CN109270056A - A kind of method and microorganism colour reagent box detecting high-throughput antibiotic residue - Google Patents
A kind of method and microorganism colour reagent box detecting high-throughput antibiotic residue Download PDFInfo
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- CN109270056A CN109270056A CN201811190620.0A CN201811190620A CN109270056A CN 109270056 A CN109270056 A CN 109270056A CN 201811190620 A CN201811190620 A CN 201811190620A CN 109270056 A CN109270056 A CN 109270056A
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- microorganism
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- reagent box
- bacteria
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Classifications
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Abstract
The present invention relates to a kind of methods and microorganism colour reagent box for detecting high-throughput antibiotic residue.Method of the present invention be it is a kind of with single or compatible use antibiotic common in animal derived food for detection target, using microorganism colour developing as principle high throughput quickly detect its remaining prescreening method and kit.The method is using escherichia coli (Escherichia coil) as indicator bacteria, optimize its microcapsule embedded condition, it is set to be more suitable for microorganism development process, and embedded particles are fixed in 96 microwell plates, the kit that can while carry out joint primary dcreening operation to different classes of Multiple Classes of Antibiotics is made, overcomes because of the problems such as actication of culture takes a long time and liquid spawn can not transport.Compared with existing method for antibiotic residue detection, operation of the present invention is easier, quick, low in cost, and as a result accurate and naked eyes can judge according to colour developing, without large-scale instrument.
Description
Technical field
The present invention relates to the detections of antibiotic residue, and in particular to a kind of method for detecting high-throughput antibiotic residue and its
Microorganism colour reagent box.
Background technique
Antibiotic plays the effects of preventing and treating disease in livestock and poultry intensive manufacture, but abuses these antibiotic and can make
Its by animal body itself transfer, enrichment enter human foods chain, eventually lead to human body and develop drug resistance, influence disease treatment with
Rehabilitation.Current antibiotic abuse condition still remains and the situation is tense；Wherein, high-throughput, quick detection method will become not
To detect development trend.Currently, main remaining antibiotic includes quinolones, Tetracyclines and aminoglycoside in livestock meat
Deng detection method mainly has chromatography, microbiological method and immunoassay etc..Microbial method is anti-as earliest detection
The raw remaining method of element has the characteristics that low in cost, easy to operate, high-throughput, and China's microorganism development process is chiefly used in milk
The detection of middle antibiotic residue, the research for detecting antibiotic residue in poultry meat with this method are less.
Main remaining antibiotic includes quinolones, Tetracyclines and aminoglycoside etc. in livestock meat at present, in order to
Enhance antibiotic curative effect, shorten the course of disease, reduces bacterial drug resistance, usually two or more Antibiotic combination is made now
With.And more common chromatography in existing method, qualitative and quantitative detection can be carried out, it is as a result sensitive and accurate, but equipment investment
Greatly, testing cost is high, and different sample pre-treatments are different and complicated, is difficult to accomplish that inhomogeneity Antibiotic combination detects, be suitable for big
Pattern synthesis laboratory carries out arbitration decision and confirmation.Though immunoassay high sensitivity, testing cost is expensive, Zhi Nengjian
Simple sample, single antibiotic are surveyed, high throughput is not easy to.Compared to first two method microbial method due to low in cost, operation
Simply, there is broad spectrum activity, do not restricted by Antibiotics, can be used as a kind of high-throughput antibiotic prescreening method and apply in agricultural production
Product security control link, and popularized in medium-sized and small enterprises.
Microbial method is the method that antibiotics leftover detection uses earliest, and there are many commercially available reagent box at present, such as
Delvotest, CharmScien etc. and China's national standard TTC method, more problems existing for national standard TTC method, as sensitivity compared with
Low, cumbersome, the preparation time of test organisms is long, and different strains drug resistances is different, sensitivity also difference etc., and external import
Kit is expensive.The kit developed both at home and abroad using microorganism colour developing principle at present is mostly to use bacillus stearothermophilus
As indicator bacteria, it is higher to the sensibility of inhibiting substances, and growth is more quick, is mainly used for beta-lactam antibiosis in milk
The remaining detection of element, but the bacterium to remain in fowl, the major antibiotics quinolones in poultry meat it is insensitive.Therefore existing method and
Strain is suitable for milk sample detection more, and the research that antibiotic residue is detected in poultry meat is less.In addition, food industry is raw
The production amount of having the characteristics that is big, batch is more, the period is short, if every part of sample all uses HPLC method to detect, testing cost is surprising, and
Sometimes enterprise does not need particularly accurate numerical value for the residual quantity of antibiotic in raw meat, only wonders in the meat bought
Whether antibiotic is contained.Therefore, develop it is a kind of it is easy to operate, expense is low, detectable fowl, in poultry meat antibiotic residue high pass
It is imperative to measure fast microbiological colour developing prescreening method.And its primary ring is screening one to Multiple Classes of Antibiotics, it is especially right
Quinolone antibiotics are sensitive and sensibility is stable, detection limit is low, the rapid indicator bacteria of growth, and how maximum study
Shorten the actication of culture time, improves the utilization rate of strain, achieve the purpose that quickly to detect.
