CN109856394B - Detection method and detection kit for cannabinoids active substances based on cell dopamine release effect - Google Patents

Abstract

The invention discloses a detection method of a cannabinoid active substance based on a cell dopamine release effect and a detection kit thereof, wherein the detection kit comprises two components of a detection kit and a control kit, the detection kit comprises a monoclonal cell line CB1/SK-N-SH and an ELISA detection kit which stably express a humanized cannabinoid receptor CB1 gene, and the control kit comprises a blank control cell line Neo/SK-N-SH and an ELISA detection kit. The invention can realize accurate and high-sensitivity rapid detection of all cannabinoids active substances, including natural cannabinoids or synthetic cannabinoids, including novel synthetic cannabinoids active substances which are layered endlessly, can solve the problem of difficult supervision of cannabinoids psychoactive substances, and can rapidly, efficiently, accurately and high-flux detect the cannabinoids active substances in various samples.

Description

Detection method and detection kit for cannabinoids active substances based on cell dopamine release effect

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for detecting a cannabinoid active substance based on a cell dopamine release effect and a detection kit thereof.

Background

The detection and supervision of the cannabis drug intake of drug addicts are always difficult problems of supervision departments in various countries. Because the current detection means mainly rely on antibody (e.g., anti-tetrahydrocannabis antibody) immunoassays, including chromatography, ELISA, and the like; and large-scale instrument analysis such as gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. However, cannabis metabolizes faster in a human body, so that the content of target detection components in a detection sample is extremely low and cannot be detected. On the other hand, the new synthetic cannabinoids are endless and have diverse molecular structures, making antibody-based immunoassays nearly impossible, even GC-MS and LC-MS.

Disclosure of Invention

In view of the above technical problems in the prior art, the present invention aims to provide a method and a kit for detecting cannabinoids active substances based on a cellular dopamine releasing effect.

The conception of the invention is as follows: both natural and a wide variety of synthetic cannabinoids act in the body to act on cannabinoid receptors as targets, thereby producing their biological effects. Cannabinoid receptors include at least two types, CB1 and CB2, CB1 is mainly present in the central nervous system. Cannabinoid receptors belong to the class of GPCRs and signal pathways are activated when acted upon by cannabinoid agonists, i.e., through IP3Ca stored in endoplasmic reticulum by DAG signal²⁺The rapid release to cytoplasm increases the concentration of free calcium ions in cytoplasm, thereby generating cell response, such as dopamine release of nerve cells. As the dopamine is measured by a mature ELISA detection kit, a cell dopamine release effect-based cannabinoid active substance detection method is established by establishing a neuroblastoma cell line CB1/SK-N-SH of stable transfer CB1, and is applied to fast, efficient, accurate and high-flux detection of the cannabinoid active substances in various samples.

The detection method of the cannabinoids active substances based on the cell dopamine release effect is characterized by comprising the following steps:

1) cloning a humanized cannabinoid receptor CB1 gene to be under a CMV promoter of a lentiviral expression vector pCDH-CMV-MCS-EF1-Neo, connecting enzyme cutting sites of EcoR I and Not I, and constructing a eukaryotic expression pCMV-CB1 plasmid;

2) transfecting the pCMV-CB1 plasmid in the step 1) to neuroblastoma cells, establishing a CB1 stably-transfected neuroblastoma cell line, and performing culture amplification to obtain a detection group cell culture solution; meanwhile, a neuroblastoma cell line transfected with a control empty vector is established by taking a lentivirus expression vector pCDH-CMV-MCS-EF1-Neo as the control empty vector, and is cultured and amplified to obtain a control cell culture solution;

3) step 2) adding tetrahydrocannabinol into the detection group cell culture solution, fully reacting, and preparing a series of standard solutions with different tetrahydrocannabinol concentrations; after the standard solution is centrifugally separated, taking supernatant, detecting an absorbance OD value under the dopamine effect by using an ELISA detection kit, drawing a standard curve by taking the measured absorbance OD value as a vertical coordinate and the concentration of the tetrahydrocannabinol in the standard solution as a horizontal coordinate, and calculating a fitting regression equation;

4) step 2) adding a sample to be detected into the cell culture solution of the detection group, fully reacting, centrifuging, taking supernatant, and detecting the absorbance OD under the dopamine effect of the supernatant of the cell culture solution of the detection group after reaction by using an ELISA detection kit₁A value; step 2) adding a sample to be detected into the cell culture solution of the control group, fully reacting, and separatingSeparating heart, collecting supernatant, and detecting absorbance OD under dopamine effect by ELISA detection kit for the supernatant of reacted control cell culture solution₂A value; calculation of the Absorbance OD₁Value and absorbance OD₂And (4) substituting the difference into the regression equation in the step 3) to calculate the effect content of the cannabinoids active substances in the sample to be detected.

