CN107037216A - The method of lincomycin in immunochromatographic measurement milk is quenched based on background fluorescence - Google Patents
The method of lincomycin in immunochromatographic measurement milk is quenched based on background fluorescence Download PDFInfo
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- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
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- 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
It is a kind of method that lincomycin in immunochromatographic measurement milk is quenched based on background fluorescence the present invention relates to milk detection technique field；This is quenched the method for lincomycin in immunochromatographic measurement milk based on background fluorescence, carries out in the steps below：The first step, inputs two-dimentional code system code device by lincomycin calibration curve equation and title, lincomycin Quick Response Code is made, then lincomycin Quick Response Code is pasted onto the Quick Response Code location for paste in lincomycin test strips, obtains lincomycin detection card；Second step, takes the μ L of detected sample 200 to being fixed with the small test glass of gold labeling antibody.The detection that the present invention determines lincomycin in milk is limited to 0.088 μ g/kg, and optimal sample-adding amount is 100 μ L, and the optimal chromatography time is 10min；Sensitivity than existing methods of the invention is high, the reaction time is short, easy to operate, and the live Quantitative detection of lincomycin in milk can be achieved, is laid a good foundation to be widely used in field of food safety.
Description
Technical field
It is a kind of be based in background fluorescence quenching-immunochromatographic measurement milk the present invention relates to milk detection technique field
The method of lincomycin.
Background technology
Lincomycin (Linconycin, LIN) belongs to lincosamide antibiotics, has stronger antibacterial for gram-positive bacteria
Effect, also there is bacteriostasis to mycoplasma, but invalid to Gram-negative bacteria.Clinically it is mainly used in staphylococcus, suppuration
Respiratory tract infection, skin soft-tissue infection, female genital tract and pelvic infection caused by property streptococcus, pneumococcus and anaerobic bacteria
With the abdominal cavity infection caused by anaerobic bacteria etc..It is the septicemia, bone and the infection of joint that can be additionally used in caused by streptococcus and staphylococcus, slow
Acute hematogenous osteomyelitis caused by the surgery auxiliary treatment of property bone and the infection of joint, staphylococcus etc..It is used as veterinary drug, Chang Beitian
It is added in feed or directly injection of breast treats the mammitis of milk cow, but the lincomycin remained in lactiferous ducts, can be through milk
Discharge, causes fresh milk antibiotic residue, allergic reaction or the resistance to the action of a drug of body is caused to consumer.Each state is all limited to its
Amount requires that it is 100ng/mL that The Ministry of Agriculture of the People's Republic of China, MOA, which announces MRL in No. 253-2002 milk,.
Imported and exported animals derived food SNT2218-2008 middle forests can sulfonamides residues quantity measuring method middle forest can be mould
The detection method of element is liquid chromatogram-string mass spectrography and mass spectrography.In National Standard of the People's Republic of China GB/T4789.27-
The method of inspection that lincomycin is remained in 2008 regulation fresh milks suppresses method for streptococcus thermophilus and bacillus stearothermophilus suppresses
Method.Document report has enzyme linked immunosorbent assay (ELISA), high performance liquid chromatography, colloidal gold immunity chromatography etc..Thermophilus
Bacterium suppresses method and bacillus stearothermophilus suppresses method and tests cheap, but sample pretreatment process is cumbersome, poor reproducibility；It is high
Effect liquid phase chromatogram method, liquid chromatography tandem mass spectrometry etc., this kind of method sensitivity are high, and specificity is good, but this quasi-instrument does not have
Portability, expensive, sample pre-treatments are complicated；ELISA method sensitivity is higher, quick, economy, but in test experience process
Multiple board-washing is needed, repeatability is poor, and reagent needs Cord blood etc., it is adaptable to laboratory great amount of samples primary dcreening operation, is not suitable for scene fast
Speed detection；Colloidal gold immunochromatographimethod because quick, easy and economical and practical, shown in the great amount of samples examination of food inspection compared with
Strong advantage, but qualitative detection is only used for, and can not be quantified, the positive sample after screening need to send laboratory to enter traveling one
Walk quantitative analysis.
