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Universal detection method for opioid active substances and detection kit thereof

A general-purpose detection technology for active substances, applied in measuring devices, material analysis through optical means, instruments, etc., can solve the problems of low component content, undetectable, impossible immunoassay, etc., to achieve high-sensitivity detection, fast Detection effect

Active Publication Date: 2019-07-12
杭州迈尔德生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, 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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  • Universal detection method for opioid active substances and detection kit thereof
  • Universal detection method for opioid active substances and detection kit thereof
  • Universal detection method for opioid active substances and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of pCMV-MOR1·EF1-copGFP plasmid

[0040] Ligase site EcoR I and not 1, the human mu opioid receptor MOR1 gene is cloned under the CMV promoter of the lentiviral expression vector pCDH-CMV-MCS-EF1-copGFP, and the eukaryotic expression plasmid pCMV-MOR1·EF1-copGFP is constructed (such as figure 1 shown).

Embodiment 2

[0041] Example 2 Establishment of MOR1·copGFP / 293 stable cell line and copGFP / 293 stable cell line

[0042] The pCMV-MOR1·EF1-copGFP plasmid, pH1 plasmid, and pH2 plasmid were co-transfected into lentiviral packaging line cell 293V to prepare CMV-MOR1·EF1-copGFP lentivirus, and transfected into HEK293 cells, and clones were picked under a fluorescent microscope to establish MOR1·copGFP / 293 stably transfected cell line. Specific steps are as follows:

[0043] 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 / ml concentration, and inoculated in a D19cm cell culture dish, 37°C, 5% CO 2 Incubate overnight.

[0044] 2) Transfection: When the cells in the culture dish grow to 70%-80% confluence, use PEI transfection reagent (refer to its standard transfection operation procedure) to inf...

Embodiment 3

[0051] Example 3 Application of Universal Detection Kit for Opioid Active Substances

[0052] 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:

[0053] 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.

[0054]

[0055] 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.

[0056] 2) Cell preparation: mix 5×10 cells one day in adv...

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Abstract

The invention discloses a universal detection method for opioid active substances and a detection kit thereof, the detection kit is divided into a detection group reagent and a control group reagent,the detection group reagent comprises a monoclonal cell line MOR1-copGFP / 293 for stably expressing a human mu opioid receptor MOR1 gene and intracellular free calcium ion detection fluorescent probe reagent Fura Red, and the reagent of the control group comprises a blank control monoclonal cell line copGFP / 293 and the intracellular free calcium ion detection fluorescent probe reagent Fura Red; thepreparation method of the blank control monoclonal cell line copGFP / 293 comprises the following steps of: performing co-transfection of pCDH-CMV-MCS-EF1-copGFP plasmids, pH1 plasmids and pH2 plasmidsto lentivirus packaging cells 293V to prepare EF1-copGFP lentivirus; and infecting human embryo kidney cells 293 with the copGFP lentivirus to obtain the blank control monoclonal cell line copGFP / 293. Accurate and high-sensitivity rapid detection of all opioid active substances can be achieved, and the problem that supervision of novel synthetic opioid psychiatric active substances is difficult can be solved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a general detection method for opioid active substances 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 immunoassays almost impossible, and even GC-MS and LC-MS are difficu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 范春雷
Owner 杭州迈尔德生物科技有限公司
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