Rapid drug sensitivity detection method based on fish tissue homogenate

Abstract

The invention discloses a rapid drug sensitivity detection method based on fish tissue homogenate, which comprises the following steps: taking fish tissues for homogenate, coating and inoculating the obtained homogenate on a flat plate, placing a drug sensitivity paper sheet, culturing for 16-24 hours at 30 DEG C, then measuring the diameter of an inhibition zone on the flat plate, and judging a drug sensitivity detection result. According to the method, the sample pretreatment and detection process is simplified, diseased fish tissue homogenate is directly used, the detection period is remarkably shortened to be within 1 d, and meanwhile the result accuracy is ensured. According to the technical scheme, the method comprises the steps of sample collection under the sterile condition, tissue homogenate preparation, homogenate coating inoculation, drug sensitive paper sheet placement and culture result interpretation. The method has the advantages of high efficiency, rapidness, simplicity and convenience in operation, accurate result and wide application range, is suitable for drug sensitivity detection of various fish pathogenic bacteria, and has important practical application value.

Description

Technical Field

[0001] The present invention relates to a method for detecting bacterial drug sensitivity, and in particular to a rapid drug sensitivity detection method based on fish tissue homogenate. Background Art

[0002] Drug sensitivity testing, as a key link in bacteriology and clinical microbiology, undertakes the important task of evaluating the sensitivity of bacteria and other microorganisms to specific antimicrobial drugs. The accuracy and timeliness of this process are directly related to the rational selection and effect evaluation of clinical antibiotic treatment, and are of immeasurable value for accurately guiding clinical treatment, effectively controlling the spread of infection, and closely monitoring the development trend of drug resistance.

[0003] In the current technical system, bacterial drug sensitivity testing mainly relies on several classic and widely recognized methods. The first is the KB paper agar diffusion method, which directly attaches a paper containing a quantitative antimicrobial drug to a solid culture medium inoculated with the bacteria to be tested, and uses the diffusion of the antimicrobial drug in the agar to form a concentration gradient, thereby visually judging the sensitivity of the bacteria to the drug by observing the diameter of the inhibition zone formed. This method is widely used because of its simple operation and intuitive results.

[0004] Secondly, the dilution method is another major type of drug sensitivity testing technology, mainly including broth dilution method and agar dilution method. They prepare a series of antimicrobial drug solutions with a concentration gradient, co-culture with the test bacteria, observe and measure the minimum drug concentration (MIC) that can completely inhibit bacterial growth, and quantitatively evaluate bacterial resistance. Although this method can provide more accurate quantitative data, the operation process is relatively complicated and time-consuming.

[0005] In addition, the E test (concentration gradient paper strip diffusion method) is an innovative combination of the dilution method and the diffusion method. It uses specially prepared paper strips containing concentration gradient antibiotics to directly read the MIC on the plate, further simplifying the operation steps and improving the detection efficiency.

[0006] Although the above methods have shown high accuracy and reliability in clinical and scientific research fields, their inherent limitations have gradually emerged in actual production applications, especially in aquaculture. On the one hand, these methods are generally cumbersome to operate, involve multiple steps, and have high requirements for experimental conditions, resulting in a long entire detection cycle, usually requiring 2 to 3 days or even longer to complete, which is difficult to meet the urgent needs of rapid clinical diagnosis and treatment of aquatic animals. On the other hand, strict experimental conditions limit the widespread application of these methods in production environments, increasing the difficulty and cost of technology promotion. Summary of the invention

[0007] One of the purposes of the present invention is to provide a rapid drug sensitivity detection method based on fish tissue homogenate. The method does not require complicated bacterial separation and purification steps, and directly uses diseased fish tissue homogenate for drug sensitivity testing, significantly shortening the detection cycle to less than 1 day, while maintaining the accuracy of the results comparable to standard operations, and is efficient, rapid and easy to operate.

[0008] The technical solution provided by the present invention is as follows: a rapid drug sensitivity detection method based on fish tissue homogenate, wherein fish tissue is homogenized under sterile conditions, the obtained homogenate is spread and inoculated on a plate, a drug sensitive paper sheet is placed, and the plate is cultured at 30°C for 16 to 24 hours, and then the diameter of the inhibition zone on the plate is measured to determine the drug sensitivity detection result.