Summary of the invention
Method of the present invention be develop it is a kind of with common single or compatible use anti-in animal derived food
Raw element is detection target, quickly detects its remaining prescreening method and kit by the high throughput of principle of microorganism colour developing.Institute
Method is stated using escherichia coli (Escherichia coil) as indicator bacteria, optimizes its microcapsule embedded condition, makes it more
It is fixed in 96 microwell plates suitable for microorganism development process, and by embedded particles, being made can be simultaneously to different classes of a variety of antibiosis
Element carries out the kit of joint primary dcreening operation, overcomes because of the problems such as actication of culture takes a long time and liquid spawn can not transport.With it is existing
There is method for antibiotic residue detection to compare, operation of the present invention is easier, quick, and low in cost, as a result accurate and naked eyes can root
Judge according to colour developing, without large-scale instrument.
Wherein, the present invention can be simultaneously to antibiotic such as quinolones common in poultry meat, aminoglycoside, Tetracyclines
Carry out the detection of joint primary dcreening operation.
A kind of method detecting high-throughput antibiotic residue provided by the invention, using escherichia coli as indicator bacteria, inspection
Survey the microorganism development process of antibiotic residue in poultry meat.
The culture presevation number of the escherichia coli (Escherichia coil) is CICC23657.
The present invention overcomes to improve strain color developing effect because actication of culture takes a long time and liquid spawn can not be transported
Problem further carries out microencapsulation processing to the indicator bacteria；The present invention has found after study, microcapsule embedded using PVA
After method carries out microencapsulation to indicator bacteria, significant effect is promoted；Especially select PVA for occlusion vehicle, boric acid-sodium tetraborate
Group is combined into embedding crosslinking agent, and on this basis, advanced optimizes the actual conditions of its embedding.
Preferably, the microcapsule embedded method specifically: preparing weight percent is 8%-15% polyvinyl alcohol (PVA)
After sterilization treatment, the bacteria suspension of escherichia coli is added, then slowly by it in bromine potassium phenol violet dextrose peptone medium solution
It instills in boric acid-sodium tetraborate solution, then in 3~5 DEG C of 5~7h of standing, filters the indicator bacteria particle of acquisition, clean spare.
It is furthermore preferred that when the weight percent of the polyvinyl alcohol is 9%~11%, the boric acid-sodium tetraborate solution
PH value be 6, embedding effect it is optimal.
Wherein, the antibiotic is selected from one of quinolones, aminoglycoside, Tetracyclines or a variety of.Especially
Antibiotics in following table, and associated with Antibiotics.
The detectable Antibiotics of table 1
Wherein, the poultry meat extracts in advance, the extraction specifically: after poultry meat is carried out homogenization, adds
Enter in the phosphate buffer solution of pH6.5-8.0 and is mixed well；Water-bath 3 at a temperature of being subsequently placed in 90~110 DEG C~
5min, high speed centrifugation take supernatant.
A kind of microorganism colour reagent box detecting high-throughput antibiotic residue provided by the invention, including microencapsulation
Indicator bacteria particle, detection liquid, poultry meat sample extracting solution and control group；
Wherein, the indicator bacteria in the indicator bacteria particle is escherichia coli.
Wherein, the detection liquid is the mixed liquor of escherichia coli bacteria culture fluid and indicator；
Preferably, the detection liquid by weight, including 8~12 parts of peptones, 2~4 portions of beef extracts, 8~12 parts
Glucose, 4~6 parts of NaCl, 0.03~0.05 part of Bromocresol purple, 800~1200 parts of water；It is described detection liquid pH value be
6.8-7.2；
Wherein, the poultry meat sample extracting solution is specially the phosphate buffer solution of pH6.5-8.0；Preferably pH7.4
Phosphate buffer solution.
Wherein, the control group includes positive controls, negative control group with blank control group；The positive controls are
Antibiotic standard solution, the negative control group are poultry meat sample extracting solution；
It wherein, is that no any antibiotic is remaining in the poultry meat sample extracting solution that the negative control group uses；
Preferably, the blank control group is physiological saline.
Preferably, the microorganism colour reagent box further includes 96 microwell plates that can be freely disassembled.
Wherein, gentamicin, streptomysin, benzyl penicillin, tetracycline, red mould is included at least in the antibiotic standard solution
Element, roxithromycin, Enrofloxacin, Norfloxacin, Ciprofloxacin；
Preferably, the concentration of the antibiotic standard solution is 0.08~0.12ug/mL.
