 mV / s respectively.

[0078] Linear, polynomial, and logarithmic models were used respectively to determine the slope of the linear fitting equation for the exponential period of the reaction kinetic curve. k (mV / s) and its corresponding Atlantic salmon epithelial cell dispersion cell concentration C Correlation analysis was performed on the (cells / L) and the Log value of the concentration, and the results are shown in Tables 8 and 9, respectively. Table 8. Slope of the linear fitting equation for the exponential phase of the reaction kinetic curve k and cell concentration in cell dispersion C Relationship ; Table 9. Slope of the linear fitting equation for the exponential phase of the reaction kinetic curve k and cell concentration in cell dispersion C The relationship between Log values .

[0079] The results summarized in Tables 4-9 show that the slope of the linear fitting equation for the exponential phase of the reaction kinetic curve is... k Cell concentration in cell dispersion C The linear regression equation between the log values ​​has the highest coefficient of determination ( R 2 =0.9970), therefore it is the best algorithm for quantitative determination.

[0080] Example 2: Quantitative working curve of K. micronesiama; Step one, preparation of uniform K. micronesiama suspension. K. micronesiama was cultured in f / 2 medium (product of Shanghai Guangyu Biotechnology Co., Ltd.) in a light incubator (RTOP-260B, product of Wuhan Ruihua Experimental Equipment Co., Ltd.) at 20°C, light intensity ~3000 lux, light and dark cycle 12h:12h to the mid-logarithmic growth phase (7d). The culture bottle was taken out and shaken gently to make the algal cells uniformly suspended. 9 mL of algal suspension was taken with a single-channel pipette (range 0-10 mL, product of Eppendorf) into a 50 mL centrifuge tube.

[0081] Step two, removal of extracellular DNA (free DNA) in K. micronesiama suspension. 1 mL of 10×DNase I Buffer was added to the 50 mL centrifuge tube containing 9 mL of algal suspension. The composition of DNase I Buffer is 40 mM Tris-HCl, 6 mM MgCl2, 2 mM CaCl2 and 6 U / mL nuclease DNase I. Mix gently with a pipette. Incubate in a constant temperature metal bath at 37°C for 30 min.

[0082] Step three, determination of concentration by hemocytometer method. 100 µL of DNase I treated algal suspension was taken and diluted 100-fold with 900 µL of ultrapure water. 10 µL of diluted algal suspension was mixed with 10 µL of Lugol's iodine solution (1%, Beijing Belo Biotechnology Co., Ltd.) in an EP tube, and left to stand for 2 min to fix the cells. 10 µL of fixed mixture was taken with a pipette and slowly and accurately filled into the counting chamber of a hemocytometer to avoid air bubbles or overflow. The counting chamber was placed under an optical microscope (10× objective) for observation. The total number of algal cells in four corner squares (each square contains 16 small squares) and the central square, a total of five squares, was counted and converted to a concentration of 5.0×10 8 cells / L.

[0083] Step four, the algal suspension with known concentration (5.0×10 8 cells / L) was diluted with ultrapure water to 1×10 6 cells / L, 1×10 5 cells / L, 1×10 4 cells / L, 1×10 3 cells / L, 1×10 2 cells / L and 1×10 1 cells / L, respectively. 1 mL of algal suspension of each concentration was taken and transferred into a glass tube with an inner diameter of 10 mm.

[0084] Step five, release the DNA in the algal cells by boiling method. Add 100 μL 10% triton X-100 into each glass tube, continue to heat until the liquid in the glass tube boils, and keep boiling for 4 min. During this process, the DNA in the cells of K. micella is released and the DNA binding proteins are denatured to separate from the DNA under the combined action of triton X-100 (a “mild” ionic surfactant that helps to dissolve the cell membrane and nuclear membrane by destroying the lipid bilayer of the cell membrane) and high temperature of 100°C. At the same time, the nuclease DNase I is denatured and inactivated during the heating process to avoid damaging the DNA released by cell lysis.

[0085] Step six, prepare the supernatant containing soluble DNA. After step five, take out each glass tube and place it in an ice water mixture to rapidly cool down to obtain the lysis mixture. Then use a pipette to take 1 mL of the lysis mixture and evenly divide it into 2 1.5 mL centrifuge tubes, and place them in a handheld centrifuge at 2000 x g for 2 min to precipitate the solid particles, cell debris, denatured proteins, etc. to the bottom of the centrifuge tube, ensuring effective precipitation without loss or damage to the DNA, to obtain supernatant containing a series of concentrations of dissolved DNA to provide templates for the LAMP reaction below.

