CN101059423A - Quick detection method for agricultural chemical residue in milk - Google Patents
Quick detection method for agricultural chemical residue in milk Download PDFInfo
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- CN101059423A CN101059423A CNA2007100853907A CN200710085390A CN101059423A CN 101059423 A CN101059423 A CN 101059423A CN A2007100853907 A CNA2007100853907 A CN A2007100853907A CN 200710085390 A CN200710085390 A CN 200710085390A CN 101059423 A CN101059423 A CN 101059423A
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Abstract
The invention relates to a quick check method of milk pesticide, as enzyme-restrain rate method, which uses colorimetry to process quick test on left pesticide, with simple operation, high automation, needed sensitivity, low cost, and wide application for testing the left pesticide in milk.
Description
Technical field:
The present invention relates to a kind of method for quick of residues of pesticides, especially relate to the method for quick of residues of pesticides in a kind of animal breast and the dairy products.
Background technology:
In today of food security growing interest, strict control product quality is avoided becoming the focus of quality control owing to the influence of medicament residue to food.Yet agricultural chemicals is as the important pollution source of food, because its most of kinds are used to prevent and treat agriculture harmful organism, so human body is had big toxic.Enter the agricultural chemicals of human body,, will cause the imbalance of normal physiological function of organism, cause pathological change and toxic hazard, so the agricultural chemicals residue problem also should obtain enough attention in the food if surpassed normal person's maximum tolerance limit.
Under the growing trend of China's dairy products consumption figure, the quality of strict control dairy products (milk) plays crucial effect to whole quality of dairy products safety.Therefore, seek a kind of sensitivity preferably the method for quick of animal Ruzhong residues of pesticides have great significance for the foodsafety of enterprise.
At present, in the exploitation of Fast Determination of Pesticide Residue method, enzyme inhibition method is to study at most and ripe relatively a kind of.Because organophosphorus and carbamate compounds are the irreversible inhibitor of nervous system acetylcholine esterase, and the people is had higher toxicity, enzyme inhibition method is the technology of the part agricultural chemicals being carried out residual fast detecting.Organophosphorus, carbamate chemicals for agriculture account for very big proportion in total pesticide dosage.Have the organophosphorus pesticide that surpasses 100 kinds worldwide to use, in the agricultural chemicals that China uses always, acephatemet, Azodrin, parathion, parathion-methyl, thimet, the contour malicious agricultural chemicals of omethoate account for the over half of total pesticide dosage.Therefore, the application of the residues of pesticides fast measuring of enzyme inhibition method has great realistic meaning.The rapid detection for pesticide residue technology that suppresses design according to enzyme all is the pesticide residue determination that is applied to fruit and vegetable, also be not applied in the cow's milk of matrix relative complex at present, at this, we with these method Application and Development in the pesticide residue determination of cow's milk.
Summary of the invention
The invention provides a kind of a kind of new method of dairy products detection range, be specially the method for quick-enzyme inhibition rate method of residues of pesticides in the dairy products, the principle of this method is: with acetylthiocholine iodide (ATCI) is substrate, two sulfo-s-dinitrobenzoic acid (DTNB) are developer, make the extract and the cholinesterase effect of sample, after the certain hour reaction, according to the degree that produces yellow substance, utilize microplate reader colorimetric under the 420-430nm wavelength, preferably colorimetric under the 425nm wavelength.By the inhibiting rate of the change calculations cholinesterase of light absorption value, thereby judge organophosphorus and carbamate pesticide residue situation.
