Detection kit for detecting phenylethanolamine A based on ILAMP and detection method thereof

Abstract

The invention discloses a detection kit for detecting phenylethanolamine A based on ILAMP and a detection method thereof, and relates to the field of biological detection.According to the method, an anti-PEAA polyclonal antibody is obtained by immunizing an animal, a PEAA indirect competitive ELISA method is established on the basis, an enzyme-linked immunosorbent assay (ELISA) and fluorescent quantitative PCR are combined, an immune-PCR detection method is established, an LAMP technology is added, and the detection kit and the detection method for detecting phenylethanolamine A based on ILAMP are obtained. The phenylethanolamine A is successfully detected; compared with indirect competitive ELISA, the linear detection range, IC50 and minimum detection limit of a standard curve of the method are improved by one order of magnitude; according to the present invention, the coefficient of variation (CV) does not exceed 10%, the method has strong reliability, and the ILAMP specific detection result shows that the method has high specificity on PEAA, and has no cross reaction (CRlt; 0.04%).

Description

Technical Field

[0001] The invention relates to the field of biological detection, in particular to a detection kit for detecting phenylethanolamine A based on ILAMP and a detection method thereof. Background Art

[0002] Phenylethanolamine A (PEAA) accumulates in the human body through the food chain, and when it reaches a certain amount, it will pose a great threat to human health. At present, the Ministry of Agriculture and Rural Affairs has explicitly prohibited the addition of this substance to animal feed and animal drinking water.

[0003] At present, there are a large number of research methods for detecting the residues of β-receptor agonists, and phenylethanolamine A is also a β-receptor agonist. The research on its detection methods can be mainly divided into the following categories: Wang Ruiguo et al.

[54] The established ultra-high performance liquid chromatography (UPLC) uses formic acid-methanol mixture to measure phenylethanolamine A in different feeds. This method has a good detection limit and quantitative line (0.3 mg / kg and 1.0 mg / kg). Liquid chromatography-mass spectrometry technology uses chromatography as a separation system and mass spectrometry as a detection system. It combines the advantages of strong chromatographic separation ability, mass spectrometry selectivity and high sensitivity. At present, liquid chromatography-mass spectrometry has been used as the main method for detecting phenylethanolamine A. It has the advantages of low detection limit, accurate detection results and fast detection speed. However, this method has high requirements for detection technology, expensive instruments and high detection costs, which are also problems that cannot be ignored.

[0004] As the times progress, people are paying more and more attention to toxic and harmful substances in food, so the accuracy of detection methods is still an area that needs to be broken through. In 1992, Sano et al. first established the antigen-antibody reaction system of immune PCR. The immune PCR technology combines the high specificity of ELISA and the amplification of PCR. Compared with traditional ELISA, its sensitivity is increased by 10 3 -10 4times, and has a wide linear dynamic range of six orders of magnitude. ImmunoPCR is to replace the signal marker from protein to nucleic acid, use specific double-stranded or single-stranded DNA fragments to label antibodies, then PCR amplify DNA reporter molecules, and finally detect. Through the quantitative relationship, the amount of PCR products can reflect the amount of antigen molecules. General immune PCR reactions are composed of four systems: antigen-antibody reaction, bridging, indication and detection system. Loop-mediated isothermal amplification technology is a new type of nucleic acid isothermal amplification method established by Notomi et al. in 2000. This method does not require expensive PCR instruments, and can complete the exponential amplification of nucleic acids at a constant temperature of 60-65°C, and it takes very little time. The realization of LAMP technology is mainly through 4 special primers and Bst DNA polymerase. Among them, the 4 special primers can specifically recognize the target sequence, and Bst DNA polymerase has the characteristics of strand displacement. The results of the LAMP reaction can be directly viewed by the naked eye, because during the reaction, a large amount of pyrophosphate ions will be produced as DNA is synthesized, and they can form white magnesium pyrophosphate precipitates with magnesium ions in the reaction solution. Due to its high specificity, high sensitivity, high amplification efficiency and simple operation, LAMP technology has been applied in various fields. For example, in the detection of bacterial diseases, there is an existing technology for Escherichia coli O 157 Primers were designed for the eight specific target regions in the LAMP reaction, which increased the sensitivity by more than 10 times compared with the PCR method; in the detection of viral diseases, Huang Shulin and others used LAMP technology to detect porcine parvovirus and found that it was 100 times more sensitive than the ordinary PCR method; in the detection of mycoplasma diseases, there is an existing technology that designed LAMP primers with the uvrC target sequence of bovine mycoplasma, which also increased the sensitivity by more than 100 times. The development and research of the above-mentioned LAMP technology reflects its broad prospects in animal pathogen detection. Therefore, it is very feasible to apply LAMP, which has the characteristics of high sensitivity, high specificity and the ability to realize visualization technology, to immune PCR to create a new detection technology immune LAMP; the present invention solves such problems. Summary of the invention

[0005] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide a detection kit and a detection method for detecting phenylethanolamine A based on ILAMP. The present invention obtains anti-PEAA polyclonal antibodies by immunizing animals, and establishes a PEAA indirect competitive ELISA method on this basis. Then, the enzyme-linked immunosorbent assay (ELISA) and fluorescent quantitative PCR are combined to establish an immune-PCR detection method, and then the LAMP technology is added to successfully detect phenylethanolamine A. The method has high reliability and high sensitivity.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] Based on ILAM 2μg / mL PEAA-OVA, 2.1mg / mL coated Bio-PEAA pAb, 28.2ng / mL Bio-DNA, 2.5μL template, 0.5μL outer primers at a concentration of 0.2mmol / L, 4μL inner primers at a concentration of 1.6mmol / L, 2μL loop primers at a concentration of 0.8mmol / L, 3.5μL dNTPs at a concentration of 1.4mmol / L, 0.5μL MnCl at a concentration of 500μmol / L 2 , 3 μL of 0.6 mol / L betaine, 1 μL of 200 μmol / L calcein, 0.5 μL of 8 mmol / L MgSO 4 , 0.5 μL 10 mmol / L KCl, 1 μL 20 mmol / L Tris-HCl, 1.0 μL Bst DNA polymerase, 0.5 μL 10 mmol / L (NH 4 ) 2 SO 4 , 0.5μL 0.1% Tween20, 4μL ddH 2 O;

[0008] The outer primer F3 is 5'-GTAACGCCAGGGTTTTCC-3' SEQ ID No: 01, and B3 is 5'CAGCTATGACCATGATTACG-3' SEQ ID No: 02;

[0009] Internal primers: FIP upstream primer is: 5'-GTACCCGGGGATCCTCTAGAG-3' SEQ ID No: 03 and FIP downstream primer is 5'-TTTTCCAAGCTATTTAGGTGACACT-3' SEQ ID No: 04; BIP upstream primer is 5'-TCGAATTCGCCCTATAGTGAGTCTTTT-3' SEQ ID No: 05 and BIP downstream primer is 5'-GTCACGACGTTGTAAAACG-3' SEQ ID No: 06;

[0010] Loop primer: LB is 5'-GCAGGCATGCAAGCTTGAG-3' SEQ ID No: 07.

[0011] The method for detecting phenylethanolamine A based on ILAMP includes:

[0012] Step 1: obtain the amino derivative PEAA-NH 2 ;

[0013] Step 2: PEAA-NH 2 The coating source PEAA-OVA and the immunogen PEAA-BSA were synthesized by diazotization reaction with ovalbumin OVA and bovine serum albumin BSA respectively;

[0014] Step 3, preparing rabbit polyclonal antibodies, and obtaining polyclonal antibody PEAA pAbs through titer determination;

[0015] Step 4, establishing an indirect competitive ELISA method for phenylethanolamine A, and then establishing a standard curve to detect phenylethanolamine A;

[0016] Step 5: Establish the LAMP method and use a fluorescence quantitative PCR instrument to detect the fluorescence start time.

