Recombinant plasmid, genetically engineered bacterium and method for detecting neonicotinoid pesticide in environment
By designing recombinant plasmids and genetically engineered bacteria, and using E. coli expression plasmids for neonicotinoid pesticide detection, the problems of complex and cost in the existing technology are solved, and a fast, simple and accurate detection effect is achieved.
Patent Information
- Application Number
- CN202510587578.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-08
- Publication Date
- 2025-08-08
AI Technical Summary
The prior art lacks simple and fast methods for the detection of neonicotinoid pesticides in the environment. The existing detection methods have problems such as expensive instruments, cumbersome operation, high cost, insufficient accuracy and precision.
The recombinant plasmid was designed, and the ligand binding domain ECD part of the α7 subunit of the nicotinic acetylcholine receptor was cloned into the expression vector, and genetically engineered bacteria were constructed. The recombinant plasmid was expressed by E. coli, and fluorescence changes were induced by the receptor conformation.
It realizes rapid, simple and accurate detection of neonicotinoid pesticides in the environment without the need for expensive instruments and professionals, improving the stability and sensitivity of detection, and is suitable for on-site inspection.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical fields of genetic engineering and pesticide detection, and in particular to a recombinant plasmid, genetically engineered bacteria and a method for detecting neonicotinoid pesticides in the environment. Background Art
[0002] Neonicotinoid pesticides, including acetamiprid (ACE), imidacloprid (IMI), thiamethoxam (THM), and nitenpyram (NTP), are widely distributed pollutants in the environment. They are highly effective in controlling microscopic lepidopteran and coleopteran pests such as aphids, whiteflies, and thrips, and are widely used in agricultural production. Due to their limited bioavailability, most neonicotinoid pesticides remain in farmland soils and may leach into groundwater through the soil profile or be transported to nearby water bodies through rainfall, leaching, and runoff, resulting in frequent detection in the environment. Neonicotinoid pesticides can act as agonists for nicotinic acetylcholine receptors (nAChRs), leading to dysregulation and various diseases during development, adulthood, and aging, posing a significant threat to ecosystem security and human health. Therefore, monitoring neonicotinoid pesticides in the environment is crucial to achieving the control of neonicotinoid pesticides in the environment and protecting the safety of aquatic ecosystems and human life and health.
[0003] Currently, there is no unified method for detecting neonicotinoid pesticides in the environment. Most of the reports are about liquid chromatography (LC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Combined with molecular imprinting, solid phase extraction and other technologies, they can achieve efficient and accurate analysis of neonicotinoid pesticides. However, they have disadvantages such as the instrument is not easy to carry, the operation is cumbersome, and the cost is high. They are not suitable for rapid screening and on-site detection. Other technologies such as enzyme-linked immunosorbent assay, fluorescence analysis, and electrochemical detection are not yet mature. Although they can be used for rapid detection, their accuracy and precision are poor and their anti-interference ability is weak. Biosensors have been a research hotspot in recent years and have also been used in the field of neonicotinoid pesticide detection. Xu YW et al. [1] A new electrochemical biosensor based on porous gold and aptamer modification was designed, which has high sensitivity, good selectivity and strong stability for the detection of ACE in fruits and vegetables, but it has many limitations and is easily interfered by other substances. Qi HJ et al. [2] Synthesized Ir(III) complexes as emitters for electrochemiluminescent biosensors, and using hybridization chain reaction, achieved enzyme-free, label-free, and highly sensitive detection of ACE, but there is still room for exploration in terms of substrates and immobilization modes to further enhance the signal. Xie W et al. [3]Magnetic nanoparticles are synthesized using Fe3O4 as a carrier, and diene ionic liquids are used as fluorescent dyes. The fluorescence quenching caused by electron transfer is used to achieve rapid detection of IMI. It has high sensitivity and strong controllability, but is easily limited by the fluorescent dye and has a complex signal response mechanism.
[0004] Therefore, in order to achieve more effective monitoring and control of neonicotinoid pesticides in the environment, it is necessary to develop simpler and faster detection methods to provide new ideas for the rapid screening and on-site detection of neonicotinoid pesticides.
