A rapid separation and detection method of pathogenic bacteria in aquaculture water body

Abstract

The application discloses a kind of quick separation and detection methods of pathogenic bacteria in aquaculture water body, belong to the field of microorganism detection.The method reduces sample amount through enrichment of microorganism, does not need complex equipment, is lysed at room temperature and isothermal amplification LAMP detection, significantly shortens detection period, meets the demand of early warning and drug instruction of aquaculture;LAMP detection is carried out using mutant Bst-WK30 DNA polymerase containing specific 7 amino acid mutation sites, with high amplification efficiency and strong specificity, can accurately identify pathogenic bacteria, and can detect a variety of common harmful pathogenic microorganisms.Kit components are synergistic, efficient lysis and specific amplification are realized;The separation device is synergistic through physical filtration and electrochemical enrichment, rapidly separates target microorganisms, reduces impurity interference, enriches microorganisms to filter membrane within 20min, has simple structure and is convenient to carry, and is suitable for on-site rapid sample processing.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection, specifically relating to a rapid isolation and detection method for pathogenic bacteria in aquaculture water. Background Technology

[0002] Aquaculture, as a vital component of the global food supply, provides humanity with a rich source of protein and plays a crucial role in ensuring food security and socio-economic development. However, with the expansion of aquaculture scale and the increase in intensification, the problem of pathogenic microorganisms in aquaculture water has become increasingly serious. Pathogenic microorganisms such as Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, and rainbow viruses are widely present in aquaculture water. They are highly pathogenic and can cause a variety of serious diseases. Vibrio parahaemolyticus is a common pathogen in shrimp farming, causing red body disease and white spot disease, leading to mass mortality. Aeromonas hydrophila can infect various fish species, causing septicemia, resulting in symptoms such as loss of appetite and bleeding on the body surface, with a high mortality rate. Rainbow viruses pose a threat to farmed organisms such as frogs, affecting their growth and reproduction. Outbreaks of these diseases are often sudden and spread rapidly, causing huge economic losses to aquaculture farmers and seriously hindering the healthy development of the aquaculture industry.

[0003] Besides directly causing the death of farmed organisms, pathogenic microorganism infections can also lead to stunted growth (reduced feed intake and decreased feed conversion rate), reproductive disorders (lower survival rate and reproductive capacity of parent organisms), and reduced commercial value (body surface damage and deformities). Antibiotics are widely used in treating aquaculture diseases, but their overuse has resulted in drug residue problems. Some pathogenic microorganisms (such as Vibrio parahaemolyticus and Listeria monocytogenes, which are foodborne pathogens) are drug-resistant, and residual antibiotics can enter the human body through the food chain, posing a potential threat to human health. Furthermore, some pathogens have zoonotic characteristics; for example, the potent exotoxins produced by Aeromonas hydrophila can be transmitted to humans and other animals, causing disease and seriously impacting public health. The spread of drug-resistant bacteria and the proliferation of pathogenic microorganisms can disrupt the balance of aquatic ecosystems. They may compete with other microorganisms for resources, affecting the composition and function of the aquatic microbial community, and thus adversely affecting the entire aquatic ecological environment. Therefore, rapid and accurate detection of pathogens has become a crucial link in disease control.

[0004] Traditional culture methods are commonly used to detect pathogens in aquaculture water, but these methods have significant limitations. The detection process typically takes 24–72 hours, which is time-consuming, and the results are not timely, limiting their value for early warning or guiding medication use in aquaculture. While colloidal gold chromatography based on immunological methods is relatively simple to operate, its detection effectiveness is affected by many factors in the complex environment of aquaculture water. Aquaculture water contains large amounts of algae, plant and animal debris, and high concentrations of ammonia and nitrate nitrogen, which can alter the physicochemical properties of the water, such as significant pH fluctuations. In such environments, colloidal gold chromatography is prone to cross-reactions, affecting the accuracy of the results. Furthermore, differences in antibody quality can lead to unstable specificity and limited sensitivity, making it difficult to meet the demand for precise detection of pathogens in aquaculture water.

[0005] Given the severe pathogen threats facing aquaculture and the limitations of existing detection technologies, developing a method that can rapidly and accurately isolate and identify pathogens in aquaculture water is of great practical significance. Summary of the Invention

[0006] To address the shortcomings of traditional detection methods in detecting pathogens in aquaculture water, such as the time-consuming nature of traditional culture methods and the susceptibility of colloidal gold chromatography to interference from complex aquatic environments, as well as unstable specificity and sensitivity, which hinder the rapid and accurate isolation and identification of pathogens and affect the current state of disease prevention and control, this invention aims to provide a method for rapidly isolating and identifying pathogens in aquaculture water, as well as a kit for identifying pathogenic microorganisms, thereby overcoming the deficiencies of traditional detection methods, such as long cycle time, poor specificity, and low sensitivity.

