CN110221062A - A kind of aquatic products Malachite Green rapid detection method - Google Patents
A kind of aquatic products Malachite Green rapid detection method Download PDFInfo
- Publication number
- CN110221062A CN110221062A CN201910591797.XA CN201910591797A CN110221062A CN 110221062 A CN110221062 A CN 110221062A CN 201910591797 A CN201910591797 A CN 201910591797A CN 110221062 A CN110221062 A CN 110221062A
- Authority
- CN
- China
- Prior art keywords
- malachite green
- aquatic products
- line
- acetonitrile
- detection method
- 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
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/405—Concentrating samples by adsorption or absorption
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
A kind of aquatic products Malachite Green rapid detection method, after fish and shrimp aquatic products homogeneous, organic reagent is extracted, and is handled with extraction column, harvests eluent, the detection of immunofluorescence card.The higher specificity of extracting method and fluorescence detection card of this detection method by extraction column quickly, easy, sensitivity combine, and provide effective guarantee for food safety.
Description
Technical field
This patent disclosure relates generally to field of detection of food safety, and in particular to a kind of quick side of detection of aquatic products Malachite Green
Method.
Background technique
Malachite green is a kind of green crystal with metallic luster also known as Viride Nitens, tight matrix be green, peacock green, very
It is early to be once used for culture fishery as fungicide, insecticide, disinfectant.For a long time, fisherman prevents the water of fish with it
Mildew, branchiomycosis, ich etc., and the fish extending life in order to keep squama impaired, during transportation and in storage pond
It is often used malachite green.But malachite green and its residues concealed malachite green has potential " three cause " effect, it is strong to the mankind
Health causes damages with environment.In view of the harmfulness of malachite green, malachite green is all classified as aquaculture disabling medicine by many countries
Object (except ornamental fish).Malachite green is also included in that " veterinary drug and its compound of food animal disabling are clear by China in May, 2002
It is single " in, forbid for all food animals.However, due to malachite green sterilization, favorable anti-corrosion effect, and it is cheap, so one
The case where a little producers are driven the Misuse in aquaculture, transport and storage by interests still remains.
Currently, the method for detection malachite green mainly has high performance liquid chromatography, thin-layered chromatography, spectrophotometry, drawing
Graceful spectroscopic methodology, immunological method.Wherein, although instrument confirmation method testing result is accurate, high sensitivity, its detection time
Instrument and equipment that is long, needing valuableness, the quick detection of unsuitable great amount of samples more demanding to operator.And existing enzyme
Although linked immunosorbent adsorption test and colloidal gold rapid detection method detection time improve more, pre-treating method compared with instrumental method
Still cumbersome or detection sensitivity is poor.It is then desired to a kind of novel aquatic products Malachite Green rapid detection method.
Summary of the invention
Existing malachite green fastly in inspection technology, extracted using a variety of salt ions and organic reagent, be net by Sample pretreatment method
Change, volatilization, redissolve, when operating cost, laborious, and the matched colloidal gold Fast Detection Technique sensitivity of the pre-treating method and immune
Detection technique of fluorescence is compared, and sensitivity is low.In order to overcome the shortcomings of existing fast inspection technology, the purpose of the present invention is to provide one kind
Simple, quick, specific good, high sensitivity the aquatic products Malachite Green rapid detection method of Sample pretreatment.
In order to achieve the above object, the present invention uses following technical scheme.
A kind of aquatic products Malachite Green rapid detection method, after fish and shrimp aquatic products homogeneous, organic reagent is extracted, with extraction
It takes column to handle, harvests eluent, the detection of immunofluorescence card.
Specifically includes the following steps:
1) fish and shrimp aquatic products sample is added saturated sodium-chloride, hydroxylamine hydrochloride and acetate, mixes to centrifuge tube after weighing homogeneous
It is even；It is acutely shaken after acetonitrile is added, adds neutral alumina, shaken；Centrifugation takes supernatant stand-by in centrifuge tube；
2) activate pillar: acetonitrile ammonium acetate mixed liquor 1 adds to extraction column；
3) it takes supernatant in step 1) that oxidant is added, mixes, extraction column is added immediately；
4) pillar is eluted with ammonium acetate, is then eluted with acetonitrile ammonium acetate mixed liquor 2, collect eluent；It is mixed to add PBS
Even is prepare liquid；
5) prepare liquid is added into immunofluorescence card well, reaction is placed on interpretation result on fluorescence chart scanner；Work as detection
Under exciting light irradiation, nature controlling line (C line) and detection line (T line) show fluorescent orange simultaneously in area, and T line ratio C line depth or
Person's colour developing is consistent, shows testing result for feminine gender；The T line ratio C line is shallow or does not show fluorescence, shows testing result for the positive.
