Culture medium for detecting staphylococcus in bovine mastitis and detection sheet thereof

Abstract

The invention discloses a culture medium for detecting staphylococcus in bovine mastitis and a detection tablet thereof.The culture medium comprises a nutrient mixture, a composite inhibitor and a composite chromogenic enzyme substrate, and the composite inhibitor comprises aztreonam, potassium tellurite, nalidixic acid and polymyxin E; the compound chromogenic enzyme substrate is prepared from a mixture of 5-bromo-6-chloro-3-indolyl-toluidine phosphate salt and 5-bromo-4-chloro-3-indole-beta-D-glucopyranoside, and the compound chromogenic enzyme substrate is prepared by mixing the 5-bromo-6-chloro-3-indolyl-toluidine phosphate salt with the 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside The compound inhibitor in the staphylococcus detection sheet can effectively inhibit growth of non-staphylococcus strains, and the compound chromogenic enzyme substrate has higher selectivity on target strains, so that the detection sheet has the characteristics of high sensitivity and high specificity, the detection accuracy of the staphylococcus strains is greatly improved, and the false positive rate and the false negative rate are reduced.

Description

[0001] This application claims priority to a Chinese patent application filed on November 22, 2024, entitled “A Staphylococcus aureus test piece and its application” and having application number 202411678075.5. Technical Field

[0002] The invention relates to the technical field of microbial detection, in particular to a culture medium and a detection sheet thereof for detecting Staphylococcus in bovine mastitis. Background Art

[0003] Cow mastitis is one of the major 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. Bovine mastitis is divided into clinical mastitis and asymptomatic latent mastitis. The pathogenic microorganisms that can cause bovine mastitis include bacteria, fungi, viruses, etc. Among them, Staphylococcus, Streptococcus and Escherichia coli dominate clinical mastitis, followed by pyogenic Corynebacterium, Pseudomonas aeruginosa, Necrotic Bacillus, Nocardia, Klebsiella, etc. The udders of diseased cows will show varying degrees of congestion, enlargement, hardening, warmth and pain. The milk production of dairy cows will decrease or even stop, and the milk will become thinner, with flocs or clots, and sometimes pus and blood can be seen. The incidence of asymptomatic latent mastitis is higher than that of clinical mastitis, and its incidence rate accounts for about 50% of the entire cattle herd. About 90% of latent mastitis is caused by streptococci and staphylococci, and latent mastitis has no obvious symptoms. If it is not discovered in time, it is easy to miss the best treatment period, causing huge economic losses to dairy cattle farming.

[0004] There are many types of environmental pathogens that cause cow mastitis. Staphylococcus aureus and Streptococcus agalactiae are contact pathogens and are the main pathogens that cause environmental cow mastitis. The incidence rate caused by them accounts for 42.37% of the total. In addition to the high detection rate of Staphylococcus aureus, the detection rate of coagulase-negative Staphylococci is also increasing, and they show different degrees of drug resistance. Therefore, it is urgent to accurately detect the types of pathogens that cause cow mastitis and provide timely and accurate treatment.

[0005] Staphylococcus Staphylococcus sp. ) is a type of Gram-positive cocci with positive catalase test, including Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus intermedius, etc. It is a common pyogenic cocci in clinical practice, widely distributed in nature, and often exists on the human body surface in the nose, pharynx, and intestines. Among them, Staphylococcus aureus is the most important pathogenic bacteria.

[0006] At present, the commonly used detection methods for diagnosing bovine mastitis on the market include PCR kits, CMT kits and pathogen identification plates. Although PCR kits are real-time, accurate and fast, this method requires high professionalism of the experimenter and has a high detection cost; CMT kits can only determine whether the cow has mastitis and the degree of inflammation, but cannot determine the type of pathogenic bacteria, which is not conducive to formulating a reasonable treatment and medication plan; pathogen identification plates can determine the type of pathogenic bacteria, but the plates are fragile, easy to contaminate, difficult to preserve, and have high transportation requirements, which increases transportation costs.

[0007] In summary, the various methods currently used to detect Staphylococcus have the disadvantages of being inconvenient to use, having high detection costs, being unable to accurately determine the type of pathogenic bacteria, and being unable to perform quantitative detection. Summary of the invention

[0008] To this end, the present invention provides a culture medium and a detection sheet thereof for detecting Staphylococcus in bovine mastitis.

[0009] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions: The invention provides a culture medium for detecting Staphylococcus in bovine mastitis, which comprises a nutrient mixture, a composite inhibitor and a composite chromogenic enzyme substrate, wherein the composite inhibitor is aztreonam, potassium tellurite, nalidixic acid and polymyxin E; and the composite chromogenic enzyme substrate is a mixture of 5-bromo-6-chloro-3-indolyl-toluidine phosphate and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside.

[0010] Preferably, the addition concentration of the 5-bromo-6-chloro-3-indolyl-toluidine phosphate salt is 0.05-0.15 g / L, and the addition concentration of 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside is 0.05-0.15 g / L; the mass ratio of 5-bromo-6-chloro-3-indolyl-toluidine phosphate salt to 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside is 1: (0.5~1.5).

[0011] Preferably, the added concentrations of the composite inhibitor are respectively 0.0001-0.0003 g / L of aztreonam, 0.01-0.04 g / L of potassium tellurite, 0.0005-0.001 g / L of nalidixic acid, and 0.0002-0.0007 g / L of polymyxin E; and the mass ratio of aztreonam, potassium tellurite, nalidixic acid, and polymyxin E is 1:(140-160):(1-5):(1-4).

