CN101545003A - Detection kit and detection method for beta-lactamase in raw milk and milk products - Google Patents
Detection kit and detection method for beta-lactamase in raw milk and milk products Download PDFInfo
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- CN101545003A CN101545003A CN200910064813A CN200910064813A CN101545003A CN 101545003 A CN101545003 A CN 101545003A CN 200910064813 A CN200910064813 A CN 200910064813A CN 200910064813 A CN200910064813 A CN 200910064813A CN 101545003 A CN101545003 A CN 101545003A
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Abstract
The invention relates to a detection kit and a detection method of raw milk and milk products, in particular to a detection kit and a detection method for beta-lactamase in raw milk and milk products. At present, no diary corporation in China can carry out production with the raw milk completely supplied by self-owned farms. All diary corporations need to purchase raw milk from dairy farmer to some extent. Thus, the detection of imparities and antibiotics becomes extremely important. The detection method adopts a standard strain of bacillus stearothermophilus absolutely sensitive to penicillin antibiotics, uses the specificity of sulbactam to inhibit the activity of the beta-lactamase, adds ampicillin as a reference and contrasts inhabitation zones of a sample with a beta-lactamase inhibitor and a sample without the inhibitor to detect the antibiotics. The detection kit comprises a sterilized culture dish with a vacuum package, a beta-lactamase standard product, a freeze-drying cillin bottle containing ampicillin and ampicillin and sulbactam, a blank filter paper sheet and a diluent. The detection kit and the detection method have the advantages of low cost, convenient and quick operation and high sensitivity.
Description
Technical field
The present invention relates to the detection kit and the detection method of a kind of raw milk and milk preparation, the detection kit and the detection method of β-Nei Xiananmei in particularly a kind of raw milk and the milk preparation.
Background technology
Milk is nutritious, readily digested absorbs, inexpensive, instant, is " near perfect food ", and person " white blood " is optimal whole food.
Protein in the milk mainly is casein, albumin, sphaeroprotein, milk-protein etc., and 8 necessary seed amino acids of human body are arranged in the 20 contained multiple amino acids, and milk proem matter is the protein of full price, and its digestibility is up to 98%.Dairy fats is high-quality fat, and quality is best, and its digestibility is more than 95%, and contains a large amount of liposoluble vitamins.Lactose in the milk is semi-lactosi and lactose, is the carbohydrate that readily digested absorbs.Mineral substance in the milk and trace element all are dissolved states, and the ratio of the content ratio of various mineral substance, particularly calcium, phosphorus is proper, is easy to digest and assimilate.
Per 100 gram milk contain moisture 87 grams, protein 3.3 grams, fat 4 grams, carbohydrate 5 grams, 120 milligrams of calcium, 93 milligrams in phosphorus, 0.2 milligram of iron, retinol1 40 international unit, vitaminB10 .04 milligram, 0.13 milligram of Wei ShengsuB2,0.2 milligram of nicotinic acid, 1 milligram of vitamins C.But 69 kilocalories of heat supplieds.
Milk can suppress tumour.The material that contains a kind of CLA in milk and the milk preparation cheese can effectively destroy the free radical that carcinogenic danger is arranged in the human body, and energy is rapid and the cytolemma combination, makes cell be in the state that the defence carcinogenic substance is invaded, thereby plays protective effect on cancer risk.And contained calcium can effectively destroy carcinogenic substance in the milk in human intestinal, makes its decomposition change over the non-carcinogenic material, and excretes.Contained vitamin A, Wei ShengsuB2, vitamins D etc. all have certain prophylactic effect to cancer of the stomach and colorectal carcinoma in the milk.And also contain the multiple immune globulin antibody that can strengthen the human body resistance against diseases in the milk, protective effect on cancer risk is also arranged.Contain a kind of enzyme in addition in the yogurt milk, can prevent effectively that the cancer patients is because of chemotherapy and the caused side effect of radiotherapy.
But milk tranquilizing and allaying excitement.The Italy scientific research personnel discovers why milk has tranquilizing effect is because contain a kind of composition that suppresses nervous excitation.When you upset, might as well remove to drink a mug milk and calm the nerves.But the milk promote sleep of having a drink before sleeping.