Detection method of the present invention comprises the steps:
1). get check sample and mix with acetone, jolting was left standstill 10-30 minute, supernatant occurred and got final product; In this step, check sample and the acetone ratio between the two does not have specific restriction, and those of ordinary skills can adjust ratio between the two according to detectability;
2). get 5-10ml supernatant (look supernatant separate out situation can suitably adjust sampling amount) and place color comparison tube, feed nitrogen, being concentrated into does not have solvent to get final product;
3). give the damping fluid that adds 2-4mL pH=7.0-8.0 in the above-mentioned color comparison tube, add 40-60 μ L cholinesterase+40-60 μ L developer two sulfo-s-dinitrobenzoic acid (DTNB) again, preferably add above-mentioned enzyme and the developer of 50 μ L respectively; Do simultaneously except that not adding all identical control group of other treatment step the sample with the sample group;
4). place 37 ℃ of constant incubators, cultivated 10-30 minute, preferably cultivated 30 minutes;
5). from constant incubator, take out culture, add 40-60 μ L acetylthiocholine iodide (ATCI, substrate), preferably add 50 these substrates of μ L;
6). the jolting reaction, move into then in ELISA Plate or the cuvette, under the 420-430nm wavelength, use microplate reader or spectrophotometric determination absorbance, preferably under 425nm, detect;
7). by the inhibiting rate of the change calculations cholinesterase of absorbance
Wherein:
Control group absorbance difference is the changing value of 3 minutes absorbances of contrast solution reaction;
Sample absorbance difference is the changing value of 3 minutes absorbances of sample solution reaction;
8) result judges:
The result represents with the repressed degree of enzyme (inhibiting rate).
When test sample during to the inhibiting rate of enzyme 〉=50%, the expression sample is positive
In detection method of the present invention, spendable damping fluid is the phosphate buffer of preferred pH=8.0.
In detection method of the present invention, the acetone that sample extraction adopts is preferably analytically pure.
In detection method of the present invention, can adopt routine instrument device known in the art, for example, can select following instrument and equipment for use:
Liquid-transfering gun, 50 μ L-200 μ L; Microplate reader (U.S. Thermo MK3); Printer (Star HL-10); 10 μ L, 100 μ L, 250 μ L microsyringes; Full temperature climate box (MLR-350H); Biochemical incubator (LRH-250-II microcomputerized control Guangdong Medical Apparatus and Instruments Factory); Blank ELISA Plate; The 10mL color comparison tube.
Detection method of the present invention, preferably higher relatively: Dichlorvos (DDVP) to the detection sensitivity of following agricultural chemicals; Methamidophos (acephatemet); Monocrotophos (Azodrin); Methidathion (methidathion).Above-mentioned several pesticide standard sample is all purchased the ChemService company in the U.S..
In detection method of the present invention, except as otherwise noted, described enzyme all refers to cholinesterase.
From following Table A as can be seen, be specially adapted to following agricultural chemicals according to enzyme inhibition rate method of the present invention for the enzyme sensitivity.Measure through microplate reader, bring the absorbance that draws into formula and calculate inhibiting rate, thus the detection limit of definite this law.This method is suitable for dimethyl dichlorovinyl phosphate commonly used, acephatemet, Azodrin, methidathion.
The contrast that the Table A agricultural chemicals is limited the quantity of
| Agricultural chemicals | Detection limit mg/kg | Maximum residue limit MRL | Median lethal dose |
| DDVP | ＜0.03 | 0.1mg/kg | 50mg/kg |
| Acephatemet | 0.28 | 0.1mg/kg | 7.5mg/kg |
| Azodrin | ＜0.07 | 0.1mg/kg | 5mg/kg |
| Methidathion | ＜0.07 | 0.1mg/kg | 26mg/kg |
Method of the present invention instrument quick and convenient, that adopt is simple relatively, and it is applicable to on-the-spot qualitative and semiquantitative determination.After the enzyme inhibition rate method detects the positive, can further detect by the reference instrument method of inspection, identify remains of pesticide kind and accurate residual quantity.This sample prescreening method is applied to both reduce the chemical examination workload in the quality control of raw material milk, and the security control to the cow's milk raw milk has great importance again.Enzyme inhibition rate method of the present invention adopts colourimetry to carry out rapid detection for pesticide residue, simple to operate, fast and automatically change the degree height, sensitivity is suitable, cost is low.
Description of drawings:
Fig. 1 has shown by after the DDVP cultivation of method of the present invention to 10 kinds of variable concentrations, the absorbance of mensuration (embodiment 1), and wherein 1 among the figure, 2,3 represents three to repeat parallel experiments.
Fig. 2 has shown by after the acephatemet cultivation of method of the present invention to 10 kinds of variable concentrations, the absorbance of mensuration (embodiment 2), and wherein 1 among the figure, 2,3 represents three to repeat parallel experiments.
