CN119177162A - Staphylococcus aureus test piece and application thereof - Google Patents
Staphylococcus aureus test piece and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganism detection, and particularly relates to a staphylococcus aureus test piece, which comprises a transparent coating, a circular culture area and a bottom plate printed with square grids from top to bottom in sequence, wherein the circular culture area comprises nutrient substances, a composite inhibitor, a composite chromogenic enzyme substrate and cold water gelable substances which are beneficial to the growth of staphylococcus aureus. The staphylococcus aureus test piece disclosed by the invention can be used for rapidly detecting staphylococcus aureus in samples such as food and processing environments thereof, medical sampling of human and animals and the like, 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
The invention belongs to the technical field of microorganism detection, and particularly relates to a staphylococcus aureus test piece and application thereof.
Background
Staphylococcus aureus (Staphylococcus aureus), a common gram-positive spherical bacterium belonging to the genus staphylococcus, is a more dangerous one of all common staphylococci, can cause infections of various organs including skin, lung, heart, bones, and even cause death in severe cases. Staphylococcus aureus is ubiquitous in nature and can be found in traces from air, water sources, dust, and even human and animal excretions, so that the risk of contamination of food products with staphylococcus aureus is relatively high, and food products can be contaminated in a number of links, such as during food processing, cooking, or sales, and staff can become carriers of bacteria.
Staphylococcus aureus is one of the main pathogens causing suppurative infection of human beings, and can cause local suppuration, severe diseases such as pneumonia, pseudomembranous enteritis, pericarditis and the like, and even systemic infection such as septicemia, sepsis and the like. Contaminated foods may produce enterotoxins sufficient to cause food poisoning by being left at a temperature of 20 ℃ to 30 ℃ for 3 to 5 hours. After ingestion of these enterotoxins, people often develop toxic symptoms such as vomiting, abdominal pain, diarrhea, etc. within 2 to 5 hours.
In view of this, in order to prevent the threat of staphylococcus aureus to human health, it is important to rapidly and accurately detect the content of staphylococcus aureus in foods. The national food microbiology test for food safety staphylococcus aureus (GB 4789.10-2016) recommends the use of a plate method for qualitative and quantitative detection, but this method has a certain complexity and cost in practical operation. At present, a plurality of staphylococcus aureus detection products exist in the markets at home and abroad. Foreign brands such as a developing medium of Lepidium meyenii (CHROMagarTM) of French family, a test piece of American 3M and Japanese DNP, and domestic brands including a Qingdao sea Boeing developing medium, a developing medium and a test piece of Guangdong Crohn, a American biological test piece, an oasis biochemical test piece and the like, have some defects in practical application. The existing test piece is still low in specificity, insufficient in sensitivity and low in accuracy, a plate needs to be poured and proper temperature is considered when a chromogenic culture medium is used, although the specificity is improved, the operation is still complicated and the cost is high, the test piece can cause false positive or false negative results due to the problem of sample applicability to influence the accuracy of detection, the individual colony growing on the test piece prepared from non-woven fabrics is unknown, the counting is influenced, the lamination is not tight, water loss and drying are easy to occur after the culture at 37 ℃ and the detection accuracy are also influenced. Therefore, developing a more efficient and accurate detection method has important significance for guaranteeing food safety and public health.
Disclosure of Invention
Therefore, the invention aims to provide a staphylococcus aureus test piece which has good specificity, high sensitivity and high accuracy and is convenient to observe.
In order to achieve the above object, the embodiment of the present invention provides the following technical solutions:
The invention aims to provide a staphylococcus aureus test piece, which sequentially comprises a transparent coating, a culture area and a bottom plate printed with square grids from top to bottom, wherein the culture area is arranged on the bottom plate, the transparent coating is covered on the bottom plate, a staphylococcus aureus selective growth culture medium is filled in the culture area, and the culture medium comprises a nutrient substance mixture, a composite inhibitor, a composite chromogenic enzyme substrate and cold water gel.
Preferably, the nutrient mixture comprises 5-50g/L tryptone, 1-10g/L yeast extract powder, 1-40g beef extract powder, 0.1-10g/L sodium pyruvate, 1-20g/L disodium hydrogen phosphate, 0.05-2g/L sodium citrate, 0.01-3g/L magnesium sulfate and 0.005-0.01g/L ferric ammonium citrate.
