A primer combination for simultaneous identification of multiple pathogenic Aeromonas species

Abstract

The present invention discloses a primer combination for simultaneously identifying multiple pathogenic Aeromonas species, and belongs to the field of molecular diagnostic technology. Specifically, the present invention provides a primer combination and detection method for simultaneously detecting Aeromonas veronii, Aeromonas caviae, Aeromonas deca and Aeromonas hydrophila based on loop-mediated isothermal amplification technology, which has the following advantages: rapid, with the amplification reaction completed in only 20 minutes; high sensitivity, which is equivalent to the lower limit of PCR detection and can reach 10 copies / reaction; standardized, with the result automatically determined based on the test data; simple, with only one step of sample processing, and the reagents are in a freeze-dried form, which is convenient to use and store. The technical solution provided by the present invention provides technical support for the diagnosis of diseases caused by pathogenic Aeromonas.

Description

Technical Field

[0001] The present invention belongs to the field of molecular diagnosis technology, and specifically relates to a primer combination and an identification method for simultaneously identifying multiple pathogenic Aeromonas species based on loop-mediated isothermal amplification technology. Background Art

[0002] Aeromonas are a genus of Gram-negative rods widely distributed in aquatic environments, soil, and the intestinal tract of animals. Some species are opportunistic pathogens, infecting humans and aquatic organisms such as fish and amphibians, causing a variety of diseases. Among them, Aeromonas veronii, Aeromonas caviae, Aeromonas deca, and Aeromonas hydrophila have attracted considerable attention due to their high pathogenicity and widespread distribution.

[0003] Infections with Aeromonas veronii typically present as wound suppuration, gastroenteritis, or rarely, hemolytic uremic syndrome. Symptoms of Aeromonas caviae include diarrhea, cholecystitis, and meningitis. Aeromonas deka is particularly virulent, often associated with severe gastroenteritis, necrotizing fasciitis, and multiple organ failure in humans. Aeromonas hydrophila is a Gram-negative, facultative anaerobic bacillus that is widely distributed in freshwater, soil, sewage, and aquatic animals. Diseases caused by pathogenic Aeromonas hydrophila not only cause serious economic losses to the aquaculture industry, but can also infect humans and livestock through aquatic animals and aquatic products, leading to diarrhea and food poisoning in humans and livestock, affecting human health. People with weakened immune systems are susceptible to infection through wounds or intestinal tracts after contact with contaminated water, causing diarrhea, skin ulcers, and even sepsis.

[0004] Currently, the diagnosis of Aeromonas infection relies on molecular biology (such as PCR), biochemical, or immunological testing. Due to the large number of Aeromonas species, biochemical identification methods require multiple biochemical reactions to identify the species. These complex and time-consuming procedures, coupled with low specificity and sensitivity, fall far short of the needs of routine rapid testing. Enzyme-linked immunosorbent assay (ELISA) can rapidly detect bacteria, but the preparation of the relevant antibodies is difficult, the procedures are complex and time-consuming, and the false positive rate is high. PCR technology, with its advantages of sensitivity, specificity, and rapidity, is widely used for testing various samples. However, PCR technology places high demands on the laboratory personnel and environment, with expensive equipment and numerous steps.

[0005] Isothermal amplification (LAMP) is a nucleic acid amplification technique widely used in scientific research and clinical testing. It can amplify trace amounts of DNA or RNA templates by 100,000 to 1 million times, visualizing the product growth process using fluorescent dyes. Among the various isothermal amplification techniques, loop-mediated isothermal amplification (LAMP) is the most commonly used. Compared with PCR, LAMP offers advantages such as faster detection, better sensitivity, and higher specificity.

