Rapid drug sensitivity detection kit for breeding site
By using a rapid drug sensitivity detection kit at aquaculture site, combining South American white shrimp polypeptide and tetrazine chloride, the problem of long and low accuracy of drug sensitivity detection on site in aquaculture was solved, and within 5 hours Obtain drug sensitivity test results, guide the use of scientific antibiotics, and reduce economic losses and bacterial resistance.
Patent Information
- Application Number
- CN202411421318.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-12
- Publication Date
- 2025-01-10
AI Technical Summary
The lack of fast and accurate drug sensitivity detection methods on aquaculture sites has led to inaccurate use of antibiotics, resulting in economic losses and increased bacterial resistance. Traditional drug sensitivity detection takes a long time and is ineffective against difficult-to-isolate culture bacteria.
Develop a fast drug sensitivity detection kit, by adding South American white prawn polypeptide as bacterial growth promoter and trityltriazole chloride as color developer, combined with drug sensitivity tablets to shorten bacteria Incubation time and improve detection sensitivity.
The drug sensitivity detection time has been shortened to 5 hours, significantly improving the growth promotion effect of common bacteria in aquaculture, providing scientific guidance on the use of antibiotics at the breeding site, and solving the problems of antibiotic abuse and long detection time.
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Figure CN119286977A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of drug sensitivity detection, and in particular relates to a rapid drug sensitivity detection kit for breeding sites. Background Art
[0002] Fish, shrimp, shellfish and other aquatic animals are rich in nutrition and delicious, and are deeply loved by consumers. Most aquatic meats are rich in protein, and the essential amino acid composition of protein is better than that of livestock and poultry meat, which is a high-quality protein. Aquatic meat is tender and easy to digest and absorb by the human body, and is more suitable for patients, the elderly and children. Aquatic meat has a low fat content and is mostly composed of unsaturated fatty acids. It is easy to digest and absorb, and is not easy to cause arteriosclerosis. It is more suitable for the elderly and cardiovascular patients. In addition, aquatic meat also contains extremely rich vitamins and trace elements. With the increase in people's demand for high-quality aquatic meat, artificial breeding of various fish, shrimps and shellfish has replaced wild aquatic fishing. With the development of intensive aquaculture, the harm of bacterial pathogens to the aquaculture industry has become increasingly serious. Due to insufficient research on the immune mechanism of aquatic animals, the lack of effective vaccine products, and the relative lack of prevention technology for aquatic diseases, aquatic diseases have caused huge economic losses to the aquaculture industry.
[0003] Bacterial diseases are one of the most harmful pathogenic factors in aquaculture. Since bacterial diseases can be treated with antibiotics, antibiotics are used in large quantities in aquaculture. The problem of antibiotic abuse has become a stubborn disease that affects the healthy development of aquaculture. Blind use of antibiotics has many hazards. First, poor efficacy affects the treatment effect; second, excessive use of antibiotics increases breeding costs; third, multi-drug resistant bacteria are produced; fourth, beneficial bacteria are killed and the ecological balance of water quality is destroyed; fifth, drug residues affect human health.
[0004] The scientific use of antibiotics is an effective method to control bacterial diseases in aquaculture. Antibiotics that pathogens are sensitive to can be screened through drug sensitivity tests, and precise drug use can quickly inhibit and kill pathogens, reducing disease losses. However, drug sensitivity tests need to be conducted in standard microbiological laboratories, which have high requirements for laboratory conditions and the professional quality of test operators. Most aquaculture sites do not have the above conditions. When diseases occur, the diseased materials need to be sent to professional testing institutions for testing, which often takes a long time, delays the best medication period, and causes significant economic losses. In addition, the widely used conventional bacterial culture drug sensitivity test method takes more than 12 hours to produce results, which is time-consuming. For a few pathogens that are difficult to separate and culture, traditional drug sensitivity test methods often have the problem of ineffective detection because the culture medium and culture method are not suitable for the growth of such bacteria. Therefore, it is necessary to optimize the drug sensitivity test culture medium and drug sensitivity test system, develop a culture medium that is compatible with the growth of bacteria that are difficult to separate and culture, and broaden the coverage of pathogens for drug sensitivity testing; and develop a more sensitive drug sensitivity test system to shorten the window period of drug sensitivity testing. What is more important is to develop drug sensitivity detection reagents that are simple and easy to operate under extensive conditions at the breeding site, so as to guide the timely and accurate use of the best antibiotics at the breeding site and effectively control bacterial diseases in aquaculture. Summary of the invention
[0005] The invention aims to provide a rapid drug sensitivity detection kit for breeding sites, belonging to the technical field of drug sensitivity detection.