[0005] [1]Xu YW, Zhang W, Shi JY, et al. Impedimetric aptasensor based on highly porous gold for sensitive detection of acetamiprid in fruits and vegetables[J].
[0006] [2]Qi HJ, Li HY, Li FN-heterocyclic Ir(III)complex targeting G-quadruplex structure to boost label-free and immobilization-freeelectrochemiluminescent sensing[J].
[0007] [3]Xie W, Ju YL, Zhang J, et al. Highly sensitive and specific determination of imidacloprid pesticide by a novel Fe3O4@SiO2@MIPILfluorescent sensor[J]. Summary of the Invention
[0008] The technical problem to be solved by the present invention is to provide a recombinant plasmid for detecting neonicotinoid pesticides in the environment in response to the deficiencies of the existing technology.
[0009] The technical problem that the present invention also aims to solve is to provide a genetically engineered bacterium for detecting neonicotinoid pesticides in the environment.
[0010] The technical problem that the present invention also aims to solve is to provide the application of the recombinant plasmid or the genetically engineered bacteria in detecting neonicotinoid pesticides in the environment.
[0011] The final technical problem to be solved by the present invention is to provide a method for detecting neonicotinoid pesticides in the environment.
[0012] The idea of the present invention is to design a recombinant plasmid and construct a genetically engineered bacterium based on the characteristics of neonicotinoid pesticides acting on nicotinic acetylcholine receptors (nAChRs) and the characteristics of protein conformation changes that trigger changes in fluorescence intensity. The sample to be tested is added to the activated bacterial solution of the genetically engineered bacteria, and the reaction is carried out under the conditions of 20℃~40℃ and 200~300r / min. The fluorescence is read and the OD is detected. 600 The unit fluorescence reduction value is calculated and the environmental neonicotinoid pesticides are detected according to the unit fluorescence reduction value.
[0013] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:
[0014] In a first aspect, the present invention provides a recombinant plasmid for detecting neonicotinoid pesticides in the environment, wherein the ligand binding domain ECD portion of the α7 subunit of the nicotinic acetylcholine receptor is cloned into an expression vector to construct the recombinant plasmid.
[0015] Wherein, the gene sequence of the recombinant plasmid for detecting neonicotinoid pesticides in the environment is shown in SEQ ID NO.1.
[0016] Wherein, the ligand binding domain ECD portion of the nicotinic acetylcholine receptor α7 subunit is derived from rat.
[0017] The gene sequence of the ligand binding domain ECD of the α7 subunit of the nicotinic acetylcholine receptor is shown in SEQ ID NO. 2.
[0018] Wherein, the expression vector is pEGFP-N1 vector.
[0019] Specifically, the gene sequence of the pEGFP-N1 vector is shown in SEQ ID NO.3.
[0020] The ligand binding domain ECD portion of the nicotinic acetylcholine receptor α7 subunit is cloned into the expression vector, specifically cloning the ligand binding domain ECD portion of the nicotinic acetylcholine receptor α7 subunit between the BtsI and BsrGI restriction sites of the expression vector.
[0021] Furthermore, the ECD portion of the ligand binding domain of the α7 subunit of the nicotinic acetylcholine receptor was cloned between the two restriction endonuclease sites of BtsI and BsrGI in the vector pEGFP-N1 (GenBank: U55762.1) that can express enhanced green fluorescent protein EGFP, and the residues between the BtsI and BsrGI restriction sites expressing EGFP (residues 227 to 466) were replaced.
[0022] In a second aspect, the present invention provides a genetically engineered bacterium for detecting neonicotinoid pesticides in the environment, wherein the genetically engineered bacterium contains the recombinant plasmid.
[0023] Wherein, the genetically engineered bacteria are obtained by transforming the recombinant plasmid into host cells.
[0024] Wherein, the host cell is Escherichia coli; preferably, it is BL21 type Escherichia coli; more preferably, the Escherichia coli is BL21 (DE3) Escherichia coli.
[0025] Specifically, the genetically engineered bacteria can be used as a single fluorescent protein bioreceptor to detect neonicotinoid pesticides in the environment.
[0026] In a third aspect, the present invention provides the use of the recombinant plasmid or the genetically engineered bacteria in detecting neonicotinoid pesticides in the environment.
