CN111273007A - A kind of kit and detection method for rapid detection of Edwardsiella channel catfish in fish - Google Patents

Abstract

The invention provides a kit and a detection method for rapidly detecting Edwardian channel catfish in fish. The kit comprises a glass slide treated with polylysine, a rabbit anti-Edwardia channel catheri polyclonal antibody, and HRP marker. goat anti-rabbit IgG, blocking solution and chromogenic solution. The invention uses the glass slide as the solid phase carrier, which greatly reduces the detection cost; the rabbit anti-Edwardia channel polyclonal antibody prepared by the invention can recognize multiple epitopes of the same antigen, has strong specificity and high sensitivity; Oxidase substrate, the present invention uses TMB as the color developing solution, which can produce a soluble light blue product, and the light blue product is adsorbed on the filter paper, so the color change can be visually observed with the naked eye. The detection method has low equipment requirements, low cost, simple operation, environmental protection and high sensitivity.

Description

A kind of kit and detection method for rapid detection of Edwardsiella channel catfish in fish

technical field

The invention belongs to the field of biotechnology, and in particular relates to a kit and a detection method for rapidly detecting Edwardsiella channel catfish.

Background technique

With the development of economy, people's pursuit of nutrients is more intense. Fish is rich in protein and is a good source of nutrition, especially the yellow catfish. The yellow catfish (pelteobagrus fulvidraco) belongs to the order Syriformes, Kueh family, and yellow catfish. It is a high-quality and precious freshwater fish in my country, commonly known as yellow drum fish. , Yellow Lading, etc. Yellow catfish is rich in protein and has the functions of maintaining potassium-sodium balance, eliminating edema, improving immunity, lowering blood pressure, and buffering anemia, which is beneficial to the growth and development of young people. At the same time, yellow catfish is rich in copper, which is an indispensable micronutrient for human health. It has important effects on the development and function of blood, central nervous system and immune system, hair, skin and bone tissue, as well as brain, liver, heart and other internal organs. , can regulate osmotic pressure, maintain acid-base balance and normal blood pressure, and enhance neuromuscular excitability.

The artificial breeding of yellow catfish in my country has a history of more than ten years. In 2018, the total output of yellow catfish in China reached 500,000 tons. Sichuan is the main breeding ground for yellow catfish. According to the "China Fishery Statistical Yearbook", the total output of yellow catfish in our province was as high as 30,500 tons in 2017, with an average market value of about 63.05 million yuan. In 2018, our province's yellow catfish The total output is as high as 34,100 tons. With the expansion of aquaculture area and aquaculture density, more and more diseases of yellow catfish occur, and the harm of Edwardella is more and more serious.

Channel catfish Edwardella was first isolated and found by HawkeJP in 1979 from the diseased channel catfish, named Edwardella "GA7752" group, and it was identified as a new species of Enterobacteriaceae Edwardsiella in 1981 and named as Edwardsiella channel catfish, short rod-shaped cells, 0.5～1μm×2～3μm in size, with periflagellate, dynamic, weak motor ability at 20～30℃, no exercise ability at 37℃, Gram’s Stain negative. It grows slowly on ordinary medium, at a temperature of 25℃～30℃, it takes 25～30 hours on BHI medium, and 48 hours on TSA medium to grow pinpoint-sized colonies. When the temperature reaches 37 ℃ does not grow. The diseased yellow catfish can show two kinds of manifestations: acute death and chronic death. Acute death is typical of sepsis; while the chronic death fish shows loss of appetite at the early stage of the disease and swims alone, but there are no obvious symptoms on the outside; Finally, congestive lesions are formed on the skull between the two eyes, which can lead to perforation on the top of the head, cracking of the skull, and even exposure of brain tissue; the diseased fish loses its balance before dying and hangs in the water in a "hanging water" shape. , that is, a spiral-like rapid flip or irregular swimming, followed by death. The disease has the characteristics of strong infectivity, acute onset, difficult prevention and treatment, high morbidity and mortality. It has caused significant economic losses to fish farming.

Traditional diagnostic methods are time-consuming, costly, complex to operate, and require high equipment. Therefore, accurate prevention and treatment of Edwardsiosis, that is, timely, rapid and convenient diagnosis and detection methods are essential.

SUMMARY OF THE INVENTION

In view of the above problems existing in the prior art, the present invention provides a kit and a detection method for rapidly detecting Edwardsiella channel catfish, which can effectively solve the high cost, complicated operation and high equipment requirements of the existing diagnostic methods. and time-consuming problems.

For realizing the above-mentioned purpose, the technical scheme that the present invention solves its technical problem adopts is:

A kit for rapid detection of Edwardsiella channeli in fish, comprising: a glass slide treated with polylysine, a rabbit anti-Edwardia channeli polyclonal antibody, HRP-labeled goat anti-rabbit IgG blocking solution and Color developing solution.

After the slides are treated with poly-lysine, poly-lysine has a positive charge, which can interact with the negative charge on the surface of E. channeli, resulting in strong adhesion, which can be avoided in the washing process. Bacteria fall off.

