CN109293776B

CN109293776B - Fipronil specific binding protein and application thereof

Info

Publication number: CN109293776B
Application number: CN201811341754.8A
Authority: CN
Inventors: 杨春江; 赵荣茂; 吴迪; 袁志波; 邰宗洋; 宋世燕; 郭秀锋
Original assignee: Beijing Nabai Bio Tech Co ltd
Current assignee: Beijing Nabai Bio Tech Co ltd
Priority date: 2018-11-12
Filing date: 2018-11-12
Publication date: 2020-10-16
Anticipated expiration: 2038-11-12
Also published as: CN109293776A

Abstract

The embodiment of the invention discloses fipronil specific binding protein and application thereof, wherein the amino acid sequence of the protein is shown as SEQ ID NO.1, or the sequence is replaced, deleted or added with one or more amino acids to form an amino acid sequence with the same function. The protein is easy to prepare, can be specifically combined with fipronil, and has high specificity and sensitivity; in addition, the method can be used for detecting fipronil in a sample.

Description

Fipronil specific binding protein and application thereof

Technical Field

The invention relates to the technical field of fipronil detection, in particular to fipronil specific binding protein and application thereof.

Background

Fipronil (also known as Fipronil, CAS number 120068-37-3) is a phenylpyrazole pesticide with broad insecticidal spectrum, mainly has stomach poisoning effect on pests, has contact killing and certain systemic action, and has the action mechanism that the Fipronil is combined with gamma-aminobutyric acid receptors of insects to block chloride ion channels controlled by gamma-aminobutyric acid and prevent chloride metabolism from interfering with the central nervous system, so that the nerve and muscle of the insects are excessively excited to death. Fipronil is also used to kill fleas and lice on cats and dogs.

The daily allowable intake (ADI) of fipronil recommended by the European food safety agency in 2006 is 0.0002mg/kg, and the ADI recommended by China in the national food safety Standard-maximum limit of pesticide residue in food (GB 2763-2016) is also 0.0002 mg/kg. In 50 kinds of pesticide maximum residual limit (survey draft) such as pyraclostrobin in national standard food for food safety issued in 2016 in 11 months in China, the maximum residual limit of fipronil in poultry meat is regulated to be 0.01mg/kg, and the maximum residual limit in eggs is 0.02 mg/kg. In 2017, in 9 months, the Ministry of agriculture in China issued No. 2583 bulletin, which prohibits fipronil and related preparations from being used for food animals.

At present, fipronil detection mainly takes an instrument analysis method as a main method, and fipronil in various samples is quantitatively detected based on a high performance liquid chromatography or liquid chromatography-mass spectrometry combined technology, so that the instrument is expensive, complicated to operate and incapable of being applied to field detection; the immunological detection method has the defects of long antibody preparation period, high screening difficulty, complex process, high cost and the like.

Disclosure of Invention

The first purpose of the embodiments of the present invention is to provide a fipronil specific binding protein, which is easy to prepare, can be specifically bound with fipronil, and has high specificity and sensitivity.

The second purpose of the embodiment of the invention is to provide an application of fipronil specific binding protein in a fipronil detection kit and a colloidal gold detection test strip, wherein the application is simple to operate and can be used for quickly and accurately detecting fipronil in a sample; in addition, the field detection of the sample is realized.

In order to achieve the above purpose, the embodiment of the present invention provides a protein specifically binding to fipronil, wherein the amino acid sequence of the protein is shown as SEQ ID No.1, or the sequence is formed by replacing, deleting or adding one or more amino acids to form an amino acid sequence with equivalent functions.

The preparation method of the protein is not strictly limited in the invention, and for example, the protein can be obtained by an artificial synthesis method, specifically, a corresponding gene sequence obtained by bioinformatics screening is shown as SEQ ID No.2, and is obtained by expressing the gene sequence.