[0086] Step seven, prepare the LAMP biochemical system targeting the K. micella marker gene ITS2 of the application Prepare 6 portions of LAMP biochemical reaction mixture with a total volume of 90 μL, each containing 20 mmol / L Tris-HCl, 10 mmol / L KCl, 10 mmol / L (NH4)2SO4, 6 mmol / L MgSO4, 0.1% Tween 20, 1.4 mmol / L dNTPs, 0.4 mol / L betaine, 8 U / µL Bst 2.0 WarmStart DNA polymerase and primers. The primer concentration is 1.6 μmol / L of FIP and BIP, and 0.2 μmol / L of F3 and B3. The sequences of each primer are shown in Table 2.

[0087] Table 2 LAMP primer base sequences targeting ITS2 the gene of the application .

[0088] 2, each of the above reaction mixtures is respectively placed in a detection tube, then 10 μL of the supernatant obtained in step six is added to each of the six detection tubes using a microsyringe, and the detection tube is sealed with a rubber plug. The detection tubes are respectively labeled as A1, A2, A3, A4, A5 and A6, corresponding to 1 x 10 11 x 10 2 1 x 10 3 1 x 10 4 1 x 10 5 1 x 10 6 1 x 10 p The positive control was 10 μg / mL genomic DNA obtained using a microalgae genomic DNA extraction kit (DP305 Plant Genomic DNA Kit, TianGen Biochemical Technology (Beijing) Co., Ltd.), and the negative control was pure water without DNA contamination. The test tubes were labeled as P n and P A1 .

[0089] Step eight, prepare the electronic gene amplification analyzer. Open the working interface of the special software "EA-Staz" on the computer controlling the analyzer, and set the parameters of the electronic gene amplification analyzer as follows: working temperature 58 ℃, excitation voltage 16V, excitation frequency 2.0 MHz, signal acquisition period 1 s, and acquisition duration 30 min. Turn on the temperature rise, and it will rise to the preset temperature in about 15 min.

[0090] Step nine, determine the LAMP reaction kinetics curve. Place the eight test tubes prepared in step six (including the negative and positive controls) into one detection channel of the electronic gene amplification analyzer that has been warmed to 58 ℃, and click the "Start" key on the working interface of the special software "EA-Staz" to start recording and displaying the kinetics curve of the LAMP reaction in each test tube in real time.

[0091] Step ten, determine whether the biochemical reaction system is normal. The determination results are shown in Figure 3 . The positive control is a semiparabolic curve, and the negative control is an approximately horizontal line, indicating that the LAMP amplification is normal, and the biochemical system is not contaminated by nucleic acids.

[0092] Step eleven, analyze the data and determine the quantitative working curve 1. Click the "Integrated Area" key on the working interface of the special software "EA-Staz" to read out the integral areas S A2 , S A3 , S A4 , S A5 and S A6250.70 V·s, 396.52 V·s, 469.60 V·s, 610.31 V·s, 708.35 V·s and 833.18 V·s, respectively.

[0093] The integral area S (V·s) and its corresponding cell concentration of K. micura suspension C (cells / L) and the Log value of the concentration were analyzed by using linear, polynomial and logarithmic models, respectively, and the results are shown in Table 10 and Table 11, respectively. Table 10 Relationship between integral area S and cell concentration of K. micura suspension C ; Table 11 Relationship between integral area S and the Log value of cell concentration of K. micura suspension C .

[0094] 2. Click the "Apparent Conductivity ΔMax" key on the working interface of the special software "EA-Staz", and read out the maximum apparent conductivity change values AC A1 , AC A2 , AC A3 , AC A4 , AC A5 and AC A6 of A1, A2, A3, A4, A5 and A6 corresponding to the LAMP reaction kinetic curve, which are 96.72 mV, 153.88 mV, 185.30 mV, 244.62 mV, 282.55 mV and 334.43 mV, respectively. The maximum apparent conductivity change values AC (mV) and its corresponding cell concentration of K. micura suspension C (cells / L) and the Log value of the concentration were analyzed by using linear, polynomial and logarithmic models, respectively, and the results are shown in Table 12 and Table 13, respectively. Table 12 Relationship between maximum apparent conductivity change value AC and cell concentration of K. micura suspension C ; Table 13 Relationship between maximum apparent conductivity change value AC and the Log value of cell concentration of K. micura suspension C .