Further, the poly-lysine-treated glass slide is prepared by the following method: diluting the poly-lysine, then evenly smearing it on the frosted surface of the glass slide, smearing it repeatedly for 2-3 times, and finally drying it naturally. have to.

Further, the rabbit anti-Edwardia channeli polyclonal antibody is prepared by the following method: culturing the E. channel catfish, making it into an inactivated vaccine, immunizing the rabbit with the inactivated vaccine, immunizing four times, and after the fourth immunization , collect blood, collect serum, and purify the serum with IgG to prepare a rabbit anti-Edwardia channeli polyclonal antibody; the specific process is: culturing E. ⁸ cfu/ml inactivated vaccine, New Zealand rabbits were immunized with the inactivated vaccine for four times. After the fourth immunization, blood was collected, serum was collected, and the serum was purified by IgG to obtain rabbit anti-Edwardia channeli polyclonal antibody. .

Further, the blocking solution is BSA with a mass concentration of 5%; the specific preparation process is: under aseptic conditions, weigh 5.0 g of bovine serum albumin (BSA), use a low-salt buffer (Tris base 24.2 g, NaCl 222.2 g) g, Tween 20 5ml, dissolve in 1L of distilled water, adjust pH to 7.3) After dissolving, dilute to 100ml to prepare, and store at 4°C.

Further, the color developing solution is a TMB solution.

The method for rapidly detecting Edwardsiella channel catfish using the above-mentioned kit includes the following steps:

(1) immobilizing the Edwardsiella channeli bacterial solution on the glass slide treated with polylysine;

(2) sealing the glass slide obtained in step (1) with a blocking solution at room temperature;

(3) smear the rabbit anti-Edwardia channel polyclonal antibody on the glass slide obtained in step (2) to react;

(4) smear HRP-labeled goat anti-rabbit IgG on the glass slide obtained in step (3) to react;

(5) The filter paper containing the color developing solution is attached to the glass slide obtained in step (4) for color development.

Further, the specific process of step (1) is as follows: smear the Edwardella channeli bacteria solution on the glass slide treated with polylysine, naturally dry or fix it with flame, and then sequentially use 10% formalin and anhydrous methanol soak the slides.

Further, in step (2), the sealing time is 20-60min; preferably 30min.

Further, in step (3), the reaction temperature is 35-40° C., and the reaction time is 40-60 min; preferably, the reaction temperature is 37° C. and the reaction time is 1 h.

Further, in step (4), the reaction temperature is 35-40° C., and the reaction time is 40-60 min; preferably, the reaction temperature is 37° C. and the reaction time is 1 h.

The kit and the detection method for the rapid detection of E. japonica channel catfish provided by the present invention have the following beneficial effects:

(1) The present invention uses a glass slide as a solid-phase carrier, which greatly reduces the detection cost; the rabbit anti-Edwardia channel catfish polyclonal antibody prepared by the present invention can recognize multiple epitopes of the same antigen, and has strong specificity. High sensitivity; TMB is a peroxidase substrate, the present invention uses TMB as a color developing solution, which can produce a soluble light blue product, and after soaking the white regular filter paper in TMB single-component dyeing solution, paste It is attached to the glass slide and develops color for 5 minutes. The light blue product will be adsorbed on the filter paper, and the color change can be observed with the naked eye. The detection method requires low equipment, low cost, simple operation and high sensitivity. , can be widely used in fishery breeding personnel, fish disease workers and researchers, to provide a guarantee for improving the economic benefits of fish, especially yellow catfish breeding and food safety.

Description of drawings

Figure 1 is a microscope image of Edwardsiella stained with Gram stain.

Figure 2 is a nucleic acid gel electrophoresis image of Edwardsiella after sequencing by PCR method.

FIG. 3 is a result diagram of detection by the method of the present invention.

Detailed ways

Example 1

A kit for rapid detection of E. channel catfish, comprising: polylysine-treated glass slides (solid phase carrier glass slides), rabbit anti-Edwardia channel catfish polyclonal antibodies, HRP marker goat anti-rabbit IgG, blocking solution and chromogenic solution.

Wherein, the blocking solution is BSA with a mass concentration of 5%, and the color developing solution is TMB solution.

Rabbit anti-Edwardia channel polyclonal antibody was prepared by the following method:

(1) Inoculate Edwardella channel catfish on the BHI plate, incubate at 27°C for 24 hours, test for purity by Gram staining, sequence by PCR method, and confirm that it is Edwardella and no other bacteria, use 0.6 % formaldehyde inactivated bacteria, the concentration is 1×10 ⁸ cfu/ml inactivated vaccine;

(2) Use the inactivated vaccine prepared in step (1) to immunize New Zealand white rabbits, immunize 4 times, and immunize once every one week. Carotid arterial bleed after immunization, blood collection, and serum collection;

(3) The serum obtained in step (2) was purified by saturated ammonium sulfate precipitation method, and the antibody titer of IgG was determined by glass slide agglutination method at 1:32.

Figure 1 shows the microscopic image of the above stained Gram stain solution. As can be seen from Figure 1, the stained Edwardsiella is observed as a red short rod under a microscope.