[0027] In a fourth aspect, the present invention provides a method for detecting neonicotinoid pesticides in the environment, which is mainly based on the quantitative detection of neonicotinoid pesticide concentrations in the environment by receptor conformation-induced fluorescence changes.
[0028] Specifically, the following steps are included:
[0029] (1) After the genetically engineered bacteria are activated, the total fluorescence intensity of the activated bacterial solution is detected, and its OD 600 value;
[0030] (2) Add the sample to be tested to the activated bacterial solution for reaction, detect the total fluorescence intensity of the reaction solution, and detect its OD 600 Value, calculate the unit fluorescence reduction value;
[0031] (3) The total fluorescence intensity and OD of the activated bacterial solution obtained in step (1) 600 value, and the total fluorescence intensity and OD of the reaction solution obtained in step (2) 600 The unit fluorescence reduction value is calculated based on the value; the unit fluorescence reduction value is then substituted into the quantitative standard curve to calculate the concentration of neonicotinoid pesticides in the sample to be tested.
[0032] Wherein, in step (1), the activation method is as follows: first, the genetically engineered strain frozen in -80°C glycerol is taken out and placed on ice, inoculated on a solid culture medium containing kanamycin, and cultured in a constant temperature incubator at 37°C overnight. The next day, a single colony is picked and placed in a liquid culture medium containing kanamycin, and cultured overnight at 37°C and 200-300 r / min to obtain an expanded culture solution. The expanded culture solution is then diluted at a ratio of 1:10 to 1:30, and cultured at 20°C to 40°C and 200-300 r / min for 4 hours to culture the strain to the logarithmic phase.
[0033] Wherein, in step (2), the amount of the sample to be tested is added as follows: 1 mL of the sample to be tested is added to every 1 mL of activated bacterial solution.
[0034] Wherein, in step (2), the reaction is carried out under the following conditions: 20-40° C., 200-300 r / min, and 1-3 h.
[0035] Wherein, in step (1) and step (2), the total fluorescence intensity is detected under the following fluorescence detection conditions: excitation light 450-500nm, emission light 500-550nm.
[0036] Wherein, in step (3), the unit fluorescence reduction value is calculated as follows:
[0037] Unit fluorescence reduction value = (total fluorescence intensity CK / OD 600 CK)-(total fluorescence intensity i / OD 600 i);
[0038] Among them, the total fluorescence intensity CK represents the total fluorescence intensity of the activated bacterial solution before the reaction, and OD 600 CK represents the OD of the activated bacterial solution before the reaction 600 value, the total fluorescence intensity i represents the total fluorescence intensity of the sample to be tested, OD 600 i represents the OD of the sample to be tested 600 value.
[0039] Wherein, in step (3), the neonicotinoid pesticide includes any one or a combination of acetamiprid, imidacloprid, thiamethoxam, and nitenpyram.
[0040] The quantitative standard curve preparation process is as follows: the fluorescence of the activated bacterial solution is read using a microplate reader, and the OD 600 The neonicotinoid pesticides imidacloprid (IMI), acetamiprid (ACE), and thiamethoxam (THM) were added to 1 mL of activated bacterial solution at concentrations of 0, 0.1, 0.5, 1, 5, 10, 50, and 100 mg / L, respectively, according to the concentration range in sewage. The reaction was shaken at 20-40°C and 200-300 r / min for 2 hours. The fluorescence was read using a microplate reader, and the OD was detected at the same time. 600 Fluorescence detection conditions are: excitation light 450-500nm, emission light 500-550nm. According to the unit fluorescence reduction value = (total fluorescence intensity CK / OD 600 CK)-(total fluorescence intensity i / OD 600i) Perform normalized fluorescence calculation. The total fluorescence intensity CK represents the total fluorescence intensity of the activated bacterial solution before the reaction, and OD 600 CK represents the OD of the activated bacterial solution before the reaction 600 value, total fluorescence intensity i represents the total fluorescence intensity of the detected sample, OD 600 i represents the OD of the test sample 600 Quantitative standard curves of concentration-unit fluorescence reduction value of neonicotinoid pesticides such as imidacloprid IMI, acetamiprid ACE, and thiamethoxam THM were drawn and their analytical performance was evaluated.
