CN102409077A - Method for detecting antibiotic residues by microplate and bacillus stearothermophilus - Google Patents

Abstract

The invention discloses a method for detecting antibiotic residues by a microbiological method on a microplate, which is characterized in that bacillus stearothermophilus is adopted as bacteria for experiments, the antibiotic residues in animal-derived food is detected on the 96-pore microplate, bromocresol purple is used as indicators, and whether the antibiotic residues exist or not is determined through the discoloration of the indicators. The method can be used in a laboratory for coarsely screening the antibiotic residues in the animal-derived food. Experiments show that compared with the traditional flat plate microbiological method, the method has the advantages that the detection efficiency is high, ingredients are saved, and in addition, the sensitivity is high. The method has main steps that: a detecting culture medium is prepared by adding distilled water into beef extract, glucose, yeast powder and bromocresol purple indicators, in addition, the pH is regulated to be 7.5, bacterium suspension containing the bacteria is added, samples to be tested are added and are cultured in a culture box for 3h, and the color change of the culture medium is observed for judging whether the antibiotic residues exist in the samples or not.

Description

A kind of microplate and residual method of bacstearothermophilus detect antibiotics of adopting

Technical field

The present invention relates to the method that antibiotic remains detects in a kind of animal-derived food that is used for, particularly fresh milk.

Background technology

Along with growth in the living standard, people more and more pay attention to self and relatives' health, mostly formed the custom of " Diet cures more than the doctors ", but the security of these tonic raw materials are more and more troubling.Since the nineties in 20th century; Drug residue exceeds standard China's outlet animal food is refused repeatedly; At the beginning of 2002; European Union announces to completely forbid the animal food and the fishery products such as shrimp, rabbit and poultry meat of import China, causes China's product water outlet decline first half of the year in 2002 more than 70% thus.In the same year, paraxin exceeds standard in the honey of outlet European Union, and having caused Zhejiang Province is the loss of export enterprise's multi-billion dollar of master.Crisis-ridden especially on human consumer's the dining table.The Ministry of Agriculture shows that to certain live pig market, city sampling observation report nearly twenty percent pork antibiotic remains exceeds standard on this market, city a few days ago.90% is used on one's body the edible animal in the annual microbiotic total amount that consumes in the whole world, and 90% all is to use as fodder additives in order to improve food conversion ratio wherein.Pertinent data shows that based on the leak that orders about and supervise of interests, abuse of antibiotics has become " malignant tumour " of farming animals aquaculture.

China starts late to the attention of microbiotic hazardness problem, in the food hygienic standard of China 1996 promulgation, the residue detection of veterinary drug also is only limited to one of Cyclomycin family antibiotic.Pollution-free food plan in 2002 comes into effect, and the Ministry of Agriculture has stipulated standard and the MRL that pollution-free food (pork, chicken, fishery products etc.) herbal medicine uses with the rules form.Confirmed to comprise that 4 kinds of FQNSs are the usage criteria of planting beastly (fishing) medicine totally surplus in the of 60 of microbiotic such as PD 160788, difloxacin, danofloxacin, sarafloxacin and R-802; But the standard detecting method of promulgation is but very limited simultaneously, and the standard test method of in meat, releasing only has less than 10 kinds.The safety-problems of animal food has become the health problem that people pay close attention to.And so far, China does not carry out complete investigation and regular monitoring to microbiotic pollution condition in the animal food as yet.Along with the whole society and government department to the exceed standard attention of problem of animal-derived food microbiotic; Research and develop that some are simple, fast, the analytical technology that can detect multiple antibiotic remains of economy and portability is the inexorable trend that develops; Especially particularly needing in the screening operation of large sample amount to carry out a kind of high-level efficiency at quality inspection organization; Microbiotic detection method cheaply, this also must be with improving China's food microbiotic detection level, for the steady progression of China's economy with society provides safeguard.

The antibiotic residues in animal-derived food detection method mainly contains the microorganism detection method, the physics and chemistry detection method, and like HPLC, vapor-phase chromatography and tlc etc. and three types of immunoassays.