Preferably, the compound inhibitor is a mixture of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium, and the compound inhibitor is 0.0001-0.0005g/L of aztreonam, 0.0005-0.003g/L of antibacterial peptide, 0.00005-0.0003g/L of trimethoprim and 0.0005-0.003g/L of fosfomycin sodium.
Preferably, in the compound inhibitor, the mass ratio of aztreonam to antibacterial peptide to trimethoprim to fosfomycin sodium is 1:1-8:0.5-3:1-15. Further preferably, the mass ratio of aztreonam to antibacterial peptide to trimethoprim to fosfomycin sodium is 1:3.5-5.5:0.5-0.8:5-6.5. Most preferably, the mass ratio of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium is 1:5:0.5:6.
Preferably, the compound chromogenic enzyme substrate is a mixture of 5-bromo-6-chloro-3-indolyl-toluamide phosphate, 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside and 5-bromo-4-chloro-3-indolyl-alpha-glucopyranoside, and the compound chromogenic enzyme substrate is 0.05-0.2g/L of 5-bromo-6-chloro-3-indolyl-toluamide phosphate, 0.05-0.3g/L of 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside and 0.05-0.1g/L of 5-bromo-4-chloro-3-indolyl-alpha-glucopyranoside.
Preferably, in the compound chromogenic enzyme substrate, the mass ratio of 5-bromo-6-chloro-3-indolyl-toluamide phosphate, 5-bromo-4-chloro-3-indole-beta-D-glucopyranoside and 5-bromo-4-chloro-3-indole-alpha-glucopyranoside is (1.3-1.5) to (1.7-1.8) to 1.
Preferably, the size of the transparent coating is 98mm multiplied by 80mm, the bonding width of the transparent coating and the bottom plate is 12mm, the thickness of the transparent coating and the thickness of the adhesive are 0.076mm, and the transparent coating is made of one of PE and PET.
Preferably, the culture area is circular, the diameter of the culture area is 50mm, the culture area is formed by coating and solidifying hot melt adhesive, the hot melt adhesive is vinyl acetate, the inner diameter of a rubber ring is 50mm, the outer diameter of the rubber ring is 50.5mm, the distance between the culture area and the left side edge of the bottom plate is 15.5mm, the distance between the culture area and the right side edge of the bottom plate is 15.5mm, the distance between the culture area and the lower side edge of the bottom plate is 19-20mm, the height is 0.70-0.78mm, square grids are arranged on the bottom plate and positioned in the culture area, and the small square grids in the square grids are 10mm multiplied by 10mm.
Preferably, the size of the bottom plate is 93mm×80mm, and the bottom plate is one of PP synthetic paper, PH synthetic paper, PET synthetic paper, PS synthetic paper, and PVC synthetic paper.
The invention also aims to provide an application of the staphylococcus aureus test piece in detecting the content of staphylococcus aureus.
The embodiment of the invention has the following advantages:
1. The staphylococcus aureus test piece provided by the invention has the structure that a transparent coating film, a circular culture area and a bottom plate printed with square grids are sequentially arranged from top to bottom, wherein culture components in the circular culture area comprise 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), a compound inhibitor (0.001-0.05 g/L), a compound chromogenic enzyme substrate (0.06-3 g/L), cold water gelable (6-40 g/L) and ferric ammonium citrate (0.008-0.01 g/L), and the test piece is a prefabricated culture system, does not need to be sterilized agar culture medium in advance, and is convenient to carry in consideration of a dumping proper temperature.
2. The composite inhibitor in the staphylococcus aureus test piece provided by the invention can effectively inhibit the growth of non-target bacteria, and the composite chromogenic enzyme substrate has stronger selectivity to the target bacteria, so that the invention has the characteristics of high sensitivity and strong specificity, greatly improves the detection accuracy, and reduces the false positive rate and the false negative rate.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the scope of the invention.
FIG. 1 is a schematic diagram of a test piece of Staphylococcus aureus of the present invention, wherein, the 1-transparent coating film, the 2-circular culture area and the 3-bottom plate;
FIG. 2 is a graph showing the comparison of the detection results of the staphylococcus aureus test piece of the present invention with the number of national standard plates;
FIG. 3 is a linear relationship between the plate (BP) count method and the detection result of the skim milk powder artificial contamination sample by the Staphylococcus aureus test piece method of the present invention.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
As shown in figure 1, the staphylococcus aureus test piece comprises a transparent coating film 1, a circular culture area 2 and a bottom plate 3, wherein the circular culture area consists of nutritional ingredients, a composite inhibitor, a composite chromogenic enzyme substrate and cold water gelable materials, the bottom plate is PP (polypropylene) synthetic paper, and the coating film is PE (polyethylene) material.