[0095] ​​​​3. On the working interface of the dedicated software "EA-Staz", click the "First-order Derivative" button to read the slope of the linear fitting equation for the exponential period of each LAMP reaction kinetic curve corresponding to A1, A2, A3, A4, A5, and A6. k A1 , k A2 , k A3 , k A4 , k A5 and k A6 The values ​​were 0.02 mV / s, 0.03 mV / s, 0.04 mV / s, 0.05 mV / s, 0.06 mV / s, and 0.07 mV / s, respectively. Linear, polynomial, and logarithmic models were used to calculate the slope of the linear fitting equation for the exponential period of the reaction kinetic curve. k (mV / s) and its corresponding Karenia mikimotoi suspension cell concentration C Correlation analysis was performed on the (cells / L) and the Log value of the concentration, and the results are shown in Tables 14 and 15, respectively: Table 14 Slope of the linear fitting equation for the exponential phase of the reaction kinetic curve k and cell concentration of Karenia mikimotoi suspension C Relationship ; Table 15 Slope of the linear fitting equation for the exponential phase of the reaction kinetic curve k and cell concentration of Karenia mikimotoi suspension C The relationship between Log values .

[0096] The results summarized in Tables 10-15 show that the slope of the linear fitting equation for the exponential phase of the reaction kinetic curve is... k and cell concentration of Karenia mikimotoi suspension C The linear regression equation between the log values ​​has the highest coefficient of determination ( R 2 =0.9939), therefore it is the best algorithm for quantitative determination.

[0097] Example 3: Quantitative working curve of Vibrio parahaemolyticus Step one, preparation of uniformly dispersed Vibrio parahaemolyticus suspension. Vibrio parahaemolyticus (ATCC 17802) was inoculated into TCBS liquid medium (product of Qingdao Haibo Biotechnology Co., Ltd.), and placed in a Herocell C1S incubator (product of Shanghai Runduo Biotechnology Co., Ltd.) for shaking culture (120 r / min) at 37°C for 18 h. After removal, it was centrifuged at 4°C for 7 min (5,000 × g) in an OptimaXL-90 centrifuge (product of Beckman Coulter), and the precipitated bacteria were resuspended and washed with physiological saline for 3 times, and then the obtained bacteria were resuspended in physiological saline. The concentration (OD 600 =0.1 corresponds to 10 8  CFU / mL) of the bacterial suspension was determined to be 10 10 cells / mL by using a DEN-1B turbidimeter (product of Dongfanghua Glass Technology Co., Ltd.). Then it was diluted with physiological saline to 1×10 6 cells / L, 1×10 5 cells / L, 1×10 4 cells / L, 1×10 3 cells / L, 1×10 2 cells / L and 1×10 1 cells / L into 6 concentration gradients, and respectively loaded into 6 glass tubes with an inner diameter of 10 mm.

[0098] Step two, removal of extracellular DNA (free DNA) in Vibrio parahaemolyticus suspension. 1 mL of 10×DNase I Buffer was added to the glass containing 9 mL of bacterial suspension. The composition of DNase I Buffer is 40 mM Tris-HCl, 6 mM MgCl2, 2 mM CaCl2 and 6 U / mL of nuclease DNase I. Lightly shake to mix, and incubate in a constant temperature metal bath at 37°C for 30 min.

[0099] Step three, release of DNA in Vibrio parahaemolyticus cells by boiling method. 100 µL of 10% triton X-100 was added to each glass tube, and the temperature was continued to rise until the liquid in the glass tube boiled, and the boiling was maintained for 4 min. During the heating process, the nuclease DNase I was denatured and inactivated to avoid damage to the released DNA.

[0100] Step four, preparation of supernatant containing soluble DNA. After step three, take out each glass tube and put it into an ice water mixture for rapid cooling to obtain the lysis mixture. Then use a pipette to take 2 mL of the lysis mixture, evenly divide it into two 1.5 mL centrifuge tubes, and put it into a handheld centrifuge (FUP 3MG, Qingdao Fute Technology Co., Ltd.) and centrifuge at 2000 x g for 2 minutes to precipitate solid particles, cell debris, denatured proteins, etc. to the bottom of the centrifuge tube, obtaining supernatant containing a series of concentrations of dissolved DNA to provide templates for the following LAMP reaction.

[0101] Step five, preparation of LAMP biochemical reaction system targeting Vibrio parahaemolyticus marker gene tlh 1. Prepare 6 portions of LAMP biochemical reaction mixture with a total volume of 90 μL, each containing 20 mmol / L Tris-HCl, 10 mmol / L KCl, 10 mmol / L (NH4)2SO4, 6 mmol / L MgSO4, 0.1% Tween 20, 1.4 mmol / L dNTPs, 0.4 mol / L betaine, 8 U / µL Bst 2.0 WarmStart DNA polymerase and primers. Primer concentration: 1.6 μmol / L of FIP and BIP, 0.8 μmol / L of LB and LF, 0.2 μmol / L of F3 and B3. The sequences of each primer are shown in Table 3; Table 3 LAMP primer base sequences targeting tlh gene .