[0017] The aforementioned method for detecting phenylethanolamine A based on ILAMP, step 1, by Raney nickel reduction method to obtain phenylethanolamine A amino derivative PEAA-NH 2 The specific steps include:

[0018] (a) Add 2.0 g of phenylethanolamine A to 10 mL of methanol and stir at room temperature for 1 h to dissolve;

[0019] (b) weighing 0.3 g of Raney nickel, adding it to the solution dissolved in step (a), and reacting it under 4 MPa hydrogen condition for 8 h;

[0020] (c) The obtained reaction product was filtered, dried by spin drying, and recrystallized with appropriate amounts of ethyl acetate and petroleum ether to obtain the amino derivative of phenylethanolamine A, PEAA-NH 2 ,use 13 C NMR, 1 The molecular structure was confirmed by H NMR and mass spectrometry.

[0021] The aforementioned method for detecting phenylethanolamine A based on ILAMP, step 2, PEAA-NH 2 The specific steps of respectively carrying out diazotization reaction with ovalbumin OVA and bovine serum albumin BSA to synthesize the coating source PEAA-OVA and the immunogen PEAA-BSA include:

[0022] (1) Weigh 18.9 mg PEAA-NH 2 , add 1.2 mL of sulfuric acid and dissolve at 4°C for 20 min;

[0023] (2) Add 0.3 mL of sodium nitrite solution dropwise to the solution obtained in step (1) on crushed ice while stirring, then add an appropriate amount of ammonium sulfamate, and detect with starch potassium iodide paper. Stop adding when the oozing circle becomes colorless, and stir at 4°C for 30 minutes.

[0024] (3) Weigh 150 mg of BSA and dissolve it in 4 mL of carbonate buffer. 2 The solution was added dropwise and stirred at 4°C overnight;

[0025] (4) The reaction product of step (3) was placed in a dialysis bag and dialyzed in 0.01 mol / L PBS buffer for 4 days, with the dialysate replaced twice a day; the dialyzed solution was stored at -20°C for later use; the same method was used to synthesize the original PEAA-OVA coating.

[0026] The aforementioned method for detecting phenylethanolamine A based on ILAMP, step 4, establishing an indirect competitive ELISA method for phenylethanolamine A, and then establishing a standard curve for detecting phenylethanolamine A. The specific method includes:

[0027] 1. Calculate the binding rate (B / B) according to the following formula (2-1): 0 value), and then draw a standard curve;

[0028] Binding rate (%) = B / B 0 ×100%(1-1),

[0029] Among them, B and B 0 are the OD values ​​measured when the standard was added and the RAC concentration was 0. 450nm ;

[0030] 2. According to formula (2-2), the average value of the experimentally measured phenylethanolamine A concentration is compared with the actual added phenylethanolamine A concentration to obtain the added recovery rate;

[0031] Recovery rate (%) = measured sample concentration / sample added concentration × 100% (1-2).

[0032] In the aforementioned method for detecting phenylethanolamine A based on ILAMP, the specific steps of establishing the LAMP method in step five include: optimizing the working concentration of coated PEAA-OVA, optimizing the working concentration of coated Bio-PEAA pAb, optimizing the working concentration of Bio-DNA, and establishing PEAA immune-LAMP after optimizing the LAMP reaction system.

[0033] The above-mentioned method for detecting phenylethanolamine A based on ILAMP, the specific method for optimizing the working concentration of coated PEAA-OVA and the working concentration of coated Bio-PEAA pAb is: using 7500, 15000, 30000 times diluted PEAA-OVA and 500, 1000, 2000, 4000 times diluted Bio-PEAA pAb in 1% glutaraldehyde treated PCR tubes for chessboard experiment: PEAA-OVA with different dilutions were coated in glutaraldehyde treated PCR tubes, 50μL / tube, incubated overnight at 4°C; the liquid in the tube was poured out and washed once with PBST; blocking solution was added, 200μL / tube, blocked at 37°C for 2h; the blocking solution was poured out, and 0ppb and 1ppb PEAA standards (50μl / tube) and different dilutions of Bio-PEAA were added respectively. pAb (50 μl / tube), incubate at 37°C for 1 h; pour out the liquid in the tube, add 2000-fold diluted SA (50 μl / tube), incubate at 37°C for 1 h; pour out the reaction liquid Bio-DNA (50 μl / tube) again, incubate at 37°C for 1 h; pour out the liquid in the tube, wash with PBST and sterile water for 5 times respectively, add LAMP system, react, and monitor in real time with fluorescent quantitative PCR instrument; the optimal antigen-antibody working concentration is the one with the smallest Tt value of 0 ppb of PEAA standard and the largest Tt value difference (ΔTt) between 0 ppb and 1 ppb.

[0034] The aforementioned method for detecting phenylethanolamine A based on ILAMP, the method for optimizing the LAMP reaction system includes: designing LAMP primers, optimizing the ratio of external primers to internal primers, optimizing the optimal LAMP reaction temperature, and optimizing the optimal amount of Bst polymerase added;

[0035] Design method of LAMP primers: Primer Explorer version 4.0 system was used, and a 209 bp fragment on the pGEM-3Z vector plasmid DNA was used as the target DNA. Primer premier 5.0 and Beacon Designer 7.9 were used to design the loop primer and inner and outer primers of LAMP respectively.

[0036] Optimization method of the ratio of external and internal primers: the concentration ratio of external and internal primers was set to 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, and the optimal ratio of external and internal primers was determined by comparing the fluorescence results;

[0037] Optimization method for the best LAMP reaction temperature: set the reaction temperature gradient to 60-66°C, and determine the best reaction temperature by comparing the time to receive the fluorescence value;

[0038] Method for optimizing the amount of Bst polymerase added: the amount of Bst enzyme added was set to 2U, 4U, 6U, 8U, 10U, and 12U respectively; the optimal amount of Bst polymerase added was determined by comparing the fluorescence results.

[0039] Based on the aforementioned method for detecting phenylethanolamine A by ILAMP, the method for establishing PEAA immuno-LAMP includes:

[0040] PEAA-OVA with the best working concentration was coated in a PCR tube pretreated with 1% glutaraldehyde, 50 μL / tube; after coating at 4°C for 24 hours, the liquid in the plate was discarded, washed once with PBST, and patted dry; blocking solution (200 μL / tube) was added; after incubation at 37°C for 2 hours, the blocking solution was poured out, washed 3 times with PBST, and patted dry; Bio-PEAA pAb with the best working concentration and PEAA standards of different concentrations were added, 50 μL / tube respectively; after incubation at 37°C for 1 hour, the reaction liquid in the tube was discarded, washed 3 times with PBST, and patted dry; SA with the best working concentration was added, 50 μL / tube; after incubation at 37°C for 1 hour, the liquid was poured out, and the plate was washed 3 times with PBST, and patted dry; Bio-DNA with the best working concentration was added, 50 μL / tube; after incubation at 37°C for 1 hour, the liquid was poured out; after washing 10 times, i.e., 5 times with PBST and sterilized ultrapure water respectively, amplified according to the LAMP reaction system and procedure;

[0041] The steps of establishing the immune LAMP method are as follows: dilute PEAA-OVA to 1:7500 (2 μg / mL) with CB coating solution, and coat in PCR tubes pretreated with 1% glutaraldehyde, 50 μL / tube; discard the liquid in the plate after coating at 4°C for 24 hours, wash once with PBST, and pat dry; add blocking solution (200 μL / tube); incubate at 37°C for 2 hours, pour off the blocking solution, wash 3 times with PBST, and pat dry; add standard and optimal working concentration of Bio-PEAA pAb (2.1 μg / mL), 50 μL / tube; after incubation at 37°C for 1 hour, discard the reaction solution in the tube, wash 3 times with PBST, and pat dry; add SA (1.5 μg / mL) with the best working concentration, 50 μL / tube; after incubation at 37°C for 1 hour, pour out the liquid and wash 3 times with PBST; add Bio-DNA (28.2 ng / mL) diluted to 1:1600, 50 μL / tube; after incubation at 37°C for 1 hour, pour out the liquid and wash 5 times with PBST and sterilized ultrapure water respectively, add LAMP system, and then react at a constant temperature of 65°C for 60 minutes, and use fluorescence quantitative PCR instrument to detect the fluorescence start time.