[0006] To achieve the above object, the present invention provides the following technical solutions:
[0007] First, the present invention provides a rapid drug sensitivity detection kit for aquaculture sites, wherein the rapid drug sensitivity detection kit is a rapid drug sensitivity detection kit composed of a tryptic soy peptone liquid culture medium, a white shrimp polypeptide as a bacterial growth promoter, and triphenyltetrazolium chloride as a color developer to form a drug sensitivity detection solution, and a drug sensitivity sheet.
[0008] Furthermore, the preparation method of the drug sensitivity detection solution is as follows: weigh 17.0g of tryptone, 3.0g of soy peptone, 5.0g of sodium chloride, 2.5g of dipotassium hydrogen phosphate, 2.5g of glucose, and 10g of triphenyltetrazolium chloride, dissolve them in 1L of deionized water, adjust the pH value to 7.3±0.2, add 75ml of white shrimp polypeptide solution, filter with a 0.22um sterilizing filter, and aseptically package.
[0009] Furthermore, the preparation method of the white shrimp polypeptide liquid is as follows:
[0010] (1) Wash 40- to 60-day-old whiteleg shrimp with pure water three times, add 5 times the amount of ultrapure water, and fully homogenize using a homogenizer;
[0011] (2) The white shrimp homogenate was frozen at -20°C for 24 hours, thawed at room temperature, and repeated freezing and thawing for 3 times, and then centrifuged at 12000 rpm / min for 10 minutes in a centrifuge;
[0012] (3) Collect the supernatant, add ammonium sulfate to a final concentration of 20% to the supernatant, let stand at 4°C for 24 hours, and centrifuge at 12000 rpm / min for 20 minutes;
[0013] (4) Collect the supernatant, filter it using a 0.45 μm filter, and then filter it using a 0.22 μm filter to sterilize it. Then, package it aseptically and store it at 2-8°C.
[0014] Secondly, the present invention provides a bacterial growth promoter, wherein the bacterial growth promoter component is a white shrimp polypeptide liquid.
[0015] Furthermore, the preparation method of the white shrimp polypeptide liquid is as follows:
[0016] (1) Wash 40- to 60-day-old whiteleg shrimp with pure water three times, add 5 times the amount of ultrapure water, and fully homogenize using a homogenizer;
[0017] (2) The white shrimp homogenate was frozen at -20°C for 24 hours, thawed at room temperature, and repeated freezing and thawing for 3 times, and then centrifuged at 12000 rpm / min for 10 minutes in a centrifuge;
[0018] (3) Collect the supernatant, add ammonium sulfate to a final concentration of 20% to the supernatant, let stand at 4°C for 24 hours, and centrifuge at 12000 rpm / min for 20 minutes;
[0019] (4) Collect the supernatant, filter it using a 0.45 μm filter, and then filter it using a 0.22 μm filter to sterilize it. Then, package it aseptically and store it at 2-8°C.
[0020] The beneficial effect of the present invention is that by preparing a white shrimp polypeptide liquid and adding it to a tryptic soytone liquid culture medium, the growth of common Vibrio parahaemolyticus, Vibrio harveyi and Edwardsiella tarda in aquaculture is significantly promoted, and the bacterial culture time can be shortened. The white shrimp polypeptide is added to the drug sensitivity detection liquid in combination with the triphenyltetrazolium chloride color developer and the drug sensitivity sheet detection system, and the prepared rapid drug sensitivity detection kit is used to perform drug sensitivity detection on clinical diseased shrimp samples. The drug sensitivity detection result can be determined in just 5 hours, and the result determination time is 4 hours earlier than that of the ordinary drug sensitivity detection kit without the addition of shrimp polypeptide, which can solve the problem of rapid drug sensitivity detection on the spot in aquaculture farms. Provide scientific guidance for the use of antibiotics on the aquaculture site, use drugs accurately, and solve the problem of antibiotic abuse. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1:Detection diagram of the effect of white shrimp peptides on the growth of Vibrio parahaemolyticus.
[0022] Figure 2 :Detection diagram of the effect of white shrimp peptides on the growth of Vibrio harveyi.
[0023] Figure 3 :Detection diagram of the effect of white shrimp peptides on the growth of Edwardsiella tarda.