Fig. 3 has shown by after the Azodrin cultivation of method of the present invention to 10 kinds of variable concentrations, the absorbance of mensuration (embodiment 3), and wherein 1 among the figure, 2,3 represents three to repeat parallel experiments.
Fig. 4 has shown by after the methidathion cultivation of method of the present invention to 10 kinds of variable concentrations, the absorbance of mensuration (embodiment 4), and wherein 1 among the figure, 2,3 represents three to repeat parallel experiments.
Embodiment:
Embodiment 1
The fast detecting experiment of Ruzhong DDVP residues of pesticides and detectability checking
Experiment agricultural chemicals: DDVP:
Its mouse oral LD
5050～92mg/kg; Rat oral 50～110mg/kg.
10 concentration, three revision test results (Fig. 1) are adopted in experiment.
The inhibiting rate of the variable concentrations that obtains is listed in following table 1, drawn Fig. 1 according to the gained inhibiting rate.
Experimental procedure:
1. the check sample of getting the variable concentrations agricultural chemicals mixes jolting with the acetone proper proportion, leaves standstill 10 minutes, supernatant occurs and gets final product.
2. get the 5ml supernatant and place color comparison tube, feeding nitrogen is concentrated into does not have solvent to get final product.
3. adding 2mL pH is 8.0 phosphate buffer in the above-mentioned color comparison tube, adds 40 μ L cholinesterases and 40 μ L developers, two sulfo-s-dinitrobenzoic acid (DTNB) again; Do simultaneously except that not adding all identical control group of other treatment step the sample with the sample group.
4. place 37 ℃ of constant incubators, cultivated 10 minutes.
5. from constant incubator, take out culture, add 40 μ L acetylthiocholine iodides (ATCI, substrate).
6. jolting reaction back moves in ELISA Plate or the cuvette, and the absorbance of using microplate reader or spectrophotometric determination 420nm place also calculates the cholinesterase inhibiting rate of agricultural chemicals under the various variable concentrations according to following formula.
7. by the inhibiting rate of the change calculations cholinesterase of light absorption value
Wherein:
Control group absorbance difference is the changing value of 3 minutes absorbances of contrast solution reaction;
Sample absorbance difference is the changing value of 3 minutes absorbances of sample solution reaction;
Table 1
The inhibiting rate of DDVP variable concentrations
| Concentration (ppm) | 0.032967 | 0.065933 | 0.131867 | 0.263733 | 0.461533 |
| Inhibiting rate (%) | 100 | 99.5708 | 98.7124 | 99.1416 | 100 |
| Concentration (ppm) | 0.659333 | 1.648333 | 3.296667 | 6.593333 | 13.18667 |
| Inhibiting rate (%) | 97.8541 | 99.1416 | 98.7124 | 99.1416 | 100 |
As can be seen from Table 1, enzyme is comparatively responsive to DDVP, and the degree of cultivating the back inhibition is big, and absorbance obviously reduces.As calculated as can be seen, 10 of interpolation inhibiting rates that concentration is calculated are all greater than 98%.So DDVP just can detect when 0.03ppm, this method is applicable to the fast detecting of DDVP.
The fast detecting experiment of Ruzhong methamidophos pesticide residue and detectability checking
Experiment agricultural chemicals: acephatemet:
Acephatemet acute toxicity: rat oral LD
507.5mg/kg; Rat sucks LC
509mg/kg; Rat is through skin LD
50: 50mg/kg.
10 concentration gradients of acephatemet design, three repeated experiments results (Fig. 2).
Experimental procedure:
1. the check sample of getting the variable concentrations agricultural chemicals mixes jolting with the acetone proper proportion, leaves standstill 20 minutes, supernatant occurs and gets final product.
2. get the 7.5ml supernatant and place color comparison tube, feed nitrogen, being concentrated into does not have solvent to get final product.
3. adding 3mL pH is 7.0 phosphate buffer in the above-mentioned color comparison tube, adds 50 μ L cholinesterases and 50 μ L developers, two sulfo-s-dinitrobenzoic acid (DTNB) again; Do simultaneously except that not adding all identical control group of other treatment step the sample with the sample group.
4. place 37 ℃ of constant incubators, cultivated 20 minutes.
5. from constant incubator, take out culture, add 50 μ L acetylthiocholine iodides (ATCI, substrate).
