CN101109735A - Fluorescence photometry for immune affinity column of aflatoxin in paddy - Google Patents
Fluorescence photometry for immune affinity column of aflatoxin in paddy Download PDFInfo
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- CN101109735A CN101109735A CNA200710141590XA CN200710141590A CN101109735A CN 101109735 A CN101109735 A CN 101109735A CN A200710141590X A CNA200710141590X A CN A200710141590XA CN 200710141590 A CN200710141590 A CN 200710141590A CN 101109735 A CN101109735 A CN 101109735A
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Abstract
The invention relates to a method for determining the fluorescent luminosity for the immunity affinity column of aflatoxicosis in rice, which comprises such procedures as extracting, cleaning, determining and calculating, etc.; and is characterized in that: the extracting procedure is to fetch 10.0g sample, add 2.0-2.5g NaCl and 60-80% methanol-water solution 50.omL, and extract by ultrasonic for 10-20 min; in the cleaning procedure, when cleaning the affinity column, first the affinity column is showered by a tween -20/PBS solution, then by a methanol: water =2:8 solution, finally by water. The invention has the advantages that the recovery rate is more near to the actual value, the accuracy is higher, the reagent is reduced, the inspection cost is reduced, the environmental pollution is reduced, and the calculation is convenient.
Description
Technical field
The invention belongs to the food safety detection technical field.
Background technology
The method of measuring aflatoxin in the cereal has thin-layered chromatography, high performance liquid chromatography, enzyme-linked immunosorbent assay, mass spectroscopy, radioimmunoassay.These methods more or less all have following weak point:
(1) in operating process, the aflatoxin that needs to use severe toxicity works the mischief to operator safety as demarcating reference material.
(2) operating process is loaded down with trivial details, complicated, the time is long, and labour intensity is big.
(3) instrument and equipment costliness, heaviness, complicated operation are difficult to realize on-the-spot express-analysis.
(4) sensitivity is relatively poor, and repeatability is difficult to obtain satisfactory result.
At present, both at home and abroad " Aspergillus flavus toxin immuno affinity post---the fluorophotometer method " that generally adopts is to be separation means with the monoclonal immune affinity column, with the rapid analysis of photofluorometer as testing tool.It has overcome TLC (thin-layered chromatography) and HPLC (high performance liquid chromatography) method use severe toxicity in operating process mycotoxin as demarcating reference material and using the organic solvent of multiple poisonous, peculiar smell in the sample pretreatment process, the shortcoming of harm operating personnel's personal safety and contaminated environment.Aspergillus flavus toxin immuno affinity post fluorophotometer method is safer, reliable than traditional HPLC method, sensitivity and accuracy height.It adopts the monoclonal antibody immunity technology, can wholesomeness aflatoxin or other mycotoxin be separated separation efficiency and recovery height.
Standard GB/T 18979-2003 (mensuration of the aflatoxin in the food---immunoaffinity chromatography purifies high performance liquid chromatography and fluorophotometric method), the method is applicable to corn, peanut and the goods thereof (mensuration of the aflatoxin in the food such as peanut butter, shelled peanut, shelled peanut, rice, vegetable fat, soy sauce, vinegar; GB/T 18980-2003 (aflatoxin M in milk and the milk powder
1Mensuration---immunoaffinity chromatography purifies high performance liquid chromatography and fluorophotometric method) aflatoxin M in the main scope of application milk, milk powder
1Determination on content.
The principle of the detection aflatoxin of immune affinity column-fluorophotometric method
Extract through methanol-water, extract is through after filtering, diluting, filtrate purifies through the immunoaffinity chromatography that contains the aflatoxin specific antibody, and this antibody has selectivity to aflatoxin B1, B2, G1, G2, and aflatoxin is crosslinked on the antibody of chromatography media.With clear water the impurity on the immune affinity column is removed, by the immune affinity column wash-out, added bromine solutions and derive, measure sensitivity to improve with methyl alcohol.Meoh eluate is by the total amount of fluorescent spectrophotometer measuring aflatoxin.
