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Cocaine nucleic acid aptamer, detection kit and application thereof

A nucleic acid aptamer and cocaine technology, which is applied in biological testing, material inspection products, DNA/RNA fragments, etc., can solve the problems of difficulty in preparing monoclonal antibodies for immunity, restrictions on the application of antibodies, and heavy workload of monoclonal antibody preparation.

Active Publication Date: 2020-02-04
SHANGHAI CRIMINAL SCI TECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection of monoclonal antibodies still has certain limitations:
[0005] First, monoclonal antibodies are derived from animals. Therefore, there will be certain restrictions in the preparation of monoclonal antibodies; for example, the toxicity of some drugs and poisons is unbearable to animals, and some small molecules with poor immunity will give a lot to the preparation of monoclonal antibodies. Resistance brings difficulties
[0006] Second, the preparation of hybridomas is limited to mice and rabbits, which greatly limits the application of antibodies
Antibodies from different sources are used for detection, which may cause false positive results due to non-specific binding
[0007] Third, the preparation of monoclonal antibodies requires a lot of work, and for some rare antibodies, a large number of clones need to be screened, which is expensive
[0008] Fourth, antibody preparation has its own defects, and cell lines must be frozen. Unexpected cell death may easily occur if frozen storage is not good.
[0011] Seven, antibody recognition is carried out under physiological conditions, and the binding kinetic parameters of antibodies and antigens cannot be changed according to needs
[0012] In summary, there is still a lack of satisfactory high-sensitivity detection methods for cocaine that are applicable to on-site detection in this area, so there is an urgent need to develop a detection technology for cocaine that has advantages such as sensitivity, fast, reliability, and good specificity.

Method used

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  • Cocaine nucleic acid aptamer, detection kit and application thereof
  • Cocaine nucleic acid aptamer, detection kit and application thereof
  • Cocaine nucleic acid aptamer, detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0142] Preparation of complete cocaine antigen:

[0143] use figure 2 In the shown process, the complete cocaine antigen coupled with BSA was synthesized using commercially available reagents.

Embodiment 2

[0145] SELEX screening

[0146] 2.1 Establish DNA aptamer library

[0147] Select a random single-stranded nucleotide sequence of 40 base pairs, including the PCR primer sequences at both ends, such a nucleic acid aptamer library will contain 1014-1015 different possible sequences. The random nucleic acid aptamer library designed in the present invention includes: (1) primers with 40 nucleotide random sequences (RND40) in the middle and (2) 18 nucleotides at both ends, with a structure such as figure 1 And shown in SEQID NO.:5:

[0148] 5-GCGGATGAAGACTGGTCT-40N-GCCCTAAATACGAGCAAC-3' (SEQ ID NO.: 5).

[0149] 2.2. Screening method:

[0150] Affinity chromatography column method was used to screen the nucleic acid aptamers combined with cocaine.

[0151] 2.3 Cloning and sequencing of aptamers

[0152] In the multi-cycle amplification screening, PCR amplification is performed in each round, and the final PCR product is cloned and DNA sequence determined.

[0153] The PCR pr...

Embodiment 3

[0163] Synthesis and identification of specific DNA aptamers:

[0164] According to the results of DNA sequencing, cocaine DNA aptamer sequences (SEQ ID NO.: 1, 2, 3 and 4) were synthesized and identified by SPR (Surface Plasmon Resonance). The specific method includes the following steps:

[0165] (a) Coating of the gold membrane: 2 mM Cys (mercaptoethylamine) was dropped on the surface of the gold membrane and incubated for 12 hours.

[0166] (b) 1ml of 15mM NHS, 75mM EDC, 6mg / ml CM-dextran mixture was dropped on the gold membrane surface and incubated for 3h.

[0167] (c) Inject cocaine complete antigen (Example 1), EA and buffer, and then add different concentrations of aptamers (4, 6, 8, 10, 12 μM).

[0168] (d) Testing the binding rate of the aptamer to the complete antigen.

[0169] The result is as Figure 4 shown. The results show that the four nucleic acid aptamers (SEQ ID NO.: 1 to 4) of the present invention can all specifically bind to cocaine (COC). Wherein...

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Abstract

The invention provides a cocaine aptamer, a detection kit and application thereof. In particular, based on a special structure ssDNA library, the inventor uses the SELEX (systematic evolution of ligands by exponential enrichment) technology and successfully screens the aptamer with high affinity and specificity on cocaine. The aptamer provided by the invention is suitable for on-site rapid and sensitive detection of cocaine.

Description

technical field [0001] The invention relates to the field of biotechnology and the field of criminal identification technology. Specifically, the present invention relates to a cocaine nucleic acid aptamer, a detection kit and applications thereof, especially in poison detection. Background technique [0002] The widespread abuse of cocaine has become one of the most serious social problems that endanger human beings. It is the most widespread drug abuse today. Its excitatory effect on the central nervous system of the human body is an important cause of abuse. Small doses of cocaine can slow the heart rate; increased doses can increase the heart rate, shortness of breath, vomiting, tremors, convulsions, and convulsions. Cocaine poses a great threat to the human body and poses a huge threat to global peace, stability and human security. Therefore, the study of new and sensitive cocaine detection methods has very important research and practical significance and application...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115G01N33/53
Inventor 曾立波张玉荣陈连康胡小龙
Owner SHANGHAI CRIMINAL SCI TECH RES INST