The other aspect of the embodiment of the invention provides a fipronil detection kit, which comprises a kit body, an ELISA plate and a reagent, wherein the ELISA plate and the reagent are detachably arranged in the kit body, and each hole of the ELISA plate is coated with the protein; the reagent comprises enzyme-labeled fipronil, fipronil standard solution, developing solution, stop solution, washing solution and sample diluent.

Preferably, the ELISA plate is prepared by the following steps:

diluting the protein to 0.5-2 mu g/ml by adopting a carbonate buffer solution, adding 50-100 mu L of the protein diluent into each hole of an enzyme label plate, and incubating overnight at the temperature of 3-5 ℃; spin-drying the protein diluent, washing the plate for 2-4 times by using a phosphate Tween buffer solution, adding a confining liquid into the ELISA plate, incubating for 1.5-2.5h at 35-39 ℃, and spin-drying the confining liquid to obtain the ELISA plate;

wherein the pH value of the carbonate buffer solution is 9.4-10; preferably 9.6; the blocking solution is 0.01M pH7.2PBS solution containing 0.05% bovine serum albumin and 5% casein or 0.01M pH7.2PBS solution containing 1% gelatin.

Preferably, the enzyme-labeled fipronil is a horseradish peroxidase-labeled fipronil hapten. More preferably, the horseradish peroxidase-labeled fipronil hapten is prepared by the following steps:

(1) dissolving fipronil hapten in N, N-dimethylformamide solution, and diluting by adopting PBS buffer solution; preferably, the mass-volume ratio of the fipronil hapten to the N, N-dimethylformamide solution is (25-28) to 1 mg/ml; the concentration of the fipronil hapten diluted by PBS buffer solution is 3.1-3.5 mg/ml;

(2) adding horseradish peroxidase into the fipronil hapten solution, and fully dissolving; preferably, the mass ratio of the addition amount of the horseradish peroxidase to the fipronil hapten is 25: 12-14;

(3) adding a glutaraldehyde solution into the dissolved mixed solution, stirring, and then dropwise adding saturated ammonium sulfate with the same volume under the stirring condition for reaction; preferably, the mass concentration of the glutaraldehyde solution is 4% -5%, and the volume ratio of the addition amount of the glutaraldehyde solution to the fipronil hapten solution is 150: 6-10; the stirring temperature is room temperature, and the stirring time is 3.5-4.5 h; the reaction temperature is 3-5 ℃, and the reaction time is 0.8-1.2 h;

(4) centrifuging the reacted solution, collecting precipitate, cleaning, dissolving the precipitate in PBS solution, dialyzing by using a dialysis bag, centrifuging, and collecting supernatant to obtain the enzyme-labeled fipronil hapten; preferably the cleaning solution is a semi-saturated ammonium sulfate solution; the pH of the PBS solution is 7.4, and the concentration is 0.15 mol/L; the molecular weight of the dialysis bag is 10 KDa; the centrifugal speed is 3000 plus 15000r/min, and the centrifugal time is 25-35 min.

Preferably, the fipronil standard solution comprises fipronil solutions with concentrations of 0ng/ml, 0.1ng/ml, 0.3ng/ml, 0.9ng/ml, 2.7ng/ml and 8.1ng/ml respectively.

Preferably, the color developing solution is a single-component TMB color developing solution.

Preferably, the stop solution is a sulfuric acid solution with the concentration of 0.2-2M.

Preferably, the washing solution is 0.01M PBS solution with pH7.2 containing 0.5% Tween-20.

Preferably, the sample dilution is 0.1M PBS solution at pH 7.2.

The embodiment of the invention also provides a fipronil colloidal gold test strip, which comprises a PVC (polyvinyl chloride) back plate, a nitrocellulose membrane, a gold-labeled pad, a sample pad and a water absorption pad, wherein the gold-labeled pad is sprayed with the protein labeled by colloidal gold; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a fipronil antigen, and the quality control line is coated with a mouse anti-His tag antibody.

