CN110006866B - General detection method and detection kit for opioid active substances - Google Patents
General detection method and detection kit for opioid active substances Download PDFInfo
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Abstract
The invention discloses a general detection method of opioid active substances and a detection kit thereof, wherein the detection kit is divided into a detection group reagent and a control group reagent, the detection group reagent comprises a monoclonal cell line MOR 1. copGFP/293 for stably expressing an anthropogenic mu-type opioid receptor MOR1 gene and an intracellular free calcium ion detection fluorescent probe reagent Fura Red, and the control group reagent comprises a blank control monoclonal cell line copGFP/293 and an intracellular free calcium ion detection fluorescent probe reagent Fura Red; the preparation method of the blank control monoclonal cell line copGFP/293 comprises the following steps: co-transfecting the pCDH-CMV-MCS-EF1-copGFP plasmid, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare an EF1-copGFP lentivirus; the blank monoclonal cell line, copGFP/293, was obtained by infecting human embryonic kidney cells 293 with EF1-copGFP lentivirus. The invention can realize the accurate and high-sensitivity rapid detection of all opioid active substances and can solve the problem of difficult supervision of novel synthesized opioid psychoactive substances.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a general detection method and a detection kit for opioid active substances.
Background
The detection and supervision of the drug addicts on taking novel opioid psychotropic substances, such as fentanyl, is a difficult problem for the regulatory departments of various countries. Because current detection means rely primarily on antibody (e.g., anti-fentanyl 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, fentanyl is metabolized rapidly 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 newly synthesized fentanyl is infinite in layer and various in molecular structure, so that antibody-based immunoassay is almost impossible, and even gas chromatography and liquid chromatography are difficult to realize supervision and detection.
Disclosure of Invention
In view of the above technical problems in the prior art, the present invention aims to provide a general detection method for opioid active substances and a detection kit thereof.
The conception of the invention is as follows: whether natural opioids or variousA wide variety of synthetic opioid psychotropic substances, such as fentanyl and its derivatives, act in the body at opioid receptors, which are targets for biological effects. The opioid receptors include at least four opioid receptors of mu type, k type, delta type and sigma type, and the opioid receptor of mu type is the most powerful receptor with affinity and action for opioid psychotics such as morphine and fentanyl. Opioid receptors belong to the GPCR class and, when activated by opioid molecular agonists, signal pathways are activated, i.e., Ca stored in the endoplasmic reticulum is signaled by IP3-DAG2+The cytoplasm is quickly released, and the concentration of free calcium ions in cytoplasm is increased, so that a cellular response is generated. Because a great number of calcium ion indicators can be used for measuring the concentration of free calcium ions in cytoplasm, a cell line which can express green fluorescent protein copGFP stably transformed MOR1 is established, the copGFP is used as an internal reference control, and Fura Red is used as a calcium ion fluorescence indicator; single-wavelength excitation and double-wavelength detection, and establishes a novel rapid, simple, efficient, accurate, high-flux and universal detection method for the opioid active substances by taking the fluorescence ratio of the fluorescent calcium to the copGFP as a measurement parameter.
The general detection method of the opioid active substances is characterized by comprising the following steps:
1) cloning the opioid receptor gene MOR1 to a CMV promoter of a vector containing EF1 for promoting expression of fluorescent protein to construct an expression plasmid;
2) transfecting the plasmid with the fluorescent protein gene constructed in the step 1) to eukaryotic immortalized cells, establishing an EF 1-fluorescent protein gene stable cell line, and performing culture amplification to obtain a detection group culture solution; similarly establishing a transfection blank control plasmid, establishing a blank control EF 1-fluorescent protein gene stable transfer cell line, and performing culture amplification to obtain a control group culture solution;
3) step 2), adding a standard substance into the detection group culture solution, wherein the standard substance is morphine or fentanyl, and preparing a series of detection group standard solutions with different standard substance concentrations; adding intracellular free calcium ion probe reagents into the standard solution of the detection group, and after full reaction, detecting the fluorescence value PF of intracellular fluorescent protein and the fluorescence value Ca of fluorescent calcium ion2+F, as measuredFluorescence value Ca2+Taking the F/fluorescence value PF ratio as a vertical coordinate, drawing a standard curve by taking the concentration of the standard substance as a horizontal coordinate, and calculating a fitting regression equation;
4) step 2) adding samples to be detected into the detection group culture solution and the control group culture solution respectively, and preparing a sample solution and a control solution respectively, wherein the concentration of the samples to be detected in the sample solution is the same as that of the samples to be detected in the control solution; adding free calcium ion probe reagent into the sample solution and the contrast solution, reacting sufficiently, and detecting the fluorescence value PF of the intracellular fluorescent protein in the reacted sample solution1And the fluorescence value Ca of the fluorescent calcium ion2+F1Detecting the fluorescence value PF of the intracellular fluorescent protein of the control solution after the reaction2And the fluorescence value Ca of the fluorescent calcium ion2+F2And calculating to obtain a fluorescence value Ca2+F1Per fluorescence value PF1Ratio and fluorescence value Ca2+F2Per fluorescence value PF2Substituting the difference of the ratios into the regression equation in the step 3) to calculate the content of the opioid active substances in the detection sample.