(TLC) is simple to operate for tlc, reliable results, but still need the complicated sample pre-treatment, sensitivity is not high.Method such as HPLC and GC has sensitivity and specificity highly than microbial method, has efficient, quick; Highly sensitive; Characteristics such as recall rate height, but apparatus expensive also has higher requirement to testing crew; Analyze time-consuming and difficult popularization, it also needs complicated loaded down with trivial details sample pre-treatments and can not screen a large amount of samples simultaneously simultaneously.Immunoassay is highly sensitive, and specificity is stronger than microbial method, and the specificity of monoclonal antibody is higher, but detecting cost has relatively high expectations, and is not easy to promote.

Microbial method is a classical way of measuring antibiotic remains, principle be according to microbiotic to the physiological function of mikrobe, metabolic restraining effect, qualitative or quantitatively confirm residues of antibiotics in the sample; Present microbial method mainly contains cotton swab method, cylinder plate method and paper disk method; These methods generally are applicable to the residual mensuration of antibacterials, operate simply relatively, and expense is low; General laboratory can both be operated; Be suitable for sample rapid screening in batches, but often lack specificity and tolerance range, and receive other antibiotic interference.Although microbial method comes with some shortcomings; All there is the method for applying mechanically antibiotic remains in microbial method rapid detection edibility animal tissues and the food in many developed countries; Especially the aminoglycoside antibiotics that ultraviolet is not had characteristic absorbance, the mikrobe method of tiring are the main method that current various countries pharmacopeia is measured such antibiotic content.Simultaneously, some companies suppress the commercialization of method test kit with mikrobe, and like BSDA, Charm&receptor assay and ECLIPSE etc., detectability possibly satisfy the maximum residue limit standard of European Union.

In China, microbial method is used to be used for detecting the antibiotic remains of animal food in national standard and the ministerial standard.But the certain methods particularity that adopts and ageing all relatively poor relatively; Can't satisfy present detection requirement, and also less to the improvement research of microbial method, therefore; Use for reference the microplate microbial method in sterilant, sanitas exploitation; With the technology in the compound risk assessment, and the advanced experience of external exploitation microorganism reagent box, efficient, sensitive, the cheap microbial method based on microplate of exploitation detects the antibiotic remains in the animal food; Be widely used in quality inspection, agricultural-food supervision and management, commodity inspection and hygiene department, and lay technical foundation for exploitation has the test kit of having intellecture property by oneself.

Summary of the invention

The technical problem that the present invention will solve is to set up a kind of simple, quick, highly sensitive, the antibiotic remains microorganism detection method that can generally apply.For solving the problems of the technologies described above, the technical scheme that the present invention adopts is:

(1) actication of culture

The preparation beef-protein medium is subsequent use, and under aseptic condition, picking one ring bacstearothermophilus is seeded to the beef extract-peptone inclined-plane with strok method, puts into 55 ℃ constant incubator and cultivates 24h.Bacterial classification after this test recovery used after 1-2 days.

(2) shake bacterium and switching

Confirm that according to the OD value of bacstearothermophilus suspension in the test shaking table the best shakes the bacterium time, cultivate and detection so that transfer in the best time.When the OD value was 0.2, the growth of bacterium was the most vigorous, was fit to switching and cultivated detection.

On the aseptic technique platform, bacstearothermophilus is received in the Erlenmeyer flask that fills 75mL common liq substratum, sealed, put into shaking table at once; At 55 ℃; Shake up under the condition of 150r/min, in time test its OD value, when the OD value is 0.2 left and right sides (this experiment is roughly 5 hours); Bacteria suspension is taken out from shaking table, wait for switching.

(3) preparation mixed bacteria liquid

Shake the bacterium time full after; Take out bacterium liquid bottle, on the aseptic technique platform, get the bacterium liquid 5mL (will fully shake up before getting) that amplification (waits to transfer) well with liquid-transfering gun and be added to mixing in the 1000mL substratum; This substratum contains glucose 1%; Yeast powder 0.2%, Carnis Bovis seu Bubali cream 0.5%, purpurum bromocresolis indicator 0.00005%.

