CN102253216A - Method, special kit and test paper strip for detecting beta-lactam antibiotics based on penicillin-binding protein - Google Patents
Method, special kit and test paper strip for detecting beta-lactam antibiotics based on penicillin-binding protein Download PDFInfo
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Abstract
The invention discloses a method, a special kit and test paper strips for detecting beta-lactam antibiotics based on penicillin-binding protein. The receptor kit used for detecting beta-lactam antibiotics comprises: protein represented by SEQ ID NO: 1, enzyme labeled ampicillin and a standard substance solution, wherein the standard substance is penicillin G. The kit and colloidal gold test paper strips provided by the invention have advantages of high sensitivity, high accuracy, high precision, low cost, simple operation, short detection period, simple storage, and long shelf-life. The kit and colloidal gold test paper strips are suitable for various work units. With the kit and colloidal gold test paper strips, simultaneous and rapid detections of large batches of samples can be realized, and on-site high flux rapid detections can be realized. Therefore, the kit, the colloidal gold test paper strips and the method provided by the invention play an important role in the detections of beta-lactam antibiotics.
Description
Technical field
The present invention relates to a kind of method and dedicated kit and test strips of the detection beta-lactam antibiotic based on PBP.
Background technology
Beta-lactam antibiotic is that a class contains the antibiotic general designation of quaternary beta-lactam ring structure, be medical at present and the maximum class microbiotic of veterinary drug sector application, be widely used in treatment and diseases such as prevention staphylococcus, pneumococcus, streptococcus, Escherichia coli, haemophilus, salmonellal animal urethra, intestines and stomach, respiratory tract infection and mastitis for milk cows.Long-term absorption contains the residual food of beta-lactam antibiotic can cause that bacterial drug resistance increases, the digestive system flora imbalance, even bring out allergic reaction.
Along with the pay attention to day by day of people to food security, European Union, China, the U.S., Japan and other countries have been made regulation to Beta-lactam medicine maximum residue limit value in the animal derived food in succession, wherein the MRL of European Union's regulation benzyl penicillin, ampicillin, Amoxicillin is 4ppb, the MRL of Oxacillin, Cloxacillin, dicloxacillin, nafcillin is 30ppb, the MRL of Ceftiofur, cefalexin is 100ppb, the MRL of cephazoline, cefoperazone is 50ppb, and the MRL of Cefquinome is 20ppb.
Immune analysis method is that present medicament residue detects one of common method, its principle is based on the specificity combination of antigen-antibody, characteristic with high sensitivity, high specific, wherein enzyme linked immunosorbent assay and colloidal gold method possess high flux again, detect quick, easy and simple to handle, be applicable to advantage such as field screening, be widely used in the medicament residue examination.The basis that immune analysis method is set up is an antibody, and conventional preparation method for antibody mainly is that immune animal obtains polyclonal serum and adopts hybridoma technology to obtain monoclonal antibody, and the cycle is long, process is complicated, expense is high.
Summary of the invention
An object of the present invention is to provide a kind of receptor agents box that is used to detect beta-lactam antibiotic.
The receptor agents box that is used to detect beta-lactam antibiotic provided by the present invention comprises: the ampicillin and the standard solution of albumen, enzyme labeling shown in the SEQ ID NO:1, described standard items are benzyl penicillin.
In the above-mentioned receptor agents box, described receptor agents box comprises that also substrate colour developing liquid, stop buffer, cleansing solution, sample diluting liquid, bag are cushioned liquid and confining liquid;
Substrate colour developing liquid is made up of A liquid and B liquid, and A liquid is the aqueous solution of 2% (quality percentage composition) urea peroxide, and B liquid is the aqueous solution of 1% (quality percentage composition) tetramethyl benzidine;
Stop buffer is the aqueous sulfuric acid of 0.2M;
Per 1 liter of described cleansing solution is prepared as follows and is obtained: 0.5ml polysorbas20,5g sodium azide and 990ml phosphate buffer are mixed, obtain described cleansing solution; The concentration of described phosphate buffer is 0.01M, and the pH value is 7.4;
Sample diluting liquid: 0.1mol/L, pH value are 7.2 phosphate buffer;
Per 1 liter of bag is cushioned liquid and is prepared as follows: with Na
2CO
31.59g and NaHCO
32.93g be dissolved in 1 premium on currency, water is settled to 1 liter, obtains described bag and is cushioned liquid;
Per 1 liter of confining liquid is prepared as follows: 50mgBSA, 1g sodium azide, 30g casein are mixed, with the phosphate buffer dissolving and be settled to 1000ml, obtain confining liquid; Wherein, the concentration of phosphate buffer is 0.02M, and the pH value is 7.2.
In above-mentioned arbitrary described receptor agents box, the ampicillin of described enzyme labeling is the ampicillin of HRP mark;
In above-mentioned arbitrary described receptor agents box, described standard solution is the following solution of benzyl penicillin concentration: 8.1 μ g/L, 2.7 μ g/L, 0.9 μ g/L, 0.3 μ g/L, 0.1 μ g/L.
In above-mentioned arbitrary described receptor agents box, described beta-lactam antibiotic is at least a in following: benzyl penicillin, ampicillin, Amoxicillin, Oxacillin, Cloxacillin, nafcillin, dicloxacillin, Carbenicillin, methicillin, cefalexin, cephazoline, cefoperazone, ceftriaxone, CTX, cefoxitin, cefadroxil, Ceftiofur, Cefquinome.
Another object of the present invention provides a kind of colloid gold test paper that detects beta-lactam antibiotic.
