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Method for Separation of Sperm in Mixed Spot Using Porous Material

A technology of porous materials and mixed spots, applied in the field of forensic separation of mixed cells, can solve the problems of widespread use in difficult laboratories, undetectable sperm cells, and high requirements for experimental conditions, avoiding the problem of mixed typing and optimizing the reagent system. , the effect of simple operation

Active Publication Date: 2022-03-08
重庆市公安局江北区分局
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The differential lysis method is currently one of the most commonly used methods to deal with the mixed spots of sperm and vaginal epithelial cells. During the lysis of this method, there are cases where the female components are not fully digested or the sperm cells are overdigested. Sometimes the DNA of the epithelial cells will adhere to the Sperm head, resulting in mixed DNA or sperm cell DNA cannot be detected during DNA typing, and this method requires repeated centrifugation and washing of epithelial cells, which is low in automation and takes a long time
[0004] Laser capture microdissection technology combines optical microscopy technology with laser cell dissection technology, which can quickly and accurately select and cut sperm cells under the direct vision of the microscope, and obtain the DNA typing of a single male. The operating platform and low-system amplification instruments require high experimental conditions, making it difficult to be widely used in laboratories

Method used

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  • Method for Separation of Sperm in Mixed Spot Using Porous Material
  • Method for Separation of Sperm in Mixed Spot Using Porous Material
  • Method for Separation of Sperm in Mixed Spot Using Porous Material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Making a Porous Material Adsorbent

[0027] To make a porous material adsorption piece, follow the steps below:

[0028] Choose a three-dimensional network porous material, the material of the porous material is stainless steel or silicon carbide or nickel or chromium or titanium, the pore size of the micropores is 1-15 microns, which can be purchased directly from the market.

[0029] First make the porous material adsorption piece, the thickness of the porous material adsorption piece is 0.5-3 mm, and can be made into a circular sheet or a square sheet. The porous material adsorption sheet is embedded in the hollow tube with both ends open to obtain the following figure 1 The porous material adsorption piece shown is mainly composed of a hollow tube 1 open at both ends and a porous material adsorption sheet 2. The porous material adsorption sheet 2 is arranged in the middle of the hollow tube 1, and the porous material adsorption sheet 2 connects the hollow...

Embodiment 2

[0030] Example 2 Application of Porous Material to Separation of Sperm in Mixed Spots

[0031] The porous material adsorption piece made in Example 1 is used to separate the sperm in the mixed spot, and the whole separation process is as follows figure 2 shown, follow the steps below:

[0032] 1) Pretreatment of mixed spots

[0033] Cut a proper amount of physical evidence mixed with sperm and other components such as female vaginal swabs, underwear, bed sheets, etc., put it into a centrifuge tube, add an appropriate amount (200-5000 μL) of cell eluent, shake for tens of seconds, discard the carrier, and centrifuge to obtain sperm The mixture with other components is 200-5000 μL. Cell eluent was 0.9% saline.

[0034] 2) Combination of sperm and porous material

[0035] Take the porous material adsorbent made of the hollow tube with both ends open in Example 1, put the porous material adsorbent into the centrifuge tube, then add the mixed solution of the sperm and other co...

Embodiment 3

[0047] Evaluation of embodiment 3 separation effect

[0048] 1. Using the method in Example 2, collect the semen of adult volunteers, count with a cytometer, and dilute the sperm to 10,000 / mL, 5,000 / mL, 3,000 / mL, 1,000 / mL, and 500 / mL , 100 cells / mL; oral epithelial cells were collected from adult volunteers, counted using a cell counting board, and the oral epithelial cells were made into 10,000 cells / mL. Different numbers of sperm suspensions and oral epithelial cells prepared above were mixed respectively to prepare different numbers of sperm-oral epithelial cell mixtures. Step 4) is not used this time, step 5) is directly performed after step 3), and then the detection is performed:

[0049] 1) The sperm DNA template obtained in step 5) is quantified by ultraviolet spectrophotometer or fluorescent real-time PCR, and the DNA concentration measured is above 0.1ng;

[0050] 2) Take 1-4 microliters of the above-mentioned DNA template, and perform multi-site multiplex amplific...

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Abstract

The invention discloses a method for separating sperm in mixed plaques using porous materials: (1) pretreatment of mixed plaques: using cell eluent to separate sperm cells and other cell components from physical evidence to obtain a suspension; ( 2) Combination of sperm and porous material: take the porous material adsorption piece and the mixed solution, pour the mixed solution on the porous material adsorption sheet, the mixed solution enters the material from the upper surface of the porous material adsorption sheet and then drips from the lower surface, after dripping (3) Removal of other cell components on the surface of the porous material: the porous material adsorption sheet obtained in step (2) is washed with TES washing solution to remove other substances other than sperm attached to the surface of the porous material. Cells and their DNA; (4) Digesting the porous material adsorption sheet obtained in step (3) with sperm digestion solution, so that the sperm attached to the micropores of the porous material are eluted into the sperm digestion solution to obtain a sperm DNA solution.

Description

technical field [0001] The invention relates to the technical field of forensic medicine separation of mixed cells, in particular to a method for separating sperm in mixed spots using porous materials. Background technique [0002] In rape cases, the mixed spot formed by semen and female vaginal fluid is one of the most common biological evidences. Successful separation of sperm in the mixed spot and DNA typing are extremely important for the identification of the suspect and the prosecution of the case. Due to the mixed presence of female vaginal epithelial cells and sperm, sperm separation, DNA extraction and typing are very difficult. According to the differences in size, specific gravity, and chemical composition of cell membranes between sperm cells and epithelial cells, forensic scientists have established a variety of separation methods for sperm cells and epithelial cells, such as differential lysis and laser capture microdissection (LCM) technology , Membrane filtr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/076
CPCC12N5/061C12N2509/00
Inventor 宁淑华王清山陈骏陈柏宇乔晓义冯双龙巩强
Owner 重庆市公安局江北区分局
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