Culture medium for detecting common pathogenic bacteria of streptococcaceae in bovine mastitis and test piece thereof

Abstract

The invention discloses a culture medium and a test piece for detecting common pathogenic bacteria of streptococcus in bovine mastitis, 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 and potassium clavulanate; the compound chromogenic enzyme substrate is 5-bromine-6-chloro-3-indole-phosphoric acid p-toluidine and 5-bromine-4-chloro-3-indole-beta-D-glucuronide, and the compound chromogenic enzyme substrate is 5-bromine-6-chloro-3-indole-phosphoric acid p-toluidine. The test strip for the common pathogenic bacteria of the streptococcus family can be used for rapidly detecting the common pathogenic bacteria of the streptococcus family in cow mastitis in a dairy farm, has the characteristics of strong specificity, high sensitivity, short detection period, easiness in observation and low detection cost, and 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 sheet thereof for detecting common pathogenic bacteria of Streptococcus family in bovine mastitis. Background Art

[0002] Cow mastitis is one of the diseases that causes the most serious economic losses in the dairy industry, seriously affecting the health, milk production and quality of dairy cows, thereby indirectly increasing the cost of breeding. The pathogens that cause cow mastitis can be divided into major pathogens and secondary pathogens. Major pathogens generally refer to bacteria that are associated with clinical mastitis and can cause obvious symptoms and economic losses. The generally recognized major pathogens are Streptococcus agalactiae, Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Escherichia coli and Acinetobacter. Among them, Streptococcus agalactiae, Streptococcus uberis and Streptococcus dysgalactiae are the core pathogens that cause bovine mastitis in the Streptococcus family.

[0003] Enterococci belong to the family Streptococcus and the genus Enterococcus. They require special attention due to their drug resistance. They are widely found in the intestines of animals, the environment (such as soil, water sources) and feces. Enterococci are pathogenic bacteria of environmental mastitis. About 80% of environmental mastitis is caused by Enterococcus faecalis, and about 10-15% is caused by Enterococcus faecium. In addition, many commonly used antimicrobial drugs fail to treat enterococcal infections due to the inherent and acquired drug resistance of enterococci. In view of this, rapid detection of pathogens of dairy cow mastitis, 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] Today's industry standards require the use of molecular biology techniques, which have high identification costs, cumbersome steps, high requirements for operators, equipment and technology, and are time-consuming and labor-intensive. They are not suitable for on-site rapid diagnosis of mastitis in dairy farms. The current conventional methods for diagnosing latent bovine mastitis include the SMT method (indirect somatic cell counting method), the BTB indicator method, and the SCC microscope counting method. The mastitis detection method (CMT) kits on the market today are subjective in the judgment of results, which can easily lead to misjudgment, and cannot locate specific pathogens, which brings certain difficulties to targeted treatment. The diagnostic dishes used for microbial diagnosis of bovine mastitis on the market involve complex bacterial species, and it is difficult to determine the specific pathogens, which can easily lead to false positive test results.

[0005] In order to overcome the problems of low specificity and sensitivity of traditional detection methods for pathogenic bacteria of cow mastitis, incomplete pathogen detection and low accuracy in the prior art, the present invention provides a test piece for detecting common pathogenic bacteria of the Streptococcus family in bovine mastitis, which is used to detect common pathogenic bacteria of the Streptococcus family in pathogenic bacteria causing cow mastitis. Summary of the invention

[0006] To this end, the present invention provides a culture medium and a test piece thereof for detecting common pathogenic bacteria of the Streptococcus family 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 common pathogenic bacteria of Streptococcus family 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 and potassium clavulanate; the composite chromogenic enzyme substrate is 5-bromo-6-chloro-3-indole-phosphate p-toluidine and 5-bromo-4-chloro-3-indole-β-D-glucuronide.

[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 0.005-0.008 g / L, and potassium clavulanate is 0.02-0.04 g / L, and the mass ratio of aztreonam, nalidixic acid, and potassium clavulanate is 1: (30-40): (150-190).

[0009] Preferably, the added concentration of the composite color-developing enzyme substrate is 0.1-0.2 g / L, wherein the mass ratio of 5-bromo-6-chloro-3-indole-toluidine phosphate to 5-bromo-4-chloro-3-indole-β-D-glucuronide is 1: (1-1.5).

