Pig-derived broad-spectrum enrichment medium and preparation method and application thereof

Abstract

The invention relates to the field of drug sensitivity diagnosis, in particular to a swine-derived broad-spectrum enrichment medium as well as a preparation method and application thereof. The pig-derived broad-spectrum enrichment culture medium is prepared from the following raw materials in parts by weight: 5.0 to 10.0 parts of peptone, 3.0 to 8.0 parts of yeast extract powder, 5.0 to 8.0 parts of sodium chloride, 5.0 to 10.0 parts of lactose, 0.5 to 2.5 parts of cholate, 0.002 to 0.005 part of crystal violet, 0.002 to 0.010 part of neutral red, 15.0 to 20.0 parts of agar, 1.0 to 3.5 parts of glucose, 1.0 to 3.5 parts of dipotassium phosphate, 2.5 to 5.0 parts of disodium hydrogen phosphate, 0.2 to 0.5 part of sodium thiosulfate and 1000 parts of distilled water. 50-100 parts of filtered and sterilized newborn calf serum and 1.0-2.5 parts of NAD (Nicotinamide Adenine Dinucleotide) with the mass fraction of 1%. According to the invention, the formula of the enrichment culture medium is optimized, the liquid culture medium is used as the enrichment culture medium for the drug sensitive test, and a new acid-base indicator is selected according to the biochemical characteristics of bacteria, so that the process of the drug sensitive test is simplified.

Description

Technical Field

[0001] The present invention relates to the field of drug sensitivity diagnosis, and in particular to a pig-derived broad-spectrum enrichment culture medium, a preparation method and an application thereof. Background Art

[0002] In recent years, bacterial diseases in pigs are often mixed with viral infections, leading to increasing morbidity and mortality rates in farms. Pigs of different ages can be affected by bacterial diseases and suffer from a variety of adverse consequences. For example, weaned piglets and nursery pigs infected with Salmonella can easily develop symptoms such as sepsis and meningitis, causing significant economic losses to the breeding industry. Traditional drug sensitivity methods have defects such as being time-consuming and highly subjective when judging results, which can lead to errors. The development of rapid diagnosis of bacterial diseases in pigs is crucial for timely control of epidemics and reducing the abuse of antibiotics. The development of rapid drug sensitivity diagnostic methods for bacterial diseases in pigs can not only shorten the course of the disease and reduce mortality through precise medication, but also effectively curb the spread of bacterial resistance genes caused by blind medication.

[0003] Existing methods for drug sensitivity diagnosis include: (1) Traditional culture method: The pathogens in the diseased material are separated and cultured in pure culture before the drug sensitivity test is performed. The drug sensitivity circle is formed by the diffusion of the drug sensitive sheet on the agar plate, and the sensitivity is determined by the size of the inhibition circle. However, this method requires the pathogens to be separated and purified before the drug sensitivity test can be performed. For pathogens with harsh growth environments or when mixed infections exist in the diseased material, separation and purification are difficult.

[0004] (2) Paper diffusion method: This method is a slight improvement on the traditional culture method. The diseased material is directly inoculated on the agar plate and then the drug sensitivity test is performed. However, this method requires judging the results based on the size of the inhibition zone. The size of the inhibition zone depends on visual observation and is easily affected by the operator's experience, resulting in deviation in the results.

[0005] (3) Dilution method: After a series of multiple dilutions of the antimicrobial drug in broth or agar, the bacteria to be tested are quantitatively inoculated and incubated at 35°C for 24 hours for observation. By observing the turbidity of the culture medium or the growth of the colonies, the lowest drug concentration that inhibits the growth of the bacteria to be tested is the minimum inhibitory concentration of the drug for the bacteria to be tested. However, this method requires multiple steps and cannot meet the needs of rapid diagnosis of severe infections; it also relies on a spectrophotometer to adjust the turbidity of the bacterial suspension. If the laboratory does not have the equipment or the equipment is not accurate enough, it will affect the result judgment. In addition, the results still need to be judged by the naked eye to see the growth of bacteria, which is subject to subjective errors. Summary of the Invention