[0012] Preferably, the nutrient mixture includes 10-20 g / L of tryptone, 1-3 g / L of yeast extract powder, 2-8 g / L of beef extract powder, 0.6-1.5 g / L of sodium pyruvate, 1-3 g / L of disodium hydrogen phosphate, 0.1-1.5 g / L of sodium citrate, 0.2-0.5 g / L of magnesium sulfate and 0.005-0.01 g / L of ammonium ferric citrate.

[0013] Preferably, the nutrient mixture further comprises cold water gelatin, and the cold water gelatin is prepared by mixing xanthan gum and guar gum in equal quantities.

[0014] Another aspect of the present invention provides a detection sheet, which comprises a base plate, and any of the above-mentioned culture media for detecting Staphylococcus in bovine mastitis prepared on the surface of the base plate.

[0015] Preferably, the bottom plate has a size of 93 mm×80 mm, is made of polyaniline (PH) synthetic paper or polyvinyl chloride resin (PVC) synthetic paper, and is provided with a culture area on the bottom plate, and a counting grid is provided at the bottom of the culture area.

[0016] The embodiments of the present invention have the following advantages: The Staphylococcus detection sheet provided by the present invention is a prefabricated culture system, which does not require the agar culture medium to be sterilized in advance by high pressure, takes into account the appropriate pouring temperature, and is easy to operate and carry; The composite inhibitor in the Staphylococcus detection sheet provided by the present invention can effectively inhibit the growth of non-Staphylococcus species, and the composite color-developing enzyme substrate has stronger selectivity for target bacteria. The detection sheet 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. The Staphylococcus detection sheet provided by the present invention can be used for the rapid detection of Staphylococcus in milk samples of cows with mastitis, can effectively distinguish the most pathogenic Staphylococcus aureus from other Staphylococcus species among a variety of bacteria, and can also quantify the detected Staphylococcus. The Staphylococcus detection sheet provided by the present invention has the characteristics of strong specificity, high sensitivity, short detection cycle, easy observation and low detection cost, and makes an important contribution to the detection of Staphylococcus species in bovine mastitis in pastures, and has extremely high development value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] 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.

[0019] Figure 1 This is a schematic diagram of the structure of the Staphylococcus detection sheet according to an embodiment of the present invention; wherein 1-transparent covering film, 2-circular culture area, 3-bottom plate; Figure 2 This is a colony characteristic diagram of Staphylococcus aureus inoculated on the detection sheet according to an embodiment of the present invention; Figure 3 This is a colony characteristic diagram of other Staphylococci inoculated on the detection sheet of the embodiment of the present invention; Figure 4 This is a colony characteristic diagram of the detection sheet of the embodiment of the present invention inoculated with Gram-positive bacteria other than Staphylococcus; Figure 5 This is a characteristic diagram of the colonies of Gram-negative bacteria inoculated on the test piece according to an embodiment of the present invention; Figure 6 A linear relationship diagram between the results of Staphylococcus aureus detection by the Staphylococcus detection sheet of the embodiment of the present invention and the results of the national standard BP flat plate detection; Figure 7 A linear relationship diagram between the results of Staphylococcus epidermidis detection by the Staphylococcus detection sheet of the embodiment of the present invention and the results of the national standard BP flat plate detection; Figure 8 It is a linear relationship diagram between the detection result of Staphylococcus xylosus by the Staphylococcus detection piece of the embodiment of the present invention and the detection result of the national standard BP plate. DETAILED DESCRIPTION

[0020] 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.

[0021] Example 1: Culture medium for detecting Staphylococcus in bovine mastitis The culture medium for detecting Staphylococcus in bovine mastitis of this embodiment includes a nutrient mixture, a composite inhibitor and a composite chromogenic enzyme substrate, wherein the composite inhibitor is aztreonam, potassium tellurite, nalidixic acid and polymyxin E; the composite chromogenic enzyme substrate is a mixture of 5-bromo-6-chloro-3-indolyl-toluidine phosphate and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside.

[0022] The concentration of the composite inhibitor was 0.01 g / L, and the mass ratio of aztreonam, potassium tellurite, nalidixic acid and polymyxin E in the composite inhibitor was 1:140:1:1; the concentration of the composite chromogenic enzyme substrate was 0.1 g / L, and the mass ratio of 5-bromo-6-chloro-3-indolyl-toluidine phosphate and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in the composite chromogenic enzyme substrate was 1:0.5.

[0023] The nutrient mixture includes 10 g / L of tryptone, 1 g / L of yeast extract powder, 2 g / L of beef extract powder, 0.6 g / L of sodium pyruvate, 1 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, and the concentration of cold water gel is 25 g / L. Among them, the cold water gel is made by mixing xanthan gum and guar gum in equal quantities.

[0024] Example 2: Culture medium for detecting Staphylococcus in bovine mastitis The culture medium for detecting Staphylococcus in bovine mastitis of this embodiment includes a nutrient mixture, a composite inhibitor and a composite chromogenic enzyme substrate, wherein the composite inhibitor is aztreonam, potassium tellurite, nalidixic acid and polymyxin E; the composite chromogenic enzyme substrate is a mixture of 5-bromo-6-chloro-3-indolyl-toluidine phosphate and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside.

[0025] The concentration of the composite inhibitor was 0.03 g / L, and the mass ratio of aztreonam, potassium tellurite, nalidixic acid and polymyxin E in the composite inhibitor was 1:150:3:2.5; the concentration of the composite chromogenic enzyme substrate was 0.25 g / L, and the mass ratio of 5-bromo-6-chloro-3-indolyl-toluidine phosphate and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in the composite chromogenic enzyme substrate was 1:1.

