Staphylococcus aureus test piece and application thereof

Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a staphylococcus aureus test piece which structurally comprises a transparent covering film, a circular culture area and a bottom plate printed with square grids from top to bottom in sequence, the circular culture area comprises nutrient substances beneficial to growth of staphylococcus aureus, a composite inhibitor, a composite chromogenic enzyme substrate and cold water gel. The staphylococcus aureus test strip disclosed by the invention can be used for rapidly detecting staphylococcus aureus in food and processing environment thereof, human and animal medical sampling samples and other samples, has the characteristics of strong specificity, high sensitivity, short detection period and easiness in observation, and has extremely high development value and wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial detection, and in particular relates to a Staphylococcus aureus test piece and application thereof. Background Art

[0002] Staphylococcus aureus ( Staphylococcus aureus ), a common Gram-positive spherical bacterium belonging to the genus Staphylococcus, is the most dangerous of all common Staphylococci and can cause infections in multiple organs including the skin, lungs, heart, and bones, and can even lead to death in severe cases. Staphylococcus aureus is ubiquitous in nature, from air and water sources to dust, and even in human and animal excrement. Therefore, the risk of food being contaminated by Staphylococcus aureus is relatively high. Food may be contaminated at multiple links: for example, during food processing, cooking or sales, workers may also become carriers of the bacteria.

[0003] Staphylococcus aureus is one of the main pathogens that cause suppurative infections in humans. It can not only cause local suppuration, but also may cause serious diseases such as pneumonia, pseudomembranous colitis, pericarditis, and even systemic infections such as sepsis and septicemia. If contaminated food is left at a temperature of 20℃ to 30℃ for 3 to 5 hours, it may produce enterotoxins sufficient to cause food poisoning. After ingesting these enterotoxins, people usually experience poisoning symptoms such as vomiting, abdominal pain, and diarrhea within 2 to 5 hours.

[0004] In view of this, in order to prevent the threat of Staphylococcus aureus to human health, it is particularly important to quickly and accurately detect the content of Staphylococcus aureus in food. The National Food Safety Standard Food Microbiology Test Staphylococcus aureus Test (GB4789.10-2016) recommends the use of plate method for qualitative and quantitative detection, but this method has certain complexity and cost in actual operation. At present, there are many Staphylococcus aureus detection products on the domestic and foreign markets. Foreign brands include the color culture medium of CHROMagar™ of France, the test pieces of 3M of the United States and DNP of Japan, while domestic brands include Qingdao Haibo color culture medium, Guangdong Huankai color culture medium and test pieces, Meizheng biological test pieces, Oasis biochemical test pieces, etc., but after verification and comparison, these products still have some shortcomings in actual application. The specificity of the existing test strips is still low, the sensitivity is insufficient, and the accuracy needs to be improved; the color development culture medium needs to be poured and the appropriate temperature is considered when using it. Although the specificity has been improved, the operation is still cumbersome and the cost is high; the test strips may cause false positive or false negative results due to sample applicability issues, affecting the accuracy of the test; the colonies grown on the test strips made of non-woven fabrics are not clear, affecting the count, and the test strips are not tightly attached to the film. After being cultured at 37°C, they are easy to lose water and dry, which also affects the accuracy of the test. Therefore, the development of more efficient and accurate detection methods is of great significance to ensuring food safety and public health. Summary of the invention

[0005] To this end, the present invention aims to provide a Staphylococcus aureus test piece with good specificity, high sensitivity, high accuracy and easy observation.

[0006] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions: An object of the present invention is to provide a Staphylococcus aureus test piece, the structure of which, from top to bottom, comprises a transparent covering film, a culture area and a bottom plate printed with a square grid, wherein the culture area is arranged on the bottom plate, and the transparent covering film covers the bottom plate; the culture area is filled with a Staphylococcus aureus selective growth medium, and the medium comprises a nutrient mixture, a composite inhibitor, a composite color developing enzyme substrate and a cold water gel.

[0007] Preferably, the nutrient mixture comprises 5-50 g / L of tryptone, 1-10 g / L of yeast extract powder, 1-40 g of beef extract powder, 0.1-10 g / L of sodium pyruvate, 1-20 g / L of disodium hydrogen phosphate, 0.05-2 g / L of sodium citrate, 0.01-3 g / L of magnesium sulfate and 0.005-0.01 g / L of ammonium ferric citrate.

