CN114507710A - Biological material evidence collecting and preserving reagent - Google Patents

Abstract

The invention relates to the technical field of forensic physical evidence DNA extraction, and discloses a biological physical evidence collection and preservation reagent, which comprises Sodium Dodecyl Sulfate (SDS), Ethylene Diamine Tetraacetic Acid (EDTA), Tris (hydroxymethyl) aminomethane (Tris) and Uric acid (Uric acid); in the reagent: the mass concentration of the Sodium Dodecyl Sulfate (SDS) is 10%, the molar concentration of the Ethylene Diamine Tetraacetic Acid (EDTA) is 0.5mol/L, the molar concentration of the Tris (hydroxymethyl) aminomethane (Tris) is 1mol/L, and the molar concentration of the Uric acid (Uric acid) is 2 mol/L. The reagent combines four components (SDS-EDTA-Tris-uric acid) to mutually cooperate and cooperate, so that the reagent can be beneficial to stripping biological material evidence from a detection material, can effectively inhibit the damage of nuclease to DNA, and can provide a microenvironment for avoiding the degradation of the DNA.

Description

Biological evidence collection and preservation reagents

technical field

The invention relates to the technical field of DNA extraction from forensic material evidence, in particular to a biological material evidence collection and preservation reagent.

Background technique

Forensic DNA technology has played an important role in the detection of many criminal cases, the settlement of civil cases, the identification of the source of corpses in major accidents, and the paternity testing of abducted women and children. With the continuous improvement of legal construction and the continuous development of DNA technology, more and more cases and physical evidences require DNA testing. Forensic DNA analysis has become a routine technology for biological evidence testing.

DNA is a macromolecule that exists in living organisms. In an in vitro environment, it is easy to be damaged, broken, and turned into small molecules, that is, DNA degradation. Successful DNA analysis begins with the correct extraction, preservation and submission of biological evidence materials. The forensic physical examination materials from the case scene play an important role in providing clues for the investigation and providing evidence for the court. Whether the extraction materials are standardized, whether they are properly stored, and whether they are submitted for inspection in a timely manner will directly affect the final identification result. Conventional biological samples include blood (spots), saliva (spots), semen (spots), hair, bones, exfoliated cells, etc. Among these biological samples, the DNA content of exfoliated cell-based samples is low. DNA is degraded seriously in a humid environment, affecting the quality and yield of DNA extraction, and easily leading to failure of DNA analysis.

"Specifications for the Extraction, Preservation and Submission of Forensic Physical Examination Materials (GA/T1162-2014)" formulates unified standard requirements for the extraction, preservation and submission of forensic physical evidence examination materials, and also provides a basis for the success of physical evidence examination. Assure. For the stained samples extracted by the wiping method, the dry and wet two-step method is often used for extraction, that is, first wipe the surface of the sample object with a wet cotton swab, and then use a dry cotton swab to dry and transfer. The most commonly used reagent for moistening cotton swabs is deionized water, but studies have shown that biological samples extracted by wiping with deionized water are easily corrupted by microorganisms, resulting in the degradation of trace amounts of DNA. At present, experts and scholars on the market have added antibiotics to deionized water as exfoliated cell collection reagents. This method can effectively inhibit the damage of microorganisms to DNA, but it also has limitations, such as the inability to strip exfoliated cells from the specimen and many more.

Therefore, there is an urgent need to develop a biological evidence collection and preservation reagent, which can effectively collect trace amounts of biological evidence DNA and provide a microenvironment that avoids DNA degradation.

SUMMARY OF THE INVENTION

In view of the above problems, the purpose of the present invention is to provide a biological evidence collection and preservation reagent. The reagent of the invention can effectively collect trace amounts of biological evidence DNA and provide a microenvironment for avoiding DNA degradation. The reagent of the invention can replace the traditional deionized water, and can be used for wiping and extracting the stain-like biological test material by dry and wet two-step method, realizing the on-site DNA extraction, and has broad development prospects in the field of forensics.

The present invention adopts the following technical solutions to realize the above-mentioned purpose:

A biological evidence collection and preservation reagent comprising sodium dodecyl sulfonate (SDS), ethylenediaminetetraacetic acid (EDTA), tris (Tris) and uric acid (Uric acid); Among the reagents: the mass concentration of the sodium dodecyl sulfonate (SDS) is 10%, the molar concentration of the ethylenediaminetetraacetic acid (EDTA) is 0.5mol/L, the tris(hydroxymethylaminomethane) The molar concentration of (Tris) is 1 mol/L, and the molar concentration of the uric acid (Uric acid) is 2 mol/L.