[0026] The nutrient mixture includes 15 g / L of tryptone, 2.5 g / L of yeast extract powder, 5 g / L of beef extract powder, 1.05 g / L of sodium pyruvate, 2 g / L of disodium hydrogen phosphate, 0.8 g / L of sodium citrate, 0.35 g / L of magnesium sulfate and 0.0075 g / L of ammonium ferric citrate, and the concentration of cold water gel is 30 g / L. Among them, the cold water gel is made by mixing xanthan gum and guar gum in equal quantities.

[0027] Example 3: Culture medium for detecting Staphylococcus in bovine mastitis The culture medium for detecting Staphylococcus in bovine mastitis of this embodiment includes a nutrient mixture, a composite inhibitor and a composite chromogenic enzyme substrate, wherein the composite inhibitor is aztreonam, potassium tellurite, nalidixic acid and polymyxin E; the composite chromogenic enzyme substrate is a mixture of 5-bromo-6-chloro-3-indolyl-toluidine phosphate and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside.

[0028] The concentration of the composite inhibitor was 0.05 g / L, and the mass ratio of aztreonam, potassium tellurite, nalidixic acid and polymyxin E in the composite inhibitor was 1:160:5:4; the concentration of the composite chromogenic enzyme substrate was 0.3 g / L, and the mass ratio of 5-bromo-6-chloro-3-indolyl-toluidine phosphate and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in the composite chromogenic enzyme substrate was 1:1.5.

[0029] The nutrient mixture includes 20 g / L of tryptone, 3 g / L of yeast extract powder, 8 g / L of beef extract powder, 1.5 g / L of sodium pyruvate, 3 g / L of disodium hydrogen phosphate, 1.5 g / L of sodium citrate, 0.5 g / L of magnesium sulfate and 0.01 g / L of ammonium ferric citrate, and the cold water gelable concentration is 35 g / L. The cold water gelable is made by mixing xanthan gum and guar gum in equal amounts.

[0030] Example 4: Preparation of test piece The structure of the Staphylococcus detection sheet provided by the present invention is as follows Figure 1 As shown, the detection sheet includes a transparent covering film 1, a bottom plate 3 provided with a culture area and counting grids, and the transparent covering film 1 can be covered on the bottom plate 3; the culture area 2 is filled with the culture medium for detecting Staphylococcus in Example 2, the bottom plate 3 is made of PH (polyaniline) synthetic paper, and the transparent covering film 1 is made of PE (polyethylene) material or PET (polyethylene terephthalate) material.

[0031] Specifically, the size of the transparent covering film 1 is 98 mm×80 mm. The transparent covering film 1 is bonded to the bottom plate 3 with a bonding width of 12 mm. The thickness of the transparent covering film 1 and the adhesive is 0.076 mm. The material of the transparent covering film 1 is PE or PET.

[0032] The size of the bottom plate 3 is 93 mm×80 mm, and the material is PH (polyaniline) synthetic paper or PVC (polyvinyl chloride resin) synthetic paper. The culture area 2 is 15.5 mm away from the left edge of the bottom plate 3, 15.5 mm away from the right edge 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.

[0033] Example 5: Detection sheet for detecting Staphylococcus in bovine mastitis The Staphylococcus detection 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 be covered on the bottom plate; wherein, the circular culture area 2 is filled with 2 mL of culture medium for detecting Staphylococcus, the bottom plate 3 is made of PH (polyaniline) synthetic paper, and the transparent covering film 1 is PET (polyethylene terephthalate).

[0034] Among them, the culture medium for detecting Staphylococcus includes a nutrient mixture, a composite inhibitor, a composite chromogenic enzyme substrate and a cold water gel. Each 100 mL of the culture medium contains 1.5 g of tryptone, 0.2 g of yeast extract powder, 0.5 g of beef extract powder, 0.1 g of sodium pyruvate, 0.25 g of disodium hydrogen phosphate, 0.05 g of sodium citrate, 0.035 g of magnesium sulfate and 0.001 g of ammonium ferric citrate, 0.0031 g of the composite inhibitor, 0.02 g of the composite chromogenic enzyme substrate and 2.6 g of the cold water gel. The composite inhibitor is composed of aztreonam, potassium tellurite, nalidixic acid and polymyxin E in a mass ratio of 1:150:3:2; the composite color-developing enzyme substrate is composed of 5-bromo-6-chloro-3-indolyl-toluidine phosphate and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in a mass ratio of 1:1; and the cold water gel is made by mixing xanthan gum and guar gum in equal amounts.

[0035] 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, and the mixture is stirred evenly. The mixture is dispensed into the culture area 2 of the test piece, dried, and affixed with a transparent covering film 1, thereby preparing a test piece for detecting Staphylococcus.

[0036] The Staphylococcus detection plate prepared in this example was verified by inoculating positive quality control strains and negative control bacteria. Figure 2-Figure 5As shown, the results showed that Staphylococcus aureus colonies presented a black core with a purple-red halo (hereinafter referred to as "purple-red"), other Staphylococcus colonies such as Staphylococcus epidermidis and Staphylococcus intermedius presented a black core without a colored halo (hereinafter referred to as "black"), enterococci such as Enterococcus faecalis and Enterococcus faecium and spore-forming bacteria such as Bacillus cereus and Bacillus subtilis presented a black core with a green halo (hereinafter referred to as "green"), and Gram-negative bacteria such as Escherichia coli did not grow.

