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Method for realizing sperm cell separation of mixed spot sample based on polypeptide modified membrane

A technology for sperm cells and modified membranes, which is applied in the field of sperm cell separation from mixed plaque samples based on polypeptide modified membranes. , to achieve the effect of suitable for large-scale promotion and application, ingenious design, and rapid separation

Pending Publication Date: 2021-03-12
宁波市博坤生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this technique of separating mixed plaques only through the difference in pore size also has some limitations, especially for old, dry, and severely degraded samples of mixed plaques, because both sperm cells and vaginal epithelial cells cannot maintain morphological integrity and Significant differences in size often result in a low recovery rate of sperm cells, which makes typing fail, or the effective separation of sperm and female epithelial cells in mixed spots cannot be achieved, and the STR spectrum is seriously mixed, which cannot be used as effective evidence for case detection and litigation

Method used

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  • Method for realizing sperm cell separation of mixed spot sample based on polypeptide modified membrane
  • Method for realizing sperm cell separation of mixed spot sample based on polypeptide modified membrane
  • Method for realizing sperm cell separation of mixed spot sample based on polypeptide modified membrane

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 DNA Extraction of Mixed Spot Males Using Poly(ε-Lysine) Physically Modified Nitrocellulose Membranes

[0045] Spray or soak the poly(ε-lysine) solution on the nitrocellulose membrane to make a layer of poly(ε-lysine) adsorb on the surface of the nitrocellulose membrane, dry it in a vacuum oven at 50°C to 100°C for 4h, press the sleeve The inner tube of the tube is cut to size and fitted to the inner tube for use.

[0046] Use 200 μL to 10 mL of epithelial cell digestion solution (containing 5% by weight to 20% by weight trishydroxymethylaminomethane, 0.5% by weight to 10% by weight of sodium chloride, 1% by weight to 10% by weight of Disodium ethylenediamine tetraacetate and 10 μL ~ 100 μL of proteinase K at a concentration of 10 mg / mL) for digestion, transfer the digested product containing sperm cells and other impurities to the above-mentioned separation sleeve, let it stand for 20 minutes, and use it if necessary Shaking, overturning and other methods pro...

Embodiment 2

[0050] Example 2 Polypropylene membrane covalently modified with polyaspartic acid for DNA extraction of mixed plaque males

[0051] Pre-spray or soak 5% to 40% by weight of dopamine and 0.5% to 20% by weight of aminosilane coupling agent solution on the polypropylene film, and pass through alkaline (pH 8 ~ 12) and heating (50 ° C ~ 100°C) to modify the surface of the polypropylene film with a transition layer modified by polydopamine and aminosilane coupling agent, and then use 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt Hydroxysuccinimide (EDC) / N-hydroxysuccinimide (NHS) method covalently coupled polyaspartic acid, after washing and drying in a vacuum oven at 50°C to 100°C for 4 hours, press the 24-well plate (including the inner plate) The size of the inner frame tube of the frame and the outer frame) is cut and assembled for use, and the number of membrane assemblies can be selected according to the number of mixed spot test materials (1 to 24).

[0052] Mix the mi...

Embodiment 3

[0056] Example 3 Use polyglutamic acid-modified carbonate microporous grooves for mixed spot male DNA extraction

[0057] Spray or soak 5% by weight to 40% by weight of dopamine and 0.5% by weight to 20% by weight of aminosilane coupling agent solution on the carbonate microporous groove, and pass through alkaline (pH 8~12) and heating (50 ℃~100℃) to modify the surface of the carbonate microporous groove with a transition layer modified by polydopamine and aminosilane coupling agent, and then use 1-(3-dimethylaminopropyl)-3-ethyl Carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) method covalently coupled polyglutamic acid, washed and dried in a vacuum oven at 50°C to 100°C for 4 hours before use.

[0058] The mixed spot sample material to be separated in Example 1 was mixed with 200 μL-10mL epithelial cell digestion solution (containing 5% by weight to 20% by weight trishydroxymethylaminomethane, 0.5% by weight to 10% by weight sodium chloride, 1% by weight ~10% by ...

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Abstract

The invention provides a method for realizing sperm cell separation of a mixed spot sample based on a polypeptide modified membrane. The method comprises the following steps: modifying a membrane by adopting polypeptide to obtain a polypeptide modified membrane; mixing and adsorbing a dispersion liquid of a mixed spot sample to be separated or a digestion product digested by an epithelial cell digestion liquid and the polypeptide modified membrane; discarding the residual solution, and washing the polypeptide modified membrane to obtain the polypeptide modified membrane with the surface enriched with male sperm cells. Preferably, the modification is physical modification or covalent modification. The membrane is loaded on the consumable or the membrane is part of the consumable. The methodfor realizing sperm cell separation of the mixed spot sample based on the polypeptide modified membrane can effectively separate male sperm cells and female epithelial cells in mixed spots to obtainmale sperm cells required by forensic DNA detection, and is ingenious in design, simple and convenient to operate, rapid in separation and suitable for large-scale popularization and application.

Description

technical field [0001] The invention relates to the technical field of cell separation, in particular to the technical field of sperm cell separation of mixed spot specimens, and specifically refers to a method for separating sperm cells from mixed spot specimens based on a polypeptide-modified membrane. Background technique [0002] At present, the application of forensic DNA testing in the field of criminal investigation is becoming more and more extensive. In addition to the collection, extraction and testing of DNA samples in major criminal cases, DNA samples are also commonly collected, Extraction and inspection. [0003] Sperm-yin mixed spot samples are common biological evidence in sexual crime cases. Effective separation of male sperm cells and female vaginal epithelial cells in mixed spots and accurate identification of the individual source of male sperm cells are the key to case detection and litigation. Since the sperm cell membrane contains a large number of th...

Claims

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Application Information

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IPC IPC(8): C12N5/076
CPCC12N5/061C12N2509/00
Inventor 李军王占海鲁斌陈德荐顾盼商锐
Owner 宁波市博坤生物科技有限公司
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