[0042] The aforementioned method for detecting phenylethanolamine A based on ILAMP, the LAMP system includes: 2.5 μL template, 0.5 μL outer primer with a concentration of 0.2 mmol / L, 4 μL inner primer with a concentration of 1.6 mmol / L, 2 μL loop primer with a concentration of 0.8 mmol / L, 3.5 μL dNTPs with a concentration of 1.4 mmol / L, 0.5 μL MnCl with a concentration of 500 μmol / L 2 , 3 μL of 0.6 mol / L betaine, 1 μL of 200 μmol / L calcein, 0.5 μL of 8 mmol / L MgSO 4 , 0.5 μL 10 mmol / L KCl, 1.0 μL 20 mmol / L Tris-HCl, 1.0 μL Bst DNA polymerase, 0.5 μL 10 mmol / L (NH 4 ) 2 SO 4 , 0.5μL 0.1% Tween 20, ddH 2 Make up to 25 μL with HO.

[0043] The present invention is beneficial in that:

[0044] The present invention obtains phenylethanolamine A amino derivative, namely PEAA-NH 2 PEAA-NH 2 The coating source PEAA-OVA and the immunogen PEAA-BSA were synthesized by diazotization reaction with ovalbumin (OVA) and bovine serum albumin (BSA), respectively. Then, New Zealand white rabbits were immunized by conventional immunization method, and polyclonal antibody PEAA pAbs (1# and 5#) were obtained by titer determination. In subsequent experiments, an indirect competitive ELISA method for phenylethanolamine A was established, and then a standard curve was established to detect phenylethanolamine A. The synergy between the various steps was achieved, making the IC of this method 50 The value is 0.401ng / mL, IC 10 The cross-reaction rate of the phenylethanolamine A was 0.035 ng / mL, and the cross-reaction rate of the phenylethanolamine A was very high, and the cross-reaction rates of the other 11 β-receptor agonists were all below 0.01%; in the actual sample detection, the recovery rate was 86.2-116.6%, the intra-plate variation coefficient was less than 4.41%, and the inter-plate variation coefficient was less than 9.58%, indicating the reliability of the present invention;

[0045] The present invention optimizes the LAMP system, including the optimization of the ratio of external and internal primers, reaction temperature and the amount of Bst polymerase added, as well as the optimization of the optimal working concentration of the coated antigen and biotin-labeled antibody ILAMP system. The optimized system is coordinated with the PEAA indirect competitive ILAMP method in terms of specificity; the experimental results show that the linear equation of the standard curve of the method is: y=1.1949x+42.251, R 2 =0.956. IC 50 The detection limit was 0.0396±0.009ng / mL, which was one order of magnitude higher than that of indirect competitive ELISA. The lowest detection limit was 0.0044ng / mL, which was also one order of magnitude higher. The specificity experiment results using ILAMP showed that ILAMP also had high specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The synthetic route of the present invention is (a) PEAA technical route; (b) PEAA-NH2 and PEAA-BSA synthesis method;

[0047] Figure 2 This is a schematic diagram of the titer of the phenylethanolamine A rabbit polyclonal antibody of the present invention;

[0048] Figure 3 The ELISA standard curve diagrams of the present invention are (A) and (B) the standard curve of the indirect ELISA method (PEAApAb1#); (C) and (D) the standard curve of the indirect ELISA method (PEAA pAb5#);

[0049] Figure 4 The figures are as follows: (a) LAMP curves of different primer ratios; (b) LAMP results of different reaction temperatures; (c) LAMP curves of different amounts of Bst polymerase added; and (d) phenylethanolamine AIPCR standard curve. DETAILED DESCRIPTION

[0050] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0051] The present invention prepares a detection kit for detection according to the following experimental materials and methods:

[0052] 1.1 Preparation of PEAA

[0053] The following method is used to prepare phenylethanolamine A (PEAA), the principle is as follows Figure 1 (a). The specific steps are as follows:

[0054] (a) Weigh 13.6 g of p-methoxybenzaldehyde, then add 100 mL of nitromethane, add 20 drops of tetramethylguanidine at 0° C. (ice-water bath), react at 0° C. for 1 h, then add 50 mL of saturated brine to quench the reaction, add 300 mL of ethyl acetate, and adjust the pH value (pH=about 5) with 5% hydrochloric acid; after separation, wash the ethyl acetate layer twice with saturated brine, dry, and spin-dry to obtain nitro alcohol.

[0055] (b) Weigh 15 g of nitro alcohol and dissolve it in 200 mL of methanol. React under 4 MPa hydrogenation for 4 h, and then filter and spin-dry the product to obtain amino alcohol.

[0056] (c) Weigh 5 g of p-nitroiodobenzene and dissolve it in 200 mL of acetonitrile, then add 1.82 g of 3-butene-2-acetic acid, 0.5 g of palladium acetate and 0.87 mL of triethylamine, and reflux for 24 h. After spin drying and purification, p-nitroketone is obtained.

[0057] (d) Weigh 6.8 g of p-nitroketone and 8.88 g of amino alcohol and dissolve them in 150 mL of dichloroethane, add 11.2 g of sodium triacetoxyborohydride and 2 mL of acetic acid at room temperature, stir overnight at room temperature, and then add saturated sodium bicarbonate to quench; after separation, extract twice with dichloromethane, dry, spin dry and add to 10 times the volume of petroleum ether PE, a small amount of solid precipitate appears, spin dry and wash twice with a small amount of ethyl acetate to obtain phenylethanolamine A. 13 C NMR, 1 The molecular structure was confirmed by H NMR and mass spectrometry.

[0058] 1.2 Preparation and identification of PEAA derivatives

[0059] The active groups on the PEAA molecule are -NO 2 and -OH, but because the steric resistance of -OH is greater, -NO is chosen 2 Transformed to -NH 2 , as a coupling site. In this study, PEAA was reduced by Raney nickel to obtain the amino derivative of phenylethanolamine A (PEAA-NH 2 )

[100] , technical routes such as Figure 1 (b). The specific steps are as follows:

[0060] (a) Add 2.0 g of phenylethanolamine A to 10 mL of methanol and stir at room temperature to dissolve for 1 h.

[0061] (b) Weigh 0.3 g of Raney nickel, add it to the above dissolved solution, and react under 4 MPa hydrogen condition for 8 h.

[0062] (c) The obtained reaction product was filtered, dried by spin drying, and recrystallized with appropriate amounts of ethyl acetate and petroleum ether to obtain the amino derivative of phenylethanolamine A (PEAA-NH 2 ),use 13 C NMR, 1 The molecular structure was confirmed by H NMR and mass spectrometry.

[0063] 1.3 Preparation of immunogen and coating

[0064] Synthesis of PEAA artificial antigen by diazotization method

[101] The specific steps are as follows:

[0065] (a) Weigh 18.9 mg PEAA-NH 2 Then, 1.2 mL of sulfuric acid was added and dissolved at 4°C for 20 min.