Example 2
The staphylococcus aureus test piece comprises a transparent coating, a circular culture area and a bottom plate, wherein the circular culture area consists of nutritional ingredients, a composite inhibitor, a composite chromogenic enzyme substrate and cold water gelable material, the bottom plate is PH (polyaniline) synthetic paper, and the coating is PE (polyethylene) material.
Example 3
The staphylococcus aureus test piece comprises a transparent coating, a circular culture area and a bottom plate, wherein the circular culture area consists of nutritional ingredients, a composite inhibitor, a composite chromogenic enzyme substrate and cold water gelable materials, the bottom plate is made of PET (polyethylene terephthalate) synthetic paper, and the coating is made of PET (polyethylene terephthalate) materials.
Example 4
The staphylococcus aureus test piece comprises a transparent coating film, a circular culture area and a bottom plate, wherein the circular culture area consists of nutritional ingredients, a composite inhibitor, a composite chromogenic enzyme substrate and cold water gelable materials, the bottom plate is PS (polystyrene) synthetic paper, and the coating film is made of PET (polyethylene terephthalate).
Example 5
The staphylococcus aureus test piece comprises a transparent coating film, a circular culture area and a bottom plate, wherein the circular culture area consists of nutritional ingredients, a composite inhibitor, a composite chromogenic enzyme substrate and cold water gelable materials, the bottom plate is made of PVC (polyvinyl chloride resin) synthetic paper, and the coating film is made of PET (polyethylene terephthalate) materials.
Example 6
A staphylococcus aureus test piece is prepared by using the material of the embodiment 1, and 100mL of culture medium in a circular culture area comprises 1.5g of tryptone, 0.5g of yeast extract powder, 1.0g of beef extract powder, 0.15g of sodium pyruvate, 0.255g of disodium hydrogen phosphate, 0.025g of sodium citrate, 0.0035g of magnesium sulfate, 0.0009g of ammonium ferric citrate, 0.00032g of a compound inhibitor, 0.045g of a compound chromogenic enzyme substrate and 2.8g of cold water gel. The compound inhibitor consists of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium with the final concentration ratio of 1:5:0.5:6, and the compound chromogenic enzyme substrate consists of 5-bromo-6-chloro-3-indolyl-toluamide salt, 5-bromo-4-chloro-3-indole-beta-D-glucopyranoside and 5-bromo-4-chloro-3-indole-alpha-glucopyranoside with the final concentration ratio of 1.3:1.7:1, wherein the cold water gelable is mixed gum of xanthan gum and guar gum in equal proportion.
And (3) fully dissolving the basic components with 100mL of solvent, adding cold water to gel, uniformly stirring, and then subpackaging and drying to prepare the staphylococcus aureus test piece.
The staphylococcus aureus test piece prepared in the example 6 is verified by inoculating a quality control strain and a negative control bacterium, and the result shows that the positive quality control bacterium shows mauve, the negative control bacterium shows bluish green, or shows no color growth or no growth at all.
The performance of the staphylococcus aureus test piece of the present invention was evaluated by using the test piece prepared in example 6:
1. Specificity experiments:
The bacterial suspensions were appropriately diluted to prepare bacterial suspensions having a concentration of 1×10 2~3×102 CFU/mL, which were inoculated into the staphylococcus aureus test pieces prepared in example 6, respectively, and inoculated into TSA plates as positive controls, and incubated at 37 ℃ for 18-24 hours, 3 listeria (ATCC 33090, ATCC 19111, ATCC 35897), 5 enterococci (ATCC 29212, ATCC 19434, ATCC 43198, ATCC 49573, ATCC 700327), 1 bacillus (CMCC 63303), and 10 gram-negative bacteria （ATCC 10031、ATCC 43864、CICC 10450、ATCC 51114、ATCC 13048、ATCC 25922、ATCC 700728、ATCC 29544、ATCC 12022、ATCC 27511）, respectively, and the bacterial suspensions were suitably diluted to prepare staphylococcus aureus test pieces having a concentration of 1×10 2~3×102 CFU/mL, which were inoculated into the staphylococcus aureus test pieces prepared in example 6, respectively, and incubated at 37 ℃ for 18-24 hours, depending on the growth conditions of the bacterial strains, and the test results were shown in table 1.48.