Preferably, the nitrocellulose membrane in the test strip is adhered to a PVC back plate; a gold label pad is adhered to the detection line (T line) end of the nitrocellulose membrane, the distance between the upper end of the gold label pad and the T line is 4-10mm, and the distance is 1-3mm coincident with the nitrocellulose membrane; the sample pad is stuck on the gold label pad and is superposed with the gold label pad by 1-3 mm; the end of the quality control line (line C) of the nitrocellulose membrane is adhered with a water absorption pad, the distance between the water absorption pad and the line C is 4-10mm, and the water absorption pad and the nitrocellulose membrane are superposed by 1-3 mm.

In the invention, the source of the colloidal gold is not strictly limited, and the colloidal gold can be purchased from the market and can also be prepared by a sodium citrate reduction method; specifically, 0.01% chloroauric acid aqueous solution is heated to boiling, 1% trisodium citrate aqueous solution is added under the condition of continuous stirring, the solution is continuously stirred and heated until the solution is in a bright wine red color, the solution is cooled at room temperature, and then deionized water is used for recovering the solution to the original volume to obtain colloidal gold particles with the particle size of 30-40 nm; wherein the volume ratio of the sodium citrate aqueous solution to the chloroauric acid aqueous solution is 1.5-2.5%.

Preferably, the colloidal gold-labeled protein is prepared by the following steps:

(1) adding K to the colloidal gold solution₂CO₃A solution; preferably, the mass concentration of the colloidal gold solution is 1% -2%; the concentration of the potassium carbonate solution is 0.08-0.12 mol/L; the volume ratio of the colloidal gold solution to the potassium carbonate solution is 500: 1-10;

(2) adding the protein solution into the colloidal gold solution for incubation; then, adding bovine serum albumin into the incubated solution, uniformly mixing, centrifuging and collecting precipitates; preferably, the concentration of the protein solution is 0.03-0.05mg/ml, and the volume ratio of the addition amount to the colloidal gold solution is 500: 5-9; the incubation temperature is room temperature, and the incubation time is 18-22 min; the concentration of the bovine serum albumin in the solution added with the bovine serum albumin is 0.2 percent g/ml; the centrifugal speed is 9000-12000r/min, and the centrifugal time is 35-45 min;

(3) re-dissolving the precipitate by using a phosphate buffer solution to obtain the protein marked by the colloidal gold particles; preferably, the phosphate buffer is a 0.02M phosphate buffer containing 1% sucrose, 1% trehalose, 2% BSA, 0.02% sodium azide.

Preferably, the preparation method of the fipronil antigen comprises the following steps:

(1) mixing fipronil, succinic anhydride and anhydrous pyridine, and stirring; preferably, the mass ratio of the fipronil to the succinic anhydride is (34-36) to 16; the mass volume ratio of the fipronil to the anhydrous pyridine is (1.7-1.8) to 40 g/ml;

(2) heating and refluxing the mixture, adding ice water, adjusting the pH value, and storing overnight; preferably, the heating reflux time is 3.5-4.5h, and is used for removing pyridine; the volume ratio of the addition amount of the ice water to the anhydrous pyridine is 3: 3-5; adjusting the pH to 2.5-3.5; the preservation temperature is 3-5 ℃;

(3) filtering the solution after the overnight, washing with ice water, filtering again, repeating the washing with ice water, and filtering again for 2-4 times to obtain fipronil hapten;

(4) dissolving fipronil hapten in anhydrous N, N '-dimethylformamide, then adding N, N' -dicyclohexylcarbodiimide into the fipronil hapten solution, stirring, adding N-hydroxysuccinimide into the fipronil hapten solution after stirring, and carrying out a light-resistant reaction at room temperature under the stirring condition; preferably, the mass-volume ratio of the fipronil hapten to the anhydrous N, N' -dimethylformamide is (80-81) to 3 mg/ml; the mass ratio of fipronil hapten to N, N' -dicyclohexylcarbodiimide to N-hydroxysuccinimide is (80-81) to 31: 20; stirring for 9-11 min; reacting for 7-9h in a dark place;

