阅读说明：本技术 一种药敏试剂盒及其制备方法以及细菌药敏检测方法 (Drug sensitivity kit, preparation method thereof and bacterial drug sensitivity detection method ) 是由 郭抗抗 李鑫鑫 刘勇 张彦明 于 2019-12-03 设计创作，主要内容包括：本发明公开了一种药敏试剂盒及其制备方法以及细菌药敏检测方法,属于药物检测技术领域。本发明试剂盒包括药敏板、液体培养基和固体培养基平板；本发明选用常规和最新兽用药物制作药敏板,并根据不同的感染细菌配制不同的培养基平板,所制备的药敏试剂盒具有试验结果精确、耗时短、操作简便且成本低廉的特点,适合于规模化养殖场及基层兽医单位进行细菌性疫病的药敏试验,从而实现动物精准用药。(The invention discloses a drug sensitivity kit, a preparation method thereof and a bacterial drug sensitivity detection method, and belongs to the technical field of drug detection. The kit comprises a drug sensitive plate, a liquid culture medium and a solid culture medium flat plate; the invention selects the conventional and latest veterinary drugs to manufacture the drug sensitive plate, and prepares different culture medium flat plates according to different infectious bacteria, and the prepared drug sensitive kit has the characteristics of accurate test result, short time consumption, simple and convenient operation and low cost, and is suitable for large-scale farms and basic veterinary units to carry out drug sensitive tests on bacterial epidemic diseases, thereby realizing accurate drug administration for animals.)

1. A veterinary bacterial drug sensitivity detection kit is characterized by comprising a drug sensitive plate, a liquid culture medium and a solid culture medium flat plate;

the drug sensitive plate is coated with drugs suitable for bacterial drug sensitive tests; the drug concentration is the minimum inhibitory concentration of the standard strain;

the liquid culture medium comprises LB liquid culture medium, meat extract broth culture medium and brain-heart extract broth culture medium;

the solid culture medium plate comprises a common nutrient agar culture medium plate, a MacConkey agar culture medium plate, a BP agar culture medium plate and a KF streptococcus agar culture medium plate.

2. The veterinary bacterial drug sensitivity detection kit according to claim 1, characterized in that the drug sensitivity plate comprises a detection well, a positive control well, a negative control well and a blank control well; the detection hole is coated with a drug suitable for a bacterial drug susceptibility test.

3. The veterinary bacterial drug sensitivity detection kit according to claim 2, characterized in that the drug sensitive plate comprises a plate body and a plate cover; the plate body comprises a detection hole, a positive control hole, a negative control hole and a blank control hole; the plate cover is marked with a detection hole corresponding to the plate body, and a drug concentration hole, a positive control hole, a negative control hole and a blank control hole.

4. The veterinary bacterial drug sensitivity detection kit according to claim 3, characterized in that the drug sensitive plates comprise an Escherichia coli drug sensitive plate, a Staphylococcus aureus drug sensitive plate and a Streptococcus drug sensitive plate;

the escherichia coli drug sensitive plate is coated with amoxicillin, florfenicol, tilmicosin, enrofloxacin, gentamicin, terramycin, streptomycin sulfate, neomycin sulfate, doxycycline, trimethoprim and ciprofloxacin;

the staphylococcus aureus drug sensitive plate is coated with penicillin, florfenicol, tilmicosin, enrofloxacin, ceftiofur sodium, streptomycin sulfate, neomycin sulfate, doxycycline, trimethoprim, lincomycin and tylosin;

the streptococcus drug sensitive plate is coated with penicillin, amoxicillin, florfenicol, enrofloxacin, ceftiofur sodium, doxycycline, trimethoprim, lincomycin, ciprofloxacin, sulfamonomethoxine and tylosin.