[0066] (b) Add 0.3 mL of sodium nitrite solution dropwise to the above solution while stirring on crushed ice. Then add an appropriate amount of ammonium sulfamate and test with starch potassium iodide paper. Stop adding when the oozing circle turns colorless. Stir at 4°C for 30 minutes.

[0067] (c) Weigh 150 mg of BSA and dissolve it in 4 mL of carbonate buffer. Add the above activated PEAA amino derivative solution dropwise and stir at 4°C to react overnight.

[0068] (d) The above reaction product was placed in a dialysis bag and dialyzed in 0.01 mol / L PBS buffer for 4 days, with the dialysate changed twice a day. The dialyzed solution was stored at -20°C for later use. The same method was used to synthesize the original PEAA-OVA coating.

[0069] 1.4 Preparation and identification of rabbit polyclonal antibodies

[0070] Five healthy male New Zealand white rabbits were selected, and after one week of feeding and observation, blood was collected from the ear vein as a negative control, and the above-mentioned immunogen (PEAA-NH2 and BSA conjugate) was used to immunize 5 rabbits (number: 1#, 2#, 3#, 4#, 5#): the immunogen (PEAA-BSA) diluted with physiological saline was emulsified with the same volume of Freund's complete adjuvant, and after complete emulsification, it was injected into the back subcutaneous of the New Zealand white rabbit according to the 5-point method. Once a week, after two weeks, the emulsification reagent was changed to Freund's incomplete adjuvant, and the immunization frequency was changed to once every 4 weeks. After 6 immunizations, venous blood was collected to determine the titer. When the titer tended to be stable, whole blood was collected and purified to obtain polyclonal antibodies. The purification steps are as follows: 50 mL of pretreated rabbit antiserum is placed in a 500 mL clean beaker, diluted with 1×PBS, and then precipitated with 100 mL of saturated ammonium sulfate. After standing at 4°C for 50 min, the precipitate is centrifuged at 4°C for 20 min to obtain a precipitate; 50 mL of 1×PBS is added to redissolve the precipitate, and the pH is adjusted to 4.6 with acetate buffer; 3.5 mL is added while stirring at room temperature, and stirring is continued for 30 min after the addition is complete; the reaction solution is centrifuged at 12000 r / min for 20 min, the supernatant is collected, and the pH is adjusted to 7.4 with NaOH; 115 mL of saturated ammonium sulfate is added to the above reaction solution, and after standing at 4°C for 50 min, the precipitate is centrifuged at 12000 r / min at 4°C for 20 min to obtain a precipitate; 25 mL of CB buffer is added to dissolve the precipitate, and the CB buffer is dialyzed at 4°C for 3 days, and the solution is changed twice a day. After completion, the rabbit polyclonal antibody is collected.

[0071] After obtaining the rabbit cloned antibody, its titer needs to be determined. This experiment uses the indirect competitive ELISA method for determination:

[0072] After PEAA-OVA was diluted with CBS buffer, 100 μL / well was added to the ELISA plate and incubated at 4°C for 24 hours; after complete coating, the liquid in the plate was discarded and the blocking solution was added at 200 μL / well and incubated at 4°C for 24 hours; after complete blocking, the liquid in the plate was discarded, serum diluted with PBS was added at 100 μL / well and incubated at 37°C for 1 hour; after complete reaction, the liquid in the plate was discarded, goat anti-rabbit IgG-HRP was added and incubated at 37°C for 1 hour; after complete reaction, the liquid in the plate was discarded, the plate was washed 3 times with 0.05% PBST and patted dry, TMB color development solution was added at 100 μL / well and incubated at 37°C for 20 minutes; after color development, the OD450 value was measured with an ELISA instrument, and the antibody titer was obtained according to the OD450 value, that is, the maximum dilution multiple corresponding to the OD450 value being 2.1 times or more of the negative serum OD450 value was selected as the antibody titer.

[0073] 1.5 Establishment of PEAA indirect competitive ELISA method

[0074] 1.5.1 Standard curve and sensitivity of PEAA indirect competitive ELISA

[0075] Add phenylethanolamine A standard in a gradient of 0.1-8.1 ng / mL, and adjust the OD value of the standard in the corresponding gradient. 450 The binding rate (B / B 0 The IC value was calculated by the established standard curve, wherein the horizontal coordinate of one standard curve is the concentration of the phenylethanolamine A standard substance, and the vertical coordinate is the binding rate of the corresponding concentration. The horizontal coordinate of the other standard curve is the logarithm of the concentration of the phenylethanolamine A standard substance, and the vertical coordinate is the binding rate of the corresponding concentration. The IC value was calculated by the established standard curve. 50 value, that is, the concentration of phenylethanolamine A standard corresponding to a binding rate of 50%, and IC 10 value, that is, the concentration of phenylethanolamine A standard corresponding to a binding rate of 90%, and the IC 10 The value was defined as the limit of detection (LOD).

[0076] Binding rate (%) = B / B 0 ×100%(1-1)

[0077] 1.5.2 Add recovery test

[0078] According to the method of Announcement No. 1486 of the Ministry of Agriculture and Rural Affairs, six kinds of pig urine samples confirmed to be negative by LC-MS / MS were pre-treated: different amounts of PEAA standard were added to the six kinds of pig urine, and centrifuged at 5000r / min for 5min, so that the concentrations of phenylethanolamine A standard in the six kinds of pig urine were 0.25, 0.5 and 1ng / mL respectively. After the pre-treatment of the pig urine samples was completed, the concentration of the standard was determined by the optimized indirect competitive ELISA method. After repeating 3 times, according to formula (1-2), the average value of the experimentally measured phenylethanolamine A concentration value was compared with the actual added phenylethanolamine A concentration value to obtain the added recovery rate.

[0079] Recovery rate (%) = sample measured concentration / sample added concentration × 100% (1-2)

[0080] 1.6 Establishment of LAMP method

[0081] 1.6.1 Design of LAMP primers

[0082] Primer Explorer version 4.0 system was used, and a 209 bp fragment on the pGEM-3Z vector plasmid DNA was used as the target DNA. Primer premier 5.0 and Beacon Designer 7.9 were used to design the loop primer and internal and external primers of LAMP, respectively.

[0083] 1.6.2 Optimization of LAMP reaction system

[0084] 1.6.2.1 Optimization of external and internal primer ratio

[0085] In the LAMP reaction mechanism, the outer primers only play a role in the initial stage of the reaction, and the self-circulation and amplification of DNA are mainly completed by the stem-loop structure continuously formed by the inner primers. Therefore, the concentration ratio of the outer primers and the inner primers is particularly important. In this experiment, the concentration ratio of the outer primers and the inner primers was set to 1:2, 1:4, 1:6, 1:8, 1:10, and 1:12, and the optimal outer primer ratio was determined by comparing the fluorescence results.

[0086] 1.6.2.2 Optimization of the optimal LAMP reaction temperature

[0087] In the LAMP reaction, the reaction temperature plays an important role in the process of primer binding to the target sequence. In this experiment, the reaction temperature gradient was set to 60-66°C, and the optimal reaction temperature was determined by comparing the time of receiving the fluorescence value.

[0088] 1.6.2.3 Optimization of the optimal amount of Bst polymerase added

[0089] In the LAMP reaction, different concentrations of Bst polymerase will affect the reaction efficiency of strand displacement. In this experiment, the addition amount of Bst enzyme was set to 2U, 4U, 6U, 8U, 10U, and 12U respectively; the optimal addition amount of Bst polymerase was determined by comparing the fluorescence results.

