Chromogenic culture medium and test plate for detecting Gram-negative bacteria in bovine mastitis

Abstract

The invention discloses a chromogenic culture medium and a test plate thereof for detecting Gram-negative bacteria in bovine mastitis, wherein the culture medium comprises a nutrient mixture, a composite inhibitor, a composite chromogenic enzyme substrate and a cold water gel, wherein the composite inhibitor is bile salt No. 3 and erythromycin, and the composite chromogenic enzyme substrate is red tetrazolium, 5-bromo-6-chloro-3-indole-β-D-galactoside and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside. The chromogenic culture medium of the invention and the test plate made therefrom can effectively inhibit the growth of non-Gram-negative bacteria, have the characteristics of strong specificity, high sensitivity, short detection cycle, easy observation and low detection cost, and have huge development potential and broad application prospects in microbial detection.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and in particular relates to a color development culture medium and a test plate thereof for detecting Gram-negative bacteria in bovine mastitis. Background Art

[0002] Cow mastitis is one of the most serious infectious diseases causing economic losses in the dairy industry. It not only leads to a decrease in milk production and milk quality, but can also be transmitted to other cows, leading to the elimination and death of chronically infected cows, and increasing breeding costs. The pathogenic microorganisms that cause cow mastitis are relatively complex, including more than 100 species of bacteria, fungi, mycoplasmas, yeasts, viruses, etc. Among these pathogens, bacteria are the most common. They are widely distributed in the living environment of dairy cows, so they pose a great threat to dairy cows. Gram-negative bacteria are one of the important pathogens causing cow mastitis. The mastitis caused by them is a disease that is difficult to treat in the modern dairy industry. Among them, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa are typical environmental pathogens that cause cow mastitis. Mastitis caused by Escherichia coli can reduce milk production by 50%, and even some sick cows have not recovered their milk production after recovery, thereby reducing the income of farmers.

[0003] In addition, since different pathogens have different drug sensitivities and different treatment methods, if the prevalence and distribution of pathogens of bovine mastitis can be statistically analyzed, the source of pathogens can be analyzed and preventive measures can be taken in time, and scientific and reasonable treatment can be achieved by referring to the existing pathogen resistance map and treatment plan, the abuse of antibiotics can be avoided and production costs can be reduced. However, although traditional bacterial biochemical experiments and ordinary identification culture media can identify pathogens to the species level, the operation is complicated, time-consuming and labor-intensive. In view of this, the development of a test plate that can quickly detect bovine pathogens has great development potential and broad application prospects.

[0004] As microbial test strips are gradually included in national standards and recognized in all walks of life, and as the technology continues to mature, its ease of operation is greatly welcomed. In order to overcome the problems of low specificity and sensitivity of traditional detection methods for pathogenic bacteria of bovine mastitis, as well as incomplete and low accuracy of pathogen detection in the prior art, the present invention provides a chromogenic culture medium and a test plate for detecting Gram-negative bacteria in bovine mastitis, which can accurately detect Gram-negative bacteria species such as Escherichia coli, Klebsiella and Pseudomonas aeruginosa among the pathogenic bacteria of bovine mastitis. Summary of the invention

[0005] To this end, the present invention aims to provide a chromogenic culture medium and a test plate thereof which have good specificity, high sensitivity, high accuracy, are easy to observe and can further accurately diagnose Gram-negative bacteria in bovine mastitis.

[0006] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0007] One object of the present invention is to provide a chromogenic culture medium for detecting Gram-negative bacteria in bovine mastitis, which comprises a nutrient mixture, a composite inhibitor and a composite chromogenic enzyme substrate. The composite inhibitor is bile salt No. 3 and erythromycin; the composite chromogenic enzyme substrate is red tetrazolium, 5-bromo-6-chloro-3-indole-β-D-galactoside and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside.

[0008] Preferably, the concentration of the composite inhibitor is 0.1-1 g / L, wherein the mass ratio of No. 3 bile salt to erythromycin is (150-200):1.

[0009] Preferably, the concentration of the composite chromogenic enzyme substrate is 0.1-0.4 g / L, wherein the mass ratio of red tetrazolium (TTC), 5-bromo-6-chloro-3-indole-β-D-galactoside and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside is 1:(8.5-9.5):(8.5-9.5).

[0010] Preferably, the nutrient mixture contains 5-15 g of tryptone, 2-8 g of yeast extract powder, 3-7 g of sodium chloride, 2-8 g of skimmed milk powder, and 0.08-0.12 g of ammonium ferric citrate per 1000 mL.

[0011] Preferably, the nutrient mixture further comprises cold water gelatinizable substances, wherein the cold water gelatinizable substances are sodium carboxymethyl cellulose, guar gum and xanthan gum.