5. The method for preparing a veterinary bacterial drug sensitivity detection kit according to claim 4, which comprises the steps of preparing a drug sensitive plate, preparing a liquid culture medium and preparing a solid culture medium plate;

the preparation method of the drug sensitive plate comprises the following steps: adding medicine suitable for bacterial drug sensitivity test and antioxidant coating liquid into the drug sensitive plate, heat drying and vacuum packaging;

the steps for preparing the liquid culture medium are as follows: preparing a liquid culture medium, sterilizing at high temperature and high pressure, aseptically packaging, and storing at 4 ℃;

the steps for preparing the solid medium plate are as follows: preparing solid culture medium, making into plate, solidifying the culture medium, vacuum packaging, and storing at 4 deg.C.

6. The preparation method of the veterinary bacterial drug sensitivity detection kit according to claim 5, wherein the drug sensitive plate is a 96-well plate, and specifically comprises the following steps:

wherein, the A1-H11 area is a detection hole, and each row is coated with different medicines suitable for bacterial drug sensitivity test; A12-B12 is a blank control hole, and liquid LB culture medium is added; D12-E12 is a negative control hole, and the inactivated bacterial liquid to be detected is added; G12-H12 is a positive control hole, and bacterial liquid to be detected is added;

respectively dissolving the medicines in the solvent according to the concentration of the dilution liquid which is 4 times of the minimum bacteriostatic concentration of the medicines to different bacteria and the concentration of the antibiotic stock solution which is hundred times of the maximum concentration of the different dilution liquid and is used by the drug sensitive plate; then diluting the antibiotic stock solution, which comprises the following specific steps:

wherein, the horizontal line in the table indicates that the plate is not coated with the medicine;

dissolving carnosol in absolute ethyl alcohol, wherein the concentration of the carnosol in the prepared solution is 0.2mg/mL, and sterilizing and filtering the solution to obtain a coating solution;

preparing a drug sensitive plate: respectively preparing drug sensitive plates aiming at escherichia coli, staphylococcus aureus and streptococcus, and coating different drugs in detection holes; adding 25 μ L of specified drug diluent into the corresponding position of the blank drug sensitive plate, adding 25 μ L of coating solution, drying at 50-60 deg.C for 12h under aseptic condition, completely drying the drug sensitive plate, vacuum packaging with vacuum packaging machine, and storing at-20 deg.C.

7. The method for preparing a veterinary bacterial drug sensitivity detection kit according to claim 6,

the LB liquid culture medium comprises the following components in concentration: 10g/L of peptone, 5g/L of yeast extract and 5g/L of sodium chloride, wherein the pH value is 7.0-7.4;

the meat extract broth medium comprises the following components in concentrations: 3g/L of beef powder, 5g/L of sodium chloride, 12g/L of peptone and 2g/L of dipotassium hydrogen phosphate, and the pH value is 7.4-7.6;

the brain heart infusion broth culture medium comprises the following components in concentration: 10g/L of peptone, 12.5g/L of dehydrated calf brain extract powder, 5g/L of dehydrated calf heart extract powder, 5g/L of sodium chloride, 2g/L of glucose and 2.5g/L of disodium hydrogen phosphate, wherein the pH value is 7.2-7.6;

the common nutrient agar culture medium comprises the following components in concentration: 10.0g/L of peptone, 5.0g/L of yeast extract, 5.0g/L of sodium chloride and 15.0g/L of agar, wherein the pH value is 7.0-7.4;

the MacconKa agar medium comprises the following components in concentration: 20g/L of peptone, 10.0g/L of lactose, 5.0g/L of bile salt, 5.0g/L of sodium chloride, 0.075g/L of neutral red and 13.0g/L of agar, wherein the pH value is 7.2-7.6;

the BP agar medium comprises the following components in concentration: 10.0g/L of peptone, 5.0g/L of beef extract, 1.0g/L of yeast extract, 10.0g/L of sodium pyruvate, 12.0g/L of glycine, 5.0g/L of lithium chloride, 15.0g/L of agar and 5ml/L of potassium tellurite, and the pH value is 7.0-7.4;

the KF streptococcus agar culture medium comprises the following components in concentration: no. 3 shows 10g/L of peptone, 20g/L of maltose, 1g/L of lactose, 10g/L of yeast extract, 5g/L of sodium chloride, 0.4g/L of sodium azide, 10g/L of sodium glycerophosphate, 0.1g/L of triphenyltetrazolium chloride, 0.015g/L of bromocresol purple and 15g/L of agar, and the pH value is 7.0-7.4.