The method for detecting the cannabinoid active substances based on the dopamine release effect of the cells is characterized in that in the step 2), neuroblastoma cells are SK-N-SH cells.

The method for detecting the cannabinoids active substances based on the dopamine release effect of the cells is characterized in that in the step 2), the process of establishing a CB1 stable-transfer neuroblastoma cell line is as follows: co-transfecting the pCMV-CB1 plasmid, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare a CMV-CB1 lentivirus; infecting SK-N-SH cells with CMV-CB1 lentivirus to obtain CB1/SK-N-SH stable transfected cells, screening the CB1/SK-N-SH stable transfected cells by using a conditioned medium containing neomycin G418, and obtaining a monoclonal cell line CB1/SK-N-SH stably expressing a humanized cannabinoid receptor CB1 gene by a picking and cloning method.

The method for detecting the cannabinoids active substances based on the dopamine release effect of the cells is characterized in that in the step 2), the process of establishing a neuroblastoma cell line transfected with a control empty vector comprises the following steps: co-transfecting the pCDH-CMV-MCS-EF1-Neo control empty vector, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare an empty vector lentivirus; infecting SK-N-SH cells with empty carrier lentivirus to obtain Neo/SK-N-SH stable transfected cells, screening the Neo/SK-N-SH stable transfected cells by using a conditioned medium containing neomycin G418, and obtaining a blank control cell line Neo/SK-N-SH by a picking and cloning method.

The detection method of the cannabinoids active substances based on the cell dopamine release effect is characterized in that in the step 3), tetrahydrocannabinol is added into a cell culture solution of a detection group and fully reacts to prepare 5 standard solutions with the concentrations of the tetrahydrocannabinol being 0.1, 0.2, 0.4, 0.8 and 1.6ng/mL respectively; wherein the process of carrying out the full reaction is as follows: and (3) uniformly mixing, and reacting for 3-10 minutes at 35-40 ℃.

The detection method of the cannabinoids active substances based on the cell dopamine release effect is characterized in that in the step 4), the process of carrying out sufficient reaction comprises the following steps: and (3) uniformly mixing, and reacting for 3-10 minutes at 35-40 ℃.

The method for detecting the cannabinoids active substances based on the cell dopamine release effect is characterized in that in the step 4), the sample to be detected is urine, blood, hair, dandruff, sweat or saliva of a drug addict, or any one of natural cannabis, natural cannabis products, synthetic cannabis products, sewage, soil and ponds.

The detection kit for the cannabinoid active substances is characterized by comprising a detection kit and a control kit, wherein the detection kit comprises a monoclonal cell line CB1/SK-N-SH and an ELISA detection kit which stably express a humanized cannabinoid receptor CB1 gene, and the control kit comprises a blank control cell line Neo/SK-N-SH and an ELISA detection kit.

Compared with the prior art, the invention has the following beneficial effects:

(1) the limit that an immunoassay method depends on an antibody is avoided, all cannabinoid active substances can be directly and universally detected, the problem of non-specific combination is avoided, and expensive manpower and material resources and complicated and long-term antibody research and development processes are not needed; meanwhile, the limit that the gas chromatography-mass spectrometry and the liquid chromatography-mass spectrometry depend on a reference substance, the limited detection limit and the complex sample pretreatment work are avoided. The technical scheme of the invention can realize accurate and high-sensitivity rapid detection of all cannabinoid active substances, including natural cannabinoids or synthetic cannabinoids, including novel synthetic cannabinoids active substances which are layered endlessly, and can solve the problem of difficult supervision of the cannabinoid psychoactive substances.

(2) The existing dopamine ELISA detection kit can be used for quantitative detection and analysis.

(3) The kit can be used for measuring by a general fluorescence microplate reader, and can realize detection in most common laboratories.