The colloid gold test paper of detection beta-lactam antibiotic provided by the present invention comprises absorption of sample pad, collaurum pad, reaction film and adsorptive pads, and it connects successively; Described collaurum pad is coated with albumen shown in the SEQ ID NO:1 that has the HIS label of colloid gold label; Contain on the described reaction film and detect band and quality control band, detect the conjugate that the band position is coated with Amoxicillin and carrier protein, the quality control band position is coated with mouse-anti HIS tag monoclonal antibody.
In above-mentioned arbitrary described colloid gold test paper, described beta-lactam antibiotic is at least a in following: benzyl penicillin, ampicillin, Amoxicillin, Oxacillin, Cloxacillin, nafcillin, dicloxacillin, Carbenicillin, methicillin, cefalexin, cephazoline, cefoperazone, ceftriaxone, CTX, cefoxitin, cefadroxil, Ceftiofur, Cefquinome.
In above-mentioned arbitrary described colloid gold test paper, described carrier protein is OVA.
Another object of the present invention provides the method for beta-lactam antibiotic in a kind of test sample.
The method of beta-lactam antibiotic in the test sample provided by the present invention, shown in following I or II:
The method of beta-lactam antibiotic comprises the steps: in I, the test sample
1) testing sample is carried out pre-treatment, obtain sample to be tested solution;
2) with above-mentioned arbitrary described receptor agents box described sample to be tested solution is detected;
Described testing sample is a milk: with milk with sample diluting liquid by dilution in 1: 4,4 ℃, 10000rpm are centrifugal, get supernatant, as sample to be tested solution;
Described testing sample is a milk powder: take by weighing 1g milk powder, be dissolved in the 5mL sample diluting liquid, mix, as sample to be tested solution.
Described testing sample is a urine: with urine directly as sample to be tested solution;
Described testing sample is muscle, fish, shrimp or egg: with the testing sample homogenized, obtain equal quality sample, the equal quality sample of every 5g is added in the 10mL acetonitrile solution, concussion 10min turns upside down, the centrifugal 10min of 5000rpm gets supernatant, and nitrogen dries up, resuspended with the 1mL sample diluting liquid, the solution that obtains is sample to be tested solution; The volume ratio of acetonitrile and water is 9: 1 in the acetonitrile solution;
Described testing sample is a honey: every 5g testing sample is added in the 15mL deionized water, mix; Described mixed solution is carried out purifying with the Oasis solid-phase extraction column, use methanol-eluted fractions, collect eluent, nitrogen dries up, and resuspended with the 1mL sample diluting liquid, the solution that obtains is sample to be tested solution.
The method of beta-lactam antibiotic comprises the steps: in II, the test sample
1) testing sample is carried out pre-treatment, obtain sample to be tested solution;
2) with above-mentioned arbitrary described colloid gold test paper described sample to be tested solution is detected;
Described sample diluting liquid is that 0.1mol/L, pH value are 7.2 phosphate buffer.
The application of albumen shown in the SEQ ID NO:1 in detecting beta-lactam antibiotic also belongs to protection scope of the present invention.
In the above-mentioned application, described beta-lactam antibiotic is at least a in following: benzyl penicillin, ampicillin, Amoxicillin, Oxacillin, Cloxacillin, nafcillin, dicloxacillin, Carbenicillin, methicillin, cefalexin, cephazoline, cefoperazone, ceftriaxone, CTX, cefoxitin, cefadroxil, Ceftiofur, Cefquinome.
That kit of the present invention and colloidal gold test paper card have is highly sensitive, accuracy is high, precision is high, cost is low, simple to operate, detection time short, be fit to various units uses, stores advantage simple, long shelf-life; The fast detecting batch samples can realize on-the-spot high flux fast detecting simultaneously.Therefore, kit of the present invention, colloid gold test paper and detection method will be brought into play significant role in the detection of beta-lactam antibiotic.
Description of drawings
Fig. 1 is PBP 2x gene PCR agarose electrophoresis figure.
Fig. 2 cuts evaluation figure for PBP 2x gene is connected the back enzyme with the pET28b carrier.
Fig. 3 induces back different time points holoprotein SDS-PAGE figure for IPTG.
Fig. 4 is PBP 2x protein purification SDS-PAGE figure.
Fig. 5 is PBP 2x albumen western blot figure.
Fig. 6 is the kit typical curve.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The ampicillin is available from U.S. Sigma-Aldrich company, and catalog number is A9518.
Expression vector pET28b is available from German Merk (Novagen) company, and catalog number is 69865; E. coli bl21 (DE3) is available from German Merk (Novagen) company, and catalog number is 69387; Protein purification His
Columns is available from German Merk (Novagen) company, and catalog number is 70971; T4 DNA Ligase is available from U.S. Promega company, and catalog number is M1801S.
(Americantype culture collection ATCC), is numbered ATCC 49619 to streptococcus pneumonia (Streptococcus pneumoniae) R6 available from the biological product of Unite States Standard (USS) collecting center
TM(http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/ta bid/452/Default.aspx).Horseradish peroxidase (HRP) is available from U.S. Sigma company.BCA protein quantification kit is available from U.S. Pierce company.
Carrier PMD19-T is available from TAKARA, and catalog number is D102A.
The sheep anti-mouse antibody of His-tag monoclonal antibody and HRP mark is available from TIANGEN Biotech (Beijing) Co., Ltd., and catalog number is respectively AB102-02 and SA101-01.
The preparation of embodiment 1, reorganization bacterium
Streptococcus pneumonia R6 genome is used following primer and is carried out pcr amplification as template, obtains the PCR product; Cut the PCR product with restriction enzyme NdeI and XhoI enzyme, the PCR product reclaims the target gene fragment (being the PBP2x genes of interest) about 2250bp through 1% agarose gel electrophoresis; Electrophoresis result as shown in Figure 1, swimming lane 1-6 is a genes of interest, swimming lane 7 is marker.