8. A method for detecting drug sensitivity of bacteria, which is characterized by using the kit of claim 7 to detect, and comprises the following specific steps:

(1) aseptically collecting pathological lesion tissues and inoculating the pathological lesion tissues to a corresponding culture medium flat plate, inoculating a bacterial sample in the pathological lesion tissues to the culture medium flat plate, culturing the inoculated flat plate at 36-38 ℃, observing the growth form of bacteria after 12-16 h, selecting bacterial colonies with the morphological characteristics meeting the requirements and inoculating the bacterial colonies to a liquid LB culture medium, culturing at 36-38 ℃ at 160-180 rpm, and obtaining a bacterial liquid after 10-12 h;

(2) diluting the bacterial liquid by using a liquid LB culture medium, adjusting the absorbance value of the bacterial liquid at 630nm to be 0.2-0.4, and then mixing 200 mu L of bacterial liquid with 9.8mL of liquid LB culture medium to obtain the bacterial concentration of about 1 multiplied by 10⁶The initial bacterial liquid for drug sensitivity detection;

(3) preparing a drug sensitive plate, adding the drug liquid into the drug sensitive plate, performing sterile heat drying at 50-60 ℃, performing vacuum packaging after drying, and storing at-20 ℃;

(4) dropping 100 mu L of diluted bacterial liquid into the corresponding detection hole and the positive control hole of the drug sensitive plate according to the detection requirement, slightly shaking to ensure that the drugs at the bottom of the drug sensitive plate are fully contacted with the bacterial liquid, culturing the drug sensitive plate at 36-38 ℃ within 15-30 min after completion, measuring the absorbance of the detection hole after 8-10 h, and directly judging the sensitivity of the bacteria in the pathological tissue to the drug components contained in the detection hole according to the absorbance value, or substituting the absorbance value into a formula for calculation to obtain the specific bacteriostasis rate of the drug components contained in the detection hole to the bacteria in the pathological tissue;

the bacteriostatic rate (%) - (A)_{Yang (Yang)}-A_{Yin (kidney)})-(A_{Sample (A)}-A_{Yin (kidney)})]/(A_{Yang (Yang)}-A_{Yin (kidney)})×100％；

And (3) judging standard: the bacteriostatic rate is greater than 90% and is sensitive to the drugs (S), the bacteriostatic rate is 70-90% and is moderately sensitive to the drugs (I), and the bacteriostatic rate is less than 70% and is insensitive to the drugs (R).

9. The method according to claim 8, wherein when calculating the inhibition rate after culturing the drug-sensitive plate, the absorbance value of the positive control in the plate is greater than that of the negative control, and the absorbance value of the blank control is less than that of the negative control, the detection result can be determined; if the absorbance value of the positive control is too small, the test thalli die naturally, the result can not be judged, and the bacteria should be shaken again and the bacteria liquid should be diluted for testing.

Technical Field

The invention relates to the technical field of drug detection, in particular to a drug sensitivity kit, a preparation method thereof and a bacterial drug sensitivity detection method.

Background

In animal production, some cases of antibiotics abuse exist in farms, for example, in order to improve the prevention and growth promotion effects of antibiotics, the antibiotics are used for a long time and in an overdose mode, so that the flora in animals is seriously disordered, and the generation and the transmission of drug-resistant strains are accelerated. With the improvement of food safety consciousness and the increasing attention on the situation of drug resistance to bacteria of people, a plurality of antibiotics are forbidden to be used in livestock and poultry breeding, so that the screening of drugs sensitive to pathogenic bacteria can realize the accurate control of animal disease medication, not only reduce the cost of drug treatment, but also slow down the generation speed of drug-resistant strains.