[0037] Example 6: Detection sheet for detecting Staphylococcus in bovine mastitis The detection sheet for detecting Staphylococcus in this embodiment is different from that in Example 5 in that the culture medium in the circular culture area 2 in the detection sheet contains 2 g of tryptone, 0.3 g of yeast extract powder, 0.8 g of beef extract powder, 0.15 g of sodium pyruvate, 0.3 g of disodium hydrogen phosphate, 0.15 g of sodium citrate, 0.05 g of magnesium sulfate and 0.001 g of ammonium ferric citrate per 100 mL of culture medium, 0.0032 g of composite inhibitor, 0.02 g of composite chromogenic enzyme substrate and 2.6 g of cold water gel. The composite inhibitor is made of aztreonam, potassium tellurite, nalidixic acid and polymyxin E in a mass ratio of 1:160:5:4; the composite color-developing enzyme substrate is composed of 5-bromo-6-chloro-3-indolyl-toluidine phosphate and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in a mass ratio of 1:1.5; and the cold water gel is made of a mixture of equal masses of xanthan gum and guar gum.

[0038] 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. After stirring evenly, the mixture can be dispensed into the culture area 2 of the detection sheet of Example 4, dried, and film-coated to prepare a detection sheet for detecting Staphylococcus.

[0039] The Staphylococcus detection sheet prepared in this embodiment was verified by inoculating positive quality control strains (target bacteria) and negative control bacteria. The results showed that the colonies of Staphylococcus aureus appeared purple-red, the colonies of other Staphylococci such as Staphylococcus epidermidis and Staphylococcus intermedius appeared black, the colonies of enterococci such as Enterococcus faecalis and Enterococcus faecium and spore-forming bacteria such as Bacillus cereus and Bacillus subtilis appeared green, and Gram-negative bacteria such as Escherichia coli did not grow. The growth rates of each target bacteria are shown in Table 1, and the growth rate PR meets the requirement of ≥0.5.

[0040] Table 1 Growth rate of various Staphylococci inoculated on the test piece

[0041] Note: "KGR detection sheet" is the Staphylococcus detection sheet provided in Example 6 of the present invention.

[0042] Example 7: Detection sheet for detecting Staphylococcus in bovine mastitis The Staphylococcus detection piece of this embodiment is different from the detection piece of Example 5 in that the culture medium in the circular culture area contains 1 g of tryptone, 0.1 g of yeast extract powder, 0.2 g of beef extract powder, 0.06 g of sodium pyruvate, 0.1 g of disodium hydrogen phosphate, 0.15 g of sodium citrate, 0.05 g of magnesium sulfate, 0.001 g of ammonium ferric citrate, 0.0032 g of composite inhibitor, 0.022 g of composite chromogenic enzyme substrate, and 2.6 g of cold water gel per 100 mL of culture medium. Among them, the composite inhibitor is made of aztreonam, potassium tellurite, nalidixic acid and polymyxin E in a mass ratio of 1:140:2:2, the composite chromogenic enzyme substrate is made of 5-bromo-6-chloro-3-indolyl-toluidine phosphate and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in a mass ratio of 1:0.5, and the cold water gel is made of a mixture of equal masses of xanthan gum and guar gum.

[0043] 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. The mixture is stirred evenly, dispensed into the culture area of ​​the test piece, dried, and covered with a transparent cover film to prepare a Staphylococcus test piece.

[0044] The Staphylococcus detection sheet prepared in this embodiment was verified by inoculating positive quality control strains (target bacteria) and negative control bacteria. The results showed that the colonies of Staphylococcus aureus appeared purple-red, the colonies of other Staphylococci such as Staphylococcus epidermidis and Staphylococcus intermedius appeared black, the colonies of enterococci such as Enterococcus faecalis and Enterococcus faecium and spore-forming bacteria such as Bacillus cereus and Bacillus subtilis appeared green, and Gram-negative bacteria such as Escherichia coli did not grow. The growth rates of each target bacteria are shown in Table 2, and the growth rate PR meets the requirement of ≥0.5.

[0045] Table 2 Growth rate of various Staphylococci inoculated on the test piece

[0046] Note: "KGR detection sheet" is the Staphylococcus detection sheet prepared in Example 7 of the present invention.

[0047] Example 8: Detection sheet for detecting Staphylococcus in bovine mastitis The Staphylococcus detection piece of this embodiment is different from that of Example 5 in that the culture medium in the circular culture area contains 1.2 g of tryptone, 0.1 g of yeast extract powder, 0.4 g of beef extract powder, 0.09 g of sodium pyruvate, 0.1 g of disodium hydrogen phosphate, 0.05 g of sodium citrate, 0.03 g of magnesium sulfate, 0.001 g of ammonium ferric citrate, 0.0032 g of composite inhibitor, 0.02 g of composite chromogenic enzyme substrate, and 2.6 g of cold water gel per 100 mL of culture medium. The composite inhibitor is made of aztreonam, potassium tellurite, nalidixic acid and polymyxin E in a mass ratio of 1:145:5:4; the composite color-developing enzyme substrate is made of 5-bromo-6-chloro-3-indolyl-toluidine phosphate and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside in a mass ratio of 1:0.8; and the cold water gel is made of a mixture of equal masses of xanthan gum and guar gum.

[0048] 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. The mixture is stirred evenly, divided into the culture area of ​​the test piece, dried, and covered with a transparent cover film to prepare a Staphylococcus test piece.

[0049] The Staphylococcus detection sheet prepared in this embodiment was verified by inoculating positive quality control strains (target bacteria) and negative control bacteria. The results showed that the colonies of Staphylococcus aureus appeared purple-red, the colonies of other Staphylococci such as Staphylococcus epidermidis and Staphylococcus intermedius appeared black, the colonies of enterococci such as Enterococcus faecalis and Enterococcus faecium and spore-forming bacteria such as Bacillus cereus and Bacillus subtilis appeared green or blue-green, and Gram-negative bacteria such as Escherichia coli and Enterobacter cloacae did not grow. The growth rates of each target bacteria are shown in Table 3, and the growth rate PR meets the requirement of ≥0.5.

