阅读说明：本技术 一种淡水鱼类细菌病药敏检测试剂盒及其应用 (Drug sensitivity detection kit for bacterial diseases of freshwater fishes and application thereof ) 是由 陈福艳 欧阳贤华 黎建斌 梁万文 陈福彩 李大列 黄彩林 雷爱莹 罗洪林 李旻 于 2019-11-05 设计创作，主要内容包括：本发明提供了一种淡水鱼类细菌病药敏检测试剂盒及其应用,属于水产疾病防治技术领域。一种淡水鱼类细菌病快速检测药敏试剂盒,包括TSA培养基、增菌液和渔类抗菌药纸片。基于所述试剂盒检测淡水鱼类细菌病药敏的方法包括：病样组织液的提取、细菌扩增、细菌接种培养和结果判断。本发明提供的试剂盒能快速筛选出对病原菌敏感的渔用药物,及时指导养殖户高效用药,提高用药治疗效果；检测方法简单方便；对实验技能要求不高,临床应用性强,同时还可实现耐药菌株的分离。(The invention provides a drug sensitivity detection kit for bacterial diseases of freshwater fishes and application thereof, belonging to the technical field of prevention and treatment of aquatic diseases. A quick detection drug sensitive kit for fresh water fish bacterial diseases comprises a TSA culture medium, a bacteria increasing liquid and a fishery antibacterial drug paper sheet. The method for detecting the bacterial disease drug sensitivity of the freshwater fishes based on the kit comprises the following steps: extracting a disease sample tissue fluid, amplifying bacteria, inoculating and culturing the bacteria and judging results. The kit provided by the invention can quickly screen out the fishing drugs sensitive to pathogenic bacteria, timely guide farmers to take high-efficiency drugs, and improve the drug treatment effect; the detection method is simple and convenient; the method has low requirement on experimental skills, strong clinical applicability and can realize the separation of drug-resistant strains.)

1. A fresh water fish bacterial disease drug sensitivity detection kit is characterized by comprising the following components:

TSA culture medium, drug sensitive paper sheet of fish antibacterial drug and enrichment liquid.

2. The freshwater fish bacterial disease drug sensitivity detection kit according to claim 1, wherein the drug sensitivity paper sheet of the fishery antibacterial drug comprises one or more of a drug sensitivity paper sheet of florfenicol, a drug sensitivity paper sheet of sulfisoxazole, a drug sensitivity paper sheet of amoxicillin, a drug sensitivity paper sheet of enrofloxacin, a drug sensitivity paper sheet of neomycin hydrochloride, a drug sensitivity paper sheet of streptomycin, a drug sensitivity paper sheet of norfloxacin and a drug sensitivity paper sheet of doxycycline.

3. The freshwater fish bacterial disease drug sensitivity detection kit according to claim 1, wherein the enrichment fluid takes a TSB culture medium as a basic culture medium, and further comprises 9-11% by volume of fish serum and 0.02-0.04% by volume of an Almartian blue solution indicator.

4. The fresh water fish bacteriosis drug sensitivity detection kit according to any one of claims 1-3, characterized by further comprising a dropper, a sealing adhesive and a culture dish for containing the TSA culture medium.

5. The freshwater fish bacterial disease susceptibility detection kit of claim 4, wherein the culture dish has a diameter of 90 mm.

6. Use of the test kit according to any one of claims 1 to 5 for screening antibiotics for treating bacterial diseases of freshwater fishes or for screening strains resistant to antibiotics.

7. A method for rapidly detecting bacterial disease drug sensitivity of freshwater fishes based on the detection kit of any one of claims 1 to 5, which is characterized by comprising the following steps:

1) taking the liver of the freshwater fish with the bacterial disease, shearing, mixing the obtained minced liver with enrichment solution, standing, dripping 2-3 drops of mixed solution onto a TSA culture medium after the mixed solution turns light red, and coating to obtain the TSA culture medium coated with the mixed solution;

2) placing a drug sensitive paper sheet of the fishery antibacterial drug on the TSA culture medium coated with the mixed solution, flattening the drug sensitive paper sheet and sealing;

3) and (3) placing the sealed TSA culture medium at 37 ℃ for constant-temperature culture for 12-24 h, observing the growth condition of bacteria, measuring the diameter of a sterile ring around a drug sensitive paper sheet, and carrying out drug sensitivity degree interpretation according to the US CLSI judgment standard.