[0006] Traditional culture, paper diffusion, and dilution methods are all routine laboratory drug sensitivity methods that require professional personnel to operate. They also have limitations that cannot be ignored, such as the inability to quickly obtain drug sensitivity results, complex experimental procedures, and certain subjective errors in the judgment of results. If the pig herd is in critical bacterial infection, it is easy to cause irreparable losses. The present invention optimizes the formula of the enrichment culture medium, allowing common pig pathogens such as Actinobacillus pleurogenus, Bordetella, Pasteurella, and porcine intestinal bacteria to multiply rapidly in the culture medium. A new composite acid-base indicator is selected to visualize the bacterial growth situation, thereby simplifying the drug sensitivity test process and meeting the needs of rapid on-site diagnosis and targeted medication. The working principle of the present invention is as follows: bacteria proliferate in the culture medium to form a large number of suspended bacteria, resulting in a decrease in the transmittance of the culture medium and a turbid state; during the proliferation process, the bacteria secrete extracellular polysaccharides, proteins, and metabolic waste (such as lactic acid, acetic acid, ammonia, etc.), and the pH of the culture medium changes. When bromocresol green-methyl red indicator is added to the culture medium, if bacteria do not grow in the culture medium, the culture medium will turn green; if bacteria proliferate in the culture medium, the culture medium will become acidic and the color will turn from green to blue. The turbidity of the culture medium combined with the color change can visually display the growth of bacteria in the diseased sample. The present invention has the following advantages:

[0007] (1) Prepare universal bacterial enrichment culture medium, and carry out drug sensitivity test without separating and purifying the bacteria in the diseased material, which shortens the time of drug sensitivity test and solves the problem of mixed infection.

[0008] (2) Common swine pathogens such as Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus, Pasteurella, Bordetella, Actinobacillus pleuropneumoniae, etc. can all grow in this culture medium.

[0009] (3) The enrichment culture medium does not need to be prepared immediately and can be stored at 4°C after preparation. It can be used to respond to sudden illnesses and maximize time for on-site medication.

[0010] (4) The production cost of the culture medium is low, and indicators are added to the culture medium according to the biochemical characteristics of the bacteria. The color change of the culture medium can be used to more intuitively compare the growth of bacteria.

[0011] (5) The culture medium can be transported in a short time. After the culture medium is placed on the pig farm, the on-site drug sensitivity test can be carried out by the on-site staff with simple training. This can not only avoid the decline of pathogen activity caused by improper transportation and storage of diseased materials, but also allow drug sensitivity testing to be carried out according to the on-site drug reserves and medication habits.

[0012] The present invention provides the following technical solutions:

[0013] A broad-spectrum enrichment medium of porcine origin, comprising the following raw materials in parts by weight: 5.0 - 10.0 parts of peptone, 3.0 - 8.0 parts of yeast extract powder, 5.0 - 8.0 parts of sodium chloride, 5.0 - 10.0 parts of lactose, 0.5 - 2.5 parts of bile salts, 0.002 - 0.005 parts of crystal violet, 0.002 - 0.010 parts of neutral red, 15.0 - 20.0 parts of agar, 1.0 - 3.5 parts of glucose, 1.0 - 3.5 parts of dipotassium hydrogen phosphate, 2.5 - 5.0 parts of disodium hydrogen phosphate, 0.2 - 0.5 parts of sodium thiosulfate, 1000 parts of distilled water, 50 - 100 parts of filtered and sterilized newborn bovine serum, and 1.0 - 2.5 parts of 1% (mass fraction) NAD (nicotinamide adenine dinucleotide).

[0014] A preparation method of a broad-spectrum enrichment medium of porcine origin, comprising the following steps: Mix 5.0 - 10.0 parts of peptone, 3.0 - 8.0 parts of yeast extract powder, 5.0 - 8.0 parts of sodium chloride, 5.0 - 10.0 parts of lactose, 0.5 - 2.5 parts of bile salts, 0.002 - 0.005 parts of crystal violet, 0.002 - 0.010 parts of neutral red, 15.0 - 20.0 parts of agar, 1.0 - 3.5 parts of glucose, 1.0 - 3.5 parts of dipotassium hydrogen phosphate, 2.5 - 5.0 parts of disodium hydrogen phosphate, 0.2 - 0.5 parts of sodium thiosulfate and 1000 parts of distilled water, and sterilize at 120 - 125 °C. After cooling, add 50 - 100 parts of filtered and sterilized newborn bovine serum and 1.0 - 2.5 parts of 1% (mass fraction) NAD to obtain the finished product.

[0015] As a further scheme of the present invention: The sterilization time is 15 - 20 minutes.