Among the GB GB/T18979-2003 in the examination of aflatoxin method among the 2.4.1 aflatoxin detection method of rice, corn, wheat, peanut as follows:
1 reagent and solution
1.1 water: the water that this method is mentioned all refers to redistilled water
1.2 methyl alcohol: chromatographically pure
1.3 methanol-water (7+3): get 70mL methanol plus water 30mL
1.4 sodium chloride (Nacl): analyze pure
1.5 PBS buffer solution: 8.0g sodium chloride, the 1.2g sodium hydrogen phosphate, the 0.2g potassium dihydrogen phosphate, 0.2g potassium chloride,
With the dissolving of 990mL pure water, regulate pH value to 7.0 with concentrated hydrochloric acid then, be diluted to 1000mL with pure water at last.
1.6 Tween-20/PBS solution (0.1%): get the 1mL Tween-20, add the PBS solution dilution to 1000mL.
1.7 bromine solutions storing solution solution (0.01%): take by weighing an amount of bromine, water-soluble, be made into 0.01% storing solution, 4 ℃ keep in Dark Place.
1.8 bromine solutions working fluid (0.002%): the storing solution of getting 10mL0.01% adds 40ml water mixing.In brown bottle, preserve standby.Need to prepare before each the use.
1.9 sulfuric acid solution (0.05mol/L): get the 2.8mL concentrated sulphuric acid, slowly add in an amount of water, cooling back constant volume is to 1000mL.1.10 fluorophotometric meter calibrating solution: take by weighing the 3.40g quinine sulfate and be diluted to 100mL with the 0.05mol/L sulfuric acid solution, this solution fluorophotometer reading is equivalent to 20 μ g/L aflatoxin standard solution.
2 instrument and equipments
2.1 fluorophotometer
2.2 high speed homogenization device: 18,000r/min～22,000r/min
2.3 Aspergillus flavus toxin immuno-affinity column
2.4 glass fibre filter paper: diameter 11cm, aperture 1.5 μ m
2.5 glass syringe: 10mL, 20 mL.
2.6 glass test tube: diameter 12mm, know 75mm, no fluorescence.
2.7 air pressure pump.
3 analytical procedures
3.1 extract
The 25.0g that accurately takes by weighing process levigate (less than 2mm) adds 5.0g sodium chloride and accurate 125.0mL (V in 250mL tool plug triangular flask
1) methanol-water (7+3), with homogenizer high-speed stirred 2min.Quantitative filter paper filters, and accurately pipettes 15.0mL (V
2) filtrate and 30.0mL (V
3) dilution.Inferior with glass fiber filter paper l～2 filtrations, to the filtrate clarification, standby.
3.2 purify
Immune affinity column is connected under the 20.0mL glass syringe.Accurately pipette 15.0mL (V
4) sample extracting solution injects under the glass syringe, and the air pressure pump is connected with glass syringe, adjusting pressure makes solution slowly pass through affinity column with about 6mL/min flow velocity, passes through cylinder until 2～3mL air.Accurately add 1.0mL (V) hplc grade methanol eluant solution, flow velocity is 1mL/min～2 mL/min, takes in whole eluents in glass test tube, uses for detecting.
3.3 measure
3.3.1 fluorophotometric meter calibrating
At laser glistening light of waves 360nm, under the emission wavelength 450nm condition, be blank with the 0.05mon/L sulfuric acid solution, regulate the reading value 0.0 μ g/L of fluorophotometer; The count value of regulating fluorophotometer in the fluorophotometric meter calibrating is 20.0 μ g/L.
3.3.2 sample liquid is measured
Get the meoh eluate 1.0mL after the above-mentioned purification, 0.002% bromine solutions leaves standstill lmin, operates the aflatoxin (B in reading in fluorophotometer by the 3.30. condition
1+ B
2+ G
1+ G
2) concentration c (μ g/L) of r.
3.3.3 blank test
Water replaces sample, does blank test by 3.3.1～3.3.2.
