CN101285834A - Method for accurately and rapidly checking aspergillus flavus toxin - Google Patents

Method for accurately and rapidly checking aspergillus flavus toxin Download PDF

Info

Publication number
CN101285834A
CN101285834A CNA2008100384190A CN200810038419A CN101285834A CN 101285834 A CN101285834 A CN 101285834A CN A2008100384190 A CNA2008100384190 A CN A2008100384190A CN 200810038419 A CN200810038419 A CN 200810038419A CN 101285834 A CN101285834 A CN 101285834A
Authority
CN
China
Prior art keywords
solution
sample
test tube
aflatoxin
preparation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CNA2008100384190A
Other languages
Chinese (zh)
Inventor
潘中华
成恒嵩
赵晓联
葛其德
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SHANGHAI QIANJIAN INSTRUMENT CO Ltd
Original Assignee
SHANGHAI QIANJIAN INSTRUMENT CO Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SHANGHAI QIANJIAN INSTRUMENT CO Ltd filed Critical SHANGHAI QIANJIAN INSTRUMENT CO Ltd
Priority to CNA2008100384190A priority Critical patent/CN101285834A/en
Publication of CN101285834A publication Critical patent/CN101285834A/en
Pending legal-status Critical Current

Links

Images

Abstract

The invention relates to a method for realizing accurate and quick detection of aflatoxin. The invention is characterized in that the method is as follows: step (1), enzyme-labelled antigen, an aflatoxin AFB1 standard solution, sample diluent and enzyme-labelled antigen diluent are made, and a substrate solution a, a substrate solution b, a stop solution (i.e. a 2mol/L sulphuric acid solution) and extract are made by a cleaning solution; step (2), to-be-measured solution pretreatment, reagent preparation and reaction plate test tube numbering are carried out, and a prepared solution, a to-be-measured sample solution and the stop solution are added in turn; meanwhile, a reaction plate is arranged, and an aflatoxin tester is used to measure the absorbance A value of each hole; then, according to the measured absorbance A value, an equation A=MC+N for regression calculation is automatically established inside the aflatoxin tester to calculate the content of AFB1 in sample. The method has the advantages of simplicity and convenience, quickness, high accuracy and high sensitivity.