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

[0008] This invention provides a rapid method for isolating and detecting pathogenic bacteria in aquaculture water, comprising:

[0009] Step 1: Enrich the microorganisms in the aquaculture water sample containing the target pathogenic bacteria;

[0010] Step 2: Add the enriched microorganisms to the lysis buffer, let them stand at room temperature for lysis, and obtain a lysed microbial mixture.

[0011] Step 3: Add the lysed microbial mixture to the PCR reaction solution for isothermal amplification and LAMP detection. Determine whether the sample contains the target pathogen based on the fluorescence signal.

[0012] The PCR reaction solution contains a mutant Bst-WK30 DNA polymerase, which contains seven amino acid mutation sites: T365S, V428A, M488T, A663R, H664P, A741M, and L847G.

[0013] The target pathogenic bacteria are one or more of Streptococcus agalactiae, carp herpesvirus type III, Pseudomonas putidae, and tilapia lake virus.

[0014] The amino acid sequence of the mutant Bst-WK30 DNA polymerase is shown in SEQ ID No. 3.

[0015] Preferably, the mutant Bst-WK30 DNA polymerase is obtained through directed evolution, with an evolutionary pressure of 0.02% to 0.2% alkylamide ethylamine and a tolerance concentration of ≥0.2%.

[0016] The lysis buffer contains Tris-HCl, ethylenediaminetetraacetic acid (EDTA), and alkylamide ethylamine.

[0017] Preferably, the molar concentration of Tris-HCl is 10 mM to 50 mM, the pH is 8.8, the molar concentration of EDTA is 0.1 mM, and the concentration of alkylamide ethylamine is 0.01% to 0.2%.

[0018] The room temperature static pyrolysis time is 5 to 10 minutes.

[0019] The PCR reaction solution also includes Tris hydrochloride (Tris-HCl), KCl, MgSO4, betaine, deoxynucleoside triphosphate (dNTP), primer combination and fluorescent indicator.

[0020] Preferably, the molar concentration of Tris-HCl is 15-20 mM, the molar concentration of KCl is 45-55 mM, the molar concentration of betaine is 0.7-0.9 M, and the molar concentration of dNTP is 1.2-1.6 mM.

[0021] The isothermal amplification temperature is 60℃~70℃, and the amplification time is 20~40min.

[0022] The present invention provides a kit for the rapid isolation and detection method of pathogenic bacteria in aquaculture water, comprising microbial lysis buffer and isothermal amplification system;

[0023] The microbial lysate contains Tris hydrochloride (Tris-HCl), ethylenediaminetetraacetic acid (EDTA), and alkylamide ethylamine.

[0024] The isothermal amplification system contains Tris hydrochloride (Tris-HCl), KCl, MgSO4, betaine, deoxynucleoside triphosphates (dNTPs), primer combinations, fluorescent indicators, and mutant Bst-WK30 DNA polymerase.

[0025] The kit also includes a positive control and a negative control.

[0026] This invention provides a rapid separation device for aquatic microorganisms, which is used to enrich microorganisms in aquaculture water samples as described in the rapid separation and detection method for pathogenic bacteria in aquaculture water as described in claim 1. The device includes a shell, a filter assembly, an external power supply, and a sample loading tank. The shell has an anode and a cathode at both ends, respectively. The filter assembly is located inside the shell near the anode end.

[0027] The filtration assembly consists of a first filter membrane, a second filter membrane, a third filter membrane, a fourth filter membrane, and filter paper, and is used to perform multi-stage filtration and separation on the added water sample to be tested.

[0028] A sample loading groove is provided near the cathode end inside the housing, and the sample loading groove is connected to the inside of the housing.

[0029] Preferably, the housing consists of a plastic insulating shell and a paper filter cartridge disposed inside the shell, used to trap large particulate impurities.

[0030] Preferably, a plastic partition is provided between the first filter membrane, the second filter membrane, the third filter membrane, the fourth filter membrane and the filter paper to fix the filter membrane.

[0031] Preferably, the paper filter cartridge is made of cellulose filter paper, and the filter membrane is made of a mixed cellulose ester material.

[0032] Preferably, the pore sizes of the first filter membrane, the second filter membrane, the third filter membrane, and the fourth filter membrane are 80-100μm, 30-50μm, 10-15μm, and 0.22μm, respectively, and the applied voltage is 2-5V / cm.