The current methods for testing drug sensitivity of microorganisms mainly comprise a paper diffusion method and a dilution method. The paper diffusion method is that a filter paper containing quantitative antibacterial drugs is pasted on the surface of agar inoculated with test bacteria, and the sensitivity of the bacteria to the drug components in the paper is judged according to the growth condition of the bacteria. The method has the defects of complex operation, limited quantity of once-detected medicines and samples and incapability of obtaining accurate bacteriostasis rate in clinical experiments. The dilution method is to prepare the antibacterial drug into a certain concentration and then dilute the antibacterial drug in a multiple ratio, add quantitative bacterial liquid into the liquid medicine and judge the Minimum Inhibitory Concentration (MIC) of the drug according to the minimum drug concentration of the visible growth of the flesh and eyes of the bacteria to be detected. The dilution method is usually carried out by using a microbial drug sensitive plate, but the drug sensitive plate of the current product has the conditions of single style, limited types of coated drugs, suitability for detecting the MIC of a small amount of samples, and incapability of meeting the drug sensitive tests of various types of bacteria and the detection requirements of a large amount of samples

Therefore, providing a drug sensitive kit, a preparation method thereof and a bacteria drug sensitive detection method is a problem that needs to be solved urgently by those skilled in the art.

Disclosure of Invention

In view of the above, the invention provides a drug sensitivity kit, a preparation method thereof and a bacterial drug sensitivity detection method, which solve the problems of old and single drug type, complex operation and excessive consumption of materials in large-scale detection in commercially available drug sensitivity detection products; the invention has the characteristics of improving the detection precision, having large sample amount in single detection, being suitable for veterinary clinic and meeting the detection requirements of various bacteria.

In order to achieve the purpose, the invention adopts the following technical scheme:

a veterinary bacterial drug sensitivity detection kit comprises a drug sensitive plate, a liquid culture medium and a solid culture medium flat plate;

the drug sensitive plate is coated with drugs suitable for bacterial drug sensitive tests; the drug concentration is the minimum inhibitory concentration of the standard strain;

the liquid culture medium comprises LB liquid culture medium, meat extract broth culture medium and brain-heart extract broth culture medium;

LB liquid culture medium is used for enrichment of Escherichia coli, meat extract broth culture medium is used for enrichment of staphylococcus aureus, and brain-heart extract broth culture medium is used for enrichment of streptococcus.

The solid culture medium plate comprises a common nutrient agar culture medium plate, a MacConkey agar culture medium plate, a BP agar culture medium plate and a KF streptococcus agar culture medium plate;

plain nutrient agar medium plate: enrichment for already established types of bacteria (i.e., after the bacteria are identified by isolation using an isolation medium, if enrichment is required, the medium can be used as an aid to reduce the use of the identification medium), on which Escherichia coli, Staphylococcus aureus and Streptococcus can grow to different extents; mackanka agar medium plate: used for separating and identifying Escherichia coli which grows into pink colonies; BP agar medium plate: the method is used for separating and identifying staphylococcus aureus, and the staphylococcus aureus can grow into gray black to black glossy colonies; KF streptococcus agar medium plate: for the isolation and identification of streptococci, which grow as colonies with a red to pink centre.

Further, the drug sensitive plate comprises a detection hole, a positive control hole, a negative control hole and a blank control hole; the detection hole is coated with a drug suitable for a bacterial drug susceptibility test.

Further, the drug sensitive plate comprises a plate body and a plate cover; the plate body comprises a detection hole, a positive control hole, a negative control hole and a blank control hole; the plate cover is marked with a detection hole corresponding to the plate body, and a drug concentration hole, a positive control hole, a negative control hole and a blank control hole.