(5) centrifuging the solution after the reaction is finished, collecting supernatant, dropwise adding the supernatant into a carrier protein solution prepared by buffer solution, and stirring overnight; preferably, the carrier protein is ovalbumin or bovine serum albumin; the buffer solution is 0.1mol/l phosphate buffer solution with pH of 8.0; the concentration of the carrier protein solution is 9-11 mg/ml; the volume ratio of the carrier protein solution to the anhydrous N, N' -dimethylformamide in the step (4) is 5: 2-4;

(6) dialyzing and purifying the overnight product by using a dialysis bag to obtain fipronil antigen; preferably, the dialysis bag has a molecular weight cut-off of 10 kDa.

The embodiment of the invention also provides an application of the kit or the test strip in detecting fipronil residues in a sample.

The embodiment of the invention has the following advantages:

(1) the fipronil specific binding protein provided by the invention is easy to prepare, can be specifically bound with fipronil, and has high specificity and sensitivity.

(2) The fipronil specific binding protein is applied to the fipronil detection kit and the colloidal gold detection test strip, the application operation is simple, the detection cost is low, and the fipronil in a sample can be quickly and accurately detected; and the field detection of the sample is realized.

Drawings

FIG. 1 is a standard curve diagram of a fipronil detection kit provided in example 4 of the present invention;

fig. 2 is a structural diagram of a fipronil colloidal gold test strip provided in embodiment 7 of the present invention.

In the figure: 1-sample pad; 2-gold label pad; 3-nitrocellulose membrane; 4-absorbent pad; 5-detection line; 6-quality control line; 7-PVC backing sheet.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

Example 1

The embodiment is a preparation method of a protein with an amino acid sequence shown as SEQ ID NO.1, which comprises the following steps:

obtaining a corresponding gene sequence shown as SEQ ID NO.2 through bioinformatics means screening;

carrying out PCR amplification on the gene sequence of SEQ ID NO.2, wherein the specific reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, 30 cycles, extension for 5min, and termination at 4 ℃;

carrying out double enzyme digestion on the PCR amplification product and pET28a respectively, mixing the products after enzyme digestion, adding T4DNA ligase at 4 ℃ overnight, then transforming the ligation product into a cloning host DH5 alpha competent cell, and naming the positive recombinant plasmid as pET28a-GABA-s alpha 1, and then transforming the cloning ligation product into an expression host BL21 competent cell for expression;

taking pET28a-GABA-s alpha 1 glycerobacteria expressing GABA receptor protein out from minus 20 ℃, adding the glycerobacteria into LB culture medium according to the proportion of 1:1000 after thawing, adding antibiotics according to the proportion of 1:1000, and then culturing for 18h at 37 ℃ and 220rpm to obtain seed liquid;

transferring the seed solution according to the proportion of 1:100, adding antibiotics according to the proportion of 1:1000, adding IPTG into the culture medium after the OD value is 0.7 according to the proportion of 1:1000, inducing overnight at 18 ℃ and 180rpm, then centrifuging the bacteria induced overnight at 5000rpm for 10min, collecting precipitates, suspending the precipitates by using Buffer A, transferring the precipitates into a vessel, adding a protease inhibitor into the bacteria, uniformly mixing, carrying out ultrasonic crushing for 30min, transferring the crushed bacteria into a centrifugal tube for balancing, and centrifuging at 15000rpm for 30min at 4 ℃; wherein, the Buffer A is 50mM Hepes500mM NaCl pH7.5;

collecting the centrifuged supernatant, filtering and removing impurities through a 0.22 mu m filter, loading the filtered supernatant onto a balanced Ni column through a peristaltic pump, performing gradient elution by using an elution Buffer B, wherein the gradient elution is 0% → 10% → 100%, the elution Buffer B is 50mM Hepes500mM NaCl 500mM imidazole pH7.5, and collecting a sample at a separation peak to obtain the protein with the amino acid sequence shown in SEQ ID NO. 1.