[0006] Patent document CN102140512A discloses a LAMP detection method for pathogenic Aeromonas hydrophila. The method involves sample DNA extraction, LAMP amplification, and colorimetric observation. After the LAMP reaction, a colorimetric solution (10% SYBR Green fluorescent dye) is added and mixed, and the color change is observed directly with the naked eye. Patent document CN103911433A discloses a LAMP detection primer set and kit for Aeromonas hydrophila. The kit features strong specificity and high sensitivity. Results can be assessed using a turbidimeter, visual observation of the color change after adding a fluorescent dye, or agarose gel electrophoresis. LAMP detection methods for Aeromonas hydrophila often rely on visual inspection to determine amplification results, which often varies widely and is difficult to assess. The method also takes a long time, often exceeding 30 minutes, making it unsuitable for rapid testing. Furthermore, LAMP detection techniques for Aeromonas veronii, Aeromonas caviae, and Aeromonas decaensis have rarely been reported. Summary of the Invention

[0007] In order to address the defects of the prior art, in the first aspect of the present invention, the present invention provides a primer combination for simultaneously detecting Aeromonas veronii, Aeromonas caviae, Aeromonas deca and Aeromonas hydrophila based on loop-mediated isothermal amplification technology, and the primer combination is the best LAMP primer for detecting target Aeromonas obtained by screening. In the second aspect of the present invention, the present invention provides the application of the LAMP primer combination in detecting target bacterial species. In the third aspect of the present invention, the present invention provides a kit and a microfluidic chip containing the LAMP primer combination. In the fourth aspect of the present invention, the present invention provides a method for simultaneously identifying target Aeromonas based on loop-mediated isothermal amplification technology.

[0008] The present invention provides a primer combination and detection method for the simultaneous detection of Aeromonas veronii, Aeromonas caviae, Aeromonas decapsidae, and Aeromonas hydrophila based on loop-mediated isothermal amplification technology. The advantages include: 1. rapidity, with the amplification reaction completing in just 20 minutes; 2. high sensitivity, comparable to the lower limit of PCR detection, reaching 10 copies / reaction; 3. standardized, with results automatically determined based on the test data; and 4. simplicity, with sample processing requiring only one step and the reagents being freeze-dried for easy use and storage. The technical solution provided by the present invention provides technical support for the diagnosis of diseases caused by pathogenic Aeromonas bacteria.

[0009] The present invention includes the following technical solutions:

[0010] In a first aspect, the present invention provides a LAMP primer combination, characterized in that the primer combination consists of the primer groups described in 1) to 4):

[0011] 1) For the detection of Aeromonas veronii ( Aeromonas veronii), the primer set consisting of primers shown in SEQ ID NO: 1 to SEQ ID NO: 4;

[0012] 2) For the detection of Aeromonas caviae ( Aeromonas caviae ), the second primer set consisting of primers shown in SEQ ID NO: 5 to SEQ ID NO: 8;

[0013] 3) For the detection of Aeromonas decaphylla ( Aeromonas dhakensis ), the primer set consisting of primers shown in SEQ ID NO: 9 to SEQ ID NO: 12;

[0014] 4) For the detection of Aeromonas hydrophila ( Aeromonas hydrophila ), the primer set consisting of primers shown in SEQ ID NO: 13-SEQ ID NO: 17.

[0015] In a specific embodiment of the present invention, the method for detecting Aeromonas veronii ( Aeromonas veronii ) The sequence information of the first LAMP primer set is as follows:

[0016] .

[0017] In a specific embodiment of the present invention, the method for detecting Aeromonas caviae ( Aeromonas caviae ) The sequence information of the second LAMP primer set is as follows:

[0018] .

[0019] In a specific embodiment of the present invention, the method for detecting Aeromonas decapsidata ( Aeromonas dhakensis ) The sequence information of the third LAMP primer set is as follows:

[0020] .

[0021] In a specific embodiment of the present invention, the method for detecting Aeromonas hydrophila ( Aeromonas hydrophila ) The sequence information of the fourth LAMP primer set is as follows:

[0022] .

[0023] In a second aspect, the present invention provides a use of the LAMP primer combination according to the first aspect of the present invention in at least one of the following:

[0024] 1) Application in the preparation of products for detecting and / or assisting in the detection of Aeromonas;

[0025] 2) Application in the preparation of drugs for screening or auxiliary screening for treating diseases caused by Aeromonas

[0026] The products include but are not limited to reagents, kits, test strips, membrane strips, chips or detection platforms.

