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Nucleic acid aptamer capable of being specifically combined with carapace arginine kinase, kit and detection method

A technology of arginine kinase and nucleic acid aptamer, which is applied in the field of food analysis, can solve the problems of strict storage conditions, high price of monoclonal antibody, and cumbersome preparation, and achieve high sensitivity, avoid false positives, and simple chemical modification.

Active Publication Date: 2018-11-23
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the price of monoclonal antibody itself is relatively high, the preparation is cumbersome and the storage conditions are relatively strict

Method used

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  • Nucleic acid aptamer capable of being specifically combined with carapace arginine kinase, kit and detection method
  • Nucleic acid aptamer capable of being specifically combined with carapace arginine kinase, kit and detection method
  • Nucleic acid aptamer capable of being specifically combined with carapace arginine kinase, kit and detection method

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Experimental program
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Effect test

Embodiment 1

[0031] Preparation of Arginine Kinase: (1) Take 50 g of Penaeus vannamei muscle, remove the head, tail, shell and gut. (2) Shrimp muscle was cut into mud with a knife, dissolved in buffer A (50mM NaCl, 2mM NaHCO 3 , 10mM EDTA) were homogenized with a homogenizer and allowed to stand at 4°C for 2h. (3) 8000r / min, centrifuge at 4°C for 20min, take the supernatant, add 70% ammonium sulfate, and let stand at 4°C for 8h. (4) 8000r / min, centrifuge at 4°C for 20min, take the supernatant, add 90% ammonium sulfate, and let stand at 4°C for 6h. (5) 8000r / min, centrifuge at 4°C for 20min, and take the precipitate. The precipitate was dissolved in Buffer B (20 mM Tris-HCL, 1 mM NaCl, pH 8.0). (6) ANX Sepharose Fast Flow anion exchange column was used for gradient elution with 0.5M NaCl solution, and the eluted product was collected to obtain arginine kinase for future use.

Embodiment 2

[0033] Screening of nucleic acid aptamers.

[0034] Construction of random ssDNA library of crustacean arginine kinase. (Library capacity is 10 14 The fragment length is 40bp, commissioned by Sangon Bioengineering (Shanghai) Co., Ltd.)

[0035] Such as figure 1 As shown, the present invention adopts three modes of alternate screening.

[0036] In the first screening, the ssDNA library was incubated with arginine kinase (prepared in Example 1) at 37° C. for 1 h, and then added to graphene solution (graphene powder, 5 mg / ml, purchased from Aladdin Reagent (Shanghai) Co., Ltd. company). The resulting mixture was incubated at 37°C to adsorb free-loose ssDNA. The mixture was centrifuged at 12,000 rpm for 15 min to separate and remove unbound ssDNA and graphene in the precipitate, and the remaining supernatant contained ssDNA bound to arginine kinase for asymmetric PCR amplification (restriction primer The ratio of non-restrictive primers is 1:50; the sequences are 5'-CAG GGG ...

Embodiment 3

[0042] First use piraha solution (H 2 SO 4 :H 2 o 2 =3:1) Soak the optical fiber probe with 600 μm quartz fiber (NA=0.22, Nanjing Chunhui) for 30 min, wash it thoroughly with ultrapure water, and dry it with nitrogen to make the surface of the probe hydroxylated; then soak it in 2% (v / v) toluene solution Clean the fiber optic probe for 1 hour with toluene and dry it with nitrogen, then soak the fiber optic probe with 5% (v / v) glutaraldehyde solution at 37°C for 2 hours, wash it with toluene, and dry it in an oven at 120°C to silanize the surface of the probe ; Soak the treated fiber optic probe in arginine kinase solution (concentration: 1mg / mL), soak overnight at 4°C to connect the coated arginine kinase; rinse the fiber optic probe with ultrapure water, and then place it in a 4mg / mL BSA (bovine serum albumin) solution for 2 h to block non-specific adsorption sites. Coated fiber optic probes can be stored at 4°C for several months.

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Abstract

The invention discloses a nucleic acid aptamer capable of being specifically combined with carapace arginine kinase, a kit and a detection method. The nucleotide sequence of the nucleic acid aptamer is shown as SEQ ID No. 1. Based on the nucleic acid aptamer, the invention establishes a method for detecting the carapace arginine kinase by utilizing a near-field optical wave targeting sensor; the method has the advantages of high specificity, high sensitivity, high stability, good repeatability and the like. The nucleic acid aptamer is easy to synthesize, has simple chemical modification and noimmunogenicity, can be specifically combined with the arginine kinase and has high affinity; the stability of a biosensor can be improved, so that high-sensitivity, strong-specificity and simple-to-operation detection is realized.

Description

technical field [0001] The invention relates to the technical fields of food analysis and food detection, in particular to a nucleic acid aptamer specifically binding to crustacean arginine kinase, a kit and a detection method. Background technique [0002] Aquatic products are rich in protein and delicious in taste. With its increasing consumption every year, aquatic products occupy an increasingly important position in people's daily life. However, as one of the eight major allergic foods, crustacean aquatic products are very likely to cause allergic reactions in certain groups of people. According to statistics, in Asian countries, about 40% of adults and children will have an allergic reaction to crustaceans. [0003] At present, countries such as the United States, the European Union, and New Zealand all require allergen labeling on allergenic foods to remind consumers to avoid eating them by mistake. Therefore, the detection of allergen content is the basis and prim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/573
CPCC12N15/115C12N2310/16G01N33/573G01N2333/9123
Inventor 傅玲琳钱一帆王彦波王翀周瑾茹王飞飞
Owner ZHEJIANG GONGSHANG UNIVERSITY
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