[0009] Fish tissues include liver, spleen, kidney and brain tissues, which can be one of them or a mixture of two or more. Usually, fish tissues are mixed with liver, spleen and kidney tissues. For bacteria that can infect the brain, such as Streptococcus dolphinus, it is also possible to consider collecting part of the brain tissue. Fish tissues are selected from dying or freshly dead fish. The collection of dead fish samples should not exceed 24 hours, and should be kept cold and dry to maintain the vitality of bacteria and the freshness of tissues to the greatest extent.

[0010] There is no clear requirement for the specific sampling amount of different tissues. Based on experience, more samples can be taken from severely infected tissues. In the absence of a more accurate weighing scale, the sampling amount can be increased or decreased as appropriate or determined based on experience. In one embodiment of the present invention, the mass of the mixed tissue sample is 0.1g±0.02g.

[0011] Columbia blood agar plates (CBA plates) are preferably used as plates.

[0012] In the present invention, the bacterial concentration in the homogenate is not less than 2.8×10 4 CFU / ml.

[0013] In the present invention, the homogenate can be ground in a grinder at 50 Hz for 1 min, followed by instant centrifugation in a centrifuge. A higher frequency may cause damage and death of bacteria in the tissue, and the insufficiently ground tissue can be thrown to the bottom of the grinding tube by instant centrifugation, which does not affect the coating in the next step. The present invention can also be ground manually, but attention should be paid to aseptic conditions and aseptic operation. Without a centrifuge, manual centrifugation or standing for a period of time is also possible, so that the tissue that has not been repeatedly ground settles but the bacteria fail to settle.

[0014] The homogenate must be used within 15 minutes.

[0015] Place the drug-sensitive paper sheets so that the distance between each sheet is no less than 24 mm and the center of the sheet is no less than 15 mm from the edge of the plate. Six sheets are preferably placed on a plate with a diameter of 90 mm.

[0016] Beneficial effects:

[0017] 1. Efficient and fast: It omits the traditional bacterial separation and purification steps, greatly shortens the detection cycle and improves the detection efficiency.

[0018] 2. Easy to operate: It simplifies the detection process and reduces the difficulty of operation, which is suitable for rapid detection needs on the production line.

[0019] 3. Accurate results: Compared with the standard operating method (National Clinical Laboratory Operating Procedures (4th Edition)), the accuracy of drug sensitivity testing can be guaranteed by constructing a database of differences in the size of inhibition zones of various strains under different conditions.

[0020] 4. Wide application: It is suitable for drug sensitivity detection of various fish pathogens and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of amplification of tissue homogenate with different concentrations of S. iniae;

[0022] +: positive control with the addition of S. iniae, -: negative control with the addition of tissue homogenate. The upper row of numbers: indicates the concentration of S. iniae mixed into the tissue homogenate.

[0023] Figure 2 These are the quality control results of two antibiotic sensitivity paper strips;

[0024] The dotted line at 6 mm in the figure is the diameter of the blank drug-sensitive tablet.

[0025] Figure 3 is the diameter of the inhibition zone at different temperatures and culture media;

[0026] Note: The 37℃MHBA in the figure is marked as the standard condition sample. The results of the standard condition sample and the results of the other condition samples are compared in terms of significance and inhibition zone diameter. The results that meet the two conditions of P<0.05 and inhibition zone difference ≥2mm indicate a significant difference, marked with "#". No "#" is marked, indicating that the difference between the condition sample and the standard condition sample is not significant. The dotted line at 6mm in the figure is the diameter of the blank drug sensitive tablet.

[0027] Figure 4 is the diameter of the inhibition zone at different culture times;

[0028] The 24h mark in the figure is the standard condition sample. The results of the standard condition sample and the results of the other condition samples are compared in terms of significance and inhibition zone diameter. If the results meet the two conditions of P < 0.05 and the difference in inhibition zone ≥ 2mm, the difference is marked with "#". If "#" is not marked, it means that the difference between the condition sample and the standard condition sample is not significant. The dotted line at 6mm in the figure is the diameter of the blank drug sensitive tablet.