8. The method according to claim 7, wherein the standing time in the step 1) is 4-6 h.

9. The method according to claim 7, wherein after the coating in step 1), the method further comprises standing the TSA culture medium coated with the mixed solution for 5-6 min to dry the water.

10. The method according to claim 7, wherein the us CLSI decision criteria in step 3) are as follows:

Technical Field

The invention belongs to the technical field of prevention and treatment of aquatic diseases, and particularly relates to a drug sensitivity detection kit for bacterial diseases of freshwater fishes and application thereof.

Background

China is a world aquaculture country and also an aquatic product trade country, the yield of aquatic products accounts for 70% of the world, and the export amount continuously occupies the top of the world for 6 years. However, in recent years, due to the rapid development of aquaculture industry, bacterial diseases occur occasionally and are not treated properly, which can cause great economic loss. At present, the most effective treatment of bacterial diseases is antibiotics, but in recent years, the treatment effect of medicines is not obvious and the phenomenon of repeated attack occurs, the reason may be related to different kinds of bacteria causing bacterial diseases, in addition, the problems of antibiotic resistance, drug residue and the like may be caused due to the frequent and irregular use of antibiotics, and particularly, the problem of drug residue can directly influence the export trade of aquatic products in China. Therefore, it is necessary to perform a drug sensitivity test before the treatment with the drug to rapidly screen out a highly effective therapeutic drug. However, only drug sensitive kits related to human-derived bacteria or mammalian-derived bacteria are currently on the market, and no products are specifically used for aquatic animal-derived bacteria; some scholars also carry out research on the aquatic animal source bacteria drug sensitive kit, but still have the problems of complicated use, high requirement on environmental conditions, long use time, disjointed antibiotic varieties and fishery medicine products and the like, and only stay in the research stage, and the popularization and the application are not achieved; in addition, antibiotic products in the fishery medicine market are various in types, unknown in content and components, and the condition of fish eyes mixing beads can exist. Therefore, a scientific, rapid and practical medication guide is urgently needed to guide farmers to timely, accurately and efficiently use antibiotic drugs for treatment, reduce the use of antibiotics and reduce the economic loss of the farmers.

Disclosure of Invention

In view of the above, the invention aims to provide a drug sensitivity detection kit for bacterial diseases of freshwater fishes and an application thereof, and the kit can be used for quickly, accurately, practically and conveniently detecting the fish drug sensitivity of fish disease samples and timely guiding farmers to scientifically take drugs.

The invention provides a drug sensitivity detection kit for bacterial diseases of freshwater fishes, which comprises the following components:

TSA culture medium, drug sensitive paper sheet of fish antibacterial drug and enrichment liquid.

Preferably, the drug sensitive paper sheets of the fishery antibacterial drugs comprise one or more of drug sensitive paper sheets of florfenicol, sulfisoxazole, amoxicillin, enrofloxacin, neomycin hydrochloride, streptomycin, norfloxacin and doxycycline.

Preferably, the enrichment fluid takes a TSB culture medium as a basic culture medium, and also comprises 9-11% of fish serum and 0.02-0.04% of Almaran blue solution indicator by volume concentration.

Preferably, a dropper, a sealing gel and a culture dish for containing the TSA culture medium are also included.

Preferably, the diameter of the culture dish is 90 mm.

The invention provides application of the detection kit in screening antibiotics for treating bacterial diseases of freshwater fishes or screening strains with antibiotic resistance.

The invention provides a method for rapidly detecting bacterial disease drug sensitivity of freshwater fishes based on the detection kit, which comprises the following steps:

1) taking the liver of the freshwater fish with the bacterial disease, shearing, mixing the obtained minced liver with enrichment solution, standing, dripping 2-3 drops of mixed solution onto a TSA culture medium after the mixed solution turns light red, and coating to obtain the TSA culture medium coated with the mixed solution;

2) placing a drug sensitive paper sheet of the fishery antibacterial drug on the TSA culture medium coated with the mixed solution, flattening the drug sensitive paper sheet and sealing;

3) and (3) placing the sealed TSA culture medium at 37 ℃ for constant-temperature culture for 12-24 h, observing the growth condition of bacteria, measuring the diameter of a sterile ring around a drug sensitive paper sheet, and carrying out drug sensitivity degree interpretation according to the US CLSI judgment standard.