The content of the invention
The invention provides a kind of method based on lincomycin in background fluorescence quenching-immunochromatographic measurement milk, gram
The deficiency of above-mentioned prior art is taken, it, which can effectively solve the method for inspection that lincomycin is remained in existing fresh milk, is present before sample
Processing procedure is cumbersome, poor reproducibility, be not suitable for field quick detection and the problem of can not carry out quantitative analysis.
The technical scheme is that realized by following measures：One kind is based on background fluorescence quenching-immunochromatography
The method for determining lincomycin in milk, is carried out in the steps below：The first step, lincomycin calibration curve equation and title is defeated
Enter two-dimentional code system code device, lincomycin Quick Response Code is made, then lincomycin Quick Response Code is pasted onto in lincomycin test strips
Quick Response Code location for paste, obtain lincomycin detection card；Second step, takes the μ L of detected sample 200 to being fixed with gold labeling antibody
In small test glass, drawn after fully mixing in the well that 100 μ L are added dropwise on lincomycin detection card, 10min is chromatographed at room temperature；
Lincomycin is detected to the card inserting mouth of card insertion fluorescence immunity analyzer after the 3rd step, chromatography, test sample, fluoroimmunoassay is clicked on
The concentration of lincomycin in detected sample title and detected sample will be shown on the display screen of instrument.
Here is the further optimization and/or improvements to foregoing invention technical scheme：
Above-mentioned lincomycin calibration curve equation is obtained as follows：The first step, the configuration of lincomycin storing solution, takes
Lincomycin standard substance, 1 μ g/mL lincomycin solution is made into PBS, lincomycin storing solution is obtained；Second step, Lin Ke
The configuration of mycin series concentration standard liquid, 10ng/mL, 20ng/ are made into by lincomycin storing solution successively with standard milk sample
ML, 30ng/mL, 40ng/mL and 60ng/mL lincomycin serial solution, obtain lincomycin series concentration standard liquid；The
Three steps, take the μ L of standard milk sample 200, each 200 μ L of lincomycin series concentration standard liquid to the lab scale for being fixed with gold labeling antibody
100 μ L are drawn in cup, after fully mixing to be added dropwise in the well in lincomycin test strips, and 10min is chromatographed at room temperature；4th
Lincomycin test strips are inserted to the card inserting mouth of fluorescence immunity analyzer after step, chromatography, test sample is clicked on, fluorescence immunity analyzer is swept
The T lines and C lines in lincomycin test strips are retouched, sample F is obtained1/F0Ratio；5th step, with the concentration of lincomycin in solution
For abscissa, corresponding F1/F0For ordinate, lincomycin calibration curve equation y=-5 × 10 are obtained-6x3+0.0004x2+
0.0016x+0.4892。
Above-mentioned standard milk sample is the milk sample for not containing lincomycin after testing.
Above-mentioned detected sample is milk to be detected.
The detection that the present invention determines lincomycin in milk is limited to 0.088 μ g/kg, and optimal sample-adding amount is 100 μ L, optimal layer
The analysis time is 10min；Sensitivity than existing methods of the invention is high, the reaction time is short, easy to operate, and milk middle forest can be achieved can be mould
Plain scene Quantitative detection, lays a good foundation to be widely used in field of food safety.
Brief description of the drawings
Accompanying drawing 1 is background fluorescence quenching-immunochromatography detects schematic diagram of existing lincomycin test strips.
Accompanying drawing 2 is lincomycin standard curve of the present invention.
Accompanying drawing 3 is the dimensional structure diagram that lincomycin of the present invention detects card and fluorescence immunity analyzer.
Embodiment
The present invention is not limited by following embodiments, can technique according to the invention scheme and actual conditions it is specific to determine
Embodiment.
Embodiment 1, is somebody's turn to do the method based on lincomycin in background fluorescence quenching-immunochromatographic measurement milk, by following steps
It is rapid to carry out：The first step, inputs two-dimentional code system code device by lincomycin calibration curve equation and title, lincomycin two dimension is made
Code, is then pasted onto lincomycin Quick Response Code the Quick Response Code location for paste in lincomycin test strips, obtains lincomycin detection
Card；Second step, takes the μ L of detected sample 200 to being fixed with the small test glass of gold labeling antibody, 100 μ L dropwise additions is drawn after fully mixing
Detected to lincomycin in the well on card, 10min is chromatographed at room temperature；Lincomycin detection card is inserted after 3rd step, chromatography
Enter the card inserting mouth of fluorescence immunity analyzer, test sample to be checked will be shown on test sample, the display screen of fluorescence immunity analyzer by clicking on
The name of an article claims the concentration with lincomycin in detected sample.