Example 2

This example is the preparation of an elisa plate, which comprises the following steps:

diluting the protein with the amino acid sequence shown as SEQ ID NO.1 to 1 mu g/ml by using a carbonate buffer solution with the pH value of 9.6, adding 100 mu L of the protein diluent into each hole of an ELISA plate, and incubating overnight at the temperature of 4 ℃; and then, spin-drying the protein diluent, washing the plate for 3 times by using a phosphate Tween buffer solution, adding a 0.01M pH7.2PBS solution containing 1% gelatin into each hole of the ELISA plate, incubating for 2 hours at the temperature of 37 ℃, and spin-drying the confining liquid to obtain the ELISA plate.

Example 3

The embodiment is a preparation method of horseradish peroxidase-labeled fipronil hapten, which comprises the following steps:

(1) dissolving 26.86mg of fipronil hapten in 1ml of N, N-dimethylformamide solution, and diluting to 8ml by adopting PBS buffer solution;

(2) adding horseradish peroxidase 50mg into the fipronil hapten solution, and stirring until the horseradish peroxidase is fully dissolved;

(3) adding 150 mu L of 4.5% glutaraldehyde solution into the dissolved mixed solution, stirring at room temperature for 4h, then continuing stirring, dropwise adding equal volume of saturated ammonium sulfate, and reacting at 4 ℃ for 1 h;

(4) centrifuging the reacted solution at 3000r/min for 30min, collecting precipitate, washing with half-saturated ammonium sulfate for 1-3 times, dissolving the precipitate in 0.15mol/LpH7.4 PBS, dialyzing with dialysis bag with molecular weight of 10KDa, centrifuging at 10000r/min for 30min, and collecting supernatant to obtain enzyme-labeled fipronil hapten.

Example 4

The embodiment is a fipronil detection kit, which comprises a kit body, a 96-hole detachable elisa plate and a reagent, wherein the elisa plate and the reagent are arranged in the kit body and are prepared in the embodiment 2; the reagent comprises the enzyme-labeled fipronil prepared in the embodiment 3, a fipronil standard solution, a developing solution, a stop solution, a washing solution and a sample diluent;

wherein the fipronil standard solution comprises fipronil solutions with the concentrations of 0ng/ml, 0.1ng/ml, 0.3ng/ml, 0.9ng/ml, 2.7ng/ml and 8.1ng/ml respectively; the color developing solution is a single-component TMB color developing solution; the stop solution is a sulfuric acid solution with the concentration of 1M; the washing solution is 0.01M PBS solution with pH7.2 containing 0.5% Tween-20; the sample dilution was 0.1M PBS solution at pH 7.2.

Example 5

The amino acid sequence of the colloidal gold particle marker in the embodiment is SEQ ID No: 1, the preparation method comprises the following steps:

(1) 10ml of 1.5% colloidal gold solution were taken into a centrifuge tube and 100. mu.l of 0.1mol/L K were added₂CO₃A solution;

(2) adding 140 μ l of the protein solution prepared in example 1 with a concentration of 0.04mg/ml to the colloidal gold solution, and incubating at room temperature for 20 min; then, adding bovine serum albumin into the incubated solution to enable the final concentration of the bovine serum albumin to be 0.2% g/ml, uniformly mixing, and centrifuging at the temperature of 4 ℃ at 10000r/min for 40min to collect precipitates;

(3) re-dissolving the precipitate by using 2ml of 0.02mol/L phosphate buffer solution containing 1% of sucrose, 1% of trehalose, 2% of BSA and 0.02% of sodium azide to obtain the colloidal gold particle labeled amino acid sequence of SEQ ID No: 1.