[0029] Figure 5 is the diameter of the inhibition zone at different concentrations;

[0030] The mark 8 in the figure is the standard condition sample. The results of the standard condition sample and the results of the other condition samples are compared in terms of significance and average inhibition zone diameter. If the results meet the two conditions of P < 0.05 and the difference in inhibition zone ≥ 2 mm, it indicates a significant difference, marked with "#". If "#" is not marked, it means that the difference between the condition sample and the standard condition sample is not significant. The dotted line at 6 mm in the figure is the diameter of the blank drug sensitive tablet.

[0031] Figure 6 is the diameter of the inhibition zone of pure bacterial solution and mixed tissue;

[0032] The pure bacterial solution marked with 8 in the figure is the standard condition sample. The results of the standard condition and diluted samples and the samples mixed with tissue homogenate solution were compared for significance and average inhibition zone diameter. The results that meet the two conditions of P<0.05 and inhibition zone difference ≥2mm indicate significant differences, marked with "#". If "#" is not marked, it means that the results of the condition sample are not significantly different from those of the standard condition sample. The dotted line at 6mm in the figure is the diameter of the blank drug sensitive tablet.

[0033] Figure 7 This is a graph of clinical sample test results;

[0034] The results of the standard condition samples and clinical condition samples were compared in terms of significance and average inhibition zone diameter. If the results met the two conditions of P < 0.05 and the inhibition zone difference ≥ 2 mm, the difference was significant and marked with "#". If "#" is not marked, it means that the results of the condition sample and the standard condition sample are not significantly different. The dotted line at 6 mm in the figure is the diameter of the blank drug sensitive tablet.

[0035] Figure 8 It is a schematic diagram of the process of the rapid drug sensitivity detection method of the present invention. DETAILED DESCRIPTION

[0036] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0037] The rapid drug sensitivity detection method based on fish tissue homogenate of the present invention is described by taking Streptococcus dolphinus of yellow catfish as an example.

[0038] 1 Materials and methods

[0039] 1.1 Experimental strains and animals

[0040] The strains were isolated from the epidemiological survey. The strains used in the inhibition zone comparison test and artificial infection case were Streptococcus dolphinus SI-11, and the other two strains used in the natural infection case were Streptococcus dolphinus SI-35 and SI-44. The experimental fish came from Guangzhou United Fish Fry Development Co., Ltd. and two yellow catfish ponds naturally infected with Streptococcus dolphinus.

[0041] 1.2 Main reagents and instruments

[0042] Agar powder and BHI broth culture medium were both from Guangdong Huankai Microbiology Co., Ltd., 5% sheep blood Columbia agar plates (hereinafter referred to as ZABP plates) and 5% sheep blood MH agar plates (which have passed the quality control of the standard strain of Streptococcus pneumoniae, hereinafter referred to as MHB plates) were both from Guangzhou Dijing Microbiology Technology Co., Ltd.; 2×M5 Hiper plus Taq HiFi PCRmix was from Polymeric Biotechnology Co., Ltd.; 6 antibiotic sensitivity papers were all from Beijing Biodi Biotechnology Co., Ltd.

[0043] Table 1 Main instrument information

[0044]

[0045]

[0046] 1.3 PCR identification of S. iniae using specific primers

[0047] PCR amplification and gel electrophoresis were used to identify S. ini. The PCR-related primers were synthesized by Guangzhou Tianyi Huiyuan Co., Ltd. (Table 2). PCR amplification was performed using different concentrations of S. ini mixed with yellow catfish tissue homogenate as a template. The PCR system and program were set according to the instructions of the 2×M5 Hiper plus Taq HiFi PCR mix product of PolyMei Company. The amplified product was electrophoresed and imaged using 1% agarose, and the target band size was 296bp. S. ini DNA was set as a positive control, and tissue homogenate as a negative control. The results are shown in Figure 1 .

