Seawater pathogenic microorganism detection kit and application

Abstract

The invention provides a seawater pathogenic microorganism detection kit and application, and belongs to the technical field of biological detection. Compared with the prior art, the method has the advantages that the requirement on professional instruments in the seawater pathogenic bacteria detection process is greatly reduced, suction filtration equipment for water pretreatment is not needed, equipment such as a centrifugal machine is also not needed, the demand quantity of a water sample is reduced from upgrading to milliliter level, and rapid microbe enrichment and in-situ nucleic acid extraction of small-volume seawater can be realized; the nucleic acid extraction yield is high, and the quality is good; through flexible adjustment of the primer group and the DNA polymerase in the reaction tank, customization of detection of target pathogens, synchronous detection of various bacteria and seawater pathogens, visualization and on-site rapid detection can be realized, and the use requirement of a low-resource environment is met.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a seawater pathogenic microorganism detection kit and application thereof. Background Art

[0002] Seawater contains a variety of pathogenic microorganisms, which can harm the marine environment and marine life, and thus affect human health. Therefore, the detection of pathogenic microorganisms in seawater is crucial to the safety of the marine environment and aquatic food.

[0003] At present, the detection of pathogenic microorganisms in seawater mainly includes plate culture method and nucleic acid-based analysis method. Although the plate culture method is cheap and reliable, for seawater samples, because pathogens are usually low in abundance, they are often not detected or the detection is one-sided and limited, and the detection time is long. Based on nucleic acid analysis, there are mainly PCR diagnostic methods and gene sequencing methods that amplify specific genes of target pathogens. However, the common problems of these two methods are: ① Limited by cumbersome and time-consuming nucleic acid extraction, generally low throughput, dependence on professional instruments and professional technicians, and often limited to laboratory operations. In particular, in order to enrich pathogenic microorganisms in water bodies, nucleic acid extraction of pathogenic microorganisms in seawater generally requires pretreatment: first, seawater is filtered, and the microorganisms are trapped on the surface of the filter membrane to achieve enrichment of microorganisms on the surface of the filter membrane; then the filter membrane is cut into pieces and soaked in lysis solution for nucleic acid extraction. The disadvantages of pretreatment are significant: the demand for water samples is large, and usually 500mL-1000mL of seawater needs to be collected for the filtration process; the pretreatment process is cumbersome, inefficient, and easy to pollute. ② The existing technology equipment is highly dependent and requires professional technicians. Common pathogen PCR diagnostic techniques, such as conventional PCR and qPCR, require thermal cyclers with precise temperature control; gene sequencing methods rely more on expensive professional sequencing instruments and require technicians with professional skills, and cannot be performed in environments other than professional laboratories (such as aquaculture areas and testing sites). ③ High R&D costs and poor adjustability. For example, handheld PCR technology based on portable chips increases integration and portability, but the chip R&D cost is high, the chip reading instrument is expensive, and the modifiability is poor. It is often used for single detection of a single pathogen and has low throughput. Therefore, the abundance of pathogenic microorganisms in seawater is low and rapid detection methods are still relatively scarce.

[0004] Therefore, it is of great significance to develop a pathogen detection method that is simple and fast to operate from sampling to determination of pathogen detection results, has low equipment dependence, and is oriented towards low resources and testing sites. Summary of the invention

[0005] The purpose of the present invention is to provide a seawater pathogenic microorganism detection kit and application. The detection method is simple and fast from sampling to determination of detection results, does not require precise instruments, and can detect multiple pathogens in a short time.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a seawater pathogenic microorganism detection kit, which is composed of the following parts:

[0008] Lysis tank, cleaning tank, reaction tank, separator capable of absorbing nucleic acid, combined solvent bottle, reagent bottle with cell wall breaking and / or protein degradation function, reagent bottle with nucleic acid protection and / or buffer adjustment function, cleaning liquid bottle, eluent bottle, reaction liquid bottle for detecting pathogenic microorganisms in seawater.

[0009] Preferably, when in use:

[0010] The lysis tank is used to hold the lysis solution;

[0011] The elution tank is used to hold the eluent;

[0012] The reaction tank is used to hold the reaction solution.