6. jolting reaction back moves in ELISA Plate or the cuvette, and the absorbance of using microplate reader or spectrophotometric determination 425nm place also calculates the cholinesterase inhibiting rate of agricultural chemicals under the various variable concentrations according to following formula.
7. by the inhibiting rate of the change calculations cholinesterase of light absorption value
Wherein:
Control group absorbance difference is the changing value of 3 minutes absorbances of contrast solution reaction;
Sample absorbance difference is the changing value of 3 minutes absorbances of sample solution reaction;
Table 2
The inhibiting rate of acephatemet variable concentrations
| Concentration (ppm) | 0.035795 | 0.07159 | 0.143181 | 0.286362 | 0.501133 |
| Inhibiting rate (%) | 9.635096 | 30.95531 | 31.93932 | 53.75154 | 72.11972 |
| Concentration (ppm) | 0.715904 | 1.78976 | 3.57952 | 5.36928 | 7.15904 |
| Inhibiting rate (%) | 81.95982 | 92.29192 | 99.01599 | 99.01599 | 98.68799 |
As can be seen from Table 2, enzyme is comparatively responsive to acephatemet, and the degree of cultivating the back inhibition is bigger, and absorbance reduces more obvious.As calculated as can be seen, 10 of interpolation inhibiting rates that concentration is calculated after the 0.28ppm inhibiting rate greater than 50%.So acephatemet can be by responsive detecting in the scope of median lethal dose.
Embodiment 3
The fast detecting experiment of Ruzhong monocron pesticide residue and detectability checking
Experiment agricultural chemicals: Azodrin:
Its mouse oral LD
505mg/kg; Rat oral LD
5021mg/kg; Rabbit is through skin LD
50149～709mg/kg
Adopt 10 concentration, three revision test results (Fig. 3).
Experimental procedure:
1. the check sample of getting the variable concentrations agricultural chemicals mixes jolting with the acetone proper proportion, leaves standstill 30 minutes, supernatant occurs and gets final product.
2. get the 10ml supernatant and place color comparison tube, feed nitrogen, being concentrated into does not have solvent to get final product.
3. adding 4mL pH is 7.5 phosphate buffer in the above-mentioned color comparison tube, adds 60 μ L cholinesterases and 60 μ L developers, two sulfo-s-dinitrobenzoic acid (DTNB) again; Do simultaneously except that not adding all identical control group of other treatment step the sample with the sample group.
4. place 37 ℃ of constant incubators, cultivated 30 minutes.
5. from constant incubator, take out culture, add 60 μ L acetylthiocholine iodides (ATCI, substrate).
6. jolting reaction back moves in ELISA Plate or the cuvette, and the absorbance of using microplate reader or spectrophotometric determination 430nm place also calculates the cholinesterase inhibiting rate of agricultural chemicals under the various variable concentrations according to following formula.
7. by the inhibiting rate of the change calculations cholinesterase of light absorption value
Wherein:
Control group absorbance difference is the changing value of 3 minutes absorbances of contrast solution reaction;
Sample absorbance difference is the changing value of 3 minutes absorbances of sample solution reaction;
The inhibiting rate of the variable concentrations that obtains is listed in following table 3, drawn Fig. 3 according to the gained inhibiting rate.
The inhibiting rate of table 3 Azodrin variable concentrations
| Concentration (ppm) | 0.070133 | 0.2104 | 0.350667 | 0.701333 | 1.402667 |
| Inhibiting rate (%) | 86.38786 | 41.77942 | 54.73555 | 75.72776 | 95.07995 |
| Concentration (ppm) | 2.805333 | 4.208 | 4.909333 | 5.610667 | 7.013333 |
| Inhibiting rate (%) | 97.70398 | 98.35998 | 98.52399 | 98.85199 | 98.68799 |
As can be seen from Table 3, enzyme is comparatively responsive to Azodrin, and the degree of cultivating the back inhibition is bigger, and absorbance obviously reduces.As calculated as can be seen, 10 of interpolation inhibiting rates that concentration is calculated are big at 0.07ppm place inhibiting rate.So Azodrin can be by the detecting of sensitivity, method of the present invention is suitable for the fast detecting of Ruzhong Azodrin.