[0050] Table 3 Growth rate of various Staphylococci inoculated on the test piece

[0051] Note: "KGR detection sheet" is the Staphylococcus detection sheet prepared in Example 8 of the present invention.

[0052] Test Example 1: Staphylococcus Detection Slice Specificity Test The performance of the Staphylococcus test sheet prepared in Example 5 of the present invention was evaluated: 5 strains of Staphylococcus aureus, 12 strains of other Staphylococci, 3 strains of Listeria, 8 strains of Streptococcus, 6 strains of Enterococcus, 1 strain of Macrococcus tyrosinase (sample separation), 12 strains of Bacillus and 10 strains of Gram-negative bacteria with strain numbers of ATCC 27217, ATCC 25923, ATCC 6538P, ATCC 6538 and NBRC 12732 were activated, and the bacterial suspension was appropriately diluted to prepare a concentration of 1×10 2 ~3×102 The bacterial suspension of CFU / mL was inoculated on the Staphylococcus detection piece and the identification area II of the quadrant dish (Staphylococcus identification area, hereinafter referred to as "quadrant dish II area") prepared in Example 5, and was inoculated on the TSA plate 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 4.

[0053] Table 4 Specificity experiments of 57 strains

[0054] Table 4 (continued)

[0055] Table 4 (continued)

[0056] Table 4 (continued)

[0057] Note: On the KGR Staphylococcus detection plate, Staphylococcus aureus colonies are purple-red, other Staphylococcus colonies are black, and negative control bacteria are blue-green or do not grow; in the quadrant II area, Staphylococcus aureus is purple-red, other Staphylococci are green, white or colorless and transparent, and negative control bacteria do not grow. "+" represents growth, and "-" represents no growth. "KGR detection plate" refers to the Staphylococcus detection plate prepared in Example 5 of the present invention.

[0058] As shown in Table 4, ① the six test bacteria, including Staphylococcus intermedius (sample isolate), Bacillus cereus, Bacillus mycoides, Bacillus licheniformis, Bacillus thuringiensis, and Proteus mirabilis, showed purple-red or pink color in the identification zone II of the quadrant dish, which could easily lead to false positive results for Staphylococcus aureus detection; ② Staphylococcus chromogenes (sample isolate) and Staphylococcus gallinarum (sample isolate) did not grow in the identification zone II of the quadrant dish, resulting in false negative results for Staphylococcus aureus detection (except Staphylococcus aureus); ③ Streptococcus thermophilus (24), Enterococcus faecalis (29), Enterococcus faecium (30), Enterococcus pyogenes (33), Enterococcus hirae (34) and Table 4 showed false negative results for Staphylococcus aureus detection. A total of 14 strains numbered 35, 37, 38, 40, 41, 42, 44, 46, and 56 in the grid appeared blue-green, white, or colorless and transparent in the identification area II of the quadrant dish, resulting in false positive results for the detection of other Staphylococci (Staphylococcus species other than Staphylococcus aureus), while the above test strains appeared blue-green or did not grow on the Staphylococcus detection sheet provided by the present invention, which can be well distinguished from Staphylococcus aureus and can also be clearly distinguished from other Gram-positive bacteria and Gram-negative bacteria, proving that the Staphylococcus detection sheet provided by the present invention has stronger specificity and can largely avoid the occurrence of false positive and false negative results.

[0059] Test Example 2: Bacteria Recovery Rate Experiment Six standard strains of Staphylococcus, including two strains of Staphylococcus aureus numbered ATCC 25923 and ATCC 6538P, Staphylococcus epidermidis numbered CMCC26069, Staphylococcus lentus numbered CICC 21602, Staphylococcus xylosus numbered ATCC 29971, and Staphylococcus saprophyticus numbered ATCC 49453, 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 2×10 1 ~2×10 2 The bacterial suspension of 1.4477 CFU / mL was inoculated on the Staphylococcus detection plate prepared in Example 5, and was also inoculated on the TSA agar plate, the identification area of ​​the quadrant II and the Baird-Parker agar plate (hereinafter referred to as the BP plate). Three parallel experiments were performed for each bacterial species and cultured at 37°C for 18-24h. The culture time of the BP plate can be extended to 48h according to the growth of the bacterial species. 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 Staphylococcus test piece prepared in Example 5 of the present invention. The denominator of the PR value is the number of colonies grown on the TSA plate.

[0062] As shown in Table 5, the six standard strains of Staphylococcus appear purple or black on the Staphylococcus detection plate provided by the present invention. Compared with the detection results of the TSA plate, the strain recovery rates (PR) are all above 0.7, which are higher than the recovery rate of the quarter dish II zone, and are comparable to the recovery rate of BP, and can be equivalent to the BP plate for quantitative detection of Staphylococcus.

[0063] Test Example 3: Minimum Detection Limit Experiment The Staphylococcus aureus subsp. aureus (ATCC 25923) was revived and activated to prepare a 10 9 CFU / mL of bacterial suspension, the bacterial content of fresh activated bacterial suspension is about 1.28×10 9 CFU / mL, the bacterial suspension was diluted 10-fold to 10 -10 From 10 -6 The gradient was inoculated, and the plates were inoculated on the national standard BP plate and the Staphylococcus detection plate prepared in Example 5 of the present invention respectively. Three parallel tests were performed for each gradient. The minimum detection limit test results of the Staphylococcus detection plate of the present invention for detecting Staphylococcus aureus are shown in Table 6.