[0012] Preferably, the concentration of the cold water gel is 25-30 g / L, wherein the mass ratio of sodium carboxymethyl cellulose, guar gum and xanthan gum is (1.5-2.5): (2.5-3.5):1.

[0013] Another object of the present invention is to provide a detection plate, which comprises a substrate, and any of the above-mentioned chromogenic culture media for detecting Gram-negative bacteria in bovine mastitis, which is arranged on the surface of the substrate.

[0014] Preferably, the test plate has a structure consisting of a transparent covering film, a circular groove culture area and a PET (polyethylene terephthalate) transparent bottom plate from top to bottom.

[0015] Preferably, the transparent coating is made of PE (polyethylene) or PET.

[0016] The embodiments of the present invention have the following advantages:

[0017] 1. The Gram-negative bacteria test plate provided by the present invention is a prefabricated culture system, which does not require high-pressure sterilization of agar culture medium in advance, takes into account the appropriate pouring temperature, and is easy to carry and operate.

[0018] 2. The composite inhibitor in the Gram-negative bacteria test plate provided by the present invention can effectively inhibit the growth of non-target bacteria, and the composite chromogenic enzyme substrate is more selective for target bacteria, and can effectively distinguish Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Therefore, the present invention has the characteristics of high sensitivity and strong specificity, greatly improves the detection accuracy, and reduces the false positive rate and false negative rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0020] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0021] Figure 1 This is a schematic diagram of the structure of a Gram-negative bacteria test plate according to an embodiment of the present invention; wherein 1-transparent covering film, 2-circular culture area, 3-bottom plate;

[0022] Figure 2 A linear relationship diagram between the results of the Gram-negative bacteria test plate of the embodiment of the present invention for detecting Escherichia coli and the results of the national standard VRBA plate detection;

[0023] Figure 3 A linear relationship diagram between the results of the Gram-negative bacteria test plate for detecting Klebsiella pneumoniae according to an embodiment of the present invention and the results of the standard MIAC plate test;

[0024] Figure 4 It is a linear relationship diagram between the detection results of Pseudomonas aeruginosa by the Gram-negative bacteria test plate of the embodiment of the present invention and the detection results of the national standard CN plate. DETAILED DESCRIPTION

[0025] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1: Chromogenic medium for detecting Gram-negative bacteria in bovine mastitis

[0027] Each 1000mL culture medium contains 5g of tryptone, 2g of yeast extract powder, 3g of sodium chloride, 2g of milk powder, 0.08g of ammonium ferric citrate, 0.1g of composite inhibitor, 0.1g of composite chromogenic enzyme substrate, and 25g of cold water gel. Among them, the composite inhibitor is composed of bile salt and erythromycin in a mass ratio of 150:1, and the composite chromogenic enzyme substrate is composed of red tetrazolium, 5-bromo-6-chloro-3-indole-β-D-galactoside and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in a mass ratio of 1:8.5:8.5. After the above basic components are fully dissolved, add cold water gel, which is composed of sodium carboxymethyl cellulose, guar gum and xanthan gum in a mass ratio of 1.5:2.5:1. The above components are dissolved in 1000mL of water, stirred evenly, divided into packages, and dried before use.

[0028] Example 2: Chromogenic medium for detecting Gram-negative bacteria in bovine mastitis

[0029] Each 1000mL culture medium contains 10g of tryptone, 5g of yeast extract powder, 4g of sodium chloride, 4.5g of milk powder, 0.09g of ammonium ferric citrate, 0.4g of composite inhibitor, 0.215g of composite chromogenic enzyme substrate, and 27g of cold water gel. Among them, the composite inhibitor is composed of bile salt and erythromycin in a mass ratio of 180:1, and the composite chromogenic enzyme substrate is composed of red tetrazolium, 5-bromo-6-chloro-3-indole-β-D-galactoside and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in a mass ratio of 1:9:9. After the above basic components are fully dissolved, add cold water gel, which is composed of sodium carboxymethyl cellulose, guar gum and xanthan gum in a mass ratio of 2:3:1. The above components are dissolved in 1000mL of water, stirred evenly, divided into packages, and dried before use.

[0030] Example 3: Chromogenic medium for detecting Gram-negative bacteria in bovine mastitis

[0031] Each 1000mL culture medium contains 15g of tryptone, 8g of yeast extract powder, 7g of sodium chloride, 8g of milk powder, 0.12g of ammonium ferric citrate, 1g of composite inhibitor, 0.4g of composite chromogenic enzyme substrate, and 30g of cold water gel. Among them, the composite inhibitor is composed of bile salt and erythromycin in a mass ratio of 200:1, and the composite chromogenic enzyme substrate is composed of red tetrazolium, 5-bromo-6-chloro-3-indole-β-D-galactoside and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in a mass ratio of 1:9.5:9.5. After the above basic components are fully dissolved, add cold water gel, which is composed of sodium carboxymethyl cellulose, guar gum and xanthan gum in a mass ratio of 2.5:3.5:1. The above components are dissolved in 1000mL of water, stirred evenly, divided into packages, and dried before use.