Wherein, in order to keep the color developing effect of its indicator bacteria more prominent, the present invention advanced optimizes the finger of its microencapsulation
Show the embedding method of bacterium particle, specifically obtain in the following way:
It is molten to prepare the bromine potassium phenol violet dextrose peptone medium that weight percent is 8%-15% polyvinyl alcohol (PVA)
After sterilization treatment, the bacteria suspension of escherichia coli is added, then be slowly dropped into boric acid-sodium tetraborate solution, then in liquid
In 3~5 DEG C of 5~7h of standing, filtering obtains the indicator bacteria particle, cleans spare.
Wherein, in order to keep its described embedding effect more excellent, preferably use PVA- boric acid-sodium tetraborate for combined embedding
Outside carrier and crosslinking agent, the weight ratio and boric acid-sodium tetraborate solution pH value of polyvinyl alcohol are further controlled.
Preferably, the boric acid-sodium tetraborate solution pH value is 6.5~7.5.
When the weight percent of the polyvinyl alcohol is 9%~11%, the boric acid-sodium tetraborate for the use of pH value being 6 is molten
Liquid, embedding effect are optimal.
The boric acid-sodium tetraborate solution can be used under type such as and prepare: sodium tetraborate solution A: accurately weigh 1.907g
Sodium tetraborate heating is dissolved in 100mL distilled water；Boric acid solution B: it accurately weighs 3.711g boric acid and dissolves by heating and distilled in 100mL
In water.PH 6: A liquid 7mL and B liquid 43mL is taken to be mixed.
The sterilization treatment is carried out using usual manner, such as 121 DEG C of sterilizing 10min；When being subsequently cooled to 30~40 DEG C,
The bacteria suspension of escherichia coli is added.
The filtering is preferably using suction filtration；It is washed after suction filtration using physiological saline, seals 0~4 DEG C and save, is spare.
Wherein, the bacteria suspension of the escherichia coli obtains in the following way:
Escherichia coli is inoculated into fluid nutrient medium, after carrying out culture 22-24h under conditions of 35-37 DEG C；It takes
Bacterium solution after 0.1mL culture is in being coated optimization secondary culture 22-24h on solid medium, continuously biography 2-3 generation lives
Change；Thallus after taking activation, after being washed, then is placed in fluid nutrient medium to get indicator bacteria bacteria suspension；
Preferably, using the liquid culture medium as blank, in wavelength be 600nm under conditions of, take absorbance value be 0.8~
1.2 indicator bacteria bacteria suspension；The optimal indicator bacteria bacteria suspension chosen absorbance value and be 1.0.
Wherein, carrying out washing after the thallus activation can be used the progress of this field usual manner；Such as: by activated bacterium
Body washes lower lawn with fluid nutrient medium, is transferred in sterile centrifugation tube, sufficiently vibrates, and is centrifuged 5-10min with 3000r/min, abandons
Supernatant is removed, fluid nutrient medium is rejoined into centrifuge tube, repeatedly after centrifuge washing 2-3 times, is filled with fluid nutrient medium
Divide dissolution precipitating, obtains indicator bacteria bacteria suspension.
Wherein, the fluid nutrient medium preferably uses (parts by weight) as following formula: 10.0 parts of peptone, 3.0 parts of powdered beef,
It 5.0 parts of sodium chloride, 1.0 parts of glucose, is dissolved in 1000 parts of distilled water, tune pH value is 7.3-7.7,121 DEG C of high pressure sterilizations
15min；
Wherein, the solid medium preferably uses (parts by weight) as following formula: 15.0 parts of tryptone, phytone
5.0 parts, 5.0 parts of sodium chloride, 15.0 parts of agar, are dissolved in 1000 parts of distilled water, and tune pH value is 7.1-7.5,121 DEG C of high pressure sterilizations
15min。
Preferred embodiment provided by the invention, the detection method of the microorganism colour reagent box are specific as follows:
1) poultry meat after taking homogeneous is placed in poultry meat sample extracting solution and mixes；It is subsequently placed in 90~110 DEG C of temperature
3~5min of lower water-bath, high speed centrifugation take supernatant；
2) the indicator bacteria particle of the microencapsulation is fixed in the micropore in microwell plate, institute is successively added into every hole
Detection liquid is stated, is then separately added into the control group and the supernatant again；The microwell plate is placed in 36~45 DEG C of temperature
It is cultivated under degree, observes color change, judge antibiotic residue situation.
Preferably, step 1) specifically: weighing homogeneous, good 5~10g poultry meat sample is placed in 50mL centrifuge tube, is added
10mL poultry meat sample extracting solution, vortex 1min, sealing, which is set, heats 3-5min in 100 DEG C of boiling water baths, 10000r/min centrifugation
10min takes supernatant for detecting.
It is further preferred that the additional amount of the detection liquid is 150 μ L；The additional amount of the control group and the supernatant
It is respectively 200 μ L.