[0008] Preferably, the composite inhibitor is a mixture of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium; the composite inhibitor is 0.0001-0.0005 g / L of aztreonam, 0.0005-0.003 g / L of antibacterial peptide, 0.00005-0.0003 g / L of trimethoprim and 0.0005-0.003 g / L of fosfomycin sodium.

[0009] Preferably, in the composite inhibitor, the mass ratio of aztreonam, bacteriostatic peptide, trimethoprim and fosfomycin sodium is 1: (1-8): (0.5-3): (1-15). Further preferably, the mass ratio of aztreonam, bacteriostatic peptide, trimethoprim and fosfomycin sodium is 1: (3.5-5.5): (0.5-0.8): (5-6.5). Most preferably, the mass ratio of aztreonam, bacteriostatic peptide, trimethoprim and fosfomycin sodium is 1: 5: 0.5: 6.

[0010] Preferably, the composite chromogenic enzyme substrate is a mixture of 5-bromo-6-chloro-3-indolyl-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-4-chloro-3-indole-α-pyranoglucoside; the composite chromogenic enzyme substrate is 0.05-0.2 g / L of 5-bromo-6-chloro-3-indolyl-toluidine phosphate, 0.05-0.3 g / L of 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside, and 0.05-0.1 g / L of 5-bromo-4-chloro-3-indole-α-pyranoglucoside.

[0011] Preferably, in the composite chromogenic enzyme substrate, the mass ratio of 5-bromo-6-chloro-3-indolyl-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-4-chloro-3-indole-α-pyranoglucoside is (1.3~1.5):(1.7~1.8):1.

[0012] Preferably, the size of the transparent coating is 98 mm×80 mm, the bonding width between the transparent coating and the base plate is 12 mm, the thickness between the transparent coating and the adhesive is 0.076 mm; the material of the transparent coating is one of PE and PET.

[0013] Preferably, the culture area is circular, the diameter of the culture area is 50 mm, the culture area 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 is 50.5 mm; the culture area is 15.5 mm away from the left edge of the bottom plate, 15.5 mm away from the right edge of the bottom plate, 19-20 mm away from the lower edge of the bottom plate, and the height is 0.70-0.78 mm; a square grid is provided on the bottom plate in the culture area, and the size of the small squares in the square grid is 10 mm×10 mm.

[0014] Preferably, the size of the base plate is 93 mm×80 mm, and the base plate is one of PP synthetic paper, PH synthetic paper, PET synthetic paper, PS synthetic paper, and PVC synthetic paper.

[0015] Another object of the present invention is to provide a use of the above-mentioned Staphylococcus aureus test piece in detecting the content of Staphylococcus aureus.

[0016] The embodiments of the present invention have the following advantages: 1. The Staphylococcus aureus test piece provided by the present invention has a structure of a transparent covering film, a circular culture area, and a bottom plate printed with a square grid from top to bottom. The culture components in the circular culture area are tryptone (5-12 g / L), yeast extract powder (1-5 g / L), beef extract powder (1-10 g), sodium pyruvate (0.1-4 g / L), disodium hydrogen phosphate (1-7 g / L), sodium citrate (0.05-0.6 g / L), magnesium sulfate (0.01-0.5 g / L), composite inhibitor (0.001-0.05 g / L), composite chromogenic enzyme substrate (0.06-3 g / L), cold water gel (6-40 g / L), and ammonium ferric citrate (0.008-0.01 g / L). The test piece is a prefabricated culture system, which does not require high-pressure sterilization of agar culture medium in advance, and is convenient to carry considering the appropriate pouring temperature.

[0017] 2. The composite inhibitor in the Staphylococcus aureus test piece provided by the present invention can effectively inhibit the growth of non-target bacteria, and the composite chromogenic enzyme substrate is more selective for target bacteria. Therefore, the present invention has the characteristics of high sensitivity and strong specificity, greatly improving the detection accuracy and reducing 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 1The schematic diagram of the structure of the Staphylococcus aureus test piece of the present invention; wherein 1-transparent covering film, 2-circular culture area, 3-bottom plate; Figure 2 This is a comparison chart of the test results of the Staphylococcus aureus test piece of the present invention and the number of national standard plates; Figure 3 The linear relationship between the plate (BP) counting method and the Staphylococcus aureus test piece method of the present invention in detecting the artificial contamination results of skim milk powder samples. DETAILED DESCRIPTION

[0021] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1 like Figure 1 As shown, the Staphylococcus aureus test piece includes a transparent coating 1, a circular culture area 2, and a bottom plate 3. The circular culture area is composed of nutrients, a composite inhibitor, a composite colorimetric enzyme substrate, and a cold water gel. The bottom plate is PP (polypropylene) synthetic paper, and the coating is made of PE (polyethylene) material.

