Culture medium for detecting streptococcus agalactiae in bovine mastitis and test piece thereof

Abstract

The invention discloses a culture medium for detecting streptococcus agalactiae in bovine mastitis and a test piece thereof, the culture medium comprises a nutritional ingredient mixture, a composite inhibitor, a composite chromogenic enzyme substrate and cold water gel, the composite inhibitor is aztreonam, nalidixic acid, potassium clavulanate and trimethoprim; the compound chromogenic enzyme substrate is 5-bromine-6-chloro-3-indole-phosphoric acid p-toluidine, 5-bromine-4-chloro-3-indole-beta-D-glucuronide, 5-bromine-6-chloro-3-indole-beta-D-glucopyranoside, and 5-bromine-6-chloro-3-indole-beta-D-galactoside, and the compound chromogenic enzyme substrate is a compound chromogenic enzyme. The composite inhibitor in the streptococcus agalactiae test piece can effectively inhibit growth of non-target bacteria, and the composite chromogenic enzyme substrate has higher selectivity on target bacteria, so that the test piece has the characteristics of high specificity, high sensitivity, short detection period, easiness in observation and low detection cost; the method has huge development potential and wide application prospect in pasture detection.

Description

Technical Field

[0001] The invention relates to the technical field of microbial detection, and in particular to a culture medium and a test piece thereof for detecting Streptococcus agalactiae in bovine mastitis. Background Art

[0002] Cow mastitis is one of the important diseases that seriously endangers the development of dairy farming. The disease has a high incidence rate and a wide range of prevalence. It seriously damages the lactation performance of dairy cows and even causes the loss of udder function of dairy cows. Streptococcus agalactiae is one of the most widespread mastitis pathogens in dairy farms. It is highly contagious and is a specialized mammary parasite. It mainly causes subclinical mastitis and mild to moderate clinical mastitis. The proportion of subclinical mastitis is relatively high, mainly manifested by normal raw milk quality and increased somatic cell count. The symptoms of cows with clinical mastitis are usually mild, only manifested as abnormal raw milk and swelling of the milk area.

[0003] Streptococcus agalactiae can be spread through milking machines, milkers' hands, towels, etc. Infected cows, including young cows, dry cows and lactating cows, can all serve as sources of infection. In view of this, rapid detection of pathogens of mastitis in dairy cows, timely intervention and treatment, and prevention of further deterioration of mastitis are of great significance to ensure the health of dairy cows and improve milk production and quality.

[0004] The commonly used methods for diagnosing latent bovine mastitis include the SMT method (indirect somatic cell counting method), the BTB indicator method, the SCC microscope counting method and the CMT kit detection method. However, these methods are subjective in judging the results, which can easily lead to misjudgment, and are unable to locate the specific pathogenic bacteria, causing certain difficulties for targeted treatment.

[0005] The diagnostic dishes currently used in the market for microbial diagnosis of bovine mastitis involve complex bacterial species, making it difficult to determine the specific pathogenic bacteria species, and easily resulting in false positive test results. In order to overcome the problems of low specificity and low sensitivity of traditional detection methods for bovine mastitis pathogens, incomplete pathogen detection in the prior art, and low accuracy, the present invention provides a culture medium for detecting Streptococcus agalactiae in bovine mastitis and a test piece thereof, which are used to accurately detect Streptococcus agalactiae in the pathogenic bacteria causing mastitis in dairy cows. Summary of the invention

[0006] To this end, the present invention provides a culture medium and a test piece thereof for detecting Streptococcus agalactiae in bovine mastitis.

[0007] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions: One object of the present invention is to provide a culture medium for detecting Streptococcus agalactiae in bovine mastitis, which comprises a nutrient mixture, a composite inhibitor, a composite chromogenic enzyme substrate and a cold water gel, wherein the composite inhibitor is aztreonam, nalidixic acid, potassium clavulanate and trimethoprim; the composite chromogenic enzyme substrate is 5-bromo-6-chloro-3-indole-phosphate p-toluidine, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-6-chloro-3-indole-β-D-galactoside.

[0008] Preferably, the added concentration of the composite inhibitor is 0.04-0.05 g / L, wherein aztreonam is 0.0001-0.0003 g / L, nalidixic acid is 0.005-0.009 g / L, clavulanate potassium is 0.02-0.04 g / L, and trimethoprim is 0.001-0.002 g / L; the mass ratio of aztreonam, nalidixic acid, clavulanate potassium and trimethoprim is 1: (30-40): (150-190): (5-8).