[0102] 2. Put each of the above reaction mixtures into a detection tube, then use a microsyringe to take 10 μL of supernatant obtained in step four and add it to the six detection tubes, respectively, and use a rubber plug to seal the detection tube. The detection tubes are labeled as B1, B2, B3, B4, B5 and B6, respectively, corresponding to 1×10 1 cells / L, 1×10 2 cells / L, 1×10 3 cells / L, 1×10 4 cells / L, 1×10 5 cells / L and 1×10 6 ​cells / L supernatant of Vibrio parahaemolyticus bacterial suspension. 10 μg / mL genomic DNA obtained by using a bacterial genomic DNA extraction kit (Bacteria Genomic DNA Kit, product of Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used as a positive control, and pure water without DNA contamination was used as a negative control. The detection tubes were labeled as P p and P n .

[0103] Step six, prepare the electronic gene amplification analyzer. Open the working interface of the special software "EA-Staz" on the computer controlling the analyzer, and set the parameters of the electronic gene amplification analyzer as follows: working temperature 58 ℃, excitation voltage 16 V, excitation frequency 2.0 MHz, signal acquisition period 1 s, and acquisition duration 30 min. Turn on the temperature rise, and it takes about 15 min to rise to the preset temperature.

[0104] Step seven, determine the LAMP reaction kinetics curve. Place the eight detection tubes (including negative and positive controls) prepared in step six into one detection channel of the electronic gene amplification analyzer which has been warmed to 58 ℃, and click the "Start" key on the working interface of the special software "EA-Staz" to start recording and displaying the kinetics curve of the LAMP reaction in each detection tube in real time.

[0105] Step eight, judge whether the biochemical reaction system is normal. The determination results are shown in Figure 4 . The positive control is a semi-parabolic curve, and the negative control is an approximately horizontal line, indicating that the LAMP amplification is normal, and the biochemical system is not contaminated by nucleic acids.

[0106] Step nine, analyze the data and determine the quantitative working curve 1. Click the "Integrated Area" key on the working interface of the special software "EA-Staz" to read out the integral areas S B1 , S B2 , S B3 , S B4 , S B5 and S B6 corresponding to the LAMP reaction kinetics curves of B1, B2, B3, B4, B5 and B6 respectively, which are 1413.80 V·s, 1174.76 V·s, 979.59 V·s, 691.04 V·s, 389.54 V·s and 164.05 V·s respectively. Using linear, polynomial and logarithmic models respectively, the integral area S (V·s) and its corresponding Vibrio parahaemolyticus bacterial suspension cell concentration C (cells / L) and the concentration Log value are correlated, and the results are shown in Tables 16 and 17 respectively; Table 16 Relationship between the integral area S and the cell concentration of V. parahaemolyticus bacterial suspension C .

[0107] Table 17 Relationship between the integral area S and the cell concentration of V. parahaemolyticus bacterial suspension C .

[0108] 2. Click the "Apparent Conductivity AMax" key on the working interface of the special software "EA-Staz", and read out the maximum apparent conductivity change values AC of B1, B2, B3, B4, B5 and B6 corresponding to the LAMP reaction kinetics curves B1 B2 B3 B4 B5 B6 are 526.91 mV, 469.38 mV, 383.39 mV, 277.06 mV, 152.82 mV and 63.37 mV, respectively. The maximum apparent conductivity change values AC (mV) and the cell concentrations of V. parahaemolyticus bacterial suspension C (cells / L) and the Log values of the concentrations corresponding to them were respectively correlated by using linear, polynomial and logarithmic models, and the results are shown in Tables 18 and 19, respectively; Table 18 Relationship between the maximum apparent conductivity change value AC and the cell concentration of V. parahaemolyticus bacterial suspension C ; Table 19 Relationship between the maximum apparent conductivity change value AC and the Log value of the cell concentration of V. parahaemolyticus bacterial suspension C .

[0109] 3. Click the "First-order Derivative" key on the working interface of the special software "EA-Staz", and read out the linear fitting equation slopes k B1 , k B2 , k B3 , k B4 , k B5 and k ​​​​​​​​​B6 0.01 mV / s, 0.03 mV / s, 0.06 mV / s, 0.08 mV / s, 0.10 mV / s and 0.12 mV / s, respectively. The linear fitting equation slope of the exponential phase of the reaction kinetics curve k (mV / s) and the corresponding Vibrio parahaemolyticus bacterial suspension cell concentration C (cells / L) and the concentration Log value were analyzed, respectively, and the results are shown in Table 20 and Table 21, respectively. Table 20 Relationship between the linear fitting equation slope of the exponential phase of the reaction kinetics curve k and the Vibrio parahaemolyticus bacterial suspension cell concentration C ; Table 21 Relationship between the linear fitting equation slope of the exponential phase of the reaction kinetics curve k and the Log value of the Vibrio parahaemolyticus bacterial suspension cell concentration C .