TABLE 1 Strain specificity experiments for 30 strains
Note that Staphylococcus aureus colonies appeared purple on KGR Staphylococcus aureus test pieces and colonies appeared purple on 3M test pieces. "+" represents growth, "-" represents no growth, and "/" represents no test. "KGR test strip" refers to a test strip of Staphylococcus aureus provided by the present invention.
As shown in Table 1,2 strains of staphylococcus epidermidis and enterococcus faecium are both purple red on the 3M test piece and are easily confused with staphylococcus aureus, but the staphylococcus aureus test piece provided by the invention is green, so that good distinction from staphylococcus aureus is realized, the detection specificity is high, and most gram negative bacteria do not grow on the test piece. Therefore, the staphylococcus aureus test piece provided by the invention has stronger detection specificity, and can avoid the occurrence of false positive detection to a large extent.
Further, after diluting part of positive control bacteria and negative control bacteria to proper gradients, adding the diluted positive control bacteria and negative control bacteria into a New Zealand milk powder sample, respectively detecting by using the test piece, the 3M test piece, the oasis test piece and the Meijian test piece provided by the invention, and comparing and analyzing colony growth and color development conditions, wherein the results are shown in Table 2.
TABLE 2 detection results of labeled New Zealand milk powder samples in four test pieces
Note that "-" indicates colony growth, "+" indicates colony growth, "less" indicates a smaller number of colonies than the positive control plate, "mauve", "green" or "bluish green" indicates a visible color of colonies, and "KGR test piece" indicates a staphylococcus aureus test piece provided by the present invention.
As can be seen from Table 2, the three competitive test pieces were inoculated with the labeled interfering bacteria, and all of them developed colonies after cultivation. The bacterial colony on the 3M test piece has clear color development and distinct individuals, but has poor specificity, so that the effective distinction of staphylococcus aureus is difficult to realize, the specificity of the oasis biochemical test piece has better effect than that of 3M test piece, but the staphylococcus aureus is difficult to distinguish from staphylococcus epidermidis, staphylococcus intermedia and bacillus cereus, and meanwhile, the bacterial colony on the test piece has fuzzy color development, distinct individuals and difficult observation. The interference is caused by colony color development after the standard milk powder is added to the staphylococcus intermedia on the beauty test piece, and good distinction is difficult to achieve. Therefore, compared with the staphylococcus aureus test piece provided by the invention, the staphylococcus aureus test piece has the advantages of specificity and accuracy, the labeled interfering bacteria are added into the milk powder, the color development of the bacterial colony after inoculation and culture can be obviously distinguished from the color development of the target bacteria, namely, the false positive of the detection result can not be caused, the color development of the bacterial colony is obvious, the counting is convenient, the accuracy is extremely high, the false positive rate is effectively reduced, and the detection accuracy is improved.
2. Recovery test
5 Staphylococcus aureus or staphylococcus aureus subspecies of strain numbers ATCC 27217, ATCC 25923, ATCC 6538P, ATCC 6538 and NBRC 12732 are recovered and activated to 3 rd generation by a recommended method of a strain collection center, the bacterial suspension is properly diluted to prepare bacterial suspension with the concentration of 2X 10 1~2×102 CFU/mL, the bacterial suspension is respectively inoculated to a staphylococcus aureus test piece prepared in the example 6, and simultaneously inoculated to a TSA agar plate, a BP agar plate, a 3M staphylococcus aureus test piece of a competing product and a DNP staphylococcus aureus test piece of a competing product, each strain is subjected to 3 parallel and is cultivated at a constant temperature of 37 ℃ for 18-24 hours, and the plate can be prolonged to 48 hours according to the growth condition of the strain, and the detection results are shown in Table 3.
TABLE 3 quality control strain detection results alignment
Note that "KGR test piece" refers to the Staphylococcus aureus test piece provided by the invention, and the denominator of PR values is the colony number grown on the TSA plate.
As shown in Table 3, compared with the TSA flat-plate detection result, the 5 staphylococcus aureus standard strains show mauve on the staphylococcus aureus test piece provided by the invention, and the strain recovery rate (PR) is above 0.7 and higher than the recovery rate of the competitive test piece, so that the strain can be equivalently utilized with the national standard Baird-Parker flat-plate.