[0033] Compared with the prior art, the present invention achieves the following technical effects:

[0034] The present invention provides a rapid isolation and detection method for pathogenic bacteria in aquaculture water. This method significantly shortens the detection cycle, and the enrichment of microorganisms can quickly concentrate target microorganisms, reduce the amount of samples to be tested in subsequent tests, and improve detection efficiency. Room temperature lysis does not require complex equipment, saving time. Isothermal amplification does not require repeated temperature changes, further shortening the detection time. The overall detection can be completed in a short time, meeting the needs of timely early warning and guidance for drug use in aquaculture. LAMP detection was performed using a mutant Bst-WK30 DNA polymerase containing seven specific amino acid mutation sites. These mutations—T365S, V428A, M488T, A663R, H664P, A741M, and L847G—gave the enzyme higher amplification efficiency and stronger specificity. In LAMP detection, this resulted in more accurate amplification of the nucleic acid of the target pathogen, reducing non-specific amplification and thus improving detection specificity. This allowed for precise identification of pathogens in aquaculture water, overcoming the instability of specificity and sensitivity issues associated with traditional methods. This facilitates timely disease control measures and reduces losses in aquaculture.

[0035] Furthermore, the method can detect a wide range of microorganisms, including common and highly pathogenic microorganisms in aquaculture, such as Streptococcus agalactiae, carp herpesvirus type III, Pseudomonas putidae, and tilapia lake virus, thus expanding the applicability of the detection method and providing an effective means for disease prevention and control of various aquaculture organisms.

[0036] Furthermore, the Tris-HCl buffer system maintains nucleic acid stability at pH 8.8 while providing a suitable environment for subsequent isothermal amplification; 0.1 mM EDTA inhibits nuclease activity by chelating metal ions, and its low concentration design avoids interference with subsequent amplification enzyme activity; alkylamide ethylamine, as a nonionic surfactant, effectively disrupts microbial cell membrane structure within a concentration range of 0.01%–0.2% without producing residues that inhibit Bst DNA polymerase. The synergistic effect of these three components overcomes the technical bottlenecks of traditional lysis buffers being prone to failure in complex aquaculture substrates and lysis products inhibiting the amplification reaction.

[0037] The kit provided by this invention utilizes a Tris-HCl buffer system in the microbial lysis buffer to maintain pH stability during lysis and prevent nucleic acid degradation; EDTA inhibits nuclease activity by chelating metal ions; and alkylamide ethylamine acts as a surfactant to disrupt the microbial cell membrane structure. The three components work together to achieve efficient lysis. In the isothermal amplification system, Tris-HCl and KCl provide an ionic environment, MgSO4 acts as a cofactor for DNA polymerase, betaine stabilizes the DNA double-strand structure, dNTPs provide raw materials for amplification, and a fluorescent indicator monitors the amplification process in real time. The primer combination is designed specifically for the target pathogenic microorganism to ensure amplification specificity; the mutant Bst-WK30 DNA polymerase has optimized enzyme activity and thermostability, enabling rapid and efficient nucleic acid amplification under isothermal conditions. The synergistic effect of each component overcomes the technical defects of traditional methods, such as insufficient sensitivity and poor specificity in complex aquatic environments, and exhibits a strong lysis effect on microorganisms. Complete lysis of pathogenic microorganisms can be achieved after standing at room temperature for 10 minutes.

[0038] This invention provides a rapid aquatic microbial separation device, which achieves overall device integration through a shell structure, providing stable physical support for filtration operations. The filtration component adopts a composite structure of four layers of filter membranes and filter paper, achieving physical retention of microorganisms through a progressively decreasing pore size gradient. The first to fourth filter membranes respectively screen for microorganisms of different particle sizes, while the filter paper acts as the final barrier to ensure microbial enrichment. The placement of the anode and cathode promotes the directional migration of microorganisms through the electric field, and combined with the specific design of the sample loading tank, effectively improves the microbial enrichment efficiency. Through the synergistic effect of physical filtration and electrochemical enrichment, this device can rapidly separate target microorganisms in complex aquatic environments, reducing interference from algae, suspended particles, and other impurities, providing high-purity microbial samples for subsequent molecular detection. It has a simple structure, is lightweight, and can enrich microorganisms from water samples onto the filter membrane within 20 minutes. No complex equipment is required, allowing for rapid on-site sample processing. Attached Figure Description

[0039] Figure 1 These are the purification results of the mutant Bst-WK30 DNA polymerase of this invention.

[0040] Figure 2 This is a schematic diagram of the rapid separation device for microorganisms in water according to the present invention, wherein, 1-filter paper, 2-first filter membrane, 3-second filter membrane, 4-third filter membrane, 5-fourth filter membrane, 6-plastic partition, 7-sample loading tank, 8-cathode, 9-anode, 10-power supply, 11-shell;

[0041] Figure 3 This is the result of the microorganisms isolated from the water sample by this invention being cultured overnight at 37°C;

[0042] Figure 4 This is a comparison of sample culture results before and after the lysis buffer treatment of this invention;

[0043] Figure 5 This is the result of detecting *Pseudomonas putida* pathogen in Example 5 of the present invention, wherein A is the detection result of microorganisms collected using conventional centrifugation technology; and B is the detection result of microorganisms rapidly enriched using the method of the present invention.

