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Cannabinoid active substance detection method based on intracellular free Ca<2+> concentration change effect and cannabinoid active substance detection kit

A technology of active substances and ion concentration, applied in the biological field, can solve the problems of low component content, undetectable, impossible immunoassay, etc., achieve rapid detection, solve difficult supervision, and highly sensitive detection effects

Active Publication Date: 2019-06-28
浙江诺迦生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, cannabinoids are metabolized quickly in the human body, resulting in extremely low levels of target detection components in the test samples, which cannot be detected
On the other hand, the endless emergence of new synthetic cannabinoids with diverse molecular structures makes antibody-based immunoassays almost impossible, even GC / LC / MS

Method used

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  • Cannabinoid active substance detection method based on intracellular free Ca&lt;2+&gt; concentration change effect and cannabinoid active substance detection kit
  • Cannabinoid active substance detection method based on intracellular free Ca&lt;2+&gt; concentration change effect and cannabinoid active substance detection kit
  • Cannabinoid active substance detection method based on intracellular free Ca&lt;2+&gt; concentration change effect and cannabinoid active substance detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Construction of pCMV-CB1 plasmid

[0031] Clone the human cannabinoid receptor CB1 gene into the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-Neo by ligating the restriction sites EcoR I and Not I to construct the eukaryotic expression plasmid pCMV-CB1 (Such as figure 1 Shown).

Embodiment 2

[0032] Example 2 Establishment of CB1 / 293 stable cell line

[0033] The pCMV-CB1 plasmid, pH1 plasmid, and pH2 plasmid were co-transfected into lentiviral packaging line cells 293V to prepare CMV-CB1 lentivirus and transfected into HEK293 cells. G418 screened and cloned to establish a CB1 / 293 stable transfected cell line. Specific steps are as follows:

[0034] 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 prepare lentiviral packaging line cells 293V into 1 ×10 6 A / ml concentration is inoculated in a D19cm cell culture dish, 37℃, 5% CO 2 Cultivate overnight.

[0035] 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-CB1 plasmid, 14 μg of pH1 plasmid, The 293V cells in the culture dish were co-transfected with 4.7 μg...

Embodiment 3

[0042] Example 3 Application of the universal detection kit for cannabinoid active substances

[0043] The universal detection kit for cannabinoid active substances developed based on the technical scheme of the present invention can be applied to the detection of various samples containing cannabinoid active substances. In this embodiment, we take the hair of a person who smokes cannabis as an example. Specific steps are as follows:

[0044] 1) Hair sample processing: Take 6 hair samples of cannabis smokers and 8 hair samples of normal people without smoking history. The numbers are shown in Table 1.

[0045] Cut 20mg / portion of hair samples within 3cm of the hair root, cut into 5ml EP tube, add 2ml HBSS buffer (pH7.4) containing 1% keratinase, add a small amount of zirconium beads and quartz sand, crusher Shake and smash for 1 minute to obtain corresponding sample liquids.

[0046]

[0047] 2) Cell preparation: 5×10 5 Single / ml monoclonal CB1 / 293 cells were seeded in a 96-well cel...

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Abstract

The invention discloses a cannabinoid active substance detection method based on an intracellular free Ca<2+> concentration change effect and a cannabinoid active substance detection kit. The detection kit comprises two components including a detection group reagent and a negative control group reagent, the detection group reagent comprises a monoclonal cell line CB1 / 293 stably expressing a human-derived cannabinoid receptor CB1 gene and an intracellular free Ca<2+> probe reagent Fluo 3-AM; the negative control group reagent comprises the monoclonal cell line CB1 / 293 stably expressing the human-derived cannabinoid receptor CB1 gene, the intracellular free Ca<2+> probe reagent Fluo 3-AM and a cannabinoid receptor antagonist. With the adoption of the detection method, accurate, high-sensitivity and rapid detection of all cannabinoid active substances including natural cannabinoid and synthetic cannabinoid as well as novel synthetic cannabinoid active substances which emerge in endlesslycan be realized, and the problem of difficulty in supervising cannabis psychoactive substances can be solved.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a detection method of cannabinoid active substances based on the effect of changing the concentration of free calcium ions in cells and a detection kit thereof. Background technique [0002] The detection and supervision of cannabis-like drugs used by drug users has always been a difficult problem for regulatory agencies in various countries. Because the current detection methods mainly rely on the immune method of antibodies (such as anti-tetrahydrocannabinoid antibodies), including chromatography, ELISA, etc.; and large-scale instrument analysis such as GC / MS and LC / MS. However, cannabis is metabolized rapidly in the human body, resulting in extremely low content of target detection components in the test samples, which cannot be detected. On the other hand, the endless emergence of new synthetic cannabinoids and their diverse molecular structures make antibody-based immunoa...

Claims

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Application Information

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IPC IPC(8): G01N33/533G01N33/577G01N21/64
Inventor 范春雷程向荣
Owner 浙江诺迦生物科技有限公司
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