[0024] Figure 4 : Schematic diagram of the operation method of the drug sensitivity detection kit. DETAILED DESCRIPTION
[0025] Example 1: Preparation of Penaeus vannamei polypeptide
[0026] (1) Wash 40- to 60-day-old whiteleg shrimp with pure water three times, add 5 times the amount of ultrapure water, and fully homogenize using a homogenizer;
[0027] (2) The white shrimp homogenate was frozen at -20°C for 24 hours, thawed at room temperature, and repeated freezing and thawing for 3 times, and then centrifuged at 12000 rpm / min for 10 minutes in a centrifuge;
[0028] (3) Collect the supernatant, add ammonium sulfate to a final concentration of 20% to the supernatant, let stand at 4°C for 24 hours, and centrifuge at 12000 rpm / min for 20 minutes;
[0029] (4) Collect the supernatant, filter it using a 0.45 μm filter, and then filter it using a 0.22 μm filter to sterilize it. Then, package it aseptically and store it at 2-8°C.
[0030] Example 2: Culture medium preparation
[0031] (1) TSB broth: Weigh 17.0 g of tryptone, 3.0 g of soy peptone, 5.0 g of sodium chloride, 2.5 g of potassium dihydrogen phosphate, and 2.5 g of glucose, dissolve in 1 L of deionized water, adjust the pH to 7.3 ± 0.2, and sterilize at 121 °C for 20 min.
[0032] (2) 2.5% shrimp polypeptide culture medium: add 25 ml of shrimp polypeptide solution to 975 ml of TSB culture medium.
[0033] (3) 5.0% shrimp polypeptide culture medium: add 50 ml of shrimp polypeptide solution to 950 ml of TSB culture medium.
[0034] (4) 7.5% Penaeus vannamei polypeptide culture medium: add 75 ml of the Penaeus vannamei polypeptide solution to 925 ml of TSB culture medium.
[0035] Example 3: Effect of Penaeus vannamei polypeptide on the growth of Vibrio parahaemolyticus
[0036] (1) The stored Vibrio parahaemolyticus strain was pre-cultured in tryptic soy broth (TSB) at 28°C for 18 h. The overnight culture was diluted to a cell density of approximately 10 6 CFU mL -1 .
[0037] (2) The above-mentioned Vibrio parahaemolyticus was inoculated into the four culture media prepared in Example 2 at a volume ratio of 1 / 100, and cultured at 28° C. for 24 h. The culture was sampled after 0, 4, 8, and 12 h of culture.
[0038] (3) By measuring the optical density (OD 650 The effect of adding different amounts of Penaeus vannamei peptides on the growth of Vibrio parahaemolyticus was monitored by 30 μg / ml. The experiment was repeated three times independently.
[0039] from Figure 1 It can be seen that adding 2.5%, 5.0%, and 7.5% of white shrimp polypeptide to TSB medium can significantly increase the culture density of Vibrio parahaemolyticus. The culture density of Vibrio parahaemolyticus in the 5.0% white shrimp polypeptide culture medium is significantly higher than that in the 2.5% group, and there is no significant difference in the culture density of Vibrio parahaemolyticus in the 5.0% and 7.5% white shrimp polypeptide culture mediums.
[0040] Example 4: Effect of Penaeus vannamei polypeptide on the growth of Vibrio harveyi
[0041] (1) The stored Vibrio harveyi strain was pre-cultured in tryptic soy broth (TSB) at 28°C for 18 h. The overnight culture was diluted to a cell density of approximately 10 6 CFU mL -1 .
[0042] (2) The above-mentioned Vibrio harveyi was inoculated into the four culture media prepared in Example 2 at a volume ratio of 1 / 100, and cultured at 28° C. for 24 h. The culture was sampled after 0, 4, 8, and 12 h of culture.
[0043] (3) By measuring the optical density (OD 650 The effect of adding different amounts of Penaeus vannamei peptides on the growth of Vibrio harveyi was monitored by 30 μg / ml. The experiment was repeated three times independently.
[0044] from Figure 2 It can be seen that adding 2.5%, 5.0% and 7.5% of Penaeus vannamei polypeptide to TSB medium can significantly increase the culture density of Vibrio parahaemolyticus. The culture density of Vibrio parahaemolyticus in the 5.0% Penaeus vannamei polypeptide medium is significantly higher than that in the 2.5% group, and the culture density of Vibrio parahaemolyticus in the 7.5% Penaeus vannamei polypeptide medium is slightly higher than that in the 5.0% group.