[0016] Application of the above-mentioned broad-spectrum enrichment medium of porcine origin in the rapid drug sensitivity diagnosis of common porcine pathogenic bacteria (Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Pasteurella multocida, Streptococcus suis, Staphylococcus aureus and intestinal pathogenic bacteria) and mixed infection samples.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] The present invention optimizes the formula of the enrichment medium, uses a liquid medium as the enrichment medium for drug sensitivity tests, selects a new acid-base indicator according to the biochemical characteristics of bacteria. The present invention can directly process mixed infection samples, overcomes the problem of difficult separation and purification of pathogenic bacteria, greatly improves the proliferation rate and efficiency of common porcine pathogenic bacteria, amplifies the indicator signal, converts the drug sensitivity of bacteria into the turbidity-color change of the medium, realizes the visualization of drug sensitivity results, and thus simplifies the drug sensitivity test process. Description of the Drawings

[0019] Figure 1 The experimental results with phenol red as the indicator.

[0020] Figure 2 These are the experimental results using bromocresol purple as an indicator.

[0021] Figure 3 These are the experimental results using bromothymol blue as an indicator.

[0022] Figure 4 These are the results of overnight culture using different culture methods.

[0023] Figure 5 This is the result of an experiment in which yeast extract powder was added to Actinobacillus pleuropneumoniae as the experimental object.

[0024] Figure 6 This is the result of an experiment using Actinobacillus pleuropneumoniae as the experimental subject and adding liver extract powder.

[0025] Figure 7 These are the results of culturing Actinobacillus pleuropneumoniae, Staphylococcus aureus, and Streptococcus without adding newborn calf serum and NAD.

[0026] Figure 8 The colors of negative control, Bordetella, Streptococcus, Staphylococcus aureus and Actinobacillus pleuroplasti in the nutrient tube.

[0027] Figure 9 It is the result of drug sensitivity testing using tissue samples from a pig farm and drugs stored on the farm.

[0028] Figure 10 These are the experimental results of negative control, Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus, Bordetella, Actinobacillus pleurogenus, and Pasteurella cultured at different times.

[0029] Figure 11 This is the experimental result of using Pasteurella as the experimental object using drug sensitivity tubes after being placed at 4℃ for one month.

[0030] Figure 12 This is a schematic diagram of the usage process of this product. DETAILED DESCRIPTION

[0031] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0032] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0033] A preparation method of a broad-spectrum enrichment medium from porcine sources, comprising the following steps: Mix 5.0 - 10.0 parts of peptone, 3.0 - 8.0 parts of yeast extract powder, 5.0 - 8.0 parts of sodium chloride, 5.0 - 10.0 parts of lactose, 0.5 - 2.5 parts of bile salts, 0.002 - 0.005 parts of crystal violet, 0.002 - 0.010 parts of neutral red, 15.0 - 20.0 parts of agar, 1.0 - 3.5 parts of glucose, 1.0 - 3.5 parts of dipotassium hydrogen phosphate, 2.5 - 5.0 parts of disodium hydrogen phosphate, 0.2 - 0.5 parts of sodium thiosulfate and 1000 parts of distilled water, and sterilize at 120 - 125 °C. After cooling, add 50 - 100 parts of filtered and sterilized newborn bovine serum and 1.0 - 2.5 parts of 1% (mass fraction) NAD to obtain the finished product. Figure 10 Experimental results of culturing different bacteria, namely negative control, Escherichia coli, Salmonella, Staphylococcus aureus, Streptococcus, Bordetella, Actinobacillus pleuropneumoniae, and Pasteurella, for different times. It can be seen from Figure 10 that the finished product can achieve color change for Escherichia coli, Salmonella, and Staphylococcus aureus after 1 hour of culture; color change for Streptococcus and Pasteurella after 3 hours of culture; and color change for Bordetella and Actinobacillus pleuropneumoniae after 8 hours of culture. The growth rate is faster than that of the traditional method of isolating and culturing bacteria, that is, inoculating on solid medium. Sterilization is to avoid the production of inhibitory substances (such as caramelization products) by the high-temperature reaction of glucose and phosphate; filtered sterilization is to avoid the destruction of the components of newborn bovine serum and NAD by moist heat sterilization.

[0034] Peptone: Hydrolyzed from animal or plant proteins, providing nitrogen sources, amino acids, and polypeptides required for bacterial growth, and is the core nutritional basis of the medium. Glucose: A carbon source that can be rapidly utilized, providing energy for bacteria through the glycolysis pathway. Lactose: A fermentable carbon source for some bacteria (such as Enterobacteriaceae bacteria), whose metabolites are acidic and can combine with pH indicators to show color, used to identify bacterial growth. Yeast extract powder: Rich in B vitamins, nucleotides, and trace elements, promoting the synthesis of metabolic coenzymes while supplementing carbon and nitrogen sources, ensuring the normal growth of some bacteria with high nutritional requirements. The above are the basic nutrient sources of the broad-spectrum enrichment medium from porcine sources.