[0032] Example 4: Test plate for detecting Gram-negative bacteria in bovine mastitis

[0033] The Gram-negative bacteria test plate of this embodiment is as follows Figure 1 As shown, from top to bottom are a transparent covering film 1, a circular culture area 2, and a bottom plate 3. The transparent covering film 1 can cover the bottom plate 3. Among them, the transparent covering film 1 is made of PET material, the circular culture area 2 is evenly adsorbed with Gram-negative bacteria colorimetric culture medium, and the bottom plate is made of transparent PET (polyethylene terephthalate) material.

[0034] The chromogenic culture medium for detecting Gram-negative bacteria contains 12g of tryptone, 5g of yeast extract powder, 5g of sodium chloride, 4.5g of milk powder, 0.09g of ammonium ferric citrate, 0.405g of composite inhibitor, 0.235g of composite chromogenic enzyme substrate, and 27g of cold water gel per 1000mL. The composite inhibitor is composed of No. 3 bile salt and erythromycin in a mass ratio of 200:1, and the composite chromogenic enzyme substrate is composed of red tetrazolium, 5-bromo-6-chloro-3-indole-β-D-galactoside and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in a mass ratio of 1:9:9. After the above basic components are fully dissolved, cold water gel is added, and the cold water gel is composed of sodium carboxymethyl cellulose, guar gum and xanthan gum in a mass ratio of 2.5:3:1.

[0035] The above ingredients are dissolved in 1000 mL of water, stirred evenly, dispensed into the circular culture area 2 of the test plate, dried, and affixed with a transparent film 1 to prepare a test plate for detecting Gram-negative bacteria.

[0036] The Gram-negative bacteria test plate prepared in this embodiment was verified by inoculating positive quality control strains and negative control bacteria. The results showed that Escherichia coli was purple-red, Klebsiella pneumoniae was blue-green, Pseudomonas aeruginosa was pink, and negative control bacteria (such as Enterococcus faecalis) showed no growth or no growth at all.

[0037] Example 5. Test plate for detecting Gram-negative bacteria in bovine mastitis

[0038] The test plate for detecting Gram-negative bacteria in this embodiment is different from the test plate in Example 4 in that the material of the transparent coating 1 is PE, and the Gram-negative bacteria culture medium in the circular culture area 2 contains 10g of tryptone, 4g of yeast extract powder, 3g of sodium chloride, 2g of milk powder, 0.01g of ammonium ferric citrate, 0.4g of composite inhibitor, 0.23g of composite chromogenic enzyme substrate, and 25g of cold water gel per 1000mL. Among them, the composite inhibitor is composed of No. 3 bile salt and erythromycin in a mass ratio of 180:1, and the composite chromogenic enzyme substrate is composed of red tetrazolium, 5-bromo-6-chloro-3-indole-β-D-galactoside and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in a mass ratio of 1:8.5:8.5. After the above basic components are fully dissolved, cold water gel is added, and the cold water gel is composed of sodium carboxymethyl cellulose, guar gum and xanthan gum in a mass ratio of 1.5:2.5:1.

[0039] The above ingredients are dissolved in 1000 mL of water, stirred evenly, dispensed into the circular culture area 2 of the test plate, dried, and affixed with a transparent film 1 to prepare a test plate for detecting Gram-negative bacteria.

[0040] The Gram-negative bacteria test plate prepared in this embodiment was verified by inoculating positive quality control strains and negative control bacteria. The results showed that Escherichia coli was purple-red, Klebsiella pneumoniae was blue-green, Pseudomonas aeruginosa was pink, and the negative control bacteria showed no color growth or no growth at all, which can avoid the appearance of false positive sample test results to a certain extent. Each positive quality control bacteria was appropriately diluted and inoculated on the Gram-negative bacteria test plate prepared in this embodiment and the TSA agar plate, respectively. The test results are shown in Table 1. The results show that the growth rate of each positive quality control bacteria is above 0.7 (PR ≥ 0.5 is required), and the negative control bacteria Staphylococcus aureus does not grow.

[0041] Table 1

[0042]

[0043] Note: "-" represents no colony growth, "+++" represents uncountable colonies, and " / " represents no such data; "KGR test plate" refers to the Gram-negative bacteria test plate provided in this example.