[0013] Preferably, the lysate comprises a reagent with cell wall disruption and / or protein degradation function and a reagent with nucleic acid protection and / or buffer adjustment function;

[0014] Preferably, the separator capable of adsorbing nucleic acid is a functional body having a polymer functional region and a rod-shaped handle; the raw materials for preparing the polymer functional region include one or more of polyacrylic acid, polyethylene glycol diacrylate, polydopamine, and silicon dioxide;

[0015] Preferably, the eluent bottle comprises an eluent capable of dissolving the nucleic acid on the separator.

[0016] Preferably, the reagent in the reagent bottle having the function of cell wall disruption and / or protein degradation comprises sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, Chelex, proteinase K, NaOH, NaHCO 3 One or more of;

[0017] Preferably, the reagents in the reagent bottle for nucleic acid protection and / or adjusting buffer function include one or more of NaCl, EDTA, Tris-HCl, mercaptoethanol, and dithiothreitol.

[0018] Preferably, the binding solvent in the binding solvent bottle includes one or more of ethanol and isopropanol;

[0019] Preferably, the cleaning solution in the cleaning solution bottle comprises 70-80% ethanol;

[0020] Preferably, the eluent in the eluent bottle comprises one or more of water, PBS buffer, and TE buffer;

[0021] Preferably, the reaction solution in the reaction solution bottle includes DNA polymerase, a primer set, and more preferably also includes an indicator.

[0022] The invention also provides an application of a seawater pathogenic microorganism detection kit in seawater pathogenic microorganism detection.

[0023] The beneficial effects of the present invention compared with the prior art are:

[0024] (1) Compared with the prior art, the present invention greatly reduces the need for professional instruments in the process of detecting pathogens in seawater. It does not require filtration equipment for water pretreatment, nor does it require equipment such as centrifuges. The demand for water samples is reduced from the milliliter level, and rapid enrichment of microorganisms and in-situ nucleic acid extraction with a minimum of 0.36 mL of seawater can be achieved. The nucleic acid extraction has a high yield and good quality. Combined with the visual LAMP technology, rapid detection of seawater pathogens can be achieved. It takes only 50 minutes from sampling to determination of test results. The entire process can be operated on-site to meet the use requirements of low-resource environments.

[0025] (2) Compared with the prior art, the present invention is simple and fast to operate, greatly saving labor and time costs, and significantly reducing the demand for water samples. At the same time, the nucleic acid extraction process avoids the cumbersome operations of the existing column separation technology that relies on centrifuges and the need to continuously suspend magnetic beads during magnetic separation, thus shortening the time of the entire process of obtaining microbial nucleic acids from water samples.

[0026] (3) The flexibility of the present invention in detecting pathogenic bacteria is greatly improved. The target bacteria for detection can be adjusted at any time by flexibly adjusting the primers and DNA polymerase to achieve customization of the detection object. Multiple pathogens can also be detected simultaneously by connecting detection holes in series or reacting simultaneously in a single hole. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0028] Figure 1 The nucleic acid separation results of the two groups of separators in Example 2 of the present invention;

[0029] Figure 2 The nucleic acid adsorption content result of the separator prepared by the third method of Example 1 in Example 2 of the present invention;

[0030] Figure 3 The results of the 7 bacteria detection under natural light in Example 3 of the present invention are as follows;

[0031] Figure 4 The results of the detection of 7 kinds of bacteria under ultraviolet light in Example 3 of the present invention are as follows;

[0032] Figure 5 The results of simultaneous detection of three bacteria in Example 4 of the present invention, M is DL2000Marker, S is the sample;

[0033] Figure 6 These are the detection results of 7 types of bacteria in seawater according to Example 5 of the present invention. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The present invention provides a seawater pathogenic microorganism detection kit, which is composed of the following parts:

[0040] Lysis tank, cleaning tank, reaction tank, separator capable of absorbing nucleic acid, combined solvent bottle, reagent bottle with cell wall breaking and / or protein degradation function, reagent bottle with nucleic acid protection and / or buffer adjustment function, cleaning liquid bottle, eluent bottle, reaction liquid bottle for detecting pathogenic microorganisms in seawater.

[0041] The separator capable of absorbing nucleic acid is a functional body having a polymer functional area and a rod-shaped handle. The raw materials for preparing the polymer functional area include one or more of polyacrylic acid, polyethylene glycol diacrylate, polydopamine, and silicon dioxide. More preferably, the surface of the separator is rough. The silicon dioxide is more preferably nano-silicon dioxide powder or quartz sand, glass, etc. whose main component is silicon dioxide. The material and shape of the rod-shaped handle area are not limited. Its function is to transfer with the polymer functional area, which can be adjusted according to the usage scenario.