The universal detection method for opioid active substances is characterized in that in the step 1), an opioid receptor gene MOR1 is a humanized mu-type opioid receptor MOR1 gene; the fluorescent protein was copGFP.
The general detection method of opioid active substances is characterized in that the plasmid constructed in the step 1) is pCMV-MOR 1.EF 1-copGFP plasmid, and the preparation process comprises the following steps: cloning the humanized mu opioid receptor MOR1 gene to the CMV promoter of a lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP, connecting enzyme cutting sites of EcoR I and Not I, and constructing a eukaryotic expression pCMV-MOR 1. EF1-copGFP plasmid.
The general detection method for opioid active substances is characterized in that in the step 2), the eukaryotic immortalized cell is a lentivirus packaging cell 293V, and the process of establishing an EF 1-fluorescent protein gene stable transfer cell line comprises the following steps: co-transfecting the pCMV-MOR 1.EF 1-copGFP plasmid, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare a CMV-MOR 1.EF 1-copGFP lentivirus, and infecting the CMV-MOR 1.EF 1-copGFP lentivirus into a human embryonic kidney cell 293; and a monoclonal cell line MOR 1. copGFP/293 which stably expresses the humanized mu opioid receptor MOR1 gene is obtained by a method of picking and cloning under a fluorescence microscope.
The general detection method for opioid active substances is characterized in that in the step 2), the blank control plasmid is pCDH-CMV-MCS-EF1-copGFP plasmid, and the process of establishing the blank control EF 1-fluorescent protein gene stable transfer cell line is as follows: co-transfecting the pCDH-CMV-MCS-EF1-copGFP plasmid, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare an EF1-copGFP lentivirus; the EF1-copGFP lentivirus was used to infect human embryonic kidney cells 293, resulting in the blank control monoclonal cell line copGFP/293.
The general detection method for the opioid active substances is characterized in that in the step 3), a standard substance is added into a detection group culture solution to prepare 5 detection group standard solutions with the concentrations of the standard substance of 0.01ng/ml, 0.05ng/ml, 0.25ng/ml, 1.25ng/ml and 6.25ng/ml respectively, wherein the standard substance is morphine.
The general detection method for the opioid active substances is characterized in that in the step 3), a standard substance is added into a detection group culture solution to prepare 5 detection group standard solutions with the concentrations of the standard substance being 1pg/ml, 3pg/ml, 9pg/ml, 27pg/ml and 81pg/ml respectively, wherein the standard substance is fentanyl.
The general detection method for opioid active substances is characterized in that intracellular free calcium ion probe reagents in the steps 3) and 4) are Fura Red; in step 3), detecting the fluorescence value PF of the intracellular fluorescent protein and the fluorescence value Ca of the fluorescent calcium ions2+The method F comprises the following steps: testing with a fluorescence microplate reader, selecting 482nm single wavelength for excitation, and detecting with 507nm and 640nm double wavelengths, wherein PF is the fluorescence value of intracellular fluorescent protein detected at 507nm, and Ca is the fluorescence value of fluorescent calcium ion detected at 640nm2+F。
The general detection method for opioid active substances 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 opioid, synthetic opioid, sewage, soil and pond.
The detection kit for the opioid active substances is characterized by comprising a detection group reagent and a control group reagent, wherein the detection group reagent comprises a monoclonal cell line MOR 1. copGFP/293 for stably expressing a humanized mu opioid receptor MOR1 gene and an intracellular free calcium ion detection fluorescent probe reagent Fura Red, and the control group reagent comprises a blank control monoclonal cell line copGFP/293 and an intracellular free calcium ion detection fluorescent probe reagent Fura Red;
the preparation method of the blank control monoclonal cell line copGFP/293 comprises the following steps: co-transfecting the pCDH-CMV-MCS-EF1-copGFP plasmid, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare an EF1-copGFP lentivirus; the blank monoclonal cell line, copGFP/293, was obtained by infecting human embryonic kidney cells 293 with EF1-copGFP lentivirus.
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 opioid active substances can be directly and universally detected, the problem of non-specific binding is avoided, and expensive manpower and material resources and complicated long-period 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 opioid active substances, including natural opioid products and synthesized opioid molecules, including infinite novel synthesized opioid active substances, such as fentanyl and various derivatives thereof, and can solve the problem of difficult supervision of the novel synthesized opioid psychoactive substances.
(2) The existing mature intracytoplasmic free calcium ion indicator is used, the green fluorescent protein which is stably expressed in a transfer mode is used as an internal reference for quality control, and qualitative and quantitative detection and analysis can be carried out on trace opioid psychoactive substances.