[0044] Example 6. Test plate for detecting Gram-negative bacteria in bovine mastitis

[0045] The Gram-negative bacteria test plate of this embodiment is different from the test plate of Example 4 in that the Gram-negative bacteria culture medium in the circular culture area 2 contains 10g of tryptone, 2g of yeast extract powder, 5g of sodium chloride, 4g of milk powder, 0.01g of ammonium ferric citrate, 0.405g of composite inhibitor, 0.235g of composite chromogenic enzyme substrate, and 27g of cold water gel per 1000mL. Among them, the composite inhibitor is composed of bile salt and erythromycin in a mass ratio of 150:1, and the composite chromogenic enzyme substrate is composed of red tetrazolium, 5-bromo-6-chloro-3-indole-β-D-galactoside and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in a mass ratio of 1:8.5:9.5. After the above basic components are fully dissolved, cold water gel is added, and the cold water gel is composed of sodium carboxymethyl cellulose, guar gum and xanthan gum in a mass ratio of 1.5:3.5:1.

[0046] The above ingredients are dissolved in 1000 mL of water, stirred evenly, dispensed into the circular culture area 2 of the test plate, dried, and affixed with a transparent film 1 to prepare a test plate for detecting Gram-negative bacteria.

[0047] The Gram-negative bacteria test plate prepared in this embodiment was verified by inoculating positive quality control strains and negative control bacteria. The results showed that Escherichia coli was purple-red, Klebsiella pneumoniae was blue-green, Pseudomonas aeruginosa was pink, and the negative control bacteria showed no color growth or no growth at all, which can avoid the occurrence of false positive sample test results to a certain extent. Each positive quality control bacteria was appropriately diluted and inoculated on the Gram-negative bacteria test plate prepared in this embodiment and the TSA agar plate, respectively. The test results are shown in Table 2. The results show that the growth rate of each positive quality control bacteria is above 0.6 (PR ≥ 0.5 is required), and the negative control bacteria Enterococcus faecalis does not grow.

[0048] Table 2

[0049]

[0050] Note: "-" represents no colony growth, "+++" represents uncountable colonies, " / " represents no such data, and "KGR test plate" refers to the Gram-negative bacteria test plate provided by the present invention.

[0051] Example 7. Test plate for detecting Gram-negative bacteria in bovine mastitis

[0052] The Gram-negative bacteria test plate of this embodiment is different from the test plate of Example 4 in that the Gram-negative bacteria culture medium in the circular culture area 2 contains 10g of tryptone, 8g of yeast extract powder, 8g of sodium chloride, 7g of milk powder, 0.01g of ammonium ferric citrate, 0.4g of composite inhibitor, 0.25g of composite chromogenic enzyme substrate, and 30g of cold water gel per 1000mL. Among them, the composite inhibitor is composed of bile salt and erythromycin with a final concentration ratio of 200:1, and the composite chromogenic enzyme substrate is composed of red tetrazolium, 5-bromo-6-chloro-3-indole-β-D-galactoside and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside with a mass ratio of 1:9.5:8.5. After the above basic components are fully dissolved, cold water gel is added, and the cold water gel is composed of sodium carboxymethyl cellulose, guar gum and xanthan gum with a final concentration ratio of 2.5:2.5:1.

[0053] The above ingredients are dissolved in 1000 mL of water, stirred evenly, dispensed into the circular culture area 2 of the test plate, dried, and affixed with a transparent film 1 to prepare a test plate for detecting Gram-negative bacteria.

[0054] The Gram-negative bacteria test plate prepared in this embodiment was verified by inoculating positive quality control strains and negative control bacteria. The results showed that Escherichia coli was purple-red, Klebsiella pneumoniae was blue-green, Pseudomonas aeruginosa was pink, and the negative control bacteria showed no color growth or no growth at all, which can avoid the occurrence of false positive sample test results to a certain extent. Each positive quality control bacteria was appropriately diluted and inoculated on the Gram-negative bacteria test plate prepared in this embodiment, and was also inoculated on the TSA agar plate. The test results are shown in Table 3. The results show that the growth rate of each positive quality control bacteria is above 0.5 (PR ≥ 0.5 is required), and the negative control bacteria Enterococcus faecalis does not grow.

[0055] Table 3

[0056]

[0057] Note: "-" represents no colony growth, "+++" represents uncountable colonies, " / " represents no such data, and "KGR test plate" refers to the Gram-negative bacteria test plate provided by the present invention.