[0035] Sodium chloride: Maintaining osmotic pressure balance and enhancing cell membrane stability. Some pathogenic bacteria (such as Staphylococcus aureus) require a specific salt concentration to grow. Dipotassium hydrogen phosphate and disodium hydrogen phosphate: As sources of phosphorus element, participating in the synthesis of bacterial nucleic acids, phospholipid membranes, and ATP; phosphate groups are essential components of some coenzymes (such as NADPH), participating in sugar metabolism (such as the pentose phosphate pathway) and redox reactions. The above are the raw materials for the broad-spectrum enrichment medium from porcine sources to achieve the regulation of osmotic pressure and ion balance.

[0036] Bile salts: Low concentrations of bile salts can stimulate intestinal pathogenic bacteria, such as Salmonella, to secrete protease and esterase, which helps break down complex nutrients in the culture medium; by dissolving lipids, it increases cell membrane permeability, promotes the uptake of hydrophobic nutrients such as fat-soluble vitamins by bacteria, and synergistically with lactose to meet the metabolic preferences of porcine intestinal bacteria. Sodium thiosulfate: The test material (especially intestinal / blood samples) contains high concentrations of hydrogen sulfide, a metabolite of Salmonella. As an antioxidant and antidote, sodium thiosulfate neutralizes the toxic substances produced by metabolism, relieves the growth inhibition of sulfur-producing pathogenic bacteria such as Salmonella on the mixed flora, and protects bacteria from oxidative damage. Newborn bovine serum: It provides growth factors (such as hormones, lipids) and binding proteins to promote the growth of fastidious bacteria such as Pasteurella. Nicotinamide adenine dinucleotide (NAD): As a coenzyme, it participates in oxidation-reduction reactions and supports energy metabolism, which is especially crucial for bacteria that require complex metabolic pathways. For example, it provides factor V necessary for the growth of Haemophilus parasuis. When used in combination with newborn bovine serum, it can meet the necessary nutritional components for the growth of fastidious bacteria. Crystal violet: Assists other acid-base indicators in identifying the growth of bacteria. Neutral red: In addition to assisting other acid-base indicators in identifying bacterial reproduction through pH indication, it can also reflect the oxidation-reduction state of bacteria through color changes. For example, it fades when anaerobic bacteria metabolize to produce reducing substances. Agar: The uniform distribution of agar can prevent the precipitation of nutrients in the culture medium due to gravity, ensuring that bacteria are exposed to uniform nutritional conditions and providing a controllable experimental environment for bacterial culture. The above are the additives that enable the cooperative effect of the porcine-source broad-spectrum enrichment medium.

[0037] Figure 12 This is a schematic diagram of the usage process of this product. The usage process of this product is as follows:

[0038] (1) Prepare the liquid medicine: Add 0.5 - 4.0 parts by weight (this part by weight is the same as that of each raw material) of methyl red ethanol solution with a mass fraction of 0.02% and 0.5 - 4.0 parts by weight of bromocresol green ethanol solution with a mass fraction of 0.01% to the porcine-source broad-spectrum enrichment medium, mix well, with a pH value of 6.8 - 7.5, and dispense it into 10 mL sterile sampling tubes as drug sensitivity tubes. Store them in a 4°C refrigerator for later use (It has been proven by experiments that the drug sensitivity tubes can still be used normally after being stored at 4°C for one month, and the test results are the same as those of freshly prepared ones). Add 1.0 mL of the liquid medicine to each drug sensitivity tube, and add 1.0 mL of normal saline to each negative control and positive control tube. Write the added drug, negative and positive controls on the label paper for each drug sensitivity tube after adding the drug, and paste it on the tube wall for easy distinction. Figure 11 is the experimental result of using the drug sensitivity tubes placed at 4°C for one month with Pasteurella as the experimental object. From Figure 11 it can be seen that with Pasteurella as the research object, the positive control changes color after three hours, and compared with Figure 11The consistent results and consistent discoloration times indicate that the drug sensitivity test tubes can still be used normally after being stored at 4°C for one month, and the test results are consistent with those of freshly prepared ones. 1.0 mL of the liquid medicine is added to each drug sensitivity test tube, and 1.0 mL of normal saline is added to each of the negative control and positive control tubes. After adding the drugs, each drug sensitivity test tube is labeled with the added drug, negative and positive controls and pasted on the tube wall for easy distinction.