Description

A kind of accurately, the method for fast detecting aflatoxin
Technical field
The present invention relates to a kind of accurately, the method for fast detecting aflatoxin, belong to immunoenzyme technics field in the bioengineering.
Background technology
Aflatoxin (being called for short AFT) is a kind of mycotoxin, it is a kind of supervirulent secondary metabolite that produces by fungi aspergillus flavus and aspergillus parasiticus, it has been established that all can evil the material of living aspergillus flavus fungi, the pollution of AFT is all arranged in seed, fruit, dry fruit, vegetables, flavouring, tobacco, jute, newborn class, dairy products, meat, fish and shrimp, fermentation category and the feed as grain, oil crops.
The AFT physicochemical property is highly stable, under 200 ℃ of high temperature and ultraviolet ray irradiation, all be not destroyed and transfer in the another kind of biosome from a kind of biosome by the interaction energy of food chain, toxin can enter human body with food and causes human poison or carcinogenic in all meat that remains in poultry, domestic animal, internal organ, egg, the milk, according to relevant departments such as the World Health Organization (WHO) and Shanghai Inst. of Tumor report aflatoxin is the first carcinogenic factor that causes liver cancer, and therefore strict restriction has all been done to aflatoxin allowance in the food by many in the world countries.The permissible value of aflatoxin is respectively less than 50PPb and 20PPb in China's regulation feed and the food, and this just requires assay method that very high sensitivity will be arranged.Assay method roughly has at present: chemical analysis, instrumental method, biological detection method and enzyme linked immunosorbent assay (ELISA).
The most commonly used in the chemical analysis with thin layer chromatography, also as the National Standard Method of China to aflatoxin mensuration in food, the feed, its detection is limited to 1-5PPb, but it must compare with known aflatoxin standard items when detecting, and confirms by doing to replenish with bioanalysis in case of necessity.
Generally with efficient TLC-scanner or high performance liquid chromatography, its automaticity high resolving power is good for instrumental method, but instrument is expensive, and technical merit requires high, is difficult to penetration and promotion and uses.
Biological detection method is generally only done the additional checking of chemical analysis because specificity is relatively poor, and sensitivity is low.More than three kinds of method sample pre-treatments all complicated, extract and purify, get rid of in the sample that other material disturbs so that specificity is not strong.
U.S. La Weilin (Lawellin) at first proposes enzyme linked immunosorbent assay analysis method and measures the strongest structure B of aflatoxicosis 1(ethyloic oxime) comes qualitative, quantitative aflatoxin content.At present both at home and abroad all with the strongest structure B of aflatoxicosis 1As the main object of prosecution.Because this method specificity height, analysis time is short, is subjected to paying attention to widely, has the people to work out " medicine box " of using enzyme linked immunosorbent assay analysis method again afterwards, and a box reagent can be surveyed a plurality of test sample, and " medicine box " has purchase in the U.S., but price is also relatively more expensive.
Summary of the invention
The purpose of this invention is to provide a kind of use simple and easy, inexpensive, incorporate accurately, the method for fast detecting aflatoxin.
For realizing above purpose, technical scheme of the present invention provide a kind of accurately, the method for fast detecting aflatoxin, it is characterized in that its method is:
One. the standard sample preparation
The first step, preparation aflatoxin AFB 1The antibody sandwich reaction plate:
With aspergillus flavus resisting toxin A FB 1Polyclonal antibody 50mmol/L-100mmol/L, Na 2CO 3-NaHCO 3The PH9.6 damping fluid is diluted to the coating buffer of 3-5 μ g/ml, join in the test tube in 48 or 96 holes of reaction plate, every hole adds 100-110 μ L, 3 ℃ of-5 ℃ of refrigerators are placed and are spent the night, discard coating buffer flushing 2 times-4 times, add 100-110mmol/L, the sealing of PH7.4 phosphate PBS damping fluid that 200-250 μ L contains 1% bovine serum albumin(BSA) BSA in every hole and put 2-4 hour for 35 ℃-40 ℃, abandon its confining liquid, will wrap by-15 ℃ of the vacuum-packed postposition of plate--25 ℃ of freezing preservations;
Second step, the preparation enzyme-labelled antigen:
Get 0.5～1mg AFB 1-ethyloic oxime AFB 1-omixe is dissolved in 20% ethanolic solution, added 20-50mg ethylenediamine 3.3-dimethylamine propyl carbodiimide hydrochloride EDCHCL stirring reaction 30 minutes-60 minutes, with phosphate PBS 0.01M, the dialysis of PH7.4 solution 2 days-4 days, obtain enzyme-labelled antigen stoste again;
The 3rd step, preparation aflatoxin AFB 1Standard solution
With the phosphate PBS PH7.4 solution of 10%-20% methyl alcohol is solvent, the AFB of preparation 0.1ng/ml-10ng/ml 1Standard serial solution;
The 4th step, the preparation sample diluting liquid
In phosphate PBS PH7.4 solution, add 10%-20% methyl alcohol;
The 5th step, preparation enzyme-labelled antigen dilution
The bovine serum albumin(BSA) BSA that in phosphate PBS PH7.4 solution, adds 0.05-0.2%;
The 6th step, preparing washing liquid
The Tween-20 TW-20 that in phosphate PBS PH7.4 solution, adds 0.03%-0.07%;
The 7th step, preparation substrate solution a:
At sodium acetate---add 0.1%-0.4% tetramethyl benzidine TMB among the citrate buffer solution PH=5.0;
The 8th step, preparation substrate solution b:
In sodium acetate-citrate buffer solution PH=5.0, add 0.01-0.03% hydrogen peroxide H 2O 2
In the 9th step, the preparation stop buffer is a 2mol/L sulfuric acid liquid;
In the tenth step, extract is a methyl alcohol: water is 1: 1;
Two, detect
The first step, liquid pre-treatment to be measured,
Take by weighing sample that 5g pulverized 20 mesh sieves in the 50ml of ground test tube, add extract 20-30ml, the vibration 5 minutes-10 minutes of jumping a queue is filtered to be and is treated sample measuring liquid;
In second step, reagent is prepared:
Add 1-2ml enzyme-labelled antigen dilute solution in the enzyme-labelled antigen stoste of standard sample preparation second step preparation, fully dissolving is made into the enzyme-labelled antigen working solution;
In the 3rd step, the test tube numbering: with the numbering of the test tube on the reaction plate framework, setting No. 1 test tube is the withered test tube of instrument according to the experiment needs, and the 2-7 test tube is aflatoxin AFB 1Standard control test tube, all the other test tubes are sample tube;
In the 4th step, add the solution that configures successively and treat sample measuring liquid
(1), in No. 1 test tube, adds 40-60 μ L sample diluting liquid, in the 2-7 test tube, add aflatoxin AFB respectively 1Standard solution 40-60 μ L adds respectively in all the other test tubes and treats sample measuring liquid;
(2), add 40-60 μ L enzyme-labelled antigen dilution in No. 1 test tube, add 40-60 μ L enzyme-labelled antigen working solution in all the other test tubes respectively, vibration makes the reactant mixing in each test tube lightly;