[0045] Example 5: Effect of Penaeus vannamei polypeptide on the growth of Edwardsiella tarda
[0046] (1) The stored Edwardsiella tarda strain was pre-cultured in tryptic soy broth (TSB) at 28°C for 18 h. The overnight culture was diluted to a cell density of approximately 10 6 CFU mL -1 .
[0047] (2) The above Edwardsiella tarda was inoculated into the four culture media prepared in Example 2 at a volume ratio of 1 / 100, and cultured at 28° C. for 24 h. The culture was sampled after 0, 4, 8, and 12 h of culture.
[0048] (3) By measuring the optical density (OD 650 The effect of adding different amounts of Penaeus vannamei peptides on the growth of Edwardsiella tarda was monitored. The experiment was repeated three times independently.
[0049] from Figure 3 It can be seen that adding 2.5%, 5.0% and 7.5% of Penaeus vannamei polypeptide to TSB medium can significantly increase the culture density of Edwardsiella tarda. The culture density of Edwardsiella tarda in the 5.0% Penaeus vannamei polypeptide medium is significantly higher than that in the 2.5% group, and the culture density of Vibrio parahaemolyticus in the 7.5% Penaeus vannamei polypeptide medium is significantly higher than that in the 5.0% group.
[0050] Therefore, the white shrimp polypeptide of the present invention has a significant promoting effect on the growth of Vibrio parahaemolyticus, Vibrio harveyi and Edwardsiella tarda which are common in aquaculture, and can shorten the bacterial culture time.
[0051] Example 6: Preparation of a common drug sensitivity test kit
[0052] (1) Preparation of drug sensitivity test solution: Weigh 17.0 g of tryptone, 3.0 g of soy peptone, 5.0 g of sodium chloride, 2.5 g of dipotassium hydrogen phosphate, 2.5 g of glucose, and 10 g of triphenyltetrazolium chloride, dissolve in 1 L of deionized water, and adjust the pH to 7.3 ± 0.2. Filter through a 0.22 μm sterilizing filter.
[0053] (2) Preparation of drug sensitivity test tubes: Take a sterile 5 ml EP tube, put one drug sensitivity tablet in each EP tube, and write the name of the drug sensitivity tablet on the tube wall.
[0054] (3) Composition of the kit: 1 sterile cotton swab, 1 sterile latex glove, 1 sterile rubber-tipped dropper, several drug sensitivity test tubes (including drug sensitivity tablets corresponding to the antibiotics that can be selected by the farm), 1 negative control tube, 1 positive control tube, and 1 bottle of drug sensitivity test solution.
[0055] Example 7: Preparation of Rapid Drug Sensitivity Detection Kit
[0056] (1) Preparation of drug sensitivity test solution: Weigh 17.0 g of tryptone, 3.0 g of soy peptone, 5.0 g of sodium chloride, 2.5 g of dipotassium hydrogen phosphate, 2.5 g of glucose, and 10 g of triphenyltetrazolium chloride, dissolve in 1 L of deionized water, adjust the pH value to 7.3 ± 0.2, add 75 ml of shrimp polypeptide solution, and filter through a 0.22 μm sterilizing filter.
[0057] (2) Preparation of drug sensitivity test tubes: Take a sterile 5 ml EP tube, put one drug sensitivity tablet in each EP tube, and write the name of the drug sensitivity tablet on the tube wall.
[0058] (3) Composition of the kit: 1 sterile cotton swab, 1 sterile latex glove, 1 sterile rubber-tipped dropper, several drug sensitivity test tubes (including drug sensitivity tablets corresponding to the antibiotics that can be selected by the farm), 1 negative control tube, 1 positive control tube, and 1 bottle of drug sensitivity test solution.
[0059] Example 8: Method of operation of drug sensitivity test kit
[0060] (1) Wear sterile gloves, open the drug sensitivity test solution, use a sterile rubber-tipped dropper to pipette 2 ml into the negative control tube, and cover the lid;
[0061] (2) taking out a sterile cotton swab to collect a sample of the diseased part of the shrimp, and putting it into the drug sensitivity detection solution and mixing it evenly;
[0062] (3) Use a sterile rubber-tipped dropper to draw up 2 ml of the mixed drug sensitivity test solution and drop it into each drug sensitivity test tube and positive control tube, ensuring that 2 ml is dropped into each tube;
[0063] (4) Place the dropwise drug sensitivity test kit in a constant temperature environment of 25°C-40°C for 12 hours;
[0064] (5) After 4 hours of incubation, take out the test tube every hour, observe the color of the liquid in the tube to determine the result, and record the time of the result determination.