[0039] (2)Sampling: If the sample for drug sensitivity test is feces or tissue sample, a sample the size of a soybean is sufficient; if it is exudate or tissue fluid, multiple sites can be taken, such as pleural effusion, blood, joint fluid, etc. for mixing, and then 0.1 - 0.5 mL is taken. If the liquid is too little to be conveniently drawn and mixed, a sterile medical cotton swab can be used to dip the tissue fluid from multiple sites and directly add it to the tube.

[0040] (3)Sample addition: Add the sample to a tube containing only enrichment medium without other drugs (if sampling with a cotton swab, it is necessary to stir well in the tube until it can be seen with the naked eye that the sample has fallen into the tube), mix well, and after mixing, use a disposable syringe to draw 0.5 mL of the liquid from this tube and add it to each drug sensitivity test tube except the negative control in step (1), and 0.5 mL of normal saline is added to the negative control.

[0041] (4)Cultivation and observation: After photographing and recording the colors of all drug sensitivity test tubes at 0 h, place them in an incubator at 37°C for 8 - 16 h. After the cultivation is completed, compare the colors of each tube with those of the 0 h, negative and positive controls, and judge whether the bacteria can grow in the drug sensitivity test tubes with drugs through the color change. There is no fading, turbidity or color change in the negative control tube, and the positive control tube changes color and becomes turbid, indicating that there are pathogenic bacteria in the sample; the color of the drug sensitivity test tube containing the effective drug should be the same as that of the negative control tube, without color change or fading. Figure 8 The left in the middle is the negative control, and the right is the color of Bordetella bronchiseptica, Streptococcus, Staphylococcus aureus, and Actinobacillus pleuropneumoniae in the nutrient tube. Figure 8 It can be seen from it that the nutrient tubes of the four bacteria all change color, indicating that Bordetella bronchiseptica, Streptococcus, Staphylococcus aureus, and Actinobacillus pleuropneumoniae can all grow in this culture medium. In addition to using the pure cultured bacteria stored in the laboratory for the test, fecal samples, blood samples, and tissue samples are also used for multiple tests, and the test results are all valid. Figure 9 This is the result of the drug sensitivity test using the tissue sample of a certain pig farm and the drugs stored in the farm. Figure 9 The negative control in it is green, and the positive control is blue. Figure 9 This shows that the drug in the drug sensitivity test tube that can effectively inhibit the growth of pathogenic bacteria is green, and the drug that cannot inhibit the growth of pathogenic bacteria is blue. Except that doxycycline has an obvious inhibitory effect on the pathogenic bacteria in this sample, the other drugs are all ineffective.

[0042] Bromocresol Green-Methyl Red Acid-Base Indicator: This mixed indicator appears orange-red under acidic conditions, green under alkaline conditions, and gray at pH = 5.1 (the color change point). Its color change range is precise and can accurately detect the pH changes caused by the growth and reproduction of bacteria. The Bromocresol Green-Methyl Red indicator is the core indicator for showing the growth and reproduction of bacteria and needs to meet the following requirements: (1) The color change range is narrow and precise, suitable for the rapid response to the acid and alkali production by bacterial metabolism; (2) Color complementarity enhances sensitivity: Before adding the indicator, the culture medium is light yellow. It is necessary that the color of the indicator remains easily distinguishable by the naked eye after neutralizing with the color of the culture medium, avoiding the interference of the gradual color change of the indicator and reducing misjudgment; (3) Strong anti-interference ability: Bacteria metabolize to produce CO2. A single indicator may cause the color change endpoint to advance due to the dissolution of CO2 and cannot adapt to the complex culture medium environment.