3.3.4 the result calculates
Testing result by formula (1) is calculated:
In the formula: X---aflatoxin (B in the sample
1, B
2, G
1Or G
2) content, μ g/kg;
C1---the content of aflatoxin B1, B2, G1 or G2 in the sample, μ g/L;
C2---the content of blank test aspergillus flavus B1, B2, G1 or G2, μ g/L;
V---final meoh eluate volume, mL;
W---the sample mass that the final purification eluent is contained, g;
M---the sample amount of taking by weighing, g;
V1---sample extracting solution volume, mL;
V2---dilution sample filtrate volume, mL;
V3---dilution volume, mL;
V4---by the sample extracting solution volume of affinity column, mL.
Result of calculation need be deducted blank value.
Summary of the invention
The assay method that the purpose of this invention is to provide the immune affinity column-fluorophotometric of aflatoxin in a kind of improved paddy, it has the recovery more near actual value, and precision is higher, reduce the use amount of reagent, reduce and detect cost, reduce pollution, advantages such as convenience of calculation environment.
Technical scheme of the present invention is: the assay method of the immune affinity column-fluorophotometric of aflatoxin in the paddy, it comprises extraction successively, purify, measure and calculation procedure, extract through methanol-water, extract is through filtering, after the dilution, filtrate is through containing the immune affinity column of aflatoxin specific antibody, chromatography purifies, this antibody is to aflatoxin B1, B2, G1, G2 has selectivity, aflatoxin is crosslinked on the antibody of affinity column chromatography medium, clean affinity column, the back, adds bromine solutions and derives by the immune affinity column wash-out with methyl alcohol, measure sensitivity to improve, with the total amount of meoh eluate by the fluorescent spectrophotometer measuring aflatoxin;
It is characterized in that:
Get the 10.0g sample in the extraction step, add 2.0-2.5g sodium chloride and 60-80% methanol-water solution 50.0mL, ultrasonic 10-20min extracts;
When cleaning affinity column in the purifying step, earlier with Tween-20/PBS solution drip washing affinity column, back methyl alcohol: the solution drip washing affinity column of water=2: 8, last water cleaning affinity column.
The assay method of the immune affinity column-fluorophotometric of aflatoxin in the aforesaid paddy is characterized in that: in the extraction step, sample filtrate is 1: 3 with its water volume ratio of dilution.
The examination of aflatoxin method is compared with GB and is mainly contained following difference in the paddy of the present invention:
1. sample and reagent dosage
Among the GB GB/T 18979-2003, sample 25.0g, methyl alcohol (7+3) aqueous solution 125.0mL; With the inventive method then be sample thief 10.0g, methanol-water solution 50.0mL.Mainly be to detect cost, reduce pollution environment in order to reduce the use amount of reagent, to reduce.
2. extracting method
Among the GB GB/T 18979-2003, be to extract with high speed agitator; Be to use ultrasonic Extraction in the detection method of the present invention.This mainly is because reagent reduces, and is more convenient with ultrasonic Extraction.
3. diluted sample ratio
Among the GB GB/T 18979-2003, get 15.0mL filtrate and add the dilution of 30.0mL water; The inventive method is to add the dilution of 30.0mL water with 10.0mL filtrate.Can make recovery height like this, and convenience of calculation.
3. purification process
GB GB/T 18979-2003 cleans pillar 2 times with 10.0mL water; And detection side's rule of the present invention is: with Tween-20/PBS solution drip washing post, use methyl alcohol (2+8) solution to clean pillar with earlier again, last water cleans pillar.This mainly helps effectively remove the impurity on the post, reduces the interference of impurity to testing result, and the detection reappearance of parallel sample is better.
Method of the present invention is compared with GB GB/T 18979-2003 method, and the recovery is more near actual value, and precision is higher, has reduced the use amount of reagent, reduces and detects cost, reduces the pollution to environment, advantages such as convenience of calculation.