[0009] Preferably, the added concentration of the composite chromogenic enzyme substrate is 0.1-0.25 g / L, and the mass ratio of 5-bromo-6-chloro-3-indole-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucopyranoside, and 5-bromo-6-chloro-3-indole-β-D-galactoside is 1:(1-1.5):(1-2):(1-2).

[0010] Preferably, the cold water gelatinizable agent is added at a concentration of 26-32 g / L, wherein the mass ratio of guar gum to xanthan gum is (1-1.5).

[0011] Preferably, the nutrient mixture comprises 10-20 g / L of tryptone, 2-5 g / L of yeast extract powder, 3-7 g of beef extract powder, 2-5 g / L of disodium hydrogen phosphate, 0.1-1 g / L of sodium citrate, 0.2-0.6 g / L of magnesium sulfate and 0.005-0.015 g / L of ammonium ferric citrate.

[0012] Another object of the present invention is to provide a test piece, comprising a base plate, and any of the above-mentioned culture media for detecting Streptococcus agalactiae in bovine mastitis prepared on the surface of the base plate.

[0013] Preferably, a culture area is provided on the bottom plate, and counting grids are provided at the bottom of the culture area.

[0014] Preferably, the test piece further includes a transparent covering film; the material of the transparent covering film is PE or PET, and the size of the transparent covering film is 98 mm×80 mm.

[0015] Preferably, the culture area is circular, with a diameter of 50 mm, a distance of 15.5 mm from the left and right edges of the bottom plate, 19-20 mm from the lower edge of the bottom plate, and a height of 0.70-0.78 mm, and a square grid is provided in the culture area of ​​the bottom plate, and the size of the small squares in the square grid is 10 mm×10 mm.

[0016] Preferably, the bottom plate has a size of 93 mm×80 mm and is made of polyaniline (PH) synthetic paper or polyvinyl chloride resin (PVC) synthetic paper.

[0017] The embodiments of the present invention have the following advantages: The Streptococcus agalactiae test piece 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, is easy to carry and operate, and has low detection cost; The composite inhibitor in the Streptococcus agalactiae test piece 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. The test piece has the characteristics of high sensitivity and strong specificity, which greatly improves the detection accuracy and reduces the false positive rate and false negative rate. It has positive guiding significance for accurately diagnosing the pathogenic bacteria of bovine mastitis and symptomatic treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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.

[0019] 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.

[0020] Figure 1 A schematic diagram of the structure of the Streptococcus agalactiae test piece provided by the present invention; wherein 1-transparent covering film, 2-circular culture area, 3-bottom plate; Figure 2 This is a linear relationship diagram between the Streptococcus agalactiae test results of the Streptococcus agalactiae test piece according to an embodiment of the present invention and the Columbia CNA blood plate test results. DETAILED DESCRIPTION

[0021] 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.

[0022] Example 1: Culture medium for detecting Streptococcus agalactiae in bovine mastitis A culture medium for detecting Streptococcus agalactiae in bovine mastitis in this embodiment comprises a nutrient mixture, a composite inhibitor, a composite chromogenic enzyme substrate and a cold water gel, wherein the composite inhibitor is aztreonam, nalidixic acid, potassium clavulanate and trimethoprim; the composite chromogenic enzyme substrate is 5-bromo-6-chloro-3-indole-phosphate toluidine, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-6-chloro-3-indole-β-D-galactoside.

[0023] The added concentration of the composite inhibitor was 0.04 g / L, wherein the mass ratio of aztreonam, nalidixic acid, clavulanate potassium and trimethoprim was 1:30:150:5; the added concentration of the composite chromogenic enzyme substrate was 0.1 g / L, wherein the mass ratio of 5-bromo-6-chloro-3-indole-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-6-chloro-3-indole-β-D-galactoside was 1:1:1:1.

[0024] The cold water gelatin addition concentration is 26 g / L, wherein the mass ratio of guar gum to xanthan gum is 1:1.

[0025] The nutrient mixture includes 10 g / L of tryptone, 2 g / L of yeast extract powder, 3 g of beef extract powder, 2 g / L of disodium hydrogen phosphate, 0.1 g / L of sodium citrate, 0.2 g / L of magnesium sulfate and 0.005 g / L of ammonium ferric citrate.