[0042] When in use, the cleaning tank is used to hold cleaning liquid; wherein, the cleaning tank comprises one or more groups, preferably 1-3 groups, which can be used to clean the separator 1-3 times; the number of tanks in each group is not limited and is adjusted according to the number of samples; the shape of the cleaning tank is not limited to adapt to the size of the separator, and the bottom functional area of ​​the separator needs to be immersed in the cleaning liquid.

[0043] When in use, the reaction tank is used to hold the reaction liquid. The reaction tank can be a single one for one pathogen in one sample, or multiple reaction tanks can be arranged side by side for multiple pathogens in one sample or for one pathogen in multiple samples.

[0044] The cleaning solution includes 70-80% alcohol, and the cleaning time is 10-60 seconds per time;

[0045] The reaction solution includes Bst DNA polymerase, LAMP primer set, and indicator, wherein the primer set includes EIP, BIP, F3 and B3, and more preferably also contains primers LP and LB; wherein the concentration of EIP / BIP is 8 to 16 μM, the concentration of F3 / B3 is 0.5 to 2 μM, and the concentration of LP / LB is 2 to 8 μM.

[0046] The indicator is calcein and hydroxy One or a mixture of two phenol blues. The concentration of calcein is: 5-50μM; hydroxy The concentration of phenol blue is: 50-500μM.

[0047] The present invention also provides a method for detecting pathogenic microorganisms in seawater, comprising the following steps:

[0048] (1) adding lysing solution into a lysing tank, then adding seawater into the lysing tank, mixing, and letting stand;

[0049] (2) adding a binding solvent to the pyrolysis tank after standing in step (1), inserting a separator, and shaking the separator;

[0050] (3) After the standing period is over, the separator is transferred to a cleaning tank containing a cleaning solution, and then cleaned and dried;

[0051] (4) the dried separator is transferred to an elution tank containing an eluent for elution;

[0052] (5) after elution, transferring the eluate to a reaction tank containing a reaction solution;

[0053] (6) Perform the reaction according to the instructions for use of the primer set, and determine the result by color change of the reaction solution, fluorescent signal or gel electrophoresis.

[0054] Preferably, in step (1), the volume ratio of seawater to lysate in the lysing tank is 1-2:8-9; the standing time is 10-30 minutes, and the temperature is 15-65°C.

[0055] Preferably, in step (2), the amount of the combined solvent added is 60-90% of the volume of seawater; and the time for shaking the separator is 10s to 5min.

[0056] Preferably, the elution time in step (4) is 5 s-5 min.

[0057] Preferably, the standing time in step (5) is 10 to 60 minutes.

[0058] Preferably, when judging the test result in step (6), the color change can be observed under natural visible light or ultraviolet light.

[0059] Table 1 Primer sequence list used in the present invention

[0060]

[0061]

[0062]

[0063] Example 1

[0064] In Example 1 of the present invention, three types of separators were prepared, and the specific steps were as follows:

[0065] Method 1: The structure of the detector array was designed using Solidworks modeling software, and commercial acrylic resin was doped with 5wt% nano-SiO 2 , the separator was prepared by DLP 3D printing with a printing layer thickness of 50μm. The functional area size was a cone with a threaded structure of 2.0mm radius and 10.00mm height. The handle was an integrally printed slender cylinder with a height of 50mm. The prepared detector was repeatedly cleaned with deionized water and anhydrous ethanol and dried for later use. ( Figure 1 a)

[0066] The second method: Use a commercially available 0.2mL or 1.5mL centrifuge tube as a mold, pour about 1cm of prepolymer solution (monomer is acrylic acid, containing 50wt% quartz sand particles (40-270μm), 0.02wt% cross-linking agent, 24.995wt% initiator) into the mold, insert a toothpick or bamboo stick, and place the mold and prepolymer solution under ultraviolet light to irradiate, using light to initiate free radical polymerization. The ultraviolet light wavelength is 280-400nm, and irradiate for 3-10 minutes on all sides to completely cure it. The long-handled detector is repeatedly washed with deionized water and anhydrous ethanol, and dried for use. ( Figure 1 b)