Example 6

This example is a method for preparing fipronil antigen, which includes the following steps:

(1) 3.4972g of fipronil, 1.6g of succinic anhydride and 81ml of anhydrous pyridine are mixed and stirred uniformly;

(2) heating and refluxing the mixture for 4h, adding 60ml of ice water, adjusting the pH value to 3.0, and storing at 4 ℃ overnight;

(3) filtering the solution after the overnight, washing with ice water, filtering again, and repeating the above operation for 2-4 times to obtain fipronil hapten;

(4) 80.58mg of fipronil hapten is dissolved in 3ml of anhydrous N, N '-dimethylformamide, then 31.0mg of N, N' -dicyclohexylcarbodiimide is added into the fipronil hapten solution and stirred for 10min, after stirring is finished, 20.0mg of N-hydroxysuccinimide is added into the fipronil hapten solution and photophobic reaction is carried out for 8h at room temperature under the stirring condition;

(5) centrifuging the solution after the reaction is finished, collecting supernatant, dropwise adding the supernatant into 5ml of 10mg/ml bovine serum albumin solution prepared by 0.1mol/l phosphate buffer solution with pH8.0, and stirring overnight at 4 ℃;

(6) and (4) dialyzing and purifying the overnight product by adopting a dialysis bag with the molecular weight of 10KDa to obtain the fipronil antigen.

Example 7

The embodiment is a fipronil colloidal gold test strip, and the test strip comprises a PVC back plate 7, a nitrocellulose membrane 3, a gold-labeled pad 2, a sample pad 1 and a water absorption pad 4, wherein the amino acid sequence of the colloidal gold particle marker sprayed on the gold-labeled pad 2 prepared in the embodiment 5 is SEQ ID No: 1; the nitrocellulose membrane 3 is provided with a detection line 5 and a quality control line 6, the fipronil antigen prepared in the embodiment 6 is coated on the detection line, and a mouse anti-His tag antibody is coated on the quality control line;

wherein, the nitrocellulose membrane 3 is stuck on the PVC backboard 7; a gold mark pad 2 is stuck at the detection line 5(T line) end of the nitrocellulose membrane 3, the distance between the upper end of the gold mark pad 2 and the T line is 6mm, and the distance is overlapped with the nitrocellulose membrane 3 by 2 mm; the sample pad 1 is stuck on the gold label pad 2 and is overlapped with the gold label pad 2 by 2 mm; the end of the quality control line 6 (line C) of the nitrocellulose membrane 3 is stuck with a water absorption pad 4, and the distance between the water absorption pad 4 and the line C is 6mm and is coincided with the nitrocellulose membrane 3 by 2 mm.

Experimental example 1

The kit of embodiment 4 of the invention is adopted to detect fipronil in milk samples;

sample treatment: after shaking the milk sample uniformly, directly diluting the milk sample by using a sample diluent according to a ratio of 1: 50 to obtain a solution to be detected;

preparation of fipronil solution to be detected: dividing the solution to be detected into 4 parts, and adding fipronil into 3 parts of the solution to be detected respectively to obtain fipronil solutions to be detected, wherein the fipronil concentrations of the fipronil solutions are 0, 0.5ng/ml, 2ng/ml and 5ng/ml respectively;

the experimental method comprises the following steps:

(1) taking out the prepared ELISA plate, and standing at room temperature for 35 min;

(2) adding the standard substance and the solution to be detected of fipronil into the hole of an enzyme-labeled plate by 50 mu L per hole, and incubating for 30min at room temperature;

(3) taking out the enzyme-linked immunosorbent assay plate, washing the plate for 3 times, adding 50 mu L of enzyme-labeled fipronil, and incubating for 15min at room temperature;

(4) taking out the enzyme label plate, washing the plate for 3 times, adding 100 mu L of TMB color development solution, and incubating for 15min at room temperature;

(5) adding 50 μ L of stop solution, measuring OD value of each well at 450nm wavelength with enzyme-labeling instrument, and the measurement results are shown in Table 1;