[0023] Example 2 The Staphylococcus aureus test piece includes a transparent covering film, a circular culture area, and a bottom plate. The circular culture area is composed of nutrients, a composite inhibitor, a composite colorimetric enzyme substrate, and a cold water gel. The bottom plate is PH (polyaniline) synthetic paper, and the covering film is made of PE (polyethylene) material.

[0024] Example 3 The Staphylococcus aureus test piece includes a transparent covering film, a circular culture area, and a bottom plate. The circular culture area is composed of nutrients, a composite inhibitor, a composite color-developing enzyme substrate, and a cold water gel. The bottom plate is PET (polyethylene terephthalate) synthetic paper, and the covering film is made of PET (polyethylene terephthalate) material.

[0025] Example 4 The Staphylococcus aureus test piece includes a transparent covering film, a circular culture area, and a bottom plate. The circular culture area is composed of nutrients, a composite inhibitor, a composite color developing enzyme substrate, and a cold water gel. The bottom plate is PS (polystyrene) synthetic paper, and the covering film is PET (polyethylene terephthalate) material.

[0026] Example 5 The Staphylococcus aureus test piece includes a transparent covering film, a circular culture area, and a bottom plate. The circular culture area is composed of nutrients, a composite inhibitor, a composite color developing enzyme substrate, and a cold water gel. The bottom plate is PVC (polyvinyl chloride resin) synthetic paper, and the covering film is PET (polyethylene terephthalate) material.

[0027] Example 6 The Staphylococcus aureus test piece was prepared using the material of Example 1. The composition of 100 mL of culture medium in the circular culture area was: 1.5 g of tryptone, 0.5 g of yeast extract powder, 1.0 g of beef extract powder, 0.15 g of sodium pyruvate, 0.255 g of disodium hydrogen phosphate, 0.025 g of sodium citrate, 0.0035 g of magnesium sulfate, 0.0009 g of ammonium ferric citrate, 0.00032 g of composite inhibitor, 0.045 g of composite chromogenic enzyme substrate, and 2.8 g of cold water gel. Among them, the composite inhibitor is composed of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium with a final concentration ratio of 1:5:0.5:6, and the composite chromogenic enzyme substrate is composed of 5-bromo-6-chloro-3-indolyl-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-4-chloro-3-indole-α-pyranoglucoside with a final concentration of 1.3:1.7:1; the cold water gel is a mixed gum of xanthan gum and guar gum in equal proportions.

[0028] The above basic components are fully dissolved in 100 mL of solvent, and then cold water is added to gel. After stirring evenly, the mixture can be packaged and dried to prepare Staphylococcus aureus test pieces.

[0029] The Staphylococcus aureus test piece prepared in Example 6 was verified by inoculating the quality control strain and the negative control bacteria. The results showed that the positive quality control bacteria appeared purple-red, and the negative control bacteria appeared blue-green, or showed no growth or no growth at all.

[0030] The performance of the Staphylococcus aureus test piece of the present invention was evaluated using the test piece prepared in Example 6: 1. Specificity experiments: Five strains of Staphylococcus aureus with strain numbers ATCC 27217, ATCC 25923, ATCC 6538P, ATCC 6538, and NBRC 12732, two strains of Staphylococcus epidermidis (ATCC 12228, CMCC 26069), one strain of Staphylococcus lentus (CICC 21602), one strain of Staphylococcus intermedius (isolated from the sample), one strain of Staphylococcus xylosus (ATCC 29971), one strain of Staphylococcus saprophyticus (ATCC 49453), three strains of Listeria monocytogenes (ATCC 33090, ATCC 19111, ATCC 35897), five strains of Enterococcus (ATCC 29212, ATCC 19434, ATCC 43198, ATCC 49573, ATCC 700327), 1 Bacillus (CMCC63303) and 10 Gram-negative bacteria (ATCC 10031, ATCC 43864, CICC 10450, ATCC 51114, ATCC13048, ATCC 25922, ATCC 700728, ATCC 29544, ATCC 12022, ATCC 27511) were activated, and the bacterial suspension was appropriately diluted to prepare a concentration of 1×10 2 ~3×10 2 The bacterial suspension of CFU / mL was inoculated on the Staphylococcus aureus test piece prepared in Example 6, 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 1.