[0041] Beneficial effects:
[0042] (1) The present invention constructs a recombinant plasmid for detecting environmental neonicotinoid pesticides by cloning the ligand binding domain ECD of the α7 subunit of the nicotinic acetylcholine receptor into an expression vector. The recombinant plasmid is further expressed in Escherichia coli to construct a genetically engineered bacterium for detecting environmental neonicotinoid pesticides. The genetically engineered bacterium can be used as a single fluorescent protein bioreceptor, effectively improving the stability of the biosensor under different environments.
[0043] (2) The method for detecting environmental neonicotinoid pesticides provided by the present invention does not require expensive instruments and professional detection personnel, and does not require cumbersome pretreatment and long-term incubation or analysis, and can quickly identify and quantitatively detect environmental neonicotinoid pesticides.
[0044] (3) The present invention utilizes the specific binding of receptor proteins to target pollutants to achieve targeted detection of environmental neonicotinoid pesticides.
[0045] (4) The present invention utilizes the rapid binding of receptor proteins to target pollutants and the fluorescence changes induced by real-time receptor conformational changes, significantly improving the response time of genetically engineered bacteria as single fluorescent protein biosensors, and achieving real-time targeted rapid detection of environmental neonicotinoid pesticides.
[0046] (5) Compared with traditional detection technologies, this single fluorescent protein biosensor technology is low-cost, easy to operate, and fast. Compared with other cells, E. coli has low environmental requirements for growth and can tolerate sewage to a certain extent. In addition, this single fluorescent protein biosensor can achieve real-time detection, avoiding the long cell culture and incubation time. Therefore, in the case of on-site detection of environmental neonicotinoid pesticides, this single fluorescent protein biosensor can be preferably used to construct a rapid detection method.
[0047] (6) The detection method based on the present invention can realize real-time in-situ detection of neonicotinoid pesticides in the water environment, promote the development of analytical detection methods for environmental neonicotinoid pesticides, and is of great significance to the subsequent development of environmental neonicotinoid pesticide control strategies and the ecological protection of the water environment. BRIEF DESCRIPTION OF THE DRAWINGS
[0048] The present invention will be further described below in detail with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more apparent.
[0049] Figure 1 The following is a diagram showing enzyme digestion verification of the recombinant plasmid GENE1. Lane 1 is the recombinant plasmid GENE1; Lane 2 is the recombinant plasmid GENE1 treated with restriction endonucleases BtsI and BsrGI; Lane M is a DNA marker. DETAILED DESCRIPTION
[0050] The present invention will be further described below in conjunction with specific embodiments, and the above and / or other advantages of the present invention will become more apparent.
[0051] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.
[0052] Example 1: Construction of recombinant plasmids and genetically engineered bacteria for detecting environmental neonicotinoid pesticides
[0053] 1. Construction of recombinant plasmid
[0054] The ECD portion of the rat nicotinic acetylcholine receptor α7 subunit α7nAChR (GenBank: AAC33136.1) was cloned into the corresponding position between the BtsI and BsrGI restriction endonuclease sites of the vector pEGFP-N1 (GenBank: U55762.1) that can express enhanced green fluorescent protein (EGFP). This replaced the residues between the BtsI and BsrGI restriction sites that express EGFP (residues 227-466) to construct a recombinant plasmid. The specific process is as follows:
[0055] The ligand-binding domain (ECD) of the α7 subunit of the rat nicotinic acetylcholine receptor (α7nAChR) (GenBank: AAC33136.1) was codon-optimized based on the codon preference of the E. coli expression system. The optimized sequence is shown in SEQ ID NO. 2. Based on the optimized sequence, target primers containing recognition sites for the BtsI and BsrGI restriction enzymes were synthesized using an automated synthesizer and PCR amplification was performed. After PCR amplification, the size of the PCR product was analyzed using agarose gel electrophoresis to confirm whether it matched the expected size of the α7nAChR ECD fragment. The target band was excised and recovered using a gel recovery kit to obtain the purified target gene fragment (α7nAChR ECD fragment).