[0010] Preferably, the cold water gelatin is added at a concentration of 25-35 g / L, specifically comprising guar gum and xanthan gum in a mass ratio of 1:(1-1.5).

[0011] Preferably, the nutrient mixture comprises 12-22 g / L of tryptone, 1.5-4.5 g / L of yeast extract powder, 3.5-7.5 g / L of beef extract powder, 1.5-3.5 g / L of disodium hydrogen phosphate, 0.1-1 g / L of sodium citrate, 0.1-0.5 g / L of magnesium sulfate, 0.5-1.5 g / L of sodium pyruvate and 0.006-0.012 g / L of ammonium ferric citrate.

[0012] Another object of the present invention is to provide a test piece, which comprises a base plate, and any of the above-mentioned culture media for detecting common pathogens of the Streptococcus family 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 test piece for detecting streptococcal pathogens commonly seen in bovine mastitis 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 test piece for detecting common pathogenic bacteria of the Streptococcus family in bovine mastitis provided by the present invention can effectively inhibit the growth of non-target bacteria, and the composite chromogenic enzyme substrate has stronger selectivity for target bacteria. Therefore, the present invention has the characteristics of high sensitivity and strong specificity, greatly improves the detection accuracy, and reduces the false positive rate and false negative rate. BRIEF DESCRIPTION OF THE DRAWINGS

[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 a test piece for detecting common pathogenic bacteria of the Streptococcus family in bovine mastitis provided by the present invention; wherein 1-transparent covering film, 2-circular culture area, 3-bottom plate; Figure 2 A linear relationship diagram between the results of the Streptococcus dysgalactiae test piece for common pathogens of the Streptococcus family provided by the present invention and the results of the Columbia blood plate test; Figure 3 A linear relationship diagram between the results of the common pathogenic bacteria test strip of the Streptococcus family provided by the present invention for detecting Enterococcus faecalis and the results of the Columbia blood plate test. 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] The culture medium of the present embodiment for detecting common pathogens of Streptococcus family in bovine mastitis 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 and potassium clavulanate; the composite chromogenic enzyme substrate is 5-bromo-6-chloro-3-indole-phosphate p-toluidine and 5-bromo-4-chloro-3-indole-β-D-glucuronide.

[0023] The added concentration of the composite inhibitor is 0.04 g / L, wherein the mass ratio of aztreonam, nalidixic acid and potassium clavulanate is 1:30:150; the added concentration of the composite chromogenic enzyme substrate is 0.1 g / L, wherein the mass ratio of 5-bromo-6-chloro-3-indole-phosphate toluidine and 5-bromo-4-chloro-3-indole-β-D-glucuronide is 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 12 g / L of tryptone, 1.5 g / L of yeast extract powder, 3.5 g of beef extract powder, 1.5 g / L of disodium hydrogen phosphate, 0.1 g / L of sodium citrate, 0.1 g / L of magnesium sulfate, 0.5 g / L of sodium pyruvate and 0.006 g / L of ammonium ferric citrate.

[0026] Example 2: Culture medium for detecting common pathogenic bacteria of the Streptococcus family in bovine mastitis The culture medium of the present embodiment for detecting common pathogens of Streptococcus family in bovine mastitis 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 and potassium clavulanate; the composite chromogenic enzyme substrate is 5-bromo-6-chloro-3-indole-phosphate p-toluidine and 5-bromo-4-chloro-3-indole-β-D-glucuronide.

[0027] The added concentration of the composite inhibitor is 0.045 g / L, wherein the mass ratio of aztreonam, nalidixic acid and potassium clavulanate is 1:35:170; the added concentration of the composite chromogenic enzyme substrate is 0.15 g / L, wherein the mass ratio of 5-bromo-6-chloro-3-indole-phosphate toluidine and 5-bromo-4-chloro-3-indole-β-D-glucuronide is 1:1.5.

[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 17 g / L of tryptone, 3 g / L of yeast extract powder, 5.5 g of beef extract powder, 2.5 g / L of disodium hydrogen phosphate, 0.5 g / L of sodium citrate, 0.3 g / L of magnesium sulfate, 1 g / L of sodium pyruvate and 0.009 g / L of ammonium ferric citrate.