(3) The detection can be realized in most common laboratories by using instruments such as a general fluorescence microplate reader, a flow cytometer and the like for detection.
(4) Because the biological effect detection based on the receptor signal channel is adopted, the result not only can correctly judge whether the opioid active substance is the opioid active substance, but also can quantitatively evaluate the activity of the opioid active substance, and provides reliable experimental basis for supervision and medical intervention.
(5) The invention provides a stable cell line which can respond to all opioid active substances including natural opioid and synthetic opioid through an IP3-DAG signal pathway of MOR1, so that the stable cell line can be applied to high-sensitivity universal detection of the opioid active substances. The stable transgenic cell line comprises a CMV-initiated high expression gene MOR 1; when the opioid active substance acts on MOR1, it can activate IP3-DAG signal path to store Ca in endoplasmic reticulum2+Rapidly released into cytoplasm and free Ca in cytoplasm2+Can be instantly improved by hundreds of times. Therefore, the mature technology for measuring the concentration of the free calcium ions in cytoplasm can be used for quantitative detection analysis. On the other hand, the invention provides a simple, rapid, efficient, sensitive (femtogram-picogram grade), accurate and high-flux universal detection kit for opioid active substances, which is based on an IP3-DAG signal channel of an MOR1 receptor and realizes rapid, sensitive and accurate detection and quantitative analysis by fluorescence detection for detecting the change of the concentration of free calcium ions in cytoplasm.
Drawings
FIG. 1 is a pCMV-MOR 1. EF1-copGFP plasmid map;
FIG. 2 shows Fura Red-Ca on the abscissa of morphine standard concentration2+The ratio of the fluorescence value to the fluorescence value of the copGFP is a standard curve drawn by a vertical coordinate;
FIG. 3 is the result of detecting morphine hair sample by the universal detection kit for opioid active substances of the present invention;
FIG. 4 is the detection results of the competitive ELISA method on morphine standards with concentrations of 0-12.8ng/ml, showing that the morphine detection limit is >0.2 ng/ml;
FIG. 5 is a standard curve of competitive ELISA for morphine detection at concentrations of 0.2-6.4 ng/ml;
FIG. 6 shows the results of the competitive ELISA method for hair samples;
FIG. 7 shows Fura Red-Ca on the abscissa of the concentration of fentanyl as a standard2+Drawing a standard curve standard by taking the ratio of the fluorescence value to the fluorescence value of the copGFP as a vertical coordinate;
FIG. 8 is the result of detection of a Fente-like sample by the universal detection kit for opioid active substances according to the present invention;
FIG. 9 is a graph of the results of a competitive ELISA assay on fentanyl standards at concentrations of 0-12.8ng/ml, showing that the detection limit for fentanyl is >0.4 ng/ml;
FIG. 10 is a standard curve for the detection of fentanyl by competitive ELISA methods at concentrations of 0.4 to 12.8 ng/ml;
FIG. 11 shows the results of a competitive ELISA assay on fentanyl 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, lentivirus packaging cells 293V were purchased from Biotech, Inc., of the Mitsuga, Beijing.
EXAMPLE 1 pCMV-MOR 1. EF1-copGFP plasmid construction
By ligation of enzyme sitesEcoRI andNoti, cloning the humanized mu opioid receptor MOR1 gene to the CMV promoter of a lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP to construct a eukaryotic expression plasmid pCMV-MOR 1. EF1-copGFP (shown in figure 1).
EXAMPLE 2 establishment of MOR 1. copGFP/293 Stable Transit cell line and copGFP/293 Stable Transit cell line
The pCMV-MOR1 & EF1-copGFP plasmid, the pH1 plasmid and the pH2 plasmid are co-transfected to a lentivirus packaging line cell 293V to prepare CMV-MOR1 & EF1-copGFP lentivirus, and then the CMV-MOR1 & EF1-copGFP lentivirus is transfected to an HEK293 cell, and the clone is picked under a fluorescence microscope to establish an MOR1 & copGFP/293 stable transfer cell line. 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 double antibody)6At a concentration of one cell/ml, and inoculated in a cell culture dish of D19cm at 37 ℃ with 5% CO2Culturing overnight。
2) Transfection: when the cells in the dish reached 70% -80% confluence, 20. mu.g of pCMV-MOR 1. EF1-copGFP plasmid, 14. mu.g of pH1 plasmid, and 4.7. mu.g of pH2 plasmid were co-transfected with PEI transfection reagent (see its standard transfection protocol) into the cells 293V in the dish.
3) Collecting viruses: culturing for 70-72 hr, and collecting supernatant; 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: taking supernatant virus liquid (about 30ml) filtered in the step 3), carrying out centrifugal separation for 90 minutes at the temperature of 4 ℃ and under the relative centrifugal force condition of 90000g, removing the supernatant to obtain a precipitate, namely CMV-MOR 1. EF1-copGFP lentivirus, and carrying out re-suspension by using 1ml of DMEM-H complete culture solution to obtain the concentrated CMV-MOR 1. EF1-copGFP lentivirus.