[0056] The pEGFP-N1 vector (GenBank: U55762.1, the gene sequence of vector pEGFP-N1 is shown in SEQ ID NO. 3) was double-digested with two restriction enzymes, BtsI and BsrGI, to remove the residues between the BtsI and BsrGI restriction sites (residues 227 to 466) that express enhanced green fluorescent protein (EGFP), thereby obtaining a linearized pEGFP-N1 vector. The purified target gene fragment was ligated with the linearized pEGFP-N1 vector, and the ligation product was transformed into Escherichia coli BL21 (DE3) competent cells. After positive clone screening and single colony selection, the recombinant plasmid GENE1 was extracted using a plasmid extraction kit, and the recombinant plasmid GENE1 was digested and sequenced to verify the successful construction of the recombinant plasmid GENE1, whose gene sequence is shown in SEQ ID NO. 1. Figure 1 This is a diagram showing enzyme digestion verification of the recombinant plasmid GENE1. The synthesis process was commissioned by Jiangsu Saisuofei Biotechnology Co., Ltd.
[0057] 2. Construction of genetically engineered bacteria
[0058] The recombinant plasmid obtained in step 1 was transformed into BL21 (DE3) Escherichia coli by heat shock method (i.e., the BL21 (DE3) competent cells were taken out and placed on ice, the recombinant plasmid was added and gently mixed, and the cells were placed on ice for 30 minutes. After heat shock at 42°C for 45 seconds, the cells were quickly returned to ice for 2 minutes without shaking, and LB liquid culture medium was added and mixed. The cells were shaken and cultured at 37°C for 1 hour). The transformed product was evenly spread on LB plate culture medium containing 50 μg / mL kanamycin using a coating rod, and the plate was inverted in a 37°C incubator for overnight culture. 3 to 6 single colonies were picked up with a sterile pipette tip and cultured in LB liquid culture medium containing 50 μg / mL kanamycin resistance. After culture, nucleic acid dye was added for electrophoresis detection. The clones with bands consistent with the expected size were selected for shake extraction of plasmids and sequencing. After enzyme digestion and sequencing verification, the genetically engineered strain gene1 was obtained.
[0059] Example 2: Detection of neonicotinoid pesticides in the environment
[0060] The genetically engineered strain gene1 constructed in Example 1 was used as a single fluorescent protein bioreceptor to detect neonicotinoid pesticides in the environment. The specific operation process is as follows.
[0061] 1. Draw a standard curve
[0062] (1) Activation of gene1 strain: The genetically engineered strain gene1 frozen in -80℃ glycerol was taken out and placed on ice, inoculated on LB solid medium containing 50μg / mL kanamycin, and cultured in a constant temperature incubator at 37℃ overnight. The next day, a single colony was picked and placed in LB liquid medium, and cultured overnight at 37℃ and 200-300r / min to obtain an expanded culture solution. The expanded culture solution was then diluted at a ratio of 1:10-30 and cultured at 20-40℃ and 200-300r / min for 4 hours to culture the strain to the logarithmic phase to obtain an activated culture solution.
[0063] (2) Establish a quantitative curve for neonicotinoid pesticides: Use a microplate reader to read the fluorescence of the activated bacterial solution and simultaneously detect the OD 600 The neonicotinoid pesticides Imidacloprid (IMI), Acetamiprid (ACE), and Thiamethoxam (THM) were added to 1 mL of activated bacterial solution at concentrations of 0, 0.1, 0.5, 1, 5, 10, 50, and 100 mg / L, respectively, according to the concentration range in sewage. The reaction was shaken at 20-40°C and 200-300 r / min for 2 hours. The fluorescence was read using a microplate reader, and the OD was detected at the same time. 600 Fluorescence detection conditions are: excitation light 450-500nm, emission light 500-550nm. According to the unit fluorescence reduction value = (total fluorescence intensity CK / OD 600 CK)-(total fluorescence intensity i / OD 600 i) Perform normalized fluorescence calculation. The total fluorescence intensity CK represents the total fluorescence intensity of the activated bacterial solution before the reaction, and OD 600 CK represents the OD of the activated bacterial solution before the reaction 600 value, total fluorescence intensity i represents the total fluorescence intensity of the detected sample, OD 600 i represents the OD of the test sample 600 Quantitative standard curves of imidacloprid IMI, acetamiprid ACE, and thiamethoxam THM concentration-unit fluorescence reduction value were drawn and their analytical performance was evaluated.