3. Minimum detection limit experiment
The staphylococcus aureus subspecies aureoviridae strain (ATCC 25923) is recovered and activated to prepare a bacterial suspension with the concentration of 10 9 CFU/mL, the bacterial content of the freshly activated bacterial suspension is about 1.1X10 9 CFU/mL, and the bacterial suspension is subjected to 10-time gradient dilution to 10 -10 gradient. Inoculation is started from 10 -6 gradients, and the inoculation is respectively carried out on a national standard BP plate and a staphylococcus aureus test piece provided by the invention, and three parallel are inoculated on each mode of each gradient, and the results are shown in Table 4.
TABLE 4 minimum detection limit test for Staphylococcus aureus
As shown in Table 4, the minimum detection limit of the staphylococcus aureus test piece provided by the invention is equivalent to that of a national standard flat plate, and can reach 1 CFU/mL.
4. Detection of staphylococcus aureus in natural sample
Referring to GB 29921-2021, food category with requirements on staphylococcus aureus content is selected pertinently, a Baird-Parker national standard plate and the staphylococcus aureus test piece provided by the invention are used for simultaneously detecting respective samples, at least 3 gradients are selected, each gradient is 3 parallel, the sampling and processing method of the samples is referred to GB 4789.1-2016, food safety national standard food microbiology inspection rule, and the analysis of detection results is shown in Table 5.
TABLE 5 detection results of Staphylococcus aureus in Natural samples
As can be seen from Table 5, the test piece for Staphylococcus aureus provided by the invention has the same detection result as the national standard plate and has the same sensitivity when detecting natural samples.
Further, different samples are treated and properly diluted by referring to national standards (GB/T4789.17-2003, GB/T4789.18-2003, GB/T4789.19-2003, GB/T4789.20-2003 and the like), 1mL of the diluted solution is respectively transferred to a Baird-Parker plate and the staphylococcus aureus test piece provided by the invention, and the diluted solution is cultured for 24 hours, wherein black nuclear colony growth is arranged on the Baird-Parker plate, but a transparent ring is not easy to observe, and the staphylococcus aureus test piece provided by the invention is provided with a mauve colony or sterile drop growth, so that the test piece is more visual in observation of detection results and has stronger specificity.
5. Detection of staphylococcus aureus in artificially contaminated samples
1ML of freshly cultured staphylococcus aureus standard strain (ATCC 25923) broth was serially diluted 10-fold in 9mL of sterile physiological saline to prepare working bacterial suspensions of 10 4 CFU/mL and 10 5 CFU/mL, respectively. Counting bacterial suspensions by GB 4789.2, wherein the concentration of the bacterial suspensions is 5.2X10. 10 4 CFU/mL and 5.5X10. 10 5 CFU/mL respectively, and preparing 1mL of the bacterial suspensions with the two concentrations into freeze-dried bacterial powder according to related requirements. Adding 5.2X10 4 CFU/mL of lyophilized powder into 1-5 high protein defatted high calcium milk powder as artificial pollution sample, adding 5.5X10 5 CFU/mL of lyophilized powder into 6-10 high protein defatted high calcium milk powder as artificial pollution sample, and storing at room temperature (20-25deg.C) for 2 weeks.
The high protein defatted high calcium milk powder artificially contaminated sample is diluted 10 times, 100 times and 1000 times. 1mL of the sample dilution was inoculated in a Baird-Parker plate and incubated at 36.+ -. 1 ℃ for 48 hours, while 1mL of the sample dilution was inoculated on the staphylococcus aureus test piece of the present invention and incubated at 36.+ -. 1 ℃ for 24 hours. The results are shown in Table 6, and the linear relationship between the plate (BP) count method and the detection result of the skim milk powder artificial contamination sample by the staphylococcus aureus test piece method of the present invention is shown in FIG. 3.
TABLE 6 detection results of high protein defatted high calcium milk powder Artificial contaminated samples
Remark 1, test samples are finished products of illite high protein defatted high calcium milk powder with production lot number 20211216B, standard bacterial strain bacterial suspension is added, and artificial pollution is carried out and detected at 2022.05.21. 2. "lg (BP)" refers to the log of the colony value of the national standard BP plate, and "lg (KGR)" refers to the log of the colony value on the KGR staphylococcus aureus test piece.
As can be seen from table 6, the 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 is not significant (P > 0.05), and further from fig. 3, the linear regression equation is y=0.9799x+0.0417, where R 2 =0.9987, that is, the fitting degree between the two is higher, and the correlation degree is high. In conclusion, the staphylococcus aureus content is detected by using the staphylococcus aureus test piece provided by the invention, and the consistency of the result of detecting the staphylococcus aureus content by using the national standard method is high, so that the staphylococcus aureus test piece can be equivalently adopted.