(4) Because the detection is based on the biological effect of the receptor signal channel, the result not only can correctly judge whether the cannabinoid active substance exists, but also can quantitatively evaluate the activity of the cannabinoid active substance, and provides reliable experimental basis for supervision and medical intervention.

(5) The invention provides a monoclonal cell line CB1/SK-N-SH for stably expressing a human cannabinoid receptor CB1 gene, which can respond to all cannabinoid active substances including natural cannabinoids and synthetic cannabinoids through an IP3-DAG signal pathway of CB1, and the cell releases dopamine, so that the monoclonal cell line can be applied to high-sensitivity universal detection of the cannabinoid active substances, the stable neuroblastoma cell line comprises a gene CB1 with high expression started by CMV, and when the cannabinoid active substances act on CB1, the IP3-DAG signal pathway can be started, so that nerve cells can quickly release dopamine; therefore, the existing dopamine detection kit can be used for quantitative detection and analysis. On the other hand, the invention provides a simple, rapid, efficient, sensitive (picogram-scale), accurate and high-flux universal detection kit for cannabinoids active substances, and the detection kit is based on an IP3-DAG signal channel of a CB1 receptor and is used for realizing rapid, sensitive and accurate quantitative detection and analysis by detecting dopamine rapidly released by cells.

Drawings

FIG. 1 is a plasmid map of pCMV-CB 1.

Fig. 2 is a standard curve plotted with the THC standard concentration as the abscissa and the OD value for dopamine detection as the ordinate.

Fig. 3 is a result of hair sample detection using the universal cannabinoid active substance detection kit of the present invention.

FIG. 4 shows the results of the competitive ELISA method for THC at concentrations of 0-12.8ng/ml, and the curves show THC detection limits >0.4 ng/ml.

FIG. 5 is a standard curve of the competitive ELISA method for THC at a concentration of 0.4-12.8 ng/ml.

FIG. 6 shows the results of the competitive ELISA method for hair samples.

Detailed Description

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

In the following examples, the lentiviral packaging line cell 293V was purchased from Biotech, Inc., of Mitsuga, Beijing.

Example 1 construction of pCMV-CB1 plasmid

By ligation of enzyme sitesEcoRI andNoti, cloning a human cannabinoid receptor CB1 gene to the CMV promoter of a lentiviral expression vector pCDH-CMV-MCS-EF1-Neo to construct a eukaryotic expression plasmid pCMV-CB1 (shown in figure 1).

EXAMPLE 2 establishment of CB1/SK-N-SH stable transfected cell line and blank control Neo/SK-N-SH stable transfected cell line

The pCMV-CB1 plasmid, the pH1 plasmid and the pH2 plasmid are co-transfected to a lentivirus packaging line cell 293V to prepare a CB1 lentivirus, and transfect an SK-N-SH cell, and a CB1/SK-N-SH stable transfer cell line is established by G418 screening and cloning. The pCDH-CMV-MCS-EF1-Neo plasmid, the pH1 plasmid and the pH2 plasmid are co-transfected to a lentivirus packaging line cell 293V to prepare an empty vector lentivirus, and transfect an SK-N-SH cell, and a blank control Neo/SK-N-SH stable transfer cell line is established by G418 screening and cloning. The method comprises the following specific steps:

1) preparing packaging line cells: one day before transfection, the lentiviral packaging line cell 293V was prepared at 1X 10 using DMEM-H complete medium (containing 10% FBS, 100U/mL penicillin, 100. mu.g/mL streptomycin diabody)⁶One cell culture dish was inoculated with D19cm at a concentration of 5% CO at 37 ℃₂The culture was carried out overnight.

2) Transfection: when the cells in the dish reached 70% -80% confluence, 20. mu.g of pCMV-CB1 plasmid, 14. mu.g of pH1 plasmid and 4.7. mu.g of pH2 plasmid were co-transfected into the cells 293V in the dish using PEI transfection reagent (see its standard transfection protocol).

3) Collecting viruses: step 2) collecting supernatant after culturing for 72 hours; centrifuging at 4 deg.C and 8000g relative centrifugal force for 15 min, removing precipitate, and filtering the supernatant with 0.45 μm filter membrane to obtain filtered supernatant.