Embodiment 2, lincomycin calibration curve equation is obtained as follows：The first step, lincomycin storing solution is matched somebody with somebody
Put, take lincomycin standard substance, 1 μ g/mL lincomycin solution is made into PBS, lincomycin storing solution is obtained；Second
Lincomycin storing solution is made into 10ng/ by step, the configuration of lincomycin series concentration standard liquid successively with standard milk sample
ML, 20ng/mL, 30ng/mL, 40ng/mL and 60ng/mL lincomycin serial solution, obtain lincomycin series concentration mark
Quasi- solution；3rd step, takes the μ L of standard milk sample 200, each 200 μ L of lincomycin series concentration standard liquid anti-to gold mark is fixed with
100 μ L are drawn in the small test glass of body, after fully mixing to be added dropwise in the well in lincomycin test strips, are chromatographed at room temperature
10min；Lincomycin test strips are inserted to the card inserting mouth of fluorescence immunity analyzer after the 4th step, chromatography, test sample is clicked on, fluorescence is exempted from
T lines and C lines in epidemic disease analyzer scanning lincomycin test strips, obtain sample F1/F0Ratio；5th step, can with solution middle forest
The concentration of mycin is abscissa, corresponding F1/F0For ordinate, lincomycin calibration curve equation y=-5 × 10 are obtained-6x3+
0.0004x2+0.0016x+0.4892。
Embodiment 3, as the optimization of above-described embodiment, standard milk sample is the milk for not containing lincomycin after testing
Sample.
Embodiment 4, as the optimization of above-described embodiment, detected sample is milk to be detected.
Background fluorescence quenching-immunochromatography detects schematic diagram of existing lincomycin test strips as shown in Figure 1, in Fig. 1,1.
Small test glass bottom is fixed with the object antibody (gold labeling antibody) of colloid gold label and the sheep anti-mouse antibody of colloid gold label, and 2. supply
The sample pad that test sample solution is chromatographed, 3. are coated with the p-wire of " object antigen-bovine serum albumin(BSA) (BSA) conjugate "
(T lines), 4. are coated with " sheep anti mouse antigen-bovine serum albumin(BSA) (BSA) conjugate " control line (C lines), and 5. carry background fluorescence
F0Nitrocellulose filter；In Fig. 1, bottom is fixed with the object antibody (gold labeling antibody) and colloid gold label of colloid gold label
Sheep anti-mouse antibody small test glass, being fixed with the nitrocellulose filter phosphor region of fluorometric reagent (can produce background fluorescence F0),
Phosphor region is coated with the p-wire (T lines) of " object antigen-bovine serum albumin(BSA) conjugate ", is coated with " sheep anti mouse antigen-ox
The control line (C lines) of seralbumin conjugate ".Sample detection principle is：By a certain amount of sample solution, addition is fixed with golden mark
(object to be measured-lincomycin antigen in sample solution is specifically bound with gold labeling antibody) in the small test glass of antibody, is mixed, is inhaled
Take a certain amount of mixing solution to be added dropwise in sample pad, chromatographed by capillarity.At mixed solution arrival T lines,
Remaining gold labeling antibody and object-lincomycin antigen binding at T lines, form antigen-gold labeling antibody compound, are trapped
On T lines, background fluorescence is quenched, F is designated as with the measured value of fluorescence intensity at T lines1, fluorescent quenching degree (F1/F0) with supplying to try
Object content to be measured is relevant in product.When target concentration is big in test sample, the amount with reference to gold labeling antibody in small test glass is just more,
Just few, the quenching journey that remaining gold labeling antibody is combined with " object antigen-bovine serum albumin(BSA) conjugate " at T lines in small test glass
Degree is with regard to weak, F1/F0Value just it is big.F1/F0Increase with testing concentration and increase, null value sample F1/F0Value is minimum.For to test strips
Quality Control is carried out, " sheep anti mouse antigen-bovine serum albumin(BSA) coupling is also secured on the nitrocellulose filter with background fluorescence
Thing " (C lines), for carrying out Quality Control to test strips, when above-mentioned mixed solution continues to be forwarded to up at C lines, gold marks sheep anti-mouse antibody
Combined with " the sheep anti mouse antigen-bovine serum albumin(BSA) conjugate " at C lines, be quenched the background fluorescence at C lines.Illustrate examination
Paper slip is up-to-standard.