[0090] 1.7 Optimization of the optimal working concentration of coated antigens PEAA-OVA and Bio-PEAA pAb1#ILAMP

[0091] The chessboard experiment was performed in 1% glutaraldehyde-treated PCR tubes using 75001, 50003, 0000 times diluted PEAA-OVA and 5001, 0002, 0004, 000 times diluted Bio-PEAA pAb respectively: PEAA-OVA with different dilutions was coated in glutaraldehyde-treated PCR tubes, 50μL / tube, incubated overnight at 4°C; the liquid in the tube was poured out and washed once with PBST; blocking solution was added, 200μL / tube, and blocked at 37°C for 2h; the blocking solution was poured out, and 0ppb and 1ppb PEAA standards (50μl / tube) and different dilutions of Bio-PEAA were added respectively. pAb (50 μL / tube), incubate at 37℃ for 1 hour; pour out the liquid in the tube, add 2000-fold diluted SA (50 μl / tube), incubate at 37℃ for 1 hour; pour out the reaction liquid Bio-DNA (50 μl / tube) again, incubate at 37℃ for 1 hour; pour out the liquid in the tube, wash with PBST and sterile water for 5 times respectively, add LAMP system, react, and monitor in real time with fluorescent quantitative PCR instrument. The optimal antigen-antibody working concentration is the one with the smallest Tt value of 0 ppb PEAA standard and the largest Tt value difference (ΔTt) between 0 ppb and 1 ppb.

[0092] 1.8 Establishment of PEAA Immuno-LAMP Method

[0093] PEAA-OVA at the optimal working concentration was coated in a PCR tube pretreated with 1% glutaraldehyde, 50 μL / tube; after coating at 4°C for 24 hours, the liquid in the plate was discarded, washed once with PBST, and patted dry; blocking solution (200 μL / tube) was added; after incubation at 37°C for 2 hours, the blocking solution was poured out, washed 3 times with PBST, and patted dry; Bio-PEAA pAb at the optimal working concentration and PEAA standards of different concentrations were added, 50 μL / tube respectively; after incubation at 37°C for 1 hour, the reaction solution in the tube was discarded, washed 3 times with PBST, and patted dry; SA at the optimal working concentration was added, 50 μL / tube; after incubation at 37°C for 1 hour, the liquid was poured out, and then washed 3 times with PBST, and patted dry; Bio-DNA at the optimal working concentration was added, 50 μL / tube; after incubation at 37°C for 1 hour, the liquid was poured out; after washing 10 times (5 times with PBST and sterilized ultrapure water respectively), amplified according to the LAMP reaction system and procedure.

[0094] Based on the optimization of the above experimental conditions, the steps of the immune LAMP method were established as follows: PEAA-OVA was diluted to 1:7500 (2 μg / mL) with CB coating solution and coated in PCR tubes pretreated with 1% glutaraldehyde, 50 μL / tube; after coating at 4°C for 24 hours, the liquid in the plate was discarded, washed once with PBST, and patted dry; blocking solution (200 μL / tube) was added; after incubation at 37°C for 2 hours, the blocking solution was poured off, PBST was washed 3 times, and patted dry; a certain concentration of standard and the optimal working concentration of Bio-PEAA were added respectively pAb (2.1 μg / mL), 50 μL / tube; incubate at 37℃ for 1 hour, discard the reaction solution in the tube, wash 3 times with PBST, and pat dry; add SA (1.5 μg / mL) of the best working concentration, 50 μL / tube; incubate at 37℃ for 1 hour, pour out the liquid, and wash 3 times with PBST; add Bio-DNA (28.2 ng / mL) diluted to 1:1600, 50 μL / tube; incubate at 37℃ for 1 hour, pour out the liquid, wash 5 times with PBST and sterilized ultrapure water, and add LAMP system, i.e. 2.5 μL template, 0.5 μL outer primer (0.2 mmol / L), 4 μL inner primer (1.6 mmol / L), 2 μL loop primer (0.8 mmol / L), 3.5 μL dNTPs (1.4 mmol / L), 0.5 μL MnCl 2 (500μmol / L), 3μL Betaine (0.6mol / L), 1μL Calcein (200μmol / L), 0.5μL MgSO 4 (8mmol / L), 0.5μL KCl (10mmol / L), 1.0μL Tris-HCl (20mmol / L), 1.0μL Bst DNA Polymerase, 0.5μL (NH 4 ) 2 SO 4 (10mmol / L), 0.5μL 0.1% Tween 20, ddH 2 O to 25 μL. Then react at 65°C for 60 min, and use a fluorescence quantitative PCR instrument to detect the fluorescence start time.

[0095] 1.8.1 PEAA Immuno-LAMP Standard Curve and Detection Sensitivity

[0096] The phenylethanolamine A standard was diluted with PBS in a 10-fold gradient, that is, the concentration gradient of the phenylethanolamine A standard was 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 ng / mL. The Tt value corresponding to each concentration was determined by the established immune LAMP method. A standard curve was prepared with the concentration of the phenylethanolamine A standard as the horizontal coordinate and the corresponding Tt value as the vertical coordinate, and with the logarithm of the concentration of the phenylethanolamine A standard as the horizontal coordinate and the corresponding Tt value as the vertical coordinate. The linear range and IC value of the method were obtained by the established standard curve. 50 The value (phenylethanolamine A standard, 0 ng / mL, the corresponding Tt value and the average value of the Tt value corresponding to the phenylethanolamine A standard, 100 ng / mL) is the corresponding phenylethanolamine A concentration. At the same time, by measuring the Tt values ​​corresponding to 8 phenylethanolamine A standards (0 ng / mL), the average value and standard deviation of these 8 Ct values ​​are calculated, and the minimum detection limit (LOD) of the immune LAMP method is calculated according to the following formula. The calculation formula is the same as formula 1-2.

[0097] 1.8.2 Specificity experiments

[0098] The IC values ​​of 11 β-agonists (clenbuterol, ractopamine, cimaterol, bambuterol, cibuterol, mabuterol, chlorprenaline, mabenterol, salbutamol, terbutaline and zilpaterol) were determined according to the established immuno-LAMP method. 50 According to the experimental IC 50 The cross-reactivity (CR) was calculated and the specificity of the method was determined by the cross-reactivity formula (1-3) as shown below.

[0099] CR (%) = IC 50(PEAA) / IC 50(其他竞争结合物) × 100% (1-3)

[0100] 1.8.3 Sample addition recovery experiment

[0101] 1.8.3.1 ILAMP Accuracy Experiment

[0102] According to the method of Announcement No. 1486 of the Ministry of Agriculture and Rural Affairs, 6 types of pig urine samples confirmed to be negative by LC-MS / MS were pretreated: different amounts of PEAA standard were added to the 6 types of pig urine, and centrifuged at 5000r / min for 5 minutes, so that the concentration of phenylethanolamine A standard in the 6 types of pig urine was 0.25, 0.5 and 1.0ng / mL in three gradients. After the pretreatment of the pig urine samples was completed, the concentration of the standard was determined by the ILAMP method established above. After repeating 3 times, the ratio of the average experimentally measured phenylethanolamine A concentration value to the actual added phenylethanolamine A concentration value was used as the added recovery value. The calculation formula (1-4) is as follows: Recovery rate (%) = sample measured concentration / sample added concentration × 100% (1-4)

[0103] 1.8.3.2 Repeatability Experiment

[0104] The precision of the experimental method is determined by the intra-plate and inter-plate coefficient of variation (CV%). PEAA standard products with concentration gradients of 0.0001, 0.001, 0.01, 0.1, 1, 10, and 100 ng / mL were added, and a certain concentration of a certain sample was tested on the ELISA plate coated at different times. Six groups of repetitions were performed to obtain different LODs, and the intra-plate and inter-plate coefficients of variation were calculated.