Upstream primer: 5 '-TT
CATATGATGAAGTGGACAAAAAGA-3 ' (5 ' end contains NdeI restriction enzyme site, protection base)
Downstream primer: 5 '-TT
CTCGAGTTAGTCTCCTAAAGTTAAT-3 ' (3 ' end contains XhoI restriction enzyme site, protection base)
Reclaim target gene fragment, it is connected with the PMD19-T carrier, connect product transformed into escherichia coli DH5 α,, positive monoclonal is carried out Liquid Culture, extract plasmid, check order by the resistance screening positive monoclonal.The result shown in SEQ ID NO:2, shows that the recombinant vector of structure is correct at the nucleotide sequence of the gene that inserts among the PMD19-T, and note is made PMD 19-T-PBP2x.
Cut PMD19-T-PBP2x with restriction enzyme NdeI and XhoI enzyme, reclaim the target gene fragment about 2250bp; Cut pET28b with restriction enzyme NdeI and XhoI enzyme, reclaim the big fragment of carrier; The big fragment of target gene fragment and carrier is connected with T4 DNA Ligase, connect product transformed into escherichia coli DH5 α, by the resistance screening positive monoclonal, positive monoclonal is carried out Liquid Culture, extract plasmid, check order and make NdeI single endonuclease digestion, XhoI single endonuclease digestion and NdeI+XhoI double digestion respectively and identify.The nucleotide sequence of result's gene that (along the direction from NdeI to XhoI) inserted between the NdeI and XhoI restriction enzyme site of pET28b is shown in SEQ ID NO:2, and the amino acid sequence of its encoded protein is (note is made PBP PBP2x) shown in SEQ ID NO:1.Show that gene direction of insertion and sequence are all correct in the recombinant vector, positive recombinant vector note is made recombinant expression carrier pET28b-PBP2x.Enzyme is cut qualification result as shown in Figure 2, the single endonuclease digestion size about 7300bp, double digestion have about 2300bp and 5000bp about band, the illustration purpose gene has correctly connected into pET28b.Among Fig. 2, swimming lane 1:NdeI single endonuclease digestion; Swimming lane 2:XhoI single endonuclease digestion; Swimming lane 3:NdeI/XhoI double digestion; Swimming lane 4:Marker.
The recombinant expression carrier pET28b-PBP2x transformed into escherichia coli BL21 (DE3) that builds, the kalamycin resistance screening obtains positive monoclonal; The positive monoclonal that obtains is inoculated in the LB fluid nutrient medium that contains kanamycins 30ug/mL cultivates, extract plasmid, order-checking, the result shows that the plasmid of extraction is correct, shows that the reorganization bacterium of structure is correct, note is made reorganization bacterium BL21-pET28b-PBP2x.
Serve as the contrast bacterium with the e. coli bl21 (DE3) that changes pET28b over to simultaneously, note is made reorganization bacterium BL21-pET28b.
The preparation of embodiment 2, albumen and purifying
One, the preparation of albumen
BL21-pET28b-PBP2x is seeded in the LB fluid nutrient medium that contains kanamycins 30ug/mL with the reorganization bacterium, 37 ℃ of joltings of 250rpm, and incubated overnight (12h) obtains seed liquor; Get the 1mL seed liquor and be inoculated in the LB fluid nutrient medium that 100mL contains kanamycins 30ug/mL, 37 ℃ of joltings of 250rpm are to the A of cultivating system
600It is 0.6 o'clock, take out 1mL bacterium liquid earlier, as not inducing contrast, all the other bacterium liquid add IPTG to final concentration 1mM, 200pm shakes bacterium for 30 ℃, from inducing 3rd hour (note is done the 0th hour when adding IPTG), per hour take out 1mL bacterium liquid, induction time is respectively 3h, 4h, 5h, 6h, 7h, 8h and spend the night (12h); The bacterium liquid collected at 4 ℃ of centrifugal 5min of following 10000rpm, is collected thalline; With the resuspended thalline of Tris-HCL (10mM Tris pH7.0), the broken thalline of 200W power ice-bath ultrasonic (ultrasonic 10s stops 10s) becomes clarification to suspension, and 4 ℃ again, the centrifugal 20min of 12000 * g collect supernatant, are the PBP extract.All substances in culture vessel note is made bacterium liquid.
Contain the preparation of the LB fluid nutrient medium of kanamycins 30ug/mL: add kanamycins in the LB fluid nutrient medium, making the concentration of kanamycins in the LB fluid nutrient medium is 30ug/mL, and the mixed solution that obtains is the LB fluid nutrient medium that contains kanamycins 30ug/mL.
The LB fluid nutrient medium is formed: be made up of yeast extract, peptone, NaCl and water, the concentration of each composition is in the solution: yeast extract 0.5% (quality percentage composition), peptone 1% (quality percentage composition), NaCl 1% (quality percentage composition).
Two, the purifying of albumen
PBP purifying: utilize histidine-tagged (His-tag) mark that has on the expression vector to pass through the nickel ion affinity chromatograph process to purify penicillin in conjunction with albumen.First binding buffer balance His with 10 times of column volumes
Columns gets above-mentioned PBP extract, uses the wash buffer wash-out foreign protein of 5 times of column volumes then, use the elution buffer wash-out target protein of 10 times of column volumes at last, collect eluent, dialyse, obtain the PBP of purifying.
Expression product with reorganization bacterium BL21-pET28b is contrast simultaneously.
Binding buffer: with 20mmol Tris, 0.5mol Nacl and the dissolving of 10mmol imidazoles water, transfer pH value to 8.0 with HCL, water is settled to 1L again, obtains 1 liter of binding buffer.