But milk beautifying face and moistering lotion.Milk is nutritious, contains high-grade fat, range protein, VITAMIN, mineral substance, particularly contains more vitamin B complex; their energy Glycerins; protection epidermis, anticracking, the wrinkle resistant skin smooth softness that makes are delicate, the pitch-black minimizing of hair is come off, thereby play the cosmetology effect.Contained iron, copper and vitamin A in the milk has effects of beautification and nourishing face can make skin keep smooth and moistens.Whey in the milk has the elimination effect to facial wrinkles.Milk can also provide the closure grease for skin, forms film in case the moisture of skin evaporation in addition, can also temporarily provide moisture, so milk is natural skin care product, also is " green skin care product ".No matter, think that can to make skin smooth so delicate so the record of external since ancient times domestic all useful milk and milk preparation beauty treatment is used in the bread that soaked in the milk every day as the roman and wipes face, the youth that seems beauty, and can suppress the growth of beard.So also contain the composition of milk or milk preparation in the makeup that have now.Milk helps fat-reducing.Milk preparation contains higher fatty acid and heat, so many people do not eat milk preparation for fat-reducing.Yet tennessee,USA university studies show that, the calcium constituent that enriches in the milk preparation is extremely important to the intravital fat acid decomposition of people.
Milk can promote child's brain development.Breast is unique food of depending on for existence and growing after the Mammals birth, and it contains and is fit to the necessary whole nutrient substances of early children development.The phosphorus that contains in the milk has important effect to promoting child's brain development; Contain Wei ShengsuB2 in the milk, help the raising of eyesight; Contain calcium in the milk, but enhances skeletal tooth intensity promotes the juvenile intelligence development.
In addition, but sour milk enhancing immunity system function, and the prevention tumour cell increases, and prevents arteriosclerosis; Contain a large amount of lactic acid and the living lactic acid bacteria that is of value to HUMAN HEALTH in the sour milk, help human consumption and absorb, activate stomach en-, strengthen digestive function, improve the specific absorption of human body mineral element calcium, phosphorus, iron.
Be to ensure cow's milk and quality of dairy products safety, milk from raw material purchase the product encapsulation, each link of dispatching from the factory carries out keeping under strict supervision close inspection, check.At present China does not also have a tame dairy industry company to accomplish all to use the former milk in own pasture to produce, and suckles to look forward to equal different ratios and need purchase dairy farmer's former milk, but like this system check and all kinds of antibiotic check of dopant is seemed particularly important.Antibiotic-free milk is meant with not containing the milk that antibiotic raw material production is come out." resist " to be meant and to be used for treating the used all kinds of microbiotic of disease ox, common have penicillin, a Streptomycin sulphate etc.Fresh herbage is milk cow food, various insect pests and disease but fresh herbage is easy to get, need laxative to remove the evil, milk cow easily suffers from mazoitis when changing according to the season every year in addition, and adopt machinery to get breast and also make the easier trouble mazoitis of cow than the hand milking, to position, cow's milk room direct injection microbiotic, milk cow can get well as early as possible.But through the milk cow of antibiotic therapy, within a certain period of time the milk that produces can be remaining a small amount of microbiotic, this milk can not carry out processing as edible milk material.
But now to the system check of dopant:
To electrolytes: salt, saltcake, lead sulfate and some chemical fertilizer, the check of materials such as NaHCO3, Na2CO3, liming, washing powder.Generally based on the check specific conductivity, in conjunction with titration acidity, milk proportion and three index detected results of lipid content analysis-by-synthesis.
To the nonelectrolyte material: as the check of mixing of materials such as urea, ox urine, sucrose, to measure the freezing point of newborn sample.In conjunction with observing titration acidity, proportion and determination of fat synthetic determination as a result, as truly have doping, carry out single qualitative reaction again.
Mix the check of colloidal type material such as thin rice gruel, soya-bean milk, soya-bean cake water, thin gelatin and doping of water etc., this type of material detects the whey specific gravity test based on newborn sample, again in conjunction with freezing point, titration acidity, determination of fat result carry out synthetic determination, as suspection colloidalmaterial is arranged, carry out the qualitative reaction of dopant again.In milk, be mixed with materials such as white bole, white shoe powder,, leave standstill or the method observation test tube bottom of centrifugation and the throw out on the wall, then reexamine and mixed which kind of material so can adopt because this class material is undissolved.To being added with sanitas, the milk of antibiotic and agricultural chemicals carries out activity test to milk earlier, if activity test is defective, milk should carry out sanitas, the qualitative test of antibiotic and agricultural chemicals.