[0048] Table 2 Primer information

[0049]

[0050] 1.4 Quality control of antibiotic sensitivity papers

[0051] The operation and quality control standards of the disk diffusion method for drug sensitivity testing refer to the relevant standards of the disk diffusion method in the "National Clinical Laboratory Operation Procedures (4th Edition)". For two antibiotics with clear resistance standards, doxycycline (DXT) and ciprofloxacin (CIP), inter-plate difference and accuracy tests were carried out. Inter-plate difference requirements: the quality control strain Escherichia coli ATCC25922 was inoculated on the MHA (Mueller-Hinton Agar, MH agar) plate, and six drug sensitivity test strips of the same antibiotic were fixed on a single agar plate, ensuring that the distance between each adjacent two pieces was at least 24 mm, and the distance from the center point of each test strip to the edge of the culture dish was not less than 15 mm. The treated plates were placed in a 37°C environment for overnight incubation. After the incubation, the diameter of the inhibition zone formed around each test strip was measured and recorded three times, and the difference between the maximum and minimum values ​​of the six inhibition zone diameters was required to be no more than 2 mm. In order to evaluate the accuracy of the test, the six diameters were averaged, and then the average inhibition zone diameter was compared with the standard quality control data of the antibiotic to judge the accuracy of the experimental results. The quality control standards for doxycycline and ciprofloxacin are shown in Table 3.

[0052] Table 3 Quality control standards

[0053]

[0054] 1.5 Effects of different temperatures and culture media on the inhibition zone

[0055] The standard conditions for the antibacterial test of Streptococcus dolphinus disk diffusion method refer to the relevant standards of B-hemolytic Streptococcus in the "National Clinical Laboratory Operation Procedure (4th Edition)", that is, 0.5 McFarland turbidity Streptococcus dolphinus suspension is inoculated on MHBA (MuellerHinton Blood Agar, MH blood agar) plates and cultured at 35-37°C and 5% CO2 for 20-24 hours. In order to understand the effect of inoculation in different culture media and different culture temperatures on the diameter of the inhibition zone, the temperature and inoculation medium conditions were changed on the basis of standard conditions, and they were changed to 30℃MHBA plate culture, 37℃CBA plate culture, 30℃CBA plate culture, 30℃BHI plate culture and 30℃MHA plate culture, and each plate was pasted with 6 different antibiotic susceptibility papers as required, including florfenicol FFC, flumequine UB, enrofloxacin ENR, ciprofloxacin CIP, doxycycline DXT and neomycin N. Six repeated plates were cultured for 24 hours and the antibacterial diameter was measured with a vernier caliper. Each inhibition zone was measured 3 times. The standard conditions were used as a comparison, and 0.5 McFarland turbidity Streptococcus indioides suspension was applied.

[0056] 1.6 Effect of different culture times on inhibition zone

[0057] In order to understand the effect of different incubation times after inoculation on the diameter of the inhibition zone, under standard conditions, 0.5 McFarland turbidity S. indica suspension was applied to MHBA plates, and the diameters of the inhibition zones were measured after incubation at 37°C for 12 h, 16 h, 20 h and 24 h, respectively. Six pieces of drug-susceptibility paper with different antibiotics were pasted on each plate as required, and six repeated plates were used, and each inhibition zone was measured three times.

[0058] 1.7 Effect of different bacterial concentrations on inhibition zone

[0059] In order to understand the effect of inoculation of different concentrations of bacterial suspension on the diameter of the inhibition zone, a 0.5 McFarland turbidity suspension of Streptococcus indica (about 2.8×10 8 CFU / mL and its gradient concentration diluted 10 times (about 2.8×10 7 CFU / mL), 100 times (about 2.8×10 6 CFU / mL), 1000 times (about 2.8×10 5 CFU / mL) and 10,000 times (about 2.8×10 4 CFU / mL) of bacterial suspension were added to MHBA plates and cultured at 37°C for 24 h, and the diameters of the inhibition zones were measured. Six different antibiotic sensitivity papers were pasted on each plate as required, and six repeated plates were used. Each inhibition zone was measured three times.

