CN102590196B - The detection method of antibacterial medicine residue in a kind of Rapid Screening animal foodstuff sample - Google Patents
The detection method of antibacterial medicine residue in a kind of Rapid Screening animal foodstuff sample Download PDFInfo
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Abstract
The invention discloses a kind of detection method of antibacterial medicine residue in Rapid Screening animal foodstuff sample, it includes：Bacterial strain is activated, prepared by bacteria suspension, and prepared by detection pipe, sample pre-treatments, sample detection；It can suppress the principle to its sensitive bacterial growth or breeding using antibacterials, draw measuring samples and add in detection pipe, cultivate certain time, whether contain antibacterial medicine residue according in the change judgement sample of culture medium color in detection pipe.Compared with the prior art, the problem of present invention maintains autochthonous microorganism in the food samples solved on the basis of TTC detection method reliabilities in this method or the false positive caused by miscellaneous bacteria interference, false negative result；With the features such as detection method is simple, the small, sensitivity of interference is high, the degree of accuracy is high, it is adaptable to the high-flux fast screening detection of antibacterial medicine residue in animal foodstuff sample.
Description
Technical field
Technical field is tested and analyzed the present invention relates to antibacterial medicine residue, specifically belongs to a kind of growth of utilization bacterium
By antibacterial Drug inhibition in test sample in Rapid Screening food samples penicillin medicament residue detection method.
Background technology
Antibacterial medicine residue is always the serious problems for perplexing food security.Penicillin medicine is the important β of a class-interior
Amide-type antibiotic, because its efficient, low toxicity is that current veterinary science clinically applies one of most popular medicine due to such medicine
Thing is widely used, and makes its residual phenomena on animal derived food increasingly severe, although penicillin medicine is right in itself
Body easily causes drug anaphylaxis without very strong toxicity, be all kinds of allergic drugs most simultaneously, if long-term consumption is low dense
Such antibiotic is spent, bacterium can be made to produce drug resistance, flora imbalance causes body immunity reduction in addition, returning animal derived
Therefore food production processing bring imponderable loss, sets up the fast detection method for Detection Of Penicillin Residues, exploitation
Moderate reagent kit product is to control animal derived food hygienic quality, guarantee consumer health, guarantee industrial production etc.
With important social and economic significance.
At present, typically there are gas chromatography (GC), high performance liquid chromatography (HPLC), light splitting for food antibiotic residual
Photometer, chemoluminescence method, inhibiting AChE, capillary electrophoresis technique (CE) etc..In aforementioned manners there is instrument and equipment and answer in detection
Miscellaneous, Sample pretreatment and the cumbersome defect of measure, equipment cost are big, and testing cost is high, and the requirement to testing staff is also high,
Be not suitable for great amount of samples screening, be unfavorable in the popularization and application of food inspection department of basic unit.A kind of easy to operate, expense of exploitation is low,
Reliably, it can use the detection method of penicillin medicament residue in a large amount of detection samples imperative.
Microbiological method be according to antibacterial material to the physiological function of microorganism and the inhibitory action of metabolism come qualitative or
The antibacterial material residual in sample is quantitatively determined, is had the advantages that quick, sensitive, cheap.The generally culture pair in tested test sample
The microorganism of antibiotic sensitive, if not having antibiotic presence in sample, because the growth of microorganism is observed that culture medium
Become muddy, opaque or cause indicator color change because microorganism produces acid；If antibiotic or other antibacterial substances are deposited
There are inhibition zone formation or culture medium still transparent on culture medium can then observe, or without color change.At present, the liquid such as milk
The antibacterial medicine residue microbiological method of body sample mainly has the nitrogen method of chlorinated triphenyl base four, and has been included in national standard.But should
In the specific operation process of method, disturbing factor is more；Food samples to be checked often with autochthonous microorganism or external microorganism,
It cannot be guaranteed that measuring samples germ-free condition, the presence of these microorganisms can have a strong impact on microbiological method.