Preferably, the pH of the reagent is adjusted to 8.0.

Preferably, the reagents must be subpackaged, sealed, sterilized, and nucleic acid-free before being used for the collection and preservation of trace biological evidence. The details are as follows: the reagents whose pH value is adjusted to 8.0 are subpackaged into nucleic acid-free reagent bottles and sealed. , after high pressure steam sterilization, take appropriate amount of reagent to extract DNA by magnetic bead method, and use kit to amplify, and electrophoresis to detect STR typing, if not detected, it is qualified.

Preferably, the high-pressure steam sterilization treatment is as follows: the temperature is 121.3° C., the pressure is 103.4 kPa, and the time is 20 minutes.

Preferably, the appropriate amount of reagent is 100 μl.

The reagent of the present invention is (SDS-EDTA-Tris-uric acid) quadruple reagent, wherein, sodium dodecyl sulfonate (SDS) is an anionic surfactant and has excellent penetration, washing, wetting, decontamination and emulsifying effects , easily soluble in water and ethanol, and its special chemical structure can be similar to the cell membrane phospholipid bilayer, so it can destroy the cell membrane and release the DNA in it. Ethylenediaminetetraacetic acid (EDTA) is an organic compound that can combine with divalent metal ions such as Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Fe ²⁺ , and is often used as a metal ion chelating agent. The active center of nuclease requires Mg ²⁺ , which is used as a nuclease inhibitor, which can inhibit the damage of nuclease to DNA. Tris (Tris), an organic compound, is widely used in the preparation of buffers in biochemical and molecular biology experiments for the dissolution of nucleic acids. Uric acid, the final product of purine metabolism, is trioxypurine, slightly soluble in water, and its chemical properties are stable, easy to form crystals, and encapsulate DNA to prevent microbial infection.

Compared with the prior art, the beneficial effects of the present invention are as follows:

The reagent of the present invention combines four components (SDS-EDTA-Tris-uric acid) to cooperate and cooperate with each other, so that the reagent can facilitate the stripping of trace cells from biological evidence, can effectively inhibit the damage of nuclease to DNA, and can provide protection against DNA degradation. microenvironment.

The reagent of the invention has a shelf life of more than 1 year at room temperature (25° C.), has no contamination by exogenous DNA, has no toxic effect, and does not affect subsequent DNA detection in the laboratory.

Fingerprint imprints are used to simulate exfoliated cells at the crime scene. The reagent of the invention is compared with deionized water. The detection rate of the reagent of the invention is high. high.

All chemical components in the reagent of the invention can be domestically produced, and the reagent is cheap, non-toxic and harmless, and environmentally friendly.

Description of drawings

Fig. 1 is the DNA genotyping map obtained by collecting simulated samples with qualified reagents after being processed in Example 2 in Example 4;

FIG. 2 is a DNA typing pattern obtained by collecting simulated samples with deionized water in Example 4. FIG.

Detailed ways

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to specific embodiments.

Example 1

A configuration of a biological evidence collection and preservation reagent, the reagent includes sodium dodecyl sulfonate (SDS), ethylenediaminetetraacetic acid (EDTA), tris (Tris) and uric acid (Uric acid). In this reagent: the mass concentration of sodium dodecyl sulfonate (SDS) is 10%, the molar concentration of ethylenediaminetetraacetic acid (EDTA) is 0.5mol/L, and the molar concentration of tris (Tris) is 1 mol/L, and the molar concentration of uric acid is 2 mol/L.

The reagent of the present invention is (SDS-EDTA-Tris-uric acid) quadruple reagent, wherein, sodium dodecyl sulfonate (SDS) is an anionic surfactant and has excellent penetration, washing, wetting, decontamination and emulsifying effects , easily soluble in water and ethanol, and its special chemical structure can be similar to the cell membrane phospholipid bilayer, so it can destroy the cell membrane and release the DNA in it. Ethylenediaminetetraacetic acid (EDTA) is an organic compound that can combine with divalent metal ions such as Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Fe ²⁺ , and is often used as a metal ion chelating agent. The active center of nuclease requires Mg ²⁺ , which is used as a nuclease inhibitor, which can inhibit the damage of nuclease to DNA. Tris, an organic compound, is widely used in the preparation of buffers in biochemical and molecular biology experiments for the dissolution of nucleic acids. Uric acid, the final product of purine metabolism, is trioxypurine, slightly soluble in water, and its chemical properties are stable, easy to form crystals, and encapsulate DNA to prevent microbial infection.

