Nucleic acid aptamer capable of being combined with prometryn and application of nucleic acid aptamer

A nucleic acid aptamer and proturon technology, which is applied in the field of environmental pollutant detection, can solve the problems of less research on nucleic acid aptamers, unfavorable proturon detection work, and no research reports.

Pending Publication Date: 2022-02-01
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are relatively few studies on nucleic acid aptamers of pesticides, and there is no report on the research on nucleic acid aptamers that can bind promethazin
At present, the detection methods of proturon usually require more complex instruments and sample pretreatment process, which is not conducive to the detection of proturon in the field environment, and the immunological detection method of proturon has the problem of low specificity. Therefore, in this field, There is an urgent need to develop a simple and fast detection method with high sensitivity and specificity

Method used

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  • Nucleic acid aptamer capable of being combined with prometryn and application of nucleic acid aptamer
  • Nucleic acid aptamer capable of being combined with prometryn and application of nucleic acid aptamer
  • Nucleic acid aptamer capable of being combined with prometryn and application of nucleic acid aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Screening of nucleic acid aptamers that can bind to promethazin

[0069] In order to obtain nucleic acid aptamers that can bind proturon with high specificity, the inventors conducted a capture SELEX experiment for screening. The screening process includes:

[0070] (1) Initial library design: The initial library sequence is 5'-ATTGGCACTCCACGCATAGG-N(40)-CCTATGCGTGCTACCGTGAA-3' (SEQ ID NO.17). Among them, N(40) is a random sequence, and both ends are fixed sequences, which is convenient for amplifying the nucleic acid aptamer and hybridizing the aptamer with a biotin-labeled primer. The primer sequences are shown in Table 1.

[0071] Table 1. Primers for nucleic acid aptamer screening

[0072]

[0073] (2) Initial screening: first use 0.1 M PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM NaCl 2 HPO 4 , 1.4 mM KH 2 PO 4 ) After dissolving 1 OD initial library in a 1 mL sterile EP tube, add 14 µL of pre-dissolved 100 µM primer S1-CS. Use a PCR instrument for sl...

Embodiment 2

[0081] Example 2: Proturon affinity and specificity experiments of candidate nucleic acid aptamers

[0082] This embodiment mainly uses nano-gold colorimetric method to measure the affinity and specificity of candidate nucleic acid aptamers to proturon, and establishes a nano-gold colorimetric method for proturon. This method utilizes nano-gold under the protection of aptamers The principle of aggregation not induced by high concentration NaCl. The specific determination process includes the following steps:

[0083] (1) Preparation of gold nanoparticles

[0084] Gold nanoparticles were prepared by sodium citrate reduction method. First, 100 mL of ultrapure water was heated to boiling, then 1 mL of 1% chloroauric acid solution was added, and 10 mL of 1% sodium citrate solution was added after 1 min, the solution turned from light yellow to gray, and finally to wine red. After heating for 5 min, turn off the heat source and cool down at room temperature.

[0085] (2) Nano-g...

Embodiment 3

[0091] Example 3: Structural prediction of nucleic acid aptamers

[0092] In order to analyze the structural principle of the binding of the candidate aptamers to promethazin and to find the nucleotide sequence binding to protamizone, the structure prediction of the candidate nucleic acid aptamers was carried out. The prediction was performed using the online prediction tool UNA-fold, where the folding temperature was 25 °C, Na + The concentration is 0.1 M.

[0093] The predicted structure of the nucleic acid aptamer P60 is as follows image 3 As shown, it was predicted that the nucleic acid aptamer P60 formed a stem-loop structure, in which nucleotides 3-8 and 30-35 formed the first stem S1, and nucleotides 11-15 and 24-28 formed the second Stem S2, positions 8-11 and 28-30 form the first loop L1, positions 15-24 form the second loop L2, and positions 35-55 form the third loop L3.

[0094]Since the deletion of the third loop L3 has no effect on the affinity of promethazin ...

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Abstract

The invention discloses a nucleic acid aptamer capable of being combined with prometryn and application of nucleic acid aptamer, belongs to the field of environmental pollutant detection, and particularly relates to a nucleic acid aptamer for detecting prometryn and a nanogold colorimetric detection method of the nucleic acid aptamer. The nucleic acid aptamer provided by the invention has relatively high affinity and specificity to prometryn, and can be used for detecting prometryn in environments, foods, medicines, raw materials or biological samples. The invention also provides a nanogold colorimetric method for detecting prometryn in a sample.

Description

technical field [0001] The invention belongs to the field of environmental pollutant detection, and in particular relates to a nucleic acid aptamer for detecting proturon and a nano-gold colorimetric detection method thereof. Background technique [0002] Promethazine is a class of triazine compounds with strong selectivity and broad herbicidal spectrum, and has become a commonly used herbicide in agriculture in my country. It has stable chemical properties and can exist in the environment for a long time. It will be collected into lakes and oceans through rainfall and surface runoff, resulting in excessive concentrations of proturin in the environment. For example, the net concentration of Laizhou Bay surface seawater is 21.2ng / L. Prometon in water will pass through the food chain and accumulate in organisms. For example, a high level of prometon has been detected in the umbilical cord blood of infants, and it has been confirmed that prometon is related to the slow develop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/53G01N21/25C07D251/52
CPCC12N15/115C12N15/1048G01N33/5308G01N21/25C07D251/52C12N2310/16C12N2310/531
Inventor 汝少国赵子昂张振忠王军
Owner OCEAN UNIV OF CHINA
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