Present invention experiment is as follows：
1. experimental section
1.1 instruments and material
Fluorescence immunity analyzer (ZC2015-0797, Shanghai Xin Pu bio tech ltd)；
Lincomycin standard substance (LIN, 10ppm, Shanghai Xin Pu bio tech ltd, lot number：20160911)；Woods
Can mycin test strips, be fixed with small test glass (the Shanghai Xin Pu bio tech ltd, lot number of lincomycin gold labeling antibody：
20160926)；
Standard milk sample (after testing without lincomycin, Shanghai Rong Hui bio tech ltd)；
Test sample, in order to verify the difference of background fluorescence quenching-immunochromatographic method and national regulations method, this experiment is in mark
A certain amount of lincomycin standard substance is artificially added in quasi- milk sample, be made 300ng/mL containing lincomycin, 220ng/mL and
50ng/mL test sample 004,005,006.
The preparation of 1.2 solution
The preparation of lincomycin storing solution：By lincomycin standard substance (10ppm), 1 μ g/mL solution is made into PBS,
Obtain lincomycin storing solution.
Lincomycin series concentration standard liquid：By 1 μ g/mL lincomycin storing solution, it is made into standard milk sample
10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL and 60ng/mL lincomycin serial solution, obtain lincomycin series dense
Spend standard liquid.
2. method and result
2.1 reaction time and the determination of sample-adding amount
2.1.1 the selection in reaction time
200 μ L standard milk samples are taken, are added in the small test glass for being fixed with gold labeling antibody, 120 μ L addition woodss are inhaled after mixing can
In well in mycin test strips, 8min and 10min is chromatographed respectively, is detected with fluorescence immunity analyzer, record F1/F0Value
It is shown in Table 1.
As it can be seen from table 1 when the chromatography time is 8min, RSD values are 6.12%, when the chromatography time is 10min, RSD values
For 4.30%；Illustrate that the RSD values when chromatography time is 10min are small, so selection chromatography 10min.
2.1.2 the selection of sample-adding amount
Take three parts of standard milk samples, every part of 200 μ L are added separately to be fixed with the small test glass of gold labeling antibody, after mixing according to
Secondary 80 μ L, 100 μ L, the 120 μ L of being inhaled from three small test glass for being fixed with gold labeling antibody are separately added into three lincomycin test strips
On well in, chromatograph 10min, lincomycin test strips inserted to after chromatography the card inserting mouth of fluorescence immunity analyzer, click on
Test sample, records F1/F0Value, be shown in Table 2.
From table 2 it can be seen that when sample-adding amount is 80 μ L, 100 μ L and 120 μ L, RSD values are followed successively by 6.35%, 1.41%,
3.68%；Illustrate that RSD values are minimum when sample-adding amount is 100 μ L, so 100 μ L sample-adding amounts of selection.
2.2 Method validation
2.2.1 curvilinear equation and scope
The μ L of standard milk sample 200,10ng/mL under 1.2 μ of lincomycin series concentration standard liquid 200 are taken respectively
L, 20ng/mL lincomycin series concentration standard liquid 200 μ L, 30ng/mL lincomycin series concentration standard liquid 200
μ L, 40ng/mL lincomycin series concentration standard liquid 200 μ L, 60ng/mL lincomycin series concentration standard liquid
200 μ L, are added separately to be fixed with the small test glass of gold labeling antibody, and each 100 μ L that inhale are separately added into lincomycin test strips after mixing
On well in, chromatograph 10min, detected with fluorescence immunity analyzer, record F1/F0, it is shown in Table 3；Can be mould with solution middle forest
The concentration of element is abscissa, corresponding F1/F0For ordinate, returned, obtain lincomycin calibration curve equation for y=-5
×10-6x3+0.0004x2+ 0.0016x+0.4892, lincomycin standard curve is as shown in Figure 2；It can be seen that from table 3 and Fig. 2
Lincomycin is in 0.102ng/mL to being in good correlation between 60ng/mL, and coefficient correlation is r=0.9923.Take standard ox
The μ L of milk sample 200, are added in the small test glass for being fixed with gold labeling antibody, and 100 μ L are inhaled after mixing and add adding in lincomycin test strips
In sample hole, 10min is chromatographed, is detected with fluorescence immunity analyzer, record F1/F0, repeat 20 times, calculate 20 times average and
Standard deviation (SD), calculates detection with average+2SD and is limited to 0.088ng/mL.