Preferably, the standing time in the step 1) is 4-6 h.

Preferably, after the coating in the step 1), standing the TSA culture medium coated with the mixed solution for 5-6 min, and airing water.

Preferably, the us CLSI criteria in step 3) are as follows:

compared with the prior art, the fresh water fish bacteriosis drug sensitivity detection kit provided by the invention has the beneficial effects that:

(1) and (3) fast: taking a fish disease sample, immediately carrying out a drug sensitivity test, and culturing for 12-48h after inoculation to obtain an experimental result;

(2) and (3) accuracy: directly measuring the inhibition zone;

(3) the operation is simple: the operation steps are simple and convenient;

(4) the technical requirement is low: the device can be operated in a laboratory or in the field, and has little influence on experimental results;

(5) simultaneously separating to obtain a drug-resistant strain;

(6) the kit is low in cost, convenient, practical, accurate and efficient, and is suitable for large-scale production.

Drawings

FIG. 1 is a schematic representation of a drug sensitivity kit of the present invention; wherein: 1 disposable culture dish with diameter of 90 mm; 2TSA medium; 3EP tube; 4 a drug sensitive paper sheet arranged in the EP tube; 5EP tube; 6 enrichment liquid filled in an EP tube; 7 coating rod; 8, a dropper; and 9, sealing glue.

Detailed Description

The invention provides a drug sensitivity detection kit for bacterial diseases of freshwater fishes, which comprises the following components:

TSA culture medium, drug sensitive paper sheet of fish antibacterial drug and enrichment liquid.

The detection kit provided by the invention comprises a TSA culture medium. The source of the TSA medium is not particularly limited, and a TSA medium known in the art may be used, for example, commercially available or self-made. The formulation of the TSA medium is as follows: tryptone 17g/L, soytone 3g/L, glucose 2.5g/L, NaCl g/L₅ g/L、K₂HPO₄2.5g/L, 20g/L agar and 7.0-7.2 of pH value. The preparation method of the TSA culture medium comprises the steps of mixing and dissolving the components in distilled water, and sterilizing at 121 ℃ for 15min for later use. The TSA culture medium is used for providing a carrier with drug sensitive bacteriostatic diameter.

The detection kit provided by the invention comprises a drug sensitive paper sheet of the fishery antibacterial drug. The drug sensitive paper sheet of the fishing antibacterial drug preferably comprises one or more of a florfenicol drug sensitive paper sheet, a sulfisoxazole drug sensitive paper sheet, an amoxicillin drug sensitive paper sheet, an enrofloxacin drug sensitive paper sheet, a neomycin hydrochloride drug sensitive paper sheet, a streptomycin drug sensitive paper sheet, a norfloxacin drug sensitive paper sheet and a doxycycline drug sensitive paper sheet. The diameter of the drug sensitive paper sheet is preferably 6mm, and the antibiotic capacity in each drug sensitive paper sheet is as follows: florfenicol (30 mu g/tablet), sulfisoxazole (300 mu g/tablet), amoxicillin (20/10 mu g/tablet), enrofloxacin (5 mu g/tablet), neomycin hydrochloride (30 mu g/tablet), streptomycin (10 mu g/tablet), norfloxacin (10 mu g/tablet), doxycycline (30 mu g/tablet). The source of the drug sensitive paper sheet of the fish antibacterial drug is not particularly limited, and the source of the drug sensitive paper sheet of the fish antibacterial drug known in the field can be adopted. In the embodiment of the invention, the drug sensitive paper sheet of the fishery antibacterial drug is purchased from Hangzhou Tian and Biotech limited. The effect of the drug sensitive paper sheet of the fishing antibacterial drug provides antibiotic with antibacterial effect to form an antibacterial ring.

The detection kit provided by the invention comprises enrichment fluid. The source of the enriched liquid is not particularly limited in the present invention, and any known sources of enriched liquids in the art may be used, for example, enriched liquids obtained from commercial sources or self-made enriched liquids. The enrichment fluid preferably takes a TSB culture medium as a basic culture medium, and also preferably comprises 9-11% of fish serum by volume concentration and 0.02% -0.04% of an Alma blue solution indicator, and more preferably comprises 10% of fish serum by volume concentration and 0.034% of an Alma blue solution indicator. The formulation of the TSB medium is as follows: tryptone 17g/L, soytone 3g/L, glucose 2.5g/L, NaCl 5g/L and K₂HPO₄2.5g/L and a pH value of 7.0-7.2.