[0026] Example 2: Culture medium for detecting Streptococcus agalactiae in bovine mastitis A culture medium for detecting Streptococcus agalactiae in bovine mastitis in this embodiment comprises a nutrient mixture, a composite inhibitor and a composite chromogenic enzyme substrate, wherein the composite inhibitor is aztreonam, nalidixic acid, potassium clavulanate and trimethoprim; the composite chromogenic enzyme substrate is 5-bromo-6-chloro-3-indole-phosphate toluidine, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-6-chloro-3-indole-β-D-galactoside.

[0027] The added concentration of the composite inhibitor was 0.045 g / L, wherein the mass ratio of aztreonam, nalidixic acid, clavulanate potassium and trimethoprim was 1:35:170:6.5; the added concentration of the composite chromogenic enzyme substrate was 0.17 g / L, wherein the mass ratio of 5-bromo-6-chloro-3-indole-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-6-chloro-3-indole-β-D-galactoside was 1:1.25:1.5:1.5.

[0028] The cold water gelatin addition concentration is 29 g / L, wherein the mass ratio of guar gum to xanthan gum is 1:1.25.

[0029] The nutrient mixture includes 15 g / L of tryptone, 3.5 g / L of yeast extract powder, 5 g of beef extract powder, 3.5 g / L of disodium hydrogen phosphate, 0.5 g / L of sodium citrate, 0.4 g / L of magnesium sulfate and 0.01 g / L of ammonium ferric citrate.

[0030] Example 3: Culture medium for detecting Streptococcus agalactiae in bovine mastitis A culture medium for detecting Streptococcus agalactiae in bovine mastitis in this embodiment comprises a nutrient mixture, a composite inhibitor and a composite chromogenic enzyme substrate, wherein the composite inhibitor is aztreonam, nalidixic acid, potassium clavulanate and trimethoprim; the composite chromogenic enzyme substrate is 5-bromo-6-chloro-3-indole-phosphate toluidine, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-6-chloro-3-indole-β-D-galactoside.

[0031] The added concentration of the composite inhibitor was 0.05 g / L, wherein the mass ratio of aztreonam, nalidixic acid, clavulanate potassium and trimethoprim was 1:40:190:8; the added concentration of the composite chromogenic enzyme substrate was 0.25 g / L, wherein the mass ratio of 5-bromo-6-chloro-3-indole-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-6-chloro-3-indole-β-D-galactoside was 1:1.5:2:2.

[0032] The cold water gelatin addition concentration is 32 g / L, wherein the mass ratio of guar gum to xanthan gum is 1:1.5.

[0033] The nutrient mixture includes 20 g / L of tryptone, 5 g / L of yeast extract powder, 7 g of beef extract powder, 5 g / L of disodium hydrogen phosphate, 1 g / L of sodium citrate, 0.6 g / L of magnesium sulfate and 0.015 g / L of ammonium ferric citrate.

[0034] Example 4: Preparation of test pieces The structure of the Streptococcus agalactiae test piece provided by the present invention is as follows Figure 1 As shown, the test piece includes a transparent covering film 1 and a bottom plate 3 with a circular culture area, the transparent covering film 1 can cover the bottom plate 3; the culture area 2 is filled with the culture medium for detecting Streptococcus agalactiae of Example 2, the bottom plate 3 is made of polyaniline (PH) synthetic paper or polyvinyl chloride resin (PVC) synthetic paper, and the transparent covering film 1 is made of PE (polyethylene) material or PET (polyethylene terephthalate) material.

[0035] Specifically, the size of the transparent film 1 is 98 mm×80 mm, the transparent film 1 is bonded to the bottom plate 3, and the bonding width is 12 mm. The size of the bottom plate 3 is 93 mm×80 mm. The culture area 2 is 15.5 mm away from the left and right edges of the bottom plate 3, 19-20 mm away from the lower edge of the bottom plate 3, and the height of the bottom plate 3 is 0.70-0.78 mm; the diameter of the culture area 2 is 50 mm, and the culture area 2 is formed by coating and curing with hot melt adhesive, the hot melt adhesive is vinyl acetate, the inner diameter of the rubber ring is 50 mm, and the outer diameter of the rubber ring is 50.5 mm; a square grid is provided at the bottom of the culture area 2, and the size of the small squares in the square grid is 10 mm×10 mm.