[0065] (6) Determine the validity of the results: If the positive control tube turns red and the negative control tube remains unchanged, the test results are considered valid. By observing the color of each tube, the sensitivity of the corresponding antibiotic to the bacterial population in the field can be determined. If the color of the drug sensitivity test tube turns red, it proves that the bacteria are growing and the antibiotic in this drug sensitivity test tube is not sensitive; if the color of the drug sensitivity test tube remains unchanged, it proves that the bacterial growth is inhibited and the antibiotic in this drug sensitivity test tube is sensitive.
[0066] As can be seen from Table 1 and Table 2, the rapid drug sensitivity test kit has a promoting effect on the growth of bacteria in diseased shrimp samples due to the addition of 7.5% of shrimp polypeptide liquid in the drug sensitivity test solution, which accelerates the proliferation rate of pathogenic bacteria in the positive control tube and the insensitive antibiotic tube. The drug sensitivity test results of the ordinary drug sensitivity test kit after 9 hours of cultivation are consistent with those after 12 hours of cultivation; the drug sensitivity test results of the rapid drug sensitivity test kit after 5 hours of cultivation are consistent with those after 12 hours of cultivation. The drug sensitivity test result determination time of the rapid drug sensitivity test kit is 4 hours earlier than that of the ordinary drug sensitivity test kit, which can solve the problem of rapid drug sensitivity detection on-site in aquaculture farms.
[0067] Table 1 Results of drug sensitivity test of diseased shrimps with common drug sensitivity test kits
[0068]
[0069] Table 2 Results of drug sensitivity test of diseased shrimps by rapid drug sensitivity test kit
[0070]
[0071]
Claims
1. A rapid drug sensitivity detection kit for breeding sites, characterized in that: The rapid drug sensitivity detection kit is a rapid drug sensitivity detection kit composed of a tryptic soy peptone liquid culture medium, a white shrimp polypeptide as a bacterial growth promoter, and triphenyltetrazolium chloride as a color developer to form a drug sensitivity detection solution, and a drug sensitivity sheet.
2. The rapid drug sensitivity detection kit according to claim 1, characterized in that: The preparation method of the drug sensitivity detection solution is as follows: 17.0 g of tryptone, 3.0 g of soy peptone, 5.0 g of sodium chloride, 2.5 g of dipotassium hydrogen phosphate, 2.5 g of glucose, and 10 g of triphenyltetrazolium chloride are weighed, dissolved in 1 L of deionized water, the pH value is adjusted to 7.3±0.2, 75 ml of white shrimp polypeptide solution is added, filtered with a 0.22 um sterilizing filter, and aseptically packaged.
3. The rapid drug sensitivity detection kit according to claim 1, characterized in that: The preparation method of the white shrimp polypeptide liquid is as follows: (1) Wash 40- to 60-day-old whiteleg shrimp with pure water three times, add 5 times the amount of ultrapure water, and fully homogenize using a homogenizer; (2) The white shrimp homogenate was frozen at -20°C for 24 hours, thawed at room temperature, and repeated freezing and thawing for 3 times, and then centrifuged at 12000 rpm / min for 10 minutes in a centrifuge; (3) Collect the supernatant, add ammonium sulfate to a final concentration of 20% to the supernatant, let stand at 4°C for 24 hours, and centrifuge at 12000 rpm / min for 20 minutes; (4) Collect the supernatant, filter it using a 0.45 μm filter, and then filter it using a 0.22 μm filter to sterilize it. Then, package it aseptically and store it at 2-8°C.
4. A bacterial growth promoter, characterized in that The bacterial growth promoter component is white shrimp polypeptide liquid.
5. The bacterial growth promoter according to claim 4, characterized in that The preparation method of the white shrimp polypeptide liquid is as follows: (1) Wash 40- to 60-day-old whiteleg shrimp with pure water three times, add 5 times the amount of ultrapure water, and fully homogenize using a homogenizer; (2) The white shrimp homogenate was frozen at -20°C for 24 hours, thawed at room temperature, and repeated freezing and thawing for 3 times, and then centrifuged at 12000 rpm / min for 10 minutes in a centrifuge; (3) Collect the supernatant, add ammonium sulfate to a final concentration of 20% to the supernatant, let stand at 4°C for 24 hours, and centrifuge at 12000 rpm / min for 20 minutes; (4) Collect the supernatant, filter it using a 0.45 μm filter, and then filter it using a 0.22 μm filter to sterilize it. Then, package it aseptically and store it at 2-8°C.