Wherein, 3 parallel controls are at least set；Result judgement are as follows: positive controls are purple；Negative control group is
Yellow；Blank control group is yellow；
If in 3 parallel samples in sample: it is positive: if wherein have 1 it is purple when, as positive model machine (suspicious sample
Product)；It is negative: when 3 Duplicate Samples detection liquid are in yellow, as negative sample (secure sample).
The present invention include at least it is following the utility model has the advantages that
One, the escherichia coli (CICC23657) that the present invention uses has the following characteristics that (1) to main in livestock meat
Remaining antibiotic (quinolones, Tetracyclines and aminoglycoside) is highly sensitive；(2) bacterial strain is stablized, easy to maintain and preparation；
(3) strain activity is strong, low to environmental condition, nutritional ingredient requirement, easily survives in capsulating material；It (4) can be in preference temperature
Lower fast-growth, it is fast that metabolism produces acid.
Two, the microorganism colour reagent box has the advantage that (1) strain microencapsulation is solid compared with traditional microbiological method
Due in micropore, facilitating storage and transport, and save the actication of culture generation time, greatly promotes detection efficiency, entirely detected
Journey 3-4h；(2) 96 microwell plates can be freely disassembled according to sample size, more flexible compared with classic flat-plate or tube manipulation, and should
Method agents useful for same by microlitre in terms of, be not necessarily to large-scale instrument and equipment, testing cost is lower.
Three, the PVA- boric acid suitable for microorganism development process-microcapsule embedded technology of sodium tetraborate is established.The embedding skill
Art overcomes since embedded material itself pH influences indicator discoloration problem in detection liquid, and polyvinyl alcohol is a kind of novel
Microorganism entrapped immobilized carrier, more existing embedding method (PVA- boric acid, carragheen, gelatin, cellulose acetate, sodium alginate)
With high mechanical strength, chemical stability is good, Resistance to microbes performance is strong, nontoxic to microorganism, cheap etc. a series of
Advantage is more suitable for microorganism development process.
Four, a kind of indicator bacteria, a kind of detection architecture and a kind of pre-treating method, can be anti-to different poultry meats, variety classes
Raw element carries out joint primary dcreening operation, both can detect single mark antibiotic or can detect combination antibiotic of the same race, not of the same race, has accomplished high throughput.
Five, the method for the present invention is respectively less than me for the detection limit of quinolones, Tetracyclines and aminoglycoside antibiotics
The Ministry of Agriculture of state 235 announces defined animal food herbal medicine maximum residue limit.
Detailed description of the invention
Fig. 1 is the appearance photo of microorganism colour reagent box provided by embodiment 1；
Fig. 2 is the colour developing situation map after embodiment 1 detects；
Fig. 3 is singly to mark the colour developing situation map that antibiotic changes with concentration in experimental example；
Fig. 4 is singly to mark the colour developing situation map that antibiotic changes with concentration in experimental example.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
In following examples
The preparation of antibiotic standard items: accurately weigh appropriate gentamicin, streptomysin, benzyl penicillin, tetracycline, erythromycin,
The standard items such as roxithromycin, Enrofloxacin, Norfloxacin, Ciprofloxacin, the mark for being 5mg/mL with deionized water dissolving and constant volume
Quasi- solution, then dilutes 0.1ug/mL.
The preparation of bacteria suspension: passage 2-3 generation is carried out by the activation requirement of bacterium, under aseptic conditions, from nutrient agar (NA)
Picking activated 5 plants of bacterium, are respectively connected in brain heart infusion broth (BHI) culture medium on culture medium, sufficiently vibrate, with
3000r/min is centrifuged 5min, discards supernatant liquid, BHI culture medium is rejoined into centrifuge tube, repeatedly centrifuge washing 2 times
Afterwards, precipitating is sufficiently dissolved with BHI culture medium, the bacteria suspension that absorbance value is 0.4~1.6 is made under wavelength 600nm.
Embodiment 1
The present embodiment provides a kind of microorganism colour reagent boxes and detection method for detecting high-throughput antibiotic residue；
The microorganism colour reagent box is as shown in Figure 1, comprising: indicator bacteria particle, the detection liquid, poultry meat of microencapsulation
Sample extracting solution and positive controls, negative control group, blank control group, 96 microwell plates that can be freely disassembled；
Wherein, the detection liquid is 10g peptone, 3g beef extract, 10g glucose, 5gNaCl, 0.04g bromocresol purple
Indicator, it is soluble in water to be settled to water 1000mL；The pH value of the detection liquid is 6.8-7.2；
The poultry meat sample extracting solution is the phosphate buffer solution of pH7.4；
The positive controls are antibiotic standard solution, and the negative control group is the remaining poultry meat sample of antibiotic-free
Product extracting solution；The physiological saline that the blank control group is 0.85%；
Wherein, the indicator bacteria particle of the microencapsulation uses immobilization embedded method, and indicator bacteria fixation support is containing weight
The bromine potassium phenol violet dextrose peptone medium solution that percentage is 10% polyvinyl alcohol (PVA) is measured, crosslinking agent is that pH value is 6
Boric acid-sodium tetraborate solution (sodium tetraborate solution A: accurately weighs the heating of 1.907g sodium tetraborate and is dissolved in 100mL distilled water；
Boric acid solution B: it accurately weighs 3.711g boric acid and dissolves by heating in 100mL distilled water.PH 6: take A liquid 7mL and B liquid 43mL into
Row mixing；).
