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Methamphetamine aptamer, detection kit and application thereof

A technology for methamphetamine and a detection kit, which is applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology and other directions, can solve the problems of unsatisfactory methamphetamine detection technology and the like

Active Publication Date: 2015-07-01
SHANGHAI CRIMINAL SCI TECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] In summary, the current methamphetamine detection technology is still unsatisfactory, therefore, there is an urgent need in this field to develop new technologies that can quickly detect methamphetamine on the spot

Method used

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  • Methamphetamine aptamer, detection kit and application thereof
  • Methamphetamine aptamer, detection kit and application thereof
  • Methamphetamine aptamer, detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Coupling of methamphetamine complete antigen to cyanogen bromide-activated agarose

[0127] Weigh 2g of cyanogen bromide-activated agarose, and after activation, incubate with 5mg of methamphetamine complete antigen overnight, with 0.1M NaHCO 3 (containing 0.5M NaCl) and centrifuged to discard the supernatant. Blocking was performed overnight with 0.2M glycine. After blocking, use Binding buffer (20mmol / L Tris-HCl, 137mmol / L NaCl, 5mmol / LKCl, 2mmol / L CaCl 2 , 1mol / l MgCl 2 ) washing, after washing, put into the affinity chromatography column. An affinity chromatography column containing BSA was prepared as a negative column as described above.

Embodiment 2

[0129] SELEX screening

[0130] Methods as below:

[0131] (1) Binding: 200pmol / L ssDNA library was heat-denatured at 95°C for 10min, then rapidly cooled to 0°C, after 5min, the ssDNA library was first added to the negative column for reverse screening, and the liquid was collected and then added to the column containing methamphetamine, using Binding Buffer was washed 3 times.

[0132] (2) Elution: adding 100 μg / ml methamphetamine for elution, and collecting the eluate for PCR.

[0133] (3) PCR reaction: PCR reaction system: 23 μl of ssDNA template, 1 μl of upstream primer, 1 μl of downstream primer, 25 μl of 10×PCR buffer. PCR reaction conditions: pre-denaturation at 95°C for 3min, 25 cycles of denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, and finally extension at 72°C for 3min. (Among them, the results of the optimization experiment are as follows: figure 1 shown. )

[0134] (4) Purification: Run the PCR product on a 2% agarose ge...

Embodiment 3

[0139] Detection of binding rate of ssDNA library to complete methamphetamine antigen in each round by SPR

[0140] (1) Coating of CM5 sensor chip: 2mM Cys1ml was dropped on the gold membrane and incubated for 12h, then 15mM NHS and 75mM EDC (freshly prepared) were mixed with 6mg / ml CM-dextran and dropped on the surface of the gold membrane for 3h.

[0141] (2) Determination of binding rate: control the flow rate at 40 μl / min, and pass through a continuous section of Binding Buffer until the baseline is stable. When the baseline is stable, inject 200 μl of amino coupling reagent containing 0.4M EDC and 0.1M NHS to activate the carboxyl group on the CM5 sensor chip. Dilute methamphetamine complete antigen to 5mg / ml with Binding Buffer, inject 200 μ l of this solution, because the amino group on the BSA and the activated ester group on the sensor chip have amino coupling reaction, methamphetamine is immobilized on the sensor chip. Inject 200 μl of 1M ethanolamine solution to bl...

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Abstract

The invention provides a methamphetamine aptamer, a detection kit and application thereof. Specifically, based on an ssDNA library with a special structure, the inventor adopts the SELEX (systematic evolution of ligands by exponential enrichment) technique to successfully screen the aptamer with high affinity and specificity to methamphetamine. The aptamer provided by the invention can be used for on-site fast and sensitive detection of methamphetamine.

Description

technical field [0001] The invention relates to the field of biotechnology and the field of criminal identification technology. Specifically, the present invention provides a methamphetamine nucleic acid aptamer, a detection kit and applications thereof, especially in poison detection. Background technique [0002] Drugs have become a more and more serious social problem. In view of the actual needs of anti-drug work, it is necessary to develop rapid drug detection technology with high sensitivity, good selectivity, portable, low energy consumption, and easy operation, so as to realize sensitive and rapid detection on the spot. [0003] At present, the main technologies for rapid on-site screening of drug addicts and drug detection in liquids at home and abroad are various products based on immune reactions, mainly including enzyme immunoassay and immune colloidal gold technology. [0004] Enzyme immunoassay involves enzyme reaction, the detection steps are cumbersome, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12Q1/68
Inventor 曾立波张玉荣陈连康胡小龙
Owner SHANGHAI CRIMINAL SCI TECH RES INST
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