[0031] Table 1 Specificity experiments of 30 strains

[0032] Note: The colonies of Staphylococcus aureus on the KGR Staphylococcus aureus test sheet are purple-red, and the colonies on the 3M test sheet are purple. "+" represents growth, "-" represents no growth, and " / " represents that the test was not performed. "KGR test sheet" refers to the Staphylococcus aureus test sheet provided by the present invention.

[0033] As shown in Table 1, the two strains of Staphylococcus epidermidis and Enterococcus faecium both appear purple-red on the 3M test piece, which is easily confused with Staphylococcus aureus, but appear green on the Staphylococcus aureus test piece provided by the present invention, which is well distinguished from Staphylococcus aureus and has high detection specificity; most Gram-negative bacteria do not grow on the test piece of the present invention. It can be seen that the Staphylococcus aureus test piece provided by the present invention has a stronger detection specificity and can largely avoid the occurrence of false positive detection.

[0034] Furthermore, some positive control bacteria and negative control bacteria were diluted to an appropriate gradient and then spiked into the New Zealand milk powder sample. The test pieces provided by the present invention, 3M test pieces, Oasis test pieces, and Meizheng test pieces were used for detection, and the colony growth and color development were compared and analyzed. The results are shown in Table 2.

[0035] Table 2 Test results of spiked New Zealand milk powder samples in four test pieces

[0036] Note: “-” means no colony growth, “+” means colony growth, “few” means the number of colonies is less than that of the positive control plate, “purple”, “green” or “blue-green” refers to the color of the colonies observed by the naked eye; “KGR test piece” refers to the Staphylococcus aureus test piece provided by the present invention.

[0037] As can be seen from Table 2, the three competing test pieces were inoculated with interfering bacteria after spiked, and all had colony color after culture. The colony color on the 3M test piece is clear and distinct, but the specificity is poor, and it is difficult to effectively distinguish Staphylococcus aureus; the specificity of the Oasis biochemical test piece is better than that of the 3M, but Staphylococcus aureus is difficult to distinguish from Staphylococcus epidermidis, Staphylococcus intermedius and Bacillus cereus. At the same time, the colony color on its test piece is fuzzy and the individual is not clear, making it difficult to observe. The colony color of Staphylococcus intermedius on the Meizheng test piece after spiked milk powder causes interference, and it is difficult to make a good distinction. Therefore, in comparison, the Staphylococcus aureus test piece provided by the present invention has the advantages of both specificity and accuracy. The interfering bacteria are spiked into the milk powder, and the colony color after inoculation and culture can be clearly distinguished from the target bacteria, that is, it will not cause a false positive test result, and the individual colonies are clearly colored, which is convenient for counting, and the accuracy is extremely high, which effectively reduces the false positive rate and improves the detection accuracy.

[0038] 2. Recovery test Five strains of Staphylococcus aureus or Staphylococcus aureus subspecies standard strains with strain numbers of ATCC 27217, ATCC 25923, ATCC 6538P, ATCC 6538, and NBRC 12732 were revived and activated to the third generation using the method recommended by the bacterial culture collection center, and the bacterial suspension was appropriately diluted to prepare a concentration of 2×10 1 ~2×10 2 The bacterial suspension of CFU / mL was inoculated on the Staphylococcus aureus test piece prepared in Example 6, and was also inoculated on TSA agar plate, BP agar plate, competitor 3M Staphylococcus aureus test piece and competitor DNP Staphylococcus aureus test piece. Three parallels were made for each bacterial species and cultured at 37°C for 18-24h. The plate culture time can be extended to 48h depending on the growth of the bacterial species. The test results are shown in Table 3.

[0039] Table 3 Comparison of quality control strain test results

[0040] Note: "KGR test piece" refers to the Staphylococcus aureus test piece provided by the present invention, and the denominator of the PR value is the number of colonies grown on the TSA plate.

[0041] As shown in Table 3, compared with the TSA plate test results, the five standard strains of Staphylococcus aureus appear purple-red on the Staphylococcus aureus test plate provided by the present invention, and the strain recovery rates (PR) are all above 0.7, which are higher than the recovery rates of competing test plates. Therefore, it can be used equivalently to the national standard Baird-Parker plate.

[0042] 3. Minimum detection limit experiment 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 1.1×10 9 CFU / mL, the bacterial suspension was diluted 10-fold to 10 -10 Gradient. From 10 -6 The gradient inoculation was started, and the national standard BP plate and the Staphylococcus aureus test piece provided by the present invention were inoculated respectively. Three parallels were inoculated in each gradient and each method. The results are shown in Table 4.