Using the liquid culture medium as blank, under conditions of wavelength is 600nm, the initial light absorption value of the bacteria suspension is
1.0。
Concrete operations are as follows: preparation is the bromine potassium phenol violet glucose proteins of 10% polyvinyl alcohol (PVA) containing weight percent
The bacteria suspension that isometric initial absorbance value is 1.0 is added in peptone culture medium solution, 121 DEG C of sterilizing 10min after being cooled to 40 DEG C,
It is uniformly mixed, is slowly dropped into boric acid-sodium tetraborate solution with No. 6 syringe needles, in 4 DEG C of standing 6h, filter out indicator bacteria embedding ball
Shape particle is cleaned with physiological saline, is sealed 0~4 DEG C and is saved, is spare；
Detection method specifically:
1) weighing homogeneous, good 10g pork sample is placed in 50mL centrifuge tube, and 10mL poultry meat sample extracting solution, rotation is added
Whirlpool 1min, sealing, which is set, heats 3-5min in 100 DEG C of boiling water baths, 10000r/min is centrifuged 10min, takes supernatant for detecting.
2) the indicator bacteria particle of the microencapsulation is fixed in the micropore in microwell plate, institute is successively added into every hole
Detection 150 μ L of liquid is stated, is then separately added into the control group and each 200 μ L of the supernatant again；The microwell plate is placed in
It is cultivated at a temperature of 42 DEG C, observes color change, judge antibiotic residue situation.As shown in Fig. 2, detection after sample and
The colour developing situation of reference substance；Wherein, A is negative control, is in yellow；B is positive control, purple；C blank control is in yellow.
Embodiment 2~3
The present embodiment and the difference of embodiment 1 are only that the weight percent of polyvinyl alcohol (PVA) replaces with for 10%
8% and 12%.
Influence of the table 2PVA content to embedding effect
It can be seen that balling-up effect is preferable when the weight percent of polyvinyl alcohol is 10%.Embodiment 4~7
The present embodiment and the difference of embodiment 1 are only that, are 6 adjustment by crosslinking agent boric acid-sodium tetraborate solution pH value
It is 9,7,5 and 3.
Sodium tetraborate solution A: it accurately weighs the heating of 1.907g sodium tetraborate and is dissolved in 100mL distilled water；Boric acid solution B:
3.711g boric acid is accurately weighed to dissolve by heating in 100mL distilled water.
PH 9:A liquid itself pH；
PH 7: A liquid 10mL and B liquid 40mL is taken to be mixed；
PH 5: A liquid 4mL and B liquid 46mL is taken to be mixed；
PH 3:B liquid itself pH.
From table 3 it can be seen that crosslinking agent pH value has certain influence to balling-up operation and reflecting time.When pH value is 9,
Sodium tetraborate is contained only in crosslinking agent, is not easy balling-up at this time, and can increase detection liquid pH value, to extend the reaction time；And work as
When pH value is too low, it is also not easy balling-up, and peracid will affect indicator bacteria activity.The boric acid that pH is 6-sodium tetraborate solution is as crosslinking
Agent effect is optimal.
Influence of 3 boric acid of the table-sodium tetraborate solution ph to embedding effect
Embodiment 8~9
The present embodiment and the difference of embodiment 1 are only that bacteria suspension initial absorbance value is adjusted to 0.6 and 1.3 for 1.0.
As can be seen from Table 4, when bacteria suspension initial absorbance is higher, antibiotic is unobvious to the inhibitory effect of bacterium, instead
It answers liquid to become yellow, testing goal is not achieved；And when bacteria suspension initial absorbance is lower, the reaction time is longer and strain is not easy
Survival.Therefore, the initial light absorption value of bacteria suspension is that 1.0 effects are more excellent.
4 bacteria suspension initial concentration of table influences detection effect
Embodiment 10~12
The present embodiment and the difference of embodiment 1 are only that the poultry meat sample extracting solution is the phosphate-buffered of pH7.4
Solution replaces with the phosphate buffer solution that pH is 6.8,7.0 and 8.0.