[0044] Figure 6 This is a comparison chart of the detection results of different Bst DNA polymerases and different sample processing methods used in Example 7 of the present invention. In the chart, A is the detection result of PBS-100℃ lysis products detected using commercially available Bst DNA polymerase; B is the detection result of PBS-100℃ lysis products detected using mutant Bst-WK30 DNA polymerase; C is the detection result of microbial lysis buffer-room temperature lysis products detected using commercially available Bst DNA polymerase; and D is the detection result of microbial lysis buffer-room temperature lysis products detected using mutant Bst-WK30 DNA polymerase. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available. Where specific techniques or conditions are not specified in the examples, they can be performed according to the techniques or conditions described in the literature or the product instructions.

[0047] The present invention uses *Pseudomonas putida* (… Pseudomonas putida ), agalactococcus ( Streptococcus agalactiae ), carp herpesvirus type III ( Cyprinid herpesvirus 3) and Tilapia Lake Virus ( Tilapia Lake virus Purchased from the China General Microbiological Culture Collection Center, it can be obtained by the public through other publicly available channels such as the China General Microbiological Culture Collection Center.

[0048] Example 1: Screening, expression, and purification of mutant Bst DNA polymerase

[0049] Using Tth DNA polymerase, an additional 1.5 mM Mn was added. 2+To increase its random mutation rate, the wild-type sequence of Bst DNA polymerase (as shown in SEQ ID No. 1-SEQ ID No. 2) was amplified using primers Bst-F: GGAATTCcatatgATGAAGAAGAAGCTAGTACTAATT (as shown in SEQ ID No. 5) and Bst-R: TGCctcgagTTATTTGGCATCATACCATGTTGGGC (as shown in SEQ ID No. 6). The obtained products were recovered by agarose gel electrophoresis to prepare a Bst DNA polymerase mutant library. Then, after double digestion with NdeI / XhoI, the polymerase was ligated into the pET28a vector to construct the pET28a-Bst recombinant plasmid library.

[0050] The pET28a-Bst recombinant plasmid library was directly transformed into Rosetta expression bacteria. After overnight culture, it was added to LB liquid medium at a ratio of 1:100 and rapidly scaled up to OD. 600 ≈0.6, then add IPTG to a final concentration of 0.5 mM, and induce at 37 ℃ for 4 h until OD... 600 ≈1.0. Centrifuge 1 mL of bacterial culture at 12000 rpm for 5 min to collect the bacterial cells. Resuspend the cells thoroughly in 300 μL of aqueous buffer (0.05% alkylamide ethylamine, 50 mM KCl, 20 mM Tris-HCl pH 8.8, 10 mM (NH4)2SO4, 2 mM MgSO4, 200 μM dNTPs, 500 nM Bst-F and 500 nM Bst-R primers) for later use. The 0.05% alkylamide ethylamine is used as the evolutionary pressure for the first round of selection.

[0051] Preparation of the emulsion system: The oil phase reagent was prepared by adding (73% Tegosoft DEC, 20% paraffin oil and 7% AbilEW09); the bacterial solution, which was fully resuspended in the aqueous buffer, was added to 700 μL of the oil phase reagent and emulsified at 42 Hz for 5 min to fully emulsify it and form water-in-oil droplets to obtain the emulsion system; at this time, Rosetta bacteria containing the pET28a-Bst plasmid were coated into the water-in-oil droplets, with one bacterium per droplet.

[0052] Add 50 μL of the above emulsification system to a PCR tube for PCR amplification. The amplification conditions are: 95℃ for 5 min; (95℃ for 20 sec, 55℃ for 20 sec, 72℃ for 2 min, 35 cycles); 72℃ for 7 min, and store at 16℃. After the reaction, all products were collected into 1.5 mL centrifuge tubes and centrifuged at 12,000 rpm for 10 min to remove the upper oil phase, retaining the emulsion layer. The emulsion layer and demulsifier were thoroughly mixed at a ratio of 1:1, allowed to stand at room temperature for 10 min, and then centrifuged at 12,000 rpm for 5 min. The upper aqueous phase was transferred to a new 1.5 mL centrifuge tube. The DNA was recovered using a PCR product purification kit. Using this DNA as a template, the selected Bst DNA polymerase sequence was further amplified using high-fidelity KOD DNA polymerase to increase its DNA concentration. The amplified product was also digested with NdeI / XhoI and ligated into the pET28a vector to construct the first-round pET28a-Bst evolutionary plasmid library. This pET28a-Bst evolutionary plasmid library was transformed into DH5α, and single colonies were picked for colony PCR. The amplification system was: 12.5 μL 2×KODPCR Mix; 1 μL 10 μM Bst-F; 10 μM... 1 μL of Bst-R; 10.5 μL of ddH2O; amplification conditions: 95℃ for 5 min; (95℃ for 20 sec, 55℃ for 20 sec, 72℃ for 2 min, 35 cycles); 72℃ for 7 min, stored at 16℃, and the obtained products were sequenced by PCR to analyze their amino acid mutation sites.