2.2.2 Precision Experiment
The lincomycin series concentration standard liquid of tri- concentration of 10ng/mL, 30ng/mL, 40ng/mL is taken, 200 μ are respectively taken
L, is added in the small test glass for being fixed with gold labeling antibody, each after mixing to inhale the sample-adding that 100 μ L are separately added into lincomycin test strips
Kong Zhong, chromatographs 10min, is detected with fluorescence immunity analyzer, records F1/F0, it is shown in Table 4；From table 4, it can be seen that 10ng/mL,
The F of the lincomycin series concentration standard liquid of tri- concentration of 30ng/mL, 40ng/mL1/F0The RSD of value is followed successively by 1.2%,
0.6%, 1.1%, the RSD of precision is less than 5%, meets the requirements.
2.2.3 recovery of standard addition
3 parts of standard milk samples are taken to put respectively in EP pipes, every part of 1mL adds lincomycin in 3 parts of standard milk samples successively
The μ L of storing solution 20,30 μ L and 40 μ L, are made into low, medium and high three concentration successively, and each concentration is parallel 3 parts, respectively takes 200 μ L, plus
To being fixed with the small test glass of gold labeling antibody, each 100 μ L that inhale are separately added into the well in lincomycin test strips after mixing,
Chromatograph 10min, detected with fluorescence immunity analyzer, record each sample middle forest can mycin detectable concentration, be shown in Table 5；From table 5
As can be seen that the average recovery of standard addition that high, medium and low lincomycin is determined in standard milk sample is respectively 103.4%,
99.7%, 98.5%, RSD are that 2.46%, RSD is less than 5%, are met the requirements.
2.2.4 replica test
The μ L of standard milk sample 995 are taken in EP pipes, the μ L of lincomycin storing solution 5 are added, mixes, is made into 5ng/mL sample
Product.Take 200 μ L to be added in the small test glass for being fixed with gold labeling antibody, mix, then inhale 100 μ L and add adding in lincomycin test strips
In sample hole, 10min is chromatographed, is detected with fluorescence immunity analyzer, the F of lincomycin detected value in record test sample1/F0, it is shown in Table 6
It is shown；As can be seen from Table 6, in lincomycin repeated experiment, F1/F0The RSD of measured value is 0.82%, concentration measurement
RSD is that 1.25%, RSD is less than 5%, is met the requirements.
2.2.5 long term repeatability is tested
Take the lincomycin series concentration mark under same batch lincomycin test strips, the μ L of standard milk sample 200 and 1.2
Quasi- each 200 μ L of solution, are added separately to be fixed with the small test glass of gold labeling antibody, after mixing it is each inhale 100 μ L be separately added into woods can be mould
In well in plain test strips, 10min is chromatographed, is detected with fluorescence immunity analyzer, is repeated once lincomycin mark every month
Directrix curve, is continuously done 5 months, and lincomycin long term repeatability experiment exam measurement result is shown in Table 7；As can be seen from Table 7,
In 5 months, standard milk sample, lincomycin series concentration standard liquid 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL and
60ng/mL, corresponding RSD are to be followed successively by 0.42%, 0.35%, 0.39%, 0.27%, 0.24% and 0.31%；RSD is less than
5%, meet the requirements.
The making of 2.3 Quick Response Codes
Above through Method validation, lincomycin calibration curve equation meets the requirements, and the lincomycin standard of acquisition is bent
Line equation and the two-dimentional code system code device of correspondence lincomycin title input, are made lincomycin Quick Response Code, are attached to lincomycin test paper
On bar, lincomycin detection card is obtained.