The raw milk such as just gathered contains bacterium, under conditions of proper temperature, and quickly, and bacterium mutually goes out bacterial reproduction
The phenomenon now alternately developed.The pH of the general raw milk just gathered is neutral, containing abundant lactose, is conducive in raw milk certainly
The lactic acid bacteria so existed, such as streptococcus lactis (Streptococcus lactis) soon breed.Their lactose fermenterses are produced
Lactogenesis acid, making the pH of cow's milk reduces, and causes proteins coagulation into cheese shape.Low pH finally also inhibits the numerous of streptococcus lactis
Grow, the strain lactobacillus (Lactobacillus) for being resistant to lower pH is replaced, they complete lactose fermentation, and make pH
More reduce.Saccharomycete is grown using lactic acid under this low ph condition.With the reduction of lactose and lactic acid, pH once again on
Rise, most of pseudomonas (Pseudomonas) and bacillus (Bacillus) growth and breeding.
Food samples to be checked can't be using the method sterilized, because can so influence food quality；Asptic technique, be
Microbiological basic demand, such reality just brings very big problem with microbiological method detection method to：These
Disturb microbe-derived unclear, species is various, the growth for having a strong impact on detection bacterium (suppresses or promoted the life of detection microorganism
It is long), or the instruction result for disturbing microorganism amount reproduction to have influence on acid-base indicator, very big is influenceed on testing result, can be produced
Raw false negative or false positive results.
The content of the invention
It is an object of the invention to overcome prior art promote the use of drawbacks described above present in process there is provided it is a kind of not
Specific large-sized analytic instrument equipment is needed, can with sensitivity is high, with a high credibility, the simple screening methods of application method
The miscellaneous bacteria interference such as autochthonous microorganism effectively in the animal foodstuff sample such as reduction milk, reduces and produces false negative or false positive results.
The purpose of the present invention is achieved through the following technical solutions：
In order to solve the above technical problems, the present invention provides one plant to penicillin drug susceptibility height and applied to penicillin medicine
Bifidobacterium breve (Bifidobacterium breve) the LJM-006 CGMCC No.5418 of analyte detection.
Bifidobacterium breve (Bifidobacterium breve) LJM-006 of the present invention, in preservation on October 28 in 2011
In China Committee for Culture Collection of Microorganisms's common micro-organisms center, it is referred to as CGMCC (addresses：Chaoyang District, Beijing City
No. 3 Institute of Microorganism, Academia Sinica of institute of North Star West Road 1, postcode 100101) preservation, Classification And Nomenclature is bifidobacterium breve
(Bifidobacterium breve), deposit number is CGMCC No.5418.
The bifidobacterium breve LJM-006 of the present invention is located away from Zhejiang Province's Healthy Youth human faecal mass.
The bifidobacterium breve LJM-006 bacterial strains of the present invention have following microbial characteristics：
(1) colonial morphology：LJM-006 bacterial strains bacterium colony on flat board is in canescence or milky, opaque, glossy, table
Face is smooth, raised, quality is soft, neat in edge, diameter 1-1.5mm.
(2) individual morphology：For irregular G+ sporeless bacteriums, there are straight-bar, knee, match head, dumbbell shape and wild goose
Shape, wherein based on wild goose shape.
(3) physiological and biochemical property：D-ribose (+)；L-arabinose (-)；Lactose (+)；Cellobiose (-)；Melezitose
(+)；Raffinose (+)；Sorbierite (-)；Starch (-)；Sodium gluconate (-)；Xylose (-)；Mannose (+)；Fructose (+)；Gala
Sugared (+)；Sucrose (+)；Maltose (+)；Trehalose (-)；Melibiose (+)；Mannitol (+)；Synanthrin (-)；Salicin (-)；
F6PPK enzymes (+)；Reaction (not grown on aerobic solid medium) to oxygen；Nitrate reduction (-)；Catalase (-)；Indole
React (-).
Do not grown in well-grown under anaerobism, aerobic environment.37-41 DEG C of optimum growth temperature；Minimum growth temperature 25-28
℃；43-45 DEG C of highest；Grow optimal pH 6.5-7.0；Do not grown in pH4.5-5.0 or 8.0-8.5.
LJM-006 bacterial strains improve more than 100 times compared with belonging to reference culture of the same race together to penicillin drug susceptibility.
