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A detection method and detection kit for opioid active substances based on the change effect of intracellular free calcium ion concentration

A technology of active substance and ion concentration, applied in the biological field, can solve the problems of low component content, undetectable, impossible immunodetection method, etc., and achieve the effect of rapid detection and high-sensitivity detection

Active Publication Date: 2022-04-19
浙江诺迦生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, fentanyl metabolizes quickly in the human body, resulting in the extremely low content of the target component in the test sample, which cannot be detected
On the other hand, the proliferation of new synthetic fentanyls with diverse molecular structures makes antibody-based immunoassays nearly impossible, and even regulatory detection by GC / LC / MS is difficult

Method used

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  • A detection method and detection kit for opioid active substances based on the change effect of intracellular free calcium ion concentration
  • A detection method and detection kit for opioid active substances based on the change effect of intracellular free calcium ion concentration
  • A detection method and detection kit for opioid active substances based on the change effect of intracellular free calcium ion concentration

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Construction of pCMV-MOR1 plasmid

[0038] Ligase site EcoR I and not 1, the human mu type opioid receptor MOR1 gene is cloned under the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-Neo, and the eukaryotic expression plasmid pCMV-MOR1 (such as figure 1 shown).

Embodiment 2

[0039] Example 2 Establishment of MOR1 / 293 Stably Transduced Cell Line

[0040] The pCMV-MOR1 plasmid, pH1 plasmid, and pH2 plasmid were co-transfected into lentiviral packaging line cell 293V to prepare MOR1 lentivirus, and transfected into HEK293 cells, G418 screening, and cloning to establish a MOR1 / 293 stable transfection cell line. Specific steps are as follows:

[0041] 1) Preparation of packaging line cells: One day before transfection, use DMEM-H complete culture medium (containing 10% FBS and 100U / ml penicillin, 100μg / ml streptomycin double antibody) to make lentiviral packaging line cells 293V into 1 ×10 6 Inoculate a D19cm cell culture dish at a concentration of 1 / ml at 37°C, 5% CO 2 Incubate overnight.

[0042] 2) Transfection: When the cells in the culture dish grow to 70%-80% confluence, use PEI transfection reagent (refer to its standard transfection procedure) to mix 20 μg of pCMV-MOR1 plasmid, 14 μg of pH1 plasmid, 293V cells in a culture dish were co-tran...

Embodiment 3

[0049] Example 3 Application of Universal Detection Kit for Opioid Active Substances

[0050] The universal detection kit for opioid active substances developed based on the technical solution of the present invention can be applied to the detection of various samples containing opioid active substances. In this embodiment, we take the hair of a person taking morphine as an example. Specific steps are as follows:

[0051] 1) Hair sample processing: Take 5 hair samples of morphine addicts and 5 hair samples of normal people without smoking history, and the numbers are shown in Table 1.

[0052] Table 1 Hair sample number

[0053]

[0054] Cut the hair sample within 3cm of the hair root at 20mg / part, cut it into pieces, put it into a 5ml EP tube, add 2ml of HBSS buffer solution (pH7.4) containing 1% keratinase, add a small amount of zirconium beads and quartz sand, and crush it Shake and pulverize for 1 minute to obtain corresponding sample liquids.

[0055] 2) Cell prepa...

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Abstract

The invention discloses a detection method and a detection kit for opioid active substances based on the change effect of intracellular free calcium ion concentration. The detection kit is divided into two components: a detection group reagent and a negative control group reagent. The detection group reagents include the monoclonal cell line MOR1 / 293 stably expressing the human μ-opioid receptor MOR1 gene and the intracellular free calcium ion probe reagent Fluo 3-AM, and the negative control group reagents include stably expressing the human μ-type opioid receptor MOR1 / 293 The monoclonal cell line MOR1 / 293 for the opioid receptor MOR1 gene, an opioid receptor antagonist, and the intracellular free calcium probe reagent Fluo 3‑AM. The detection method of the invention can realize the accurate, highly sensitive and rapid detection of all opioid active substances, whether it is a natural opioid product or a synthetic opioid molecule, can be accurately and rapidly detected, and can solve the difficulty in the supervision of new synthetic opioid psychoactive substances The problem.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting opioid active substances based on the change effect of intracellular free calcium ion concentration and a detection kit thereof. Background technique [0002] The detection and supervision of drug addicts taking new opioid psychoactive substances, such as fentanyl, is a difficult problem for the regulatory authorities of various countries. Because the current detection methods mainly rely on antibody (such as anti-fentanyl antibody) immunoassays, including chromatography, ELISA, etc.; and large-scale instrumental analysis such as GC-MS and LC-MS. However, fentanyl metabolizes quickly in the human body, resulting in the extremely low content of the target detection component in the test sample, which cannot be detected. On the other hand, new synthetic fentanyls emerge in endlessly, and their molecular structures are diverse, making antibody-based i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867
Inventor 范春雷程向荣
Owner 浙江诺迦生物科技有限公司
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