Penicillin is a kind of high-efficiency broad spectrum antibiotics, is widely used in prevention and treatment cow disease in the veterinary drug.But cause its residual in animal derived foods such as milk because of a large amount of the use, badly influenced China's people's health and foreign trade.So-called residue of veterinary drug is meant the parent compound and the metabolite thereof of the contained veterinary drug of any edible portion of animal product, and the impurity relevant with veterinary drug is residual.Residue of veterinary drug had both comprised that former medicine also comprised medicine meta-bolites in animal body, and wherein because the anaphylaxis of penicillin is the most extensive, the meaning of its residue detection is very great.Bioelectrochemical sensor is current electrochemistry and electroanalytical chemistry ten minutes active research field, has been applied to many aspects such as life science, environmental science, analysis science, Materials science at present.Because quick, highly sensitive, the specificity of its peculiar detection is strong, than the residual traditional detection method of penicillin, be hopeful more to realize that the scene of this antibiotics residue detects.With the different modifying method Bacillus cereus and penicillinase are solidified, adopted different base electrode preparation microbiological sensor and Enzyme sensor is that the penicillin measured in the milk of response signal is residual with current potential or electric current.This method more is hopeful to realize the scene detection of this antibiotics residue.Or with the different modifying method with penicillinase or the immobilization of penicillin polyclonal antibody, with the glass-carbon electrode is that basal electrode has prepared Enzyme sensor and immunosensor, with the electric current is that response signal is measured the residual penicillin in phosphate buffer solution and the milk, but this detection method complexity is difficult for popularizing." fresh cow's milk microbiotic decomposition agent " appearred in the market, this decomposition agent residual penicillin and cynnematin in the alternative decomposing milk after diluting.The main component of " fresh cow's milk microbiotic decomposition agent " is a β-Nei Xiananmei, be to a kind of extracellular enzyme of the drug-fast bacterium excretory of Penicillin antibiotics, residual Penicillin antibiotics in the alternative decomposing milk of this enzyme, it acts on Penicillin antibiotics, by hydrolysis beta-lactam nucleus is opened and is lost anti-microbial activity.According to up-to-date " food safety method ", forbid this material joined and be used to decompose microbiotic in the antibiotic milk.Up to now, the standard that the Ministry of Health recommends to use has three kinds, is respectively the Peni-ase Sensor test kit that adopts Unisensor company to produce and carries out rapid screening method, liquid phase method and cylinder plate method, and wherein cylinder plate method is expected to be proposed as the national standard use.Need experiment in advance shortcoming such as to grope but the cylinder plate method of its recommendation has the experimental implementation more complicated, need to be equipped with a series of pharmaceutical standards solution, need prepare substratum separately and be made into check with dull and stereotyped, bacterial concentration, be unfavorable for basic unit's laboratory employing.
Summary of the invention
The present invention overcomes above-mentioned deficiency, and develops a kind of novel test kit that can be used for β-Nei Xiananmei rapid detection in cow's milk and the milk-product, uses for each correlation detection unit.
The present invention has adopted the absolute responsive bacstearothermophilus reference culture of Penicillin antibiotics, utilize the Sulbactam specificity to suppress the activity of β-Nei Xiananmei, and add the Ampicillin Trihydrate in contrast, add beta-lactamase inhibitor by comparison and come the indirect measurement sample whether to contain beta-lactam enzyme medicine with the size that does not add the inhibition circle that sample produced of inhibitor.
For achieving the above object, the present inventor has carried out a large amount of research, has developed a kind of test kit that is used for the β-Nei Xiananmei qualitative detection, and described test kit comprises following compositions:
1. vacuum-packed sterilization culture dish with long preservation period;
2. β-Nei Xiananmei standard substance;
3. contain Ampicillin Trihydrate freeze-drying cillin bottle and Ampicillin Trihydrate+Sulbactam freeze-drying cillin bottle;
4. blank filter paper sheet;
5. diluent.
Described sterilization culture dish is the disposable plate of 50-120mm, pours into calibrating substratum (pH value 7.8-8.5) 10-30mL, and preservation condition is 4-6 ℃, keeps in Dark Place.
Described β-Nei Xiananmei standard substance are 100-400 ten thousand units, and preservation condition is 4-7 ℃.
Described contain Ampicillin Trihydrate freeze-drying cillin bottle and contain Ampicillin Trihydrate+Sulbactam freeze-drying cillin bottle respectively contain Ampicillin Trihydrate 50ng and contain Ampicillin Trihydrate 50ng and the lyophilized products cillin bottle of Sulbactam 10-30 μ g, 4-6 ℃ of preservation.
The detection kit of β-Nei Xiananmei in described raw milk and the milk preparation is characterized in that diluent is aseptic, pH8.0-10.0 phosphate buffered saline buffer.
Detection method of the present invention may further comprise the steps:
1) the β-Nei Xiananmei standard substance is diluted;
2) get vacuum-packed sterilization culture dish with long preservation period, the blank filter paper sheet is affixed on the culture dish surface according to the order of A, B, C, D;
3) get each two bottles of the freeze-drying cillin bottles that contain Ampicillin Trihydrate medicament freeze-drying cillin bottle and contain Ampicillin Trihydrate+sulbactam medicine respectively, add milk to be measured respectively, the former is labeled as A, C, and the latter is labeled as B, D; β-Nei Xiananmei standardized solution with dilution adds respectively in C, the D bottle again, with above-mentioned freeze-drying cillin bottle mixing, puts in the incubator and hatches.And establish the pure water sample and do negative control;
4) by milk sample and pure water sample in the freeze-drying cillin bottle of getting the above-mentioned A of being labeled as, B, C, D respectively, corresponding joining on the blank filter paper sheet that is labeled as A, B, C, D;
5) treat milk and pure water fully by after the filter paper absorption, with each culture dish cultivation 6h;
6) filter paper of each culture dish takes out gently with tweezers, with inhibition zone survey meter or vernier caliper measurement antibacterial circle diameter.