5) HEK293 cell preparation: 1 is multiplied by 10 in advance5HEK293 cells at individual/ml concentration were seeded into 1 well of 12-well cell culture plates at 37 ℃ with 5% CO2Cultured to 50% cell confluence.
6) Transfection of HEK293 cells: sucking out the culture solution for culturing HEK293 cells in the 12-well plate in the step 5), and adding 1ml of CMV-MOR 1. EF1-copGFP lentivirus concentrated in the step 4); 37 ℃ and 5% CO2Culturing for 22-24 hr, and replacing with fresh DMEM-H complete culture solution.
7) Establishing a single-clone stable-transfer cell line of MOR 1. copGFP/293: culturing the transfected cells in the step 6) for more than one week to prepare 1 × 103Inoculating the culture medium into 6-well cell culture plates at the concentration of 2 ml/well; after 10 days of culture, single clone cell masses with different fluorescence intensities are picked up by a 50-microliter pipette under a fluorescence microscope and transferred to a new 6-well cell culture plate for culture, and 1 clone/well is obtained; after amplification, the MOR1 gene is detected by PCR, the MOR1 protein is detected by WB, so that MOR 1. copGFP/293 cell clone lines with high, medium and low different expression levels are obtained, and the clone lines are used for detecting opioid active substances with different activity effect degrees.
8) Establishing a copGFP/293 monoclonal stable transfer cell line: the procedure of 1) -7) above was followed to establish a copGFP/293 monoclonal stable cell line, except that: replacing the pCMV-MOR 1-EF 1-copGFP plasmid in the step 2) with a pCDH-CMV-MCS-EF1-copGFP plasmid with the same mass to finally obtain a copGFP/293 monoclonal stable cell line.
EXAMPLE 3 general detection kit for opioid actives
The universal detection kit for the opioid active substances, which is developed based on the technical scheme of the invention, can be applied to the detection of various samples containing the opioid active substances. In this example we take the example of the hair of a person who takes morphine. The method comprises the following specific steps:
1) hair sample treatment: hair samples from 5 morphine feeders and 5 normal persons with no history of feeding were taken and numbered as in table 1.
Clipping hair sample with hair root less than 3cm 20 mg/part, placing into 5ml EP tube after clipping, adding 2ml HBSS buffer solution (pH7.4) containing 1% keratinase, adding small amount of zirconium beads and quartz sand, and vibrating and pulverizing for 1 min on pulverizer to obtain corresponding sample solutions respectively.
2) Cell preparation: 5X 10 in advance one day5The single clone MOR 1. copGFP/293 cells and the blank control copGFP/293 cells with each concentration per ml were inoculated in a 96-well cell culture plate at 200. mu.l/well, respectively; 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: MOR 1. copGFP/293 cells in 6 wells, each containing Morphine (MOP) standard at final concentrations of 0, 0.01, 0.05, 0.25, 1.25, 6.25ng/ml and 100. mu.l/well, respectively; the blank control copGFP/293 cells were treated similarly.
Sample group: MOR 1. copGFP/293 cells with 30 wells (3 repeat wells each) were added to the sample solution obtained in step 1) at 100. mu.l/well; the blank control copGFP/293 cells were treated similarly.
4) And (3) detection: for the standard substance group and the sample group processed in the step 3), respectivelyAnd (3) detecting the fluorescence value of the intracellular fluorescent protein and the fluorescence value of the fluorescent calcium ions, 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: adding Fura Red working solution into the standard substance group treated in the step 3), and culturing at 37 ℃ for 20 minutes; adding 3 times of volume of HBSS buffer solution containing 1% FBS, and continuing to culture for 40 minutes; the cells were washed 3 times with HBSS buffer, and centrifuged each time with a 96-well plate to prevent cell loss; culturing at 37 deg.C for 10 min, detecting intracellular fluorescent calcium ion with fluorescence microplate reader, exciting at 482nm single wavelength, detecting at 507nm and 640nm dual wavelengths, and detecting the fluorescence value of intracellular fluorescent calcium ion at 640nm (labeled as Fura Red-Ca)2+Fluorescence value), the fluorescence value of the fluorescent protein in the cell was detected at 507nm (labeled as the fluorescent value of copGFP).
5) As a result: drawing a standard curve for the detection result of the standard substance group in the step 3), taking the concentration of the morphine standard substance as the abscissa, and using Fura Red-Ca2+Fluorescence value (640 nm detection)/copGFP fluorescence value (507 nm detection) ratio is used as the ordinate to draw a standard curve, and the regression equation is y = 0.4375x + 0.6012, R2= 0.9922, standard curve plotted as shown in fig. 2. Step 3) response values of the sample group to CB 1. copGFP/293 cells and blank control copGFP/293 cells are shown in figure 3, and results can obviously distinguish that positive samples and negative samples are obvious; and (3) calculating according to a standard curve drawing equation, wherein the morphine components cannot be detected in the negative samples, and the morphine contents in the positive samples are respectively (ng/mg): m1:0.049, M2:0.090, M3:0.048, M4:0.02 and M5: 0.019.