[0058] Test Example 1: Gram-negative bacteria test plate specificity test

[0059] The specific performance evaluation was performed using the Gram-negative bacteria detection plate prepared in Example 4 of the present invention: 4 strains of Escherichia coli with strain numbers ATCC 25922, ATCC 700728, CICC 24187, and CMCC 44102, 5 strains of Klebsiella pneumoniae with strain numbers SY2P and SY1P, 2 strains of Pseudomonas aeruginosa with strain numbers ATCC27853 and ATCC 9027, 1 strain of Staphylococcus epidermidis (CMCC26069), 1 strain of Staphylococcus lentus (CICC 21602), 1 strain of Staphylococcus aureus (ATCC 6538P), 1 strain of Staphylococcus xylosus (ATCC 29971), 1 strain of Staphylococcus saprophyticus (ATCC 49453), 3 strains of Listeria monocytogenes (ATCC 33090, ATCC19111, ATCC 35897), 2 strains of enterococci (ATCC 29212, ATCC 19434), and 1 strain of Bacillus (CMCC 63303) were activated, and their suspensions were appropriately diluted to prepare a suspension with a concentration of 100-300 CFU / mL, which were inoculated on the Gram-negative bacteria test plate prepared in Example 4 and the TSA plate (positive control), respectively, and cultured at 37°C for 18-24h. According to the growth of the bacteria, the plate culture time can be extended to 48h. The test results are shown in Table 4.

[0060] Table 4 Specificity experiments of 22 strains

[0061]

[0062] Note: "+" represents growth, "-" represents no growth. "KGR test plate" refers to the Gram-negative bacteria test plate of Example 4.

[0063] As can be seen from Table 4, the positive quality control bacteria appear purple, blue-green or pink on the Gram-negative bacteria test plate provided by the present invention, and most Gram-positive bacteria do not grow or do not grow at all. It can be seen that the Gram-negative bacteria test plate provided by the present invention has a strong detection specificity and can largely avoid the occurrence of false positive and false negative results.

[0064] Furthermore, after diluting some positive control bacteria and negative control bacteria to an appropriate gradient, they were inoculated on the Gram-negative bacteria test plate of this embodiment and on the IV zone of the AccuMast mastitis pathogen rapid diagnosis quadrilateral dish (hereinafter referred to as the quadrilateral dish). Gram-negative bacteria appeared purple-red, blue-green, white or transparent on the IV zone of the quadrilateral dish. The colony growth and color development of the two were compared and analyzed, and the results are shown in Table 5.

[0065] Table 5 Summary of growth of typical quality control strains

[0066]

[0067] As can be seen from Table 5, Enterococcus faecalis, Enterococcus faecium, Streptococcus thermophilus, and Stenotrophomonas xylosus will all grow in zone IV of the quadrant, and the color development is consistent with that of Gram-negative bacteria, which can easily cause false positive test results. However, they do not grow on the Gram-negative bacteria test plate provided in this embodiment. Therefore, the product of the present invention has stronger specificity and can avoid the occurrence of false positive test results to a large extent.

[0068] Test Example 2: Recovery Rate Experiment

[0069] The sensitivity of the Gram-negative bacteria detection plate prepared in Example 4 of the present invention was evaluated: four strains of Escherichia coli with strain numbers ATCC 25922, ATCC 700728, CICC 24187, and CMCC 44102, and four strains of Escherichia coli with strain numbers ATCC 10031, ATCC 25955, ATCC 13883, 3 strains of Klebsiella pneumoniae, 2 strains of Pseudomonas aeruginosa, 2 strains of ATCC27853 and ATCC9027, were revived and activated to the third generation using the method recommended by the culture collection center, and the bacterial suspension was appropriately diluted to prepare a suspension with a concentration of 20-200 CFU / mL, which was inoculated on the Gram-negative bacteria test plate, TSA agar plate and the identification area IV of the four-piece dish prepared in Example 4, respectively. Three parallel tests were performed for each strain, and the plates were cultured at a constant temperature of 37°C for 18-24h. Depending on the growth of the strains, the plate can be extended to 48h. The test results are shown in Table 6.

[0070] Table 6 Comparison of quality control strain test results

[0071]

[0072] Note: "KGR test plate" refers to the Gram-negative bacteria test plate of Example 4. The denominator of the PR value is the number of colonies grown on the TSA plate, and the numerator is the number of colonies on the test plate or quadruplicate dish.

[0073] It can be seen from Table 6 that, compared with the TSA plate test results, the 8 positive quality control bacteria standard strains are purple, blue-green or pink on the Gram-negative bacteria test plate provided by the present invention, and the strain recovery rates (PR) are all above 0.5, and are all higher than the recovery rate of zone IV of the four-divided dish to varying degrees. This shows that the Gram-negative bacteria test plate provided by the present invention has higher detection sensitivity and can avoid the occurrence of missed detection to a certain extent.