[0030] Example 3: Culture medium for detecting common pathogenic bacteria of the Streptococcus family in bovine mastitis The culture medium of the present embodiment for detecting common pathogens of Streptococcus family in bovine mastitis 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 and potassium clavulanate; the composite chromogenic enzyme substrate is 5-bromo-6-chloro-3-indole-phosphate p-toluidine and 5-bromo-4-chloro-3-indole-β-D-glucuronide.

[0031] The added concentration of the composite inhibitor is 0.05 g / L, wherein the mass ratio of aztreonam, nalidixic acid and potassium clavulanate is 1:40:190; the added concentration of the composite chromogenic enzyme substrate is 0.2 g / L, wherein the mass ratio of 5-bromo-6-chloro-3-indole-phosphate toluidine and 5-bromo-4-chloro-3-indole-β-D-glucuronide is 1:2.

[0032] The cold water gelatin addition concentration is 29 g / L, wherein the mass ratio of guar gum to xanthan gum is 1:1.5.

[0033] The nutrient mixture includes 22 g / L of tryptone, 4.5 g / L of yeast extract powder, 7.5 g / L of beef extract powder, 3.5 g / L of disodium hydrogen phosphate, 1 g / L of sodium citrate, 0.5 g / L of magnesium sulfate, 1.5 g / L of sodium pyruvate and 0.012 g / L of ammonium ferric citrate.

[0034] Example 4: Preparation of test pieces The structure of the test piece for common pathogenic bacteria of Streptococcus provided by the present invention is as follows: Figure 1As 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 common pathogens of the Streptococcus family in 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 common pathogenic bacteria of the Streptococcus family in bovine mastitis The test sheet for common pathogens of the Streptococcus family in 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 common pathogens of the Streptococcus family, 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 used for detecting common pathogens of the Streptococcus family 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 trypsin, 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, 0.09 g of sodium pyruvate, 0.045 g of the composite inhibitor, 0.013 g of the composite chromogenic enzyme substrate and 3.0 g of cold water gel. Among them, the composite inhibitor is composed of aztreonam, nalidixic acid and potassium clavulanate in a mass ratio of 1:38:175, the composite chromogenic enzyme substrate is composed of 5-bromo-6-chloro-3-indole-phosphate p-toluidine and 5-bromo-4-chloro-3-indole-β-D-glucuronide in a mass ratio of 1:1.5; and the cold water gel is composed of guar gum and xanthan gum in a mass ratio of 1:1.

[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, and the mixture is stirred evenly. The mixture is dispensed into the culture area of ​​the test piece, dried and covered with a transparent covering film to prepare a test piece for detecting common pathogens of the Streptococcus family in bovine mastitis.

[0039] The test piece for common pathogens of the Streptococcus family prepared in this example was verified by inoculating positive quality control strains and negative control bacteria. The results showed that Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis, as the core pathogens of bovine mastitis caused by the Streptococcus family, appeared green or blue-green, enterococci and other streptococci appeared purple-red, and Gram-negative bacteria such as Escherichia coli and Salmonella did not grow.

[0040] Example 6: A test piece for detecting common pathogenic bacteria of the Streptococcus family in bovine mastitis The test piece for common pathogens of the Streptococcus family in this embodiment is different from the test piece in Example 5 in the material of the test piece and the culture medium in its circular culture area. The bottom plate material of the test piece in this embodiment is PVC (polyvinyl chloride resin) synthetic paper, and the transparent coating is PET (polyethylene terephthalate). Each 100 mL of culture medium contains 2.2 g of tryptone, 0.45 g of yeast extract powder, 0.75 g of beef extract powder, 0.35 g of disodium hydrogen phosphate, 0.1 g of sodium citrate, 0.05 g of magnesium sulfate, 0.15 g of sodium pyruvate, 0.0012 g of ammonium ferric citrate, 0.04 g of composite inhibitor, 0.015 g of composite colorimetric enzyme substrate, and 3.5 g of cold water gel. Among them, the composite inhibitor is composed of aztreonam, nalidixic acid and potassium clavulanate in a mass ratio of 1:30:155, the composite chromogenic enzyme substrate is composed of 5-bromo-6-chloro-3-indole-phosphate p-toluidine and 5-bromo-4-chloro-3-indole-β-D-glucuronide in a mass ratio of 1:1.5; and the cold water gel is composed of guar gum and xanthan gum in a mass 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, and the mixture is stirred evenly. The mixture is dispensed into the culture area of ​​the test piece, dried, and covered with a transparent cover film to prepare a test piece for detecting common pathogens of the Streptococcus family in bovine mastitis.