Concrete embodiment
Specific embodiments of the invention are as follows.The present invention is through overtesting and research, and the method for this prior art is improved and optimized, and the examination of aflatoxin method follows these steps to carry out in the paddy after the improvement:
1 reagent and solution
1.1 water: the water that the method for the embodiment of the invention is mentioned all refers to redistilled water
1.2 methyl alcohol: chromatographically pure
1.3 60-80% methanol-water solution: get 60mL methanol plus water 40mL, tired according to this pushing away, preparation 70% or methanol solution.
1.4 methanol-water (2+8): get 20mL methanol plus water 80mL
1.5 sodium chloride (Nacl): analyze pure
1.6 PBS buffer solution: 8.0g sodium chloride, the 1.2g sodium hydrogen phosphate, the 0.2g potassium dihydrogen phosphate, 0.2g potassium chloride with the dissolving of 990mL pure water, is regulated pH value to 7.0 with concentrated hydrochloric acid then, is diluted to 1000mL with pure water at last.
1.7 Tween-20/PBS solution (0.1%): get the 1mL Tween-20, add the PBS solution dilution to 1000mL.
1.8 original-pack chromophoric solution: inspection Vicon Co., Ltd. provides in Beijing.
1.9 preparation chromophoric solution: get the original-pack chromophoric solution of 1mL, add 9ml water mixing, need each preceding preparation of using.
1.10 fluorophotometric meter calibrating solution: inspection Vicon Co., Ltd. provides in Beijing.Reading is equivalent to 22 μ g/L aflatoxin standard solution in the red calibration liquid fluorophotometer, and the green liquid of demarcating is equivalent to-0.100 μ g/L aflatoxin standard solution, and the yellow liquid of demarcating is equivalent to 11 μ g/L aflatoxin standard solution.
2 instrument and equipments
2.1 VICAM-4 fluorophotometer
2.2 Extraction by Ultrasound device
2.3 Aspergillus flavus toxin immuno-affinity column
2.4 glass fibre filter paper: diameter 11cm, aperture 1.5 μ m
2.5 glass syringe: 10mL, 20 mL.
2.6 glass test tube: diameter 12mm, long 75mm, no fluorescence.
2.7 air pressure pump.
3 analytical procedures
3.1 extract
The 10.0g that accurately takes by weighing process levigate (less than 2mm) adds 2.0g sodium chloride and accurate 50.0mL (V in 150mL tool plug triangular flask
1) 60-80% methanol-water solution, ultrasonic 10min.Quantitative filter paper filters, and accurately pipettes 10.0mL (V
2) filtrate and 30.0mL (V
3) dilution.Filter 1～2 time with glass fiber filter paper, to the filtrate clarification, standby.
3.2 purify
Immune affinity column is connected under the 10.0mL glass syringe.Accurately pipette 10.0mL (V
4) sample extracting solution injects under the glass syringe, and the air pressure pump is connected with glass syringe, adjusting pressure makes solution slowly pass through affinity column with about 6mL/min flow velocity, passes through cylinder until 2～3mL air.Earlier with 10.0mL Tween-20/PBS solution drip washing post 1～2 time, affinity column is cleaned with 10.0mL water at last with 10.0mL methanol-water (2+8) solution drip washing pillar in the back, discards whole effluent, and makes liquid emptying in the post.Accurately add 1.0mL (V) hplc grade methanol eluant solution, flow velocity is 1mL/min～2mL/min, takes in whole eluents in glass test tube, uses for detecting.
3.3 measure
3.3.1 fluorophotometric meter calibrating
According to the operation instructions of instrument, instrument is proofreaied and correct.Demarcate with red calibrating tube earlier, key in correct reading 44ppb; Demarcate with green calibrating tube again, key in correct reading-0.1ppb; Detect yellow calibrating tube at last, reading should be 22 ± 2ppb.
3.3.2 sample liquid is measured
The meoh eluate of getting after the above-mentioned purification adds the 1.0mL chromophoric solution, and mixing leaves standstill 1min, operates the aflatoxin (B in reading in fluorophotometer by the 3.3.1 condition
1+ B
2+ G
1+ G
2) concentration c (μ g/kg).
3.3.3 blank test
Water replaces sample, does blank test by 4.3.1～3.3.2.