Wash buffer: with 20mmol Tris, 0.5mol NaCl and the dissolving of 20mmol imidazoles water, transfer pH value to 8.0 with HCL, water is settled to 1L again, obtains 1 liter of wash buffer.
Elution buffer: with 20mmol Tris, 0.5mol Nacl and the dissolving of 500mmol imidazoles water, transfer pH value to 8.0 with HCL, water is settled to 1L again, obtains 1 liter of elution buffer.
Three, albumen checking
1, induce the front and back product to carry out the SDS-PAGE detection product before and after the purifying and IPTG, the result as shown in Figure 3 and Figure 4.
Fig. 3 is the result of reorganization bacterium BL21-pET28b-PBP2x, swimming lane 1: do not induce control group; Swimming lane 2～8: be respectively recombinant bacterial strain through 3,4,5,6,7,8 hours and spend the night induce after protein expression figure; Swimming lane 9:Marker.
Fig. 4 is the result of reorganization bacterium BL21-pET28b-PBP2x, swimming lane 1:Marker; Swimming lane 2: the unpurified recombinant bacterial strain holoprotein of not inducing; Swimming lane 3: induce but unpurified recombinant bacterial strain holoprotein; Swimming lane 4: induce and purifying after albumen.
The result shows, induces down at IPTG, and containing in the reorganization bacterium BL21-pET28b-PBP2x that IPTG induces has tangible band about 80kD, and is consistent with the expection size of destination protein, the success of PBP2x protein expression.And do not contain the purpose band among the reorganization bacterium BL21-pET28b-PBP2x that induces without IPTG.
No matter whether induce, do not contain the purpose band in the expression product of reorganization bacterium BL21-pET28b.
2, albumen checking Western blot:
Collect each stage product in the above-mentioned preparation process, carry out the 10%SDS-PAGE electrophoresis; The electrophoretic band trace to the NC film, is hybridized the DAB colour developing more successively with the sheep anti-mouse antibody of His-tag monoclonal antibody and HRP mark.
The result as shown in Figure 5.Among Fig. 5, swimming lane 1:Marker; Swimming lane 2: the unpurified reorganization bacterium BL21-pET28b-PBP2x holoprotein of not inducing; Swimming lane 3: induce but unpurified reorganization bacterium BL21-pET28b-PBP2x holoprotein; Swimming lane 4: reorganization bacterium BL21-pET28b-PBP2x induce and purifying after albumen.
The result shows: it is about 80kD that reorganization bacterium BL21-pET28b-PBP2x expresses the molecular weight of albumen that obtains, consistent with the molecular weight of albumen of expection.Do not contain destination protein in the expression product of reorganization bacterium BL21-pET28b.
The kit of embodiment 3, detection beta-lactam antibiotic
One, kit is made up of following substances:
1, bag is cushioned liquid with bag and is configured to 1ug/mL by the PBP of preparation purifying among the ELISA Plate of PBP: the embodiment 2; Every hole 100uL.
2, enzyme mark thing working fluid: the ampicillin working fluid of HRP mark, its working concentration 1ug/mL, the ampicillin of diluting the HRP mark with sample diluting liquid obtains.
3, beta-lactam antibiotic standard items: standard items are the benzyl penicillin freeze-dried powder, and benzyl penicillin is available from U.S. Sigma-Aldrich company; Catalog number is 46616; Benzyl penicillin is dissolved with sample diluting liquid, obtain the standard solution of following variable concentrations: 8.1 μ g/L, 2.7 μ g/L, 0.9 μ g/L, 0.3 μ g/L, 0.1 μ g/L.
4, substrate colour developing liquid: be made up of A liquid and B liquid, A liquid is the aqueous solution of 2% (quality percentage composition) urea peroxide, and B liquid is the aqueous solution of 1% (quality percentage composition) tetramethyl benzidine;
5, stop buffer: 0.2M aqueous sulfuric acid;
6, cleansing solution: per 1 liter of described cleansing solution is prepared as follows and is obtained: 0.5ml polysorbas20,5g sodium azide and 990ml phosphate buffer are mixed, obtain described cleansing solution; The concentration of described phosphate buffer is 0.01M, and the pH value is 7.4;
7, the phosphate buffer of sample diluting liquid: 0.1mol/L, pH value are 7.2.
8, bag is cushioned aqueous solution, the pH9.6 of the sodium carbonate of liquid: 0.05mol/L.
9, confining liquid: per 1 liter of confining liquid is prepared as follows: 50mgBSA, 1g sodium azide, 30g casein are mixed, with the phosphate buffer dissolving and be settled to 1000ml, obtain confining liquid; Wherein, the concentration of phosphate buffer is 0.02M, and the pH value is 7.2.
0.01M, the pH value is the preparation of 7.4 PBS damping fluid: per 1 liter of phosphate buffer (PBS) is prepared as follows: with 8g NaCl, 0.2g KCl, 3.53g Na
2HPO
4.12H
2O, 0.24g KH
2PO
4Mix with deionized water, be settled to 1 liter with deionized water.The concentration of the phosphate buffer that so obtains is 0.01M, and the pH value is 7.4.
0.1M, the pH value is the preparation of 7.2 PBS damping fluid: dispose earlier 0.1mol/L Na respectively
2HPO
4Solution (takes by weighing Na
2HPO
4.12H
2O 35.8g adds deionized water to 1000m L) and 0.1mol/L NaH
2PO
4Solution (takes by weighing NaH
2PO
4.2H
2O 15.6g adds deionized water to 1000m L); Get 0.1mol/L Na
2HPO
4Solution 72m L and 0.1mol/LNaH
2PO
4Solution 28m L adds 8.5g NaCL, mixes.