The present invention also provides a kind of detection method of antibacterial medicine residue in food, the described method comprises the following steps：
(1) bacterial strain is activated：By bifidobacterium breve LJM-006 inoculations on nutrient agar slant medium, regulation should
The pH value of culture medium is to 6.8~7.2, in optimizing culture at 35~37 DEG C, and carries out transferred species squamous subculture；
(2) prepared by bacteria suspension：Bifidobacterium breve LJM-006 obtained by taking step (1) is inoculated in nutrient agar slant medium,
35 ± 2 DEG C of Anaerobic culturels 24 hours, lawn is washed down, suspension is made, adjust its OD with sterile saline600Value to 0.25~
0.35, put in 2-8 DEG C of refrigerator and preserve；
(3) prepared by detection pipe：By weight, peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, acetic acid are taken
Sodium 5g, K2HPO42g, MgSO4·7H2O 0.5g, MnSO4·4H2O 0.2g, FOS 3g, dibasic ammonium citrate 2g, tween-
80 1mL, distilled water 1L, dissolve by heating correction pH value to 6.5,115 DEG C of autoclavings 15-20 minute, chilling temperature is 50~65
DEG C, and take rapidly in 10mL aforesaid liquids, injection 10mL test tubes, it is stand-by as test bacterium solution；
(4) sample pre-treatments：Animal foodstuff sample homogenization tissue 5g to be checked is taken, with sterile 1% phosphate buffer
20mL is diluted, and whirlpool is mixed, and in 80 DEG C of heating water bath 5min, cooling centrifuges 15min after 5,000r/min, takes supernatant conduct
Test sample liquid；
(5) sample detection：With reference to TTC methods, the residual quantity of antibiotic in animal foodstuff is examined.Take obtained by step (4) for examination
The μ L of sample liquid 20~100 are added in step (3) described detection pipe, closed sealing after 35 ± 2 DEG C of Anaerobic culturels 8~10 hours,
Plus TTC 0.3mL, 36 ± 1 DEG C of water-bath culture 30min, judge result of the test according to color status：It is when sample is in newborn primary colors
The positive, takes on a red color as feminine gender.
The work bacterial strain of the present invention is bifidobacterium breve LJM-006, on October 28th, 2011 in China Microbiological bacterium
The common micro-organisms center preservation of preservation administration committee is planted, deposit number is CGMCCNo.5418.
Animal food sample of the present invention includes：Milk, pork, beef, chicken, egg etc..
The color status criterion of table 1
Determination methods：Accurate culture 30min observation results, as being the positive, are cultivated for 30min and make second of observation.
It is rapid during observation, it is to avoid illumination is disturbed too long.With the presence of antibiotic in animal foodstuff sample, though bacterium is then added in sample
Liquid culture, but because the breeding of bacterium is suppressed, thereby indicate that agent TTC is not reduced, do not develop the color.In contrast, if do not had
Antibiotic is present, then adds bacterium solution and breed at once, TTC is reduced and aobvious red, that is to say, that sample be in it is colourless when for the positive,
Take on a red color as feminine gender.
The present invention is to the sensitivity of Penicillin Residues in animal foodstuff sample between 2-3ng/mL.
The present invention has advantages below and effect：
1st, the present invention is intrinsic in the food samples during this method is solved on the basis of maintaining TTC detection method reliabilities
The problem of microorganism or the caused false positive of miscellaneous bacteria interference, false negative result；Detection bacterium used in the present invention is strictly anaerobic
Bacterium, the necessary anaerobic state of fermentation process, the autochthonous microorganism of food samples or miscellaneous bacteria are aerobic bacteria, it is impossible to raw under anaerobic state
It is long, it so effectively prevent the interference to testing result；
2nd, high to antibacterial drug test specificity, sensitivity height, the degree of accuracy are good, the spirit of Penicillin Residues in detection plain chocolate
Sensitivity is between 2-3ng/mL, less than national MRL standard, meets residue detection requirement；
3rd, sample is with little need for pre-treatment, and the tissue such as muscle, liver can take animal tissue's liquid to examine by the mode such as squeezing
Survey.
Embodiment
It is noted that detailed description below is all exemplary, it is intended to provide the present invention further invention.Unless another
Be described, all scientific and technical terms used herein have with the technical field of the invention personnel be generally understood that it is identical
Implication.
With reference to specific embodiment, the invention will be further described, but illustrated embodiment is not as the limit to the present invention
It is fixed.Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.