[0042] The test piece for common pathogens of the Streptococcus family prepared in this example was verified by inoculating positive quality control strains and negative control bacteria. The results showed that Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis, as the core pathogens of bovine mastitis caused by the Streptococcus family, appeared green or blue-green, enterococci and other streptococci appeared purple-red, and Gram-negative bacteria such as Escherichia coli and Salmonella did not grow.

[0043] Each positive quality control bacteria was appropriately diluted and inoculated on the common pathogen test piece of Streptococcus prepared in this example and the TSA agar plate, respectively. The test results are shown in Table 1. The results show that the recovery rate (PR) of each positive quality control bacteria is above 0.6 (PR ≥ 0.5 is required to be qualified), and the negative control bacteria such as Staphylococcus aureus and Escherichia coli do not grow.

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

[0045] Note: "KGR test piece" refers to the test piece for common pathogens of the Streptococcus family provided in this embodiment.

[0046] Example 7: A test piece for detecting common pathogenic bacteria of the Streptococcus family in bovine mastitis The test piece for common pathogens of the Streptococcus family of this embodiment is different from the test piece of Example 6 in that the culture medium in the circular culture area contains 1.2 g of tryptone, 0.15 g of yeast extract powder, 0.35 g of beef extract powder, 0.15 g of disodium hydrogen phosphate, 0.01 g of sodium citrate, 0.01 g of magnesium sulfate, 0.05 g of sodium pyruvate, 0.0008 g of ammonium ferric citrate, 0.05 g of composite inhibitor, 0.02 g of composite chromogenic enzyme substrate, and 2.5 g of cold water gel per 100 mL of culture medium. Among them, the composite inhibitor is composed of aztreonam, nalidixic acid and potassium clavulanate in a mass ratio of 1:40:185, the composite chromogenic enzyme substrate is composed of 5-bromo-6-chloro-3-indole-phosphate p-toluidine and 5-bromo-4-chloro-3-indole-β-D-glucuronide in a mass ratio of 1:1; the cold water gel is composed of guar gum and xanthan gum in a final concentration ratio of 1:1.

[0047] The nutrient mixture, the composite inhibitor and the composite colorimetric 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 of ​​the test piece, dried, and covered with a transparent cover film to prepare a test piece for detecting common pathogens of the Streptococcus family in bovine mastitis.

[0048] The test piece for common pathogens of the Streptococcus family prepared in this example was verified by inoculating positive quality control strains and negative control bacteria. The results showed that Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis, as the core pathogens of bovine mastitis caused by the Streptococcus family, appeared green or blue-green, enterococci and other streptococci appeared purple-red, and Gram-negative bacteria such as Escherichia coli and Salmonella did not grow.

[0049] Each positive quality control bacteria was appropriately diluted and inoculated on the common pathogen test piece of Streptococcus prepared in this example and the TSA agar plate, respectively. The test results are shown in Table 2. The results show that the recovery rate (PR) of each positive quality control bacteria is above 0.6 (PR ≥ 0.5 is required to be qualified), and the negative control bacteria such as Staphylococcus aureus and Escherichia coli do not grow.

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

[0051] Note: The KGR test piece refers to the common pathogenic bacteria test piece of the Streptococcus family provided in this embodiment.