[0074] Experimental Example 3: Detection of Gram-negative bacteria in natural samples

[0075] Milk from cows with mastitis in different areas of a pasture was collected, and each cow was numbered. The milk samples were appropriately diluted and inoculated on the Gram-negative bacteria test plate provided in Example 4, the IV identification area of ​​the quadrant dish, and the IV identification area of ​​the quadrant dish commonly used in the veterinary station (hereinafter referred to as the veterinary dish), and 3 dilution gradients were selected, and 3 parallel tests were performed for each gradient. The sampling and processing methods of the samples were referred to GB 4789.1-2016 "General Rules for Food Microbiology Examination of National Food Safety Standards", and the test results are shown in Tables 7 and 8.

[0076] Table 7 Natural sample test results

[0077]

[0078] Table 8 Comparison of natural sample test results (partial data)

[0079]

[0080] Table 8 (continued)

[0081]

[0082] Note: “ / ” means the experiment was not conducted, “√” means it meets the requirements, “×” means it does not meet the requirements, and “None” means no colony growth.

[0083] As can be seen from Table 7, a total of 300 samples were compared with the four-plate dish, 120 samples were compared with the four-plate dish, and the compliance rate was 90.00%, and a total of 180 samples were compared with the veterinary dish, and the compliance rate was 76.11%; as can be seen from Table 5, the specific comparison data shows that the test results of the Gram-negative bacteria test plate provided by the present invention are mostly consistent with the four-plate dish and the veterinary dish, and some individual samples are only consistent with one of them, and the other has no colony growth. This phenomenon also shows that the detection sensitivity of the present invention is higher (for example, samples of cows numbered 201981, 195115 and 207950); for those with inconsistent detected strains, molecular biological identification shows that they are indeed the strains detected by the present invention (for example, samples of cows numbered 175479, 171894, 158063, and 182392), indicating that the Gram-negative bacteria test plate provided by the present invention has higher detection specificity and accuracy.

[0084] Test Example 4: Detection of Escherichia coli in artificially contaminated samples

[0085] The Escherichia coli numbered ATCC 25922 was activated according to the method recommended by the Culture Collection Administration Center. The fresh activated bacterial suspension was counted according to the method in GB 4789.2. The live bacterial content of the bacterial suspension was about 3.15×10 9CFU / mL, take 1mL of freshly cultured Escherichia coli (ATCC 25922) suspension and add 9mL of sterile saline to make 10-fold gradient dilutions to 10 -2 , 10 -4 , 10 -6 Then, 1 mL of each of the three gradient bacterial solutions was added to 9 mL of sterile milk to make a volume of 10 mL with a bacterial content of approximately 3.15 × 10 6 CFU / mL, 3.15×10 4 CFU / mL, 3.15×10 2 The artificially contaminated milk samples with different CFU / mL were named as high, medium and low concentration artificially contaminated samples respectively; according to the above method, three parallel contaminated samples were made for the three gradient spiked samples, and they were numbered 1, 2, 3, 4, 5, 6, 7, 8 and 9 from high to low.

[0086] Estimate the bacterial content in each spiked sample, perform gradient dilutions on high, medium, and low artificial contamination samples, and select 1 / 2×10 -4 (and 1 / 4×10 -4 )、1 / 2×10 -2 (and 1 / 4×10 -2 ), 1 mL of each of the 1 / 2 (and 1 / 4) gradient dilutions were taken and inoculated on the Gram-negative bacteria test plate and VRBA agar plate of Example 4 (based on: "GB 4789.3-2016 National Food Safety Standard Food Microbiology Test Coliform Group Count"), and cultured at 36°C±1°C for 16-24 hours. The VRBA agar plate culture time can be extended to 48 hours as needed. The test results are shown in Table 9. The linear relationship between the test results of the Gram-negative bacteria test plate provided by the present invention and the national standard VRBA plate results is shown in Table 9. Figure 2 .

[0087] Table 9 Detection results of Escherichia coli in sterile milk artificial contamination samples

[0088]

[0089] Notes: 1. All test samples were fresh sterile milk with standard bacterial strain suspension. 2. "KGR test plate" refers to the Gram-negative bacteria test plate in Example 4, "lg (VRBA)" refers to the logarithmic value of the colony count on the national standard VRBA plate, and "lg (KGR)" refers to the logarithmic value of the colony count on the KGR Gram-negative bacteria test plate.

[0090] As shown in Table 9, there was no significant difference in the results of the two methods for detecting the live Escherichia coli content in artificially contaminated samples No. 1-9 (i.e., P>0.05). Figure 2It can be seen that the linear regression equation between the two is y = 0.9982x -0.0021, where R 2 =1, that is, the degree of fit between the two is good. In summary, the Gram-negative bacteria test plate provided by the present invention is used to detect the content of Escherichia coli in bovine milk, which is highly consistent with the national standard method for testing the content of Escherichia coli in bovine milk, and can be equally applied to the preliminary detection of the content of Escherichia coli in bovine mastitis.