Result report: the pure water sample result should be: (A), (B), (D) all should produce inhibition zone; (A) inhibition zone is compared with the inhibition zone of (B), and with interior (containing 2mm), and repeatability is good at 2mm for difference; (C) inhibition zone is less than the inhibition zone of (D), and difference (contains 2mm) more than 2mm, and repeatability is good.As result for this reason, can carry out following judgement to sample result:
If in the sample result (B) and, (D) all produce inhibition zone, and when (C) and (D) inhibition zone difference (contains 2mm) more than 2mm, can be by (1), (2) result of determination:
(1) inhibition zone (A) (contains 2mm) less than the inhibition zone difference of (B) more than 2mm, and repeatability is good, should judge that this sample is added with β-Nei Xiananmei, and report beta-lactam enzyme drug abuse test is the positive as a result.
(2) less than 2mm, and repeatability is good, should judge that this sample is not added with β-Nei Xiananmei with the inhibition zone difference of (B) for inhibition zone (A), and report beta-lactam enzyme drug abuse test is feminine gender as a result.
(3) if (A) He (B) all do not produce inhibition zone, the amount that β-Nei Xiananmei in this sample is described should will detect behind the diluted sample than higher again.
Detection method of the present invention can also may further comprise the steps:
1) dilution of β-Nei Xiananmei standard substance is 3000U/mL;
2) get vacuum-packed sterilization culture dish with long preservation period, 10-13mm blank filter paper sheet is affixed on the culture dish surface according to the order of A, B, C, D;
3) vacuum-packed sterilization culture dish with long preservation period has been coated with bacstearothermophilus;
4) contain Ampicillin Trihydrate medicine 50ng in the freeze-drying cillin bottle respectively; Contain Ampicillin Trihydrate 50ng, Sulbactam 10-30 μ g;
5) get each two bottles of the freeze-drying cillin bottles that contain Ampicillin Trihydrate medicament freeze-drying cillin bottle and contain Ampicillin Trihydrate+sulbactam medicine respectively, add 1mL milk to be measured respectively, the former is labeled as A, C, and the latter is labeled as B, D; The β-Nei Xiananmei 10ul that adds 3000U/mL more respectively in C, D bottle with above-mentioned freeze-drying cillin bottle mixing, puts in the incubator 36 ℃ ± 1 ℃ and hatches.And establish the pure water sample and do negative control;
6) get milk sample and each 50 μ L of pure water sample in the freeze-drying cillin bottle of the above-mentioned A of being labeled as, B, C, D, join on the blank test-paper that is labeled as A, B, C, D;
7) treat that milk is fully absorbed by filter paper after, each culture dish is put 55-60 ± 1 ℃ cultivates 6h;
8) scraps of paper with each culture dish take out gently with tweezers, with inhibition zone survey meter or vernier caliper measurement antibacterial circle diameter.
Result report: the pure water sample result should be: (A), (B), (D) all should produce inhibition zone; (A) inhibition zone is compared with the inhibition zone of (B), and with interior (containing 2mm), and repeatability is good at 2mm for difference; (C) inhibition zone is less than the inhibition zone of (D), and difference (contains 2mm) more than 2mm, and repeatability is good.As result for this reason, can carry out following judgement to sample result:
If in the sample result (B) and, (D) all produce inhibition zone, and when (C) and (D) inhibition zone difference (contains 2mm) more than 2mm, can be by (1), (2) result of determination:
(1) inhibition zone (A) (contains 2mm) less than the inhibition zone difference of (B) more than 2mm, and repeatability is good, should judge that this sample is added with β-Nei Xiananmei, and report beta-lactam enzyme drug abuse test is the positive as a result.
(2) less than 2mm, and repeatability is good, should judge that this sample is not added with β-Nei Xiananmei with the inhibition zone difference of (B) for inhibition zone (A), and report beta-lactam enzyme drug abuse test is feminine gender as a result.
(3) if (A) He (B) all do not produce inhibition zone, the amount that β-Nei Xiananmei in this sample is described should will detect behind the diluted sample than higher again.
The invention has the beneficial effects as follows: the detection kit of β-Nei Xiananmei is with low cost in raw milk of the present invention and the milk preparation, the check using method is convenient to operation, the result is highly sensitive, can be widely used in the check of antibiotic content in raw milk and the milk preparation, can guarantee that people take healthy milk.