3.3.4 the result calculates
Testing result by formula (2) is calculated:
X＝c
1-c
0 ………(2)
In the formula: X---the content of aflatoxin in the sample, μ g/kg;
c
1---the content of aflatoxin in the sample liquid, μ g/L;
c
0---blank sample aflatoxin content, μ g/L.
Six, the examination of aflatoxin method is compared with GB and is mainly contained following difference in the paddy of the present invention:
2. sample and reagent dosage
Among the GB GB/T 18979-2003, sample 25.0g, methyl alcohol (6+4) solution 125.0mL; With the inventive method then be sample thief 10.0g, methyl alcohol (6+4) solution 50.0mL.Mainly be to detect cost, reduce pollution environment in order to reduce the use amount of reagent, to reduce.
2. extracting method
Among the GB GB/T 18979-2003, be to extract with high speed agitator; Be to use ultrasonic Extraction in the detection method of the present invention.This mainly is because reagent reduces, and is more convenient with ultrasonic Extraction.
3. diluted sample ratio
Among the GB GB/T 18979-2003, get 15.0mL filtrate and add the dilution of 30.0mL water; The inventive method is to add the dilution of 30.0mL water with 10.0mL filtrate.This is mainly convenience of calculation.
3. purification process
GB GB/T 18979-2003 cleans pillar 2 times with 10.0mL water; And detection side's rule of the present invention is: with 10.0mL Tween-20/PBS solution drip washing post 1～2 time, use 10.0mL methyl alcohol (2+8) solution to clean pillar with earlier again, clean pillar with 10.0mL water at last.This mainly helps effectively remove the impurity on the post, reduces the interference of impurity to testing result, and the detection reappearance of parallel sample is better, and testing result is more near actual value.
4. compare with GB GB/T 18979-2003, aflatoxin average recovery and precision that the inventive method detects in the paddy compare.
GB GB/T 18979-2003 detects paddy, and average recovery is between 110%～250%, and average recovery rate is 155%, and parallel sample precision is 31.6%;
The inventive method detects paddy, and average recovery is 85%～92%, and average recovery rate is 89%, and parallel sample precision is 4.4%.
Seven. compare with GB GB/T 18979-2003, the aflatoxin that the inventive method detects in the paddy has the following advantages
The present invention compares with GB GB/T 18979-2003 method by amended method, and the rate of recovery is more near actual value, and precision is higher, has reduced the use amount of reagent, reduces testing cost, reduces the pollution to environment, the advantages such as convenience of calculation.
Claims (2)
1. the assay method of the immune affinity column-fluorophotometric of aflatoxin in the paddy, it comprises extraction successively, purify, measure and calculation procedure, extract through methanol-water, extract is through filtering, after the dilution, filtrate is through containing the immune affinity column of aflatoxin specific antibody, chromatography purifies, this antibody is to aflatoxin B1, B2, G1, G2 has selectivity, aflatoxin is crosslinked on the antibody of affinity column chromatography medium, clean affinity column, the back, adds bromine solutions and derives by the immune affinity column wash-out with methyl alcohol, measure sensitivity to improve, with the total amount of meoh eluate by the fluorescent spectrophotometer measuring aflatoxin;
It is characterized in that:
Get the 10.0g sample in the extraction step, add 2.0-2.5g sodium chloride and 60-80% methanol-water solution 50.0mL, ultrasonic 10-20min extracts;
When cleaning affinity column in the purifying step, earlier with Tween-20/PBS solution drip washing affinity column, back methyl alcohol: the solution drip washing affinity column of water=2: 8, last water cleaning affinity column.
2. the assay method of immune affinity column one fluorophotometric of aflatoxin in the paddy as claimed in claim 1 is characterized in that: in the extraction step, sample filtrate is 1: 3 with its water volume ratio of dilution.