[0105] The calculation (1-5) is as follows, represents the mean OD value, Sd represents the standard deviation, and CV represents the coefficient of variation.

[0106]

[0107] 2 Experimental results

[0108] 2.1 Identification of PEAA rabbit cloned antibodies

[0109] The rabbit cloned antibody was tested for titer by indirect competitive ELISA method. Figure 2 The results showed that the antiserum titers of the five New Zealand white rabbits were all at a high level, among which the titers of rabbits 1# and 5# were the highest. Therefore, rabbit clone antibodies 1# and 5# were selected for the next experiment.

[0110] 2.2 Standard curve and sensitivity of PEAA indirect competitive ELISA

[0111] The standard curve of the indirect competitive ELISA method was established by adding different concentrations of PEAA standards, and the IC 50 Value and IC 10 The sensitivity of this detection method was evaluated by using the value. Figure 3As shown, the linear range of this method is 0.1-10 ng / mL, and the formula of the standard curve of PEAA pAb1# is y=-37.61x+35.06(R 2 =0.968); the formula of PEAA pAb5# is y=-36.43x+33.41, R 2 =0.956. According to the formula of the standard curve, the concentration of the standard added at different inhibition rates can be calculated. Therefore, the IC value of PEAA pAb1# can be obtained through the standard curve. 50 The value was 0.401ng / mL, and the minimum detection limit (IC 10 ) is 0.035ng / ml; IC of PEAA pAb5# 50 The value was 0.391 ng / mL, and the minimum detection limit (IC 10 ) was 0.028 ng / mL. The direct competitive detection of phenylethanolamine A residues established by Zhang et al.: IC 50 The minimum detection limit (IC 10 ) is 0.022ng / m, the indirect competitive ELISA method established in this study can also meet the needs of actual PEAA detection.

[0112] 2.3 Addition recovery test

[0113] The results of the addition experiment of 6 kinds of pig urine samples are shown in Table 1. The results show that the recovery rate of the indirect ELISA method of phenylethanolamine A established in this experiment is between 86.2% and 116.6%, and the coefficient of variation is below 10%, which shows that the method meets the requirements of actual sample detection and has high accuracy.

[0114] Table 1 Pig urine addition recovery test

[0115]

[0116]

[0117] 2.4 Design of LAMP primers

[0118] The outer primers F3 and F4 are 5'-GTAACGCCAGGGTTTTCC-3' SEQ ID No: 01B, and 3 are 5'CAGCTATGACCATGATTACG-3' SEQ ID No: 02; the amplified product is 209 bp;

[0119] Inner primers: FIP upstream primer is: 5'-GTACCCGGGGATCCTCTAGAG-3' SEQ ID No: 03 and FIP downstream primer is 5'-TTTTCCAAGCTATTTAGGTGACACT-3' SEQ ID No: 04; BIP upstream primer is 5'-TCGAATTCGCCCTATAGTGAGTCTTTT-3' SEQ ID No: 05 and BIP downstream primer is 5'-GTCACGACGTTGTAAAACG-3' SEQ ID No: 06; loop primer: LB is 5'-GCAGGCATGCAAGCTTGAG-3' SEQ ID No: 07.

[0120] 2.5 Optimization of LAMP reaction system

[0121] 2.5.1 Optimization of external-internal primer ratio

[0122] The experimental results show that Figure 4 As shown in (a), as the ratio of inner and outer primer concentrations increases, the time of initial fluorescence onset begins to be significantly advanced, but after the ratio reaches 1:8, the effect change becomes insignificant. Considering the cost of the experiment, 1:8 was finally determined as the optimal outer and inner primer ratio for the LAMP reaction system.

[0123] 2.5.2 Optimization of reaction temperature

[0124] The experimental results show that Figure 4 As shown in (b), the amplification efficiency of the primers did not change significantly, so the recommended temperature of 65°C in the instructions of Bst polymerase was selected as the optimal reaction temperature of the LAMP reaction system.

[0125] 2.5.3 Optimization of the optimal amount of Bst polymerase added

[0126] In this experiment, the addition amount of Bst polymerase was 2U-12U. The experimental results showed that Figure 4 As shown in (c), with the increase of the amount of Bst polymerase added, the efficiency of the LAMP reaction is significantly improved, but after the amount of Bst polymerase added reaches 8 U, the reaction efficiency does not change significantly. Considering the cost of the experiment, 8 U was finally used as the optimal amount of Bst polymerase added for the LAMP reaction system.

[0127] 2.6 Optimal working concentration selection of PEAA-OVA and Bio-PEAA pAb1#ILAMP

[0128] The chessboard method was tested with 75001, 50003, 0000 times diluted PEAA-OVA and 5001, 0002, 0004, 000 times diluted Bio-PEAA pAb. The results are shown in Table 2. When the dilution multiples of PEAA-OVA and Bio-PEAA pAb1# were 1:7500 (2μg / mL) and 1:1000 (2.1μg / mL), respectively, the Tt value corresponding to the PEAA standard with a concentration of 0ppb (39.50) had the largest difference with the Tt value corresponding to the PEAA standard concentration of 1ppb (43.02). Therefore, the combination of PEAA-OVA with a concentration of 2μg / mL and Bio-PEAA pAb1# with a concentration of 2.1μg / mL can be used as the optimal working concentration in IPCR.

[0129] Table 2 Antigen and antibody optimization

[0130]

[0131] 2.7 Linear range and detection sensitivity

[0132] Through indirect competitive immune LAMP reaction, the starting reaction time Tt value is the ordinate and the standard concentration is the abscissa. The standard curve is shown in the following figure. The starting reaction time Tt value is the ordinate and the logarithm of the standard concentration is the abscissa. Figure 4 (d) According to the standard curve, the linear range and detection sensitivity of the ILAMP method were obtained:

[0133] (1) Detection sensitivity: Based on the ILAMP standard curve Figure 4 (d) Calculate IC 50 The value is 0.0396±0.009ng / mL, which is an order of magnitude higher than that of the indirect competitive ELISA method; the detection sensitivity is 0.0001-100ng / mL, which is 100 times that of the ELISA method.

[0134] (2) Linear range: The linear equation of ILAMP standard curve 4 (d) is y = 1.1949x + 42.251, R 2 =0.956, indicating that the linear range of IPCR reached 0.0001-100 ng / mL, which was improved by two orders of magnitude both forward and backward compared with the ELISA in Part 3.

[0135] 2.8 Specificity

[0136] The IC values ​​of the 11 β-agonists listed in Table 3 were determined by the established ILAMP method. 50 value, based on the obtained IC 50The cross-reaction rates of the detection method with these β-agonists were calculated using the values. The results (Table 3) showed that the cross-reaction rates of the phenylethanolamine A indirect ELISA method established in this experiment with these 11 β-agonists were all below 0.04%, indicating that the specificity of the method met the actual detection requirements.

[0137] Table 3 Immune LAMP cross-reaction

[0138]

[0139] 2.9 Accuracy

[0140] The results of the spike recovery experiments of the six pig urine samples are shown in Table 4. The results showed that the average recovery was 84-119% and the relative standard deviation was 2.01-9.25%, proving that the ILAMP method established in this study has good accuracy.

[0141] 2.10 Precision

[0142] The repeatability of the method was evaluated by calculating the intra-plate and inter-plate coefficients of variation (CV). From the results (Table 5), it can be seen that the intra-plate coefficient of variation (CV%) was between 8.5% and 9.5%, the average coefficient of variation was 9.06%, and the inter-plate coefficient of variation was 9.58%. The coefficients of variation were all below 10%, which shows that the IPCR method established in this chapter has high accuracy.