Example 2

The sub-packaging, sealing, sterilization and nucleic acid-free treatment of a biological evidence collection and preservation reagent (as described in Example 1) are as follows: the reagent whose pH value is adjusted to 8.0 is distributed into a nucleic acid-free reagent bottle, sealed , after high pressure steam sterilization (temperature 121.3°C, pressure 103.4kPa, time 20min), take 100μl of reagent to extract DNA by magnetic bead method, and use the kit to amplify, detect STR typing by electrophoresis, if not detected, it is qualified .

Example 3

Fingerprint imprints were used to simulate the exfoliated cells at the crime scene, and a comparative experiment was conducted between the qualified reagents treated in Example 2 and deionized water. After collecting 90 fingerprint exfoliated cells, DNA was extracted by magnetic bead method, amplified by kit, and STR typing was detected by electrophoresis. In this experiment, more than 100 fluorescence units were used for interpretation. The detection rate of DNA loci in the group experiment:

In Example 2, 90 fingerprints were collected from qualified reagents after treatment, and the detection rate of DNA loci was: 76.9%.

Deionized water was used to collect 90 fingerprints, and the detection rate of DNA loci was 42.9%.

Example 4

Diluted blood was used to simulate the trace biological samples at the crime scene, and the qualified reagents treated in Example 2 were compared with deionized water. DNA was extracted by magnetic bead method, amplified by kit, and STR typing was detected by electrophoresis. In this experiment, more than 100 fluorescence units were used for interpretation, and statistics were not performed below this standard. The detection rate of DNA loci in the two groups of experiments was calculated:

90 mock samples were collected with qualified reagents after the treatment in Example 2. The detection rate of DNA loci was 100%, and the heterozygotes of all samples were well balanced; Figure 1 is the collection of qualified reagents after treatment with Example 2. The DNA typing map obtained from the simulated samples.

90 samples were collected in deionized water, and the detection rate of DNA loci was 95.2%, and all samples showed a trend of long fragment degradation.

Example 5

The qualified reagents treated in Example 2 were placed at room temperature (25° C.) for preservation, and the influence of the preservation time was investigated:

It can be seen from the above table that the reagent of the present invention has a shelf life of more than 1 year at room temperature (25° C.), has no exogenous DNA pollution, has no toxic effect, does not affect laboratory DNA testing, meets the requirements of forensic material evidence testing standards, and its technical level can reach international standards. advanced level.

The technical solutions provided by the embodiments of the present invention have been described in detail above. The principles and implementations of the embodiments of the present invention are described in this paper by using specific examples. The descriptions of the above embodiments are only applicable to help understand the embodiments of the present invention. At the same time, for those of ordinary skill in the art, according to the embodiments of the present invention, there will be changes in the specific implementation and application scope. To sum up, the contents of this specification should not be construed as limitations of the present invention.

Claims (5)

1. A biological evidence collection and preservation reagent, wherein the reagent comprises Sodium Dodecyl Sulfate (SDS), Ethylene Diamine Tetraacetic Acid (EDTA), Tris (hydroxymethyl) aminomethane (Tris), and Uric acid (Uric acid); in the reagent: the mass concentration of the Sodium Dodecyl Sulfate (SDS) is 10%, the molar concentration of the Ethylene Diamine Tetraacetic Acid (EDTA) is 0.5mol/L, the molar concentration of the Tris (hydroxymethyl) aminomethane (Tris) is 1mol/L, and the molar concentration of the Uric acid (Uric acid) is 2 mol/L.

2. The biological material evidence collection and preservation reagent of claim 1 wherein the pH of the reagent is adjusted to 8.0.

3. The biological material evidence collection and preservation reagent according to claim 2, wherein the reagent is used for the micro-biological material evidence collection and preservation which is subjected to subpackaging, sealing, sterilization and nucleic acid-free treatment, and comprises the following specific steps: and (3) subpackaging the reagent with the pH value adjusted to 8.0 into nucleic acid-free reagent bottles, sealing, carrying out high-pressure steam sterilization treatment, taking a proper amount of reagent, extracting DNA by a magnetic bead method, carrying out amplification by using a kit, and carrying out electrophoresis detection on STR typing, wherein the STR typing is qualified if the STR typing is not detected.

4. The biological material evidence collecting and preserving reagent according to claim 3, wherein the autoclaving process is as follows: the temperature is 121.3 ℃, the pressure is 103.4kPa, and the time is 20 min.

5. The biological material evidence collection and preservation reagent of claim 3, wherein the appropriate amount of reagent is 1 ml.

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