[0064] In the same manner as above, Staphylococcus epidermidis with strain number CMCC 26069 and Staphylococcus xylosus with strain number ATCC 29971 were prepared into a 10 8 CFU / mL of bacterial suspension, from 1×10 -5 The inoculation started with a CFU / mL gradient, and was inoculated on the national standard BP plate and the Staphylococcus detection piece prepared in Example 5 of the present invention, respectively. Three parallel tests were performed for each gradient. The minimum detection limit test results of the Staphylococcus detection piece of the present invention for detecting Staphylococcus epidermidis and Staphylococcus xylosus are shown in Tables 7 and 8, respectively.

[0065] Table 6 Minimum detection limit test for Staphylococcus aureus (ATCC 25923)

[0066] Table 7 Minimum detection limit test of Staphylococcus epidermidis (CMCC 26069)

[0067] Table 8 Minimum detection limit test of Staphylococcus xylosus (ATCC 29971)

[0068] It can be seen from Table 6, Table 7 and Table 8 that the minimum detection limit of the Staphylococcus detection sheet provided by the present invention for Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus xylosus can reach 1 CFU / mL.

[0069] Test Example 4: Detection of Staphylococcus aureus and other Staphylococcus species in natural samples by the test piece of the present invention Different dairy cows with bovine mastitis from different feeding areas were selected from a ranch, and freshly squeezed milk samples were numbered, and the numbers were simplified to 1, 2, 3, etc. The milk samples were appropriately diluted and inoculated on the Staphylococcus detection piece prepared in Example 5 of the present invention, the competitor's three-element veterinary dish, and the II identification area of ​​the four-element dish. Three gradients were selected for detection, and three 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 analysis of the test results is shown in Table 9.

[0070] Table 9 Comparison of detection results of Staphylococcus aureus in natural samples

[0071] As can be seen from Table 9, the Staphylococcus detection strip provided by the present invention was compared with 204 samples in total, 108 samples were compared with the three-element veterinary dish, and the compliance rate was 91.67%; a total of 96 samples were compared with the four-element dish, and the compliance rate was 94.79%. Specific data are shown in Table 10 (partial data).

[0072] Table 10

[0073] The samples that did not match the test results of the competing products were identified as bacterial species. After biomolecular identification, sample No. 5 was Bacillus cereus, which means that the test results of both competing products were false positive. The bacterial species detected by sample No. 17 was Escherichia coli, which means that the test result of the three-element veterinary dish was false positive. The bacterial species detected by sample No. 18 was Staphylococcus aureus, which means that the test results of both competing products were false negative. The bacterial species detected by samples No. 20, 22, and 24 were all Enterococcus faecalis, which means that the test result of the four-element dish was false positive. The bacterial species detected by sample No. 23 was Streptococcus equine, which means that the test result of the four-element dish was false positive. The bacterial species detected by sample No. 29 was Staphylococcus aureus, which means that the test result of the four-element dish was false negative. Other identification results are omitted here.

[0074] The above results show that the Staphylococcus detection piece provided by the present invention is more accurate in detecting Staphylococcus aureus infected in bovine mastitis and can avoid false positive and false negative test results to a large extent.

[0075] Experimental Example 5: Detection of Staphylococcus aureus in artificially contaminated samples by the test piece of the present invention According to the method recommended by the Center for Microbial Culture Collection, Staphylococcus aureus numbered ATCC 25923 (hereinafter referred to as the test strain) was revived and activated. The fresh activated bacterial suspension was counted according to GB 4789.2. The live bacterial content of the bacterial suspension was about 1.18×10 8CFU / mL, take 1mL of freshly cultured test strain broth and dilute it in 9mL of sterile saline for 10-fold serial gradient dilution 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 approximately 1.18×10 6 CFU / mL, 1.18×10 4 CFU / mL, 1.18×10 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.

[0076] In order to facilitate the counting of colonies and estimate the bacterial content in each spiked sample, the high, medium and low artificial contamination samples were diluted 10 times, and 10 -4 , 10 -2 , the original multiple gradient dilution bacterial solution, 1 mL of the sample dilution solution was respectively transferred and inoculated on the Staphylococcus detection piece provided by the present invention and the Baird-Parker agar plate (hereinafter referred to as "BP") (based on: "GB 4789.10-2016 National Food Safety Standard Food Microbiology Test Staphylococcus aureus Test", "NYT2962-2016 Isolation and Identification Method of Staphylococcus aureus, Coagulase-negative Staphylococci and Streptococcus agalactiae in Mastitis Milk of Dairy Cows"), and cultured at 36°C±1°C for 18-24h. The BP plate can extend the culture time to 48h as needed. The test results are shown in Table 11, and the linear relationship between the test results of the Staphylococcus detection piece provided by the present invention and the BP plate is shown in Table 12. Figure 6 .

[0077] Table 11 Detection results of Staphylococcus aureus in sterile milk artificial contamination samples

[0078] Notes: 1. All test samples were artificially contaminated. 2. "KGR test piece" is the Staphylococcus test piece provided in Example 5 of the present invention, "lg(BP)" refers to the logarithmic value of the colony count on the national standard BP plate, and "lg(KGR)" refers to the logarithmic value of the colony count on the KGR Staphylococcus test piece.

[0079] As shown in Table 11, the colony count values ​​of samples 1-9 inoculated on the BP plate and the Staphylococcus detection plate provided by the present invention were calculated by the national standard GB 4789.10-2016 plate counting method, and it was found that there was no significant difference between the two (P>0.05). Figure 6 It can be seen that the linear regression equation is y=0.9841x+0.0404, where R 2 =0.9998, that is, the degree of fit between the two is high. In summary, the Staphylococcus detection piece provided by the present invention detects the content of Staphylococcus aureus in milk, which has a high consistency with the national standard method for testing the content of Staphylococcus aureus in milk, and can be used for the preliminary detection of Staphylococcus aureus in bovine mastitis.