[0091] Experimental Example 5: Detection of Klebsiella pneumoniae in artificially contaminated samples

[0092] Klebsiella pneumoniae subspecies pneumoniae numbered ATCC 25955 (hereinafter referred to as the “test strain”) was activated according to the method recommended by the Center for Culture Collection Administration. The fresh activated bacterial suspension was counted according to GB 4789.2. The live bacterial content of the bacterial suspension was about 2.92×10 9 CFU / mL, take 1 mL of freshly cultured test strain suspension and dilute it 10 times in 9 mL of sterile saline to 10 -2 , 10 -4 , 10 -6 Then, 1 mL of each of the three gradient bacterial solutions was added to 9 mL of sterile milk to make a volume of 10 mL with a bacterial content of approximately 2.92 × 10 6 CFU / mL, 2.92×10 4 CFU / mL, 2.92×10 2 The artificially contaminated milk samples with different CFU / mL were named as high, medium and low concentration artificially contaminated samples respectively; according to the above method, three parallel contaminated samples were made for the three gradient spiked samples, and they were numbered 1, 2, 3, 4, 5, 6, 7, 8 and 9 from high to low.

[0093] According to the estimation of bacterial content of each spiked sample, the samples with high, medium and low artificial contamination were diluted in series, and 1 / 2×10 -4 (and 1 / 4×10 -4 )、1 / 2×10 -2 (and 1 / 4×10 -2 ), 1 mL of 1 / 2 (and 1 / 4) gradient dilutions were inoculated on the Gram-negative bacteria test plate of Example 4 and the MacConkey inositol adonol carbenicillin agar (MIAC) plate (based on: "SN-T 2552.9-2010 Microbiological Test Methods for Milk and Dairy Products Part 9: Klebsiella Test"), and cultured at 36°C ± 1°C for 16-24h. The test results are shown in Table 10. The linear relationship between the test results of the Gram-negative bacteria test plate provided by the present invention and the results of the MIAC plate is shown in Table 10. Figure 3 .

[0094] Table 10 Detection results of Klebsiella pneumoniae in artificially contaminated samples of sterile milk

[0095]

[0096] Notes: 1. All test samples are fresh sterile milk with standard bacterial strain suspension. 2. "KGR test plate" refers to the Gram-negative bacteria test plate in Example 4, "lg (MIAC)" refers to the logarithm of the colony value of the standard MIAC plate, and "lg (KGR)" refers to the logarithm of the colony value on the KGR Gram-negative bacteria test plate.

[0097] As shown in Table 10, there was no significant difference in the results of the two methods for detecting the live bacteria content of Klebsiella pneumoniae in artificially contaminated samples No. 1-9 (i.e., P>0.05), and further Figure 3 It can be seen that the linear regression equation between the two is y = 0.9973x + 0.0088, where R 2 =0.9999, that is, the degree of fit between the two is good. In summary, the Gram-negative bacteria test plate provided by the present invention is used to detect the content of Klebsiella pneumoniae in bovine milk, which is highly consistent with the standard method for detecting the content of Klebsiella pneumoniae in bovine milk, and can be equally applied to the preliminary detection of the content of Klebsiella pneumoniae in bovine mastitis.

[0098] Experimental Example 6: Detection of Pseudomonas aeruginosa in artificially contaminated samples

[0099] Pseudomonas aeruginosa numbered ATCC 27853 (hereinafter referred to as the “test strain”) was activated according to the method recommended by the Culture Collection Administration Center. The fresh activated bacterial suspension was counted according to GB 4789.2. The live bacterial content of the bacterial suspension was about 6.75×10 8 CFU / mL, take 1 mL of freshly cultured test strain suspension and dilute it in 9 mL of sterile saline in a 10-fold gradient to 10 -2 , 10 -4 , 10 -6 Then, 1 mL of each of the three gradient bacterial solutions was added to 9 mL of sterile milk to make a volume of 10 mL with a bacterial content of about 6.75 × 10 5 CFU / mL, 6.75×10 3 CFU / mL, 6.75×10 1 The artificially contaminated milk samples with different CFU / mL were named as high, medium and low concentration artificially contaminated samples respectively; according to the above method, three parallel contaminated samples were made for the three gradient spiked samples, and they were numbered 1, 2, 3, 4, 5, 6, 7, 8 and 9 from high to low.