[0052] Test Example 1: Specificity test of test pieces for common pathogens of Streptococcus family The performance evaluation was performed using the test piece for common pathogenic bacteria of the Streptococcus family prepared in Example 5: two strains of Streptococcus agalactiae with strain numbers ATCC 13813 and ATCC12403, Streptococcus uberis, Streptococcus dysgalactiae, Streptococcus Macedonianus, Streptococcus Parisianus, Streptococcus pyogenes with strain number CMCC 32210, Enterococcus faecalis with strain number ATCC 29212, Enterococcus faecium with strain number ATCC 51559, Enterococcus cecum with strain number ATCC 43198, Enterococcus gallinarum with strain number ATCC49573, etc., three strains of isolated strains from the samples and 11 standard strains purchased from the bacterial culture collection management center were used as negative control bacteria (see Table 3 for details), and 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 CFU / mL was inoculated on the common pathogenic bacteria test piece of Streptococcus prepared in Example 5, and was inoculated on the TSA plate as a positive control, and cultured at 37°C for 18-24h. Depending on the growth of the bacteria, the plate culture time can be extended to 48h. The test results are shown in Table 3.

[0053] Table 3 Specificity experiments of 25 strains

[0054] Note: "+" represents growth, "-" represents no growth. "KGR test piece" refers to the test piece for common pathogenic bacteria of Streptococcus prepared in Example 5.

[0055] As shown in Table 3, on the test piece for common pathogens of the Streptococcus family, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis appear green or blue-green, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus faecium and other Streptococcus (family) species appear purple-red, and most of the negative control bacteria such as Staphylococcus species and Gram-negative bacteria do not grow. It can be seen that the test piece for common pathogens of the Streptococcus family provided by the present invention has a strong detection specificity and can effectively avoid the missed detection of pathogenic Streptococcus family species.

[0056] Furthermore, some positive control bacteria and negative control bacteria were diluted to an appropriate gradient and inoculated on the common pathogenic bacteria test piece of the Streptococcus family provided by the present invention and the competitive bovine mastitis diagnosis dish area III (hereinafter referred to as the four-dish area III), 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] Note: In zone III of the quadrant, Streptococcus agalactiae and Streptococcus dysgalactiae appear light blue, Streptococcus uberis appears dark blue, Enterococcus faecalis and other Enterococcus colonies appear larger purple or purple-red, and other streptococci appear blue-green, purple (or purple-red), white or transparent.

[0059] As can be seen from Table 4, Streptococcus agalactiae and Streptococcus dysgalactiae subsp. equine are blue-green on the Streptococcus test piece and the quad dish III zone provided by the present invention, and Streptococcus uberis (isolated from the sample) is green on the Streptococcus test piece of the present invention, but does not grow on the competing product, that is, causing a false negative test result of the competing product; the test Enterococcus faecalis and the Macedonian Streptococcus isolated from the sample are purple-red or light purple on the Streptococcus test piece and the competing product quad dish III zone provided in this embodiment; the test strains Shigella sonnei, Escherichia coli and the light green aerococcosis isolated from the sample are used as negative control bacteria. They do not grow on the common pathogenic bacteria test piece of the Streptococcus family of the present invention, but are blue-green or light blue in the quad dish III zone, causing a false positive test result. Therefore, the common pathogenic bacteria test piece of the Streptococcus family of the present invention has a stronger detection specificity for Streptococcus and Enterococcus species, can largely avoid the situation of false positive or false negative test results, and can quickly and accurately screen out pathogenic bacteria.

[0060] Test Example 2: Recovery Rate Experiment Five quality control bacteria, including two strains of Streptococcus agalactiae with strain numbers ATCC 13813 and ATCC 12403, Streptococcus dysgalactiae subsp. equine with strain number ATCC 35666, Streptococcus uberis isolated from the sample, and Enterococcus faecalis with strain number ATCC 29212, were revived and activated to the third generation using the method recommended by the strain collection center. The bacterial suspension was appropriately diluted to prepare a concentration of 3×10 1 ~3×102 The bacterial suspension of CFU / mL was inoculated on the common pathogenic bacteria test piece of Streptococcus prepared in Example 5, and was inoculated on the TSA agar plate and the third area of ​​the four-divided dish at the same time. Each strain was made into 3 parallels and cultured at 37°C for 18-24h. The plate culture can be extended to 48h according to the growth of the strain. The test results are shown in Table 5.

[0061] Table 5 Comparison of quality control strain test results

[0062] Note: "KGR test piece" refers to the test piece for common pathogens of Streptococcus prepared 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 for common pathogens of Streptococcus prepared in Example 5 or on the III zone of the quadrant.