[0053] Multiple rounds of directed evolution were performed. The first round of pET28a-Bst evolution plasmid library was amplified using Tth DNA polymerase and primers Bst-F: GGAATTCcatatgATGAAGAAGAAGCTAGTACTAATT (as shown in SEQ ID No. 5) and Bst-R: TGC ctcgagTTATTTGGCATCATACCATGTTGGGC (as shown in SEQ ID No. 6). The second round of directed evolution was then performed with a pressure of 0.1% alkylamide acetaminophen. Similarly, a third round of directed evolution was performed with a pressure of 0.2% alkylamide acetaminophen.

[0054] After multiple rounds of directed evolution, the resulting Bst DNA polymerase mutants were sequenced, ultimately yielding a Bst DNA polymerase containing seven amino acid mutation sites: T365S, V428A, M488T, A663R, H664P, A741M, and L847G, named the mutant Bst-WK30 DNA polymerase (as shown in SEQ ID No. 3-SEQ ID No. 4). pET28-Bst-WK30 was then transformed into Rosetta and induced overnight at 16°C with 0.5 mM IPTG; subsequently, it was subjected to Ni... 2+ Purification was performed using an affinity chromatography column, and the results were as follows: Figure 1 We obtained high-purity mutant Bst-WK30 DNA polymerase.

[0055]

[0056] Example 2

[0057] This embodiment provides a rapid separation device for microorganisms in water. See attached document. Figure 2 The rapid separation device for microorganisms in water includes a housing 11, inside which a filter assembly is provided. The filter assembly consists of a first filter membrane 2, a second filter membrane 3, a third filter membrane 4, a fourth filter membrane 5, and filter paper 6, and is used to perform multi-stage filtration and separation on the added water sample to be tested. An anode 9 and a cathode 8 are respectively provided at both ends of the housing 11. When an external power supply 10 applies voltage, an electric field can be formed inside the housing 11 to assist in the separation of microorganisms in the water sample.

[0058] The housing 11 is provided with a sample loading groove 7, which is in communication with the interior of the housing 11.

[0059] The paper filter cartridge has a pore size of 100-200 μm, a diameter of 1 cm, and a length of 5-10 cm, trapping large particles such as algae, aquatic plants, and silt. The pore sizes of the four filter membranes, from the inside out, are as follows: the first filter membrane 2 has pore sizes of 80-100 μm, 30-50 μm, 10-15 μm, and 0.22 μm, respectively. Bacteria are ultimately enriched on the 0.22 μm filter membrane. The four filter membranes are separated by plastic partitions 6, with a spacing of 2-5 mm.

[0060] How to use this rapid separation device for microorganisms in water:

[0061] The collected water sample is slowly added into the shell 11 through the sample addition tank 7. The flow rate must be controlled during addition to prevent overflow or air bubbles from affecting the separation effect. The external power supply 10 is turned on, applying a set voltage between the anode 9 and cathode 8 to create an electric field inside the shell 11. Under the influence of the electric field, microorganisms in the water sample will move directionally according to their electrical properties and charge. After entering the shell 11, the water sample first passes through a paper filter cartridge with a pore size of 100-200 μm, a diameter of 1 cm, and a length of 5-10 cm, which can trap large particles such as algae, aquatic plants, and silt. It then passes sequentially through four layers of filter membranes with pore sizes of 80-100 μm, 30-50 μm, 10-15 μm, and 0.22 μm, separated by plastic partitions 6 with a spacing of 2-5 mm. Microorganisms and impurities of different particle sizes are progressively intercepted at filter membranes with different pore sizes, ultimately enriching bacteria on the 0.22μm fourth filter membrane, achieving multi-stage filtration and separation of microorganisms in the water. After the separation process reaches the predetermined time or achieves the expected separation effect, the external power supply 10 is turned off, stopping the electric field effect. The 0.22μm fourth filter membrane enriched with bacteria is then removed and placed in PBS buffer for further microbial LAMP detection, depending on subsequent testing requirements, to determine the types and concentrations of pathogenic bacteria in the water.

[0062] Example 3

[0063] This embodiment is based on the rapid aquatic microorganism separation device of Example 2. The device was used to collect samples and compared with traditional separation methods to examine the impact of different methods on the detection results. Experimental group: 4 mL of water sample was taken from a pond where farmed fish had already developed disease and placed in a sample loading tank with a diameter of 1 cm and a length of 5 cm. The sample was run at 25V for 20 min. Then, a 0.22 μm filter membrane was removed, placed in PBS buffer, and then spread onto LB solid medium.

[0064] Control group: Bacteria in the water were collected by centrifugation at 10,000 g for 10 min using the conventional method. The bacteria were then resuspended in 200 μL PBS and spread onto LB solid medium. Finally, the medium was incubated at 37°C for 2 days.

