CN111273007B - Kit for rapidly detecting Edwardsiella ictaluri and detection method - Google Patents
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Abstract
The invention provides a kit for rapidly detecting Edwardsiella ictaluri of fish and a detection method, wherein the kit comprises a glass slide treated by polylysine, a rabbit anti-Edwardsiella ictaluri polyclonal antibody, an HRP-marked sheep anti-rabbit lgG, a sealing liquid and a color development liquid. The invention takes the glass slide as the solid phase carrier, thereby greatly reducing the detection cost; the rabbit anti-Edwardsiella ictaluri polyclonal antibody prepared by the invention can recognize a plurality of epitopes of the same antigen, and has strong specificity and high sensitivity; TMB is a peroxidase substrate, and TMB is used as a color development liquid, so that a soluble light blue product can be generated, and the light blue product is adsorbed on filter paper, so that the color change can be visually observed. The detection method has the advantages of low equipment requirement, low cost, simple and convenient operation, environmental protection and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for rapidly detecting Edwardsiella ictaluri and a detection method.
Background
With the development of economy, the pursuit of nutrients is more intense. The fish is rich in protein and is a good nutrition source, in particular to yellow catfish, the yellow catfish (pelteobagrus fulvidraco) belongs to catfish order, family of the Cryptotavernaculaceae, the yellow catfish belongs to high-quality and noble freshwater fish in China, commonly called yellow catfish, huang Lading and the like. The pelteobagrus fulvidraco is rich in protein, has the effects of maintaining potassium-sodium balance, eliminating edema, improving immunity, regulating hypotension and buffering anemia, and is beneficial to the growth and development of teenagers. Meanwhile, the pelteobagrus fulvidraco is rich in copper, which is an essential trace nutrition cable for human health, has important influence on development and functions of blood, central nervous and immune system, hair, skin and skeletal tissue, brain, liver, heart and other viscera, can regulate osmotic pressure, maintain acid-base balance and normal blood pressure, and strengthen neuromuscular excitability.
The artificial culture of pelteobagrus fulvidraco in China has been in the past ten years, and the total yield of pelteobagrus fulvidraco in 2018 nationwide reaches 50 ten thousand tons. Sichuan is the main breeding place for pelteobagrus fulvidraco breeding, according to statistics of Chinese fishery statistics annual authentication, the total yield of pelteobagrus fulvidraco in China in 2017 is up to 3.05 ten thousand tons, the average market value is about 6305 ten thousand yuan, and the total yield of pelteobagrus fulvidraco in China in 2018 is up to 3.41 ten thousand tons. Along with the expansion of the cultivation area and cultivation density, pelteobagrus fulvidraco diseases are more and more increased, and the damage of Edwardsiella is also more and more serious.
Edwardsiella ictaluri was first isolated from diseased channel catfish in 1979 by HawkeJP, and was identified as Edwardsiella group "GA7752" in 1981 as a new strain of Edwardsiella of Enterobacteriaceae and designated as Edwardsiella ictaluri, which has a short rod-like shape with a size of 0.5-1 μm×2-3 μm, with periflagella, with a dynamic force, with weak exercise ability at 20-30 ℃, without exercise ability at 37 ℃, and gram staining negative. The growth is slow on the common culture medium, the culture is needed for 25-30 hours on the BHI culture medium at the temperature of 25-30 ℃, the colony with the needle point size can be grown only by 48 hours on the TSA culture medium, and the colony does not grow when the temperature reaches 37 ℃. Diseased pelteobagrus fulvidraco can exhibit 2 manifestations of acute death and chronic death. Acute death manifests as typical sepsis; the chronic dead fish body shows anorexia, is isolated and has no obvious symptoms when the fish body is in the initial stage of disease; along with the development of the disease, congestion points appear at the top of the head of the fish with disease, the head is enlarged continuously, and finally, a congestive focus is formed on the skull between two eyes, and when the congestion is serious, perforation of the top of the head, cracking of the skull and even exposure of brain tissues can be caused; the fish is out of balance just before death, hangs in water, is in a shape of hanging water, and turns over quickly or moves irregularly in a spiral shape when stimulated, so that death occurs. The disease has the characteristics of strong infectivity, urgent disease, difficult prevention and treatment, high disease rate and death rate, and the like. Causing great economic loss for fishery cultivation.
The traditional diagnosis method has long time consumption, high cost, complex operation and high equipment requirement. Therefore, accurate prevention and treatment of Edwardsiellosis, i.e. a timely, rapid and convenient diagnostic detection method is indispensable.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the kit and the detection method for rapidly detecting the Edwardsiella ictaluri, which can effectively solve the problems of high cost, complex operation, high equipment requirement and long time consumption of the conventional diagnosis method.
In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a kit for rapid detection of edwardsiella ictaluri in fish, comprising: the method comprises the steps of a glass slide treated by polylysine, a rabbit anti-Edwardsiella ictaluri polyclonal antibody, an HRP-marked goat anti-rabbit lgG blocking solution and a color development solution.