It the results are shown in Table 5.This experiment is used to detect liquid, and color change interval is pH 6.8 (purple) -5.6 (yellow), and commercially available
For the pH value of fresh meat generally between 6.6-5.8, gravy acid-base property itself is affected to detection liquid color changeable effect.Experiment is chosen not
Phosphate buffer with pH value carries out pre-treatment to sample, and when pH of cushioning fluid is less than 7.4, fresh meat extracting solution is added to inspection
In survey system, it can make to detect the flavescence of liquid moment；When pH of cushioning fluid is higher, then it can extend the reaction time.Therefore, pH value 7.4
Phosphate buffer solution to extract effect to the antibiotic in sample optimal.
Influence of 5 different pH buffer of table to detection effect
Comparative example 1~4
The difference of this comparative example and embodiment 1 is only that, respectively by bacillus subtilis (FSCC115036), waxy gemma
Bacillus (FSCC115002), bacillus stearothermophilus (CICC 10392), streptococcus thermophilus (CICC20379) replace large intestine angstrom
Uncommon Salmonella (CICC23657)
Sensitivity tests
150uL detection liquid, 50uL bacteria suspension and 100uL antibiotic standard solution are sequentially added into 96 orifice plates, in each bacterium
Optimum temperature is cultivated, observation detection liquid discoloration and reaction time.
It the results are shown in Table 6, escherichia coli (CICC23657) is to common antibiotics in livestock meat as can be seen from Table 6
Quinolones, aminoglycoside, Tetracyclines have sensibility and the reaction time is shorter.
Table 6 detects bacterium screening
Comparative example 5~8
The difference of this comparative example and embodiment 1 is only that occlusion vehicle PVA- boric acid-sodium tetraborate replaces with PVA- boron
Acid, gelatin, carragheen and cellulose acetate.
The results are shown in Table 7, it can be seen that using gelatin, carragheen make carrier embedding strain mechanical strength it is lower, particle is easily
It is broken so that the cell that is embedded releases, and easy adhesion when gelatin stripping and slicing, it is not easy to operate.Carrier is made using cellulose acetate,
Although high mechanical strength, survival rate is low after bacterial strain water activation, and reason may be the acetone added in cellulose acetate
It is toxic to strain cell.Carrier is made using the present invention, mechanical strength is very high, squeezes the bead that is embedded with 100g counterweight, small
Ball shape is substantially unchanged, and nontoxic to microorganism, and survival rate is up to 85% after bacterial strain rehydration and purple is presented in embedding bead, helps
It is observed in result.And PVA- boric acid embedding bead trailing phenomenon is serious, and bead turns yellow influence reaction solution color.With grace promise
Husky star standard antibiotic gradient carries out detection limit measurement, it can be seen that the method for the present invention detection limit is lower.
The difficulty or ease situation and appearance features of 7 different carriers of table preparation embedding strain
Experimental example
Microorganism colour reagent box described in embodiment 1 is subjected to antibiotics leftover detection verifying
1, single mark antibiotic detection limit determines
With deionized water by Enrofloxacin, Ciprofloxacin, Sparfloxacin, marbofloxacin, Orbifloxacin, Danofloxacin, two
Flucloxacillin, flumequine, fleraxacin, sarafloxacin, Enoxacin, Norfloxacin, amikacin standard reserving solution difference
It is diluted to 0.02,0.04,0.06,0.08,0.10,0.12,0.14,0.16,0.18,0.20 μ g/mL totally 10 gradients, Fourth Ring
Element, aureomycin, terramycin, fortimicin, gentamicin, neomycin, streptomysin, apramycin stock solution be diluted to 0.02,
0.04,0.06,0.08,0.10 μ g/mL totally 5 gradients, are detected with kit of the present invention, determine various antibiotic detections
Limit.It the results are shown in Table 8.The kit is respectively less than the Ministry of Agriculture of China regulation to the detection limit of single mark antibiotic as can be seen from Table 8
Animal food herbal medicine maximum residue limit, can reach the purpose for sifting out exceeded sample.As shown in Figure 3 and Figure 4, to reagent
The antibiotic standard solution of gradient concentration is added in box, antibiotic concentration successively becomes larger from right to left, and color also becomes from yellow
Purple.
The single mark antibiotic detection limit of table 8
2, combination antibiotic detection limit determines
The quinolone antibiotics and aminoglycoside antibiotics of normal compatible use are mixed, compatibility program and series
Each antibiotic final concentration is shown in Table 9 in gradient.By in table 96 respectively combined mix antibiotic gradient kit of the present invention into
Row detection, determines various antibiotic detection limits, is shown in Table 10.Combine the relatively single mark of detection limit of antibiotic as can be seen from Table 10
It is low, illustrate that quinolones and aminoglycoside antibiotics are used in combination and indicator bacteria inhibitory effect is reinforced, there is synergistic effect.