[0027] In the present invention, the Aeromonas is Aeromonas veronii ( Aeromonas veronii ), Aeromonas caviae ( Aeromonas caviae ), Aeromonas decaphylla ( Aeromonas dhakensis ) and Aeromonas hydrophila ( Aeromonas hydrophila ) combination.

[0028] The related diseases caused by Aeromonas are known in the art and are caused by Aeromonas veronii ( Aeromonas veronii ), Aeromonas caviae ( Aeromonas caviae ), Aeromonas decaphylla ( Aeromonas dhakensis ) and Aeromonas hydrophila ( Aeromonas hydrophila ) diseases caused by infection. For example, diseases caused by Aeromonas veronii infection include wound suppuration, gastroenteritis, or the rare hemolytic uremic syndrome; diseases caused by Aeromonas caviae infection include diarrhea, cholecystitis, and meningitis; diseases caused by Aeromonas decaphylla infection include gastroenteritis, necrotizing fasciitis, and multiple organ failure; diseases caused by Aeromonas hydrophila infection include diarrhea, skin ulcers, and even sepsis.

[0029] In a third aspect, the present invention provides a kit, characterized in that the kit comprises the LAMP primer combination described in the first aspect of the present invention.

[0030] The kit provided by the present invention is a rapid test product for simultaneously detecting Aeromonas veronii, Aeromonas caviae, Aeromonas decalca and Aeromonas hydrophila based on loop-mediated isothermal amplification technology. It can quickly and accurately detect whether the sample to be tested contains Aeromonas veronii, Aeromonas caviae, Aeromonas decalca and Aeromonas hydrophila. The kit also includes other conventional reagents required for LAMP technology.

[0031] In some embodiments of the present invention, the other conventional reagents include Bst enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer, and magnesium ions. Preferably, the buffer contains an RNase inhibitor.

[0032] In some embodiments of the present invention, the kit further includes a sample lysis solution, a sample diluent, and a sample DNA extraction reagent / kit. The above reagents can be purchased from commercial sources or prepared by the user.

[0033] In one embodiment of the present invention, the sample lysate contains a surfactant, wherein the surfactant is composed of one or more of NP-40, Triton X-100, PEG 6000, Tween-20, Tween-80, and sodium dodecyl sulfate (SDS). The mass concentration of each surfactant component in the sample lysate is between 0.2% and 4.3%.

[0034] In order to read the amplification results efficiently and accurately, the kit also includes a fluorescent dye, which is a dye with a fluorescent group commonly used by those skilled in the art, and has a fluorescence excitation wavelength and emission wavelength between 485-530 nm. In a specific embodiment, the fluorescent dye is Eva Green.

[0035] Preferably, the LAMP primer combination, fluorescent dye and other conventional reagents in the kit are packaged separately.

[0036] In a preferred embodiment of the present invention, the LAMP primer combination, fluorescent dye, and other conventional reagents in the kit are stored in the form of freeze-dried beads in a vacuum-packed aluminum foil bag. Specifically, the freeze-dried beads are prepared by the following method:

[0037] S1) preparing a buffer containing Bst polymerase, reverse transcriptase, fluorescent dye, and dNTPs, and adding the LAMP primer combination described in the first aspect of the present invention to form a LAMP amplification reaction solution;

[0038] S2) dropping the LAMP amplification reaction solution into liquid nitrogen to form a sphere;

[0039] S3) Place in freeze-drying equipment and freeze-dry overnight to form freeze-dried beads.

[0040] In the LAMP amplification reaction solution described in step S1), primer P v1 、P v2 、P c1 、P c2 、P d1 、P d2 、P h1 、P h2 、P h5 The final concentration of primer P is 0.1-0.2 μM. v3 、P v4 、P c3 、P c4 、P d3 、P d4 、P h3 、P h4 The final concentration is 0.3-1.0 μM.

[0041] In step S2), each droplet preferably has a volume of 25-30 μl. In a specific operation, each droplet has a volume of 25 μl.

[0042] Furthermore, the kit also includes a positive quality control product, which is a plasmid containing genes of Aeromonas veronii, Aeromonas caviae, Aeromonas decaphycus and Aeromonas hydrophila.