Further, the drug sensitive plates comprise an escherichia coli drug sensitive plate, a staphylococcus aureus drug sensitive plate and a streptococcus drug sensitive plate;

the escherichia coli drug sensitive plate is coated with amoxicillin, florfenicol, tilmicosin, enrofloxacin, ceftiofur sodium, oxytetracycline, streptomycin sulfate, neomycin sulfate, doxycycline, trimethoprim and ciprofloxacin;

the staphylococcus aureus drug sensitive plate is coated with penicillin, florfenicol, tilmicosin, enrofloxacin, ceftiofur sodium, streptomycin sulfate, neomycin sulfate, doxycycline, trimethoprim, lincomycin and tylosin;

the streptococcus drug sensitive plate is coated with penicillin, amoxicillin, florfenicol, enrofloxacin, ceftiofur sodium, doxycycline, trimethoprim, lincomycin, ciprofloxacin, sulfamonomethoxine and tylosin.

Further, a preparation method of the veterinary bacterial drug sensitivity detection kit comprises the steps of preparing a drug sensitive plate, preparing a liquid culture medium and preparing a solid culture medium flat plate;

the preparation method of the drug sensitive plate comprises the following steps: adding medicine suitable for bacterial drug sensitivity test and antioxidant coating liquid into the drug sensitive plate, heat drying and vacuum packaging;

the steps for preparing the liquid culture medium are as follows: preparing a liquid culture medium, sterilizing at high temperature and high pressure, aseptically packaging, and storing at 4 ℃;

the steps for preparing the solid medium plate are as follows: preparing solid culture medium, making into plate, solidifying the culture medium, vacuum packaging, and storing at 4 deg.C.

Further, the drug sensitive plate is a 96-well plate, and is specifically as follows:

wherein, the A1-H11 area is a detection hole, and each row is coated with different medicines suitable for bacterial drug sensitivity test; A12-B12 is a blank control hole, and liquid LB culture medium is added; D12-E12 is a negative control hole, and the inactivated bacterial liquid to be detected is added; G12-H12 is a positive control hole, and bacterial liquid to be detected is added;

positive control: for the detection requirement of multiple strains, bacterial liquids of the strains to be detected can be mixed and then added into the positive control holes (for example, when the drug resistance of 8 escherichia coli strains is simultaneously detected, after the corresponding bacterial liquids are added into the detection holes, 200 mu L of each bacterial liquid is absorbed into a clean centrifugal tube or other sterilized containers and then fully and uniformly mixed, and then 200 mu L of the mixed bacterial liquid is absorbed and respectively added into the two positive control holes).

Negative control: the method is used for eliminating the influence of dead bacteria precipitation on absorbance and calculating the bacteriostasis rate, so that bacteria liquid obtained after the secondary detection of the inactivated strains is added (the bacteria liquid is the same as a positive control, if a plurality of bacteria are detected, 200 mu L of each bacteria liquid is sucked out, mixed and inactivated, and then 200 mu L of the bacteria liquid is sucked out from the inactivated mixed bacteria liquid and respectively added into two negative control holes).

The blank control hole is added with culture medium for shaking bacteria, so as to eliminate the influence of the liquid culture medium polluted by improper operation on the experimental result.

Respectively dissolving the medicines in a solvent according to the concentration of the dilution liquid which is 4 times of the minimum inhibitory concentration (namely 4MIC) of the medicines to different bacteria and the concentration of antibiotic stock solution (storage concentration) which is hundred times of the maximum value in different dilution liquid concentrations (aiming at different bacterial strains) and is used for the drug sensitive plate; then diluting the antibiotic stock solution, which comprises the following specific steps:

wherein, the horizontal line in the table indicates that the plate is not coated with the medicine;

dissolving carnosol in absolute ethyl alcohol, wherein the concentration of the carnosol in the prepared solution is 0.2mg/mL, and sterilizing and filtering the solution to obtain a coating solution;

the corresponding solvents for each drug are as follows:

medicine	Solvent(s)	Medicine	Solvent(s)	Medicine	Solvent(s)
						Amoxicillin	Water (W)	Ceftiofur sodium	Water (W)	Doxycycline	Dilute hydrochloric acid
Florfenicol	Dimethyl sulfoxide	Enrofloxacin	Dilute hydrochloric acid	Trimethoprim	Dilute hydrochloric acid
						Oxytetracycline	Dilute hydrochloric acid	Streptomycin sulfate	Water (W)	Ciprofloxacin	Water (W)
Tilmicosin	Water (W)	Neomycin sulfate	Water (W)	Penicillin	Water (W)
						Lincomycin	Water (W)	Tylosin	Dilute hydrochloric acid	Gentamicin	Water (W)
Sulfamonomethoxine	Dilute hydrochloric acid

Preparing a drug sensitive plate: respectively preparing drug sensitive plates aiming at escherichia coli, staphylococcus aureus and streptococcus, and coating different drugs in detection holes; adding 25 μ L of specified drug diluent into the corresponding position of the blank drug sensitive plate, adding 25 μ L of coating solution, drying at 50-60 deg.C for 12h under aseptic condition, completely drying the drug sensitive plate, vacuum packaging with vacuum packaging machine, and storing at-20 deg.C.

Blank 96-well drug-sensitive plates were prepared using the same well positions as conventional 96-well plates. The drug dilution and sample adding positions in the drug sensitive plate are as follows:

further, the LB liquid medium includes the following components at the following concentrations: 10g/L of peptone, 5g/L of yeast extract and 5g/L of sodium chloride, wherein the pH value is 7.0-7.4;

the meat extract broth medium comprises the following components in concentrations: 3g/L of beef powder, 5g/L of sodium chloride, 12g/L of peptone and 2g/L of dipotassium hydrogen phosphate, and the pH value is 7.4-7.6;

the brain heart infusion broth culture medium comprises the following components in concentration: 10g/L of peptone, 12.5g/L of dehydrated calf brain extract powder, 5g/L of dehydrated calf heart extract powder, 5g/L of sodium chloride, 2g/L of glucose and 2.5g/L of disodium hydrogen phosphate, wherein the pH value is 7.2-7.6;

the common nutrient agar culture medium comprises the following components in concentration: 10.0g/L of peptone, 5.0g/L of yeast extract, 5.0g/L of sodium chloride and 15.0g/L of agar, wherein the pH value is 7.0-7.4;

the MacconKa agar medium comprises the following components in concentration: 20g/L of peptone, 10.0g/L of lactose, 5.0g/L of bile salt, 5.0g/L of sodium chloride, 0.075g/L of neutral red and 13.0g/L of agar, wherein the pH value is 7.2-7.6;

the BP agar medium comprises the following components in concentration: 10.0g/L of peptone, 5.0g/L of beef extract, 1.0g/L of yeast extract, 10.0g/L of sodium pyruvate, 12.0g/L of glycine, 5.0g/L of lithium chloride, 15.0g/L of agar and 5ml/L of potassium tellurite, and the pH value is 7.0-7.4;

the KF streptococcus agar culture medium comprises the following components in concentration: no. 3 shows 10g/L of peptone, 20g/L of maltose, 1g/L of lactose, 10g/L of yeast extract, 5g/L of sodium chloride, 0.4g/L of sodium azide, 10g/L of sodium glycerophosphate, 0.1g/L of triphenyltetrazolium chloride, 0.015g/L of bromocresol purple and 15g/L of agar, and the pH value is 7.0-7.4.

Further, a method for detecting drug sensitivity of bacteria by using the kit comprises the following specific steps:

(1) aseptically collecting pathological lesion tissues and inoculating the pathological lesion tissues to a corresponding culture medium flat plate, inoculating a bacterial sample in the pathological lesion tissues to the culture medium flat plate, culturing the inoculated flat plate at 36-38 ℃, observing the growth form of bacteria after 12-16 h, selecting bacterial colonies with the morphological characteristics meeting the requirements and inoculating the bacterial colonies to a liquid LB culture medium, culturing at 36-38 ℃ at 160-180 rpm, and obtaining a bacterial liquid after 10-12 h;