[0110] From the results summarized in Table 16-Table 21, the polynomial regression equation between the linear fitting equation slope of the exponential phase of the reaction kinetics curve k and the Log value of the Vibrio parahaemolyticus bacterial suspension cell concentration C has the highest determination coefficient (R2=0.9981), and therefore is the best algorithm for quantitative determination. R 2

[0111] Example A method for simultaneously determining the number of Atlantic salmon epithelial cells, K. micronesica and Vibrio parahaemolyticus in seawater, which specifically comprises the following steps: Step one, the eDNA in seawater is collected on the filter membrane by suction filtration method. 2 L of target seawater is filtered by negative pressure suction filtration method based on suction filtration bottle, Buchner funnel, filter membrane and vacuum pump, and the particulate matter and cells including plant and animal debris, algae and microorganisms in seawater are intercepted on the surface of the filter membrane. The filter membrane is a mixed cellulose ester filter membrane with a pore size of 0.45 μm (diameter 47 mm), which ensures the interception of bacteria and eukaryotic cells; the filtration pressure is ≤-0.08 MPa to prevent mechanical damage to the cells. After filtering the seawater, 1 L of pure water is added to the Buchner funnel and continues to be filtered to remove soluble inorganic and organic salts and eliminate the negative effects of high salt content on subsequent operations.

[0112] ​​​Step two, removing extracellular DNA (free DNA). The filter membrane was taken out of the suction filtration device, and was folded with the help of sterile tweezers, and then was put into a glass tube (inner diameter 10 mm) preloaded with 10 mL DNase I Buffer. The composition of DNase I Buffer was 40 mM Tris-HCl, 6 mM MgCl2, 2 mM CaCl2and 6 U / mL nuclease DNase I. The glass tube was inserted into the heating hole of the metal bath, and was incubated at 36℃ for 10 min, and was vortexed for 5 s every 2 min. The purpose of this operation was to remove the extracellular DNA (free DNA) attached to the filter membrane by enzymatic hydrolysis.

[0113] Step three, releasing DNA in the cells of animals, plants, algae and microorganisms by boiling method. After step two, 1 mL 10% triton X-100 was added to the glass tube, and the liquid in the glass tube was boiled, and was kept boiling for 4 min. During this process, under the combined action of triton X-100 (a “mild” ionic surfactant, which helps to dissolve the cell membrane and nuclear membrane by destroying the lipid bilayer of the cell membrane) and high temperature of 100℃, the DNA in the intact cells of animals, plants, algae and microorganisms was released, and the DNA-binding proteins were denatured, so that the two were separated. At the same time, the nuclease DNase I was denatured and inactivated during the heating process, so as to avoid damaging the DNA released by cell lysis.

[0114] Step four, preparing supernatant containing soluble DNA. After step three, the glass tube was taken out and was quickly cooled in an ice-water mixture to obtain a lysis mixture. Then 2 mL of the lysis mixture was taken by using a pipette, and was evenly divided into two 1.5 mL centrifuge tubes, and was centrifuged at 2000 x g for 2 min in a handheld centrifuge, so that the solid particles, cell debris, denatured proteins, etc. were precipitated at the bottom of the centrifuge tube, and the DNA was effectively precipitated without loss or damage, and the supernatant containing dissolved DNA was obtained to provide a template for the following LAMP reaction.

[0115] Step five, preparing LAMP reaction mixtures respectively targeting the marker genes of Atlantic salmon Cytb , karen cluster ITS2 and Vibrio parahaemolyticus tlhLAMP biochemical system. In each disposable glass capillary detection tube (outer diameter 2.6 mm, inner diameter 1 mm, hereinafter referred to as detection tube), a total volume of 90 μL of LAMP biochemical reaction mixture was preloaded, and the mixture contained 20 mmol / L Tris-HCl, 10 mmol / L KCl, 10 mmol / L (NH4)2SO4, 6 mmol / L MgSO4, 0.1% Tween 20, 1.4 mmol / L dNTPs, 0.4 mol / L betaine, 8 U / µL Bst 2.0 WarmStart DNA polymerase and primers. Among them, Bst 2.0 WarmStart DNA polymerase is inactive at room temperature and is activated at 58°C to avoid non-specific amplification; betaine is used to stabilize the secondary structure of DNA and promote strand displacement. Primers are targeted at the marker genes of Cytb K. micronesica ITS2 and V. parahaemolyticus tlh.