[0043] Table 4 Minimum detection limit test for Staphylococcus aureus

[0044] As shown in Table 4, the minimum detection limit of the Staphylococcus aureus test plate provided by the present invention is equivalent to that of the national standard plate, both of which can reach 1 CFU / mL.

[0045] 4. Detection of Staphylococcus aureus in natural samples With reference to GB 29921-2021 "National Food Safety Standard Limits of Pathogenic Bacteria in Pre-packaged Foods", food categories with requirements for Staphylococcus aureus content were selected in a targeted manner, and the natural samples were tested simultaneously using the Baird-Parker national standard plate and the Staphylococcus aureus test piece provided by the present invention. At least 3 gradients were selected, and 3 parallel tests were performed for each gradient. The sampling and processing methods of the samples were referred to GB 4789.1-2016 "National Food Safety Standard General Rules for Food Microbiology Examination", and the test results analysis is shown in Table 5.

[0046] Table 5 Detection results of Staphylococcus aureus in natural samples

[0047] It can be seen from Table 5 that when testing natural samples, the test results of the Staphylococcus aureus test piece provided by the present invention are consistent with those of the national standard plate, and the sensitivity is comparable.

[0048] Further, different samples were processed with reference to national standards (GB / T4789.17-2003, GB / T4789.18-2003, GB / T4789.19-2003, GB / T4789.20-2003, etc.) and then appropriately diluted. 1 mL of the dilution was respectively transferred to a Baird-Parker plate and a Staphylococcus aureus test piece of the present invention and incubated for 24 hours. Black-core colonies grew on the Baird-Parker plate, but transparent rings were not easily observed. Purple-red colonies or no colonies grew on the Staphylococcus aureus test piece of the present invention. The test piece was more intuitive in observing the test results and had stronger specificity.

[0049] 5. Detection of Staphylococcus aureus in Artificially Contaminated Samples Take 1 mL of freshly cultured Staphylococcus aureus standard strain (ATCC 25923) broth and dilute it 10-fold in 9 mL of sterile saline to make 10 4 CFU / mL and 10 5 CFU / mL working bacterial suspension. The bacterial suspension was counted according to GB4789.2, and the concentration of the bacterial suspension was 5.2×10 4 CFU / mL and 5.5×10 5 CFU / mL, take 1mL of the two concentrations of bacterial suspension and make freeze-dried bacterial powder according to relevant requirements. 4 CFU / mL of freeze-dried bacterial powder was added to the high-protein skim and high-calcium milk powders with serial numbers 1-5 as artificial contamination samples, and another sample with a concentration of 5.5×10 5 CFU / mL of freeze-dried bacterial powder was added to high-protein skim high-calcium milk powder with serial numbers 6-10 as artificial contaminated samples to prepare samples containing 10 CFU / mL of Staphylococcus aureus. 4 CFU / 25g and 10 5 Artificially contaminated samples with CFU / 25g were stored at room temperature (20~25℃) for 2 weeks.

[0050] The artificial contamination samples of high-protein skim and high-calcium milk powder were selected at 10-fold, 100-fold, and 1000-fold dilutions. 1 mL of the sample dilution was inoculated in a Baird-Parker plate and cultured at 36°C ± 1°C for 48 hours. At the same time, 1 mL of the sample dilution was inoculated on the Staphylococcus aureus test piece of the present invention and cultured at 36°C ± 1°C for 24 hours. The results are shown in Table 6. The linear relationship between the plate (BP) counting method and the Staphylococcus aureus test piece method of the present invention in detecting artificial contamination samples of skim milk powder is shown in Table 6. Figure 3 .

[0051] Table 6 Test results of artificially contaminated samples of high-protein skim high-calcium milk powder

[0052] Notes: 1. The test samples are all finished products of "Yili high-protein skim high-calcium milk powder" with production batch number 20211216B and standard strain bacterial suspension, which were artificially contaminated and tested on May 21, 2022. 2. "lg (BP)" refers to the logarithm of the colony value of the national standard BP plate, and "lg (KGR)" refers to the logarithm of the colony value on the KGR Staphylococcus aureus test piece.

[0053] As shown in Table 6, there is no significant difference between the values ​​of samples 1-10 detected by the national standard GB 4789.10-2016 plate count method and the Staphylococcus aureus test piece provided by the present invention (P>0.05). Figure 3 It can be seen that the linear regression equation is y=0.9799x+0.0417, where R 2 =0.9987, that is, the fitting degree between the two is high and the correlation is high. In summary, the results of testing the content of Staphylococcus aureus using the Staphylococcus aureus test piece provided by the present invention are highly consistent with the results of testing the content of Staphylococcus aureus using the national standard method, and can be equivalently adopted.