After the glass slide is treated by polylysine, the polylysine has positive charges, can interact with negative charges on the surface of Edwardsiella ictaluri to generate stronger adhesive force, and can avoid bacterial shedding in the washing process.
Further, the polylysine-treated slide was prepared by the following method: diluting polylysine, uniformly smearing on the frosted surface of a glass slide, repeatedly smearing for 2-3 times, and naturally drying.
Further, rabbit anti-Edwardsiella ictaluri polyclonal antibody passesThe preparation method comprises the following steps: culturing Edwardsiella ictaluri, preparing the Edwardsiella ictaluri into an inactivated vaccine, immunizing a rabbit with the inactivated vaccine for four times, collecting blood after the fourth immunization, collecting serum, and purifying the serum by IgG to prepare the polyclonal antibody of the Edwardsiella ictaluri; the specific process is as follows: edwardsiella ictaluri is cultured, and 0.6% formaldehyde is used to inactivate bacteria to obtain a concentration of 1×10 8 cfu/ml inactivated vaccine, the inactivated vaccine is immunized on New Zealand rabbits for four times, blood is collected after the fourth immunization, serum is collected, igG is purified on the serum, and the polyclonal antibody of the rabbit against Edwardsiella ictaluri is prepared.
Further, the blocking solution is BSA with the mass concentration of 5%; the preparation process comprises the following steps: under aseptic conditions, 5.0g Bovine Serum Albumin (BSA) was weighed, dissolved in 1L distilled water with a low salt buffer (Tris base 24.2g,NaCl 222.2g, tween 20 5ml, pH was adjusted to 7.3) and then fixed to 100ml, and stored at 4 ℃.
Further, the color developing solution is TMB solution.
The method for rapidly detecting the Edwardsiella ictaluri by adopting the kit comprises the following steps of:
(1) Fixing Edwardsiella ictaluri bacterial liquid on a glass slide treated by polylysine;
(2) Sealing the glass slide obtained in the step (1) by using sealing liquid at normal temperature;
(3) Smearing rabbit anti-Edwardsiella ictaluri polyclonal antibody on the slide obtained in the step (2) for reaction;
(4) Smearing the HRP-marked goat anti-rabbit lgG on the slide obtained in the step (3) for reaction;
(5) And (3) attaching filter paper containing a color developing solution to the slide obtained in the step (4) for color development.
Further, the specific process of the step (1) is as follows: the Edwardsiella ictaluri bacterial liquid is smeared on a slide glass treated by polylysine, naturally dried or fixed by flame, and then 10% formalin and absolute methanol are adopted to soak the slide glass in sequence.
Further, the closing time in the step (2) is 20-60min; preferably 30min.
Further, in the step (3), the reaction temperature is 35-40 ℃ and the reaction time is 40-60min; the reaction temperature is preferably 37℃and the reaction time is 1h.
Further, in the step (4), the reaction temperature is 35-40 ℃ and the reaction time is 40-60min; the reaction temperature is preferably 37℃and the reaction time is 1h.
The kit and the detection method for rapidly detecting the Edwardsiella ictaluri of the pelteobagrus fulvidraco have the following beneficial effects:
(1) The invention takes the glass slide as the solid phase carrier, thereby greatly reducing the detection cost; the rabbit anti-Edwardsiella ictaluri polyclonal antibody prepared by the invention can recognize a plurality of epitopes of the same antigen, and has strong specificity and high sensitivity; TMB is a peroxidase substrate, TMB is used as a color development liquid, a soluble light blue product can be generated, white regular filter paper sheets are soaked in TMB single-component dyeing liquid and then attached to a glass slide for color development for 5min, the light blue product is adsorbed on the filter paper, and the color change can be visually observed through naked eyes.
Drawings
FIG. 1 is a microscopic image of Edwardsiella after staining with a gram stain.
FIG. 2 is a diagram showing the electrophoresis of nucleic acid gel after sequencing by PCR method of Edwardsiella.
FIG. 3 is a graph showing the results of the detection using the method of the present invention.
Detailed Description
Example 1
A kit for rapid detection of edwardsiella ictaluri in pelteobagrus fulvidraco, comprising: polylysine treated slide (solid phase carrier slide), rabbit anti-Edwardsiella ictaluri polyclonal antibody, HRP labeled goat anti-rabbit lgG, blocking solution and color development solution.
Wherein the blocking solution is BSA with the mass concentration of 5%, and the developing solution is TMB solution.
The rabbit anti-Edwardsiella ictaluri polyclonal antibody is prepared by the following method:
(1) Inoculating Edwardsiella ictaluri to BHI plate, culturing in incubator at 27deg.C for 24 hr, purifying by gram staining method, sequencing by PCR method, inactivating bacteria with 0.6% formaldehyde to obtain a concentration of 1×10 8 cfu/ml inactivated vaccine;
(2) Immunizing a New Zealand white rabbit with the inactivated vaccine prepared in the step (1), immunizing 4 times, immunizing 1 time every other week, wherein the dosage of the vaccine for the first 3 times is 1ml, the dosage of the vaccine for the 4 th time is 0.5ml, bleeding the carotid artery after the 4 th immunization, collecting blood and collecting serum;
(3) Purifying the serum obtained in the step (2) by using a saturated ammonium sulfate precipitation method, and determining the antibody titer 1 of the IgG by adopting a slide agglutination method: 32.
the microscopic view of the gram stained with the gram stain is shown in FIG. 1, and it is clear from FIG. 1 that the stained Edwardsiella is observed as a red short bar under a microscope.