[0065] The results are as follows Figure 3 As shown, this invention can collect more bacteria. Therefore, compared with traditional centrifugation methods, this invention has the advantages of simple operation and higher detection sensitivity.

[0066] Example 4: Rapid lysis of samples

[0067] A 4 mL water sample was taken from a pond where farmed fish had already developed the disease and placed in a sample loading trough with a diameter of 1 cm and a length of 5 cm. The sample was run at 25 V for 20 min. Then, a 0.22 μm filter membrane was removed and placed in PBS buffer and the lysis buffer of this invention, respectively. After standing at room temperature for 10 min, it was spread onto LB solid medium and incubated at 37 °C for 2 days. The results are as follows: Figure 4 As shown, after treatment with 0.2% alkylamide ethylamine at room temperature for 10 minutes, the bacteria were fully lysed. The lysis buffer of this invention can fully lyse bacteria in a short time at room temperature, which is highly efficient and convenient compared to lysis methods that require specific temperatures, longer treatment times, or other complex conditions. This rapid lysis method can quickly release the components within the bacteria, facilitating subsequent detection and analysis, and helping to improve the efficiency of the entire detection process. It has significant application value in the rapid detection of pathogenic bacteria in aquaculture water.

[0068] Example 5

[0069] Twenty-four samples of microorganisms from the water were collected using traditional centrifugation and rapid enrichment methods, placed in PBS, and heated at 100°C for 5 minutes to release their DNA for later use.

[0070] LAMP technology was used to detect microorganisms in water collected by two different methods, and Pseudomonas putida was detected in the samples using LAMP technology.

[0071] The LAMP reaction system contained 20 mM Tris-HCl (pH 8.8), 50 mM KCl, 8 mM MgSO4, 0.8 M betaine, 1.4 mM dNTPs, 1.6 μM primers FIP / BIP, 0.8 μM primers LF / LB, 0.4 μM primers F3 / B3, 150 μM hydroxynaphthol blue (HNB) solution, 1.5 × Gelgreen solution, and commercially available Bst DNA polymerase. The primer sequences used are shown in Table 2.

[0072] Table 2: Primer set information for LAMP assay of *Pseudomonas putida*

[0073]

[0074] The processed sample DNA was added to the prepared LAMP reaction system and reacted at 65℃ for 30 min. The presence of *Pseudomonas putida* DNA amplification was determined by observing the color changes or fluorescence signals of HNB and Gelgreen. The results are as follows: Figure 5As shown, among the 24 samples collected by the traditional centrifugation method, only 7 were positive, with a positive rate of only 29.2%. However, among the 24 samples enriched by the rapid aquatic microorganism separation device of this invention, 19 were positive, with a positive rate of 79.2%. This indicates that the rapid aquatic microorganism separation device has a better effect in collecting aquatic microorganisms and can more effectively enrich the target microorganism (Pseudomonas putida), thereby improving the positive detection rate of LAMP detection.

[0075] Overall, the experimental results indicate that a rapid isolation and detection method for pathogenic bacteria in aquaculture water has higher efficiency and accuracy in detecting *Pseudomonas putida* in water, providing an effective method for the rapid detection of pathogenic microorganisms in aquaculture water.

[0076] Example 6

[0077] This embodiment, based on Embodiments 1 to 5, focuses on the presence of agalactococci (Streptococcus agalactiae) in water samples. Streptococcus agalactiae ), carp herpesvirus type III ( Cyprinid herpesvirus 3) and Tilapia Lake Virus ( Tilapia Lake virus The difference lies in the different detection primers used, and the specific primer sequences are shown in Table 3.

[0078] Table 3: LAMP Primer Set Information

[0079]

[0080] The results are shown in Table 4. Using the rapid isolation and detection method for pathogenic bacteria in aquaculture water of the present invention to enrich pathogenic microorganisms in the samples can significantly improve the positive rate of the detection results.

[0081] Table 4: Comparison of detection results between two sample pretreatment methods (n=24)

[0082]

[0083] Comparison of Streptococcus agalactiae detection results: The traditional centrifugation method detected only 3 positive samples out of 24 samples, with a positive rate of 12.5%. The enrichment method of this invention detected 5 positive samples, with a positive rate of (5 / 24) × 100% ≈ 20.8%. This indicates that the enrichment method of this invention can improve the positive detection rate of Streptococcus agalactiae compared to the traditional centrifugation method, demonstrating that the enrichment method more effectively enriches Streptococcus agalactiae in water samples, making them easier to detect.

[0084] Comparison of carp herpesvirus type III detection results: The traditional centrifugation method detected only 1 positive sample out of 24 samples, with a positive rate of 4.2%. The enrichment method of this invention detected 4 positive samples, with a positive rate of 16.7%. It can be seen that the enrichment method significantly improves the positive detection rate of carp herpesvirus type III.