(4) making of microplate

Get above mixed bacteria liquid 270 μ L with liquid-transfering gun, add in the microplate hole, do aseptic blank with the substratum that does not add bacterium liquid simultaneously, in test sample microplate hole, add the extracting solution of 30 μ L milk samples or other animal-derived food again.

(5) detect

Prepared detection microplate is put into 55 ℃ of constant incubators cultivate observations behind the 3h.Do not contain the detection limit that microbiotic or antibiotic content are lower than method in the microplate hole in the substratum its colour changed into yellow interpret sample.If the color of substratum is a purple still in the microplate hole, antibiotic remains is arranged in the interpret sample.

The detectability of method (ppb):

Penicillin G 1-2

Amoxycilline Trihydrate bp 1-4

Cephalexin Monohydrate Micro/Compacted 25-50

Cephapirin 2-4

Terramycin 20-80

Tsiklomitsin 20-80

Oxacyclotetradecane,erythromycin deriv 100-200

Paraxin 3000

Application example one

The single bacterium colony of bacstearothermophilus of picking cultivation previous day is put into liquid nutrient medium in Bechtop, puts into shaking table, and 55 ℃ shake cultivation 7h down, and the scope of survey OD600 is between 0.2-0.4.

Only add 270 μ L substratum at microplate the 1st row, what between 2～12 examples, add is the mixing solutions (wherein detecting substratum 87.5%, bacteria suspension 12.5%) that 270 μ L detect substratum and bacteria suspension; Then the 1st; 3～6 list continuation adds 30 μ L milk samples, lists the adding sterilized water the 2nd, adds milk and antibiotic mixtures 7～12; What wherein A～D added is the mixture of penicillium mould and milk, and 7～12 routine concentration are followed successively by 10～10 ^-5μ g/mL, what E～H added is the mixture of paraxin and milk, 7～12 concentration are followed successively by 1～10 ^-5μ g/mL.In 55 ℃ of constant incubators, cultivate 3h and observe, the minimum concentration that adds variable color in the penicillium mould sample is 0.001 μ g/mL, and the minimum concentration that adds the variable color of paraxin sample is 1 μ g/mL, and this just meets country to these two kinds of antibiotic safety standardss.All do not add the equal yellowing of substratum of antibiotic milk sample, explain that present commercially available milk all is antibiotic-free milk.

Application example two

The single bacterium colony of bacstearothermophilus of picking cultivation previous day is put into liquid nutrient medium in Bechtop, puts into shaking table, and 55 ℃ shake cultivation 7h down, and the scope of survey OD600 is between 0.2-0.4.

Only add 270 μ L substratum at microplate the 1st row; What between 2～12, add is that the mixing solutions that 270 μ L detect substratum and bacteria suspension (wherein detects substratum 87.5%; Bacteria suspension 12.5%), continue to add the egg white of 20 times of 30 μ L feed eggs dilutions then at the A～D of the 1st row, the E of the 1st row～H adds the egg white of 20 times of Countryside Egg dilutions; List the adding sterilized water the 2nd; Add the feed egg at 3～12 routine A～D and dilute 20 times egg white, add Countryside Egg at E～H and release 20 times egg white, observations behind the cultivation 3h in 55 ℃ of constant incubators.In 11 samples of Countryside Egg, 1 not variable color of sample is arranged, and 4 not variable colors are arranged in 11 samples of feed egg, explain that these samples might have residues of antibiotics, but need to adopt chromatography further to confirm.

Claims (3)

1. one kind is used for the microbial process that antibiotic remains in the animal-derived food, particularly fresh milk detects, and it is characterized in that test experience carries out on 96 hole microplates.

2. the mikrobe in the claim 1 is characterized in that adopting bacstearothermophilus (Bacillus stearothermophilus) for supplying the examination bacterium.

3. the detection method in the claim 1 is characterized in that with the purpurum bromocresolis being color indicator.