0.02M, the pH value is the preparation of 7.2 PBS damping fluid: get 0.1M, pH value and be 7.2 PBS damping fluid 200mL, add deionized water 800mL, mixing is promptly.
0.05mol/L, the pH value is the preparation of the aqueous solution of 9.6 sodium carbonate: Na
2CO
31.59g, NaHCO
32.93g, add deionized water and be settled to 1 liter.
Two, the preparation of kit
(1) is coated with the ELISA Plate and the preparation thereof of PBP
Be coated with the polystyrene ELISA Plate of PBP: be cushioned liquid with bag PBP is diluted to 1.0 μ g/mL, bag is by 96 hole polystyrene ELISA Plate, every hole 100 μ L, 4 ℃ are spent the night, and dry liquid in the hole, with cleansing solution (after 20 times of dilutions) washing 1 time, pat dry, in every hole, add 200 μ L confining liquids, 37 ℃ of incubation 2h then, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
(2) preparation of standard items
It is 8.1 μ g/L that the benzyl penicillin standard items are mixed with concentration with cryopreserving liquid (contain the 0.01M PBS of 5%BSA, pH 7.4), and every freeze-drying bottle packing 1mL places the freeze dryer freeze-drying, seals back 4 ℃ of preservations.
(3) preparation of enzyme mark thing
With the EDC method haptens ampicillin is coupled on the HRP.
A. get the 50mg ampicillin, (0.05mol/L is pH7.0) in the solution to be dissolved in 5mL PBS;
B. add 40mg EDC, 4 ℃ of stirring reaction 1h;
C. getting the HRP of 100mg and 10mg EDC is dissolved in 10mLPBS (0.05mol/L pH7.0) in the solution, mixes 4 ℃ of stirring reaction 48h with above-mentioned solution;
D. the bag filter of packing into, (0.05mol/L, pH7.0) dialysis obtain the conjugate of ampicillin and HRP to PBS, are enzyme and mark thing.
0.05M, the pH value is the preparation of 7.0 PBS damping fluid: dispose earlier 0.05mol/L Na respectively
2HPO
4Solution (takes by weighing Na
2HPO
4.12H
2O 17.9g adds deionized water to 1000m L) and 0.05mol/L KH
2PO
4Solution (takes by weighing KH
2PO
4.2H
2O 8.6g adds deionized water to 1000m L); Get 0.05mol/L Na
2HPO
4Solution 60m L and 0.05mol/LKH
2PO
4Solution 40m L adds 8.5g NaCL, mixes.
Three, kit test method
1, the making of typical curve
The benzyl penicillin standard solution 50 μ L that in the ELISA Plate micropore that is coated with PBP, add variable concentrations, add enzyme mark thing working fluid 50 μ L again, with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens, dry liquid in the hole, every hole adds 350 μ L cleansing solutions, pours out liquid in the hole behind the 30s, so repetitive operation is washed plate 4 times altogether, pats dry with thieving paper.Every hole adds substrate colour developing liquid, the mixing that vibrates gently, and 37 ℃ of constant temperature oven lucifuge colour developing 10min, every hole adds stop buffer 50 μ L, and the mixing that vibrates is gently used microplate reader, measures every hole absorbance.
With the absorbance mean value (B) of the standard solution of each concentration absorbance (B divided by first standard solution (0 standard)
0) multiply by 100% again, obtain the percentage absorbance.Semilog value with standard items concentration (μ g/L) is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map.The typical curve that obtains as shown in Figure 6.The functional relation of this typical curve is y=-18.95Ln
(x)+ 43.087, R
2=0.9918.
Percentage absorbance (%)=(B/B
0) * 100%
2, the mensuration of beta-lactam antibiotic concentration in the sample
Basic identical in the preparation of method and typical curve, different is to replace standard solution with sample to be tested solution.Measure every hole absorbance.With the absorbance mean value (B) of each test sample solution absorbance (B divided by first standard solution (0 standard)
0) multiply by 100% again, obtain the percentage absorbance.With the functional relation of percentage absorbance substitution typical curve, promptly obtain the residual quantity of beta-lactam antibiotic in the sample solution.
3, the pre-treating method of test sample
The detection sample is milk, milk powder: milk is centrifugal by 1: 4 back 4 ℃ of 10000rpm of dilution with sample diluting liquid, gets 50 μ L supernatants and is used for detecting.Take by weighing 1g milk powder, be dissolved in the 5mL sample diluting liquid, mix, get 50 μ L and be used for detecting.
The detection sample is a urine: directly get 50 μ L urines and be used for detecting.
The detection sample is muscle, fish, shrimp and eggs: take by weighing the equal quality sample of 5g, add 10mL acetonitrile solution (V
Second Nitrile: V
Water=9: 1), the concussion 10min that turns upside down, the centrifugal 10min of 5000rpm gets supernatant, and nitrogen dries up, and is resuspended with the 1mL sample diluting liquid, gets 50 μ L and be used for detecting.
The detection sample is a honey: take by weighing the 5g sample, add the 15mL deionized water, on the vortex instrument, fully mix 1min, (utilize ion-exchange behind the Oasis solid-phase extraction column excessively, with object enrichment and purifying), with 5mL deionization washing post, drain under the negative pressure earlier, use the 3mL methanol-eluted fractions again, collect eluent, nitrogen dries up, and is resuspended with the 1mL sample diluting liquid, gets 50 μ L and be used for detecting.
Four, the accuracy of kit
(1) accuracy test
In the sample that does not contain beta-lactam antibiotic, add the benzyl penicillin standard items, make the final concentration of benzyl penicillin standard items in sample be respectively 4 μ g/L, 8 μ g/L; Sample after adding is carried out pre-treatment according to method described in the experiment three respectively, obtain test sample solution.