4) And (3) virus concentration: and (3) centrifuging the virus solution (about 30mL) filtered in the step 3) for 90 minutes at the temperature of 4 ℃ and under the relative centrifugal force of 90000g, removing the supernatant to obtain a precipitate, namely the CMV-CB1 lentivirus, and carrying out resuspension on the virus solution by using 1mL of DMEM-H complete culture solution to obtain the concentrated CMV-CB1 lentivirus.

5) SK-N-SH cell preparation: 1 is multiplied by 10 in advance⁵SK-N-SH cells at a concentration of one/mL are inoculated into 1 well of a 12-well cell culture plate, and the plate is incubated at 37 ℃ and 5% CO₂Cultured to 50% cell confluence.

6) SK-N-SH cell transfection: sucking off a culture solution for culturing SK-N-SH cells in the 12-well plate in the step 5), and adding 1mL of CMV-CB1 lentivirus concentrated in the step 4); 37 ℃ and 5% CO₂Culturing for 22-24 hr, and replacing with fresh DMEM-H complete culture solution.

7) Screening of stable transfected cells of CB 1/SK-N-SH: culturing the SK-N-SH cells transfected in the step 6) for more than one week to prepare 1 × 10⁴The culture medium is inoculated to 6-well cell culture plates at the concentration of 2 mL/well; the culture solution is aspirated after the cells adhere to the wall the next day, and 2mL of conditioned medium (DMEM-H complete culture solution containing 600. mu.g/mL of G418) is added; the conditioned medium (DMEM-H complete medium containing 600. mu.g/mL of G418) was replaced 1 time every three days, and the DMEM-H complete medium containing 200. mu.g/mL of G418 was replaced after 21-30 days.

8) Establishment of a monoclonal CB1/SK-N-SH stable transfer cell line: preparing the CB1/SK-N-SH stable cell of the step 7) into 1 × 10³The culture medium is inoculated to 6-well cell culture plates at the concentration of 2 mL/well; after 10 days of culture, single clones were picked under a microscope with a 50. mu.L pipette and transferred to a new 6-well cell culture plate for culture, 1 clone/well; after amplification, the CB1 gene is detected by PCR, the CB1 protein is detected by WB, so that a CB1/SK-N-SH cell clone line with high, medium and low different expression levels is obtained and is used for detecting the cannabinoids active substances with different active effect degrees.

Establishment of a blank control Neo/SK-N-SH stable transgenic cell line: establishing a blank control Neo/SK-N-SH stable cell line according to the steps 1) to 7) except that: replacing the pCMV-CB1 plasmid in the step 2) with a pCDH-CMV-MCS-EF1-Neo plasmid with the same quality, and finally obtaining a blank control Neo/SK-N-SH stable cell line.

Example 3 application of Universal detection kit for cannabinoids active substances

The universal detection kit for the cannabinoids active substances, which is developed based on the technical scheme of the invention, can be applied to the detection of various samples containing the cannabinoids active substances. In this example, the hair of a person who sucks hemp is taken as an example. The method comprises the following specific steps:

1) hair sample treatment: hair samples from 6 cannabis smokers and 8 normal persons with no history of smoking were taken and numbered as in table 1.

A hair sample of 20 mg/part of hair having a root length of 3cm or less was cut, and then cut into pieces and put into a 5mL EP tube, 2mL of 1% keratinase-containing HBSS buffer solution (pH7.4) was added, and a small amount of zirconium beads and quartz sand were added, followed by pulverization with shaking on a pulverizer for 1 minute.

2) Cell preparation: 5X 10 in advance one day⁵Monoclonal CB1/SK-N-SH cells and blank control Neo/SK-N-SH cells with the concentration of one cell/mL are respectively inoculated on a 96-well cell culture plate, and the cell culture plate is 200 mu L/well; 37 ℃ and 5% CO₂The culture was carried out for 24 hours.

3) Sample adding treatment: the samples were classified into a standard sample group and a sample group, and the treatment method was as follows.

Standard substance group: CB1/SK-N-SH cell 6 holes, each hole is added with THC standard substance with final concentration of 0.1, 0.2, 0.4, 0.8, 1.6ng/mL, 100 muL/hole; blank control Neo/SK-N-SH cells were treated similarly.

Sample group: CB1/SK-N-SH cells total 42 holes (each sample has 3 repeated holes), the sample liquid obtained in the step 1) is added, 100 mu L/hole; blank control Neo/SK-N-SH cells were treated similarly.