[0043] Furthermore, the kit also includes a negative quality control product, which is pure water treated with diethyl pyrocarbonate (DEPC).

[0044] In a fourth aspect, the present invention provides a microfluidic chip, characterized in that the microfluidic chip is composed of 2-4 reaction detection areas, each reaction detection area includes an injection pool, a distribution pool, a capillary microvalve and an amplification pool connected in sequence, and the amplification pool is coated with the LAMP primer combination described in the first aspect of the present invention.

[0045] In a specific embodiment of the present invention, the LAMP primer combination is fixed in the amplification pool by a vacuum drying method.

[0046] Those skilled in the art know that the injection pool is used to load the reaction liquid, the distribution pool is used to evenly distribute the reaction liquid to the amplification pool, and the amplification pool is used to perform LAMP amplification reaction. The capillary microvalve controls the movement of the fluid in the microfluidic chip by utilizing the blocking effect of the capillary on the liquid.

[0047] In a fifth aspect, the present invention provides a method for simultaneously identifying multiple Aeromonas species based on loop-mediated isothermal amplification technology, wherein the method is not intended for disease treatment or diagnosis, and is characterized in that the method comprises the following steps:

[0048] S1: Extract genomic DNA or RNA from the sample to be tested, reverse transcribe the RNA into cDNA, and combine the DNA and cDNA as sample DNA;

[0049] S2: Add the sample DNA to the loop-mediated isothermal amplification reaction solution and amplify at 65°C for 20-25 minutes;

[0050] S3: Read the fluorescence value every 20-30 seconds and judge the result based on the amplification curve.

[0051] The loop-mediated isothermal amplification reaction solution described in step S2 is a buffer solution containing the LAMP primer combination described in the first aspect of the present invention, Bst polymerase, reverse transcriptase, fluorescent dye and dNTPs.

[0052] Furthermore, the loop-mediated isothermal amplification reaction solution also includes freeze-dried beads prepared according to the loop-mediated isothermal amplification reaction solution.

[0053] When the loop-mediated isothermal amplification reaction solution is freeze-dried beads prepared therefrom, step S2 specifically comprises: mixing the sample DNA with the freeze-dried beads, and amplifying at 65° C. for 20-25 minutes, preferably for 20 minutes.

[0054] The sample to be tested is selected from samples known to those skilled in the art that may contain Aeromonas veronii, Aeromonas caviae, Aeromonas decapsidae and / or Aeromonas hydrophila, and the sample includes but is not limited to feces.

[0055] The multiple Aeromonas species are selected from Aeromonas veronii ( Aeromonas veronii ), Aeromonas caviae ( Aeromonas caviae ), Aeromonas decaphylla ( Aeromonas dhakensis ) and Aeromonas hydrophila ( Aeromonas hydrophila ) combination.

[0056] In a specific embodiment of the present invention, the result judgment method is: when the amplification curve is S-shaped, it is a positive curve, indicating that the Aeromonas is present; when the amplification curve is a horizontal straight line, it is a negative curve, indicating that the Aeromonas is not present.

[0057] The technical solution provided by the present invention has the following technical advantages:

[0058] 1) The loop-mediated isothermal amplification technology provided by the present invention can simultaneously detect four common Aeromonas, including Aeromonas veronii, Aeromonas caviae, Aeromonas decalca, and Aeromonas hydrophila, filling the gap in the existing technology for LAMP detection technology for Aeromonas veronii, Aeromonas caviae, and Aeromonas decalca.

[0059] 2) The method for detecting Aeromonas based on loop-mediated isothermal amplification technology provided by the present invention has a fast detection speed, taking only 20 minutes, which is significantly faster than conventional detection methods and is very suitable for rapid on-site inspection.

[0060] 3) The method for detecting Aeromonas based on loop-mediated isothermal amplification technology provided by the present invention has good specificity and high sensitivity, and can achieve 10 copies / reaction.