(6) the OD value of a well containing 0ng/ml fipronil standard solution is positioned B₀(ii) a Positioning the OD values of the other fipronil standard solution holes B; with B/B₀Is a vertical coordinate, the logarithm C of the concentration C of the corresponding standard substance is a horizontal coordinate, and a fipronil standard curve is drawn as shown in figure 1; and the regression equation for obtaining the standard curve is that y is 0.0845x³+0.0707x²-0.4071 x + 0.3616; and the coefficient of correlation R²＝0.9973。

(7) And substituting the OD value of the to-be-detected solution hole containing the fipronil into a regression equation of a standard curve to qualitatively and quantitatively determine the concentration of the fipronil in the sample, wherein the detection result is shown in a table 2.

TABLE 1

TABLE 2

Fipronil concentration (ng/ml)	Test results (ng/ml)	Recovery (%)
			0	—	—
0.5	0.461	92.2
			2	1.754	87.7
5	4.819	96.38

As can be seen from tables 1 and 2: the detection sensitivity of the detection kit reaches 0.5ng/ml, and the recovery rate reaches 87.7-96.38%.

Experimental example 2

The fipronil colloidal gold test strip in the embodiment 7 is adopted to detect fipronil residues in the milk sample, and the specific operations are as follows:

the experimental method comprises the following steps:

(1) preparing the prepared colloidal gold test strip and the micropore reagent, taking out and placing at room temperature for 35 min;

(2) adding 200 mu L of the solution to be detected added with the fipronil into the micropore reagent, sucking and beating the solution up and down for three times by using a pipettor, fully and uniformly mixing the solution, and standing the solution for 5min at room temperature;

(3) taking out the prepared fipronil colloidal gold test strip, inserting the sample pad end into the uniformly mixed micropore reagent solution, enabling the solution to be detected to creep towards the water absorption pad end along the sample pad end of the test strip under the siphon action, and standing for 5min at room temperature;

(4) and (3) shearing off the end of the sample pad by using scissors or sucking the residual solution to be detected on the sample pad by using absorbent paper, and judging the detection result according to the color development of the C line and the T line on the colloidal gold test strip.

When the color of the C line on the colloidal gold test strip is the same as that of the T line or is deeper than that of the T line, the result is positive; when the color of the line C is lighter than that of the line T, the result is negative; if the C line is not developed, the result is invalid and a new test strip is required for retesting.

The detection results of the four additive concentrations of the sample to be detected are shown in table 3;

TABLE 3

Fipronil concentration (ng/ml)	Test results (ng/ml)
		0	—————
0.5	—————
		2	————+
5	+++++

As can be seen from Table 3: when the adding concentration is 5ng/ml, the detection results of the fipronil colloidal gold test strip are all positive, namely the detection limit of the fipronil colloidal gold test strip on fipronil can be determined to be 5 ng/ml.

Example 3

The fipronil detection kit and the colloidal gold detection test strip of the invention have specific detection

The procedure of example 9 was followed using the fipronil colloidal gold test strip of example 7 to detect imidacloprid with a concentration of 100 μ g/ml, dieldrin with a concentration of 100 μ g/ml, chlorpyrifos with a concentration of 100 μ g/ml, endrin with a concentration of 100 μ g/ml, and cypermethrin standard solution with a concentration of 100 μ g/ml, and the detection results are shown in table 4;

TABLE 4

Medicine and concentration (mug/ml)	The result of the detection
		Imidacloprid 100 microgram/ml	—————
100 mug/ml dieldrin	—————
		100 mu g/ml chlorpyrifos	—————
100 mug/ml endrin	—————
		Cypermethrin 100 mug/ml	—————

As can be seen from Table 4, the test strip of the present invention has no significant cross reaction to the above-mentioned unrelated drugs; namely, the specificity of the protein and the fipronil adopted by the invention is good.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