[0054] Example 7 The Staphylococcus aureus test piece was prepared using Example 1, and the composition of 100 mL of culture medium in the circular culture area was: 1.0 g of tryptone, 0.5 g of yeast extract powder, 0.8 g of beef extract powder, 0.1 g of sodium pyruvate, 0.25 g of disodium hydrogen phosphate, 0.03 g of sodium citrate, 0.003 g of magnesium sulfate, 0.001 g of ammonium ferric citrate, 0.00025 g of composite inhibitor, 0.025 g of composite chromogenic enzyme substrate, and 2.5 g of cold water gelatin. Among them, the composite inhibitor is composed of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium with a final concentration ratio of 1:4.5:0.6:6; the composite chromogenic enzyme substrate is composed of 5-bromo-6-chloro-3-indolyl-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-4-chloro-3-indole-α-pyranoglucoside with a final concentration of 1.5:1.8:1; and the cold water gel is composed of xanthan gum and carrageenan with a final concentration ratio of 2:1.

[0055] The above basic components are fully dissolved in 100 mL of solvent, and then cold water is added to gel. After stirring evenly, the mixture can be packaged and dried to prepare Staphylococcus aureus test pieces.

[0056] The Staphylococcus aureus test piece prepared in Example 7 was inoculated with bacteria. The results showed that the inoculated target bacteria (positive quality control bacteria) were all purple-red, and the interfering bacteria (negative quality control bacteria) were blue-green or did not grow, and could be clearly distinguished from the target bacteria. The growth rates of the target bacteria are shown in Table 7, and the growth rate PR meets the requirement of ≥0.7.

[0057] Table 7

[0058] Example 8 The Staphylococcus aureus test piece was prepared using Example 1, and the composition of 100 mL of culture medium in the circular culture area was: 1.2 g of tryptone, 0.4 g of yeast extract powder, 0.6 g of beef extract powder, 0.15 g of sodium pyruvate, 0.2 g of disodium hydrogen phosphate, 0.035 g of sodium citrate, 0.0025 g of magnesium sulfate, 0.0008 g of ammonium ferric citrate, 0.00028 g of composite inhibitor, 0.028 g of composite chromogenic enzyme substrate, and 2.6 g of cold water gel. Among them, the composite inhibitor is composed of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium with a final concentration ratio of 1:4.5:0.6:6, the composite chromogenic enzyme substrate is composed of 5-bromo-6-chloro-3-indolyl-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-4-chloro-3-indole-α-pyranoglucoside with a final concentration of 1.4:1.75:1; the cold water gel is composed of sodium carboxymethyl cellulose and guar gum with a final concentration ratio of 1:1.

[0059] The above basic components are fully dissolved in 100 mL of solvent, and then cold water is added to gel. After stirring evenly, the mixture can be packaged and dried to prepare Staphylococcus aureus test pieces.

[0060] The Staphylococcus aureus test piece prepared in Example 8 was inoculated with bacteria. The results showed that the interfering bacteria Enterococcus gallus had no colony growth on the test piece, and did not affect the detection of the target bacteria (positive quality control bacteria) in the sample. Other interfering bacteria showed blue-green or no color and no growth. The growth rate of the target bacteria is shown in Table 8. The growth rates of target bacteria No. 1, 2, and 4 met the requirement of ≥0.7, and the growth rate of target bacteria No. 3 was slightly less than 0.7.

[0061] Table 8

[0062] Example 9 The Staphylococcus aureus test piece was prepared using Example 1, and the composition of 100 mL of culture medium in the circular culture area was: 1.4 g of tryptone, 0.5 g of yeast extract powder, 1.0 g of beef extract powder, 0.25 g of sodium pyruvate, 0.3 g of disodium hydrogen phosphate, 0.04 g of sodium citrate, 0.0035 g of magnesium sulfate, 0.0009 g of ammonium ferric citrate, 0.00032 g of composite inhibitor, 0.025 g of composite chromogenic enzyme substrate, and 2.7 g of cold water gel. Among them, the composite inhibitor is composed of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium with a final concentration ratio of 1:4.5:0.6:6, the composite chromogenic enzyme substrate is composed of 5-bromo-6-chloro-3-indolyl-toluidine phosphate, 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-4-chloro-3-indole-α-pyranoglucoside with a final concentration of 1.3:1.7:1; the cold water gel is composed of xanthan gum and carrageenan with a final concentration ratio of 1:1.