[0036] Example 5: A test piece for detecting Streptococcus agalactiae in bovine mastitis The Streptococcus agalactiae test sheet of this embodiment includes a transparent covering film 1, a culture area 2 and a bottom plate 3. The bottom of the culture area 2 has a square grid, and the transparent covering film 1 can cover the bottom plate; wherein, the circular culture area 2 is filled with 2 mL of culture medium for detecting streptococci, the bottom plate 3 is made of PH (polyaniline) synthetic paper, and the transparent covering film 1 is PE (polyethylene).

[0037] Among them, the culture medium for detecting Streptococcus agalactiae includes a nutrient mixture, a composite inhibitor, a composite chromogenic enzyme substrate and a cold water gel. The composition of each 100 mL of the culture medium is: 1.5 g of tryptone, 0.25 g of yeast extract powder, 0.5 g of beef extract powder, 0.25 g of disodium hydrogen phosphate, 0.05 g of sodium citrate, 0.04 g of magnesium sulfate, 0.045 g of the composite inhibitor, 0.02 g of the composite chromogenic enzyme substrate and 3.0 g of cold water gel. The composite inhibitor is composed of aztreonam, nalidixic acid, potassium clavulanate and trimethoprim in a final concentration ratio of 1:36:180:7.5; the composite chromogenic enzyme substrate is composed of 5-bromo-6-chloro-3-indole-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-6-chloro-3-indole-β-D-galactoside in a final concentration ratio of 1:1.25:1.5:1.5; and the cold water gel is composed of guar gum and xanthan gum in a final concentration ratio of 1:1.

[0038] The above basic components are fully dissolved in 100 mL of water, and then cold water is added to gel. After stirring evenly, the mixture can be packaged, dried, and film-coated to prepare a test piece for detecting Streptococcus agalactiae in bovine mastitis.

[0039] The Streptococcus agalactiae test piece prepared in this example was verified by inoculating positive quality control strains and negative control bacteria. The results showed that Streptococcus agalactiae appeared blue-green, while negative control bacteria such as Streptococcus uberis, Streptococcus dysgalactiae, Atropha viridis, and Escherichia coli did not grow or grew purple-red, which did not interfere with the interpretation of Streptococcus agalactiae.

[0040] Example 6: A test piece for detecting Streptococcus agalactiae in bovine mastitis The Streptococcus agalactiae test piece of this embodiment is different from the test piece of Example 5 in the material of the test piece and the culture medium in the circular culture area. The base material of the test piece of this embodiment is PVC (polyvinyl chloride resin) synthetic paper, and the transparent coating is PET (polyethylene terephthalate) material. The composition of each 100mL culture medium is: 1.0g tryptone, 0.3g yeast extract powder, 0.7g beef extract powder, 0.3g disodium hydrogen phosphate, 0.08g sodium citrate, 0.06g magnesium sulfate, 0.04g composite inhibitor, 0.025g composite color enzyme substrate, and 3.2g cold water gel. The composite inhibitor is composed of aztreonam, nalidixic acid, potassium clavulanate and trimethoprim in a mass ratio of 1:30:165:5; the composite chromogenic enzyme substrate is composed of 5-bromo-6-chloro-3-indole-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-6-chloro-3-indole-β-D-galactoside in a mass ratio of 1:1.5:2:2; and the cold water gel is composed of guar gum and xanthan gum in a final concentration ratio of 1:1.

[0041] The above-mentioned nutrient mixture, composite inhibitor and composite color developing enzyme substrate are fully dissolved in 100 mL of water, and then cold water is added to gel, stirred evenly, dispensed into the culture area of ​​the test piece, dried, and covered with a transparent cover film to prepare a Streptococcus agalactiae test piece.

[0042] The Streptococcus agalactiae test piece prepared in this embodiment was verified by inoculating positive quality control strains and negative control bacteria. The results showed that Streptococcus agalactiae appeared green or blue-green, and negative control bacteria such as Streptococcus uberis, Streptococcus dysgalactiae, Atropha viridis, and Escherichia coli did not grow or grew purple-red, which did not interfere with the interpretation of Streptococcus agalactiae.

[0043] Each positive quality control bacteria was appropriately diluted and inoculated on the Streptococcus agalactiae test piece and TSA agar plate prepared in this example. The test results are shown in Table 1. The results show that the recovery rates (PR) of the two Streptococcus agalactiae strains were both above 0.6 (PR≥0.5, qualified).

[0044] Table 1 Recovery rate of each positive quality control bacteria inoculated

[0045] Note: "KGR test piece" refers to the Streptococcus agalactiae test piece provided in this example.