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gaaaacgaaa ccgactacca ggacgctttc tgctctatcg tttcttgctc tccgcagacc 360

gttcacccgg aaaccatcat cgacgtttct cgtcgtcgtt cttctctgat ctgcgacgtt 420

tacggtgtta acgaatctgt ttcttacgaa cgtggttcta tgaccgtttc tgttacctct 480

aaaccgtcta ccatggaacg tcagacccag accgaaatct ggatcccgaa atggcgtcag 540

ttcctgtact gcctggctgg tgacgaatct ttccgtaaac gtcgtcagcg tgaagctgct 600

ggtaactgcc gtaaacgtgg tgaaaaaatc gctcgtcaca tcaactctgt ttcttacatc 660

gacaaagttg ctcgtatcgt tttcccggct tctttcggtc tgctgaacgt ttgctactgg 720

aagctt 726

Claims

1. A protein specifically binding fipronil is characterized in that the protein is obtained by expression of a gene sequence shown as SEQ ID No. 2.

2. A fipronil detection kit comprises a kit body, a detachable ELISA plate and a reagent, wherein each hole of the ELISA plate is coated with the protein of claim 1; the reagent comprises enzyme-labeled fipronil, fipronil standard solution, developing solution, stop solution, washing solution and sample diluent.

3. The kit of claim 2, wherein the microplate is prepared by steps comprising:

the blocking solution is 0.01M PBS (pH7.2) solution containing 0.05% bovine serum albumin and 5% casein or 0.01M PBS solution containing 1% gelatin and pH7.2.

4. The kit of claim 2, wherein the enzyme-labeled fipronil is a horseradish peroxidase-labeled fipronil hapten.

5. The kit according to claim 4, wherein the horseradish peroxidase-labeled fipronil hapten is prepared by the following steps:

(1) dissolving fipronil hapten in N, N-dimethylformamide solution, and diluting by adopting PBS buffer solution;

(2) adding horseradish peroxidase into the fipronil hapten solution, and fully dissolving;

(3) adding a glutaraldehyde solution into the dissolved mixed solution, stirring, and then dropwise adding saturated ammonium sulfate with the same volume under the stirring condition for reaction;

(4) and centrifuging the reacted solution, collecting precipitate, cleaning, dissolving the precipitate in a PBS solution, dialyzing by using a dialysis bag, centrifuging and collecting supernatant to obtain the enzyme-labeled fipronil hapten.

6. A fipronil colloidal gold test strip, comprising a PVC back plate, a nitrocellulose membrane, a gold-labeled pad, a sample pad and a water absorption pad, wherein the gold-labeled pad is sprayed with the colloidal gold-labeled protein of claim 1; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a fipronil antigen, and the quality control line is coated with a mouse anti-His tag antibody.

7. The test strip of claim 6, wherein the colloidal gold-labeled protein is prepared by the steps comprising:

(1) adding K to the colloidal gold solution₂CO₃A solution;

(2) adding the protein solution into the colloidal gold solution for incubation; then, adding bovine serum albumin into the incubated solution, uniformly mixing, centrifuging and collecting precipitates;

(3) and re-dissolving the precipitate by using a phosphate buffer solution to obtain the protein marked by the colloidal gold particles.

8. The test strip of claim 6, wherein the fipronil antigen is prepared by the steps of:

(1) mixing fipronil, succinic anhydride and anhydrous pyridine, and stirring;

(2) heating and refluxing the mixture, adding ice water, adjusting the pH value, and storing overnight;

(3) filtering the solution after the overnight, reducing pressure, washing with ice water, and filtering to obtain fipronil hapten;

(4) dissolving fipronil hapten in anhydrous N, N '-dimethylformamide, then adding N, N' -dicyclohexylcarbodiimide into the fipronil hapten solution, stirring, adding N-hydroxysuccinimide into the fipronil hapten solution after stirring, and carrying out a light-resistant reaction at room temperature under the stirring condition;

(5) centrifuging the solution after the reaction is finished, collecting supernatant, dropwise adding the supernatant into a carrier protein solution prepared by buffer solution, and stirring overnight;

(6) and purifying the overnight product to obtain the fipronil antigen.