[0063] As can be seen from Table 5, compared with the TSA plate test results, the 5 positive quality control bacteria standard strains appear blue-green or purple-red on the Streptococcus common pathogen test piece provided by the present invention, and the strain recovery rate (PR) is above 0.7 (quality control requires PR ≥ 0.5), and is higher than the recovery rate of the same strain in the quadrant III zone to varying degrees, which proves that the Streptococcus common pathogen test piece provided by the present invention has higher detection sensitivity and can avoid the occurrence of missed detection to a large extent.

[0064] Test Example 3: Detection of common pathogens of Streptococcus in natural samples Milk from cows with mastitis in different feeding areas of a ranch was collected and numbered. After the milk samples were appropriately diluted, they were inoculated on the test piece for common pathogens of the Streptococcus family and the third area of ​​the four-dish provided by the present invention. At least three gradients were selected, and three parallels were made 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". The specific results are shown in Table 6 (partial data).

[0065] Table 6

[0066] Note: This table only lists part of the sample test data, and other data are omitted here; " / " means that the experiment was not conducted, "√" means that it meets the requirements, "×" means that it does not meet the requirements, and "None" means that there is no colony growth.

[0067] As can be seen from Table 6, the test piece for common pathogens of the Streptococcus family provided by the present invention was compared with the quadrant dish III zone for a total of 121 samples, of which 12 were inconsistent, with a compliance rate of about 90.08%. The strains were identified by molecular biology, and the test results of the quadrant dish III zone had false negatives or false positives. Therefore, the test piece for common pathogens of the Streptococcus family provided by the present invention has stronger specificity, and compared with the quadrant dish, the test piece for common pathogens of the Streptococcus family provided by the present invention has lower cost, and can replace the quadrant dish for the detection of common pathogens of the Streptococcus family in bovine mastitis in pastures.

[0068] Test Example 4: Detection of Streptococcus dysgalactiae in artificially contaminated samples The Streptococcus dysgalactiae subsp. equine (hereinafter referred to as the test strain) numbered ATCC 35666 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 3.76×10 7 CFU / mL, take 1mL of the broth of the freshly cultured test bacteria standard strain (ATCC 35666) and dilute it in 9mL of sterile saline for 10-fold series 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 3.76 × 10 5 CFU / mL, 3.76×10 3 CFU / mL, 3.76×10 1 The artificially contaminated milk samples with different CFU / mL were named as high, medium and low concentration artificially contaminated samples respectively; according to the above method, three parallel contaminated samples were made for the three gradient spiked samples, and they were numbered 1, 2, 3, 4, 5, 6, 7, 8 and 9 from high to low.

[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 -3 (and 1 / 4×10 -3 )、1 / 2×10 -1 (and 1 / 4×0 -1 ), the original gradient dilution, 1 mL of the sample dilution was respectively taken and inoculated on the common pathogenic bacteria test piece of Streptococcus prepared in Example 5 and Columbia blood agar plate (hereinafter referred to as "blood plate") (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 7. The linear relationship between the test results of the common pathogenic bacteria test piece of Streptococcus provided by the present invention and the blood plate results is shown in Table 7. Figure 2 .

[0070] Table 7 Test results of artificially contaminated samples of sterile milk

[0071] Notes: 1. All test samples are fresh sterile milk with standard strain suspension. 2. "KGR test piece" refers to the test piece for common pathogens of Streptococcus prepared in Example 5. 3. "lg (blood plate)" refers to the logarithmic value of the colony value on the national standard Columbia blood agar plate. 4. "lg (KGR)" refers to the logarithmic value of the colony value on the test piece for common pathogens of Streptococcus prepared in Example 5.

[0072] As shown in Table 7, there was no significant difference in the results of the two methods for detecting the live bacteria content of Streptococcus dysgalactiae 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 = 1.0045x-0.0458, where R 2 =0.9997, that is, the degree of fit between the two is good and extremely correlated. In summary, the content of Streptococcus dysgalactiae in bovine milk detected by the common pathogenic bacteria test piece of the Streptococcus family provided by the present invention is highly consistent with the national standard plate method for detecting the content of Streptococcus dysgalactiae in bovine milk, and can be equally applied to the preliminary detection of the content of Streptococcus dysgalactiae in bovine mastitis.