[0063] The above basic components are fully dissolved in 100 mL of solvent, and then cold water is added to gel. After stirring evenly, the mixture can be packaged and dried to prepare Staphylococcus aureus test pieces.

[0064] The Staphylococcus aureus test piece prepared in Example 9 was inoculated with bacteria for verification. The results showed that the interfering bacteria (negative quality control bacteria) enterococci were blue-green, and other interfering bacteria did not show color or did not grow, which did not affect the detection of the target bacteria (positive quality control bacteria) in the sample; after inoculation and cultivation of the target bacteria, the growth rate is shown in Table 9, and the growth rate of each target bacteria meets the requirement of ≥0.7.

[0065] Table 9

[0066] Example 10 The Staphylococcus aureus test piece is prepared using the material of Example 1. The composition of the culture medium is the same as that of Example 6 except for the composite inhibitor and the composite chromogenic enzyme substrate. In this embodiment, the composite inhibitor is composed of aztreonam, antibacterial peptide, trimethoprim, and fosfomycin sodium at a final concentration ratio of 1:5.5:0.8:6.5, and the composite chromogenic enzyme substrate is composed of 5-bromo-6-chloro-3-indolyl-phosphotoluidine salt, 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-4-chloro-3-indole-α-pyranoglucoside at a final concentration of 1.4:1.75:1. The preparation method is the same as that of Example 6.

[0067] The Staphylococcus aureus test piece prepared in Example 10 was verified by inoculation. The results showed that the interfering bacteria Bacillus cereus showed blue-green color, the sample isolated bacteria Staphylococcus intermedius did not grow, and the color and growth conditions were not changed after being added to the milk sample, that is, they did not affect the detection of the target bacteria (positive quality control bacteria) in the sample. After inoculation and cultivation of the target bacteria, the growth rate is shown in Table 10, and the growth rate of each target bacteria meets the requirement of ≥0.7.

[0068] Table 10

[0069] Embodiment 11 The Staphylococcus aureus test piece is prepared using the test piece material of Example 1. The culture medium composition is the same as Example 6 except for the composite inhibitor and the composite chromogenic enzyme substrate. In this embodiment, the composite inhibitor is composed of aztreonam, antibacterial peptide, trimethoprim, and fosfomycin sodium at a final concentration ratio of 1:3.5:0.5:5, and the composite chromogenic enzyme substrate is composed of 5-bromo-6-chloro-3-indolyl-phosphotoluidine salt, 5-bromo-4-chloro-3-indole-β-D-pyranoglucoside and 5-bromo-4-chloro-3-indole-α-pyranoglucoside at a final concentration of 1.4:1.75:1. The preparation method is the same as Example 6.

[0070] The results showed that the quality control bacteria with colony growth had clear color and distinct individuals; the interfering bacteria (negative control bacteria) were blue-green, or grew without color, or did not grow at all. The growth rate of the target bacteria (positive quality control bacteria) is shown in Table 11. The growth rate of the two target bacteria met the requirement of ≥0.7.

[0071] Table 11

[0072] Comparative Example 1 The Staphylococcus aureus test piece of this comparative example is the same as the Staphylococcus aureus test piece of Example 6 in other structures, components and compositions, except that the composition of the composite inhibitor is different. The composite inhibitor used in this comparative example is composed of sodium azide, aztreonam, deferoxamine and lithium chloride, and the final concentration ratio of sodium azide, aztreonam, deferoxamine and lithium chloride is 1:5:0.5:6.

[0073] The Staphylococcus aureus test piece prepared in Comparative Example 1 was verified by inoculation. The results showed that the interfering bacteria Staphylococcus epidermidis and Bacillus cereus could grow normally and appear purple-red, which was consistent with the color of the positive quality control bacteria, and could easily cause false positive test results.

[0074] Comparative Example 2 The Staphylococcus aureus test piece of this comparative example is the same as the Staphylococcus aureus test piece of Example 6 in other structures, components and compositions, except that the composite inhibitor composition is different. The composite inhibitor used in this comparative example is composed of aztreonam, nisin, trimethoprim and nitrofurantoin. The final concentration ratio of aztreonam, nisin, trimethoprim and nitrofurantoin is 1:5:0.5:6.

[0075] The Staphylococcus aureus test piece prepared in Comparative Example 2 was verified by inoculation. The results showed that the interfering bacteria Bacillus cereus was not completely inhibited, and colonies would grow and appear purple-red. The sample isolated bacteria Staphylococcus intermedius grew normally and appeared purple-red. After being spiked into the milk sample, the inoculated colonies grew more. Staphylococcus xylosus ATCC 29971 would have colonies growing around the test piece and appear purple-red, which could easily cause false positive test results.