[0046] Example 7: A test piece for detecting Streptococcus agalactiae in bovine mastitis The Streptococcus agalactiae test piece of this embodiment is different from the test piece of Example 6 in that the culture medium in the circular culture area, per 100 mL of the culture medium, is composed of: 2.0 g of tryptone, 0.2 g of yeast extract powder, 0.4 g of beef extract powder, 0.2 g of disodium hydrogen phosphate, 0.02 g of sodium citrate, 0.02 g of magnesium sulfate, 0.05 g of composite inhibitor, 0.022 g of composite chromogenic enzyme substrate, and 3.2 g of cold water gelatin. The composite inhibitor is composed of aztreonam, nalidixic acid, potassium clavulanate and trimethoprim in a mass ratio of 1:40:185:8; the composite chromogenic enzyme substrate is composed of 5-bromo-6-chloro-3-indole-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-6-chloro-3-indole-β-D-galactoside in a mass ratio of 1:1:2:2; and the cold water gel is composed of guar gum and xanthan gum in a final concentration ratio of 1:1.

[0047] The above-mentioned nutrient mixture, composite inhibitor and composite color developing enzyme substrate are fully dissolved in 100 mL of water, and then cold water is added to gel, stirred evenly, dispensed into the culture area of ​​the test piece, dried, and covered with a transparent cover film to prepare a Streptococcus agalactiae test piece.

[0048] The Streptococcus agalactiae test piece prepared in this embodiment was verified by inoculating positive quality control strains and negative control bacteria. The results showed that Streptococcus agalactiae appeared green or blue-green, and negative control bacteria such as Streptococcus uberis, Streptococcus dysgalactiae, Atropha viridis, and Escherichia coli did not grow or grew purple-red, which did not interfere with the interpretation of Streptococcus agalactiae.

[0049] Each positive quality control bacteria was appropriately diluted and inoculated on the Streptococcus agalactiae test piece and TSA agar plate prepared in this example. The test results are shown in Table 2. The results show that the recovery rates (PR) of the two strains of Streptococcus agalactiae were both above 0.6 (PR≥0.5, qualified).

[0050] Table 2 Recovery rate of each positive quality control bacteria inoculated

[0051] Note: "KGR test piece" refers to the Streptococcus agalactiae test piece provided in this example.

[0052] Test Example 1: Specificity test of Streptococcus agalactiae test piece The performance of the Streptococcus agalactiae test piece prepared in Example 5 was evaluated: two strains of Streptococcus agalactiae with strain numbers ATCC13813 and ATCC12403 were used as positive control bacteria, and Streptococcus uberis, Streptococcus dysgalactiae, Atropha viridis, Streptococcus macedoniae, drug-resistant Escherichia coli and 14 standard strains purchased from the bacterial culture collection management center were used as negative control bacteria (see Table 1 for details). The above strains were activated by appropriate methods, and the bacterial suspension was appropriately diluted to prepare a concentration of 3×10 1 ~3×10 2 The bacterial suspension of 100 CFU / mL was inoculated into the Streptococcus agalactiae test piece prepared in Example 5, and the TSA plate was inoculated as a positive control. The plates were cultured at 37°C for 18-24 hours. The plate culture time can be extended to 48 hours according to the growth of the bacteria. The test results are shown in Table 3.

[0053] Table 3 Specificity experiments of 21 strains

[0054] Note: "+" represents growth, "-" represents no growth. "KGR test piece" refers to the Streptococcus agalactiae test piece provided in Example 5.

[0055] As shown in Table 3, positive quality control bacteria appear blue-green on the Streptococcus agalactiae test piece provided by the present invention, while other Streptococci, Enterococci and Gram-negative bacteria appear purple-red or do not grow. It can be seen that the Streptococcus agalactiae test piece provided by the present invention has a strong detection specificity and can largely avoid the occurrence of false positive detection.

[0056] Furthermore, some positive control bacteria and negative control bacteria were diluted to an appropriate gradient and inoculated on the Streptococcus agalactiae test piece provided by the present invention and on the competing bovine mastitis diagnosis dish area I (hereinafter referred to as the quarter dish area I), and the colony growth and color development were compared and analyzed. The results are shown in Table 4.