(3), then reaction plate being put into 35 ℃ of-40 ℃ of constant incubators hatched 20 minutes-40 minutes;
(4), take out reaction plate, get rid of reactant liquor, pat dry, every test tube adds 200 μ L-300 μ L cleansing solutions, place 1 minute-3 minutes after, get rid of cleansing solution, on thieving paper, pat dry, repeat to wash plate 3-5 time;
(5), in every test tube, add each 50 μ L of substrate solution a and substrate solution b respectively, shake up, reaction plate is put into 35 ℃ of-40 ℃ of constant incubators, developed the color 10 minutes-20 minutes;
(6), take out reaction plate, in each test tube, add stop buffer 40-60 μ L respectively, measure the absorbance A value in each hole with the aflatoxin analyzer at 360mm-450mm wavelength place;
(7),, set up equation A=MC+N regression Calculation in the aflatoxin analyzer automatically, calculate testing sample hole A value and A the absorbance A value that records 0ng/mlRatio, look into typical curve, can obtain the common logarithm value LgC of corresponding sample liquid concentration C to be measured, to its logarithm of negating, can try to achieve the AFB1 concentration C for the treatment of sample measuring liquid, calculate the content of AFB1 in the sample by following formula:
Figure A20081003841900081
In the formula: the AFB that C----checks in from typical curve 1Content (ng/ml)
V----sample extracting solution volume (ml);
D----diluted sample multiple;
The quality of m----sample (g)
Described aflatoxin analyzer comprises single-chip microcomputer and stabilized voltage supply, described single-chip microcomputer is connected with ROM (read-only memory), keyboard, display, random access memory, interface and digital converter respectively, digital converter is connected with analog to digital converter by prime amplifier, analog to digital converter is connected with printer with print drive by interface, stabilized voltage supply is connected with interference filter by light source, interference filter is connected with silicon photocell by sample cell, and silicon photocell is connected with prime amplifier.
The present invention is attached to carrier surface with the immunoglobulin (Ig) that contains the aflatoxin specific antibody earlier, contain antigenic solution and enzyme-labelled antigen solution mixes in varing proportions, carrier surface with sensitization is incubated then, the amount of hydrolysis of the special substrate by enzyme determines whether aflatoxin and content are arranged in the unknown solution, the enzymatic substrate hydrolysis amount is just counted content by determining instrument again.
Atom in the material and molecule contained energy produce the absorption effect to light with several different methods and interaction, and the material transmittance changes with measured matter concentration certain functional relation is arranged, and it meets youth primary---law of Beer within the specific limits.Therefore computing machine can be according to youth primary---law of Beer in determining instrument, be provided with the calculation procedure of an equation of linear regression A=MC+N, so as long as M, N coefficient just can directly be measured the concentration position of unknown concentration sample, mensuration wavelength range of choice 450mm～360mm in input concentration value of standard sample or the A=MC+N equation.This instrument microcomputer adopts state-of-the-art MCS-51 series monolithic in 8 machines, and the operator is as long as tell single-chip microcomputer with the thing that oneself will do by keyboard, and single-chip microcomputer is told the operator with various results by display or printer.
Major technique of the present invention is the preparation of standard sample, and this is the key that aflatoxin is measured accuracy, and gordian technique is two aspects in the standard sample preparation: aspergillus flavus resisting toxin B 1The preparation of antibody and the preparation of enzyme-labelled antigen.
1, the preparation of aspergillus flavus resisting toxin antibody.
This antibody must possess the characteristics of high effect, sensitivity, high specificity, because aflatoxin is a haptens, does not have immunogenicity, need at first make complex antigen, utilize the antigen immune rabbit to obtain to contain antibody serum then, contain aspergillus flavus resisting toxin A F B through biochemical purification, separation 1The immunoglobulin (Ig) of antibody, again with the immunoglobulin (Ig) bag by on carrier, and make bag by reaction plate.
2, the preparation of enzyme-labelled antigen
Because aflatoxin (AFB 1) can not directly combine with enzyme, must be earlier with AFB 1Introduce a reactive group,, make its activity, the chromogenic reaction that can dissolve substrate with enzyme then with enzyme reaction.
The serve as a mark enzyme of antigen of horseradish peroxidase (HRP) has successfully synthesized enzyme-labelled antigen (AF B 1-O-HRP) satisfied in the euzymelinked immunosorbent assay (ELISA) (ELISA) requirement to desmoenzyme in the enzyme-labelled antigen.
Advantage of the present invention is:
1. easy, quick
The present invention is simpler than the extraction and the purification step of chemical analysis, instrumental method, biological detection method pre-treatment, a pre-treatment only spent to finish in 10～20 minutes, sample determination also only needed to finish in 5～10 minutes, increase work efficiency 30～40 times than present National Standard Method, and its result and National Standard Method match;
2. accurate, highly sensitive
The present invention with instrument and equipment supporting kit, its accuracy and sensitivity all than single reagent box ocular estimate height, repeatability might as well, limit detection of the present invention reaches 5PPb, 20PPb, 50PPb, reach food, the minimum detection limit that allows of feed national regulation, minimum limiting the quantity of to 2.5Pg surpasses National Standard Method, its sensitivity is 160 times of National Standard Method;
3. inexpensive
Because the present invention is that reagent and determining instrument are integrated, its specificity, sensitivity, degree of accuracy are all than National Standard Method height, and its cost is significantly less than National Standard Method, only is 1/30 of National Standard Method;
4, the present invention shows than the superiority of U.S.'s " kit ":
1. analyse that speed is faster, specificity strengthens, stability increases.AFB 1-ethyloic oxime maximum output reaches 84%, AFB 1The highest the tiring of antibody reaches 1: 800000, and the recovery is more than 90%;
2. improved marker enzyme-horseradish peroxidase and aflatoxin is crosslinked;
Because the crosslinking chemical of selecting (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC.HCl)) more helps marker enzyme-horseradish peroxidase and aflatoxin is crosslinked data accuracy increasing, the sensitivity of mensuration being improved, is 160 times of National Standard Method sensitivity;
3. the control of Bao Wen time and temperature is better, has improved repeatability, the degree of accuracy of testing result;
4. the integrated production of reagent and detecting instrument not only improves analysis efficiency and has also reduced cost, adapts to on-the-spot the detection.
Description of drawings