The preparation method of the immunofluorescence card, comprising the following steps: preparation sample pad --- preparing bonding pad --- system
Standby reaction film --- preparing water absorption pad --- assembling detection card.Wherein, the malachite green of fluorescent microsphere label is applied on bonding pad
Antibody sprays malachite green conjugate antigenic solution on reaction film, is formed detection line (T line), is sprayed on the reaction film
It identifies the two corresponding anti-solution of malachite green antibody, is formed nature controlling line (C line).
In the present invention, sample to be tested is extracted first with organic reagent maximal efficiency, then utilizes strong cation exchange
It extracts column purification, determinand in sample is concentrated, while eluting pillar with salt ion liquid before eluting column, be eliminated as much as residual in column
The organic reagent stayed avoids organic reagent from blocking colour developing to detection and generates adverse effect, and prepare liquid can also be carried out quickly after eluting column
Detection.Sample pretreatment process uses strong cation exchange extraction column, and surface is seen similar with instrument detection pre-treating method but real
It is significantly innovated on border.Instrument pre-treatment extracts concentration, but extracting solution in the present invention using a variety of organic reagents
Without being concentrated, purifying.Meanwhile detection card colour developing is influenced in order to guarantee the organic reagent in eluting liquid not, it is living using mixed liquor
Change pillar, then reaches better extract effect using salt ion and organic reagent mixed liquor elution pillar.
Compared with prior art, the beneficial effects of the present invention are:
1, the organic reagent that the sub- extraction column of strong cat ion exchange column replaces tradition cumbersome volatilizees, redissolves process, does not need
Operation can be completed in instrument and equipment.
2, it after the elution of sample Malachite Green combination extraction column, does not need organic on consumption long period volatilization extraction column
Reagent directly elutes.
3, Immunofluorescence test card replaces immune colloid gold detection card, substantially increases the sensitivity of detection.With extraction column
Advantage carry out complementary, inventing a kind of simple pre-treatment, high sensitivity, specificity, good aquatic products Malachite Green quickly detects
Method.
Detailed description of the invention
Fig. 1 is the side view of specific embodiment Malachite Green fluorescence detection test strip.
Fig. 2 is the top view of specific embodiment Malachite Green fluorescence detection test strip.
Wherein, each label respectively represents: 1. sample pad, 2. bonding pad, 3. reaction film, 4. detection line, 5. nature controlling line 6. is inhaled
Water cushion .7. bottom plate.
Specific embodiment
Below by a kind of aquatic products Malachite Green rapid detection method of specific embodiment detailed description of the present invention.This
Field is it will be appreciated by the skilled person that the embodiments described below is only to make to exemplary illustration of the invention not for it
Any restrictions out.
1, malachite green extracts
After the aquatic products such as 1.1 fishes and shrimps remove the peel (shell), meat 20-30g or so is taken, after homogenizer homogeneous, weighs 3g to 50ml
In centrifuge tube.0.5ml saturated sodium-chloride, 0.1ml hydroxylamine hydrochloride and 1ml acetate is added, mixes 1min.After 4ml acetonitrile is added
Acutely concussion 3min adds 1g neutral alumina, shakes 1min.4000rpm or more is centrifuged 5min, take 3ml supernatant in
It is stand-by in 10ml centrifuge tube.
Saturated sodium-chloride: weighing sodium chloride 200g, adds water 500ml, and ultrasound dissolves it sufficiently.
Hydroxylamine hydrochloride: weighing 2.5g hydroxylamine hydrochloride, and 10ml is dissolved and be diluted to water, mixes.
Acetate: weighing 4.95g anhydrous sodium acetate and the p- toluenesulfonic acid of 0.95g is dissolved in 950ml water, uses glacial acetic acid
Adjusting pH value of solution is 4.5, is diluted with water to 1L, is mixed.