[0143] Table 4 Recovery of Phenylethanolamine A in Pig Urine

[0144]

[0145]

[0146] Table 5 ILAMP intra-plate and inter-batch coefficient of variation

[0147]

[0148] From the above experiments, it can be seen that the present invention combines fluorescent quantitative LAMP with enzyme-linked immunosorbent assay (ELISA) to establish a detection technology for phenylethanolamine A. The results obtained are as follows: The amino derivative of phenylethanolamine A, namely PEAA-NH 2 PEAA-NH 2The coating source PEAA-OVA and the immunogen PEAA-BSA were synthesized by diazotization reaction with ovalbumin (OVA) and bovine serum albumin (BSA), respectively. New Zealand white rabbits were immunized by conventional immunization methods, and polyclonal antibody PEAA pAbs (1# and 5#) were obtained by titer determination. Based on the prepared PEAA-OVA artificial antigen and PEAA polyclonal antibody, an indirect competitive ELISA method for detecting PEAA residues was established. The IC value of this method for detecting PEAA was obtained by making a standard curve. 50 The value was 0.401ng / mL, the lowest detection limit (LOD) was 0.035ng / mL, the cross-reaction rate to phenylethanolamine A was very high, and the cross-reaction rates to the other 11 β-receptor agonists were all below 0.01%. In the actual sample detection, the intra-plate recovery rate was 86.2-116.6%, and the relative standard deviation was 1.66-4.52%. At the same time, the intra-plate coefficient of variation (CV%) was between 4.5-5.5%, the average coefficient of variation was 4.41%, the inter-plate coefficient of variation was 9.58%, and the CV was less than 10%, proving that this method is reliable. After completing the design of LAMP primers for the target sequence, optimizing the efficient LAMP system, determining the ratio of external and internal primers to 1:8, the reaction temperature to 65℃, and the amount of Bst polymerase added to 8U, as well as optimizing the optimal working concentrations of the coated antigen and biotin-labeled antibody ILAMP system (2μg / mL and 2.1μg / mL), the immune LAMP detection method for PEAA was established. The experimental results show that the linear equation of the standard curve of this method is: y = 1.1949x + 42.251, R 2 =0.956. The linear detection range is 0.0001-100ng / mL. IC 50 The minimum detection limit was 0.0396±0.009ng / mL, which was an order of magnitude higher than that of indirect competitive ELISA. The minimum detection limit was 0.0044ng / mL, which was also an order of magnitude higher. The precision experiment results showed that the coefficient of variation (CV) did not exceed 10%, proving the reliability of the method. The ILAMP specificity test results showed that the method had high specificity for PEAA and had no cross-reaction with other 11 β-receptor agonist substances (CR<0.04%). Due to the characteristics of LAMP technology, immune LAMP technology has broken away from the limitations of instruments, shortened the reaction time, and provided the possibility for rapid detection at the grassroots level.

[0149] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A detection kit for detecting phenylethanolamine A based on ILAMP, characterized in that: include: 2μg / mL PEAA-OVA, 2.1mg / mL coated Bio-PEAA pAb, 28.2ng / mL Bio-DNA, 2.5μL template, 0.5μL outer primer at a concentration of 0.2mmol / L, 4μL inner primer at a concentration of 1.6mmol / L, 2μL loop primer at a concentration of 0.8mmol / L, 3.5μL dNTPs at a concentration of 1.4mmol / L, 0.5μL MnCl2 at a concentration of 500μmol / L, 3μL betaine at a concentration of 0.6mol / L, 1μL calcein at a concentration of 200μmol / L, 0.5μL MgSO4 at a concentration of 8mmol / L, 0.5μL KCl at a concentration of 10mmol / L, 1μL Tris-HCl at a concentration of 20mmol / L, 1.0μL BstDNA polymerase, 0.5μL (NH4)2SO4 at a concentration of 10mmol / L, 0.5μL 0.1% Tween 20, 4 μL ddH2O; The outer primer F3 is 5'-GTAACGCCAGGGTTTTCC-3' SEQ ID No: 01, and B3 is 5'CAGCTATGACCATGATTACG-3' SEQ ID No: 02; The inner primers: FIP upstream primer is: 5'-GTACCCGGGGATCCTCTAGAG-3' SEQ ID No: 03 and FIP downstream primer is 5'-TTTTCCAAGCTATTTAGGTGACACT-3' SEQ ID No: 04; BIP upstream primer is 5'-TCGAATTCGCCCTATAGTGAGTCTTTT-3' SEQ ID No: 05 and BIP downstream primer is 5'-GTCACGACGTTGTAAAACG-3' SEQ ID No: 06; The loop primer: LB is 5'-GCAGGCATGCAAGCTTGAG-3' SEQ ID No:

07.

2. A method for detecting phenylethanolamine A based on ILAMP, characterized in that: include: Step 1, obtaining phenylethanolamine A amino derivative PEAA-NH2 by Raney nickel reduction method; Step 2, PEAA-NH2 is subjected to diazotization reaction with ovalbumin OVA and bovine serum albumin BSA to synthesize the coating source PEAA-OVA and the immunogen PEAA-BSA respectively; Step 3, preparing rabbit polyclonal antibodies, and obtaining polyclonal antibody PEAA pAbs through titer determination; Step 4, establishing an indirect competitive ELISA method for phenylethanolamine A, and then establishing a standard curve to detect phenylethanolamine A; Step 5: Establish the LAMP method and use a fluorescence quantitative PCR instrument to detect the fluorescence start time.

3. The method for detecting phenylethanolamine A based on ILAMP according to claim 1, characterized in that: The specific steps of obtaining the amino derivative PEAA-NH2 of phenylethanolamine A by Raney nickel reduction method include: (a) Add 2.0 g of phenylethanolamine A to 10 mL of methanol and stir at room temperature for 1 h to dissolve; (b) weighing 0.3 g of Raney nickel, adding it to the solution dissolved in step (a), and reacting it under 4 MPa hydrogen condition for 8 h; (c) filtering the obtained reaction product, drying it by spin drying, and recrystallizing it with appropriate amount of ethyl acetate and petroleum ether to obtain the amino derivative of phenylethanolamine A PEAA-NH2, and using 13 C NMR, 1 The molecular structure was confirmed by H NMR and mass spectrometry.

4. The method for detecting phenylethanolamine A based on ILAMP according to claim 1, characterized in that: The specific steps of step 2, respectively performing diazotization reaction on PEAA-NH2 with ovalbumin OVA and bovine serum albumin BSA to synthesize the coating source PEAA-OVA and the immunogen PEAA-BSA, include: (1) Weigh 18.9 mg PEAA-NH2, add 1.2 mL sulfuric acid, and dissolve at 4 °C for 20 min; (2) Add 0.3 mL of sodium nitrite solution dropwise to the solution obtained in step (1) on crushed ice while stirring, then add ammonium sulfamate, and detect with starch potassium iodide paper. Stop adding when the oozing circle becomes colorless, and stir at 4°C for 30 minutes; (3) Weigh 150 mg of BSA and dissolve it in 4 mL of carbonate buffer. Add the activated PEAA-NH2 solution dropwise and stir at 4 °C for overnight reaction. (4) The reaction product of step (3) was placed in a dialysis bag and dialyzed in 0.01 mol / L PBS buffer for 4 days, with the dialysate replaced twice a day; the dialyzed solution was stored at -20°C for later use; the same method was used to synthesize the original PEAA-OVA coating.