Embodiment 1：The separation and identification of bifidobacterium breve LJM-006 bacterial strains
Separation sample, on MRS agar mediums, 37 DEG C of anaerobism bars are used as using the excrement of Zhejiang Province's healthy young people
Under part, 48h coatings are separately cultured, and are obtained bifidobacterium breve LJM-006 of the present invention, are accredited as bifidobacterium breve, the bacterium
Strain is on October 28th, 2011 in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address：Beijing
No. 3 Institute of Microorganism, Academia Sinica of institute of Chaoyang District North Star West Road 1, postcode 100101) preservation, Classification And Nomenclature is short bifid
Bacillus (Bifidobacterium breve), preserving number is CGMCC No.5418.
The formula of MRS agar mediums：Peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium acetate 5g,
K2HPO42g, MgSO4·7H2O 0.5g, MnSO4·4H2O 0.2g, FOS 3g, dibasic ammonium citrate 2g, Tween-80
1mL, agar 15g, distilled water 1L, adjust pH to 7.0, and 115 DEG C sterilize 15 minutes.
The LJM-006 bacterial strains of the present invention have following microbial characteristics：
(1) colonial morphology：LJM-006 bacterial strains bacterium colony on flat board is in canescence or milky, opaque, glossy, table
Face is smooth, raised, quality is soft, neat in edge, diameter 1-1.5mm.
(2) individual morphology：For irregular G+ sporeless bacteriums, there are straight-bar, knee, match head, dumbbell shape and wild goose
Shape, wherein based on wild goose shape.
(3) physiological and biochemical property：D-ribose (+)；L-arabinose (+)；Lactose (+)；Cellobiose (-)；Melezitose
(+)；Raffinose (+)；Sorbierite (-)；Starch (-)；Sodium gluconate (-)；Xylose (+)；Mannose (-)；Fructose (+)；Gala
Sugared (+)；Sucrose (+)；Maltose (+)；Trehalose (-)；Melibiose (+)；Mannitol (+)；Synanthrin (-)；Salicin (-)；F6PPK
Enzyme (+)；Reaction (not grown on aerobic solid medium) to oxygen；Nitrate reduction (-)；Catalase (-)；Indole reacts
(-)。
Do not grown in well-grown under anaerobism, aerobic environment.37-41 DEG C of optimum growth temperature；Minimum growth temperature 25-28
℃；43-45 DEG C of highest；Grow optimal pH 6.5-7.0；Do not grown in pH4.5-5.0 or 8.0-8.5.
Experiment 1：Sensitivity tests
Using scraps of paper AGP test (K-B) method, the penicillin titer of various concentrations is chosen, with modified MRS agar culture
Base, culture medium prescription is shown in embodiment 1, using bifidobacterium breve LJM-006 of the present invention as test bacterium, is placed in anaerobism in 37 DEG C of incubators
The growing state that LJM-006 bacterial strains are observed after 24h is cultivated, penicillin sensitivity tests is made of agar diffusion method of the paper, determines blue or green
Bacteriostasis property of the mycin to LJM-006 bacterial strains.
3 repetitions are done in above-mentioned experiment, to belong to bifidobacterium breve reference culture of the same race together as control.
Table 2：Sensitivity tests
| Bacterial strain | The susceptibility of penicillin | The bacteriostatic diameter (mm) of 2ng/mL penicillin |
| The LJM-006 bacterial strains of the present invention | ≤1ng/mL | 19 |
| Bifidobacterium breve reference culture | ＞ 2ng/mL | 13 |
Result of the test：Bifidobacterium breve LJM-006 of the present invention has the sensitiveness of height to penicillin, and it is with belonging to mark together
Quasi- bacterial strain is compared, and improves more than 100 times to the sensitiveness of penicillin, and bifidobacterium breve LJM-006 penicillin-susceptible degree≤
2-3ng/mL, less than the residual quantity of national standard, completely can as screening Penicillin Residues work bacterial strain.