Embodiment 4
The fast detecting experiment of Ruzhong methidathion residues of pesticides and detectability checking
Experiment agricultural chemicals: methidathion:
Mouse acute oral toxicity LD great and mighty or powerful
50Be 26mg/kg, female rat LD
50Be 43.8mg/kg; To rat acute percutaneous toxicity LD
50Be 1546mg/kg, rabbit LD
50Be 200mg/kg.
Adopt 10 concentration, three revision test results (Fig. 4).
Experimental procedure:
1. the check sample of getting the variable concentrations agricultural chemicals mixes jolting with the acetone proper proportion, leaves standstill 10-30 minute, supernatant occurs and gets final product;
2. get the 10ml supernatant and place color comparison tube, feed nitrogen, being concentrated into does not have solvent to get final product;
3. adding 4mL pH is 7.2 phosphate buffer in the above-mentioned color comparison tube, adds 50 μ L cholinesterases and 50 μ L developers, two sulfo-s-dinitrobenzoic acid (DTNB) again; Do simultaneously except that not adding all identical control group of other treatment step the sample with the sample group;
4. place 37 ℃ of constant incubators, cultivated 30 minutes;
5. from constant incubator, take out culture, add 50 μ L acetylthiocholine iodides (ATCI, substrate);
6. jolting reaction back moves in ELISA Plate or the cuvette, and the absorbance of using microplate reader or spectrophotometric determination 425nm place also calculates the cholinesterase inhibiting rate of agricultural chemicals under the various variable concentrations according to following formula;
7. by the inhibiting rate of the change calculations cholinesterase of light absorption value
Wherein:
Control group absorbance difference is the changing value of 3 minutes absorbances of contrast solution reaction;
Sample absorbance difference is the changing value of 3 minutes absorbances of sample solution reaction;
The inhibiting rate of the variable concentrations that obtains is listed in following table 4, drawn Fig. 4 according to the gained inhibiting rate.
Table 4
The inhibiting rate of methidathion variable concentrations
| Concentration (ppm) | 0.0768 | 0.1536 | 0.3072 | 0.5376 | 0.768 |
| Inhibiting rate (%) | 93.76794 | 93.27593 | 91.63592 | 98.19598 | 99.836 |
| Concentration (ppm) | 1.92 | 3.84 | 7.68 | 15.36 | 19.2 |
| Inhibiting rate (%) | 99.672 | 38.99139 | 99.836 | 100 | 100 |
As can be seen from Table 4, enzyme is extremely responsive to methidathion, and the degree of cultivating the back inhibition is very big, and absorbance reduces obviously.As calculated as can be seen, 10 of interpolation inhibiting rates that concentration is calculated are all greater than 90%.So methidathion can detecting by sensitivity.
Sum up
By utilizing this method above 4 kinds of agricultural chemicals are tested, drawing this method can be applied in the residual fast detecting of milk conventional pesticide, primary dcreening operation means as residues of pesticides, in the quality control of raw material milk, play important effect, provide a kind of quick, effective control device for food security is rigid in checking up.
Claims (7)
1. the method for quick of Ruzhong residues of pesticides is characterized in that, it comprises the steps:
1). get check sample and mix with acetone, jolting is left standstill, and supernatant occurs and gets final product;
2). get the 5-10ml supernatant and place color comparison tube, feed nitrogen, concentrate;
3). give the damping fluid that adds 2-4mL pH=7.0-8.0 in the above-mentioned color comparison tube, add 40-60 μ L cholinesterase+40-60 μ L developer two sulfo-s-dinitrobenzoic acid (DTNB) again; Do simultaneously except that not adding all identical control group of other treatment step the sample with the sample group;
4). above-mentioned sample group and control group are placed 37 ℃ of constant incubators, cultivate;
5). from constant incubator, take out culture, add the substrate of 40-60 μ L: acetylthiocholine iodide (ATCI);
6). with above-mentioned sample group and control group jolting reaction, move into then in ELISA Plate or the cuvette, under the 420-430nm wavelength, use microplate reader or spectrophotometric determination absorbance;
7). by the inhibiting rate of the change calculations cholinesterase of absorbance
Wherein:
Control group absorbance difference is the changing value of 3 minutes absorbances of contrast solution reaction;
Sample absorbance difference is the changing value of 3 minutes absorbances of sample solution reaction;
8) result judges:
The result is with the repressed degree of enzyme (inhibiting rate) expression,
When test sample during to the inhibiting rate of enzyme 〉=50%, the expression sample is positive.