2.4 samples are determined
Each 200 μ L in test sample 004,005 and 006 are taken respectively to being fixed with the small test glass of gold labeling antibody, fully after mixing
Draw in the well that 100 μ L are added dropwise on lincomycin detection card, 10min is chromatographed at room temperature；Lincomycin is examined after chromatography
The card inserting mouth of card insertion fluorescence immunity analyzer is surveyed, test sample is clicked on, fluorescence immunity analyzer first scans lincomycin Quick Response Code, obtained
The lincomycin calibration curve equation stored in Quick Response Code, then the T lines and C lines on scanning lincomycin detection card successively are obtained, is obtained
Obtain sample F1/F0Ratio, softwares of Simple 1 in fluorescence immunity analyzer are by F1/F0Ratio substitute into lincomycin standard
Curvilinear equation is calculated, and display screen will show the concentration of detected sample title and detected sample after calculating.Take respectively again
Test sample 004,005 and 006 is measured by national standard (GB 29685-2013)；Using the softwares of SPSS 17.0, to the present invention and
Measured value makees t check analyses after National Standard Method is determined；Lincomycin measurement result in test sample (N=3) it is shown in Table 8,
As can be seen from Table 8, t assays P>0.05, illustrate there is no significant difference using bFQICA methods of the present invention and National Standard Method.
Background fluorescence quenching-immunochromatographic method (Background fluorescence quenching that the present invention is applied
Immunochromatographic assay, bFQICA) mainly include lincomycin detection card and fluorescence immunity analyzer, this
The dimensional structure diagram of invention lincomycin detection card and fluorescence immunity analyzer is as shown in Figure 3；Fluorescence immunity analyzer is pacified
Equipped with the software systems of Simple 1.
The present invention is based on background fluorescence quenching-immunochromatography technique, sets up the methodology of lincomycin in detection milk, this
Lincomycin calibration curve equation and title are inputted two-dimentional code system code device by invention, lincomycin Quick Response Code are made, then woods
Can mycin Quick Response Code be pasted onto Quick Response Code location for paste in lincomycin test strips, obtain lincomycin detection card；Lincomycin
Detection card is used cooperatively with fluorescence immunity analyzer, for the quick measure of actual sample, is compared with National Standard Method without conspicuousness
Difference.
3. brief summary
The lincomycin detection card that present invention application has been developed, establishes the detection method of lincomycin in detection milk.
The concentration range that the present invention determines lincomycin in milk is 0.102ng/mL to 60.0ng/mL, and lowest detection is limited to
0.088ng/mL.The average recovery of standard addition that high, medium and low lincomycin is determined in milk sample of the present invention is respectively 103.4%,
99.7%, 98.5%, RSD are 2.46% (n=9).
The lincomycin determined with the present invention in milk can be mould with national standard method (GB 29685-2013) measure milk middle forest
The method of element is compared, and the present invention is the reaction based on antigen, antibody, so specificity is strong；Nitrocellulose filter in the present invention
It is fixed with the upper T lines marked on have powerful connections fluorescence, collaurum quencher and antibody binding, nitrocellulose filter corresponding anti-
Original, when liquid flows in test strips, Ag-Ab-collaurum is combined at T lines, causes background fluorescence at T lines to be quenched, quenching
Degree F1/F0Represent, quantitative analysis is carried out, so detection sensitivity is high.The inspection of lincomycin in milk is determined with the present invention
It is the detection that the LC-MS in 0.088 μ g/kg, national standard method (GB 29685-2013) determines lincomycin in milk to survey limit
Limit is 8 μ g/kg；The lincomycin in milk is determined with the present invention, it is not necessary to sample pre-treatments, and the present invention will be detected in milk
The information of lincomycin standard curve, is stored in lincomycin Quick Response Code, testing staff does mark song without scene, becomes detection
It is convenient, fast, every part of sample only needs 10min.And the LC-MS in national standard method (GB 29685-2013) is determined in milk
Lincomycin not only needs sample pre-treatments, in addition it is also necessary to which testing staff does at scene mark song, and the detection of every part of sample needs 55min；
The present invention is compared with GICA methods, and GICA can only carry out qualitative analysis, is adapted to the initial screening of great amount of samples, the sample filtered out
Originally also need to detect again using quantitative method.And the present invention can not only be combined according to Ag-Ab at T lines-collaurum and drawn
The color change risen carries out qualitative analysis, can also carry out quantitative analysis.