Respectively extract 3 kits and detect from the kit of three different batches, each experiment repeats 5 times, calculates the coefficient of variation respectively.The result sees Table 1 respectively.
The computing method of the recovery: the ratio of RG=measured value and actual value * 100%;
The computing method of the coefficient of variation: CV=(ratio of the standard deviation of each parallel samples and each parallel samples mean value) * 100%;
The computing method of variation within batch coefficient: CV=is with the mean value of the coefficient of variation of each parallel samples in once measuring in batch.
The computing method of interassay coefficient of variation: the same sample of CV=is got its mean value in the coefficient of variation of different batches measurement result between batch.
The result shows: the interpolation recovery of all samples is 72.2%～97.5%, and the variation within batch coefficient is 6.4%～9.9%, and interassay coefficient of variation is 9.8%～13.0%.
The measurement result of table 1, benzyl penicillin accuracy
(2) specific detection
Detection method is basic identical with the preparation method who tests typical curve in three, and different is to use the beta-lactam antibiotic of variable concentrations to replace standard items respectively, obtains 50% inhibition concentration (IC50) of every kind of beta-lactam antibiotic.
Beta-lactam antibiotic is as follows: benzyl penicillin, ampicillin, Amoxicillin, Oxacillin, Cloxacillin, dicloxacillin, nafcillin, Carbenicillin, methicillin, cefalexin, cefadroxil, cephazoline, cefoperazone, ceftriaxone, CTX, cefoxitin, Ceftiofur, Cefquinome.
3 repetitions are established in experiment.
The result is as follows:
1) each standard items concentration of being added of absorbance and every hole is inversely proportional to, prove that the PBP that expressed purifying obtains has the characteristic of the multiple beta-lactam antibiotic of identification, and present linear relationship, illustrate that this PBP can be used for the detection of multiple beta-lactam antibiotic.
2) lowest detectable limit of various medicines: the IC80 with various medicines is a lowest detectable limit: the absorbance of positive control hole (promptly not adding the hole of standard solution) is B
0, the absorbance of each experimental port is B, works as B/B
0The concentration that is 80% o'clock pairing standard solution is lowest detectable limit (IC80).The results are shown in Table 2.
The result is as shown in table 2, and PBP 2x albumen can be discerned at least 18 kinds of Beta-lactam medicines that comprise penicillin, Amoxicillin, ampicillin, cefalexin etc., and all can satisfy the detection requirement for the medicine that European Union is set with MRL.
Ppb in the literary composition is ng/mL.
The lowest detectable limit of many kinds of beta-lactam medicines of table 2
(3) kit storage life
The kit preservation condition is 2-8 ℃, and through 12 months mensuration, the maximum absorbance value (zero standard) of kit, 50% inhibition concentration, beta-lactam antibiotic added the practical measurement value all within normal range.Consider in transportation and the use, have improper preservation condition and occur, kit was placed 8 days that carry out the accelerated deterioration experiment, the result shows that every index of this kit meets the requirements fully under the condition of 37 ℃ of preservations.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 8 days, measurement result shows that also the every index of kit is normal fully.Can draw kit from above result can preserve more than 12 months at least at 2-8 ℃.
The test strips of embodiment 4, detection beta-lactam antibiotic
One, the structure of test paper
Described test paper is made up of absorption of sample pad, collaurum pad, reaction film and adsorptive pads;
Described absorption of sample pad, collaurum pad, reaction film and adsorptive pads are linked in sequence successively, and the end of absorption of sample pad links to each other with the top of collaurum pad, and the end of collaurum pad links to each other with the top of reaction film, and the end of reaction film links to each other with the top of adsorptive pads;
Be coated with the PBP that has the HIS label (embodiment 2 preparation purifying obtain) of colloid gold label on the described collaurum pad;
Detection zone and Quality Control district are arranged on the described reaction film, and detection zone (C line) is the ribbon vertical with the appearance of described test paper with Quality Control district (T line); Detection zone is positioned at a side that is bordering on collaurum pad end; The Quality Control district is positioned at the side away from collaurum pad end; Detection zone is coated with coating antigen, and the Quality Control district is coated with mouse-anti HIS tag monoclonal antibody; Coating antigen is the conjugate of Amoxicillin and OVA.
The absorption of sample pad is a cellulose filter membrane, and reaction film is nitrocellulose filter (a NC film); Adsorptive pads is a thieving paper; The collaurum pad is the glass fibre membrane that is coated with the streptomysin antibody of colloid gold label; Sample well is positioned at the test paper top.
Two, the preparation of test paper
1, the preparation of coating antigen
With the EDC method haptens Amoxicillin is coupled on the oralbumin OVA.
A. get the 150mg Amoxicillin, (0.05mol/L is pH7.0) in the solution to be dissolved in 40mL PBS;
B. add 100mg EDC, 4 ℃ of stirring reaction 1h;
C. add 100mg OVA again, 4 ℃ of stirring reaction 24h; The bag filter of packing into, (0.05mol/L, pH7.0) dialysis obtain the conjugate of Amoxicillin and OVA to PBS, are coating antigen.
2, the PBP behind colloid gold label antibody-colloid gold label purifying:
The preparation of colloidal gold solution: get 0.01% aqueous solution of chloraurate 100mL and be heated to boiling with the constant temperature magnetic stirrer, continue to add 1% trisodium citrate aqueous solution 2.5mL under the condition of stirring, continue agitating heating 20min, solution is bright redness.The room temperature cooling returns to original volume with deionized water, 2-8 ℃ of preservation.