The nucleic acid gel electrophoresis diagram after sequencing by the PCR method is shown in FIG. 2, wherein the molecular weight of a protein Marker is 2000bp (2000 bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom), the Edwardsiella after PCR amplification is positioned in lane 1, the Edwardsiella after PCR amplification is positioned in lane 2, and the Edwardsiella after PCR amplification is positioned in lane 3.
The method for rapidly detecting Edwardsiella ictaluri by adopting the kit specifically comprises the following steps:
(1) Diluting polylysine and sterile water according to a mass ratio of 1:10, uniformly smearing on a frosted surface of a slide, repeatedly smearing for 2 times, and naturally drying to obtain the slide treated by polylysine;
(2) Smearing Edwardsiella ictaluri liquid on a slide treated by polylysine, naturally drying or fixing by flame for 5min, then soaking and fixing the slide by 10% formalin for 10min, soaking and fixing by absolute methanol at-20 ℃ for 10min, and finally sealing by using a sealing liquid with mass fraction of 5%, namely 5% BSA at normal temperature for 0-60min;
(3) Mixing the rabbit anti-Edwardsiella ictaluri polyclonal antibody and 0.9% physiological saline in a mass ratio of 1:5, smearing on the slide treated in the step (2), placing in a wet box to keep the humidity of the reaction environment, then treating for 1h at 37 ℃, and then washing with distilled water for 3-5 times;
(4) Mixing HRP-marked goat anti-rabbit lgG with 0.9% physiological saline in a mass ratio of 1:5, smearing the mixture on the slide treated in the step (3), treating the slide at 37 ℃ for 1h, and finally washing the slide with distilled water for 3-5 times;
(5) And (3) soaking a white regular filter paper sheet in TMB single-component staining solution, then attaching the white regular filter paper sheet to the glass slide obtained in the step (4), and developing for 5 minutes.
In the detection method, the closing time is 0, 5, 10, 15, 30 and 60min, when the closing time is 5 to 10min, color cannot be developed, the color development effect is poor in 15min, and the color development effects in 30min and 60min are consistent, so that the optimal time is determined to be 30min; in the step (3), the reaction time of the Edwardsiella ictaluri and the rabbit anti-Edwardsiella ictaluri polyclonal antibody is 0, 5, 10, 15, 30 and 60 minutes, and the color cannot be developed when the reaction time is 5 to 30 minutes, and the color development effect is optimal when the reaction time is 60 minutes.
The detection results are shown in fig. 3, wherein the graph A in fig. 3 shows the results of an experimental group, and the graph B shows the results of a control group, namely the Edwardsiella ictaluri control graph which is not treated by the double antibody sandwich method.
As can be seen from FIG. 3, the single-component TMB staining solution is soaked by a white regular filter paper sheet, attached to Edwardsiella ictaluri subjected to double-antibody sandwich treatment on a glass slide, and shows a bright blue result graph.
Claims (7)
1. The method for rapidly detecting the Edwardsiella ictaluri of fish is characterized by comprising the following steps of:
(1) Smearing Edwardsiella ictaluri bacterial liquid on a slide treated by polylysine, naturally drying or fixing by flame, and then soaking the slide by adopting 10% formalin and absolute methanol in sequence;
(2) Sealing the glass slide obtained in the step (1) by using sealing liquid at normal temperature;
(3) Smearing rabbit anti-Edwardsiella ictaluri polyclonal antibody on the slide obtained in the step (2) for reaction;
(4) Smearing the HRP-marked goat anti-rabbit lgG on the slide obtained in the step (3) for reaction;
(5) And (3) attaching filter paper containing TMB color development liquid to the slide glass obtained in the step (4) for color development.
2. The method of claim 1, wherein the polylysine-treated slide is prepared by: and diluting polylysine, uniformly smearing on the frosted surface of the glass slide, and finally naturally drying to obtain the product.
3. The method according to claim 1, wherein the rabbit anti-edwardsiella ictaluri polyclonal antibody is prepared by the following method: culturing Edwardsiella ictaluri, preparing the Edwardsiella ictaluri into an inactivated vaccine, immunizing a rabbit with the inactivated vaccine for four times, collecting blood after the fourth immunization, collecting serum, and purifying the serum by IgG to prepare the anti-Edwardsiella ictaluri polyclonal antibody.
4. The method according to claim 1, wherein the blocking solution is 5% by mass BSA.
5. The method according to claim 1, wherein the closing time in step (2) is 20-60min.
6. The process according to claim 1, wherein the reaction temperature in step (3) is 35-40 ℃ and the reaction time is 40-60min.
7. The process according to claim 1, wherein the reaction temperature in step (4) is 35-40 ℃ and the reaction time is 40-60min.
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