[0100] According to the estimation of bacterial content of each spiked sample, the samples with high, medium and low artificial contamination were diluted in series, and 1 / 4×10 -3 (and 10 -4 )、1 / 4×10 -2 (and 10 -1 ), 1 mL of 1 / 4 (and stock solution) gradient dilution, inoculated on the Gram-negative bacteria test plate and CN agar plate of Example 4 (according to: "GB 8538-2022 National Food Safety Standard Drinking Natural Mineral Water Test Method 57 Pseudomonas aeruginosa"), and cultured at 36°C ± 1°C for 16-24h. The culture time of the CN agar plate can be extended to 48h as needed. The test results are shown in Table 11. The linear relationship between the test results of the Gram-negative bacteria test plate provided by the present invention and the CN plate results is shown in Figure 4 .

[0101] Table 11 Detection results of Pseudomonas aeruginosa in sterile milk artificial contamination samples

[0102]

[0103] Notes: 1. All test samples are fresh sterile milk with standard strain suspension. 2. "KGR test plate" refers to the Gram-negative bacteria test plate provided by the present invention, "lg (CN)" refers to the logarithmic value of the colony value of the national standard CN plate, and "lg (KGR)" refers to the logarithmic value of the colony value on the KGR Gram-negative bacteria test plate.

[0104] As shown in Table 11, there was no significant difference in the results of the two methods for detecting the live bacteria content of Pseudomonas aeruginosa in artificially contaminated samples 1-9 (i.e., P>0.05), and there was further evidence that Figure 4 It can be seen that the linear regression equation between the two is y = 0.9991x - 0.0169, where R 2 =0.9999, that is, the degree of fit between the two is good. In summary, the Gram-negative bacteria test plate provided by the present invention is used to detect the content of Pseudomonas aeruginosa in bovine milk, which is highly consistent with the national standard method for detecting the content of Pseudomonas aeruginosa in bovine milk, and can be equally applied to the preliminary detection of the content of Pseudomonas aeruginosa in bovine mastitis.

[0105] In summary, the Gram-negative bacteria test plate provided by the present invention has a high detection rate for bacteria in natural samples and artificially contaminated samples, and has the characteristics of high sensitivity. Compared with other identification dishes on the market, it has the advantages of strong specificity, easy observation, high accuracy of test results, and low cost. The Gram-negative bacteria test plate of the present invention overcomes the existing technical barriers, has obvious advantages, can greatly reduce the cost of pasture detection, and has extremely high application value in the diagnosis of bovine mastitis.

[0106] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A chromogenic medium for detecting Gram-negative bacteria in bovine mastitis, characterized in that: The invention comprises a nutrient mixture, a composite inhibitor and a composite color-developing enzyme substrate, wherein the composite inhibitor is bile salt No. 3 and erythromycin; and the composite color-developing enzyme substrate is red tetrazolium, 5-bromo-6-chloro-3-indole-β-D-galactoside and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside.

2. The chromogenic medium for detecting Gram-negative bacteria in bovine mastitis according to claim 1, characterized in that: The concentration of the composite inhibitor is 0.1-1 g / L, wherein the mass ratio of No. 3 bile salt to erythromycin is (150-200):

1.

3. The chromogenic medium for detecting Gram-negative bacteria in bovine mastitis according to claim 1, characterized in that: The concentration of the composite color-developing enzyme substrate is 0.1-0.4 g / L, wherein the mass ratio of red tetrazolium, 5-bromo-6-chloro-3-indole-β-D-galactoside and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside is 1:(8.5-9.5):(8.5-9.5).

4. A chromogenic medium for detecting Gram-negative bacteria in bovine mastitis as described in any one of claims 1 to 3, characterized in that: Each 1000 mL of the nutrient mixture contains 5-15 g of tryptone, 2-8 g of yeast extract powder, 3-7 g of sodium chloride, 2-8 g of skimmed milk powder, and 0.08-0.12 g of ammonium ferric citrate.

5. The chromogenic medium for detecting Gram-negative bacteria in bovine mastitis according to claim 4, characterized in that: The nutritional ingredient mixture also includes a cold water gelable, which is sodium carboxymethylcellulose, guar gum and xanthan gum.

6. The chromogenic medium for detecting Gram-negative bacteria in bovine mastitis according to claim 5, characterized in that: The concentration of the cold water gel is 25-30 g / L, wherein the mass ratio of sodium carboxymethyl cellulose, guar gum and xanthan gum is (1.5-2.5): (2.5-3.5):

1.

7. A test plate comprising a substrate, and the chromogenic culture medium for detecting Gram-negative bacteria in bovine mastitis according to any one of claims 1 to 6 adsorbed on the surface of the substrate.

8. The test board according to claim 7, characterized in that: The substrate comprises a transparent covering film, a circular groove culture area and a PET transparent bottom plate from top to bottom.

9. The test board according to claim 8, characterized in that The material of the transparent coating is PE or PET.

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