Example 7
A staphylococcus aureus test piece was prepared by using example 1, and 100mL of the medium in the circular culture area consisted of 1.0g of tryptone, 0.5g of yeast extract, 0.8g of beef extract, 0.1g of sodium pyruvate, 0.25g of disodium hydrogen phosphate, 0.03g of sodium citrate, 0.003g of magnesium sulfate, 0.001g of ferric ammonium citrate, 0.00025g of a compound inhibitor, 0.025g of a compound chromogenic enzyme substrate and 2.5g of a cold water gelable gel. The compound inhibitor consists of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium with the final concentration ratio of 1:4.5:0.6:6, the compound chromogenic enzyme substrate consists of 5-bromo-6-chloro-3-indolyl-methylbenzylamine salt, 5-bromo-4-chloro-3-indole-beta-D-glucopyranoside and 5-bromo-4-chloro-3-indole-alpha-glucopyranoside with the final concentration ratio of 1.5:1.8:1, and the cold water gelable consists of xanthan gum and carrageenan with the final concentration ratio of 2:1.
And (3) fully dissolving the basic components with 100mL of solvent, adding cold water to gel, uniformly stirring, and then subpackaging and drying to prepare the staphylococcus aureus test piece.
The test pieces of staphylococcus aureus prepared in example 7 are subjected to inoculation verification, and the results show that the inoculated target bacteria (positive quality control bacteria) are all purple red, each interference bacteria (negative quality control bacteria) is blue-green or does not grow, the interference bacteria can be obviously distinguished from the target bacteria, the growth rate of each target bacteria is shown in Table 7, and the growth rate PR meets the requirement of more than or equal to 0.7.
TABLE 7
Example 8
A staphylococcus aureus test piece was prepared by using example 1, and 100mL of the medium in the circular culture area consisted of 1.2g of tryptone, 0.4g of yeast extract powder, 0.6g of beef extract powder, 0.15g of sodium pyruvate, 0.2g of disodium hydrogen phosphate, 0.035g of sodium citrate, 0.0025g of magnesium sulfate, 0.0008g of ferric ammonium citrate, 0.00028g of a complex inhibitor, 0.028g of a complex chromogenic enzyme substrate and 2.6g of cold water gelable gel. The compound inhibitor consists of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium with the final concentration ratio of 1:4.5:0.6:6, the compound chromogenic enzyme substrate consists of 5-bromo-6-chloro-3-indolyl-toluamide salt, 5-bromo-4-chloro-3-indole-beta-D-glucopyranoside and 5-bromo-4-chloro-3-indole-alpha-glucopyranoside with the final concentration ratio of 1.4:1, and the cold water gelable consists of carboxymethyl cellulose sodium and guar gum with the final concentration ratio of 1:1.
And (3) fully dissolving the basic components with 100mL of solvent, adding cold water to gel, uniformly stirring, and then subpackaging and drying to prepare the staphylococcus aureus test piece.
The test piece of staphylococcus aureus prepared in the embodiment 8 is subjected to inoculation verification, and the result shows that the enterococcus gallinarum asepsis drop growth of the interference bacteria on the test piece does not influence the detection of target bacteria (positive quality control bacteria) in a sample, other interference bacteria show bluish green or do not develop or grow, the growth rate of the target bacteria is shown in tables 8,1, 2 and 4, the growth rate of the target bacteria meets the requirement of more than or equal to 0.7, and the growth rate of the target bacteria of No. 3 is slightly less than 0.7.
TABLE 8
Example 9
A staphylococcus aureus test piece was prepared by using example 1, and 100mL of the medium in the circular culture area consisted of 1.4g of tryptone, 0.5g of yeast extract, 1.0g of beef extract, 0.25g of sodium pyruvate, 0.3g of disodium hydrogen phosphate, 0.04g of sodium citrate, 0.0035g of magnesium sulfate, 0.0009g of ferric ammonium citrate, 0.00032g of a complex inhibitor, 0.025g of a complex chromogenic enzyme substrate and 2.7g of a cold water gelable gel. The compound inhibitor consists of aztreonam, antibacterial peptide, trimethoprim and fosfomycin sodium with the final concentration ratio of 1:4.5:0.6:6, the compound chromogenic enzyme substrate consists of 5-bromo-6-chloro-3-indolyl-phosphate toluamide salt, 5-bromo-4-chloro-3-indole-beta-D-glucopyranoside and 5-bromo-4-chloro-3-indole-alpha-glucopyranoside with the final concentration ratio of 1.3:1, and the cold water gelable consists of xanthan gum and carrageenan with the final concentration ratio of 1:1.