[0043] When optimizing the culture medium formula of this technology, the color change ranges of a large number of acid-base indicators have been searched to cover the key pH range (pH 4.6 - 5.2) of bacterial metabolism. Phenol Red, Bromocresol Purple, and Bromothymol Blue indicators have been selected. The experimental results are shown in Figures 1 - 3 . Figure 1 The experimental results with Phenol Red as the indicator, Figure 1 from left to right in [figure] are the negative control, the addition of Salmonella, and the addition of fecal samples. The colors have not changed. There is obvious turbidity only in the tube with the addition of fecal samples. Therefore, this indicator is not adopted. Figure 2 The experimental results with Bromocresol Purple as the indicator, Figure 2 from left to right, 1 - 7 in [figure] are the color changes of Pasteurella multocida after adding different drugs, 8 is the positive control, and 9 is the negative control. In the theoretical state, the positive control should be yellow and the negative control should be purple. However, if the growth condition of the bacteria is general and the yellow color is not obvious enough, the solution will show a wine-red color, such as 3, 4, 5. The color change is not intuitive enough. Therefore, this indicator is not adopted. Bordetella bronchiseptica can cause respiratory diseases in pigs, such as infectious atrophic rhinitis. The nasal secretions of pigs suffering from atrophic rhinitis are collected, and the color changes of Bromothymol Blue are tested with the purified Bordetella and nasal secretions as the research objects respectively, Figure 3 The experimental results with Bromothymol Blue as the indicator, Figure 3 from left to right in [figure] are the purified bacteria positive control, the negative control, and the nasal secretion positive control. Theoretically, the negative control is green and the positive control is yellow. However, due to the influence of the color of the nasal secretions themselves, the color change is not intuitive enough. Therefore, this indicator is not adopted.

[0044] Taking Bordetella bronchiseptica as the research object, the culture methods are compared. The drug sensitivity test tubes are placed in a 37°C constant temperature incubator and a 37°C constant temperature shaking incubator respectively to observe whether there are differences in the growth and reproduction rates of Bordetella bronchiseptica. It is found that the growth and reproduction rate of bacteria is slightly faster when cultured in the shaking incubator than in the constant temperature incubator, but it is easy to cause contamination during the culture process.Figure 4 It is the result after overnight culture, Figure 4 from left to right are the shaker negative control, shaker positive control, incubator positive control, and incubator negative control. It can be seen that the negative control was contaminated during shaker culture. Considering that this drug sensitivity tube is used for drug sensitivity testing on the pig farm site, it is more convenient to use an incubator. Therefore, an incubator was used for culture.

[0045] The raw material selection of the porcine broad-spectrum enrichment medium was also experimented. It was considered to use liver extract powder instead of yeast extract powder. The role of liver extract powder in the medium is similar to that of yeast extract powder, but liver extract powder is yellowish-brown, and replacing yeast extract powder results in the medium being yellowish-green, making it impossible to observe the growth of bacteria. Figure 5 and Figure 6 using Actinobacillus pleuropneumoniae as the experimental object, Figure 5 is the experimental result with the addition of yeast extract powder, Figure 6 is the experimental result with the addition of liver extract powder, and the rightmost is the positive control, Figure 5 and 6 By comparing, it was found that the addition of yeast extract powder had a better effect. Components similar to yeast extract powder in the medium also include yeast extract, brain heart infusion powder, malt extract powder, etc., but they have problems such as dark color and high price. Therefore, yeast extract powder was finally used as the core nutrient component.

[0046] It was also considered not to add newborn bovine serum and NAD, and using Actinobacillus pleuropneumoniae, Staphylococcus aureus, and Streptococcus as research objects for culture respectively to explore the growth of bacteria after the addition of newborn bovine serum and NAD. Figure 7 From left to right are the negative control, Actinobacillus pleuropneumoniae, Staphylococcus aureus, and Streptococcus. The experiment proved that Actinobacillus pleuropneumoniae did not grow without adding serum and NAD, proving that the growth of fastidious porcine bacteria depends on special factors.

[0047] The present invention also considered alternative schemes for the indicator system. Acid-base indicator combinations: such as bromocresol purple + neutral red, can cover a wider or different pH range to adapt to the differences in acid / base production characteristics of different bacterial species during metabolism. Redox indicators or fluorescent indicators: such as methylene blue to detect metabolic activity through redox reactions; after adding fluorescein, the fluorescence is strong without metabolism, and the fluorescence quenches during bacterial proliferation. However, the medium is light yellow, and the color change of the indicator after adding it to the medium is not intuitive, and it goes against the original intention of this technology of low cost, simple operation, and rapid result output.

[0048] The present invention also considered alternative schemes for the medium components. Replacement of selective inhibitors: such as replacing bile salts with sodium selenite. Diversification of carbon sources: lactose can be replaced with mannitol or glycerol. Diversification of nitrogen sources: yeast extract powder can be replaced with brain heart infusion. But it goes against the original intention of this technology of low cost, simple operation, and rapid result output.