Comparative example 1: competitive ELISA method for detecting hair sample of morphine absorber
At present, the opioid detection mainly depends on an immunoassay method, including a colloidal gold immunochromatography method and a competitive ELISA method. 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. This comparative example is a sample of hair from morphine smokers tested by ELISA. The method comprises the following specific steps:
1) hair sample treatment: the samples were treated in the same manner as in step 1) of example 3 to obtain respective sample liquids (hair sample numbers are the same as those in Table 1).
2) Coating antigen: the MOP-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 MOP standard substances are prepared into 9 groups with final concentrations of 0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8ng/ml respectively, each group contains 5 mu g/ml (the concentration is determined by the titer of the antibody), and after mixing, the MOP-BSA coated 96-well plate is added into 1 well and 100 mu l/well respectively.
Sample group: adding MOP-Mab monoclonal antibody into the sample liquid extracted from the hair, wherein the final concentration of the MOP-Mab monoclonal antibody is 5 mu g/ml, uniformly mixing, adding 1 hole and 100 mu l/hole of a 96-well plate coated by MOP-BSA, 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: the detection results of the standard curve set are shown in FIG. 4, and the results show that the detection limit of the competitive ELISA method for MOP>0.2 ng/ml; a standard curve with MOP concentration of 0.2-6.4ng/ml is shown in FIG. 5, and the curve equation is y = -0.2458x + 1.6281, R2= 0.9444. The results of the panel are shown in FIG. 6, with 4 of 5 positive samples detected and 1 undetected. And (3) calculating according to a standard curve equation, wherein the morphine components can not be detected in all the negative samples, and the morphine contents in the detected 4 positive samples are respectively (ng/mg): m1:0.049, M2:0.078, M3:0.063 and M4: 0.059.
As can be seen from example 3 and comparative example 1, the competitive ELISA method for detecting morphine-absorbed hair samples is far less sensitive and accurate than the system of the present invention. Moreover, the ELISA method relies on specific antibodies, only can detect a single component, and cannot help various kinds of synthesized opioid novel psychotropic substances, but the technology of the invention well solves the problem. 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.
EXAMPLE 4 use of the Universal test kit for opioid actives-fentanyl detection
The universal detection kit for the opioid active substances, which is developed based on the technical scheme of the invention, can be applied to the detection of various samples containing the opioid active substances. In this example we take a sample of the fentanyl-containing species as an example. The method comprises the following specific steps:
1) rabbit hair sample treatment: 3 samples of hair from rabbits injected intravenously with fentanyl and 3 samples of hair from normal rabbits are numbered as in Table 2.
20 mg/part of rabbit hair sample is taken, cut into pieces and put into a 5ml EP tube, 2ml HBSS buffer solution (pH7.4) containing 1% keratinase is added, a small amount of zirconium beads and quartz sand are added, the mixture is vibrated and crushed for 1 minute on a crusher, and each sample is diluted by 10 times by using sterile PBS buffer solution (pH7.4) to obtain corresponding sample liquid respectively.
2) Urban sewage: dividing the municipal sewage into 3 parts, wherein 2 parts are respectively added with fentanyl and carfentanil to make the final concentrations respectively 3pg/ml and 0.3pg/ml, and are respectively F4 and F5 of the municipal sewage added in the table 2; another 1 part was municipal sewage (NF4) of a blank control without any drug. Each sample was centrifuged at 1200g for 15 minutes under a relative centrifugal force, and the supernatant was sterilized with a 0.22 μm filter and dialyzed overnight with a sterile PBS buffer (pH7.4) to obtain the corresponding sample solutions.
3) Cell preparation: 5X 10 in advance one day5The single clone MOR 1. copGFP/293 cells and the blank control copGFP/293 cells with each concentration per ml were inoculated in a 96-well cell culture plate at 200. mu.l/well, respectively; 37 ℃ and 5% CO2The culture was carried out for 24 hours.
4) 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: MOR 1. copGFP/293 cells total 6 wells, each well contains a fentanyl standard, and the final concentration of fentanyl is 1, 3, 9, 27, 81pg/ml and 100 mul/well respectively; the blank control copGFP/293 cells were treated similarly.
Sample group: MOR 1. copGFP/293 cells in 27 wells (3 replicate wells each) were added to the sample solutions obtained in step 1) or step 2), 100. mu.l/well, respectively; the blank control copGFP/293 cells were treated similarly.