[0061] 4) The method for detecting Aeromonas based on loop-mediated isothermal amplification technology provided by the present invention can prepare the amplification solution used for LAMP amplification into the form of freeze-dried beads for use, which is convenient for use and storage, simplifies experimental operations, and is suitable for rapid on-site inspection. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Fluorescence amplification curves of four Aeromonas positive samples;

[0063] Figure 2 Gradient amplification curve of Aeromonas veronii quality control product;

[0064] Figure 3 Gradient amplification curve of Aeromonas guinea pigs quality control product;

[0065] Figure 4 Gradient amplification curve of Aeromonas decaphylla quality control product;

[0066] Figure 5 Gradient amplification curve of Aeromonas hydrophila quality control product;

[0067] Figure 6 Specific test results for Aeromonas salmonicida;

[0068] Figure 7 Specific detection results for Aeromonas veronii;

[0069] Figure 8 Specific test results for Aeromonas caviae;

[0070] Figure 9 Specific detection results for Aeromonas decaphylla;

[0071] Figure 10 Specific test results for Aeromonas hydrophila;

[0072] Figure 11 Specific test results for Aeromonas salmonicida;

[0073] Figure 12 Specific detection results for Aeromonas hydrophila. DETAILED DESCRIPTION

[0074] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0075] Example 1 Design of LAMP primer combination

[0076] The loop-mediated isothermal amplification (LAMP) reaction is a novel nucleic acid amplification technology based on chain displacement cycling. The reaction is performed at a constant temperature of 60-65°C. LAMP uses four to six primers, each recognizing six to eight regions on the target gene, giving the reaction greater specificity. Because the LAMP reaction produces a large amount of a white magnesium pyrophosphate precipitate as a byproduct, the amplified product can be directly identified by visual observation or turbidimetry without electrophoresis analysis. The present invention incorporates a fluorescent dye into the amplification system, enabling more real-time and intuitive observation of the amplification process using a fluorescent thermostated amplification instrument or a fluorescent PCR instrument, enabling accurate interpretation of the results.

[0077] In detection methods based on loop-mediated isothermal amplification (LAMP) technology, the design of LAMP primers is crucial for accurate detection. The LAMP primer combinations provided by the present invention are determined by the following method: screening the target gene sequence to be detected; performing a Blast comparison on the target gene sequence similarity; and using online software to design multiple pairs of primers for six regions of the target gene (F3c, F2c, and F1c at the 3' end of the target gene, and B1, B2, and B3 at the 5' end of the target gene). After manual modification, the two to three LAMP primer sets with the best theoretical parameters and the highest success rate are selected for experimental verification, and the LAMP primers with the best experimental results are selected.

[0078] After screening and optimization, the present invention is used to detect Aeromonas veronii ( Aeromonas veronii ), Aeromonas caviae ( Aeromonas caviae ), Aeromonas decaphylla ( Aeromonas dhakensis ) and Aeromonas hydrophila ( Aeromonas hydrophila ) of the LAMP primers were P v1 -P v4 、P c1 -P c4 、P d1 -P d4 、P h1 -P h5 The sequence information of the LAMP primers is as follows:

[0079] Table 1 Sequence information of LAMP primer combinations

[0080]

[0081] Example 2 Kit for detecting Aeromonas based on LAMP technology

[0082] The kit comprises the following parts:

[0083] (1) Lyophilized beads;

[0084] (2) Sample lysis buffer, sample diluent, negative quality control product, and positive quality control product;

[0085] (3) Boxes for separating and centrally packaging these reagent bottles or tubes.

[0086] The freeze-dried beads were prepared by the following method:

[0087] S1) Prepare a buffer containing Bst polymerase, reverse transcriptase, fluorescent dye and dNTPs, add the LAMP primer combination described in Table 1 to form a LAMP amplification reaction solution; in the LAMP amplification reaction solution, primer Pv1 、P v2 、P c1 、P c2 、P d1 、P d2 、P h1 、P h2 、P h5 The final concentration of primer P is 0.1-0.2 μM. v3 、P v4 、P c3 、P c4 、P d3 、P d4 、P h3 、P h4 The final concentration is 0.3-1.0 μM;

[0088] S2) dropping the LAMP amplification reaction solution into liquid nitrogen to form a sphere, with each drop being 25 μl;

[0089] S3) Place in freeze-drying equipment and freeze-dry overnight to form freeze-dried beads.