[0049] The present invention has also considered alternative solutions for the buffer system, and phosphate can be replaced with Tris-HCl buffer. However, this goes against the original intention of the present technology, which is to be low-cost, easy to operate, and produce results quickly.

[0050] The present invention has also considered alternative solutions for the drug concentration. The gradient dilution method can be used to replace the fixed 10-fold therapeutic dose. However, the operation is cumbersome, there are certain errors in the dilution process, and reagent consumables are wasted.

[0051] The present invention has also considered alternative solutions for the storage of nutritional tubes. For example, the culture medium can be made into freeze-dried powder and then sub-packed, and re-dissolved during use; or the drug sensitivity tubes can be encapsulated with inert gas (such as nitrogen). However, this goes against the original intention of the present technology, which is to be low-cost, easy to operate, and produce results quickly.

[0052] In summary, the present invention has the following advantages:

[0053] (1) The present invention optimizes the formula of the general enrichment medium

[0054] ① Compared with "An Enrichment Medium and Its Preparation Method and Application" (CN118389320A) and "An Enrichment Medium and Its Preparation Method, Enrichment Culture Method" (CN106047781B), although both involve general enrichment, the first two patents only cover poultry pathogens and food pathogens, and the target flora of the two is significantly different from that of swine diseases. Fastidious bacteria such as Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, and Pasteurella multocida, which are common swine pathogens, have not been covered in the comparative patents.

[0055] ② Existing similar patents all focus on "enrichment" itself, while the goal of the present invention is to obtain drug sensitivity results more efficiently through the means of rapid enrichment. A mixed indicator is added to the optimized high-efficiency enrichment medium, and the color fluctuations caused by the turbidity and pH changes of the medium are integrated with the drug sensitivity process, which belongs to application innovation.

[0056] ③ The formula of this enrichment medium fully reflects the metabolic characteristics of swine pathogens, and the components thereof are not involved in similar patents. For example, newborn bovine serum + NAD: is a necessary component to support the growth of fastidious bacteria such as Actinobacillus pleuropneumoniae and Haemophilus parasuis; bile salts + lactose target the metabolic preferences of swine intestinal bacteria; sodium thiosulfate, as a reducing agent and detoxifying agent, can neutralize the residual halogen disinfectants in the medium, eliminate their inhibition of the target bacteria, and in addition, it also acts as an electron donor to create a suitable growth environment for facultative anaerobes or microaerophiles. In the early stage of the present invention, a pre-experiment was carried out using the indicator formula published in "An Enrichment Medium and Its Preparation Method and Application" (CN118389320A) (VI. Specific Embodiments - Figure 6 ) When culturing a single bacterium, the color change of the medium is obvious. If the diseased material is blood, pleural effusion, nasal secretion, etc., due to the properties and color of the diseased material will cause certain interference to the color of the medium, the color change effect is not ideal.

[0057] ④ Different from the "traditional culture method" and the "disk diffusion method", the present invention uses a liquid medium as the enrichment medium for the drug sensitivity test, increases the oxygen solubility and the diffusion efficiency of nutrients, significantly improves the bacterial metabolic activity; there is no need for an agar solidification step, and it can be directly subpackaged, reducing the operation steps and the risk of contamination; the liquid medium has a longer shelf life after sterilization, reducing the waste of consumables for repeated preparation and lowering the cost.

[0058] ⑤ Ensure the normal proliferation of bacteria with harsh growth conditions, accelerate the bacterial proliferation rate, improve the detection sensitivity of the target bacteria, and quickly obtain the drug sensitivity results for guiding on-site drug use.

[0059] ⑥ Data from the Ministry of Agriculture and Rural Affairs and the Animal Disease Control Center from 2024 to 2025 reflect the severe situation of current mixed infections. And compared with the patent (CN118389320A) which only targets single disease materials, the medium prepared by the present invention achieves the technical effect of the "disk diffusion method", can judge the sensitivity of pathogens to different drugs on the farm without separating and purifying the pathogens, has a certain ability to directly process mixed infection samples, solves the problem of low efficiency in isolating and culturing pathogens caused by mixed infections, and better adapts to the pathogen epidemic situation.

[0060] ⑦ The nutrient components of the medium are stable. By pre-subpackaging the medium into sterile sampling tubes, long-term storage of the drug sensitivity tubes and batch detection of drug sensitivity are realized.