5) And (3) detection: and (3) respectively detecting the fluorescence value of intracellular fluorescent calcium ions of the standard substance group and the sample group treated in the step 4), wherein the detection method of the standard substance group and the detection method of the sample group have the same steps. The detection process of the standard group comprises the following steps: adding Fura Red working solution into the standard substance group treated in the step 4), and culturing at 37 ℃ for 20 minutes; adding 3 times of volume of HBSS buffer solution containing 1% FBS, and continuing to culture for 40 minutes; the cells were washed 3 times with HBSS buffer, and centrifuged each time with a 96-well plate to prevent cell loss; culturing at 37 deg.C for 10 min, detecting intracellular fluorescent calcium ion with fluorescence enzyme labeling instrument, exciting at single wavelength of 482nm, and detecting at double wavelengths of 507nm and 640 nm.
6) As a result: with fentanyl standard concentration as abscissa, Fura Red-Ca2+Fluorescence value (640 nm detection)/copGFP fluorescence value (507 nm detection) ratio is plotted on ordinate to obtain standard curve standard, regression equation is y = 0.0305x + 0.7985, R2= 0.9806, as shown in fig. 7. The detection results of the sample group are shown in FIG. 8, and the positive samples and the negative samples can be obviously distinguished; and (3) calculating according to a standard curve drawing equation, wherein the fentanyl component cannot be detected in the negative samples, and the fentanyl content in the positive rabbit hair samples is (pg/mg): f1: 19.80, F2:33.78 and F3: 46.69. Average Fura Red-Ca of sewage sample containing 3pg/ml fentanyl in urban sewage sample2+The ratio of fluorescence (640 nm detected)/copgFP fluorescence (507 nm detected) (n =3) was 0.889 ± 0.062, which is 5.08 times that of the negative sample; average Fura Red-Ca of wastewater samples containing 0.3pg/ml carfentanil2+The ratio of fluorescence (640 nm detected)/copgFP fluorescence (507 nm detected) (n =3) was 1.625 ± 0.085, which is 9.28 times that of the negative sample.
Comparative example 2: competitive ELISA method for detecting component sample containing fentanyl substance
At present, the opioid detection mainly depends on an immunoassay method, including a colloidal gold immunochromatography method and a competitive ELISA method. 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 a sample for testing fentanyl-containing components by ELISA. The method comprises the following specific steps:
1) sample treatment: the same treated samples as in example 4 were used. However, the rabbit hair samples were not diluted with sterile PBS buffer (pH7.4) after preparation, and the stock solutions were used as they were to obtain the corresponding sample solutions (the rabbit hair samples were numbered as in Table 2).
2) Coating antigen: Fentanly-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: fentanyl standard was prepared into 9 groups of final concentrations of 0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 12.8ng/ml with PBS buffer (pH7.4), each group containing 5. mu.g/ml Fentanly-Mab, and after mixing, 1 well and 100. mu.l/well of a Fentanly-BSA-coated 96-well plate were added.
Sample group: Fentanly-Mab (Fentanly-Mab) is added into sample liquid extracted from rabbit hair, the final concentration of the Fentanly-Mab is 5 mu g/ml, and after uniform mixing, Fentanly-BSA coated 96-well plates are added, wherein each of the 96-well plates is 1 well, 100 mu l/well, and each sample is provided with 3 multiple wells.
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. 9, the results indicate the detection limit of fentanyl (fentanyl) by the competitive ELISA method>0.4 ng/ml; fentanylThe standard curve of absorbance (OD) values at concentrations of 0.4-12.8ng/ml is shown in FIG. 10, and the regression equation is y = -0.1465x + 1.7861, R2 = 0.9031. The results of the sample group are shown in FIG. 11, and fentanyl component was not detected in both 5 positive samples and 5 negative samples.