In summary, the detection of lincomycin is limited to 0.088 μ g/kg in present invention measure milk, and optimal sample-adding amount is 100
μ L, the optimal chromatography time is 10min；Sensitivity than existing methods of the invention is high, the reaction time is short, easy to operate, and ox can be achieved
Lincomycin scene Quantitative detection, lays a good foundation to be widely used in field of food safety in milk.
Above technical characteristic constitutes embodiments of the invention, and it has stronger adaptability and implementation result, can basis
The non-essential technical characteristic of increase and decrease is actually needed, to meet the demand of different situations.
The selection of the differential responses time of table 1
The selection of the different sample-adding amounts of table 2
Lincomycin standard curve data in the milk of table 3
The precision test of table 4 (n=6)
Lincomycin recovery of standard addition in the milk of table 5
The replica test of the lincomycin of table 6
The lincomycin long term repeatability experiment exam measurement result of table 7
Lincomycin measurement result in the test sample of table 8 (N=3)
Claims (5)
1. a kind of method based on lincomycin in background fluorescence quenching-immunochromatographic measurement milk, it is characterised in that by following
Step is carried out：The first step, inputs two-dimentional code system code device by lincomycin calibration curve equation and title, lincomycin two dimension is made
Code, is then pasted onto lincomycin Quick Response Code the Quick Response Code location for paste in lincomycin test strips, obtains lincomycin detection
Card；Second step, takes the μ L of detected sample 200 to being fixed with the small test glass of gold labeling antibody, 100 μ L dropwise additions is drawn after fully mixing
Detected to lincomycin in the well on card, 10min is chromatographed at room temperature；Lincomycin detection card is inserted after 3rd step, chromatography
Enter the card inserting mouth of fluorescence immunity analyzer, test sample to be checked will be shown on test sample, the display screen of fluorescence immunity analyzer by clicking on
The name of an article claims the concentration with lincomycin in detected sample.
2. the method according to claim 1 based on lincomycin in background fluorescence quenching-immunochromatographic measurement milk, its
It is characterised by that lincomycin calibration curve equation is obtained as follows：The first step, the configuration of lincomycin storing solution, takes woods can
Mycin standard substance, 1 μ g/mL lincomycin solution is made into PBS, lincomycin storing solution is obtained；Second step, lincomycin
The configuration of series concentration standard liquid, by lincomycin storing solution be made into successively with standard milk sample 10ng/mL, 20ng/mL,
30ng/mL, 40ng/mL and 60ng/mL lincomycin serial solution, obtain lincomycin series concentration standard liquid；3rd
Step, takes the μ L of standard milk sample 200, each 200 μ L of lincomycin series concentration standard liquid to the small test glass for being fixed with gold labeling antibody
In, draw 100 μ L after fully mixing and be added dropwise in the well in lincomycin test strips, 10min is chromatographed at room temperature；4th
Lincomycin test strips are inserted to the card inserting mouth of fluorescence immunity analyzer after step, chromatography, test sample is clicked on, fluorescence immunity analyzer is swept
The T lines and C lines in lincomycin test strips are retouched, sample F is obtained1/F0Ratio；5th step, with the concentration of lincomycin in solution
For abscissa, corresponding F1/F0For ordinate, lincomycin calibration curve equation y=-5 × 10 are obtained-6x3+0.0004x2+
0.0016x+0.4892。
3. the side according to claim 1 or 2 based on lincomycin in background fluorescence quenching-immunochromatographic measurement milk
Method, it is characterised in that standard milk sample is the milk sample for not containing lincomycin after testing.
4. the side according to claim 1 or 2 based on lincomycin in background fluorescence quenching-immunochromatographic measurement milk
Method, it is characterised in that detected sample is milk to be detected.
5. the method according to claim 3 based on lincomycin in background fluorescence quenching-immunochromatographic measurement milk, its
It is milk to be detected to be characterised by detected sample.
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