1.2 cross column
1.2.1 the selection of extraction column
PRS solid-phase extraction column is selected, producer: German SimonAldrich, specification: quality 100mg, volume 1ml, average grain
45 μm of diameter, filler title: propyl sulfonic acid, using silica gel as the strong cation exchange extraction column of matrix.The extraction column combination malachite
Green ability is stronger, is very suitable to the extraction of malachite green.
1.2.2 activation pillar condition is determined
1.2.2.1 determining optimal activation solution
Choose single solution activation respectively: concentration is 0.01M PBST, acetonitrile and 7.2 concentration of pH are 0.1M ammonium acetate
1000 μ l activate pillar；It chooses acetonitrile and concentration and activates pillar for the 1000 μ l of ammonium acetate mixed liquor of 0.1M, acetonitrile and concentration are
The ammonium acetate volume ratio of 0.1M is respectively 0:1,1:1,1:2,1:3,3:1 and 2:1.Determine pillar optimal activation solution.
1.2.2.2 determining activated solution volume.250 μ l, 500 μ l, 1000 μ l and 1500 μ are set separately in optimal activation solution
l.Liquid, which is added dropwise to detect, before and after being activated by detection blocks upper negative findings colour developing situation and activation time, final determining 500 μ l pH
7.2 0.1M ammonium acetates and acetonitrile equal proportion activation effect are best.
1.2.3 determined column flow rate of liquid: take 1ml concentration be 1ppm malachite green liquid, flow velocity by 0.5 drop/2s, 1 drop/
2s, 2 drops/2s, 3 drops/2s cross column, and as a result it is preferable to cross column effect by 1 drop/2s.
1.2.4 determine elution process: taking 1ml concentration is 1ppm malachite green liquid, crosses column by 1 drop/2s speed rapidly,
Then with different volumes (250 μ l, 500 μ l, 1000 μ l and 1500 μ l) pH7.2 0.1M ammonium acetate elution pillar or without
Elution directly elutes, and as a result 500 μ l pH7.2 0.1M ammonium acetates elution pillar effect is best, green malachite green elution on column
Most completely.
1.2.5 effluent volume is determined: respectively with 500 μ l pH of different volumes ratio (1:0,1:1,1:2,2:1,3:1)
7.0 0.1M ammonium acetates and acetonitrile mixture elution, as a result 500 μ l pH, 7.2 0.1M ammonium acetate and acetonitrile press 2:1 volume ratio
Mixing elution effect is best.
1.2.6 after pillar is by optimum way activation, supernatant is obtained from 1.1, and 100 μ l oxidants are added and mix, immediately
Column is crossed by 1 drop/2s speed, then elution removes organic reagent, 500 μ l pH, 7.2 0.1M ammonium acetate and acetonitrile in an optimal manner
It mixes and elutes by 2:1 volume ratio.
The detection of 1.3 immunofluorescence cards
1.3.1 the preparation method of fluorescence detection card, comprising the following steps:
(1) sample pad 1 is prepared, the glass fibre element film of 1.2cm × 18cm is cut, it is uniformly soaked in confining liquid
20min, vacuum drying, it is spare to obtain sample pad 1；
(2) it prepares bonding pad 2: cutting the glass fibre membrane of 0.6cm × 15cm, take the peacock of suitable fluorescent microsphere label
Malachite green antibody 0.8ml is coated on glass fibre membrane, and 37 DEG C of dry 2h obtain bonding pad 2；
(3) it prepares reaction film 3: taking the reaction film 3 of wide 20mm to spray malachite green conjugate antigenic solution, form detection line
4, the two corresponding anti-solution of spraying identification malachite green antibody, forms nature controlling line 5 on the reaction film 3；
(4) it prepares water absorption pad 6: cutting the water absorption pad 6 of 1.4cm × 18cm；
(5) assembling detection card: the 60mm bottom plate 7 bought in the market is taken, the sample pad 1, institute are successively pasted on bottom plate 7
State bonding pad 2, the reaction film 3 and the water absorption pad 6；
(6) be loaded: big plate being cut into 4mm wide fluorescence detection test strip and is put into card bottom, covers Ka Gai, detection card.