[0085] Comparison of tilapia lake virus detection results: The traditional centrifugation method detected 1 positive sample out of 24 samples, with a positive rate of 4.2%; the enrichment method of this invention detected 4 positive samples, with a positive rate of 16.7%, showing a higher positive rate in the detection of tilapia lake virus, further proving its effectiveness in enriching pathogenic microorganisms.

[0086] Based on the above comparison of the detection results of the three pathogenic microorganisms, the rapid separation device for aquatic microorganisms of the present invention can significantly improve the positive rate of detection results for Streptococcus agalactiae, carp herpesvirus type III, and tilapia lake virus by enriching pathogenic microorganisms in samples. It can collect target pathogenic microorganisms more efficiently, reduce interfering substances in samples, and thus improve the sensitivity and accuracy of detection, providing a more effective method for the rapid and accurate detection of multiple pathogenic microorganisms in aquaculture water.

[0087] Example 7

[0088] Based on Examples 1 to 5, this embodiment uses the enrichment device of the present invention to collect 24 samples of microorganisms in water, which are placed in PBS and microbial lysis buffer respectively. The microorganisms in PBS are heated at 100°C for 5 minutes to release their DNA; the microorganisms in microbial lysis buffer are treated at room temperature for 10 minutes to release their DNA for later use.

[0089] LAMP assays were performed using commercially available Bst DNA polymerase and Bst-WK30 DNA polymerase, respectively, to compare the positive detection rates of *Pseudomonas putida*. The detection system and primers were the same as in Example 5.

[0090] The results are as follows Figure 6As shown, using commercially available Bst DNA polymerase to detect PBS-100℃ lysis products, 20 samples were positive, with a positive rate of 83.3%. However, using the mutant Bst-WK30 DNA polymerase, 22 samples were positive, with a positive rate of 91.7%, indicating that the mutant Bst-WK30 DNA polymerase has higher detection sensitivity than the commercially available Bst DNA polymerase. Using commercially available Bst DNA polymerase to detect microbial lysis buffer-room temperature lysis products, 10 samples were positive, with a positive rate of 41.7%. This is mainly because the microbial lysis buffer contains a certain amount of surfactant alkylamide ethylamine, which has a certain inhibitory effect on the reaction. However, using the mutant Bst-WK30 DNA polymerase to detect microbial lysis buffer-room temperature lysis products, 24 samples were positive, with a positive rate of 100%, indicating that the mutant Bst-WK30 DNA polymerase has higher tolerance to the surfactant alkylamide ethylamine than the commercially available Bst DNA polymerase.

[0091] Example 8

[0092] The method of Example 7 was used to treat agalactococci in water samples. Streptococcus agalactiae ), carp herpesvirus type III ( Cyprinid herpesvirus 3) and Tilapia Lake Virus ( Tilapia Lake virus The microorganisms were enriched and then divided into two groups for different lysis treatments. One group was placed in PBS buffer and heated at 100°C for 5 min for lysis. The high temperature destroyed the microbial cell structure and released DNA. The other group was treated with the microbial lysis buffer of the present invention at room temperature for 10 min to release DNA. The difference between the two groups was that they used different detection primers. The specific primer sequences are shown in Table 3. LAMP detection was performed using commercially available Bst enzyme and the mutant Bst-WK30 DNA polymerase of the present invention, respectively. Under the same conditions, the effect of different DNA polymerases on the detection results was compared. The detection results are shown in Table 5.

[0093] Table 5: Comparison of detection effects of different sample processing methods combined with DNA polymerase (n=24)

[0094]

[0095] Table 5 shows that in the PBS buffer + 100℃ heating lysis group: when using commercially available Bst enzyme, the number of positive samples was 18, with a positive rate of 75.0%; when using mutant Bst-WK30 DNA polymerase, the number of positive samples increased to 21, with a positive rate of 87.5%. This indicates that mutant Bst-WK30 DNA polymerase, under conventional lysis methods, can improve the detection positive rate of Streptococcus agalactiae compared to commercially available Bst enzyme. In the microbial lysis buffer + room temperature lysis group: when using commercially available Bst enzyme, the number of positive samples was only 8, with a positive rate of 33.3%. This is because the surfactant alkylamide ethylamine in the microbial lysis buffer has an inhibitory effect on commercially available Bst enzyme; while when using mutant Bst-WK30 DNA polymerase, the number of positive samples reached 23, with a positive rate of 95.8%, indicating that mutant Bst-WK30 DNA polymerase has higher tolerance to surfactants and can function better under these lysis conditions.