[0073] Experimental Example 5: Detection of Enterococcus faecalis in artificially contaminated samples According to the method recommended by the Center for Microbial Culture Collection, Enterococcus faecalis numbered ATCC 29212 (hereinafter referred to as the “test strain”) was activated. The freshly activated bacterial suspension was counted according to GB 4789.2. The live bacterial content of the bacterial suspension was about 1.36×10 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 1.36 × 10 6 CFU / mL, 1.36×10 4 CFU / mL, 1.36×10 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.

[0074] 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 10 -4 , 10 -2 , the original multiple gradient dilution, 1 mL of the sample dilution was respectively taken and inoculated on the common pathogenic bacteria test piece of Streptococcus prepared in Example 5 and Columbia blood agar plate (hereinafter referred to as "blood plate") (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 common pathogenic bacteria test piece of Streptococcus provided by the present invention and the blood plate results is shown in Table 8. Figure 3 .

[0075] Table 8 Test results of Enterococcus faecalis content in sterile milk artificial contamination samples

[0076] Notes: 1. All test samples are fresh sterile milk with standard strain suspension. 2. "KGR test piece" refers to the test piece for common pathogens of Streptococcus prepared in Example 5. 3. "lg (blood plate)" refers to the logarithmic value of the colony value of the national standard Columbia blood plate. 4. "lg (KGR)" refers to the logarithmic value of the colony value on the test piece for common pathogens of Streptococcus prepared in Example 5.

[0077] As shown in Table 8, there was no significant difference in the results of the two methods for detecting the live bacteria content of Enterococcus faecalis in artificially contaminated samples No. 1-9 (i.e., P>0.05), and further Figure 3 It can be seen that the linear regression equation between the two is y = 0.9862x +0.0274, where R 2 =0.9997, that is, the degree of fit between the two is good and extremely correlated. In summary, the test piece for common pathogenic bacteria of the Streptococcus family provided by the present invention is used to detect the content of Enterococcus faecalis in bovine milk, which is highly consistent with the national standard method for testing the content of Enterococcus faecalis in bovine milk, and can be equally applied to the preliminary detection of the content of Enterococcus faecalis in bovine mastitis.

[0078] In summary, the test piece for detecting common pathogenic bacteria of the Streptococcus family in bovine mastitis provided by the present invention has a high detection rate for bacteria in natural samples and artificially contaminated samples. Compared with other diagnostic dishes 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 detection and has extremely high application value in the diagnosis of bovine mastitis.

[0079] 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 common pathogens of Streptococcus family 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 is aztreonam, nalidixic acid and potassium clavulanate; and the composite chromogenic enzyme substrate is 5-bromo-6-chloro-3-indole-phosphate p-toluidine and 5-bromo-4-chloro-3-indole-β-D-glucuronide.

2. A culture medium for detecting common pathogenic bacteria of Streptococcus family in bovine mastitis as claimed in 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 and clavulanate potassium is 1: (30-40): (150-190).

3. A culture medium for detecting common pathogenic bacteria of Streptococcus family in bovine mastitis as claimed in claim 1, characterized in that: The added concentration of the composite color-developing enzyme substrate is 0.1-0.2 g / L, wherein the mass ratio of 5-bromo-6-chloro-3-indole-toluidine phosphate to 5-bromo-4-chloro-3-indole-β-D-glucuronide is 1:(1-2).

4. A culture medium for detecting common pathogenic bacteria of Streptococcus family in bovine mastitis as claimed in 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 common pathogenic bacteria of Streptococcus family in bovine mastitis as described in any one of claims 1 to 4, characterized in that: The nutrient mixture includes 12-22 g / L of tryptone, 1.5-4.5 g / L of yeast extract powder, 3.5-7.5 g / L of beef extract powder, 1.5-3.5 g / L of disodium hydrogen phosphate, 0.1-1 g / L of sodium citrate, 0.1-0.5 g / L of magnesium sulfate, 0.5-1.5 g / L of sodium pyruvate and 0.006-0.012 g / L of ammonium ferric citrate.

6. A test piece, comprising a transparent covering film, a base plate, and a culture medium for detecting common pathogenic bacteria of the Streptococcus family 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 synthetic paper or polyvinyl chloride resin 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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