[0061] (2) The present invention selects bromocresol green-methyl red as the acid-base indicator of the medium according to the biochemical characteristics of bacteria

[0062] ① A two-color system indicator with a color change threshold ΔpH not greater than 0.3, a narrow color change range, and high sensitivity. The color contrast between the negative and positive controls is obvious. Referring to the result determination standard of the "dilution method", the innovation combines the turbidity change caused by bacterial proliferation and the color change of the medium caused by pH change to realize the "turbidity-color change" dual-signal visualization detection for measuring the bacterial growth situation.

[0063] ② The bromocresol green-methyl red indicator changes from green to blue under the acidic metabolites of bacteria. Experiments have proved that the color change of this composite indicator is more obvious than that of phenol red, bromocresol purple, and bromothymol blue during the proliferation of swine pathogenic bacteria. Combining turbidity to achieve dual-signal interpretation, compared with the traditional drug sensitivity method which takes 24 - 48h, the present invention shortens the drug sensitivity time to 8 - 16h, can meet the enterprise requirements for quickly guiding on-site drug use, and has a certain promotion value.

[0064] ③ By comparing the color changes before and after cultivation, and setting negative and positive controls at the same time, the interference of the colors of the samples and the drugs themselves is excluded by color contrast, eliminating the subjectivity of individual interpretation and improving the objectivity and accuracy of the results.

[0065] (3)The present invention simplifies the drug sensitivity test process

[0066] ① Compared with the prior art, through simple training, the operator can master the experimental operation, significantly reducing the operation threshold of pig farms or grass-roots laboratories, which conforms to the simplification trend of veterinary detection equipment.

[0067] ② Without the need for large-scale experimental instruments, on-site drug sensitivity can be achieved, respecting the drug use habits and drug reserve differences of each farm; at the same time, the freshness of the diseased material is ensured, solving the pain points of easy contamination and easy degradation of clinical samples, and responding to the country's call for "reducing the abuse of antibiotics".

[0068] ③ The pre-installed drug sensitivity tubes can be stored at 4°C for 1 month with unchanged activity, and the single detection cost is significantly reduced compared with the traditional drug sensitivity method, meeting the batch screening needs of pig farms.

[0069] In addition, it should be understood that although this specification is described according to the embodiments, not each embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A broad-spectrum bacteria-enriching medium derived from pigs, characterized in that, It comprises the following raw materials in parts by weight: 5.0 - 10.0 parts of peptone, 3.0 - 8.0 parts of yeast extract powder, 5.0 - 8.0 parts of sodium chloride, 5.0 - 10.0 parts of lactose, 0.5 - 2.5 parts of bile salts, 0.002 - 0.005 parts of crystal violet, 0.002 - 0.010 parts of neutral red, 15.0 - 20.0 parts of agar, 1.0 - 3.5 parts of glucose, 1.0 - 3.5 parts of dipotassium hydrogen phosphate, 2.5 - 5.0 parts of disodium hydrogen phosphate, 0.2 - 0.5 parts of sodium thiosulfate, 1000 parts of distilled water, 50 - 100 parts of filtered and sterilized newborn bovine serum, and 1.0 - 2.5 parts of 1% NAD by mass fraction.

2. A preparation method of a broad-spectrum bacteria-enriching medium from pigs, characterized in that, It comprises the following steps: Mix 5.0 - 10.0 parts of peptone, 3.0 - 8.0 parts of yeast extract powder, 5.0 - 8.0 parts of sodium chloride, 5.0 - 10.0 parts of lactose, 0.5 - 2.5 parts of bile salts, 0.002 - 0.005 parts of crystal violet, 0.002 - 0.010 parts of neutral red, 15.0 - 20.0 parts of agar, 1.0 - 3.5 parts of glucose, 1.0 - 3.5 parts of dipotassium hydrogen phosphate, 2.5 - 5.0 parts of disodium hydrogen phosphate, 0.2 - 0.5 parts of sodium thiosulfate and 1000 parts of distilled water, sterilize at 120 - 125 °C, and after cooling, add 50 - 100 parts of filtered and sterilized newborn bovine serum and 1.0 - 2.5 parts of 1% NAD by mass fraction to obtain the finished product.

3. The preparation method of the broad-spectrum enrichment medium from porcine sources according to claim 2, wherein, The sterilization time is 15 - 20 minutes.

4. Application of the porcine - source broad - spectrum enrichment medium as claimed in claim 1 in rapid drug sensitivity diagnosis of common porcine pathogenic bacteria and mixed - infection samples.

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