[0090] Example 3 Method for detecting Aeromonas based on LAMP technology

[0091] The detection steps are as follows:

[0092] S1: Use a stool swab as the specimen to be tested. Place the collected stool swab in a dry, dedicated small box. It can be sent directly for testing or stored in a 4°C refrigerator for 7 natural days.

[0093] S2: Place the swab into a plastic tube containing sample lysis solution, break the swab, cover the tube, and shake vigorously for 20 seconds;

[0094] S3: Take out one drop, add it to the plastic tube containing the diluent, and shake for 20 seconds to mix;

[0095] S4: Take one drop of the diluent or pipette 25 μl into the reaction tube and mix with the lyophilized beads (prepared in Example 2) in the tube. Directly pipette 25 μl of the negative and positive quality control products into the reaction tube.

[0096] S5: Place the reaction tube on an oscillator for 10 seconds, centrifuge briefly, and then place it in a dedicated fluorescence device for amplification (fluorescence thermostat amplification instrument or fluorescence PCR instrument);

[0097] S6: Program settings: 65°C, 30 seconds to read FAM fluorescence, 40 cycles, total duration 20 minutes;

[0098] S7: After amplification, carefully remove the reaction tube, wrap it with gloves, tie a knot and discard it to avoid aerosol contamination;

[0099] S8: Result determination: 1) If there is no S-shaped curve in the amplification, the sample is determined to be negative; 2) If the amplification curve is clearly S-shaped, the sample is determined to be positive.

[0100] When the test sample is a positive sample of Aeromonas veronii, Aeromonas caviae, Aeromonas deca, and Aeromonas hydrophila, the amplification curve is as follows: Figure 1 shown.

[0101] Example 4 Sensitivity Detection

[0102] A certain amount of plasmid containing specific gene fragments of Aeromonas veronii, Aeromonas caviae, Aeromonas decapsidae, and Aeromonas hydrophila was dissolved in an appropriate amount of ultrapure water. The OD value was measured and the concentration was calculated using the formula OD260×50µg / ml. The copy number per microliter was calculated based on the molar mass of the plasmid. The solution was then diluted with ultrapure water to 2.0 copies / µl, 1.0 copies / µl, 0.5 copies / µl, and 0.2 copies / µl, respectively.

[0103] In the reaction tube, 25µl of plasmid template of various gradients was added, and each gradient was repeated three times. Together with the negative control, the experiment was performed on a Macrostone SLAN-96S fluorescence quantitative PCR instrument using a temperature control program of 65°C for 30 seconds (FAM fluorescence was read) and 40 cycles.

[0104] The experimental results are as follows Figure 2-5 As shown in the figure, three replicates were positive for 2.0 copies / µl, 1.0 copies / µl, and 0.5 copies / µl, while the negative control showed a negative result. This fully demonstrates that the LAMP method provided by the present invention can detect Aeromonas veronii, Aeromonas caviae, Aeromonas decagenes, and Aeromonas hydrophila with a sensitivity of 0.5 copies / µl.

[0105] Example 5 Specificity Detection

[0106] According to the method provided in Example 3, Aeromonas salmonicida ( Aeromonas salmonicida ) DNA 25µl was added to the freeze-dried bead reaction tube containing Aeromonas veronii, Aeromonas caviae, Aeromonas deca, and Aeromonas hydrophila, and amplified by LAMP. No amplification curve was found ( Figure 6 ).

[0107] According to the method provided in Example 3, 25 μl of the positive control sample of Aeromonas veronii was added to the freeze-dried bead reaction tube containing the test Aeromonas veronii, Aeromonas caviae, Aeromonas deca, and Aeromonas hydrophila. After LAMP amplification, only the DNA of Aeromonas veronii showed positive ( Figure 7 ).

[0108] According to the method provided in Example 3, 25 μl of the positive control sample of Aeromonas caviae was added to the freeze-dried bead reaction tube containing Aeromonas veronii, Aeromonas caviae, Aeromonas deca, and Aeromonas hydrophila. After LAMP amplification, only the DNA of Aeromonas caviae showed positive ( Figure 8 ).