Fig. 1 be a kind of accurately, the method program process flow diagram of fast detecting aflatoxin;
Fig. 2 is an aflatoxin analyzer structural representation;
Fig. 3 is light channel structure figure;
Fig. 4 is an aflatoxin analyzer calculation procedure process flow diagram.
Embodiment
The invention will be further described below in conjunction with drawings and Examples.
Embodiment
As shown in Figure 1, for a kind of accurately, the method program process flow diagram of fast detecting aflatoxin, a kind of accurately, the method for fast detecting aflatoxin is:
One. the standard sample preparation
The first step, preparation aflatoxin AFB 1The antibody sandwich reaction plate:
With aspergillus flavus resisting toxin A FB 1Polyclonal antibody 70mmol/L, Na 2CO 3-NaHCO 3The PH9.6 damping fluid is diluted to the coating buffer of 4 μ g/ml, join in the test tube in 48 or 96 holes of reaction plate, every hole adds 100 μ L, 4 ℃ of refrigerators are placed and are spent the night, discard coating buffer flushing 3 times, add 100mmol/L, the sealing of PH7.4 phosphate PBS damping fluid that 200 μ L contain 1% bovine serum albumin(BSA) BSA in every hole and put for 37 ℃ and abandoned its confining liquid in 3 hours and will wrap by-20 ℃ of freezing preservations of the vacuum-packed postposition of plate;
Second step, the preparation enzyme-labelled antigen:
Get the 1mg AFB 1---ethyloic oxime AFB 1-omixe is dissolved in and added 30mg ethylenediamine 3.3-dimethylamine propyl carbodiimide hydrochloride EDCHCL stirring reaction in 20% ethanolic solution 40 minutes, with phosphate PBS 0.01M, the dialysis of PH7.4 solution 3 days, obtains enzyme-labelled antigen stoste again;
The 3rd step, preparation aflatoxin AFB 1Standard solution
With the phosphate PBS PH7.4 solution of 20% methyl alcohol is the AFB of solvent preparation 0.1ng/ml～10ng/ml 1Standard serial solution;
The 4th step, preparation sample diluting liquid: in phosphate PBS PH7.4 solution, add 20% methyl alcohol;
The 5th step, preparation enzyme-labelled antigen dilution
The bovine serum albumin(BSA) BSA of adding 0.1% in phosphate PBS PH7.4 solution;
The 6th step, preparing washing liquid
The Tween-20 TW-20 of adding 0.05% in phosphate PBS PH7.4 solution;
The 7th step, preparation substrate solution a:
At sodium acetate---add 0.2% tetramethyl benzidine TMB among the citrate buffer solution PH=5.0;
The 8th step, preparation substrate solution b:
At sodium acetate---add 0.01% hydrogen peroxide H among the citrate buffer solution PH=5.0 2O 2
In the 9th step, the preparation stop buffer is a 2mol/L sulfuric acid liquid;
In the tenth step, extract is a methyl alcohol: water is 1: 1;
Two, detect
The first step, liquid pre-treatment to be measured,
Take by weighing sample that 5g pulverized 20 mesh sieves in the 50ml of ground test tube, add extract 20-30ml, the vibration 5 minutes-10 minutes of jumping a queue is filtered to be and is treated sample measuring liquid;
In second step, reagent is prepared:
Add 1.5ml enzyme-labelled antigen dilute solution in the enzyme-labelled antigen stoste of standard sample preparation second step preparation, fully dissolving is made into the enzyme-labelled antigen working solution;
In the 3rd step, the test tube numbering: with the numbering of the test tube on the reaction plate framework, setting No. 1 test tube is the withered test tube of instrument according to the experiment needs, and the 2-7 test tube is aflatoxin AFB 1Standard control test tube, all the other test tubes are sample tube;
In the 4th step, add the solution that configures successively and treat sample measuring liquid
(1), in No. 1 test tube, adds 50 μ L sample diluting liquids, in the 2-7 test tube, add aflatoxin AFB respectively 1Standard solution 50 μ L add respectively in all the other test tubes and treat sample measuring liquid;
(2), add 50 μ L enzyme-labelled antigen dilutions in No. 1 test tube, add 50 μ L enzyme-labelled antigen working solutions in all the other test tubes respectively, vibration makes the reactant mixing in each test tube lightly;
(3), then reaction plate being put into 37 ℃ of constant incubators hatched 30 minutes;
(4), take out reaction plate, get rid of reactant liquor, pat dry, every test tube adds 250 μ L cleansing solutions, places after 2 minutes, gets rid of cleansing solution, pats dry on thieving paper, repeats to wash plate 4 times;
(5), in every test tube, add each 50 μ L of substrate solution a and substrate solution b respectively, shake up, reaction plate is put into 37 ℃ of constant incubators, developed the color 15 minutes;
(6), take out reaction plate, in each test tube, add stop buffer 50 μ L respectively, measure the absorbance A value in each hole with the aflatoxin analyzer at 450nm wavelength place;
(7),, set up equation A=MC+N regression Calculation in the aflatoxin analyzer automatically, calculate testing sample hole A value and A the absorbance A value that records 0ng/mlRatio, look into typical curve, can obtain the common logarithm value LgC of corresponding sample liquid concentration C to be measured, to its logarithm of negating, can try to achieve the AFB1 concentration C for the treatment of sample measuring liquid, calculate the content of AFB1 in the sample by following formula:
Figure A20081003841900121
In the formula: the AFB that C----checks in from typical curve 1Content (ng/ml)
V----sample extracting solution volume (ml);
D----diluted sample multiple;
The quality of m----sample (g)
As shown in Figure 2, be aflatoxin analyzer structural representation, described aflatoxin analyzer comprises single-chip microcomputer and stabilized voltage supply, described single-chip microcomputer is connected with ROM (read-only memory), keyboard, display, random access memory, interface and digital converter respectively, digital converter is connected with analog to digital converter by prime amplifier, analog to digital converter is connected with printer with print drive by interface, stabilized voltage supply is connected with interference filter by light source, interference filter is connected with silicon photocell by sample cell, and silicon photocell is connected with prime amplifier.
Aflatoxins enzyme mark measuring instrument is according to the work of relative measurement principle, promptly selected a certain solvent (distilled water, air or sample) is as standard solution, and the transmittance τ (being transmittance T) that sets it is 100.0%, and the transmittance τ of tested sample (being transmittance T) obtains with respect to standard solution, the variation of transmittance τ (being transmittance T) and the concentration of measured matter have-funtcional relationship, within the specific limits, it meets lambert-beer law.
τ(T)＝I/I 0
A＝KCL＝-lg I/I 0
Wherein: τ-transmittance
The A-absorbance
C-concentration
The absorptivity of K-solution
The length of L-liquid layer in light path
Shine the intensity on the photoelectric commutator after the I-light transmission tested sample
I 0Shine the intensity on the photoelectric commutator behind the-light transmission standard sample