[0067] The third method: insert a 0.2mL centrifuge tube into a commercially available 1.5mL centrifuge tube, insert a toothpick into the sandwich formed by the two tubes, inject the silica-doped polyacrylic acid-quartz sand prepolymer solution (acrylic acid monomer contains 50wt% quartz sand particles (40-270μm), 0.02wt% cross-linking agent, 24.995wt% initiator), irradiate with ultraviolet light, and make a wall-attached separator that matches the 1.5mL centrifuge tube, wash with water and ethanol, and dry. ( Figure 1 c)

[0068] Example 2

[0069] Example 2 of the present invention detects the nucleic acid adsorption effect of the separator, and the specific steps are as follows:

[0070] (1) According to the third method in Example 1, a 0.2 mL centrifuge tube was placed in a 1.5 mL centrifuge tube, a toothpick was inserted into the sandwich formed by the two tubes, and a polyacrylic acid-quartz sand material doped with silica (acrylic acid monomer containing 50 wt% quartz sand particles (40-270 μm), 0.02 wt% cross-linking agent, and 24.995 wt% initiator) was injected and irradiated with ultraviolet light to make a wall-attached separator matching the 1.5 mL centrifuge tube, and then washed with water and ethanol and dried.

[0071] (2) Add 360 μL of seawater collected from the aquaculture pond to each of two 1.5 mL centrifuge tubes (lysis tanks) pre-filled with 40 μL of high-concentration lysis solution (1.0 M Tris-HCl, 250 mM of EDTA. 5 M of NaCl, 10 wt% SDS, 100 μg / mL proteinase K) and mix well.

[0072] Place at 60℃ for 10min until the sample tissue is completely dissolved and the solution becomes clear; add 200μL of anhydrous ethanol to the centrifuge tube in the lysis tank, insert the separator, and gently shake the separator for 1min; transfer the separator to a cleaning tank pre-filled with 300μL of 75% ethanol, shake it up and down for 30s to clean the separator, then take it out and repeat this operation twice; transfer the separator to an elution tank containing 100μL of TE elution solution and shake it up and down for 1min to elute the nucleic acid from the device. The obtained nucleic acid sample is used for UV spectrophotometry to test its nucleic acid adsorption content. Figure 2 shown.

[0073] Depend on Figure 2 It can be seen that compared with the prior art, the present invention greatly reduces the demand for professional instruments in the process of detecting seawater pathogens. It does not require filtration equipment for water pretreatment, nor does it require equipment such as centrifuges. The demand for water samples is reduced from the upgrade level to the milliliter level. The nucleic acid extracted by the separator has a high concentration, which can fully meet subsequent needs.

[0074] Example 3

[0075] Example 3 of the present invention detects the detection effect of the seawater pathogenic microorganism detection kit of the present invention, and the specific steps are as follows:

[0076] (1) Preparation of reagents:

[0077] Lysis buffer: 0.1M Tris-HCl, 25mM of EDTA, 500mM of NaCl, 1wt% SDS, 10μg / mL proteinase K

[0078] Eluent: TE buffer (30 mM Tris·Cl, 1 mM EDTA, pH 8.0)

[0079] (2) The cells of seven kinds of bacteria frozen in the laboratory were added to a centrifuge tube pre-filled with 100 μL of lysis solution and allowed to stand at 60°C for 15 min; 60 μL of isopropanol was added, and the 3D printed separator prepared by the first method of Example 1 was inserted into it, and stirred up and down 3-4 times for 1 min; the separator was taken out and transferred to a centrifuge tube pre-filled with 150 μL of 75% ethanol, and the separator was shaken up and down for 30 seconds to clean; the cleaning process was repeated once; the separator was taken out, the excess liquid was shaken off, and it was inverted on the laboratory bench to air dry for 5 min; the dried separator was placed in a centrifuge tube pre-filled with 80 μL of eluent, and the separator was disturbed up and down for 1 min-1.5 min. The separator was removed and the eluent was quantified by a UV spectrophotometer. Each bacterial experiment was repeated 3 times. The concentration of bacterial DNA extracted by the separator was 36.48-142.02 ng / μL, A260 / 280>2, and there was no protein contamination, which met the requirements of subsequent LAMP experiments.

[0080] (3) The eluate was transferred to a reaction tank for LAMP reaction.