0.1mol/L K
2CO
3Aqueous solution is regulated colloidal gold solution pH 8.2.To add in the 10mL colloidal gold solution in the 50mL beaker, magnetic stirrer 250r/min stirs, and dropwise adds 1mL and contains 1mg PBP solution.Dropwise add 3mL 5%BSA aqueous solution, continue to stir 10min.With the centrifugal 20min of solution normal temperature low speed (1500r/min), discard the precipitation that forms by the gold grain that condenses.Get red supernatant solution, 2-8 ℃ of red supernatant solution, the centrifugal 40min of 11000r/min.Solution is divided into three layers, transparent supernatant, the gold grain layer of black densification on flowable kermesinus precipitation in the pipe end and the pipe diapire.Flowable kermesinus precipitation is transferred in the another one centrifuge tube, be suspended to original volume with the 0.01mol/L phosphate buffer that contains 1%BSA.Equilibrate overnight, the same repeated centrifugation 2 times.Use the 0.01mol/L phosphate buffer of 1%BSA (to contain 0.02%NaN at last
3) will to precipitate suspendible be 1/40 of original volume, promptly obtains the PBP of colloid gold label, 2-8 ℃ of preservation.
3, metal spraying: the PBP of colloid gold label is sprayed onto on the glass fibre, makes the collaurum pad;
4, spray film: T line on the reaction film and C line position are sprayed coating antigen and mouse-anti HIS tag monoclonal antibody respectively;
5, assembling: sample pad, collaurum pad, nitrocellulose filter, thieving paper are assembled according to a conventional method, and slitting is then packed test card in the plastics fabrication into, forms test card.
Three, detect with test card
(1) sample pre-treatments and detection
Milk sample: need not pre-treatment, directly detect.
Take out test card, lie against desktop behind the Kaifeng, draw liquid to be measured and dropwise add 4 in sample well; 42 ℃ of reactions, the 10min judged result, the result behind the 20min is invalid.
(2) result judges
Negative: the colour developing of C line, T line naked eyes as seen, no matter shade all is judged to feminine gender.
Positive: the colour developing of C line, the T line does not develop the color, and is judged to the positive.
Invalid: the C line does not develop the color, and no matter whether the T line develops the color, and it is invalid that this test card all is judged to.
The detection principle of this colloid gold test paper: when containing too much beta-lactam antibiotic in the sample, the PBP of colloid gold label is fully by the beta-lactam antibiotic combination in the sample, so the Amoxicillin on the detection line can not combine with the PBP of colloid gold label, therefore do not develop the color on the detection line, testing result is positive; When not containing beta-lactam antibiotic in the sample, the PBP of colloid gold label only combines with Amoxicillin on the detection line, so develops the color on the detection line, and testing result is negative.
Four, the effect of test card
(1) false positive rate and false negative rate
Former milk sample directly picks up from the farm milk cow.Detect benzyl penicillin content in the milk sample with LC-MS/MS, LC-MS/MS carries out according to GB/T 22975-2008.
Feminine gender milk sample (benzyl penicillin content is less than the 4ng/ml) 100 parts of the LC-MS/MS that learns from else's experience conclusive evidence, positive milk sample (benzyl penicillin content is greater than the 4ng/ml) 100 parts of the LC-MS/MS that learns from else's experience conclusive evidence.Sample is detected with test card respectively, calculate false positive rate and false negative rate.
The result: in 100 parts of negative milk samples were measured, test card detected 5 parts of positive (13#, 32#, 58#, 66#, 67#), and false positive rate is 6%.In 100 parts of positive milk samples were measured, test card detected 0 part of negative sample, and false negative rate is 0%.
Detect the milk that the supermarket is bought by the same way.Detect benzyl penicillin content in the milk sample with LC-MS/MS, LC-MS/MS carries out according to GB/T 22975-2008.
15 parts in the negative finished milk sample (benzyl penicillin content is less than 4ng/ml) of the LC-MS/MS that learns from else's experience conclusive evidence, positive milk sample (benzyl penicillin content is greater than the 4ng/ml) 10 parts of the LC-MS/MS that learns from else's experience conclusive evidence.Sample is detected with test card respectively, calculate false positive rate and false negative rate.
The result: in 15 parts of negative finished milk samples were measured, test card detected 1 part of positive (8#), and false positive rate is 6.67%.In 10 parts of positive milk samples were measured, test card detected 0 part of negative sample, and false negative rate is 0%.
(2) test card storage life
Stability test is the result show, this test card can be preserved 1 year at shady and cool dry place under 2-8 ℃ or room temperature condition.
Claims (10)
1. receptor agents box that is used to detect beta-lactam antibiotic, comprising: the ampicillin and the standard solution of albumen, enzyme labeling shown in the SEQ ID NO:1, described standard items are benzyl penicillin.
2. receptor agents box according to claim 1 is characterized in that: described receptor agents box comprises that also substrate colour developing liquid, stop buffer, cleansing solution, sample diluting liquid, bag are cushioned liquid and confining liquid;
Substrate colour developing liquid is made up of A liquid and B liquid, and A liquid is the aqueous solution of 2% (quality percentage composition) urea peroxide, and B liquid is the aqueous solution of 1% (quality percentage composition) tetramethyl benzidine;
Stop buffer is the aqueous sulfuric acid of 0.2M;
Per 1 liter of described cleansing solution is prepared as follows and is obtained: 0.5ml polysorbas20,5g sodium azide and 990ml phosphate buffer are mixed, obtain described cleansing solution; The concentration of described phosphate buffer is 0.01M, and the pH value is 7.4;
Sample diluting liquid: 0.1mol/L, pH value are 7.2 phosphate buffer;
Per 1 liter of bag is cushioned liquid and is prepared as follows: with Na
2CO
31.59g and NaHCO
32.93g be dissolved in 1 premium on currency, water is settled to 1 liter, obtains described bag and is cushioned liquid;
Per 1 liter of confining liquid is prepared as follows: 50mgBSA, 1g sodium azide, 30g casein are mixed, with the phosphate buffer dissolving and be settled to 1000ml, obtain confining liquid; Wherein, the concentration of phosphate buffer is 0.02M, and the pH value is 7.2.