[0060] 1.8 Effect of tissue homogenate on inhibition zone

[0061] In order to understand the effect of bacterial suspension mixed with tissue homogenate on the diameter of the inhibition zone, a 1 McFarland turbidity suspension of Streptococcus indica (coating count of about 5.6×10 8 CFU / mL and 10 times (about 5.6×10 7 CFU / mL), 100 times (about 5.6×10 6 CFU / mL), 1000 (about 5.6×10 5 CFU / mL) times and 10,000 times (about 5.6×10 4 CFU / mL) gradient dilution, mixed with the same volume of yellow catfish liver, spleen and kidney tissue homogenate and coated on CBA plates, cultured at 30℃ for 24h and measured the diameter of the inhibition zone respectively. Each plate was pasted with 6 pieces of different antibiotic sensitivity paper as required, 6 repeated plates, and each inhibition measurement was repeated 3 times. A blank control was set up with only tissue homogenate coated.

[0062] 1.9 Clinical sample testing

[0063] In order to understand the difference between the mixed tissue samples in contact with the production line and the standard operation, the drug sensitivity test was carried out on yellow catfish artificially infected with Streptococcus dolphinus or naturally infected with Streptococcus dolphinus. The standard operation results were obtained by conventional separation and purification of Streptococcus dolphinus in yellow catfish. The standard operation results were inoculated on CBA plates with the mixed homogenate of liver, spleen and kidney tissues of yellow catfish and cultured at 30℃ for 16h. Each plate was pasted with 6 pieces of drug sensitivity paper of different antibiotics as required, 3 repeated plates, and each antibacterial measurement was performed 3 times. Among them, there were 3 cases of artificial infection and 2 cases of natural infection, totaling 5 cases.

[0064] 1.10 Establishment of rapid drug sensitivity testing method

[0065] Based on the above test data, the standard conditions were replaced with conditions that are more convenient for clinical testing operations, and a flow chart was drawn. 1.10 Application of rapid drug sensitivity testing methods

[0066] The rapid drug sensitivity test method was used to detect yellow catfish artificially infected with S. iniae or naturally infected with S. iniae, and the rapid drug sensitivity test results were compared with the standard operation results of conventional isolation and purification of S. iniae in yellow catfish. There were 1 artificial infection case and 2 natural infection cases, a total of 3 cases.

[0067] 1.12 Data Processing

[0068] The test results were preliminarily analyzed and collated using Microsoft Excel software, and the OD-corresponding colony count curve and growth curve were drawn; SPSS26.0 software was used to calculate the MIC of antimicrobial drugs. 50 (MIC required to inhibit 50% of the test strains in the same batch), MIC 90 (MIC required to inhibit 90% of the test strains in the same batch) and generate a cluster diagram of drug sensitivity results; use GraphPadPrism9.5 to draw strain proportions, monthly distribution results, drug sensitivity result heat maps, bacterial load group diagrams, etc., draw drug sensitivity result pictures, and calculate and annotate significant differences; use Microsoft PowerPoint software to combine the drug sensitivity result cluster diagram and heat map into a cluster heat map, and draw a rapid drug sensitivity test flow chart.

[0069] 2 Results

[0070] 2.1 PCR identification of S. iniae using specific primers

[0071] The specific PCR test was performed on the yellow catfish tissue homogenate mixed with different concentrations of bacterial suspension. The results showed that the DNA of Streptococcus iniae as a positive control and the final bacterial concentration were 2.8×10 9 CFU / mL, 2.8×10 8 CFU / mL, 2.8×107 CFU / mL and 2.8×10 6 The samples with CFU / mL showed a band of about 300 bp, which was consistent with the target band size, and 2.8×10 8 CFU / mL, 2.8×10 7 CFU / mL and 2.8×10 6 The brightness of the CFU / mL sample strips gradually decreases. 5 CFU / mL, 2.8×10 4 CFU / mL and pure tissue homogenate have no obvious bands ( Figure 1 ).

[0072] The above results show that this detection method can effectively detect the concentration of bacteria carried by 2.8×10 6 CFU / mL and above of tissue homogenate samples of S. iniae.