Embodiment
The present invention is based on and uses the absolute responsive reference culture bacstearothermophilus of β-Nei Xiananleikangshengsu, utilize the Sulbactam specificity to suppress the activity of β-Nei Xiananmei, and add penicillin medicine in contrast, add beta-lactamase inhibitor by comparison and come the indirect measurement sample whether to contain beta-lactam enzyme medicine with the size that does not add the inhibition circle that sample produced of inhibitor.
Embodiment 1
1) blank milk is 5 parts, adds β-Nei Xiananmei, is mixed with the sample of following content: 3U/mL, 5U/mL, 10U/mL, 15U/mL, 30U/mL.And establish pure water as blank.
2) dilution of β-Nei Xiananmei standard substance is 3000U/mL.
3) take out vacuum-packed sterilization culture dish with long preservation period, vacuum-packed sterilization culture dish with long preservation period has been coated with bacstearothermophilus, according to the order of A, B, C, D the blank test-paper of 13mm is affixed on out vacuum-packed sterilization culture dish with long preservation period surface.
4) get each two bottles of freeze-drying cillin bottles that contain Ampicillin Trihydrate medicine 50ng and contain Ampicillin Trihydrate 50ng+ Sulbactam 10ug medicine respectively, add and added the milk 1mL that contains β-Nei Xiananmei of various dose, the freeze-drying cillin bottle that wherein contains the medicine of Ampicillin Trihydrate is labeled as A, C respectively, the freeze-drying cillin bottle that contains the medicine of Ampicillin Trihydrate+Sulbactam is labeled as B, D, adds the β-Nei Xiananmei 10ul of 3000U/mL more respectively in C, D bottle.Put in 36 ℃ ± 1 ℃ incubator and hatch half an hour.And establish the pure water sample and do negative control.
5) by getting the freeze-drying cillin bottle content 50 μ L that are labeled as A, B, C, D respectively, join on the blank test-paper that is labeled as A, B, C, D.
6) treat that milk is fully absorbed by filter paper after, each vacuum-packed sterilization culture dish with long preservation period is put 55 ± 1 ℃ cultivates 6h.
7) scraps of paper with each culture dish take out gently with tweezers, with inhibition zone survey meter or vernier caliper measurement antibacterial circle diameter.
8) pure water sample (A), (B), (D) all produce inhibition zone; (A) inhibition zone is compared with the inhibition zone of (B), difference at equal 2mm with interior and repeated good; (C) inhibition zone is less than the inhibition zone of (D), and difference (contains 2mm) more than 2mm, and repeatability is good, is reported as the β-Nei Xiananmei feminine gender.
9) contain the milk sample of 3U/mL, 5U/mL, 10U/mL, 15U/mL, 30U/mL β-Nei Xiananmei, inhibition zone (A) is all less than the inhibition zone of (B), and difference is all more than 2mm, and repeatability is good, all is reported as the β-Nei Xiananmei assay positive.
Embodiment 2
1) dilution of β-Nei Xiananmei standard substance is 3000U/mL.
2) get 5 parts in blank milk, add β-Nei Xiananmei, be mixed with the sample of following content: 3U/mL, 10U/mL, 30U/mL, 100U/mL, 300U/mL.And establish pure water as blank.
3) but get the culture dish of vacuum packaging prolonged preservation sterilization, vacuum-packed sterilization culture dish with long preservation period has been coated with bacstearothermophilus, according to the order of A, B, C, D blank test-paper is affixed on the culture dish surface.
4) get each two bottles of freeze-drying cillin bottles that contain Ampicillin Trihydrate medicine 50ng and contain Ampicillin Trihydrate 50ng+ Sulbactam 20 μ g respectively, add and added the β-Nei Xiananmei milk 1mL of various dose, the cillin bottle that wherein contains the medicine of Ampicillin Trihydrate is labeled as A, C respectively, the cillin bottle that contains the medicine of Ampicillin Trihydrate+Sulbactam is labeled as B, D, adds the β-Nei Xiananmei 10ul of 3000U/mL more respectively in C, D bottle.And blank get freeze-drying cillin bottle content 50 μ L and the pure water sample that is labeled as A, B, C, D, join on the blank test-paper that is labeled as A, B, C, D.
5) treat that milk and pure water are fully absorbed by filter paper after, each plate is put 55 ± 1 ℃ cultivates 6h.
6) scraps of paper with each culture dish take out gently with tweezers, with inhibition zone survey meter or vernier caliper measurement antibacterial circle diameter.
7) pure water sample (A), (B), (D) all produce inhibition zone; (A) inhibition zone is compared with the inhibition zone of (B), difference at equal 2mm with interior and repeated good; (C) inhibition zone is less than the inhibition zone of (D), and difference (contains 2mm) more than 2mm, and repeatability is good, is reported as the β-Nei Xiananmei feminine gender.