1.3.2 prepare liquid sample-adding detection: eluent is diluted with pH7.2 0.01M PBS by 1:3 volume ratio as to be measured
Liquid.Prepare liquid is added into immunofluorescence card well, reaction 10min is placed on interpretation result on fluorescence chart scanner.Work as detection zone
Under exciting light irradiation, nature controlling line (C line) and detection line (T line) show fluorescent orange simultaneously, and T line ratio C line depth or
Colour developing is consistent, shows testing result for feminine gender；The T line ratio C line is shallow or does not show fluorescence, shows testing result for the positive.
1.4 detection limits determine
By adding the malachite green standard items of the μ g/kg of final concentration of 0,0.5,1,2,3 and 4 respectively in negative sample,
It is mark-on sample that mixing, which fullys shake,.Then sample extraction is carried out according to 1.1,1.2 steps, is then examined according to 1.3.2 step
Test sample sheet, malachite green are determined as positive findings in 2 μ g/kg and concentrations above.Therefore, this fast detecting method detection is limited to 2 μ
g/kg。
Claims (6)
1. a kind of aquatic products Malachite Green rapid detection method, which is characterized in that after fish and shrimp aquatic products homogeneous, organic reagent
It extracts, is handled with extraction column, harvest eluent, the detection of immunofluorescence card.
2. aquatic products Malachite Green rapid detection method according to claim 1, specifically includes the following steps:
1) fish and shrimp aquatic products sample is added saturated sodium-chloride, hydroxylamine hydrochloride and acetate, mixes to centrifuge tube after weighing homogeneous；
It is acutely shaken after acetonitrile is added, adds neutral alumina, shaken；Centrifugation takes supernatant stand-by in centrifuge tube；
2) activate pillar: acetonitrile ammonium acetate mixed liquor 1 adds to extraction column；
3) it takes supernatant in step 1) that oxidant is added, mixes, extraction column is added immediately；
4) pillar is eluted with ammonium acetate, is then eluted with acetonitrile ammonium acetate mixed liquor 2, collect eluent；PBS is added to mix i.e.
For prepare liquid；
5) prepare liquid is added into immunofluorescence card well, reaction is placed on interpretation result on fluorescence chart scanner；When detection zone exists
Under exciting light irradiation, nature controlling line (C line) and detection line (T line) show fluorescent orange simultaneously, and the T line ratio C line is deep or aobvious
Color is consistent, shows testing result for feminine gender；The T line ratio C line is shallow or does not show fluorescence, shows testing result for the positive.
3. aquatic products Malachite Green rapid detection method according to claim 1, it is characterised in that: this method detection limit
For 2 μ g/kg.
4. aquatic products Malachite Green rapid detection method according to claim 1, it is characterised in that: the immunofluorescence
Card sensitivity is 0.1 μ g/kg.
5. aquatic products Malachite Green rapid detection method according to claim 2, which is characterized in that acetonitrile ammonium acetate is mixed
Closing liquid 1 is 7.2 0.1M ammonium acetate of pH and the mixing of acetonitrile equal proportion.
