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Kit, preparation method and application thereof in detecting animal trichinosis

A kit and trichinella technology, applied in kits, detection of animal trichinosis, and preparation field, can solve problems such as inability to meet serological antibody detection requirements for pathogenic infection, limit of application scope of non-nucleic acid targets, etc.

Pending Publication Date: 2022-06-28
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application field of functional nucleic acid biosensors depends on the research progress of aptamers, and its application scope for non-nucleic acid targets is strictly limited.
Furthermore, hybridization chain reaction biosensors relying solely on aptamers cannot meet the growing demand for serological antibody detection of pathogenic infections.

Method used

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  • Kit, preparation method and application thereof in detecting animal trichinosis
  • Kit, preparation method and application thereof in detecting animal trichinosis
  • Kit, preparation method and application thereof in detecting animal trichinosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Preparation of competitive monoclonal antibody Ts-WN10-1H9

[0061] Mouse ascites was prepared with the hybridoma cell line WN10-1H9 with the microbial deposit number of CGMCC No. 18316, and the ascites was harvested and purified by Protein G to obtain the competitive monoclonal antibody Ts-WN10-1H9, followed by quantum dot fluorescent microsphere coupling mark. The specific method is as follows:

[0062] Inject 12-week-old healthy BALB / c mice intraperitoneally with paraffin oil, 0.5 mL / mice, and inject 1×10 intraperitoneally after 1 week 6 Hybridoma cell WN10-1H9 (microorganism deposit number: CGMCC No. 18316), after 7 to 10 days, when the abdominal cavity of the mouse is extremely distended, the ascites is extracted, and the ascites is extracted every 2 days. Precipitate and store the supernatant in aliquots at -20°C. Refer to Antibody Purification 125 of the GE Healthcare manual Antibody Purification 125 for the antibody purification process of the AKTA ...

Embodiment 2

[0063] Example 2 Reduction of the I1 disulfide bond of the thiolated oligonucleotide chain

[0064] Take 2OD of the synthesized SH-I1 DNA lyophilized powder, add 400 μL of 10 mM TCEP solution, fully dissolve and let stand in the dark for 10 min for subsequent gold nanoparticle coupling labeling.

[0065] The SH-modified oligonucleotide chain I1 was designed with the following sequence:

[0066] I1: 5'-AGTCTAGGATTCGGCGTGGGTTAATTTT-(CH2)3-SH-3' (as shown in SEQ ID No. 1)

Embodiment 3

[0067] Example 3 Preparation of 13nm gold nanoparticles

[0068] The citric acid reduction method was used to control the addition amount of the reducing agent and the reaction conditions to prepare 13nm gold nanoparticles, and their size and concentration were characterized by UV-Vis absorption spectroscopy for subsequent gold nanoparticles coupling labeling. The specific method is as follows:

[0069] Weigh 0.228g of trisodium citrate dihydrate into a 50mL sterile centrifuge tube, add 20mL of deionized water to fully dissolve, and prepare a 38.8mM trisodium citrate solution. Add 126.9 mL of chloroauric acid solution to the siliconized 250 mL Erlenmeyer flask, and heat the liquid to boiling on an induction cooker. Quickly add 10 mL of trisodium citrate solution to the Erlenmeyer flask while shaking, and continue to heat and shake the Erlenmeyer flask. After the solution changed from bright yellow to wine red, it was heated to boil for 10 min, cooled to room temperature and ...

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Abstract

The invention relates to the field of biological detection, in particular to a kit, a preparation method and application of the kit to detection of animal trichinosis. The invention provides a kit, comprising: a gold nanoparticle probe, the gold nanoparticle probe comprising a gold nanoparticle coupled with a monoclonal antibody Ts-WN10-1H9 and an oligonucleotide chain I1; the monoclonal antibody Ts-WN10-1H9 is secreted by a hybridoma cell strain WN10-1H9; the preservation number of the hybridoma cell is CGMCC (China General Microbiological Culture Collection Center) No.18316. The kit prepared by the invention has the advantages of simplicity in operation, high sensitivity and the like which are not possessed by ELISA (Enzyme-Linked Immuno Sorbent Assay), can be used for simultaneously detecting pig and human serum samples, and is not limited by host species. Meanwhile, cross reaction with other parasite positive serum is avoided during detection, the specificity and the sensitivity are high, and market development value is achieved.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a kit, a preparation method and application thereof in detecting animal trichinosis. Background technique [0002] Trichinosis is a very serious zoonotic disease, which can not only cause huge economic losses to animal husbandry, but also pose a huge threat to human health. Caterpillar meat (mainly pork) and disease. [0003] For the inspection of trichinosis in slaughtered animals, the regulatory inspection methods of the International Organization for Animal Health (OIE) are microscopic inspection method and sample digestion method. At present, my country is also using these two methods. However, the above two methods have certain drawbacks. Microscopic examination is time-consuming and labor-intensive and has poor sensitivity. The sensitivity can only be detected when the density of worms in meat reaches 3 worms per gram. Although the collection digestion method can great...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/543C12Q1/6888C12Q1/682C12Q1/6804
CPCG01N33/6854G01N33/577G01N33/54326G01N33/54346C12Q1/6888C12Q1/6804C12Q1/682G01N2333/4353C12Q2563/107C12Q2563/143C12Q2563/149Y02A50/30
Inventor 刘晓雷刘明远徐凝杨勇白雪丁静唐斌李辰刘琰
Owner JILIN UNIV