8) contain the milk sample of 3U/mL, 5U/mL, 10U/mL, 15U/mL, 30U/mL β-Nei Xiananmei, inhibition zone (A) is all less than the inhibition zone of (B), and difference is all more than 2mm, and repeatability is good, all is reported as the β-Nei Xiananmei assay positive.
Embodiment 3
1) dilution of β-Nei Xiananmei standard substance is 3000U/mL.
2) get 5 parts of blank sour milks, its pH is transferred to 7.0, add the β-Nei Xiananmei of dilution, be mixed with the sample of following content: 3U/mL, 30U/mL, 100U/mL, 300U/mL, 3000U/mL.And establish pure water as blank.
3) but get the culture dish of vacuum packaging prolonged preservation sterilization, vacuum-packed sterilization culture dish with long preservation period has been coated with bacstearothermophilus, but blank test-paper is affixed on the culture dish surface of vacuum packaging prolonged preservation sterilization according to the order of A, B, C, D.
4) get each two bottles of freeze-drying cillin bottles that contain Ampicillin Trihydrate 50ng and contain Ampicillin Trihydrate 50ng+ Sulbactam 20 μ g medicines respectively, add and added the β-Nei Xiananmei sour milk 1mL of various dose, the freeze-drying cillin bottle that wherein contains the medicine of Ampicillin Trihydrate is labeled as A, C respectively, the freeze-drying cillin bottle that contains the medicine of Ampicillin Trihydrate+Sulbactam is labeled as B, D, adds the β-Nei Xiananmei 10ul of 3000U/mL more respectively in C, D bottle.Put in 36 ℃ ± 1 ℃ incubator and hatched 30 minutes.And establish the pure water sample and do negative control.
5) get freeze-drying cillin bottle content (milk sample and pure water sample) the 50 μ L that are labeled as A, B, C, D respectively, join on the blank test-paper that is labeled as A, B, C, D.
6) treat that sour milk and pure water are fully absorbed by filter paper after, each plate is put 55 ± 1 ℃ cultivates 6h.
7) scraps of paper with each culture dish take out gently with tweezers, with inhibition zone survey meter or vernier caliper measurement antibacterial circle diameter.
8) pure water sample (A), (B), (D) all produce inhibition zone; (A) inhibition zone is compared with the inhibition zone of (B), difference at equal 2mm with interior and repeated good; (C) inhibition zone is less than the inhibition zone of (D), and difference (contains 2mm) more than 2mm, and repeatability is good, is reported as the β-Nei Xiananmei feminine gender.
9) contain the milk sample of 3U/mL, 5U/mL, 10U/mL, 15U/mL, 30U/mL β-Nei Xiananmei, inhibition zone (A) is all less than the inhibition zone of (B), and difference is all more than 2mm, and repeatability is good, all is reported as the β-Nei Xiananmei assay positive.
10) contain the milk sample of 3000U/mL β-Nei Xiananmei, (A), (B), (C), (D) bacterium do not have inhibition zone and produce, the excessive concentration of β-Nei Xiananmei in the interpret sample needs that sample is further diluted the back and detects.
Embodiment 4
1) dilution of β-Nei Xiananmei standard substance is 3000U/mL.
2) get from the milk cattle cultivating family and each 5 parts of raw milks that milking station is gathered, be labeled as R1～R5, R6～R10 respectively, and establish pure water as negative control.
3) but get the culture dish of vacuum packaging prolonged preservation sterilization, vacuum-packed sterilization culture dish with long preservation period has been coated with bacstearothermophilus, but the blank test-paper of 13mm is affixed on the culture dish surface of vacuum packaging prolonged preservation sterilization according to the order of A, B, C, D.
4) get the freeze-drying cillin bottle that contains Ampicillin Trihydrate 50ng and contain Ampicillin Trihydrate 50ng+ Sulbactam 20 μ g medicines, add each milk sample respectively, the cillin bottle that contains the medicine of Ampicillin Trihydrate is labeled as A, C respectively, the cillin bottle that contains the medicine of Ampicillin Trihydrate+Sulbactam is labeled as B, D, in C, D bottle, add the β-Nei Xiananmei 10ul of 3000U/mL more respectively, put in 36 ℃ ± 1 ℃ incubator and hatch half an hour.And establish the pure water sample and do negative control.
5) get freeze-drying cillin bottle content 50 μ L and the pure water sample that is labeled as A, B, C, D respectively, join on the blank test-paper that is labeled as A, B, C, D, treat that milk and pure water are fully absorbed by filter paper after, each culture dish is put 55 ± 1 ℃ cultivates 6h.
6) scraps of paper with each culture dish take out gently with tweezers, with inhibition zone survey meter or vernier caliper measurement antibacterial circle diameter.