And (3) fully dissolving the basic components with 100mL of solvent, adding cold water to gel, uniformly stirring, and then subpackaging and drying to prepare the staphylococcus aureus test piece.
The test pieces of staphylococcus aureus prepared in the example 9 are subjected to inoculation verification, and the result shows that each enterococcus of the interfering bacteria (negative quality control bacteria) shows blue-green color, the growth of other interfering bacteria does not develop color or grow, the detection of target bacteria (positive quality control bacteria) in a sample is not affected, the growth rate after the inoculation of the target bacteria is shown in the table 9, and the growth rate of each target bacteria meets the requirement of more than or equal to 0.7.
TABLE 9
Example 10
The staphylococcus aureus test piece is prepared by using the material of the example 1, and the composition of the culture medium is the same as the example 6 except for the composite inhibitor and the composite chromogenic enzyme substrate. In this example, the complex inhibitor consisted of aztreonam, antibacterial peptide, trimethoprim, and fosfomycin sodium in a final concentration ratio of 1:5.5:0.8:6.5, and the complex chromogenic enzyme substrate consisted of 5-bromo-6-chloro-3-indolyl-phosphate toluamide salt, 5-bromo-4-chloro-3-indol-beta-D-glucopyranoside, and 5-bromo-4-chloro-3-indol-alpha-glucopyranoside in a final concentration ratio of 1.4:1.75:1. The preparation method is the same as in example 6.
The test piece of staphylococcus aureus prepared by using the embodiment 10 is verified by inoculation, and the result shows that the bacillus cereus of the interfering bacteria shows bluish green, the staphylococcus in the middle of the sample separation bacteria does not grow, the color development and the growth condition are not changed after the sample separation bacteria are added to the milk sample, namely the detection of target bacteria (positive quality control bacteria) in the sample is not affected, the growth rate is shown in table 10 after the inoculation of the target bacteria for culture, and the growth rate of each target bacteria meets the requirement of more than or equal to 0.7.
Table 10
Example 11
The staphylococcus aureus test piece is prepared by adopting the test piece material of the example 1, and the composition of the culture medium is the same as the example 6 except for the composite inhibitor and the composite chromogenic enzyme substrate. In this example, the complex inhibitor consisted of aztreonam, antibacterial peptide, trimethoprim, fosfomycin sodium in a final concentration ratio of 1:3.5:0.5:5, and the complex chromogenic enzyme substrate consisted of 5-bromo-6-chloro-3-indolyl-phosphate toluamide salt, 5-bromo-4-chloro-3-indole-beta-D-glucopyranoside, and 5-bromo-4-chloro-3-indole-alpha-glucopyranoside in a final concentration ratio of 1.4:1.75:1. The preparation method is the same as in example 6.
The results show that the quality control bacteria colonies with colony growth are clear in color development and individual definition, and each interference bacteria (negative control bacteria) is blue-green in color development, or does not develop at all, the growth rate of target bacteria (positive quality control bacteria) is shown in Table 11, and the growth rates of two target bacteria meet the requirement of more than or equal to 0.7.
TABLE 11
Comparative example 1
The staphylococcus aureus test piece of the comparative example has the same structure, components and composition as the rest of the staphylococcus aureus test piece of the example 6, and is different in composition from the composite inhibitor, wherein the composite inhibitor adopted in the comparative example consists of sodium azide, aztreonam, deferoxamine and lithium chloride, and the final concentration ratio of the sodium azide to the aztreonam to the deferoxamine to the lithium chloride is 1:5:0.5:6.
The staphylococcus aureus test piece prepared by the comparative example 1 is verified by inoculation, and the result shows that the staphylococcus epidermidis and bacillus cereus which are interfering bacteria can grow normally and have purple red color, and the color is consistent with that of positive quality bacteria control, thus being easy to cause false positive of detection results.
Comparative example 2
The staphylococcus aureus test piece of the comparative example has the same structure, components and composition as the rest of the staphylococcus aureus test piece of the example 6, except that the composition of the composite inhibitor is different, and the composite inhibitor adopted in the comparative example consists of aztreonam, nisin, trimethoprim and nitrofurantoin. Wherein the final concentration ratio of aztreonam, nisin, trimethoprim and nitrofurantoin is 1:5:0.5:6.