2. the method for quick of claim 1 is characterized in that, wherein said damping fluid is a phosphate buffer.
3. the method for quick of claim 1 is characterized in that, wherein time of repose is 10-30 minute in the step 1), and the incubation time in the constant incubator is 10-30 minute.
4. the method for quick of claim 2 is characterized in that, wherein the pH=8.0 of phosphate buffer.
5. the described detection method of above-mentioned each claim is characterized in that wherein acetone is analytically pure.
6. the described detection method of above-mentioned each claim is characterized in that wherein the detection wavelength in the step 6) is 425nm.
7. each described detection method among the claim 1-4 is characterized in that, wherein said agricultural chemicals is DDVP, acephatemet, Azodrin, methidathion.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134559A (en) * | 2010-11-15 | 2011-07-27 | 广州吉家庄生物科技有限公司 | Bacillus brevis, method for extracting bacillus brevis esterase and application of same |
| CN102304558A (en) * | 2011-05-25 | 2012-01-04 | 青岛大学 | Method for analyzing inhibition of immobilized flow injection enzyme |
| CN104597039A (en) * | 2014-12-28 | 2015-05-06 | 福建医科大学 | Chemiluminescence sensing detection method for organophosphorus pesticide residues and application thereof |
| CN106053454A (en) * | 2016-05-30 | 2016-10-26 | 西华大学 | Quick sensibilization detecting method of organophosphorus pesticide |
| CN106093032A (en) * | 2016-08-31 | 2016-11-09 | 孟月志 | A kind of Fast Determination of Pesticide Residue eliminates false-positive detection method |
| CN106222235A (en) * | 2016-07-13 | 2016-12-14 | 温州医科大学 | A kind of Rapid Screening and the method for assessment Pesticide Residues in Tea |
| CN106591422A (en) * | 2016-11-11 | 2017-04-26 | 广西壮族自治区水牛研究所 | Kit for rapid detection of pesticide residues in buffalo milk and use thereof |
| CN108693126A (en) * | 2017-04-05 | 2018-10-23 | 中国科学院大连化学物理研究所 | A kind of rapid detection method of carbamate chemicals for agriculture |
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2007
- 2007-03-02 CN CNA2007100853907A patent/CN101059423A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102134559A (en) * | 2010-11-15 | 2011-07-27 | 广州吉家庄生物科技有限公司 | Bacillus brevis, method for extracting bacillus brevis esterase and application of same |
| CN102134559B (en) * | 2010-11-15 | 2012-12-19 | 广州吉家庄生物科技有限公司 | Bacillus brevis, method for extracting bacillus brevis esterase and application of same |
| CN102304558A (en) * | 2011-05-25 | 2012-01-04 | 青岛大学 | Method for analyzing inhibition of immobilized flow injection enzyme |
| CN104597039A (en) * | 2014-12-28 | 2015-05-06 | 福建医科大学 | Chemiluminescence sensing detection method for organophosphorus pesticide residues and application thereof |
| CN106053454A (en) * | 2016-05-30 | 2016-10-26 | 西华大学 | Quick sensibilization detecting method of organophosphorus pesticide |
| CN106222235A (en) * | 2016-07-13 | 2016-12-14 | 温州医科大学 | A kind of Rapid Screening and the method for assessment Pesticide Residues in Tea |
| CN106093032A (en) * | 2016-08-31 | 2016-11-09 | 孟月志 | A kind of Fast Determination of Pesticide Residue eliminates false-positive detection method |
| CN106591422A (en) * | 2016-11-11 | 2017-04-26 | 广西壮族自治区水牛研究所 | Kit for rapid detection of pesticide residues in buffalo milk and use thereof |
| CN108693126A (en) * | 2017-04-05 | 2018-10-23 | 中国科学院大连化学物理研究所 | A kind of rapid detection method of carbamate chemicals for agriculture |
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Open date: 20071024 |