Computing machine in this instrument is provided with the calculation procedure of an equation of linear regression A=MC+N according to lambert-beer law, adopt the establishment of C language, as shown in Figure 4, so as long as the concentration value of input standard sample or coefficient M and the N in the equation of linear regression, just can directly measure the concentration position of unknown concentration sample, can be widely used in fields such as aflatoxins, immunopathogenesis, microbial antigen and antibody test, parasitic disease diagnosis, blood disease diagnosis, the research of phytopathy worm.
Its key technical indexes
1, wavelength coverage: 330-900nM (this instrument is only joined the 450nM interference filter in instrument), the user also can order 340,380,405,492,510,546,578 to manufacturer, the interference filter of 650nM;
2, the half width of interference filter is: 10nM;
3, light current stability :≤0.3 τ (T)/5 minute;
4, dark current stability :≤0.2% τ (T)/5 minute;
5, printing opacity is than scope: 0.0% τ (T)-110.0% τ (T);
6, absorbance scope :-0.041-1.999 (A);
7, concentration range: 0.000-9999
8, (T) conversion accuracy :≤± 0.004A
9, environment for use:
A, temperature: 5-35 ℃
B, humidity :≤85%
10, working power: 220V ± 22V, frequency 20HZ
As shown in Figure 3, be light channel structure figure, be provided with catoptron 3, collector lens 4 and light hurdle 5 in the described sample cell, this instrument adopts bromine tungsten filament lamp as light source, and stabilized voltage supply provides the voltage of high stability for light source, avoid the energy of the influence of fluctuations light source of external voltage, the continuous radiation light beam that light source 1 sends is incident upon reflective mirror 3 behind optical filter 2, be incident upon on the collector lens 4 through reflective mirror 3, become the reaction plate in the light directive sample cell of specific wavelength then, be incident upon silicon photocell 7 at last.
State-of-the-art MCS-51 series monolithic in 8 machines is adopted in the single-chip microcomputer outsourcing of this instrument, random access memory (RAM) and interface PIO0 adopt 8155, analog to digital converter (A/D) is 14433, digital converter (D/A) is 7520, ROM (read-only memory) (ROM) is 2764, prime amplifier is 7650, and printer is 16 row printers.
Photoelectric cell transfers light signal to electric signal, and through the amplification of prime amplifier, signal enters analog to digital converter, and analog to digital converter is that digital signal is sent to single-chip microcomputer and carries out data processing with analog signal conversion.Single-chip microcomputer is according to different reference signals, give an order to analog to digital converter, thereby change the enlargement factor of prime amplifier, be automatic zero set (AZS), accent full scale, the operator is as long as tell single-chip microcomputer with the thing that oneself will do by keyboard, and single-chip microcomputer is told the operator with various results by display or printer.
Instrument can be set up accounting equation A=MC+N according to two kinds of methods, and this is an equation of linear regression, in case set up this equation in the instrument, the operator just can directly obtain the concentration value of sample.
First method operator disposes 1-n different serfs' standard sample, and they are changed over to light path one by one,
Import simultaneously the concentration value of corresponding sample one by one by keyboard, as long as the concentration value of (1-n) individual sample is imported, just sets up equation A=MC+N in the instrument automatically, then the unknown being pushed light path just can direct-reading concentration.
Second method is coefficient M and the N among the equation A=MC+N of known sample to be tested, if by keyboard with in M and the N input instrument, instrument also can be set up the concentration accounting equation A=MC+N of this sample immediately.
1, sets up equation by cultivating standard sample
A) concentration value input range 0.000-9999
B) standard sample mostly is 99 most.
2, by input coefficient M with set up equation
A) must import M earlier, and then input N;
B) Shu Ru M, N value, the linear relationship between coefficient R reflection concentration C and the absorbance A, R approaches 1 more, and linear relationship is good more between concentration value C and the absorbance A, otherwise poor more.
First kind is every: the input standard specimen, set up the A=MC+N equation of linear regression automatically, if only import its concentration curve of some standard specimens be exactly 0 with this standard specimen set up curve, be referred to as one point method, we just are referred to as two point method to import 2 standard specimens.By that analogy, can cultivate 1-n different standard sample according to user's needs, measurement requirement is not high, and the standard specimen of joining can lack, and the higher sample number of joining of measurement requirement also the more.
The present invention is by detecting:
1. nutrition of Chinese Disease Control and Prevention Center and food security institute, on October 21st, 2007 testing cassete and detecting instrument are carried out the technical performance index quality inspection, quality inspection analysis report (numbering 20070705) shows with this method and measures its result and national standard thin layer chromatography (TLC) indifference that its measurement result is effectively, reliably.
Figure A20081003841900151
B:1ng/mlAFB 1The OD value average of gauge orifice (2-4 hole)
B0:0ng/mlAFB 1The OD value average (following blank) of gauge orifice (5-12 hole)
Therefore in daily detection, can replace loaded down with trivial details National Standard Method to carry out the scene and detect, conveniently promote the use of.
Tianjin, Shanghai City product quality supervision and testing institute respectively on Dec 23rd, 2006, on February 15th, 2006 its quality inspection analysis report also all meet national standard and TLC measurement result indifference.
Sequence number Technical performance index Checking result (Tianjin) Checking result (Shanghai)
1 Preparation of product principle and using method Meet the concerned countries standard Meet the concerned countries standard
2 Detection sensitivity ＜0.1ug/kg 0.01ug/kg
3 Detection time ≤ 2 hours 1.5 hour
4 Detect repeatability CV＜10％ CV＝7％
5 Sensing range 0.1～10ug/kg 0.1～10ug/kg
6 The term of validity 6 months (4 ℃) 6 months (4 ℃)
7 Detection accuracy Detect Through check, with HPLC method there was no significant difference
3. Jiangxi, Jiangsu, the supervision of Sichuan veterinary drug feed are observed and predicted announcement on April 29th, 2006, on October 8th, 2006, daily test in October 27 in 2006 respectively.
Sequence number Technical performance index Checking result (Jiangxi) Checking result (Jiangsu) Checking result (Sichuan)
1 Preparation of product principle and using method Meet the concerned countries standard Meet GB/T17480-1998 Meet the concerned countries standard
2 Detection sensitivity ＜0.1ug/kg ＜0.1ug/kg ＜0.1ug/kg
3 Detection time 1.5 hour 1 hour 40 minutes 1.5 hour
4 Detect repeatability CV＝8.9％ CV＝7.4％ CV＝8.9％
5 Sensing range 0.1～10ug/kg 0.1～10ug/kg 0.1～10ug/kg
6 The term of validity 6 months (4 ℃) 6 months (4 ℃) 6 months (4 ℃)
7 Detection accuracy There is not the conspicuousness difference with TLC, HPLC method Through check, there is not the conspicuousness difference with the TLC method Through check, there is not the conspicuousness difference with the HPLC method, the TLC method is not tested