As can be seen from example 4 and comparative example 2, the competitive ELISA method for detecting fentanyl samples is far less sensitive and accurate than the system of the present invention. Moreover, the ELISA method relies on specific antibody, only can detect single component, and has no effect on various kinds of synthetic fentanyl with infinite layers, but the technology of the invention well solves the problem, and the biological effect of the detected substance can be truly reflected by taking the biological activity degree of the detected substance as the detection basis. 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 universal detection method for opioid active substances, characterized by comprising the following steps:
1) cloning the opioid receptor gene MOR1 to a CMV promoter of a vector containing EF1 for promoting expression of fluorescent protein to construct an expression plasmid;
2) transfecting the plasmid with the fluorescent protein gene constructed in the step 1) to eukaryotic immortalized cells, establishing an EF 1-fluorescent protein gene stable cell line, and performing culture amplification to obtain a detection group culture solution; similarly establishing a transfection blank control plasmid, establishing a blank control EF 1-fluorescent protein gene stable transfer cell line, and performing culture amplification to obtain a control group culture solution;
3) step 2), adding a standard substance into the detection group culture solution, wherein the standard substance is morphine or fentanyl, and preparing a series of detection group standard solutions with different standard substance concentrations; adding intracellular free calcium ion probe reagent into the standard solution of the detection group, reacting sufficiently, and detecting intracellular fluorescenceFluorescent value PF of photoprotein and fluorescent value Ca of fluorescent calcium ion2+F, measured as fluorescence value Ca2+Taking the F/fluorescence value PF ratio as a vertical coordinate, drawing a standard curve by taking the concentration of the standard substance as a horizontal coordinate, and calculating a fitting regression equation;
4) step 2) adding samples to be detected into the detection group culture solution and the control group culture solution respectively, and preparing a sample solution and a control solution respectively, wherein the concentration of the samples to be detected in the sample solution is the same as that of the samples to be detected in the control solution; adding free calcium ion probe reagent into the sample solution and the contrast solution, reacting sufficiently, and detecting the fluorescence value PF of the intracellular fluorescent protein in the reacted sample solution1And the fluorescence value Ca of the fluorescent calcium ion2+F1Detecting the fluorescence value PF of the intracellular fluorescent protein of the control solution after the reaction2And the fluorescence value Ca of the fluorescent calcium ion2+F2And calculating to obtain a fluorescence value Ca2+F1Per fluorescence value PF1Ratio and fluorescence value Ca2+F2Per fluorescence value PF2Substituting the difference of the ratios into the regression equation in the step 3) to calculate the content of the opioid active substances in the detection sample;
in the step 1), the opioid receptor gene MOR1 is an anthropogenic mu-type opioid receptor MOR1 gene; the fluorescent protein is copGFP;
the plasmid constructed in the step 1) is a pCMV-MOR1, EF1-copGFP plasmid, and the preparation process comprises the following steps: cloning a humanized mu opioid receptor MOR1 gene to the CMV promoter of a lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP, connecting enzyme cutting sites of EcoR I and Not I, and constructing a eukaryotic expression pCMV-MOR 1. EF1-copGFP plasmid;
in the step 2), the eukaryotic immortalized cell is a lentivirus packaging cell 293V, and the process of establishing the EF 1-fluorescent protein gene stable transfer cell line comprises the following steps: co-transfecting the pCMV-MOR 1.EF 1-copGFP plasmid, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare a CMV-MOR 1.EF 1-copGFP lentivirus, and infecting the CMV-MOR 1.EF 1-copGFP lentivirus into a human embryonic kidney cell 293; and a monoclonal cell line MOR 1. copGFP/293 which stably expresses the humanized mu opioid receptor MOR1 gene is obtained by a method of selecting and cloning under a fluorescence microscope;
in the step 2), the blank control plasmid is pCDH-CMV-MCS-EF1-copGFP plasmid, and the process of establishing the blank control EF 1-fluorescent protein gene stable transfer cell line is as follows: co-transfecting the pCDH-CMV-MCS-EF1-copGFP plasmid, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare an EF1-copGFP lentivirus; the EF1-copGFP lentivirus was used to infect human embryonic kidney cells 293, resulting in the blank control monoclonal cell line copGFP/293.
2. The universal test method for opioid active substances according to claim 1, wherein in step 3), 5 test group standards with standard concentrations of 0.01ng/ml, 0.05ng/ml, 0.25ng/ml, 1.25ng/ml and 6.25ng/ml are added to the test group culture solution, and the standard is morphine.
3. The method for the universal detection of opioid actives according to claim 1, wherein in step 3), 5 test-group standard solutions with concentrations of 1pg/ml, 3pg/ml, 9pg/ml, 27pg/ml and 81pg/ml are prepared by adding standard to the test-group culture solution, wherein the standard is fentanyl.
4. The universal test method for opioid active substances according to claim 1, wherein the intracellular free calcium ion probe reagents of step 3) and step 4) are Fura Red; in step 3), detecting the fluorescence value PF of the intracellular fluorescent protein and the fluorescence value Ca of the fluorescent calcium ions2+The method F comprises the following steps: testing with a fluorescence microplate reader, selecting 482nm single wavelength for excitation, and detecting with 507nm and 640nm double wavelengths, wherein PF is the fluorescence value of intracellular fluorescent protein detected at 507nm, and Ca is the fluorescence value of fluorescent calcium ion detected at 640nm2+F。
5. The universal test method for opioid active substances according to claim 1, wherein in step 4), the sample to be tested is urine, blood, hair, dandruff, sweat or saliva of drug addicts, or any one of natural opioid, synthetic opioid, sewage, soil and pond.