[0073] 2.2 Quality Control

[0074] The inter-plate difference and accuracy tests were conducted for doxycycline and ciprofloxacin with clear drug resistance standards. The results showed that after E. coli ATCC25922 was inoculated on the MHA plate and cultured overnight at 37°C, the difference between the maximum and minimum inhibition zone diameters of the two antibiotic susceptibility plates was within 2 mm, which met the inter-plate difference requirements. The average inhibition zone diameter of doxycycline was 20.92±0.39 mm ( Figure 2 ), which is within the range of 18 to 24 mm in the quality control standard; similarly, the average inhibition zone diameter of ciprofloxacin was 31.09 ± 0.78 mm ( Figure 2 ), within the quality control standard range of 30 to 40 mm, it meets the accuracy test requirements.

[0075] The above results show that both antibiotic susceptibility strips meet the requirements for inter-strip difference and accuracy detection.

[0076] 2.3 Effects of different temperatures and culture media on inhibition zones

[0077] The standard conditions for the drug sensitivity paper method of Streptococcus ini was to inoculate a 0.5 McFarland turbidity Streptococcus ini suspension on an MHBA plate and culture it at 37°C and 5% CO2 for 24 hours as a control. Figure 3 The sample is marked as 37℃ MHBA plate sample. The standard condition sample is compared with other samples with changed culture temperature and culture medium conditions. If the difference in the diameter of the inhibition zone between the two is >2mm and P<0.05, the difference between the conditions is considered significant.

[0078] The results showed that flumequine tablets had no inhibition zone under various culture media and temperature conditions; no inhibition zone was observed for neomycin tablets on CBA plates at 30°C, but there was an inhibition zone on CBA plates and MHBA plates at 37°C, but the inhibition zone was very close to the diameter of the blank tablet (6mm). Except flumequine, the inhibition zone results of the other five tablets on MHA and BHI plates at 30°C were P<0.05. At the same time, except for the difference of 1.14mm in the average diameter of the inhibition zone of neomycin on BHI plates compared with the samples under standard conditions, the difference was less than 2mm, and the inhibition zones of the other four tablets on BHI plates and the five tablets on MHA plates were ≥2mm compared with the samples under standard conditions. Except for neomycin, which did not form a non-inhibitory zone on the 30°C CBA plate, the inhibition zones formed by the other five antibiotic susceptibility tablets on the 30°C MHBA plate, the 37°C CBA plate, and the 30°C CBA plate were compared with the diameters of the inhibition zones on the 37°C MHBA plate under standard conditions (37°C MHBA plate). The results of the inhibition zone diameters were P ≥ 0.05 or the diameter difference was < 2 mm ( Figure 3 ).

[0079] 2.4 Effect of different culture times on inhibition zone

[0080] The results of the comparison test of the diameter of the inhibition zone at different culture times showed that ( Figure 4 ), there was no inhibition zone for flumequine susceptibility tablets at different incubation times. For florfenicol, enrofloxacin, doxycycline and ciprofloxacin, after 12-24h incubation under standard conditions, the results of the diameter of the inhibition zone were P ≥ 0.05 or the diameter difference was < 2mm. As for neomycin susceptibility tablets, after 16-24h incubation under standard conditions, the results of the diameter difference of the inhibition zone were < 2mm, and when the incubation was 12h, the size of the inhibition zone was P < 0.05 and the diameter difference was ≥ 2mm compared with the diameter of the inhibition zone after 24h incubation.

[0081] 2.5 Effect of different bacterial concentrations on inhibition zone

[0082] The results of the inhibition zones of different bacterial concentrations under standard conditions showed that ( Figure 5 ), the inhibition zone results of florfenicol, enrofloxacin, doxycycline and ciprofloxacin drug sensitive tablets at each concentration were all P < 0.05 and the diameter difference was ≥ 2 mm. For neomycin drug sensitive tablets, at a bacterial concentration of 2.8×10 7 CFU / mL, 2.8×10 8 CFU / mL and 2.8×10 6 The difference in average diameter on the CFU / mL plate was less than 2 mm, but the inhibition zone results were all P < 0.05; 8 CFU / mL, 2.8×10 5CFU / mL and 2.8×10 4 The difference in CFU / mL plate diameter was ≥2mm, and the inhibition zone diameter results were all P<0.05. Flumequine tablets had no inhibition zone at different bacterial concentrations.