9 antibiotic of table often uses compatibility table
The mixed mark antibiotic detection limit of the combination of table 10
3, stabilization of kit is tested
The kit prepared, which is sealed, is placed on 4 DEG C of preservations 1,3,5,10,15,18,20,25d, is carried out with Enrofloxacin
Reaction time and detection limit verifying, the results are shown in Table 11.Since thallus has the process of a decaying, thalline quantity and activity can all have
It is reduced, therefore, detection time and detection limit can be varied with the extension of time.Kit in 15d as can be known from the results
Reaction time and detection limit can keep stable state.
The test of 11 stabilization of kit of table
| Resting period (d) | Reaction time (h) | Detection limit (μ g/kg) |
| 1 | 3.2 | 60 |
| 3 | 3.2 | 60 |
| 5 | 3.2 | 60 |
| 10 | 3.2 | 60 |
| 15 | 3.5 | 60 |
| 18 | 3.5 | 80 |
| 20 | 4.0 | 80 |
| 25 | 4.5 | 100 |
4, kit false positive, false negative test
By 100 parts of fresh meat samples (30 parts of pork, 20 parts of beef, 20 parts of mutton, 15 parts of chicken, 15 parts of duck) from 1-100
It is numbered.To guarantee there is certain positive rate, using artificial addition manner, blind sample test is carried out, chromatography is as a result used
It is verified, determines the kit false positive and false negative.The results are shown in Table 12, kit detection shares 24 samples and is positive,
Through chromatography repetition measurement, wherein 20 positive samples contain the antibiotic residue of the above addition, remaining 4 are false positive sample, greatly
Intestines Escherichia has broad spectrum activity, and other than the antibiotic sensitive studied the present invention, there is also to other antibiotic sensitives
Possibility, therefore there is the possibility containing other antibiotic residues in 4 false positive samples.And microbial method detection is negative 76
A sample, is verified through chromatography, does not measure the above medicament residue, illustrates that the microorganism development process result is relatively reliable, though in the presence of
Certain false positive results, but as long as antibiotic residual quantity is more than its method detection limit, no false negative result exists.
12 kit false positive of table, false negative test
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1. a kind of method for detecting high-throughput antibiotic residue, which is characterized in that using escherichia coli as indicator bacteria, detect fowl
The microorganism development process of antibiotic residue in poultry meat.
2. the method according to claim 1, wherein the method uses the micro- glue of PVA- boric acid-sodium tetraborate
Capsule investment carries out microencapsulation to indicator bacteria；
Preferably, the microcapsule embedded method specifically: prepare the bromine potassium that weight percent is 8%-15% polyvinyl alcohol (PVA)
After sterilization treatment, the bacteria suspension of escherichia coli is added, then be slowly dropped into phenol violet dextrose peptone medium solution
In boric acid-sodium tetraborate solution, then in 3~5 DEG C of 5~7h of standing, the indicator bacteria particle of acquisition is filtered, is cleaned spare；
It is furthermore preferred that when the weight percent of the polyvinyl alcohol is 9%~11%, the boric acid-sodium tetraborate solution pH
Value is 6, and embedding effect is optimal.
3. method according to claim 1 or 2, which is characterized in that the antibiotic is selected from quinolones, aminoglycoside
One of class, Tetracyclines are a variety of；
Preferably, the poultry meat extracts in advance, the extraction specifically: after poultry meat is carried out homogenization, is added
The phosphate buffer solution of pH6.5-8.0 is mixed well；3~5min of water-bath at a temperature of being subsequently placed in 90~110 DEG C is high
Fast centrifuging and taking supernatant.
4. a kind of microorganism colour reagent box for detecting high-throughput antibiotic residue, which is characterized in that the finger including microencapsulation
Show bacterium particle, detection liquid, poultry meat sample extracting solution and control group；
Wherein, the indicator bacteria in the indicator bacteria particle is escherichia coli.
5. microorganism colour reagent box according to claim 4, which is characterized in that the detection liquid by weight, wraps
Include 8~12 parts of peptones, 2~4 portions of beef extracts, 8~12 parts of glucose, 4~6 parts of NaCl, 0.03~0.05 part of bromocresol purple
Indicator, 800~1200 parts of water；The pH value of the detection liquid is 6.8-7.2；
And/or the poultry meat sample extracting solution is specially the phosphate buffer solution of pH6.5-8.0, the preferably phosphorus of pH7.4
Hydrochlorate buffer solution.
6. microorganism colour reagent box according to claim 4 or 5, which is characterized in that the control group includes positive right
According to group, negative control group with blank control group；The positive controls are antibiotic standard solution, and the negative control group is fowl
Poultry meat sample extracting solution；
Preferably, the blank control group is physiological saline.
7. microorganism colour reagent box according to claim 6, which is characterized in that in the antibiotic standard solution at least
It is husky including gentamicin, streptomysin, benzyl penicillin, tetracycline, erythromycin, roxithromycin, Enrofloxacin, Norfloxacin, cyclopropyl
Star；
Preferably, the solubility of the antibiotic standard solution is 0.08~0.12ug/mL.