Embodiment 2：The detection of Pig Liver penicillin medicament residue
(1) bacterial strain is activated：By bifidobacterium breve LJM-006 inoculations on nutrient agar slant medium, regulation should
The pH value of culture medium is to 6.8~7.2, in optimizing culture at 35~37 DEG C, and carries out transferred species squamous subculture；
(2) prepared by bacteria suspension：Bifidobacterium breve LJM-006 obtained by taking step (1) is inoculated in nutrient agar slant medium,
35 ± 2 DEG C of Anaerobic culturels 24 hours, lawn is washed down, suspension is made, adjust its OD with sterile saline600Value to 0.25~
0.35, put in 2-8 DEG C of refrigerator and preserve；
(3) prepared by detection pipe：By weight, peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, acetic acid are taken
Sodium 5g, K2HPO42g, MgSO4·7H2O 0.5g, MnSO4·4H2O 0.2g, FOS 3g, dibasic ammonium citrate 2g, tween-
80 1mL, distilled water 1L, dissolve by heating correction pH value to 6.5,115 DEG C of autoclavings 15-20 minute, chilling temperature is 50~65
DEG C, and take rapidly in 10mL aforesaid liquids, injection 10mL test tubes, it is stand-by as test bacterium solution；
(4) sample pre-treatments：Pig Liver homogenised tissue 5g to be checked is taken, it is dilute with sterile 1% phosphate buffer 20mL
Release, whirlpool is mixed, in 80 DEG C of heating water bath 5min, cooling centrifuges 15min after 5,000r/min, take supernatant as supplying sample
Product liquid；
(5) sample detection：With reference to TTC methods, the residual quantity of antibiotic in animal foodstuff is examined.Take obtained by step (4) for examination
The μ L of sample liquid 20~100 are added in step (3) described detection pipe, closed sealing after 35 ± 2 DEG C of Anaerobic culturels 8~10 hours,
Plus TTC 0.3mL, 36 ± 1 DEG C of water-bath culture 30min, observation is as being the positive, and culture 30min makees second of sight in water-bath
Examine, every part of sample makees two parts.Negative and each portion of positive control, the animal foodstuff of the effective antibiotic-free of positive control are remake in addition
Sample added with antibiotic and bacterium solution and TTC.The effective antibiotic-free animal foodstuff sample of negative control adds bacterium solution and TTC.
4% 2, the nitrogen of 3,5 monochlor(in)ate triphen four (TTC) aqueous solution：1g TTC are weighed, are melted into 5mL sterile purified waters,
Fill in brown bottle and to be preserved in 7 DEG C of refrigerators, facing the used time is diluted to 5 times with sterile purified water.In case of solution is changed into jade green or filbert,
It can not then use again.
Experiment 1：False negative rate is tested
Blank each 5g of animal foodstuff sample of homogeneous is weighed, penicillin titer is added, makes penicillin concn 2- in food
3ng/mL, assay method be the same as Example 2, replication 100 times, the probability that observation negative findings occurs calculates false negative rate.
Result of the test：When Penicillin Residues in Milk is 2-3ng/mL, false negative rate is 0；Penicillin Residues are in pork
During 2-3ng/mL, false negative rate is 2%.
Experiment 2：Stability test
Detection tubule is prepared in being preserved successively at 4 DEG C 1 week, 2 weeks, 3 weeks, 4 weeks by the method in embodiment 2, is entered respectively
Row, which is detected, simultaneously records its negative detection time, 10 hours, 10 hours, 10 hours, 10.5 hours, negative detection time can with when
Between extension be varied from, the change of negative detection time is smaller, illustrates that stability is good.For the accuracy of guarantee test, every time
Experiment should do a negative control, and it detects that the judgement time is defined by negative control exactly.It is blue or green in the inventive method detection food
Be stable in 10 hours within 3 weeks before the stability of mycin residues of pesticides, be 10.5 hours by the 4th week, detection in this approach it is steady
Qualitative is one month.
Experiment 3：The determination of test limit
Use respectively containing various concentrations (1ng/mL, 2ng/mL, 4ng/mL, 8ng/mL, 16ng/mL) penicillin standard items
PBS solution is added dropwise in testing tube as analyte sample fluid, carries out sample detection, analyzes testing result, it is determined that present invention detection
The detection of method is limited to 2-3ng/mL.
Experiment 4：Specific assay
Corresponding concentration is 1.5ng/L (IC when the inhibiting rate that penicillin suppresses Bifidobacterium LJM-006 is 50%50=
1.5mg/L), penicillin can specificity suppression, the Bifidobacterium LJM-006 with other antibiotic pair to Bifidobacterium LJM-006
Cross reacting rate % is respectively less than 0.5%, the results are shown in Table 3.