(2) diluting the bacterial liquid by using a liquid LB culture medium, adjusting the absorbance value of the bacterial liquid at 630nm to be 0.2-0.4, and then mixing 200 mu L of bacterial liquid with 9.8mL of liquid LB culture medium to obtain the bacterial concentration of about 1 multiplied by 10⁶The initial bacterial liquid for drug sensitivity detection;

(3) preparing a drug sensitive plate, adding the drug liquid into the drug sensitive plate, performing sterile heat drying at 50-60 ℃, performing vacuum packaging after drying, and storing at-20 ℃;

(4) according to the detection requirement, 100 mu L of diluted bacterial liquid is dripped into a corresponding detection hole and a positive control hole of the drug sensitive plate (25 mu L of specified drug diluent is added into the drug sensitive plate, the concentration of the drug diluent is 4MIC, after drying, 100 mu L of diluted bacterial liquid is added, the concentration of the drug in the drug sensitive plate is the minimum inhibitory concentration MIC for each strain), and the drug at the bottom of the drug sensitive plate is fully contacted with the bacterial liquid by slight oscillation;

after the bacteria liquid is added into the drug sensitive plate, the concentration of the drugs in the plate is as follows: unit (μ g/mL)

Escherichia coli drug sensitive plate:

staphylococcus aureus drug sensitive plate:

streptococcus drug sensitive plate:

within 15-30 min after sample adding, culturing the drug sensitive plate at 36-38 ℃, measuring the absorbance of the detection hole after 8-10 h, directly judging the sensitivity of bacteria in the pathological tissue to the drug components contained in the detection hole according to the absorbance value, or substituting the absorbance value into a formula for calculation to obtain the specific bacteriostasis rate of the drug components contained in the detection hole to the bacteria in the pathological tissue;

the formula for calculating the bacteriostasis rate is as follows:

the bacteriostatic rate (%) - (A)_{Yang (Yang)}-A_{Yin (kidney)})-(A_{Sample (A)}-A_{Yin (kidney)})]/(A_{Yang (Yang)}-A_{Yin (kidney)})×100％；

And (3) judging standard: the bacteriostatic rate is greater than 90% and is sensitive to the drugs (S), the bacteriostatic rate is 70-90% and is moderately sensitive to the drugs (I), and the bacteriostatic rate is less than 70% and is insensitive to the drugs (R).

Further, when the antibacterial rate is calculated after the drug sensitive plate is cultured, the absorbance value of a positive control in the plate is greater than that of a negative control, and the absorbance value of a blank control is less than that of the negative control, the detection result can be judged; if the absorbance value of the positive control is too small, the test thalli die naturally, the result can not be judged, and the bacteria should be shaken again and the bacteria liquid should be diluted for testing.

According to the technical scheme, compared with the prior art, the invention discloses a drug sensitive kit, a preparation method thereof and a bacteria drug sensitive detection method, wherein a conventional and latest veterinary drug is selected to prepare a drug sensitive plate, and different culture medium flat plates are prepared according to different infected bacteria, so that the prepared drug sensitive kit has the characteristics of accurate test result, short time consumption, simplicity and convenience in operation and low cost, and is suitable for carrying out drug sensitive test on bacterial epidemic diseases in large-scale farms and basic veterinary units, thereby realizing accurate administration of animals. The kit comprises a liquid culture medium and a solid culture medium plate which are sterilized and packaged, and can be taken at any time, so that the time waste caused by the existing preparation is avoided; and the invention prepares three kinds of drug sensitive boards at the same time: the escherichia coli drug sensitive plate, the staphylococcus aureus drug sensitive plate and the streptococcus drug sensitive plate are coated with 11 drugs, 3 kinds of bacteria can be identified simultaneously, the best drugs can be screened simultaneously, time can be fully saved, a drug administration scheme can be rapidly formulated, related bacterial blight can be prevented and treated timely, and loss of a large-scale farm is reduced.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.