5. The method for detecting phenylethanolamine A based on ILAMP according to claim 1, characterized in that, The specific method of step 4, establishing an indirect competitive ELISA method for phenylethanolamine A, and then establishing a standard curve to detect phenylethanolamine A includes:

1. Calculate the binding rate (B / B0 value) according to the following formula (1-1), and then draw a standard curve; Binding rate (%) = B / B0×100% (1-1), B and B0 are the OD values ​​measured when the standard and RAC concentration are 0, respectively. 450nm 2. According to formula (1-2), the average value of the experimentally measured phenylethanolamine A concentration is compared with the actual added phenylethanolamine A concentration to obtain the added recovery rate; Recovery rate (%) = measured sample concentration / sample added concentration × 100% (1-2).

6. The method for detecting phenylethanolamine A based on ILAMP according to claim 1, characterized in that: The specific steps of establishing the LAMP method in step 5 include: optimizing the working concentration of coated PEAA-OVA, optimizing the working concentration of coated Bio-PEAA pAb, optimizing the working concentration of Bio-DNA, and establishing PEAA immune-LAMP after optimizing the LAMP reaction system.

7. The method for detecting phenylethanolamine A based on ILAMP according to claim 6, characterized in that: The specific method for optimizing the working concentration of coated PEAA-OVA and the working concentration of coated Bio-PEAA pAb is as follows: using 7500, 15000, and 30000 times diluted PEAA-OVA and 500, 1000, 2000, and 4000 times diluted Bio-PEAA pAb to perform a chessboard experiment in a 1% glutaraldehyde-treated PCR tube: coating PEAA-OVA with different dilution multiples in a glutaraldehyde-treated PCR tube, 50 μL / tube, and incubating overnight at 4°C; pouring out the liquid in the tube and washing once with PBST; adding blocking solution, 200 μL / tube, and blocking at 37°C for 2h; pouring out the blocking solution, adding 0ppb and 1ppb PEAA quasi-samples (50 μl / tube) and Bio-PEAApAb with different dilution multiples (50 μl / tube), respectively, and incubating at 37°C for 1h; pouring out the blocking solution, and adding 0ppb and 1ppb PEAA quasi-samples (50 μl / tube) and Bio-PEAApAb with different dilution multiples (50 μl / tube), respectively, and incubating at 37°C for 1h; pouring out the blocking solution, and adding 0ppb and 1ppb PEAA quasi-samples (50 μl / tube) and Bio-PEAApAb with different dilution multiples (50 μl / tube), respectively, and incubating at 37°C for 1h. After the liquid in the tube, add 2000-fold diluted SA (50 μl / tube) and incubate at 37°C for 1 h; pour out the reaction liquid Bio-DNA (50 μl / tube) again and incubate at 37°C for 1 h; after pouring out the liquid in the tube, wash with PBST and sterile water for 5 times respectively, add LAMP system, react, and monitor in real time with fluorescent quantitative PCR instrument; the optimal antigen-antibody working concentration is the one with the smallest Tt value of 0 ppb PEAA standard concentration and the largest Tt value difference (ΔTt) between 0 ppb and 1 ppb.

8. The method for detecting phenylethanolamine A based on ILAMP according to claim 6, characterized in that: The method for optimizing the LAMP reaction system includes: designing LAMP primers, optimizing the ratio of external and internal primers, optimizing the optimal LAMP reaction temperature, and optimizing the optimal amount of Bst polymerase added; The design method of the LAMP primers: using the Primer Explorer version 4.0 system, taking a 209 bp fragment on the pGEM-3Z vector plasmid DNA as the target DNA, and using Primer premier 5.0 and BeaconDesigner7.9 to design the loop primer and the inner and outer primers of LAMP respectively; The method for optimizing the ratio of outer and inner primers is as follows: the concentration ratio of the outer and inner primers is set to 1:2, 1:4, 1:6, 1:8, 1:10, 1:12, and the best outer and inner primer ratio is determined by comparing the fluorescence results; The optimal LAMP reaction temperature optimization method: the reaction temperature gradient is set to 60-66°C, and the optimal reaction temperature is determined by comparing the time when the fluorescence value is received; Method for optimizing the optimal amount of Bst polymerase added: set the amount of Bst enzyme added to 2U, 4U, 6U, 8U, 10U, and 12U respectively; determine the optimal amount of Bst polymerase added by comparing the fluorescence results.

9. The method for detecting phenylethanolamine A based on ILAMP according to claim 6, characterized in that: The method for establishing PEAA immune-LAMP comprises: PEAA-OVA with the best working concentration was coated in a PCR tube pretreated with 1% glutaraldehyde, 50 μL / tube; after coating at 4°C for 24 hours, the liquid in the plate was discarded, washed once with PBST, and patted dry; blocking solution (200 μL / tube) was added; after incubation at 37°C for 2 hours, the blocking solution was poured out, washed 3 times with PBST, and patted dry; Bio-PEAA pAb with the best working concentration and PEAA standards of different concentrations were added, 50 μL / tube respectively; after incubation at 37°C for 1 hour, the reaction liquid in the tube was discarded, washed 3 times with PBST, and patted dry; SA with the best working concentration was added, 50 μL / tube; after incubation at 37°C for 1 hour, the liquid was poured out, and the plate was washed 3 times with PBST, and patted dry; Bio-DNA with the best working concentration was added, 50 μL / tube; after incubation at 37°C for 1 hour, the liquid was poured out; after washing 10 times, i.e., 5 times with PBST and sterilized ultrapure water respectively, amplified according to the LAMP reaction system and procedure; The steps of establishing the immune LAMP method are as follows: dilute PEAA-OVA to 1:7500 (2 μg / mL) with CB coating solution, and coat in PCR tubes pretreated with 1% glutaraldehyde, 50 μL / tube; discard the liquid in the plate after coating at 4°C for 24 hours, wash once with PBST, and pat dry; add blocking solution (200 μL / tube); incubate at 37°C for 2 hours, pour off the blocking solution, wash 3 times with PBST, and pat dry; add standard and optimal working concentration of Bio-PEAA pAb (2.1 μg / mL), 50 μL / tube; after incubation at 37°C for 1 hour, discard the reaction solution in the tube, wash 3 times with PBST, and pat dry; add SA (1.5 μg / mL) with the best working concentration, 50 μL / tube; after incubation at 37°C for 1 hour, pour out the liquid and wash 3 times with PBST; add Bio-DNA (28.2 ng / mL) diluted to 1:1600, 50 μL / tube; after incubation at 37°C for 1 hour, pour out the liquid and wash 5 times with PBST and sterilized ultrapure water respectively, add LAMP system, and then react at a constant temperature of 65°C for 60 minutes, and use fluorescence quantitative PCR instrument to detect the fluorescence start time.

10. The method for detecting phenylethanolamine A based on ILAMP according to claim 6, characterized in that: The LAMP system includes: 2.5 μL template, 0.5 μL outer primer with a concentration of 0.2 mmol / L, 4 μL inner primer with a concentration of 1.6 mmol / L, 2 μL loop primer with a concentration of 0.8 mmol / L, 3.5 μL dNTPs with a concentration of 1.4 mmol / L, 0.5 μL MnCl2 with a concentration of 500 μmol / L, 3 μL betaine with a concentration of 0.6 mol / L, 1 μL calcein with a concentration of 200 μmol / L, 0.5 μL MgSO4 with a concentration of 8 mmol / L, 0.5 μL KCl with a concentration of 10 mmol / L, 1.0 μL Tris-HCl with a concentration of 20 mmol / L, 1.0 μL Bst DNA polymerase, 0.5 μL (NH4)2SO4 with a concentration of 10 mmol / L, 0.5 μL 0.1% Tween 20, and ddH2O supplemented to 25 μL.

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