The cross reaction of the penicillin of table 3 and other organophosphorus insecticides
| Compound | IC50(ng/mL) | Cross reacting rate (%) |
| Penicillin | 1.5 | 100 |
| Tetracycline | 132 | ＜ 0.5 |
| Terramycin | ＞ 260 | ＜ 0.5 |
| Ofloxacin | ＞ 260 | ＜ 0.5 |
| Chloramphenicol | ＞ 260 | ＜ 0.5 |
| Streptomysin | ＞ 260 | ＜ 0.5 |
The detection of the other samples of embodiment 3
The tissue such as meat, liver, kidney can press extracting juice after weighing and add appropriate phosphate buffer to adjust pH to 7.0；
To different animal samples, different buffer solutions are added, to control pH.Other operating procedure be the same as Examples 2.
Claims (1)
1. the detection method of Penicillin Residues in a kind of Rapid Screening animal foodstuff sample, it is characterised in that methods described includes
Following steps：
(1) bacterial strain is activated：By bifidobacterium breve (Bifidobacterium breve) LJM-006 bacterial strains, deposit number is
CGMCC No.5418, are inoculated on nutrient agar slant medium, adjust the pH value of the culture medium to 6.8~7.2, in 35~
Culture is optimized at 37 DEG C, and carries out transferred species squamous subculture；
(2) prepared by bacteria suspension：Bifidobacterium breve obtained by taking step (1) is inoculated in nutrient agar slant medium, 35 ± 2 DEG C of anaerobism
Culture 24 hours, lawn is washed down, suspension is made, adjust its OD with sterile saline600Value puts 2-8 DEG C to 0.25~0.35
Preserved in refrigerator；
(3) prepared by detection pipe：By weight, peptone 10g, beef extract 10g, yeast extract 5g, glucose 20g, sodium acetate 5g are taken,
K2HPO42g, MgSO4·7H2O 0.5g, MnSO4·4H2O 0.2g, FOS 3g, dibasic ammonium citrate 2g, Tween-80
1mL, distilled water 1L, dissolve by heating correction pH value to 6.5,115 DEG C of autoclavings 15-20 minute, chilling temperature is 50~65 DEG C,
And take out rapidly in bacteria suspension of the 10mL liquid simultaneously prepared by blend step (2), injection 10ml test tubes, it is stand-by as testing；
(4) sample pre-treatments：Animal foodstuff sample homogenization tissue 5g to be checked is taken, it is dilute with sterile 1% phosphate buffer 20mL
Release, whirlpool is mixed, in 80 DEG C of heating water bath 5min, cooling centrifuges 15min after 5,000r/min, take supernatant as supplying sample
Product liquid；
(5) sample detection：With reference to TTC methods, the residual quantity of penicillin in animal foodstuff is examined, test sample obtained by step (4) is taken
The μ L of liquid 20~100 are added in detection pipe prepared by step (3), closed sealing after 35 ± 2 DEG C of Anaerobic culturels 8~10 hours, plus
TTC0.3mL, 36 ± 1 DEG C of water-bath culture 30min, result of the test is judged according to color status：It is when detection sample is in newborn primary colors
The positive, takes on a red color as feminine gender.
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| CN102517230B (en) * | 2011-12-12 | 2013-06-19 | 温州医学院 | Bifidobacterium breve and detection method of methamidopho pesticide residue in foodstuff |
| CN105713859B (en) * | 2016-03-02 | 2021-01-01 | 温州医科大学 | Method for detecting multiple antibiotic residues in bifidobacterium breve and milk and application |
| 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 |
| CN106319026A (en) * | 2016-08-19 | 2017-01-11 | 界首市科创食品检测中心 | Method for detecting antibiotic in milk |
| CN109971669A (en) * | 2019-02-01 | 2019-07-05 | 温州医科大学 | The detection method and application of bifidobacterium pseudocatenulatum and Residue of Antibiotics in Milk |
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| CN102268471B (en) * | 2011-06-25 | 2014-06-11 | 温州医学院 | Microbiological method for detecting acephate residues in food |
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