CN103134923A

CN103134923A - Aflatoxin M1 detection kit

Info

Publication number: CN103134923A
Application number: CN2011103812835A
Authority: CN
Inventors: 赵春城; 赵黎明; 王晓东; 单金莲; 钱汝文
Original assignee: Wuxi Xresearch Product Design and Research Co Ltd
Current assignee: Wuxi Xresearch Product Design and Research Co Ltd
Priority date: 2011-11-26
Filing date: 2011-11-26
Publication date: 2013-06-05

Abstract

The invention relates to an aflatoxin M1 detection kit, and belongs to the technical field of enzyme-linked immunosorbent assay (ELISA), wherein the aflatoxin M1 detection kit is used for detecting aflatoxin M1 (short for AFM1) content in milk products. The aflatoxin M1 detection kit comprises a box body, a 24-cell AFM1 coated reaction plate inside the box, a reaction cover plate, 12 bottles of reagents, concave bottle positions for placing the reagents, an ice bag, a frame, an instruction manual, a packet of water absorption paper, two disposable droppers and a quality detection report. The aflatoxin M1 detection kit is characterized in that the coated reaction plate adopts a 24-well reagent plate as a solid phase carrier, and the 12 bottles of the reagents comprise 6 bottles of AFM1 standard substance solutions, enzyme labeled antigen, an enzyme labeled antigen dilution buffer solution, a concentration washing solution, a substrate solution, a coloration solution and a termination solution, and the number of the concave bottle positions is 16. The kit has characteristics of simple structure, easy use, low price, and high sensitivity, wherein the sensitivity can be more than 0.l ng/ml.

Description

A kind of detection aflatoxin M 1Kit

Technical field

A kind of detection aflatoxin M ₁Kit, belong to Enzyme Linked Immunoadsorbent Assay (ELISA) technical field, be used for the dairy products aflatoxin M ₁(be called for short AFM ₁) detection of content.

Background technology

Aflatoxin (AFM ₁) be the secondary metabolite of one group of strong Ai of fungi aspergillus flavus Hspergillus flavus and the generation of aspergillus parasiticus A.parasilicus bacterial strain, aflatoxin (AFT) has found that at present more than 20 plant, and wherein aflatoxin B1 is toxicity and the strongest material of carcinogenicity.Aflatoxin M l is the hydroxylation metabolism product of aflatoxin B1, is also a kind of strong carcinogen.When mammal is taken in AFB ₁After the feed that pollutes, in vivo can be with AFM through hydroxylation ₁Secrete in the middle of milk.Milk cow is through taking in AFB ₁Just contain aflatoxin M in the milk of output after the feed that pollutes ₁Due to aflatoxin M ₁Quite stable, pasteurization also can't be killed it, so detect aflatoxin M ₁Not only the animal feed raw material to be detected, and final products will be detected.Aflatoxin M in cow's milk and goods thereof has all been stipulated in countries in the world for this reason ₁Maximum allow content and with it as compulsory standard, as aflatoxin M in Chinese Government's regulation cow's milk and goods (sterilized milk, fresh raw milk, full-fat cow milk, evaporated milk, condensed milk, cream) thereof ₁The highest permission content be 0. 5 μ g/ml.

Determination Technology of Aft commonly used in prior art mainly contains thin layer chromatography (TLC), high performance liquid chromatography (HPLC), immune analysis method.The above two have the sample pre-treatments complex steps, time and effort consuming, instrument is expensive, the shortcoming that needs the professional to operate etc., and the immune analysis method has high specificity, sample pre-treatments is simple, cost is low, to the advantage such as the contamination hazard of experimenter and environment is little.The most frequently used immune analysis method has enzyme linked immunosorbent assay, nm of gold immunochromatographic method etc.

Summary of the invention

The object of the present invention is to provide a kind of detection AFM ₁Kit, be used for dairy products AFM ₁The detection of content.

The technical solution adopted for the present invention to solve the technical problems is: a kind of detection aflatoxin M ₁Kit, comprise 24 hole AFM in box body and box ₁Coated reaction plate, a reaction cover plate, 12 bottles of reagent and the recessed bottle position of putting reagent, an ice bag, a framework, a instructions, one bag thieving paper, 2 disposable droppers and a quality inspection report is characterized in that: coated reaction plate is to adopt 24 hole agent plate as solid phase carrier, 6 bottles of AFM of 12 bottles of reagent difference ₁Standard solution, enzyme-labelled antigen, enzyme-labelled antigen dilution buffer liquid, concentrated cleaning solution, substrate solution, nitrite ion, stop buffer, recessed bottle totally 16.

Box body is carton box; The AFM in 24 holes ₁Coated reaction plate is put in the vacuum aluminium foil bag; Framework is white 48 hole plastic frames, frame aperture size and AFM ₁Coated reaction plate micropore size matches; The reaction cover plate is the plastics hardcoats, and size matches with frame size; AFM ₁Standard solution is all used the Brown Glass Brown glass bottles and jars only of black caps, enzyme-labelled antigen is with the Brown Glass Brown glass bottles and jars only of white cap, enzyme-labelled antigen dilution buffer liquid is with the white PE plastic bottle of green cap, concentrated cleaning solution is with the white PE plastic bottle of white cap, substrate solution is with the white PE plastic bottle of blue cap, nitrite ion is with the black PE plastic bottle of black caps, and stop buffer is with the white PE plastic bottle of yellow cap; Recessed bottle position is made by plastic foam.