[0076] In summary, the Staphylococcus aureus test piece of the present invention can effectively distinguish Staphylococcus aureus and has the characteristics of high specificity. Compared with other test pieces on the market, it has the advantages of high specificity, easy observation, and high accuracy of test results. In addition, the minimum detection limit of this test piece is 1 CFU / mL, and the sensitivity is high. The Staphylococcus aureus test piece of the present invention overcomes the defects of the prior art, has obvious advantages, and has a very high application prospect in food detection.

[0077] 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 Staphylococcus aureus test piece, characterized in that: Its structure from top to bottom includes a transparent covering film, a culture area and a bottom plate printed with a square grid, wherein the culture area is arranged on the bottom plate, and the transparent covering film covers the bottom plate; the culture area is filled with a Staphylococcus aureus selective growth culture medium, and the culture medium includes a nutrient mixture, a composite inhibitor, a composite color-developing enzyme substrate and a cold water gel.

2. The Staphylococcus aureus test piece according to claim 1, wherein: The nutrient mixture comprises 5-50 g / L of tryptone, 1-10 g / L of yeast extract powder, 1-40 g of beef extract powder, 0.1-10 g / L of sodium pyruvate, 1-20 g / L of disodium hydrogen phosphate, 0.05-2 g / L of sodium citrate, 0.01-3 g / L of magnesium sulfate and 0.005-0.01 g / L of ammonium ferric citrate.

3. The Staphylococcus aureus test piece according to claim 1, wherein: The composite inhibitor is a mixture of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium; the composite inhibitor is 0.0001-0.0005 g / L of aztreonam, 0.0005-0.003 g / L of antibacterial peptide, 0.00005-0.0003 g / L of trimethoprim and 0.0005-0.003 g / L of fosfomycin sodium.

4. The Staphylococcus aureus test piece according to claim 3, wherein: In the composite inhibitor, the mass ratio of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium is 1: (3.5-5.5): (0.5-0.8): (5-6.5).

5. The Staphylococcus aureus test piece according to claim 1, wherein: The composite color-developing enzyme substrate is a mixture of 5-bromo-6-chloro-3-indolyl-toluidine phosphate salt, 5-bromo-4-chloro-3-indolyl-β-D-pyranoglucoside and 5-bromo-4-chloro-3-indolyl-α-pyranoglucoside; the composite color-developing enzyme substrate is 0.05-0.2 g / L of 5-bromo-6-chloro-3-indolyl-toluidine phosphate salt, 0.05-0.3 g / L of 5-bromo-4-chloro-3-indolyl-β-D-pyranoglucoside and 0.05-0.1 g / L of 5-bromo-4-chloro-3-indolyl-α-pyranoglucoside.

6. The Staphylococcus aureus test piece according to claim 5, wherein: In the composite color-developing enzyme substrate, the mass ratio of 5-bromo-6-chloro-3-indolyl-toluidine phosphate, 5-bromo-4-chloro-3-indolyl-β-D-pyranoglucoside and 5-bromo-4-chloro-3-indolyl-α-pyranoglucoside is (1.3-1.5): (1.7-1.8):

1.

7. The Staphylococcus aureus test piece according to claim 1, wherein: The size of the transparent coating is 98 mm×80 mm, the bonding width between the transparent coating and the base plate is 12 mm, and the thickness of the transparent coating and the adhesive is 0.076 mm; the material of the transparent coating is one of PE and PET.

8. The Staphylococcus aureus test piece according to claim 1, wherein: The culture area is circular, with a diameter of 50 mm. The culture area 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 is 50.5 mm; the culture area is 15.5 mm away from the left edge of the bottom plate, 15.5 mm away from the right edge of the bottom plate, 19-20 mm away from the lower edge of the bottom plate, and the height is 0.70-0.78 mm; a square grid is provided on the bottom plate in the culture area, and the size of the small squares in the square grid is 10 mm×10 mm.

9. The Staphylococcus aureus test piece according to claim 1, wherein: The size of the base plate is 93 mm×80 mm, and the base plate is one of PP synthetic paper, PH synthetic paper, PET synthetic paper, PS synthetic paper, and PVC synthetic paper.

10. Use of the Staphylococcus aureus test piece according to any one of claims 1 to 9 in detecting the content of Staphylococcus aureus.

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