4) And (3) detection: and (3) respectively carrying out dopamine concentration test on the standard substance group and the sample group treated in the step 3), wherein the detection method steps of the standard substance group and the sample group are the same. The detection process of the standard group comprises the following steps: and 3) uniformly mixing the standard substance groups processed in the step 3), reacting for 5-10 minutes at 37 ℃, centrifuging for 5 minutes under the condition of 800g relative centrifugal force, sucking supernatant liquid, and adding the supernatant liquid into a pore plate of a dopamine ELISA detection kit for quantitative determination and analysis.

5) As a result: drawing a standard curve by taking the concentration of the THC standard substance as an abscissa and the OD value detected by dopamine as an ordinate, wherein the regression equation is y = 1.6053x + 0.6044, R²= 0.9835, as shown in fig. 2. The OD value results of dopamine detection in the sample group are shown in FIG. 3, and positive samples and negative samples can be obviously distinguished; calculating according to a standard curve equation, wherein no cannabinoid component can be detected in the negative samples, and the relative content of the cannabinoid in the positive samples is (ng/mg): c1: 0.0101, C2:0.0537, C3:0.0344, C4:0.0341, C5:0.0230 and C6: 0.0756.

Comparative example 1: competitive ELISA method for detecting hair sample of hemp sucking person

At present, the cannabis detection mainly depends on immunoassay, including colloidal gold immunochromatography and competitive ELISA. The colloidal gold method is suitable for quick detection, but the ELISA method is 1 order of magnitude higher than the colloidal gold method in sensitivity. The comparative example is the hair sample of hemp suckers detected by ELISA method. The method comprises the following specific steps:

1) hair sample treatment: samples were treated in the same manner as in example 3 to obtain corresponding sample solutions (hair sample numbers are the same as those in table 1).

2) Coating antigen: the THC-BSA antigen protein was formulated to a concentration of 1. mu.g/mL with 0.05mol/L carbonate buffer (pH9.6) and coated on a 96-well plate at 100. mu.l/well, and after overnight at 4 ℃, the plate was washed 5 times with PBS buffer (pH7.4) to remove non-specific binding as much as possible.

3) Grouping experiments: the processing method of the standard curve group and the sample group comprises the following steps:

standard curve set: the THC standard substance is prepared into 9 groups with final concentration of 0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8ng/mL by PBS buffer solution (pH7.4), each group contains 5 mu g/mL THC-Mab, and after mixing, 1 hole and 100 mu L/hole of a 96-hole plate coated by THC-BSA are added.

Sample group: adding THC-Mab monoclonal antibody into the sample liquid extracted from the hair, wherein the final concentration of the THC-Mab monoclonal antibody is 5 mug/mL, uniformly mixing, adding 1 hole of each 96-pore plate coated by THC-BSA, 100 mug/hole of each 96-pore plate, and repeating 3 holes of each sample.

4) Competitive binding and assay: incubating each group at 37 ℃ for 30 minutes, pouring out the solution, and washing the plate 5 times with PBS (pH7.4); adding a secondary goat anti-mouse antibody labeled with HRP, incubating at 37 ℃ for 30 minutes, pouring out the solution, and washing the plate 5 times with PBS (pH7.4); after the reaction was developed and terminated, the absorbance (OD) was measured at a wavelength of 450nm using a microplate reader.

5) As a result: as shown in FIG. 4, the results indicate the detection limit of THC by the competitive ELISA method>0.4 ng/mL; the standard curve of absorbance (OD) values at THC concentrations of 0.4-12.8ng/mL is shown in FIG. 5, and the curve equation is y = -0.164x + 2.1684, R²= 0.9323. The results of the sample set are shown in fig. 6, only 2 positive samples were detected, and the cannabinoid component was not detected in all the negative samples, as calculated by drawing a standard curve equation, and the tetrahydrocannabinol content in the detected 2 positive samples was (ng/mg): 0.020 part by weight of C2 and 0.062 part by weight of C6.

As can be seen from example 3 and comparative example 1, the competitive ELISA method for detecting hair samples of marijuana smokers is far less sensitive and accurate than the system of the present invention. Moreover, ELISA relies on specific antibodies and can only detect a single component, which is ineffective for the infinite variety of synthetic cannabinoids, and the present technology solves this problem well. In addition, the method of the invention is a direct method, and the method has sensitivity, simple and convenient operation, linear range of dose-effect, accuracy of quantification and the like which are obviously superior to the competitive ELISA method.