[0096] Results of carp herpesvirus type III detection: PBS buffer + 100℃ heating lysis group: 16 positive samples were detected with commercially available Bst enzyme, a positive rate of 66.7%; 19 positive samples were detected with mutant Bst-WK30 DNA polymerase, a positive rate of 79.2%, demonstrating the advantage of mutant Bst-WK30 DNA polymerase in improving detection sensitivity. Microbial lysis buffer + room temperature lysis group: Only 6 positive samples were detected with commercially available Bst enzyme, a positive rate of 25.0%; 22 positive samples were detected with mutant Bst-WK30 DNA polymerase, a positive rate of 91.7%, further proving that mutant Bst-WK30 DNA polymerase has stronger tolerance to inhibitors in microbial lysis buffer.

[0097] Tilapia lake virus detection results: PBS buffer + 100℃ heating lysis group: 19 positive samples were detected by commercially available Bst enzyme, with a positive rate of 79.2%; 22 positive samples were detected by mutant Bst-WK30 DNA polymerase, with a positive rate of 91.7%, demonstrating the high sensitivity of mutant Bst-WK30 DNA polymerase. Microbial lysis buffer + room temperature lysis group: 9 positive samples were detected by commercially available Bst enzyme, with a positive rate of 37.5%; 24 positive samples were detected by mutant Bst-WK30 DNA polymerase, with a positive rate of 100%, indicating that mutant Bst-WK30 DNA polymerase can completely and effectively detect tilapia lake virus under these lysis conditions.

[0098] The combined detection results of the three pathogens show that the method of this invention, by enriching pathogenic microorganisms in samples and treating them with the microbial lysis buffer of this invention, can rapidly and efficiently release the DNA of the microorganisms. When used in conjunction with the mutant Bst-WK30 DNA polymerase of this invention, the positive rate of the detection results can be significantly improved, whether under conventional lysis conditions of PBS buffer + 100°C heating or under lysis conditions of microbial lysis buffer containing inhibitors + room temperature. This is due to the high sensitivity and strong tolerance to surfactants of the mutant Bst-WK30 DNA polymerase, providing a more reliable technical means for the rapid and accurate detection of various pathogenic microorganisms in aquaculture water.

[0099] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A rapid method for isolating and detecting pathogenic bacteria in aquaculture water, characterized in that, include: Step 1: Enrich the microorganisms in the aquaculture water sample containing the target pathogenic bacteria; Step 2: Add the enriched microorganisms to the lysis buffer, let them stand at room temperature for lysis, and obtain a lysed microbial mixture. Step 3: Add the lysed microbial mixture to the PCR reaction solution for isothermal amplification and LAMP detection. Determine whether the sample contains the target pathogen based on the fluorescence signal. The PCR reaction solution contains a mutant Bst-WK30 DNA polymerase, the amino acid sequence of which is shown in SEQ ID No. 3; the target pathogenic bacteria are one or more of Streptococcus agalactiae, carp herpesvirus type III, Pseudomonas putidae, and tilapia lake virus.

2. The rapid separation and detection method according to claim 1, characterized in that, The lysis buffer contains Tris-HCl, ethylenediaminetetraacetic acid, and alkylamide ethylamine.

3. The rapid separation and detection method according to claim 1, characterized in that, The room temperature static pyrolysis time is 5 to 10 minutes.

4. The rapid separation and detection method according to claim 1, characterized in that, The PCR reaction solution also includes Tris-HCl, KCl, MgSO4, betaine, deoxyribonucleoside triphosphate, primer combination and fluorescent indicator.

5. The rapid separation and detection method according to claim 1, characterized in that, The isothermal amplification temperature is 60℃~70℃, and the amplification time is 20~40min.

6. The rapid separation and detection method according to claim 1, characterized in that, Microorganisms in aquaculture water samples are enriched using a rapid microbial separation device. The rapid microbial separation device includes a shell (11), a filter assembly, an external power supply (10), and a sample loading tank (7). The shell (11) is provided with an anode (9) and a cathode (8) at both ends. The filter assembly is located inside the shell (11) near the anode (9). The filtration assembly consists of a first filter membrane (2), a second filter membrane (3), a third filter membrane (4), a fourth filter membrane (5), and filter paper (1), and is used to perform multi-stage filtration and separation on the added water sample to be tested; A sample loading groove (7) is provided inside the housing (11) near the cathode (8), and the sample loading groove (7) is connected to the inside of the housing (11).

7. A kit for implementing the rapid separation and detection method according to any one of claims 1 to 6, characterized in that, This includes microbial lysis buffers and isothermal amplification systems; The microbial lysate contains Tris hydrochloride, ethylenediaminetetraacetic acid, and alkylamide ethylamine. The isothermal amplification system comprises Tris hydrochloride, potassium chloride, magnesium sulfate, betaine, deoxyribonucleoside triphosphate, primer combinations with sequences as shown in SEQ ID NO.7-SEQ ID NO.28, fluorescent indicator, and mutant Bst-WK30 DNA polymerase.

8. The reagent kit according to claim 7, characterized in that, The kit also includes a positive control and a negative control.

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