Claims (3)

  1. One kind accurately, the method for fast detecting aflatoxin, it is characterized in that its method is
    One. the standard sample preparation
    The first step, preparation aflatoxin AFB 1The antibody sandwich reaction plate:
    With aspergillus flavus resisting toxin A FB 1Polyclonal antibody 50mmol/L-100mmol/L, Na 2CO 3-NaHCO 3The PH9.6 damping fluid is diluted to the coating buffer of 3-5 μ g/ml, join in the test tube in 48 or 96 holes of reaction plate, every hole adds 100-110 μ L, 3 ℃ of-5 ℃ of refrigerators are placed and are spent the night, discard coating buffer flushing 2 times-4 times, add 100-110mmol/L, the sealing of PH7.4 phosphate PBS damping fluid that 200-250 μ L contains 1% bovine serum albumin(BSA) BSA in every hole and put for 35 ℃-40 ℃ and abandoned its confining liquid in 2-4 hour and will wrap by-15 ℃ of the vacuum-packed postposition of plate--25 ℃ of freezing preservations;
    Second step, the preparation enzyme-labelled antigen:
    Get 0.5～1mg AFB 1-ethyloic oxime AFB 1-omixe is dissolved in and added 20-50mg ethylenediamine 3.3-dimethylamine propyl carbodiimide hydrochloride EDCHCL stirring reaction in 20% ethanolic solution 30 minutes-60 minutes, with phosphate PBS 0.01M, the dialysis of PH7.4 solution 2 days-4 days, obtains enzyme-labelled antigen stoste again;
    The 3rd step, preparation aflatoxin AFB 1Standard solution
    With the phosphate PBS PH7.4 solution of 10%-20% methyl alcohol is the AFB of solvent preparation 0.1ng/ml-10ng/ml 1Standard serial solution;
    The 4th step, the preparation sample diluting liquid
    In phosphate PBS PH7.4 solution, add 10%-20% methyl alcohol;
    The 5th step, preparation enzyme-labelled antigen dilution
    The bovine serum albumin(BSA) BSA that in phosphate PBS PH7.4 solution, adds 0.05-0.2%;
    The 6th step, preparing washing liquid
    The Tween-20 TW-20 that in phosphate PBS PH7.4 solution, adds 0.03%-0.07%;
    The 7th step, preparation substrate solution a:
    At sodium acetate---add 0.1%-0.4% tetramethyl benzidine TMB among the citrate buffer solution PH=5.0;
    The 8th step, preparation substrate solution b:
    In sodium acetate-citrate buffer solution PH=5.0, add 0.01-0.03% hydrogen peroxide H 2O 2
    In the 9th step, the preparation stop buffer is a 2mol/L sulfuric acid liquid;
    In the tenth step, extract is a methyl alcohol: water is 1: 1;
    Two, detect
    The first step, liquid pre-treatment to be measured,
    Take by weighing sample that 5g pulverized 20 mesh sieves in the 50ml of ground test tube, add extract 20-30ml, the vibration 5 minutes-10 minutes of jumping a queue is filtered to be and is treated sample measuring liquid;
    In second step, reagent is prepared:
    Add 1-2ml enzyme-labelled antigen dilute solution in the enzyme-labelled antigen stoste of standard sample preparation second step preparation, fully dissolving is made into the enzyme-labelled antigen working solution;
    In the 3rd step, the test tube numbering: with the numbering of the test tube on the reaction plate framework, setting No. 1 test tube is the withered test tube of instrument according to the experiment needs, and the 2-7 test tube is aflatoxin AFB 1Standard control test tube, all the other test tubes are sample tube;
    In the 4th step, add the solution that configures successively and treat sample measuring liquid
    (1), in No. 1 test tube, adds 40-60 μ L sample diluting liquid, in the 2-7 test tube, add aflatoxin AFB respectively 1Standard solution 40-60 μ L adds respectively in all the other test tubes and treats sample measuring liquid;
    (2), add 40-60 μ L enzyme-labelled antigen dilution in No. 1 test tube, add 40-60 μ L enzyme-labelled antigen working solution in all the other test tubes respectively, vibration makes the reactant mixing in each test tube lightly;
    (3), then reaction plate being put into 35 ℃ of-40 ℃ of constant incubators hatched 20 minutes-40 minutes;
    (4), take out reaction plate, get rid of reactant liquor, pat dry, every test tube adds 200 μ L-300 μ L cleansing solutions, place 1 minute-3 minutes after, get rid of cleansing solution, on thieving paper, pat dry, repeat to wash plate 3-5 time;
    (5), in every test tube, add each 50 μ L of substrate solution a and substrate solution b respectively, shake up, reaction plate is put into 35 ℃ of-40 ℃ of constant incubators, developed the color 10 minutes-20 minutes;
    (6), take out reaction plate, in each test tube, add stop buffer 40-60 μ L respectively, measure the absorbance A value in each hole with the aflatoxin analyzer at 360mm-450mm wavelength place;
    (7),, set up equation A=MC+N regression Calculation in the aflatoxin analyzer automatically, calculate testing sample hole A value and A the absorbance A value that records 0ng/mlRatio, look into typical curve, can obtain the common logarithm value LgC of corresponding sample liquid concentration C to be measured, to its logarithm of negating, can try to achieve the AFB1 concentration C for the treatment of sample measuring liquid, calculate the content of AFB1 in the sample by following formula:
    Figure A20081003841900041
    In the formula: the AFB that C----checks in from typical curve 1Content (ng/ml)
    V----sample extracting solution volume (ml);
    D----diluted sample multiple;
    The quality of m----sample (g).
  2. 2. according to claim 1 a kind of accurate, the method of fast detecting aflatoxin, it is characterized in that, described aflatoxin analyzer comprises single-chip microcomputer and stabilized voltage supply, described single-chip microcomputer respectively with ROM (read-only memory), keyboard, display, random access memory, interface is connected with digital converter, digital converter is connected with analog to digital converter by prime amplifier, analog to digital converter is connected with printer with print drive by interface, stabilized voltage supply is connected with interference filter by light source, interference filter is connected with silicon photocell by sample cell, and silicon photocell is connected with prime amplifier.
  3. 3. according to claim 1 a kind of accurately, the method for fast detecting aflatoxin, it is characterized in that, be provided with catoptron (3), collector lens (4) He Guanglan (5) in the described sample cell, the continuous radiation light beam that stabilized voltage supply (1) is sent, behind optical filter (2), be incident upon reflective mirror (3), be incident upon on the collector lens (4) through reflective mirror (3), the reaction plate (6) in the directive sample cell is incident upon silicon photocell (7) at last then.
CNA2008100384190A 2008-06-02 2008-06-02 Method for accurately and rapidly checking aspergillus flavus toxin Pending CN101285834A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CNA2008100384190A CN101285834A (en) 2008-06-02 2008-06-02 Method for accurately and rapidly checking aspergillus flavus toxin