7) pure water sample (A), (B), (D) all produce inhibition zone; (A) inhibition zone is compared with the inhibition zone of (B), difference at equal 2mm with interior and repeated good; (C) inhibition zone is less than the inhibition zone of (D), and difference (contains 2mm) more than 2mm, and repeatability is good, is reported as the β-Nei Xiananmei feminine gender.R1～R6, R9～R10 milk sample A) inhibition zone is compared with the inhibition zone of (B), and difference with interior and repeated good, is reported as the β-Nei Xiananmei feminine gender at equal 2mm; The inhibition zone of R7～R8 milk sample (A) is all less than the inhibition zone of (B), and difference is all more than 2mm, and repeatability is good, all is reported as the β-Nei Xiananmei assay positive.
Here explanation a bit is that example of the present invention is a practical example and unrestricted, and the same idea design should be at the row of the present invention's protection.In addition, it is on probation that embodiment 1, embodiment 2, embodiment 3 can be understood as the laboratory, actual measurement when embodiment 4 is practical.Technical term does not have special feature among the present invention, and those skilled in the art can both understand.
Milk of the present invention and milk preparation are equal to cow's milk and milk-product.
The present invention the invention still further relates to other milk and milk preparation with milk and milk preparation explanation, and for example goat milk, mare's milk, camel milk, yak milk etc. reach milk preparation, and these domain classes should be at the row of the present invention's protection like the method for inspection.
Claims (7)
1, the detection kit of β-Nei Xiananmei in a kind of raw milk and the milk preparation is characterized in that described test kit comprises following compositions:
1. vacuum-packed sterilization culture dish with long preservation period;
2. β-Nei Xiananmei standard substance;
3. contain Ampicillin Trihydrate freeze-drying cillin bottle and contain Ampicillin Trihydrate+Sulbactam freeze-drying cillin bottle;
4. blank filter paper sheet;
5. diluent.
2, the detection kit of β-Nei Xiananmei in raw milk according to claim 1 and the milk preparation, it is characterized in that vacuum-packed sterilization culture dish with long preservation period is the disposable plate of 50-120mm, pour into calibrating substratum (pH value 7.8-8.5) 10-30mL, preservation condition is 4-6 ℃, keeps in Dark Place.
3, the detection kit of β-Nei Xiananmei in raw milk according to claim 1 and the milk preparation is characterized in that described β-Nei Xiananmei standard substance are 100-400 ten thousand units, and preservation condition is 4-7 ℃.
4, the detection kit of β-Nei Xiananmei in raw milk according to claim 1 and the milk preparation, it is characterized in that described contain Ampicillin Trihydrate freeze-drying cillin bottle and contain Ampicillin Trihydrate+Sulbactam freeze-drying cillin bottle respectively contain Ampicillin Trihydrate 50ng, Ampicillin Trihydrate 50ng+ Sulbactam 10-30 μ g, 4-6 ℃ of preservation.
5, the detection kit of β-Nei Xiananmei in raw milk according to claim 1 and the milk preparation is characterized in that diluent is aseptic, pH8.0-10.0 phosphate buffered saline buffer.
6, the detection method of the detection kit of β-Nei Xiananmei in raw milk according to claim 1 and the milk preparation is characterized in that its detection method may further comprise the steps:
1) the β-Nei Xiananmei standard substance is diluted;
2) get vacuum-packed sterilization culture dish with long preservation period, the blank filter paper sheet is affixed on the culture dish surface according to the order of A, B, C, D;
3) get each two bottles of the freeze-drying cillin bottles that contain Ampicillin Trihydrate medicament freeze-drying cillin bottle and contain Ampicillin Trihydrate+sulbactam medicine respectively, add milk to be measured respectively, the former is labeled as A, C, and the latter is labeled as B, D; β-Nei Xiananmei standardized solution with dilution adds respectively in C, the D bottle again, with above-mentioned freeze-drying cillin bottle mixing, puts in the incubator and hatches, and establish the pure water sample and do negative control;
4) by milk sample and pure water sample in the freeze-drying cillin bottle of getting the above-mentioned A of being labeled as, B, C, D respectively, corresponding joining on the blank filter paper sheet that is labeled as A, B, C, D;
5) treat milk and pure water fully by after the filter paper absorption, with each plate cultivation 6h;
6) filter paper of each culture dish takes out gently with tweezers, with inhibition zone survey meter or vernier caliper measurement antibacterial circle diameter.