[0109] According to the method provided in Example 3, 25 μl of the positive control sample of Aeromonas decalca was added to the freeze-dried bead reaction tube containing Aeromonas veronii, Aeromonas caviae, Aeromonas decalca and Aeromonas hydrophila. After LAMP amplification, only Aeromonas decalca DNA showed positive ( Figure 9 ).

[0110] According to the method provided in Example 3, 25 μl of the Aeromonas hydrophila positive control product was added to the freeze-dried bead reaction tube containing Aeromonas veronii, Aeromonas caviae, Aeromonas deca, and Aeromonas hydrophila. After LAMP amplification, only Aeromonas hydrophila DNA showed positive ( Figure 10 ).

[0111] Example 6 Screening process of LAMP primer combinations

[0112] The LAMP primers provided by the present invention for the target Aeromonas bacteria are prepared by the following method: determining the target gene sequence; and designing the LAMP primers using primer design software. While the software-designed primers are theoretically capable of amplifying the target gene, preliminary experimental results indicate that the software-designed primers do not necessarily meet the requirements of the present invention in terms of specificity and sensitivity. Therefore, base substitutions are performed on the sequences of the two paired primers, and the amplification effects of different primer pairs are repeatedly tested to achieve better specificity and sensitivity.

[0113] Taking Aeromonas veronii and Aeromonas decacata as examples, the target genes to be tested screened in the present invention are gyrB gene and rpoD gene, respectively. Multiple sets of LAMP primers are designed using online LAMP primer design software. Tables 2 and 3 respectively show the LAMP amplification primers for Aeromonas veronii and Aeromonas decacata with better comprehensive parameters provided by the software.

[0114] Table 2 Sequence information of LAMP primer sets for Aeromonas veronii

[0115] .

[0116] Table 3 Sequence information of LAMP primer sets for Aeromonas decapsidata

[0117] .

[0118] Tables 2 and 3 provide primer combinations with good theoretical parameters provided by primer design software. However, experimental results show that both sets of primers have specificity problems.

[0119] According to the method provided in Example 3, Aeromonas salmonicida ( Aeromonas salmonicida ) DNA 25µl was added to the reaction tubes containing freeze-dried beads for detecting Aeromonas veronii, Aeromonas caviae, Aeromonas deca, and Aeromonas hydrophila. The LAMP primers in the freeze-dried beads for detecting Aeromonas veronii are shown in Table 2, and the LAMP primers in the other three freeze-dried beads are shown in Table 1. After LAMP amplification, it was found that the reaction tube for detecting Aeromonas veronii showed amplification ( Figure 11 ).

[0120] According to the method provided in Example 3, 25 μl of the positive control DNA of Aeromonas hydrophila was added to the reaction tube containing the freeze-dried beads for detecting Aeromonas veronii, Aeromonas caviae, Aeromonas deca, and Aeromonas hydrophila. The LAMP primers in the freeze-dried beads for detecting Aeromonas deca are shown in Table 3, and the LAMP primers in the other three freeze-dried beads are shown in Table 1. After LAMP amplification, it was found that amplification was observed for both Aeromonas deca and Aeromonas hydrophila ( Figure 12 ).

[0121] The above two test results demonstrate that software-designed primers for LAMP amplification, even if theoretically optimized, can routinely achieve specific amplification of target genes, but the LAMP amplification system involves multiple primers, and the Tm value of each primer must be strictly controlled to avoid excessive mismatches and redundancies. Therefore, the inventors manually modified the software-designed primers to improve their specificity. Based on this, the present invention ultimately preferred the LAMP primer combination shown in Table 1 for simultaneous detection of Aeromonas veronii, Aeromonas caviae, Aeromonas decacornis, and Aeromonas hydrophila as the optimal LAMP primer combination.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A LAMP primer combination, characterized in that: The primer combination consists of the primer groups described in 1)-4): 1) For the detection of Aeromonas veronii ( Aeromonas veronii ), the primer set consisting of primers shown in SEQ ID NO: 1 to SEQ ID NO: 4; 2) For the detection of Aeromonas caviae ( Aeromonas caviae ), the second primer set consisting of primers shown in SEQ ID NO: 5 to SEQ ID NO: 8; 3) For the detection of Aeromonas decaphylla ( Aeromonas dhakensis ), the primer set consisting of primers shown in SEQ ID NO: 9 to SEQ ID NO: 12; 4) For the detection of Aeromonas hydrophila ( Aeromonas hydrophila ) is a fourth primer set, which consists of primers shown in SEQ ID NO: 13-SEQ ID NO:

17.