6. The detection kit is characterized by comprising a detection group reagent and a control group reagent, wherein the detection group reagent comprises a monoclonal cell line MOR 1. copGFP/293 for stably expressing a humanized mu opioid receptor MOR1 gene and an intracellular free calcium ion detection fluorescent probe reagent Fura Red, and the control group reagent comprises a blank control monoclonal cell line copGFP/293 and an intracellular free calcium ion detection fluorescent probe reagent Fura Red;
the preparation method of the blank control monoclonal cell line copGFP/293 comprises the following steps: co-transfecting the pCDH-CMV-MCS-EF1-copGFP plasmid, the pH1 plasmid and the pH2 plasmid to a lentivirus packaging cell 293V to prepare an EF1-copGFP lentivirus; the blank monoclonal cell line, copGFP/293, was obtained by infecting human embryonic kidney cells 293 with EF1-copGFP lentivirus.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1315547A (en) * | 2000-03-28 | 2001-10-03 | 上海博德基因开发有限公司 | Polypeptide-human phosphatidase 10 and polynucleotide for coding it |
| CN1860364A (en) * | 2003-04-02 | 2006-11-08 | 诺丁汉大学 | Fluorescently tagged ligands |
| AU2005336514A8 (en) * | 2004-09-25 | 2008-12-11 | Novartis Ag | TORC polynucleotides and polypeptides, and methods of use |
| CN201429601Y (en) * | 2009-07-07 | 2010-03-24 | 宁波博奥生物工程有限公司 | Up-converting phosphor detection test paper strip capable of checking morphine quantitatively and rapidly |
| CN102072894A (en) * | 2009-11-25 | 2011-05-25 | 欧普图斯(苏州)光学纳米科技有限公司 | Nano-structure-based spectrum detecting method for detecting chemical and biochemical impurities |
| CN103937836A (en) * | 2014-04-04 | 2014-07-23 | 天津医科大学总医院 | Detection method of aquaporin-4 autoantibody, fusion expression virus vector and application thereof |
| AU2014210856A1 (en) * | 2013-02-01 | 2015-07-30 | Selexis S.A. | Enhanced transgene expression and processing |
| CN105492621A (en) * | 2013-03-28 | 2016-04-13 | 英属哥伦比亚大学 | Microfluidic devices and methods for use thereof in multicellular assays of secretion |
| CN107345909A (en) * | 2017-06-23 | 2017-11-14 | 浙江诺迦生物科技有限公司 | The detection method of trace morphine and the SPR chips used in a kind of saliva sample |
| EP3612088A1 (en) * | 2017-04-21 | 2020-02-26 | AnaBios Corporation | Method for the identification of sensory neuron subtypes in ex vivo preparations |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE602004013280T2 (en) * | 2003-02-18 | 2009-02-19 | Astrazeneca Ab | SCREENING TESTS FOR CANNABINOID-LIGAND ARTIFICIAL MODULATORS OF GPR55 |
| AU2018337737A1 (en) * | 2017-09-19 | 2020-04-02 | Cannametrix, Llc | Cell-based assay for quantifying the potency and efficacy of cannabinoids and/or terpenoids, and methods of use thereof |
-
2019
- 2019-04-16 CN CN201910305617.7A patent/CN110006866B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1315547A (en) * | 2000-03-28 | 2001-10-03 | 上海博德基因开发有限公司 | Polypeptide-human phosphatidase 10 and polynucleotide for coding it |
| CN1860364A (en) * | 2003-04-02 | 2006-11-08 | 诺丁汉大学 | Fluorescently tagged ligands |
| AU2005336514A8 (en) * | 2004-09-25 | 2008-12-11 | Novartis Ag | TORC polynucleotides and polypeptides, and methods of use |
| CN201429601Y (en) * | 2009-07-07 | 2010-03-24 | 宁波博奥生物工程有限公司 | Up-converting phosphor detection test paper strip capable of checking morphine quantitatively and rapidly |
| CN102072894A (en) * | 2009-11-25 | 2011-05-25 | 欧普图斯(苏州)光学纳米科技有限公司 | Nano-structure-based spectrum detecting method for detecting chemical and biochemical impurities |
| AU2014210856A1 (en) * | 2013-02-01 | 2015-07-30 | Selexis S.A. | Enhanced transgene expression and processing |
| CN105492621A (en) * | 2013-03-28 | 2016-04-13 | 英属哥伦比亚大学 | Microfluidic devices and methods for use thereof in multicellular assays of secretion |
| CN103937836A (en) * | 2014-04-04 | 2014-07-23 | 天津医科大学总医院 | Detection method of aquaporin-4 autoantibody, fusion expression virus vector and application thereof |
| EP3612088A1 (en) * | 2017-04-21 | 2020-02-26 | AnaBios Corporation | Method for the identification of sensory neuron subtypes in ex vivo preparations |
| CN107345909A (en) * | 2017-06-23 | 2017-11-14 | 浙江诺迦生物科技有限公司 | The detection method of trace morphine and the SPR chips used in a kind of saliva sample |
Non-Patent Citations (2)
| Title |
|---|
| "Opioid elevation of intracellular free calcium: Possible mechanisms and physiological relevance";Damien S.K. Samways.et al;《Cellular Signalling》;20050930;第151-156页 * |
| "PH对心脏k-阿片受体调节ca2+瞬变作用的影响";裴建明;《第四军医大学学报》;20021231;全文 * |
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