[0081] The experimental conditions of LAMP are as follows: each 25 μL reaction system contains: 2×BcaBEST Buffer 12.5 μL, EIP (100 pM) 0.4 μL, BIP (100 pM) 0.4 μL, F3 (100 pM) 0.1 μL, B3 (100 pM) 0.1 μL, LP (100 pM) 0.2 μL, LB (100 pM) 0.2 μL, 0.85 μL color developer, 2 μL DNA sample obtained in step (2), 0.25 μL sterile deionized water. Negative reference DNA is replaced by water. The reaction conditions are: incubate at 62°C for 25 min, and heat at 90°C for 3 min. Determine the LAMP results under natural light or ultraviolet light, such as Figure 2 and 3 As shown, Figures 1-3 are 3 repetitions of LAMP experiments for each bacteria, and NC is a negative reference. Figure 3 In natural light, positive results appear blue, and negative results appear purple. Figure 4 In the figure, positive under ultraviolet light is green and negative is pink.

[0082] The present invention is applicable to the detection of various bacteria, has high detection sensitivity, and can realize visualization of result determination. The detection process is simple to operate, has low dependence on equipment in the whole process, and is applicable to low-resource environments.

[0083] Example 4

[0084] Example 4 of the present invention tests the effect of the seawater pathogenic microorganism detection kit of the present invention on simultaneous detection of multiple bacteria, and the specific steps are as follows:

[0085] (1) The separator was prepared using the second method in Example 1.

[0086] (2) Add the frozen bacterial cells in the laboratory into a centrifuge tube pre-filled with 100 μL lysis solution (0.1 M Tris-HCl, 25 mM of EDTA. 500 mM of NaCl, 1 wt% SDS, 20 μg / mL proteinase K), and let it stand at 60°C for 15 min; add 70 μL of anhydrous ethanol, insert the separator into it, stir it up and down 3-4 times, and maintain it for 1 min; take out the separator, transfer it to a cleaning tank pre-filled with 150 μL of 75% ethanol, and shake the separator up and down to clean it for 30 seconds; repeat the cleaning process once; take out the separator, shake off the excess liquid, and turn it upside down on the laboratory bench to air dry for 5 min; put the dried separator into an elution tank pre-filled with 80 μL of elution solution, disturb the separator up and down, maintain it for 1 min-1.5 min, and remove the separator.

[0087] (3) Transfer 5 μL of the eluate obtained in step (2) to a reaction tank pre-filled with 45 μL of reaction solution (a total system of 50 μL containing 29.6 μL of ultrapure water, 5 μL of 10× buffer, 4 μL of dNTPMixture, 1 μL of each primer SEQ ID NO.39-44 (1 μL of each of 5 sets of DNA primers), and 0.4 μL of TakaRa Taq), and react under the following conditions: 94°C for 10 min; 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min for 40 cycles, and 72°C for extension for 10 min. After the reaction, the detection result was determined by gel electrophoresis results ( Figure 5 ).

[0088] Depend on Figure 5 It can be seen that in the same detection hole, three bacteria, namely, Vibrio harveyi, Vibrio parahaemolyticus and Vibrio alginolyticus, were detected simultaneously, which indicates that the present invention can detect multiple pathogens simultaneously with high detection efficiency.

[0089] Example 5

[0090] Example 4 of the present invention uses a seawater pathogenic microorganism detection kit to detect seawater, and the specific steps are as follows:

[0091] A seawater sample collected from the seaside park was placed in a 50mL centrifuge tube pre-filled with 1.5mL 1M TRIS-HCl (pH=8.0), 0.75mL 1M EDTA (PH=8.0), 0.4383g NaCl, 7.5μL proteinase K (20mg / mL), and 1.5mL 10wt% SDS. 12mL of the seawater sample was added, and the second separator with a long handle prepared in Example 1 was added to disturb the separator back and forth in the liquid. After about 1 minute, 9mL of isopropanol was added, and the long handle was continued to be held in the hand to disturb the device back and forth in the liquid. After 1 minute, the device was removed and the separator was placed in a 200μL centrifuge tube containing 150μL 75% ethanol. The separator was immersed in the liquid and moved up and down several times for cleaning and then taken out. The separator was placed in air to dry for 10 minutes and transferred to 80μL 1×TE Soak in a centrifuge tube containing Buffer (pH=8.0) for 1.5 min to elute the nucleic acid and then remove from the separator. The nucleic acid eluate is quantified by UV, and the nucleic acid concentration is 14.150 ng / μL.