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNA2008100384190A CN101285834A (en) 2008-06-02 2008-06-02 Method for accurately and rapidly checking aspergillus flavus toxin

Publications (1)

Publication Number Publication Date
CN101285834A true CN101285834A (en) 2008-10-15

Family

ID=40058146

Family Applications (1)

Application Number Title Priority Date Filing Date
CNA2008100384190A Pending CN101285834A (en) 2008-06-02 2008-06-02 Method for accurately and rapidly checking aspergillus flavus toxin

Country Status (1)

Country Link
CN (1) CN101285834A (en)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101881771A (en) * 2010-05-15 2010-11-10 福建农林大学 Kit and method for detecting aflatoxin (AF)B1 of gene engineering single chain antibody
CN102331414A (en) * 2011-08-29 2012-01-25 西南大学 Aflatoxin B1 fluorescent sensitizer and applications thereof
CN102453686A (en) * 2010-10-27 2012-05-16 中国科学院植物研究所 Method for inhibiting generation of aflatoxin by using D-glucal
CN102818740A (en) * 2012-09-10 2012-12-12 广东省农业科学院作物研究所 Identification method of resistance to aspergillus flavus infection of peanuts and application thereof
CN103175874A (en) * 2013-02-19 2013-06-26 上海师范大学 Aflatoxin B1 detecting method
CN103808935A (en) * 2012-11-07 2014-05-21 江苏维赛科技生物发展有限公司 Preparation of ELISA kit for detecting ochratoxins
CN104569380A (en) * 2015-01-23 2015-04-29 天津伯克生物科技有限公司 Method for detecting aflatoxin B1 and enzyme-linked immunosorbent assay kit
CN104965075A (en) * 2015-07-16 2015-10-07 深圳德夏科技发展有限公司 Chemiluminiscence enzyme immunoassay fluorescence comprehensive detector
CN105806921A (en) * 2016-03-16 2016-07-27 济南大学 Preparation method and application of aflatoxin photoelectrochemical sensor without external light source
CN109776672A (en) * 2019-02-12 2019-05-21 国家食品安全风险评估中心 Aflatoxin B1 haptens and preparation method thereof, artificial antigen and its application