6. aquatic products Malachite Green rapid detection method according to claim 2, it is characterised in that: acetonitrile ammonium acetate is mixed
Closing liquid 2 is that 7.2 0.1M ammonium acetate of pH and acetonitrile are mixed in 2:1 ratio.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910591797.XA CN110221062A (en) | 2019-07-01 | 2019-07-01 | A kind of aquatic products Malachite Green rapid detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910591797.XA CN110221062A (en) | 2019-07-01 | 2019-07-01 | A kind of aquatic products Malachite Green rapid detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110221062A true CN110221062A (en) | 2019-09-10 |
Family
ID=67815595
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910591797.XA Pending CN110221062A (en) | 2019-07-01 | 2019-07-01 | A kind of aquatic products Malachite Green rapid detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110221062A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1767947A1 (en) * | 2005-09-22 | 2007-03-28 | Randox Laboratories Ltd. | An immunoassay method and kit to leucomalachite green and malachite green |
| US20070254323A1 (en) * | 2006-04-21 | 2007-11-01 | Jun Wang | Malachite green derivatives for immunoassay reagents to detect malachite green |
| CN102353775A (en) * | 2011-06-13 | 2012-02-15 | 清华大学深圳研究生院 | Immunochromatographic test strip for detecting malachite green (MG) and preparation process thereof |
| CN107255690A (en) * | 2017-07-06 | 2017-10-17 | 佛山科学技术学院 | A kind of method of liquid chromatogram measuring malachite green |
| CN108152493A (en) * | 2017-12-19 | 2018-06-12 | 广州安诺科技股份有限公司 | The method for detecting aquatic products Malachite Green |
-
2019
- 2019-07-01 CN CN201910591797.XA patent/CN110221062A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1767947A1 (en) * | 2005-09-22 | 2007-03-28 | Randox Laboratories Ltd. | An immunoassay method and kit to leucomalachite green and malachite green |
| US20070254323A1 (en) * | 2006-04-21 | 2007-11-01 | Jun Wang | Malachite green derivatives for immunoassay reagents to detect malachite green |
| CN102353775A (en) * | 2011-06-13 | 2012-02-15 | 清华大学深圳研究生院 | Immunochromatographic test strip for detecting malachite green (MG) and preparation process thereof |
| CN107255690A (en) * | 2017-07-06 | 2017-10-17 | 佛山科学技术学院 | A kind of method of liquid chromatogram measuring malachite green |
| CN108152493A (en) * | 2017-12-19 | 2018-06-12 | 广州安诺科技股份有限公司 | The method for detecting aquatic products Malachite Green |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101930006B (en) | High-sensitivity immunochromatographic test strip for detecting total aflatoxin content quickly and preparation method thereof | |
| CN103048455B (en) | Herbicide 2, 4-D and pesticide CHI bigeminy detection card and preparation method thereof | |
| US7759128B2 (en) | Histamine detection method and histamine detection kit | |
| CN105203535B (en) | A kind of method of paper substrate visualization molecular imprinting biosensor detection pesticide residue | |
| CN101539578A (en) | Colloidal gold test strip for testing melamine content | |
| CN102680714B (en) | Colloidal gold immune test paper for rapid detection of dibutyl phthalate as well as preparation method and application thereof | |
| CN1509409A (en) | In-line test device and methods of use | |
| CN102507564A (en) | Kit for quickly detecting nitrite in edible bird's nest and application of kit in edible bird's nest detection | |
| CN106153770A (en) | A kind of Solid-Phase Extraction liquid chromatography mass detection method of aquatic products glyphosate | |
| CN108700583B (en) | Device for detecting neurotoxin and method for manufacturing same | |
| CN101915841B (en) | Ternary detection test bar of beta-stimulants albuterol, clenobuterol hydrochloride and ractopamine and preparation method thereof | |
| CN105675874A (en) | Colloidal gold test strip for detection of imidacloprid and application thereof | |
| CN103499690A (en) | Olaquindox metabolite immunochromatography test paper card and preparation method thereof | |
| CN104975107A (en) | Method for detecting contaminants in water employing mussel | |
| CN107101999A (en) | The method that sxemiquantitative quickly recognizes THC content in cannabis plants | |
| CN110221062A (en) | A kind of aquatic products Malachite Green rapid detection method | |
| ZHANG et al. | A portable photoelectric sensor based on colloidal gold immunochromatographic strips for rapid determination of clenbuterol in pig urine | |
| CN106896094B (en) | Method for simultaneously detecting CLE, RAC and SBL and special paper chip thereof | |
| CN102590488A (en) | Method for regulating and controlling vitellogenin level of zebra fishes | |
| CN104897908A (en) | Colloidal gold immunochromatographic test strip for detecting clenobuterol hydrochloride colloidal gold and preparation method of test strip | |
| CN108663513A (en) | A method of reducing Sidestream chromatography test paper detection limit | |
| CN200982971Y (en) | Penicillin quick half quantitative detection test paper | |
| CN103713133A (en) | Test strip for detecting spiramycin, streptomycin, gentamycin and neomycin, and method | |
| CN1232825C (en) | Reference immuno chromatographic test paper for detecting clenbuterol hydrochloride and detection method thereof | |
| Bellon‐Humbert et al. | Localization of serotonin‐like immunoreactivity in the eyestalk of the prawn Palaemon serratus (Crustacea, Decapoda, Natantia) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190910 |
|
| RJ01 | Rejection of invention patent application after publication |