[0092] Take 2 μL of each and add it to 8 tubes pre-installed with 2×BcaBEST Buffer 12.5 μL, EIP (100 pM) 0.4 μL, BIP (100 pM) 0.4 μL, F3 (100 pM) 0.1 μL, B3 (100 pM) 0.1 μL, LP (100 pM) 0.2 μL, LB (100 pM) 0.2 μL, 0.85 μL colorimetric agent, and 0.25 μL sterile deionized water. The primers in tubes 1-7 are respectively directed against pathogens: Vibrio splendidus (Vsp), Vibrio parahemolyticus (Vpa), Vibrio harveyi (Vha), Vibrio alginolyticus (Val), Vibrio rotificularis (Vro), Vibrio corallilyticus (Vro), Vibrio splendidus (Vsp), Vibrio harveyi (Vha), Vibrio alginolyticus (Val), Vibrio rotificularis (Vro), Vibrio splendidus (Vsp), Vibrio splendidus (Vpa), Vibrio splendidus (Vha), Vibrio splendidus (Vsp), Vibrio splendidus (Vsp), Vibrio splendidus (Vpa), Vibrio splendidus (Vha), Vibrio splendidus (Val), Vibrio splendidus (Vro), Vibrio splendidus (V corallinolyticus.Vco), Shewanella (She), and No. 8 was the negative reference.

[0093] Cover the 8-tube strips and place them in a 62° constant temperature module for 25 minutes. Figure 6 As shown, No. 1 is green and No. 2-8 are pink, indicating that Aspergillus splendens contamination was detected in the collected seawater.

[0094] Compared with the prior art, the present invention greatly reduces the need for professional instruments in the process of detecting pathogens in seawater. It does not require filtration equipment for water pretreatment, nor does it require equipment such as centrifuges, reducing the demand for water samples from the upgrade to the milliliter level. Combined with the visual LAMP technology, it can achieve rapid detection of seawater pathogens, and it takes only 50 minutes from sampling to the determination of test results. The whole process can be operated on-site to meet the use requirements of low-resource environments. By connecting multiple bacterial detection holes in series, it is possible to simultaneously detect 7 types of bacteria and improve detection efficiency.

[0095] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A seawater pathogenic microorganism detection kit, characterized in that: It consists of the following parts: Lysis tank, cleaning tank, reaction tank, separator capable of absorbing nucleic acid, combined solvent bottle, reagent bottle with cell wall breaking and / or protein degradation function, reagent bottle with nucleic acid protection and / or buffer adjustment function, cleaning liquid bottle, eluent bottle, reaction liquid bottle for detecting pathogenic microorganisms in seawater.

2. The seawater pathogenic microorganism detection kit according to claim 1, characterized in that: In use, The lysis tank is used to hold the lysis solution; The elution tank is used to hold the eluent; The reaction tank is used to hold the reaction solution.

3. The seawater pathogenic microorganism detection kit according to claim 1, characterized in that: The lysate includes a reagent with cell wall disruption and / or protein degradation functions and a reagent with nucleic acid protection and / or buffer adjustment functions; The separator capable of adsorbing nucleic acid is a functional body having a polymer functional area and a rod-shaped handle; the raw materials for preparing the polymer functional area include one or more of polyacrylic acid, polyethylene glycol diacrylate, polydopamine, and silicon dioxide; The eluent bottle includes an eluent capable of dissolving the nucleic acid on the separator.

4. The seawater pathogenic microorganism detection kit according to claim 3, characterized in that: The reagent in the reagent bottle having the function of cell wall disruption and / or protein degradation comprises one or more of sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, Chelex, proteinase K, NaOH, and NaHCO3; The reagents in the reagent bottle for nucleic acid protection and / or adjusting buffer function include one or more of NaCl, EDTA, Tris-HCl, mercaptoethanol, and dithiothreitol.

5. The seawater pathogenic microorganism detection kit according to claim 1, characterized in that: The combination solvent in the combination solvent bottle includes one or more of ethanol and isopropanol.

6. The seawater pathogenic microorganism detection kit according to claim 1, characterized in that: The cleaning solution in the cleaning solution bottle includes 70-80% ethanol.

7. The seawater pathogenic microorganism detection kit according to claim 2, characterized in that: The eluent in the eluent bottle includes one or more of water, PBS buffer, and TE buffer.

8. The seawater pathogenic microorganism detection kit according to claim 2, characterized in that: The reaction solution in the reaction solution bottle includes DNA polymerase and a primer set.

9. Use of the seawater pathogenic microorganism detection kit according to any one of claims 1 to 8 in the detection of seawater pathogenic microorganisms.

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