[0080] Experimental Example 6: Detection of Staphylococcus epidermidis in artificially contaminated samples by the test piece of the present invention According to the method recommended by the Center for Culture Collection, Staphylococcus epidermidis numbered CMCC 26069 (hereinafter referred to as the test strain) was revived and activated. The fresh activated bacterial suspension was counted according to GB 4789.2. The live bacterial content of the bacterial suspension was about 5.46×10 8 CFU / mL, take 1mL of freshly cultured test strain broth and dilute it in 9mL of sterile saline for 10-fold serial gradient dilution 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 5.46 × 10 6 CFU / mL, 5.46×10 4 CFU / mL, 5.46×10 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.

[0081] In order to facilitate the counting of colonies and estimate the bacterial content in each spiked sample, the samples with high, medium and low artificial contamination were diluted in a gradient manner, and 10 -5 (and 1 / 4×10 -4 )、10 -3 (and 1 / 4×10 -2 )、10 -1(and 1 / 4) gradient dilution of the bacterial solution, 1 mL of the sample dilution solution was respectively transferred and inoculated on the Staphylococcus detection piece and BP agar plate provided by the present invention (according to: "GB 4789.10-2016 National Food Safety Standard Food Microbiology Inspection Staphylococcus aureus Inspection", "NYT2962-2016 Isolation and Identification Method of Staphylococcus aureus, Coagulase-negative Staphylococci and Streptococcus agalactiae in Mastitis Milk of Dairy Cows"), and cultured at 36°C±1°C for 18-24h. The BP plate can extend the culture time to 48h as needed. The test results are shown in Table 12, and the linear relationship between the test results of the Staphylococcus detection piece provided by the present invention and the BP plate is shown in Table 13. Figure 7 .

[0082] Table 12 Detection results of Staphylococcus epidermidis in sterile milk artificial contamination samples

[0083] Notes: 1. All test samples were artificially contaminated. 2. "KGR test piece" is the Staphylococcus test piece provided in Example 5 of the present invention, "lg(BP)" refers to the logarithmic value of the colony count on the national standard BP plate, and "lg(KGR)" refers to the logarithmic value of the colony count on the KGR Staphylococcus test piece.

[0084] As shown in Table 12, the colony count values ​​of samples 1-9 inoculated on BP agar plates and the Staphylococcus detection plate provided by the present invention were calculated by the national standard GB 4789.10-2016 plate counting method, and there was no significant difference between the two (P>0.05). Figure 7 It can be seen that the linear regression equation is y=1.0014x-0.0172, where R 2 =1, that is, the degree of fit between the two is high. In summary, the Staphylococcus detection piece provided by the present invention detects the content of Staphylococcus epidermidis in milk, which has a high consistency with the national standard method for testing the content of Staphylococcus epidermidis in milk, and can be used for the preliminary detection of the content of Staphylococcus epidermidis in bovine mastitis.

[0085] Experimental Example 8: Detection of Staphylococcus xylosus in artificially contaminated samples by the test piece of the present invention According to the method recommended by the Center for Culture Collection, Staphylococcus xylosus numbered ATCC 29971 (hereinafter referred to as the test strain) was revived and activated. The fresh activated bacterial suspension was counted according to GB 4789.2. The live bacterial content of the bacterial suspension was about 3.12×10 8 CFU / mL, take 1mL of freshly cultured test strain broth and dilute it in 9mL of sterile saline for 10-fold serial gradient dilution to 10 -1 , 10 -3 , 10 -5Then, 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 3.12 × 10 6 CFU / mL, 3.12×10 4 CFU / mL, 3.12×10 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] In order to facilitate the counting of colonies and estimate the bacterial content in each spiked sample, the samples with high, medium and low artificial contamination were diluted in a gradient manner, and 1 / 2×10 -4 (and 1 / 4×10 -4 )、1 / 2×10 -2 (and 1 / 4×10 -2 ), 1 / 2 (and 1 / 4) gradient dilution of bacterial solution, 1 mL of sample dilution solution was respectively transferred and inoculated on the Staphylococcus detection piece and BP agar plate provided by the present invention (according to: "GB 4789.10-2016 National Food Safety Standard Food Microbiology Inspection Staphylococcus aureus Inspection", "NYT2962-2016 Isolation and Identification Method of Staphylococcus aureus, Coagulase-negative Staphylococci and Streptococcus agalactiae in Mastitis Milk of Dairy Cows"), and cultured at 36°C±1°C for 18-24h. The culture time of BP agar plate can be extended to 48h as needed. The test results are shown in Table 13, and the linear relationship between the test results of Staphylococcus detection piece provided by the present invention and BP plate is shown in Table 14. Figure 8 .

[0087] Table 13 Detection results of Staphylococcus xylosus in artificially contaminated samples of sterile milk

[0088] Notes: 1. All test samples were artificially contaminated. 2. "KGR test piece" is the Staphylococcus test piece provided in Example 5 of the present invention, "lg(BP)" refers to the logarithmic value of the colony count of the national standard BP plate, and "lg(KGR)" refers to the logarithmic value of the colony count of the KGR Staphylococcus test piece (on the plate).

[0089] As shown in Table 13, the colony count values ​​of samples 1-9 on the BP agar plate and the Staphylococcus detection plate provided by the present invention were calculated by the national standard GB 4789.10-2016 plate counting method, and there was no significant difference between the two (P>0.05). Figure 8 It can be seen that the linear regression equation is y=1.0002x-0.0325, where R 2=0.9997, that is, the fitting degree between the two is high. In summary, the Staphylococcus detection piece provided by the present invention detects the content of xylosus Staphylococcus in milk, which has a high consistency with the national standard method for testing the content of xylosus Staphylococcus in milk, and can be used for the preliminary detection of xylosus Staphylococcus in bovine mastitis.