The invention has the beneficial effects as follows: this kit is simple in structure, and is easy to use, cheap, highly sensitive, more than can reaching 0.lng/ml.

Description of drawings

Fig. 1 is schematic appearance of the present invention.

Embodiment

Below in conjunction with accompanying drawing, further illustrate the specific embodiment of the present invention:

First sample is processed:

Milk

Fat-contg milk is cooled to below 10 ℃, 3500 turn/min under centrifugal 10min, discard the upper strata fat deposit with suction pipe, lower floor's milk liquid is treats sample measuring liquid.(skim milk is directly measured).

Milk powder

Take 5g milk powder in the 100ml triangular flask, add 40ml distilled water, jolting 5min makes it dissolve (skim milk make milk powder curing ratio be that every 40ml skim milk is made 5 gram milk powder) fully.Following steps are with above-mentioned milk treatment process.

Preparatory work of experiment

1, concentrated cleaning solution with 20 times of distilled water dilutings, dilutes before using.(for example: 3ml concentrated cleaning solution+57ml distilled water, use in enough 24 holes)

2, AFM ₁Enzyme-labelled antigen working solution compound method: before use, get 1 bottle of AFM ₁Enzyme-labelled antigen accurately adds 1.5ml enzyme-labelled antigen dilution, and mixing is mixed with experiment AFM ₁The enzyme-labelled antigen working solution, enough 24 hole reaction plates use.

Experimental procedure

1, reagent balance: testing cassete is taken out, and more than placing 15min, balance is to room temperature.

2, aperture numbering: pipette on required micropore placing response board mount, every hole adds 250 μ l cleansing solutions, and washing lotion must not be overflowed, and places 1 minute, gets rid of washing lotion, pats dry on thieving paper, washs altogether 2 times.Setting No. 1 hole is microplate reader zeroing hole, and No. 2-7 is AFM ₁The standard solution control wells, all the other are sample well.

3, immune response: by shown in series of steps, add successively the solution and the sample liquid that prepare.

The first step: No. 1 the hole adds 50 μ l AFM ₁Standard solution 0ppb, the 2-7 hole adds respectively the AFM of 50 μ l series concentration (0,0.1,0.25,0.5,1,2 ppb) ₁Standard solution, all the other holes add corresponding sample extracting solution.

Second step: add 50 μ l enzyme-labelled antigen dilutions in No. 1 hole, add 50 μ l AFM in other all micropores ₁The enzyme-labelled antigen working solution.

The 3rd step: jolting gently makes reactant mixing in each hole.

The 4th step: 37 ℃, hatch 30min.

Washing: outwell liquid in plate, every hole adds 250 μ l cleansing solutions, and washing lotion must not be overflowed, and places after 1 minute, gets rid of washing lotion, pats dry on thieving paper, washs altogether 5 times.

Colour developing: add 50 μ l substrate solutions in each aperture, then add 50 μ l nitrite ions, mixing, 15min develops the color in 37 ℃ of insulation cans.Stop: add 50 μ l stop buffers in each micropore.Measure: shake gently microwell plate, the inherent 450nm of 30min reads optical density value (OD value) in the place.

Should be noted that at last: above embodiment is only in order to illustrate that technical scheme of the present invention is not intended to limit; Although with reference to preferred embodiment, the present invention is had been described in detail, those of ordinary skill in the field are to be understood that: still can modify or the part technical characterictic is equal to replacement the specific embodiment of the present invention; And not breaking away from the spirit of technical solution of the present invention, it all should be encompassed in the middle of the technical scheme scope that the present invention asks for protection.

Claims

1. one kind is detected aflatoxin M ₁Kit, comprise 24 hole AFM in box body and box ₁Coated reaction plate, a reaction cover plate, 12 bottles of reagent and the recessed bottle position of putting reagent, an ice bag, a framework, a instructions, one bag thieving paper, 2 disposable droppers and a quality inspection report is characterized in that: coated reaction plate is to adopt 24 hole agent plate as solid phase carrier, 6 bottles of AFM of 12 bottles of reagent difference ₁Standard solution, enzyme-labelled antigen, enzyme-labelled antigen dilution buffer liquid, concentrated cleaning solution, substrate solution, nitrite ion, stop buffer, recessed bottle totally 16.

2. a kind of detection aflatoxin M according to claim 1 ₁Kit, it is characterized in that: box body is carton box; The AFM in 24 holes ₁Coated reaction plate is put in the vacuum aluminium foil bag; Framework is white 48 hole plastic frames, frame aperture size and AFM ₁Coated reaction plate micropore size matches; The reaction cover plate is the plastics hardcoats, and size matches with frame size; AFM ₁Standard solution is all used the Brown Glass Brown glass bottles and jars only of black caps, enzyme-labelled antigen is with the Brown Glass Brown glass bottles and jars only of white cap, enzyme-labelled antigen dilution buffer liquid is with the white PE plastic bottle of green cap, concentrated cleaning solution is with the white PE plastic bottle of white cap, substrate solution is with the white PE plastic bottle of blue cap, nitrite ion is with the black PE plastic bottle of black caps, and stop buffer is with the white PE plastic bottle of yellow cap; Recessed bottle position is made by plastic foam.