[0083] 2.6 Effect of mixing tissue homogenate on the inhibition zone

[0084] In order to further explore the effect of mixing with tissue homogenate on the diameter of the inhibition zone, tissue homogenate was mixed into bacterial solutions of different concentrations to compare the differences in the inhibition zones. Figure 6 ), the results of pure bacterial solution and tissue mixing of florfenicol, enrofloxacin, doxycycline and ciprofloxacin at each concentration were P<0.05, and the average diameter of the inhibition zone of pure bacterial solution was 2.3-3.0 mm larger than that of tissue homogenate. In addition, no inhibition zone was formed when the pure bacterial solution of each concentration and tissue homogenate of flumequine and neomycin were cultured on CBA plates.

[0085] 2.7 Clinical case sample testing

[0086] In order to more fully understand the differences between samples collected from the production line and standard operations, clinically infected yellow catfish tissues were used to compare the inhibition zones of various concentrations. Among them, three artificially injected infected samples were marked as artificial 1 (mixed sample bacterial load of about 1.3×10 9 CFU / mL), artificial 2 (mixed sample bacterial load of about 2.2×10 9 CFU / mL) and artificial 3 (mixed sample bacterial load of about 2.8×10 9 CFU / mL); two naturally infected samples, natural 1 (mixed sample bacterial load of about 2.5×10 8 CFU / mL) and natural 2 (mixed sample bacterial load of about 6.2×10 8 CFU / mL). The results of inhibition zone showed ( Figure 7 ), the results of enrofloxacin, ciprofloxacin and neomycin samples in the three artificially infected samples were significantly different from those of the standard treatment samples (P<0.05 and the diameter difference was ≥2mm), the results of florfenicol in artificial 2 and artificial 3 samples and doxycycline in artificial 2 samples were significantly different from the standard operation (P<0.05 and the diameter difference was ≥2mm), and the differences between the other samples and the standard operation were not significant (P≥0.05 or the diameter difference was <2mm). In addition, the flumequine drug sensitive tablets on the two blood agar plates and the neomycin drug sensitive tablets on the CBA agar plates did not form an inhibition zone.

[0087] 2.8 Rapid drug susceptibility test method for Streptococcus iniae under simplified conditions

[0088] Based on the previous experimental data, the standard conditions are simplified to be closer to the actual operation in the production environment. The optimized rapid drug sensitivity test method is described as follows:

[0089] Take 0.1±0.02g of liver, spleen and kidney tissues of dying or freshly dead yellow catfish, add about ten times the weight of sterile PBS buffer, and add 2 sterile steel balls with a diameter of about 3mm. Grind the tissue for 1min at 50Hz, and centrifuge the large tissue fragments to the bottom of the tube by instantaneous centrifugation. Next, use a sterile cotton swab to dip into the upper tissue homogenate and evenly spread it on the CBA plate. Stick the corresponding drug-susceptible patch as required. Finally, incubate at 30℃ for 16-24h, and then measure the diameter of the inhibition zone ( Figure 8 ). The method of the present invention is not only more suitable for production environment, but also can effectively improve the detection efficiency.

[0090] 2. Embodiment

[0091] 1. Tissue Sample Collection

[0092] 1.1 Choose yellow catfish that are dying or freshly dead. The dead fish samples should not be collected for more than 24 hours, and should be stored in a dry and refrigerated environment to maintain the vitality of bacteria and the freshness of tissues to the greatest extent.

[0093] 1.2 Dissection should be performed under sterile conditions. It should be noted that the environment and operation conditions will have a certain impact on the results. Fish should be dissected in a sterile environment, using sterile instruments and sterile operations.

[0094] 1.3 Weigh 0.1g (±0.02g) of the liver, spleen and kidney tissues of yellow catfish and put them into a 2mL sterile grinding tube. To facilitate operation and comparison of results, the sampling tissues and sampling volumes are unified. For bacteria that can infect the brain, such as Streptococcus iniae, it is also possible to consider collecting part of the brain tissue. There is no clear requirement for the specific sampling volume of different tissues. Based on experience, more parts can be taken from severely infected tissues as appropriate. In the absence of a more accurate weighing scale, the sampling volume can be increased or decreased as appropriate or determined based on experience.