[0116] Cytb Gene primer concentration: 1.6 μmol / L of FIP and BIP, 0.8 μmol / L of LB and LF, and 0.2 μmol / L of F3 and B3. The sequences of each primer are listed in Table 1.

[0117] ITS2 Gene primer concentration: 1.6 μmol / L of FIP and BIP, 0.2 μmol / L of F3 and B3. The sequences of each primer are listed in Table 2.

[0118] tlh Gene primer concentration: 1.6 μmol / L of FIP and BIP, 0.8 μmol / L of LB and LF, and 0.2 μmol / L of F3 and B3. The sequences of each primer are listed in Table 3.

[0119] Step six, prepare the detection tube. Use a microsyringe to take 10 μL of the supernatant obtained in step four, and add it to the LAMP biochemical detection tube targeting the marker genes of Cytb K. micronesica ITS2 and V. parahaemolyticus tlh prepared in step five, and use a rubber plug to seal the detection tube. The detection tubes are labeled as T f , T a and T b , respectively. Atlantic salmon genomic DNA, K. micronesica genomic DNA and V. parahaemolyticus genomic DNA are used as positive controls, and the detection tubes are labeled as P f , P a and P b; the pure water without DNA contamination as negative control, and the test tube was marked as N c .

[0120] Step seven, prepare the electronic gene amplification analyzer. Open the working interface of the special software "EA-Staz", and set the parameters of the electronic gene amplification analyzer as follows: working temperature 58 ℃, excitation voltage 16 V, excitation frequency 2.0 MHz, signal acquisition period 1 s, and acquisition time length 30 min. Turn on the temperature rise, and it takes about 15 min to rise to the preset temperature.

[0121] Step eight, determine the LAMP reaction kinetics curve. Put each test tube prepared in step six (including negative and positive controls) into a detection channel of the electronic gene amplification analyzer which has been warmed to 58 ℃, and click the "Start" key on the working interface of the special software "EA-Staz" to start recording and displaying the LAMP reaction kinetics curve in each test tube in real time. The working principle of this step is as follows: since LAMP uses at least 2 pairs of primers to recognize 6 sites of base sequences, it has high specificity and can accurately identify target genes to achieve qualitative determination. During the LAMP reaction, every pair of base doublets synthesized generates 4 H + with strong ion conductivity, causing an increase in the electrical conductivity of the reaction mixture. This increase is recorded by the sensitive component of the electronic gene amplification analyzer, the capacitive coupling non-contact conductivity sensor, and plotted against the reaction time to generate a biochemical reaction kinetics curve. The kinetics curve parameters and the copy number of the template gene and the number of biological cells have a stable functional relationship, which can achieve quantitative detection.

[0122] Step nine, calculate the number of intact cells to be tested. After the LAMP reaction kinetics curves of T f , T a , T b , P f , P a , P b and N c tubes are determined, click the "First-order Derivative" key on the working interface of the special software "EA-Staz" to read the linear fitting equation slope of the exponential phase of the LAMP reaction kinetics curve of T f , T a and T b respectively. k f , k a and k b . Substitute the linear fitting equation slope into the formula Log N f (cells / L)= 52.96 k f (mV / s) + 0.27, Log N a (cells / L) = 88.13 k a (mV / s) - 0.54 and Log N b (cells / L) = 70.79 k b 2 (mV / s) + 35.67 k b (mV / s) + 0.70, the number of Atlantic salmon epithelial cells, K. micronesiaca and V. parahaemolyticus intact cells in the measured seawater sample N f , N a and N b are calculated. Note: the positive controls P f , P a and P b should be a front half parabolic curve, and the negative control N c should be an approximate horizontal line. If the positive control is an approximate horizontal line, it means that the LAMP biochemical reaction system is abnormal; if the negative control is a front half parabolic curve, it indicates that the LAMP biochemical reaction system is contaminated by non-target genes. In these two cases, the results of this determination are invalid, and should be re-determined after the cause is found.

[0123] Example 5: Monitoring of Atlantic salmon epithelial cells, K. micronesiaca and V. parahaemolyticus in the net cage seawater of the aquaculture work ship "Guoxin No. 1" Step one, sample collection. In July 2025, 2 L of surface seawater was collected in the net cage of the aquaculture work ship "Guoxin No. 1".