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101881771B (en) * 2010-05-15 2013-02-27 福建农林大学 Kit and method for detecting aflatoxin (AF)B1 of gene engineering single chain antibody
CN101881771A (en) * 2010-05-15 2010-11-10 福建农林大学 Kit and method for detecting aflatoxin (AF)B1 of gene engineering single chain antibody
CN102453686A (en) * 2010-10-27 2012-05-16 中国科学院植物研究所 Method for inhibiting generation of aflatoxin by using D-glucal
CN102453686B (en) * 2010-10-27 2013-12-18 中国科学院植物研究所 Method for inhibiting generation of aflatoxin by using D-glucal
CN102331414B (en) * 2011-08-29 2014-06-11 西南大学 Aflatoxin B1 fluorescent sensitizer and applications thereof
CN102331414A (en) * 2011-08-29 2012-01-25 西南大学 Aflatoxin B1 fluorescent sensitizer and applications thereof
CN102818740A (en) * 2012-09-10 2012-12-12 广东省农业科学院作物研究所 Identification method of resistance to aspergillus flavus infection of peanuts and application thereof
CN102818740B (en) * 2012-09-10 2014-06-25 广东省农业科学院作物研究所 Identification method of resistance to aspergillus flavus infection of peanuts and application thereof
CN103808935A (en) * 2012-11-07 2014-05-21 江苏维赛科技生物发展有限公司 Preparation of ELISA kit for detecting ochratoxins
CN103175874A (en) * 2013-02-19 2013-06-26 上海师范大学 Aflatoxin B1 detecting method
CN103175874B (en) * 2013-02-19 2014-12-10 上海师范大学 Aflatoxin B1 detecting method
CN104569380A (en) * 2015-01-23 2015-04-29 天津伯克生物科技有限公司 Method for detecting aflatoxin B1 and enzyme-linked immunosorbent assay kit
CN104965075A (en) * 2015-07-16 2015-10-07 深圳德夏科技发展有限公司 Chemiluminiscence enzyme immunoassay fluorescence comprehensive detector
CN105806921A (en) * 2016-03-16 2016-07-27 济南大学 Preparation method and application of aflatoxin photoelectrochemical sensor without external light source
CN105806921B (en) * 2016-03-16 2018-07-31 济南大学 A kind of preparation method and application of the aflatoxin optical electro-chemistry sensor of no peripheral hardware light source
CN109776672A (en) * 2019-02-12 2019-05-21 国家食品安全风险评估中心 Aflatoxin B1 haptens and preparation method thereof, artificial antigen and its application

Similar Documents

Publication Publication Date Title
CN101285834A (en) Method for accurately and rapidly checking aspergillus flavus toxin
CN102955031B (en) Enzyme-linked immunosorbent assay kit for detecting aflatoxin B1-containing medicine and application for same
Bueno et al. Determination of mycotoxins in food: A review of bioanalytical to analytical methods
CN101451999B (en) Directly competitive ELISA kit for detecting implicit malachite green
CN103018458B (en) Ultra-sensitive aflatoxin B1 enzyme-linked immunosorbent assay kit
CN104655846A (en) Enzyme linked immunosorbent assay kit for detecting progesterone and detection method thereof
Guo et al. Highly sensitive and simultaneous detection of melamine and aflatoxin M1 in milk products by multiplexed planar waveguide fluorescence immunosensor (MPWFI)
CN102967709B (en) Detect enzyme linked immunological kit and the application thereof of zearalenone medicine
CN103808934A (en) Preparation of ELISA kit for detecting deoxynivalenol
CN103439503B (en) The enzyme linked immunological kit of Sparfloxacin and establishment thereof and detection method
CN108169497A (en) Human prolactin detection kit and preparation method and application
CN107271682A (en) A kind of dog c reactive protein fluorescence detection test strip
CN102621297A (en) Fluorescence polarization immunoassay detection method for sarafloxacin
Di Giovanni et al. Sweet sensor for the detection of aflatoxin M1 in whole milk
CN101424686A (en) ELISA reagent for detecting malachite green and method
CN103288872A (en) Methyl parathion hapten, and preparation method and application thereof
Zachariasova et al. Cross-reactivity of rapid immunochemical methods for mycotoxins detection towards metabolites and masked mycotoxins: the current state of knowledge
CN103308685B (en) Nonylphenol polyoxyethylene ether detection kit, and preparation and using methods thereof
CN105334323A (en) Method and test strip for detecting zilpaterol, and application of test strip
CN102033130A (en) Enzyme-linked immunological detection kit and method for detecting T-2 toxin in samples
CN103288661A (en) Preparation method and application of malachite green hapten
CN101162230A (en) Kit for quantitative determining enrofloxacin content in food product and testing method thereof
CN102943066A (en) Human apolipoprotein B100 (ApoB100) monoclonal antibody and chemiluminescence immune assay determination kit adopting the human ApoB100 monoclonal antibody
CN103102319A (en) Melamine semiantigen, preparation method and application thereof
CN110045108B (en) Capsaicin index-based illegal cooking oil enzyme-linked immunosorbent assay kit and detection method thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C02 Deemed withdrawal of patent application after publication (patent law 2001)
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20081015