[0064] 2. Detection of neonicotinoid pesticides in the environment
[0065] According to the method in step 1 (1), the activated bacterial solution was obtained. 1 mL of the sample to be tested was added to 1 mL of the activated bacterial solution from the water environment of the Yangtze River tributary in Hai'an County, Nantong City, Jiangsu Province. The reaction was shaken at 37°C and 200 r / min for 2 h. The fluorescence was read and the OD was detected. 600 The unit fluorescence reduction value is calculated based on the concentration-unit fluorescence reduction value quantitative standard curve of different neonicotinoid pesticides. The unit fluorescence reduction value is respectively substituted to calculate the concentration of neonicotinoid pesticides IMI, ACE, and THM in the sample. The IMI, ACE, and THM concentration information of the test sample can be quickly detected within 3 hours.
[0066] The present invention provides a recombinant plasmid, genetically engineered bacteria, and a method for detecting neonicotinoid pesticides in the environment. While there are numerous methods and approaches for implementing this technical solution, the foregoing merely represents a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.
Claims
1. A recombinant plasmid for detecting neonicotinoid pesticides in the environment, characterized in that: The ligand binding domain ECD portion of the nicotinic acetylcholine receptor α7 subunit is cloned into an expression vector to construct the recombinant plasmid.
2. The recombinant plasmid according to claim 1, characterized in that The gene sequence of the ligand binding domain ECD portion of the nicotinic acetylcholine receptor α7 subunit is shown in SEQ ID NO.
2.
3. The recombinant plasmid according to claim 1, characterized in that The expression vector is pEGFP-N1 vector.
4. The recombinant plasmid according to claim 3, characterized in that The ligand binding domain ECD portion of the nicotinic acetylcholine receptor α7 subunit is cloned into the expression vector, specifically cloning the ligand binding domain ECD portion of the nicotinic acetylcholine receptor α7 subunit between the BtsI and BsrGI restriction sites of the expression vector.
5. A genetically engineered bacterium for detecting neonicotinoid pesticides in the environment, characterized in that: The genetically engineered bacteria contains the recombinant plasmid according to any one of claims 1 to 4.
6. Use of the recombinant plasmid according to any one of claims 1 to 4 or the genetically engineered bacteria according to claim 5 in detecting neonicotinoid pesticides in the environment.
7. A method for detecting neonicotinoid pesticides in the environment, characterized in that: The steps include: (1) After the genetically engineered bacteria are activated, the total fluorescence intensity of the activated bacterial solution is read and its OD is detected 600 value; (2) Add the sample to be tested to the activated bacterial solution for reaction, read the total fluorescence intensity of the reaction solution, and detect its OD 600 Value, calculate the unit fluorescence reduction value; (3) The total fluorescence intensity and OD of the activated bacterial solution obtained in step (1) 600 value, and the total fluorescence intensity and OD of the reaction solution obtained in step (2) 600 Value, calculate the unit fluorescence reduction value; The unit fluorescence reduction value is then substituted into the quantitative standard curve to calculate the concentration of neonicotinoid pesticides in the sample to be tested.
8. The method according to claim 7, characterized in that In step (2), the amount of the sample to be tested is: 1 mL of the sample to be tested is added to every 1 mL of the activated bacterial solution; and the reaction conditions are: 20-40° C., 200-300 r / min, and 1-3 h.
9. The method according to claim 7, characterized in that In step (3), the unit fluorescence reduction value is calculated as follows: Unit fluorescence reduction value = (total fluorescence intensity CK / OD 600 CK)-(total fluorescence intensity i / OD 600 i); Among them, the total fluorescence intensity CK represents the total fluorescence intensity of the activated bacterial solution before the reaction, and OD 600 CK represents the OD of the activated bacterial solution before the reaction 600 value, the total fluorescence intensity i represents the total fluorescence intensity of the sample to be tested, OD 600 i represents the OD of the sample to be tested 600 value.
10. The method according to claim 7, characterized in that In step (3), the neonicotinoid pesticide includes any one or a combination of acetamiprid, imidacloprid, thiamethoxam, and nitenpyram.