[0124] Step two, eDNA collection by suction filtration. On site, a negative pressure suction filtration method based on a suction filtration bottle, a Buchner funnel, a filter membrane and a vacuum pump was used to filter seawater, and particles and cells including animal and plant debris, algae and microorganisms in seawater were intercepted on the surface of the filter membrane. The filter membrane was a 0.45 μm mixed cellulose ester filter membrane (diameter 47 mm) to ensure the interception of bacteria and eukaryotic cells; the filtration pressure was -0.08 MPa to prevent mechanical damage to the cells. After filtering the seawater, 1 L of pure water was added to the Buchner funnel and continued to be filtered to remove soluble inorganic and organic salts and eliminate the negative effects of high salinity on subsequent operations.

[0125] Step three, removing extracellular DNA (free DNA). The filter membrane was taken out of the filtration device, and was folded with the help of sterile forceps, and then was put into a glass tube (10 mm in inner diameter) preloaded with 10 mL DNase I Buffer. The DNase I Buffer was composed of 40 mM Tris-HCl, 6 mM MgCl2, 2 mM CaCl2, and 6 U / mL nuclease DNase I. The glass tube was inserted into the heating hole of a metal bath, and was incubated at 36℃ for 10 min, and was vortexed for 5 s every 2 min. The purpose of this operation was to remove the extracellular DNA (free DNA) attached to the filter membrane by enzymatic hydrolysis.

[0126] Step four, lysing the intact cells to release DNA. After step three, 1 mL of 10% Triton X-100 was added to the glass tube, and the liquid in the glass tube was boiled, and was kept boiling for 4 min. In this process, under the combined action of Triton X-100 (a “mild” ionic surfactant that helps to dissolve the cell membrane and nuclear membrane by destroying the lipid bilayer of the cell membrane) and 100℃ high temperature, the DNA in the intact cells of animals, plants, algae and microorganisms was released, and the DNA-binding proteins were denatured, so that the two were separated. At the same time, the nuclease DNase I was denatured and inactivated during the heating process, so as to avoid damaging the DNA released by cell lysis.

[0127] Step five, preparing the supernatant containing soluble DNA. The glass tube treated in step three was taken out and was quickly cooled in an ice-water mixture to obtain a cell lysis mixture. Then 2 mL of the lysis mixture was taken with a pipette, and was evenly divided into two 1.5 mL centrifuge tubes, and was centrifuged at 2000 x g for 2 min in a handheld centrifuge, so that the solid particles, cell debris, denatured proteins, etc. were precipitated at the bottom of the centrifuge tube, and the DNA was effectively precipitated without loss or damage, and the supernatant containing dissolved DNA was obtained to provide a template for the LAMP reaction below.

[0128] Step six, preparing LAMP biochemical systems respectively targeting the Atlantic salmon marker gene Cytb , the K. micronesiaca marker gene ITS2 , and the V. parahaemolyticus marker gene tlh . In each detection tube, 90 μL of LAMP biochemical reaction mixture was preloaded, and the mixture contained 20 mmol / L Tris-HCl, 10 mmol / L KCl, 10 mmol / L (NH4)2SO4, 6 mmol / L MgSO4, 0.1% Tween 20, 1.4 mmol / L dNTPs, 0.4 mol / L betaine, 8 U / µL Bst2.0 WarmStart DNA Polymerase and primers. Among them, Bst 2.0 WarmStart DNA Polymerase is inactive at room temperature and activated at 58℃, avoiding non-specific amplification; betaine is used to stabilize the secondary structure of DNA and promote strand displacement. The concentrations and sequences of primers targeting Atlantic salmon marker gene Cytb , Karenia mikimotoi marker gene ITS2 and Vibrio parahaemolyticus marker gene tlh are the same as the first three examples.

[0129] Step seven, prepare the detection tube. Use a microsyringe (50 µL, Shanghai Gaoge Trading Co., Ltd. product) to take 10 µL of the supernatant obtained in step five, and add it to the LAMP biochemical reaction detection tube prepared in step six targeting Atlantic salmon marker gene Cytb , Karenia mikimotoi marker gene ITS2 and Vibrio parahaemolyticus marker gene tlh , and use a rubber plug to seal the detection tube. The detection tubes are labeled as T f , T a and T b . 10 μg / mL of Atlantic salmon genomic DNA, Karenia mikimotoi genomic DNA and Vibrio parahaemolyticus genomic DNA are used as positive controls, and the detection tubes are labeled as N f , N a and N b . Pure water without DNA contamination is used as a negative control, and the detection tube is labeled as N c .

[0130] Step eight, prepare the electronic gene amplification analyzer. Open the working interface of the special software "EA-Staz" and set the parameters of the electronic gene amplification analyzer as follows: working temperature 58 ℃, excitation voltage 16 V, excitation frequency 2.0 MHz, signal acquisition period 1 s, and acquisition time 30 min. Turn on the temperature rise, and it will rise to the preset temperature in about 15 min.