The test piece of staphylococcus aureus prepared by using the comparative example 2 is verified by inoculation, and the result shows that the interference bacteria bacillus cereus is not thoroughly inhibited, bacterial colony growth exists, the bacterial colony grows and shows purple red, staphylococcus in the middle of sample separation bacteria normally grows to show purple red, inoculated bacterial colony grows more after a milk sample is marked, staphylococcus xylosus ATCC 29971 can have bacterial colony growth at the periphery of the test piece and shows purple red, and false positive of a detection result is easily caused.
In conclusion, the staphylococcus aureus test piece provided by the invention can effectively distinguish staphylococcus aureus and has the characteristic of high specificity. Compared with other test pieces on the market, the test piece has the advantages of high specificity, easiness in observation and high accuracy of detection results, and in addition, the lowest detection limit of the test piece is 1CFU/mL, and the sensitivity is high. The staphylococcus aureus test piece overcomes the defects of the prior art, has obvious advantages and has extremely high food detection application prospect.
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
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| CN119552939A (en) * | 2025-02-01 | 2025-03-04 | 北京纳百生物科技有限公司 | Chromogenic medium for detecting gram-negative bacteria in bovine mastitis and test board thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002079486A2 (en) * | 2001-03-30 | 2002-10-10 | Biomerieux S.A. | Milieux de detection specific de staphylococcus aureus |
| WO2011090662A2 (en) * | 2009-12-30 | 2011-07-28 | 3M Innovative Properties Company | Rapid detection of molds that produce glucose oxidase |
| CN103290094A (en) * | 2013-05-30 | 2013-09-11 | 广州绿洲生化科技股份有限公司 | Staphylococcus aureus chromogenic medium and test piece thereof |
| WO2017131062A1 (en) * | 2016-01-29 | 2017-08-03 | 大日本印刷株式会社 | Medium for detecting staphylococcus aureus, sheet for detecting s. aureus comprising same, and method for detecting s. aureus using same |
| CN109371101A (en) * | 2018-11-23 | 2019-02-22 | 黑龙江省绿色食品科学研究院 | A kind of staphylococcus aureus chromogenic culture medium and detection testing piece |
-
2024
- 2024-11-22 CN CN202411678075.5A patent/CN119177162A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002079486A2 (en) * | 2001-03-30 | 2002-10-10 | Biomerieux S.A. | Milieux de detection specific de staphylococcus aureus |
| CN1518602A (en) * | 2001-03-30 | 2004-08-04 | �������˹��ĭ�ۺ���ɷ�����˾ | Staphylococcus aureus-specific detection media and identifying and/or counting method using the same |
| WO2011090662A2 (en) * | 2009-12-30 | 2011-07-28 | 3M Innovative Properties Company | Rapid detection of molds that produce glucose oxidase |
| CN102713617A (en) * | 2009-12-30 | 2012-10-03 | 3M创新有限公司 | Rapid detection of molds that produce glucose oxidase |
| CN103290094A (en) * | 2013-05-30 | 2013-09-11 | 广州绿洲生化科技股份有限公司 | Staphylococcus aureus chromogenic medium and test piece thereof |
| WO2017131062A1 (en) * | 2016-01-29 | 2017-08-03 | 大日本印刷株式会社 | Medium for detecting staphylococcus aureus, sheet for detecting s. aureus comprising same, and method for detecting s. aureus using same |
| CN109371101A (en) * | 2018-11-23 | 2019-02-22 | 黑龙江省绿色食品科学研究院 | A kind of staphylococcus aureus chromogenic culture medium and detection testing piece |
Non-Patent Citations (4)
| Title |
|---|
| 汤光: "《实用外科药物手册》", 31 January 2001, 中国医药科技出版社, pages: 87 * |
| 汤光: "《现代药物学》", 31 March 1997, 中国医药科技出版社, pages: 81 * |
| 胡功政，邱银生: "《家禽常用药物及其合理使用》", 31 August 2021, 中国医药科学技术出版社, pages: 401 - 94 * |
| 郑继方等: "《兽医药物临床配伍与禁忌》", 30 September 2006, 金盾出版社, pages: 108 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119552939A (en) * | 2025-02-01 | 2025-03-04 | 北京纳百生物科技有限公司 | Chromogenic medium for detecting gram-negative bacteria in bovine mastitis and test board thereof |
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