[0090] Comparative Example 1 The Staphylococcus aureus detection sheet of this comparative example is substantially the same as the structure, basic components of the culture area, and composition of the Staphylococcus detection sheet of Example 5, with the obvious difference being the different composition of the composite inhibitor. This comparative example uses the composite inhibitors sodium azide, aztreonam, deferoxamine, and lithium chloride in the Staphylococcus aureus detection sheet, and the optimal mass ratio thereof, which is 2:1:5:12 for the mass ratio of sodium azide, aztreonam, deferoxamine, and lithium chloride, and the total amount of the composite inhibitor added is 0.031 g / L, to prepare the Staphylococcus detection sheet.

[0091] The Staphylococcus detection sheet prepared in Comparative Example 1 was verified by inoculation, and the test results showed that Staphylococcus epidermidis could grow on the culture medium but could not be distinguished from Staphylococcus aureus; interfering bacteria, Bacillus cereus, Bacillus mycoides, etc. could all grow normally, and the positive purple-red color was also indistinguishable from Staphylococcus aureus and other Staphylococci, which could easily cause false positive test results. Therefore, it is not suitable for detecting Staphylococcus species in bovine mastitis.

[0092] Comparative Example 2 The Staphylococcus detection piece of this comparative example is substantially the same as the Staphylococcus detection piece of Example 5 in structure, basic components of the culture area, and composition, with the obvious difference being the different composition of the composite inhibitor. The composite inhibitor used in this comparative example consists of aztreonam, nisin, ceftriaxone, and nitrofurantoin, and the added amount is 0.032 g / L, wherein the final concentration ratio of aztreonam, nisin, trimethoprim, and nitrofurantoin is 1:5:0.5:6.

[0093] The Staphylococcus detection sheet prepared by Comparative Example 2 was verified by inoculation. The test results showed that the interfering bacteria Bacillus cereus would grow and appear purple-red, resulting in a false positive test result. The sample isolated bacteria Staphylococcus intermedius, Staphylococcus xylosus ATCC 29971, and Staphylococcus epidermidis CMCC 26069 grew purple-red and could not be distinguished from Staphylococcus aureus, which could easily cause a false positive result for Staphylococcus aureus. Slow Staphylococcus growth appeared green and could not be distinguished from negative control bacteria such as enterococci, which could easily cause a false negative result for Staphylococcus detection. Therefore, this formula test sheet is not suitable for the detection of Staphylococcus species in bovine mastitis in pastures.

[0094] The Staphylococcus detection piece provided by the present invention overcomes the defects of the prior art. It can not only detect most of the common Staphylococcus species in bovine mastitis, but also effectively distinguish the most pathogenic Staphylococcus aureus from other Staphylococci. In addition, the minimum detection limit of the detection piece is 1 CFU / mL, and it has the characteristics of high sensitivity and strong specificity, and has extremely high application value in the fields of pasture and food detection.

[0095] 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 culture medium for detecting Staphylococcus in bovine mastitis, characterized in that: The invention comprises a mixture of nutrient components, a composite inhibitor and a composite chromogenic enzyme substrate, wherein the composite inhibitor comprises aztreonam, potassium tellurite, nalidixic acid and polymyxin E; and the composite chromogenic enzyme substrate comprises 5-bromo-6-chloro-3-indolyl-toluidine phosphate and 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside.

2. The culture medium for detecting Staphylococcus in bovine mastitis according to claim 1, characterized in that: The concentration of the composite color-developing enzyme substrate is 0.1-0.3 g / L, wherein the mass ratio of the 5-bromo-6-chloro-3-indolyl-toluidine phosphate salt to 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside is 1: (0.5-1.5).

3. The culture medium for detecting Staphylococcus in bovine mastitis according to claim 1, characterized in that: The concentration of the composite inhibitor is 0.01-0.05 g / L; wherein the mass ratio of aztreonam, potassium tellurite, nalidixic acid and polymyxin E is 1:(140-160):(1-5):(1-4).

4. A culture medium for detecting Staphylococcus in bovine mastitis as described in any one of claims 1 to 3, characterized in that: The nutrient mixture comprises 10-20 g / L of tryptone, 1-3 g / L of yeast extract powder, 2-8 g / L of beef extract powder, 0.6-1.5 g / L of sodium pyruvate, 1-3 g / L of disodium hydrogen phosphate, 0.1-1.5 g / L of sodium citrate, 0.2-0.5 g / L of magnesium sulfate and 0.005-0.01 g / L of ammonium ferric citrate.

5. The culture medium for detecting Staphylococcus in bovine mastitis according to claim 4, characterized in that: The nutritional ingredient mixture also includes a cold water gellable.

6. The cold water gelable composition according to claim 5, characterized in that The cold water gel has a concentration of 25-35 g / L; xanthan gum and guar gum are mixed in equal amounts.

7. A detection sheet comprising a base plate, and the culture medium for detecting Staphylococcus in bovine mastitis according to any one of claims 1 to 6 prepared on the surface of the base plate.

8. The detection sheet according to claim 7, characterized in that: The base plate is made of polyaniline synthetic paper or polyvinyl chloride resin synthetic paper, and a culture area is arranged on the base plate, and a counting grid is arranged at the bottom of the culture area.

9. The detection sheet according to claim 8, characterized in that: The detection sheet also includes a transparent covering film for covering the bottom plate, and the material of the transparent covering film is PE or PET.

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