[0095] 2. Tissue Sample Processing

[0096] 2.1 Add 1 mL of sterile PBS to the weighed tissue and add two 3 mm sterile steel beads. The amount of sterile PBS added is about 10 times the weight volume of the tissue, which is equivalent to diluting the bacterial load of the tissue ten times.

[0097] 2.2 Grind the tissue at 50Hz for 1min, and then centrifuge it instantly in a centrifuge. Higher frequencies may cause damage and death to the bacteria in the tissue. Instant centrifugation can throw the insufficiently ground tissue to the bottom of the grinding tube without affecting the coating in the next step. Manual grinding is also possible without a grinding machine, but attention should be paid to aseptic conditions and aseptic operation. Manual centrifugation or static for a period of time is also possible without a centrifuge, so that the tissue is not repeatedly ground and the bacteria are not settled.

[0098] 3. Spreading culture

[0099] 3.1 Use a sterile cotton swab to take the tissue homogenate from the upper layer of the grinding tube and spread it evenly on the CBA plate. The prepared inoculation solution must be used within 15 minutes.

[0100] 3.2 Place the drug-sensitive paper strips. The distance between each paper strip should be no less than 24 mm, and the center of the paper strip should be no less than 15 mm from the edge of the plate. It is best to paste 6 pieces on a plate with a diameter of 90 mm. After pasting the paper strips, turn the plate upside down within 15 minutes and place it in an incubator at 30°C for cultivation.

[0101] 3.3 Incubate for 16 to 24 hours. It is best to place the CBA plates individually in the incubator, with no more than two plates stacked together. Otherwise, the plate in the middle will not reach the incubator temperature and will cause pre-diffusion. After the CBA plates have been incubated for 16 to 24 hours, read the results.

[0102] 4. Read the results

[0103] After incubation, take out the CBA plate and measure the diameter of the inhibition zone. Use a vernier caliper to measure the diameter of the inhibition zone. If there is no vernier caliper, you can also use a ruler. Measure three times and take the average value. The edge of the inhibition zone is limited to the point where no obvious bacterial growth can be seen with the naked eye. Some strains may spread and grow into the inhibition zone, and sulfonated drugs may grow slightly in the inhibition zone. These are not considered the edge of the inhibition zone.

[0104] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. A rapid drug sensitivity detection method based on fish tissue homogenate, characterized in that: Take fish tissue and homogenize it. Spread the homogenate on a plate, place a drug-susceptibility paper, and culture it at 30℃ for 16 to 24 hours. Then measure the diameter of the inhibition zone on the plate to determine the drug sensitivity test results.

2. The rapid drug sensitivity detection method based on fish tissue homogenate according to claim 1 is characterized in that: The fish tissue is one or a mixture of two or more of the liver, spleen, kidney and brain, and the sampling amount is 0.1g±0.02g.

3. The rapid drug sensitivity detection method based on fish tissue homogenate according to claim 1 is characterized in that: The fish tissue is selected from dying or freshly dead fish. The dead fish samples should not be collected more than 24 hours and should be stored in a dry and cold condition.

4. The rapid drug sensitivity detection method based on fish tissue homogenate according to claim 1 is characterized in that: The plate used was Columbia blood agar plate.

5. The rapid drug sensitivity detection method based on fish tissue homogenate according to claim 1 is characterized in that: The bacterial concentration of the homogenate is not less than 2.8×10 4 CFU / ml.

6. The rapid drug sensitivity detection method based on fish tissue homogenate according to claim 1 is characterized in that: The homogenate can be made by grinding the tissue at 50 Hz for 1 min in a grinder and centrifuging it instantly in a centrifuge.

7. The rapid drug sensitivity detection method based on fish tissue homogenate according to claim 1 is characterized in that: The homogenate was used within 15 min.

8. The rapid drug sensitivity detection method based on fish tissue homogenate according to claim 1 is characterized in that: When placing the drug-sensitive paper sheets, the distance between each drug-sensitive paper sheet is not less than 24 mm, and the center of the paper sheet is not less than 15 mm from the edge of the plate.

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