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| CN102692469A (en) * | 2011-03-21 | 2012-09-26 | 上海市食品药品检验所 | Method for using liquid phase chromatography-tandem mass spectrometry method to determine content of mycotoxin in ginseng |
| CN103116024A (en) * | 2012-11-30 | 2013-05-22 | 中国农业科学院油料作物研究所 | Application and method of anti-aflatoxin universal monoclonal antibody 1C11 in aflatoxin B1 fluorescence quenching |
| CN103217528A (en) * | 2012-11-30 | 2013-07-24 | 中国农业科学院油料作物研究所 | Non-labeled immunization analysis method for detecting content of aflatoxin B1 |
| CN103604902A (en) * | 2013-12-06 | 2014-02-26 | 张淑金 | Method for quickly detecting content of aflatoxin in cereals and foods |
| CN103792358A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Preparation and detection method of aflatoxin G1 colloidal gold immunochromatograohic assay detection board |
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| CN105651899A (en) * | 2016-04-08 | 2016-06-08 | 云南健牛生物科技有限公司 | Method for high-sensitivity detection of aflatoxin and application of aflatoxin |
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| CN106370736A (en) * | 2016-08-15 | 2017-02-01 | 舟山市食品药品检验检测研究院 | Method for simultaneous SMART column on-line purification and HPLC/UVE fluorescent detection of four aflatoxins in peanut product |
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| CN102692469B (en) * | 2011-03-21 | 2016-03-30 | 上海市食品药品检验所 | The method of mycotoxin levels in LC-MS/MS ginseng |
| CN102692469A (en) * | 2011-03-21 | 2012-09-26 | 上海市食品药品检验所 | Method for using liquid phase chromatography-tandem mass spectrometry method to determine content of mycotoxin in ginseng |
| CN103792358A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Preparation and detection method of aflatoxin G1 colloidal gold immunochromatograohic assay detection board |
| CN103116024A (en) * | 2012-11-30 | 2013-05-22 | 中国农业科学院油料作物研究所 | Application and method of anti-aflatoxin universal monoclonal antibody 1C11 in aflatoxin B1 fluorescence quenching |
| CN103217528A (en) * | 2012-11-30 | 2013-07-24 | 中国农业科学院油料作物研究所 | Non-labeled immunization analysis method for detecting content of aflatoxin B1 |
| CN103116024B (en) * | 2012-11-30 | 2014-04-09 | 中国农业科学院油料作物研究所 | Application and method of anti-aflatoxin universal monoclonal antibody 1C11 in aflatoxin B1 fluorescence quenching |
| CN103217528B (en) * | 2012-11-30 | 2014-04-09 | 中国农业科学院油料作物研究所 | Non-labeled immunization analysis method for detecting content of aflatoxin B1 |
| CN103604902A (en) * | 2013-12-06 | 2014-02-26 | 张淑金 | Method for quickly detecting content of aflatoxin in cereals and foods |
| CN103604902B (en) * | 2013-12-06 | 2016-02-17 | 张淑金 | The method of aflatoxin content is detected fast in cereal and food |
| CN104007255A (en) * | 2014-05-16 | 2014-08-27 | 中南林业科技大学 | Aflatoxin B1 rapid detection test paper strip and preparation method and application thereof |
| CN104007255B (en) * | 2014-05-16 | 2016-01-06 | 中南林业科技大学 | A kind of aflatoxin B1 Rapid detection test strip and preparation method thereof and application |
| CN104678039A (en) * | 2015-01-30 | 2015-06-03 | 湖南中烟工业有限责任公司 | Method for simultaneously measuring contents of four aflatoxins in tobacco and tobacco products by liquid chromatography-tandem mass spectrometry combination |
| CN106153785A (en) * | 2015-04-09 | 2016-11-23 | 上海磐合科学仪器股份有限公司 | A kind of online sample introduction of aflatoxin analyzes method |
| CN105651899A (en) * | 2016-04-08 | 2016-06-08 | 云南健牛生物科技有限公司 | Method for high-sensitivity detection of aflatoxin and application of aflatoxin |
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| CN106370736A (en) * | 2016-08-15 | 2017-02-01 | 舟山市食品药品检验检测研究院 | Method for simultaneous SMART column on-line purification and HPLC/UVE fluorescent detection of four aflatoxins in peanut product |
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