8. the microorganism colour reagent box according to claim 4~7, which is characterized in that the indicator bacteria of the microencapsulation
Particle obtains in the following way:
Prepare the bromine potassium phenol violet dextrose peptone medium solution that weight percent is 8%-15% polyvinyl alcohol, sterilization treatment
Afterwards, the bacteria suspension of escherichia coli is added, then is slowly dropped into boric acid-sodium tetraborate solution, it is then quiet in 3~5 DEG C
5~7h is set, the indicator bacteria particle of acquisition is filtered, cleans spare；
Preferably, the weight percent of the polyvinyl alcohol is 9%~11%, and the boric acid-sodium tetraborate solution pH value is
6。
9. the microorganism colour reagent box according to shown in claim 8, which is characterized in that the bacteria suspension of the escherichia coli
It obtains in the following way:
Escherichia coli is inoculated into fluid nutrient medium, after carrying out culture 22-24h under conditions of 35-37 DEG C；It takes
Bacterium solution after 0.1mL culture is in being coated optimization secondary culture 22-24h on solid medium, continuously biography 2-3 generation lives
Change；Thallus after taking activation, after being washed, then is placed in fluid nutrient medium to get indicator bacteria bacteria suspension；
Preferably, using the liquid culture medium as blank, under conditions of wavelength is 600nm, taking absorbance value is 0.8~1.2
Indicator bacteria bacteria suspension.
10. according to the detection method of any one of the claim 4~9 microorganism colour reagent box, which is characterized in that specific
Are as follows:
1) poultry meat after taking homogeneous is placed in poultry meat sample extracting solution and mixes；90~110 DEG C of temperature is subsequently placed in be lauched
3~5min is bathed, high speed centrifugation takes supernatant；
2) the indicator bacteria particle of the microencapsulation is fixed in the micropore in microwell plate, the inspection is successively added into every hole
Liquid is surveyed, is then separately added into the control group and the supernatant again；The microwell plate is placed at a temperature of 36~45 DEG C
It is cultivated, observes color change, judge antibiotic residue situation.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102964612A (en) * | 2012-08-30 | 2013-03-13 | 北京爱美客生物科技有限公司 | Polyvinyl alcohol-borax microsphere and preparation method thereof |
| CN105675604A (en) * | 2016-03-31 | 2016-06-15 | 山东五洲检测有限公司 | Method for detecting antibiotic residues in milk |
| CN105694319A (en) * | 2016-01-18 | 2016-06-22 | 袁暾 | Medical polyvinyl alcohol gel and preparing method thereof |
| CN106636294A (en) * | 2017-02-28 | 2017-05-10 | 滨海瀚鸿生化有限公司 | Process for producing unnatural amino acid products through coupling reaction of immobilized bi-enzyme |
| CN107179357A (en) * | 2017-03-23 | 2017-09-19 | 苏州农业职业技术学院 | The detection method of antibiotic residue in poultry meat |
| CN107917911A (en) * | 2017-11-15 | 2018-04-17 | 中华人民共和国龙口出入境检验检疫局 | The remaining method of antibiotic in one kind detection meat product |
-
2018
- 2018-10-12 CN CN201811190620.0A patent/CN109270056B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102964612A (en) * | 2012-08-30 | 2013-03-13 | 北京爱美客生物科技有限公司 | Polyvinyl alcohol-borax microsphere and preparation method thereof |
| CN105694319A (en) * | 2016-01-18 | 2016-06-22 | 袁暾 | Medical polyvinyl alcohol gel and preparing method thereof |
| CN105675604A (en) * | 2016-03-31 | 2016-06-15 | 山东五洲检测有限公司 | Method for detecting antibiotic residues in milk |
| CN106636294A (en) * | 2017-02-28 | 2017-05-10 | 滨海瀚鸿生化有限公司 | Process for producing unnatural amino acid products through coupling reaction of immobilized bi-enzyme |
| CN107179357A (en) * | 2017-03-23 | 2017-09-19 | 苏州农业职业技术学院 | The detection method of antibiotic residue in poultry meat |
| CN107917911A (en) * | 2017-11-15 | 2018-04-17 | 中华人民共和国龙口出入境检验检疫局 | The remaining method of antibiotic in one kind detection meat product |
Non-Patent Citations (6)
| Title |
|---|
| 刘元坤 等: "PVA固定化微生物方法及应用", 《科技导报》 * |
| 吕圭源 等: "《药理学》", 31 March 2017, 中国中医药出版社 * |
| 李凤美 等: "不同载体包埋食用菌菌种的实验研究", 《中国食用菌》 * |
| 李梅芝: "应用嗜热脂肪芽胞杆菌检测原料奶中抗生素残留的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 范维 等: "动物源性食品中喹诺酮类药物残留的检测", 《肉类研究》 * |
| 陈宜鸿 等: "《新编家庭常用药物手册》", 31 January 2001, 知识出版社 * |
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