[0057] Table 4 Summary of growth of typical quality control strains

[0058] As can be seen from Table 4, as the target bacteria, Streptococcus agalactiae appears blue-green on the Streptococcus agalactiae test piece and the quadrant I zone provided by the present invention. A total of 8 negative control bacteria, including Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus cecum, Enterococcus pyogenes, Escherichia coli, and light green aerococcosis and Streptococcus Parisiensis separated from the sample, appear purple-red or do not grow on the Streptococcus agalactiae test piece provided by the present invention, and are easily distinguished from the target bacteria. However, in Zone I of the quadrant I of the competitor, the color appears blue, light blue or blue-green, which can easily cause weak false positive or false positive results in the interpretation. Therefore, the Streptococcus agalactiae test piece provided by the present invention has a stronger detection specificity for Streptococcus agalactiae, and can largely avoid the occurrence of false positive test results.

[0059] Test Example 2: Recovery Test Two strains of Streptococcus agalactiae, ATCC 13813 and ATCC 12403, were revived and activated to the third generation using the method recommended by the Culture Collection Center. The bacterial suspension was appropriately diluted to prepare a concentration of 3×10 1 ~3×10 2 The bacterial suspension of CFU / mL was inoculated on the Streptococcus agalactiae test piece prepared in Example 5, and was inoculated on the TSA agar plate and the area I of the four-divided dish at the same time. Three parallels were made for each strain. The culture was kept at 37°C for 18-24 hours. Depending on the growth of the strain, the plate culture can be extended to 48 hours. The test results are shown in Table 5.

[0060] Table 5 Comparison of quality control strain test results

[0061] Note: "KGR test piece" refers to the Streptococcus agalactiae test piece provided in Example 5. 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 piece or the quadrant plate I.

[0062] As shown in Table 5, compared with the TSA plate test results, the two positive quality control bacteria standard strains showed blue-green on the Streptococcus agalactiae test piece provided by the present invention, and the strain recovery rate (PR) was above 0.5, and was higher than the recovery rate of the quarter dish I area to varying degrees. It is proved that the Streptococcus agalactiae test piece provided by the present invention has higher detection sensitivity and can avoid the occurrence of missed detection to a certain extent.

[0063] Test Example 3: Detection of Streptococcus agalactiae in natural samples Milk squeezed from mastitis cows in different feeding areas of a ranch was collected and numbered. After the milk samples were appropriately diluted, they were inoculated on the Streptococcus agalactiae test piece provided by the present invention, the CHROMagar Streptococcus agalactiae culture medium and the I zone of the quadrant dish. At least 3 gradients were selected, and 3 parallels were made for each gradient. The sampling and processing methods of the samples were referred to GB4789.1-2016 "National Food Safety Standard General Rules for Food Microbiology Examination". The test results are shown in Table 6, and the specific results are shown in Table 7 (partial data).

[0064] Table 6 Comparison of natural sample test results

[0065] Table 7

[0066] Table 7 (Continued)

[0067] As shown in Table 6, the Streptococcus agalactiae test piece provided by the present invention was compared with 75 samples in total, 41 samples were compared with CHROMagar culture medium, and the results were all negative, with a compliance rate of 100%; 42 samples were compared with the quarter dish I area, of which 2 were inconsistent, with a compliance rate of about 95.24%. The strains were identified by molecular biology, and the results were all light green balloon bacteria, that is, the test results of the quarter dish I area were false positive. Therefore, the Streptococcus agalactiae test piece provided by the present invention has stronger detection specificity, which can effectively avoid the occurrence of false positive test results.

[0068] Test Example 4: Detection of Streptococcus agalactiae in artificially contaminated samples The Streptococcus agalactiae numbered ATCC 13813 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 1.72×10 8 CFU / mL, take 1mL of fresh cultured Streptococcus agalactiae standard strain (ATCC 13813) broth and dilute it 10-fold in 9mL sterile saline to 10 -1 , 10 -3 , 10 -5 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 1.72 × 10 6 CFU / mL, 1.72×10 4 CFU / mL, 1.72×10 2The 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.

[0069] 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 10 -4 )、1 / 2×10 -2 (and 10 -2 ), 1 / 2 (and original times) gradient dilution, 1 mL of sample dilution was respectively taken and inoculated on the Streptococcus agalactiae test piece prepared in Example 5 and the Columbia CNA blood agar plate (hereinafter referred to as "CNA") (according to: "GB 4789.11-2014 National Food Safety Standard Food Microbiology Test β-Hemolytic Streptococcus Test"), and cultured at 36°C±1°C for 18-24h. The test results are shown in Table 8. The linear relationship between the test results of the Streptococcus agalactiae test piece provided by the present invention and the test results of the CNA blood agar plate is shown in Table 8. Figure 2 .