The statements in this specification merely set forth a list of implementations of the inventive concept and the scope of the present invention should not be construed as limited to the particular forms set forth in the examples.

Claims (6)

1. A method for detecting cannabinoids active substances based on cell dopamine release effect is characterized by comprising the following steps:

1) cloning a humanized cannabinoid receptor CB1 gene to be under a CMV promoter of a lentiviral expression vector pCDH-CMV-MCS-EF1-Neo, connecting enzyme cutting sites of EcoR I and Not I, and constructing a eukaryotic expression pCMV-CB1 plasmid;

2) transfecting the pCMV-CB1 plasmid in the step 1) to neuroblastoma cells, establishing a CB1 stably-transfected neuroblastoma cell line, and performing culture amplification to obtain a detection group cell culture solution; meanwhile, a neuroblastoma cell line transfected with a control empty vector is established by taking a lentivirus expression vector pCDH-CMV-MCS-EF1-Neo as the control empty vector, and is cultured and amplified to obtain a control cell culture solution;

3) step 2) adding tetrahydrocannabinol into the detection group cell culture solution, fully reacting, and preparing a series of standard solutions with different tetrahydrocannabinol concentrations; after the standard solution is centrifugally separated, taking supernatant, detecting an absorbance OD value under the dopamine effect by using an ELISA detection kit, drawing a standard curve by taking the measured absorbance OD value as a vertical coordinate and the concentration of the tetrahydrocannabinol in the standard solution as a horizontal coordinate, and calculating a fitting regression equation;

4) step 2) adding a sample to be detected into the cell culture solution of the detection group, fully reacting, centrifuging, taking supernatant, and detecting the absorbance OD under the dopamine effect of the supernatant of the cell culture solution of the detection group after reaction by using an ELISA detection kit₁A value; step 2) adding a sample to be detected into the cell culture solution of the control group, fully reacting, centrifuging, taking supernatant, and detecting the absorbance OD under the dopamine effect of the supernatant of the cell culture solution of the control group after reaction by using an ELISA detection kit₂A value; calculation of the Absorbance OD₁Value and absorbance OD₂Substituting the difference into the regression equation in the step 3) to calculate the effect content of the cannabinoid active substances in the sample to be detected;

in step 2), the process of establishing the CB 1-stable neuroblastoma cell line is as follows: co-transfecting the pCMV-CB1 plasmid, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare a CMV-CB1 lentivirus; infecting SK-N-SH cells with CMV-CB1 lentivirus to obtain CB1/SK-N-SH stable transfected cells, screening the CB1/SK-N-SH stable transfected cells by using a conditioned medium containing neomycin G418, and obtaining a monoclonal cell line CB1/SK-N-SH stably expressing a humanized cannabinoid receptor CB1 gene by a picking and cloning method.

2. The method according to claim 1, wherein in step 2), the neuroblastoma cells are SK-N-SH cells.

3. The method for detecting cannabinoid active ingredients based on dopamine releasing effect in cells according to claim 2, wherein in step 2), the neuroblastoma cell line transfected with control empty vector is established by: co-transfecting the pCDH-CMV-MCS-EF1-Neo control empty vector, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare an empty vector lentivirus; infecting SK-N-SH cells with empty carrier lentivirus to obtain Neo/SK-N-SH stable transfected cells, screening the Neo/SK-N-SH stable transfected cells by using a conditioned medium containing neomycin G418, and obtaining a blank control cell line Neo/SK-N-SH by a picking and cloning method.

4. The method according to claim 1, wherein in step 3), the THC is added to the cell culture solution of the test group and fully reacted to prepare 5 standard solutions with THC concentrations of 0.1, 0.2, 0.4, 0.8 and 1.6 ng/mL; wherein the process of carrying out the full reaction is as follows: and (3) uniformly mixing, and reacting for 3-10 minutes at 35-40 ℃.

5. The method according to claim 1, wherein the sufficient reaction is performed in step 4) by: and (3) uniformly mixing, and reacting for 3-10 minutes at 35-40 ℃.

6. The method according to claim 1, wherein in step 4), the sample is urine, blood, hair, dandruff, sweat or saliva of drug addicts, or any one of natural hemp, natural hemp product, synthetic hemp product, sewage, soil and pond.

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