The nucleic acid gel electrophoresis after sequencing by PCR method is shown in Figure 2, in which the molecular weight of the protein marker is 2000bp (from top to bottom: 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp), the first position of the lane after PCR amplification Edwardsiella, lane 2 PCR-amplified Edwardsiella, lane 3 for control.

The method for rapidly detecting E. japonica by using the above-mentioned kit specifically includes the following steps:

(1) Dilute poly-lysine and sterile water at a mass ratio of 1:10, then evenly smear it on the frosted surface of the glass slide, apply it twice, and finally dry it naturally. glass slide;

(2) Smear the Edwardella channel catfish solution on the polylysine-treated glass slide, dry naturally or fix it with flame for 5 minutes, then soak the glass slide in 10% formalin for 10 minutes, and then use no The solution was soaked in water and methanol for 10 minutes at -20°C, and finally sealed with 5% blocking solution, that is, 5% BSA at room temperature for 0-60 minutes at room temperature;

(3) Mix the rabbit anti-Edwardia channel polyclonal antibody and 0.9% normal saline in a mass ratio of 1:5, apply it to the glass slide treated in step (2), and place it in a wet box to maintain the reaction Ambient humidity, then treated at 37℃ for 1h, then washed with distilled water for 3-5 times;

(4) Mix HRP-labeled goat anti-rabbit IgG and 0.9% normal saline at a mass ratio of 1:5, apply it to the glass slide treated in step (3), treat at 37° C. for 1 hour, and finally wash with distilled water 3-5 times;

(5) Soak the white regular filter paper in TMB single-component staining solution, and then attach it to the glass slide obtained in step (4), and develop color for 5 minutes.

In the above detection method, the sealing time is 0, 5, 10, 15, 30, 60min. When the sealing time is 5 to 10min, the color cannot be developed. The color rendering effect is not good at 15min. The color rendering effect is the same at 30min and 60min. Therefore, it is determined that the optimal time is 30min; in step (3), the reaction time of Edwardella channel and rabbit anti-Edwardia channel polyclonal antibody is 0, 5, 10, 15, 30, 60min, and when the reaction time is 5 to 30min , can not develop color, the best color rendering effect is 60min.

The above detection results are shown in Fig. 3, in Fig. 3, picture A is the result of the experimental group, and B is the result of the control group, that is, the control picture of Edwardian channel catfish without the double-antibody sandwich method.

It can be seen from Figure 3 that the TMB single-component staining solution was infiltrated with a white regular filter paper, and then attached to the glass slide and the double-antibody sandwich-treated Edwardian channel catfish showed a bright blue result.

Claims (10)

1. a test kit for the rapid detection of fish Edwardian channel catfish is characterized in that, comprising: the glass slide that polylysine is processed, the rabbit anti-Edwardia channel catfish polyclonal antibody, the sheep marked by HRP Anti-rabbit IgG blocking solution and chromogenic solution. 2. the test kit that is used for the rapid detection of Edwards channel catfish according to claim 1, is characterized in that, the described glass slide that polylysine is processed is obtained by the following method: by polylysine The amino acid is diluted, then evenly spread on the frosted surface of the glass slide, and finally dried naturally. 3. the test kit that is used for the rapid detection of fish Edwardian channel according to claim 1, is characterized in that, the rabbit anti-Edwardia channel polyclonal antibody is prepared by the following method: cultivate the channel Edwardian, and An inactivated vaccine is prepared, and the inactivated vaccine is immunized in rabbits for four times. After the fourth immunization, blood is collected, serum is collected, and the serum is purified by IgG to obtain a rabbit anti-Edwardia channel polyclonal antibody. 4. The test kit for rapidly detecting E. channel catfish according to claim 1, wherein the blocking solution is BSA with a mass concentration of 5%. 5. the test kit for rapidly detecting Edwardsiella channel catfish according to claim 1, is characterized in that, the color developing solution is TMB solution. 6. adopt the method for the rapid detection of Edwardsiella channel catfish of the test kit described in any one of claim 1-5, is characterized in that, comprises the following steps: (1) immobilizing the Edwardsiella channeli bacterial solution on the glass slide treated with polylysine; (2) sealing the glass slide obtained in step (1) with a blocking solution at room temperature; (3) smear the rabbit anti-Edwardia channel polyclonal antibody on the glass slide obtained in step (2) to react; (4) smear HRP-labeled goat anti-rabbit IgG on the glass slide obtained in step (3) to react; (5) The filter paper containing the color developing solution is attached to the glass slide obtained in step (4) for color development. 7. detection method according to claim 6 is characterized in that, the concrete process of step (1) is: smear on the glass slide processed through polylysine by Edwards channelella bacteria liquid, naturally dry or through flame After fixation, slides were soaked in 10% formalin and anhydrous methanol in turn. 8. detection method according to claim 6 is characterized in that, in step (2), sealing time is 20-60min. 9. detection method according to claim 6 is characterized in that, in step (3), reaction temperature is 35-40 ℃, and reaction time is 40-60min. 10 . The detection method according to claim 6 , wherein the reaction temperature in step (4) is 35-40° C., and the reaction time is 40-60 min. 11 .

Images