[0070] Table 8 Detection results of artificial contamination of Streptococcus agalactiae in sterile milk

[0071] Notes: 1. All test samples are fresh sterile milk with standard strain suspension. 2. "KGR test piece" refers to the Streptococcus agalactiae test piece provided in Example 5, "lg (CNA)" refers to the logarithmic value of the colony value on the national standard Columbia CNA blood agar plate, and "lg (KGR)" refers to the logarithmic value of the colony value on the Streptococcus agalactiae test piece prepared in Example 5.

[0072] As shown in Table 8, there was no significant difference in the results of the two methods for detecting the live bacteria content of Streptococcus agalactiae in artificially contaminated samples No. 1-9 (i.e., P>0.05). Figure 2 It can be seen that the linear regression equation between the two is y = 0.9902x+0.0201, where R 2 =0.9999, that is, the fitting degree between the two is good and extremely correlated. In summary, the Streptococcus agalactiae test piece provided by the present invention is used to detect the content of Streptococcus agalactiae in bovine milk, which is highly consistent with the national standard plate method for detecting the content of Streptococcus agalactiae in bovine milk, and can be equally applied to the preliminary detection of the content of Streptococcus agalactiae in bovine mastitis.

[0073] In summary, the Streptococcus agalactiae test strip provided by the present invention has a high detection rate for bacteria in natural samples and artificially contaminated samples. Compared with other identification plates on the market, it has the advantages of strong specificity, high sensitivity, high accuracy of test results, easy interpretation, and low cost. It overcomes the existing technical barriers and has obvious advantages. It can greatly reduce the cost of pasture testing and has extremely high application value in the diagnosis of bovine mastitis.

[0074] 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 on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A culture medium for detecting Streptococcus agalactiae in bovine mastitis, characterized in that: The invention comprises a nutrient mixture, a composite inhibitor, a composite color-developing enzyme substrate and a cold water gel, wherein the composite inhibitor is aztreonam, nalidixic acid, potassium clavulanate and trimethoprim; the composite color-developing enzyme substrate is 5-bromo-6-chloro-3-indole-phosphate p-toluidine, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-6-chloro-3-indole-β-D-galactoside.

2. A culture medium for detecting Streptococcus agalactiae in bovine mastitis according to claim 1, characterized in that: The added concentration of the composite inhibitor is 0.04-0.05 g / L, wherein the mass ratio of aztreonam, nalidixic acid, clavulanate potassium and trimethoprim is 1: (30-40): (150-190): (5-8).

3. A culture medium for detecting Streptococcus agalactiae in bovine mastitis according to claim 1, characterized in that: The added concentration of the composite color-developing enzyme substrate is 0.1-0.3 g / L, wherein the mass ratio of 5-bromo-6-chloro-3-indole-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-glucuronide, 5-bromo-6-chloro-3-indole-β-D-pyranoglucopyranoside and 5-bromo-6-chloro-3-indole-β-D-galactoside is 1:(1-1.5):(1-2):(1-2).

4. A culture medium for detecting Streptococcus agalactiae in bovine mastitis according to claim 1, characterized in that: The cold water gelatinizable agent is added at a concentration of 26-32 g / L, wherein the mass ratio of guar gum to xanthan gum is 1: (1-1.5).

5. A culture medium for detecting Streptococcus agalactiae in bovine mastitis as described in any one of claims 1 to 4, characterized in that: The nutrient mixture comprises 10-20 g / L of tryptone, 2-5 g / L of yeast extract powder, 3-7 g of beef extract powder, 2-5 g / L of disodium hydrogen phosphate, 0.1-1 g / L of sodium citrate, 0.2-0.6 g / L of magnesium sulfate and 0.005-0.015 g / L of ammonium ferric citrate.

6. A test piece, comprising a transparent covering film, a base plate, and a culture medium for detecting Streptococcus agalactiae in bovine mastitis according to any one of claims 1 to 5 prepared on the surface of the base plate.

7. The test piece according to claim 6, wherein: The bottom plate is polyaniline (PH) synthetic paper or polyvinyl chloride resin (PVC) synthetic paper, and a culture area is arranged on the bottom plate, and a counting grid is arranged at the bottom of the culture area.

8. The test piece according to claim 6, wherein: The transparent coating is made of PE or PET.

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