7, the detection method of the detection kit of β-Nei Xiananmei in raw milk according to claim 6 and the milk preparation is characterized in that its detection method may further comprise the steps:
1) dilution of β-Nei Xiananmei standard substance is 3000U/mL;
2), 10-13mm blank filter paper sheet is affixed on the culture dish surface according to the order of A, B, C, D with vacuum packaging sterilization culture dish with long preservation period;
3) culture dish has been coated with bacstearothermophilus;
4) contain Ampicillin Trihydrate medicine 50ng in the freeze-drying cillin bottle respectively; Contain Ampicillin Trihydrate 50ng, Sulbactam 10-30 μ g;
5) get each two bottles of the freeze-drying cillin bottles that contain above-mentioned Ampicillin Trihydrate medicament freeze-drying cillin bottle and contain Ampicillin Trihydrate+sulbactam medicine respectively, add 1mL milk to be measured respectively, the former is labeled as A, C, and the latter is labeled as B, D; The β-Nei Xiananmei 10ul that adds 3000U/mL more respectively in C, D bottle with above-mentioned cillin bottle mixing, puts in the incubator 36 ℃ ± 1 ℃ and hatches, and establish the pure water sample and do negative control;
6) get milk sample and each 45-60 μ L of pure water sample in the freeze-drying cillin bottle of the above-mentioned A of being labeled as, B, C, D, join on the blank test-paper that is labeled as A, B, C, D;
7) treat that milk is fully absorbed by filter paper after, each plate is put 55-60 ± 1 ℃ cultivates 6h;
8) scraps of paper with each culture dish take out gently with tweezers, with inhibition zone survey meter or vernier caliper measurement antibacterial circle diameter.
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| CN2009100648136A CN101545003B (en) | 2009-05-07 | 2009-05-07 | Detection kit and detection method for beta-lactamase in raw milk and milk products |
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| CN2009100648136A CN101545003B (en) | 2009-05-07 | 2009-05-07 | Detection kit and detection method for beta-lactamase in raw milk and milk products |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102042980A (en) * | 2010-10-29 | 2011-05-04 | 北京物资学院 | Detection of beta-lactamase in milk and milk products by iodometry |
| CN102115780A (en) * | 2010-12-15 | 2011-07-06 | 黑龙江省乳品工业技术开发中心 | Kit for detecting beta-lactamase as well as preparation and detection method thereof |
| CN104483363A (en) * | 2015-01-05 | 2015-04-01 | 天津商业大学 | Method for manufacturing electrochemical biosensor for measuring antibiotic |
| CN106153613A (en) * | 2016-06-20 | 2016-11-23 | 内蒙古蒙牛乳业（集团）股份有限公司 | The detection method of sulbactam in milk |
| CN106554989A (en) * | 2015-09-29 | 2017-04-05 | 北京华益精点生物技术有限公司 | A kind of test kit and its detection method for detecting beta-lactamase in milk |
| CN108037286A (en) * | 2017-12-12 | 2018-05-15 | 北京市饲料监察所（北京市饲料质量监督检验站） | A kind of method and kit for detecting beta-lactamase |
| CN108330158A (en) * | 2018-04-09 | 2018-07-27 | 江苏省产品质量监督检验研究院 | Exogenous analysis method in a kind of fresh milk beta-lactamase |
| CN109342531A (en) * | 2018-10-16 | 2019-02-15 | 吉林大学 | A kind of electrochemical method detecting beta-lactam antibiotic |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20050069901A1 (en) * | 2003-09-29 | 2005-03-31 | Eppendorf Ag | Method for detecting microbial antibiotic resistance |
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- 2009-05-07 CN CN2009100648136A patent/CN101545003B/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102042980A (en) * | 2010-10-29 | 2011-05-04 | 北京物资学院 | Detection of beta-lactamase in milk and milk products by iodometry |
| CN102042980B (en) * | 2010-10-29 | 2013-02-27 | 北京物资学院 | Detection of beta-lactamase in milk and milk products by iodometry |
| CN102115780A (en) * | 2010-12-15 | 2011-07-06 | 黑龙江省乳品工业技术开发中心 | Kit for detecting beta-lactamase as well as preparation and detection method thereof |
| CN104483363A (en) * | 2015-01-05 | 2015-04-01 | 天津商业大学 | Method for manufacturing electrochemical biosensor for measuring antibiotic |
| CN106554989A (en) * | 2015-09-29 | 2017-04-05 | 北京华益精点生物技术有限公司 | A kind of test kit and its detection method for detecting beta-lactamase in milk |
| CN106153613A (en) * | 2016-06-20 | 2016-11-23 | 内蒙古蒙牛乳业（集团）股份有限公司 | The detection method of sulbactam in milk |
| CN108037286A (en) * | 2017-12-12 | 2018-05-15 | 北京市饲料监察所（北京市饲料质量监督检验站） | A kind of method and kit for detecting beta-lactamase |
| CN108330158A (en) * | 2018-04-09 | 2018-07-27 | 江苏省产品质量监督检验研究院 | Exogenous analysis method in a kind of fresh milk beta-lactamase |
| CN109342531A (en) * | 2018-10-16 | 2019-02-15 | 吉林大学 | A kind of electrochemical method detecting beta-lactam antibiotic |
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