3. receptor agents box according to claim 1 and 2 is characterized in that: the ampicillin of described enzyme labeling is the ampicillin of HRP mark;
Described standard solution is the following solution of benzyl penicillin concentration: 8.1 μ g/L, 2.7 μ g/L, 0.9 μ g/L, 0.3 μ g/L, 0.1 μ g/L.
4. according to arbitrary described receptor agents box among the claim 1-3, it is characterized in that: described beta-lactam antibiotic is at least a in following: benzyl penicillin, ampicillin, Amoxicillin, Oxacillin, Cloxacillin, nafcillin, dicloxacillin, Carbenicillin, methicillin, cefalexin, cephazoline, cefoperazone, ceftriaxone, CTX, cefoxitin, cefadroxil, Ceftiofur, Cefquinome.
5. a colloid gold test paper that detects beta-lactam antibiotic comprises absorption of sample pad, collaurum pad, reaction film and adsorptive pads, and it connects successively; Described collaurum pad is coated with albumen shown in the SEQ IDNO:1 that has the HIS label of colloid gold label; Contain on the described reaction film and detect band and quality control band, detect the conjugate that the band position is coated with Amoxicillin and carrier protein, the quality control band position is coated with mouse-anti HIS tag monoclonal antibody.
6. colloid gold test paper according to claim 5 is characterized in that: described beta-lactam antibiotic is at least a in following: benzyl penicillin, ampicillin, Amoxicillin, Oxacillin, Cloxacillin, nafcillin, dicloxacillin, Carbenicillin, methicillin, cefalexin, cephazoline, cefoperazone, ceftriaxone, CTX, cefoxitin, cefadroxil, Ceftiofur, Cefquinome.
7. according to claim 5 or 6 described colloid gold test papers, it is characterized in that: described carrier protein is OVA.
8. the method for beta-lactam antibiotic in the test sample, shown in following I or II:
The method of beta-lactam antibiotic comprises the steps: in I, the test sample
1) testing sample is carried out pre-treatment, obtain sample to be tested solution;
2) with arbitrary described receptor agents box among the claim 1-4 described sample to be tested solution is detected;
Described testing sample is a milk: with milk with sample diluting liquid by dilution in 1: 4,4 ℃, 10000rpm are centrifugal, get supernatant, as sample to be tested solution;
Described testing sample is a milk powder: take by weighing 1g milk powder, be dissolved in the 5mL sample diluting liquid, mix, as sample to be tested solution.
Described testing sample is a urine: with urine directly as sample to be tested solution;
Described testing sample is muscle, fish, shrimp or egg: with the testing sample homogenized, obtain equal quality sample, the equal quality sample of every 5g is added in the 10mL acetonitrile solution, concussion 10min turns upside down, the centrifugal 10min of 5000rpm gets supernatant, and nitrogen dries up, resuspended with the 1mL sample diluting liquid, the solution that obtains is sample to be tested solution; The volume ratio of acetonitrile and water is 9: 1 in the acetonitrile solution;
Described testing sample is a honey: every 5g testing sample is added in the 15mL deionized water, mix; Described mixed solution is carried out purifying with the Oasis solid-phase extraction column, use methanol-eluted fractions, collect eluent, nitrogen dries up, and resuspended with the 1mL sample diluting liquid, the solution that obtains is sample to be tested solution.
The method of beta-lactam antibiotic comprises the steps: in II, the test sample
1) testing sample is carried out pre-treatment, obtain sample to be tested solution;
2) with arbitrary described colloid gold test paper among the claim 5-7 described sample to be tested solution is detected;
Described sample diluting liquid is that 0.1mol/L, pH value are 7.2 phosphate buffer.
9.SEQ the application of albumen shown in the ID NO:1 in detecting beta-lactam antibiotic.
10. application according to claim 9 is characterized in that: described beta-lactam antibiotic is at least a in following: benzyl penicillin, ampicillin, Amoxicillin, Oxacillin, Cloxacillin, nafcillin, dicloxacillin, Carbenicillin, methicillin, cefalexin, cephazoline, cefoperazone, ceftriaxone, CTX, cefoxitin, cefadroxil, Ceftiofur, Cefquinome.
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| CN103864908A (en) * | 2013-08-02 | 2014-06-18 | 吉林省农业科学院 | Molecular modified penicillin-binding protein (PBP) capable of detecting beta-lactam antibiotic residues |
| CN103983775A (en) * | 2014-06-10 | 2014-08-13 | 无锡杰圣杰康生物科技有限公司 | Receptor-based immunochromatography test strip for detecting Beta-lactam antibiotics and preparation method of test strip |
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| CN103983775A (en) * | 2014-06-10 | 2014-08-13 | 无锡杰圣杰康生物科技有限公司 | Receptor-based immunochromatography test strip for detecting Beta-lactam antibiotics and preparation method of test strip |
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| CN110187117A (en) * | 2019-06-18 | 2019-08-30 | 清华大学深圳研究生院 | The detection kit and its application of beta-Lactam antibiotic |
| CN111961128A (en) * | 2020-05-28 | 2020-11-20 | 深圳市金阅科技有限责任公司 | Amoxicillin complete antigen and preparation method thereof, and reagent strip and preparation method thereof |
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