The detection kit provided by the invention preferably further comprises a dropper, a sealing adhesive and a culture dish for containing the TSA culture medium. The diameter of the culture dish is preferably 90 mm. The culture dish is preferably a disposable culture dish. The dropper is used for sucking the mixed liquid after enrichment culture, and the specification of the dropper is not particularly limited and can be a dropper specification well known in the field. The sealing glue is used for sealing the culture dish filled with the TSA culture medium. The sources of the dropper, the sealing glue and the culture dish are not particularly limited by the invention, and the dropper, the sealing glue and the culture dish can be obtained by adopting the conventional purchasing method well known in the field.

The invention provides application of the detection kit in screening antibiotics for treating bacterial diseases of freshwater fishes or screening strains with antibiotic resistance. In the application, the method for screening the antibiotics for treating the bacterial diseases of the freshwater fishes comprises the steps of culturing pathogenic bacteria in the infected freshwater fishes on a TSA culture medium, judging the sensitivity degree of the pathogenic bacteria to the antibiotics loaded on the drug sensitive paper sheets according to the size of an inhibition zone formed by the pathogenic bacteria to the drug sensitive paper sheets, mastering the drug resistance and the sensitivity of the pathogenic bacteria to the common antibiotics, and selecting the antibiotics with strong sensitivity according to the result so as to guide farmers to take medicines efficiently in time, improve the medication effect or screen strains with strong drug resistance to the antibiotics for subsequent technical research.

The invention provides a method for rapidly detecting bacterial disease drug sensitivity of freshwater fishes based on the detection kit, which comprises the following steps:

1) taking the liver of the freshwater fish with the bacterial disease, shearing, mixing the obtained minced liver with enrichment solution, standing, dripping 2-3 drops of mixed solution onto a TSA culture medium after the mixed solution turns light red, and coating to obtain the TSA culture medium coated with the mixed solution;

2) placing a drug sensitive paper sheet of the fishery antibacterial drug on the TSA culture medium coated with the mixed solution, flattening the drug sensitive paper sheet and sealing;

3) and (3) placing the sealed TSA culture medium at 37 ℃ for constant-temperature culture for 12-24 h, observing the growth condition of bacteria, measuring the diameter of a sterile ring around a drug sensitive paper sheet, and carrying out drug sensitivity degree interpretation according to the US CLSI judgment standard.

In the present invention, the kind of the freshwater fish is not particularly limited, and freshwater fish known in the art may be used. The types of pathogenic bacteria of the bacterial diseases are not particularly limited, and the common pathogenic bacteria of the freshwater fish can be determined by adopting the method provided by the invention.

In the invention, the volume ratio of the mass of the minced liver to the enrichment fluid is preferably 0.5-1 g: 2.5-3.0 mL, more preferably 1 g: 3 mL. The standing time is preferably 4-6 h, and more preferably 5 h. And (3) standing, carrying out enrichment culture on bacteria in the enrichment solution to obtain a bacteria solution, when the mixed solution turns light red, indicating that the bacteria can be proliferated, carrying out next inoculation and drug sensitivity test, but if the enrichment solution does not turn color, indicating that the bacteria are not proliferated, and stopping the test.

In the present invention, after coating, the method further preferably comprises allowing the TSA medium coated with the mixed solution to stand for 5 to 6min and air-drying the water.

In the present invention, the pieces of drug-sensitive paper for the fishery antibacterial drugs are placed on the TSA medium coated with the mixed solution, and preferably, 4 pieces of the same or different pieces of drug-sensitive paper for the fishery antibacterial drugs are placed on each medium.

In the invention, the constant temperature culture time is preferably 15-22 h, and more preferably 18 h. The time for the isothermal culture varies depending on the kind of pathogenic bacteria. The method for measuring the diameter of the sterile ring around the drug sensitive paper sheet preferably measures the diameter of the sterile ring for 3 times, and an average value is taken.

In the present invention, the us CLSI criteria are preferably as follows:

the present invention provides a fresh water fish bacterial disease drug sensitive detection kit and its application in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.