2. Use of the LAMP primer combination according to claim 1 in at least one of the following: 1) Application in the preparation of products for detecting and / or assisting in the detection of Aeromonas; 2) Application in the preparation of drugs for screening or assisting in screening for the treatment of diseases caused by Aeromonas; The Aeromonas is Aeromonas veronii ( Aeromonas veronii ), Aeromonas caviae ( Aeromonas caviae ), Aeromonas decaphylla ( Aeromonas dhakensis ) and Aeromonas hydrophila ( Aeromonas hydrophila ) combination.

3. A kit, characterized in that The kit comprises the LAMP primer combination according to claim 1.

4. The kit according to claim 3, wherein The kit also includes other conventional reagents required for LAMP technology, including Bst enzyme, reverse transcriptase, dNTPs, ultrapure water, buffer, and magnesium ions.

5. The kit according to claim 4, characterized in that The kit also includes a sample lysis solution, a sample diluent, and a sample DNA extraction reagent / kit.

6. The kit according to claim 5, characterized in that The kit also includes a fluorescent dye, which is a dye with a fluorescent group, and the fluorescence excitation wavelength and emission wavelength are both between 485-530 nm.

7. The kit according to any one of claims 3 to 6, characterized in that The LAMP primer combination, fluorescent dye and other conventional reagents in the kit are packaged separately; or The LAMP primer combination, fluorescent dye and other conventional reagents in the kit are stored in the form of freeze-dried beads in a vacuum-packed aluminum foil bag; the freeze-dried beads are prepared by the following method: S1) preparing a buffer containing Bst polymerase, reverse transcriptase, fluorescent dye, and dNTPs, and adding the LAMP primer combination of claim 1 to form a LAMP amplification reaction solution; S2) dropping the LAMP amplification reaction solution into liquid nitrogen to form a sphere; S3) Place in freeze-drying equipment and freeze-dry overnight to form freeze-dried beads.

8. The kit according to claim 7, characterized in that In the LAMP amplification reaction solution described in step S1), the final concentration of the primers represented by SEQ ID NO: 1-2, SEQ ID NO: 5-6, SEQ ID NO: 9-10, SEQ ID NO: 13-14, and SEQ ID NO: 17 is 0.1-0.2 μM, and the final concentration of the primers represented by SEQ ID NO: 3-4, SEQ ID NO: 7-8, SEQ ID NO: 11-12, and SEQ ID NO: 15-16 is 0.3-1.0 μM.

9. A microfluidic chip, characterized in that: The microfluidic chip consists of 2-4 reaction detection areas, each reaction detection area includes a sampling pool, a distribution pool, a capillary microvalve and an amplification pool connected in sequence, and the amplification pool is coated with the LAMP primer combination according to claim 1.

10. A method for simultaneously identifying multiple Aeromonas species based on loop-mediated isothermal amplification technology, wherein the method is not intended for disease treatment or diagnosis, and is characterized in that: The method comprises the following steps: S1: Extract genomic DNA or RNA from the sample to be tested, reverse transcribe the RNA into cDNA, and combine the DNA and cDNA as sample DNA; S2: Add the sample DNA to the loop-mediated isothermal amplification reaction solution and amplify at 65°C for 20-25 minutes; S3: Read the fluorescence value every 20-30 seconds and judge the result based on the amplification curve; The loop-mediated isothermal amplification reaction solution described in step S2 is a buffer solution containing the LAMP primer combination, Bst polymerase, reverse transcriptase, fluorescent dye and dNTPs described in claim 1; alternatively, the loop-mediated isothermal amplification reaction solution also includes freeze-dried beads prepared according to the loop-mediated isothermal amplification reaction solution.

Images