[0131] Step nine, determine the LAMP reaction kinetics curve. Place the seven detection tubes prepared in step eight (including negative and positive controls) into the electronic gene amplification analyzer which has been warmed to 58℃ in one detection channel. Click the "Start" key on the working interface of the special software "EA-Staz" to record and display the kinetics curve of the LAMP reaction in each detection tube in real time.

[0132] Step ten, judge whether the biochemical reaction system is normal. The determination results are shown in Figure 5 . All three positive controls are parabolic curves, and one negative control is an approximate horizontal line, indicating that the LAMP amplification is normal and the biochemical system is not contaminated by nucleic acids.

[0133] Step eleven, click the "First-order Derivative" key on the working interface of the special software "EA-Staz", read out the T f , T a and T b respectively c LAMP reaction kinetics curve exponential phase linear fitting equation slope k f , k a and k b . The linear fitting equation slope is respectively substituted into the formula Log N f (cells / L) = 52.96 k f (mV / s) + 0.27, Log N a (cells / L) = 88.13 k a (mV / s) - 0.54 and Log N b (cells / L) = 70.79 k b 2 (mV / s) + 35.67 k b (mV / s) + 0.70, the number of the Atlantic salmon epithelial cells, Karenia mikimotoi and Vibrio parahaemolyticus intact cells in the measured seawater sample N f , N a and N b are respectively 37 cells / L, 50 cells / L and 108 cells / L.

Claims

1. A method for simultaneous quantitative determination of Atlantic salmon epithelial cells, Karenia mikimotoi, and Vibrio parahaemolyticus in seawater, wherein the method is for non-disease treatment and prevention purposes, characterized in that, Environmental DNA, or eDNA, is extracted and then added to cells containing targeted Atlantic salmon marker genes. Cytb Marker genes of Karenia mikimotoi ITS2 and Vibrio parahaemolyticus marker genes tlh The LAMP reaction system was placed in a reaction tube, and then the reaction tube was placed in a fully automated electronic gene amplification analyzer to detect the target Atlantic salmon marker gene in real time. Cytb Marker genes of Karenia mikimotoi ITS2 and Vibrio parahaemolyticus marker genes tlh From the LAMP biochemical reaction kinetic curves, read the slope of the linear fitting equation corresponding to the exponential phase of the kinetic curve. k f , k a and k b Substitute them into the formula Log N f = 52.96 k f + 0.27, Log N a =88.13 k a -0.54 and Log N b =70.79 k b 2 +35.67 k b +0.70, calculate the number of intact Atlantic salmon epithelial cells, Karenia mikimotoi, and Vibrio parahaemolyticus cells in the tested seawater sample. N f , N a and N b .

2. The method for simultaneous quantitative determination of Atlantic salmon epithelial cells, Karenia mikimotoi, and Vibrio parahaemolyticus in seawater according to claim 1, characterized in that, The method for preparing the eDNA is as follows: Step 1: Collect eDNA from seawater onto a filter membrane using a vacuum filtration method. Remove the filter membrane from the vacuum filtration device and place it into a detection tube pre-filled with DNase I Buffer. Incubate in a 36°C water bath with shaking to remove extracellular DNA attached to the filter membrane through enzymatic digestion. Step 2: Add 10% by volume of Triton 100 to the detection tube after Step 1, continue to heat in a water bath until the liquid in the detection tube boils, and maintain boiling for 4 minutes. Step 3: After processing in Step 2, remove the test tube and quickly cool it to obtain a lysis mixture. Centrifuge the mixture to obtain a supernatant containing dissolved DNA.

3. The method for simultaneous quantitative determination of Atlantic salmon epithelial cells, Karenia mikimotoi, and Vibrio parahaemolyticus in seawater according to claim 1, characterized in that, The LAMP reaction system is as follows: the mixture contains 20 mmol / L Tris-HCl, 10 mmol / L KCl, 10 mmol / L (NH4)2SO4, 6 mmol / L MgSO4, 0.1% Tween 20, 1.4 mmol / L dNTPs, 0.4 mol / L betaine, 8 U / µL DNA polymerase and primers.

4. The method for simultaneous quantitative determination of Atlantic salmon epithelial cells, Karenia mikimotoi, and Vibrio parahaemolyticus in seawater according to claim 1, characterized in that, The reaction parameters of the fully automated electronic gene amplification analyzer are: operating temperature 58 ℃, excitation voltage 16 V, excitation frequency 2.0 MHz, signal acquisition period 1 s, and acquisition duration 30 min.

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