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

Biological material evidence collecting and preserving reagent Download PDF

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CN114507710A
CN114507710A CN202210162254.8A CN202210162254A CN114507710A CN 114507710 A CN114507710 A CN 114507710A CN 202210162254 A CN202210162254 A CN 202210162254A CN 114507710 A CN114507710 A CN 114507710A
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reagent
tris
uric acid
dna
biological material
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吴雅兰
陈灿球
曹丹
贾霄
孙华明
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Qingyuan Public Security Bureau
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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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 material evidence collecting and preserving reagent
Technical Field
The invention relates to the technical field of forensic physical evidence DNA extraction, in particular to a biological physical evidence collection and preservation reagent.
Background
The forensic DNA technology plays an important role in the investigation of a plurality of criminal cases, the solution of civil cases, the identification of serious accident cadaver sources, the paternity test of people such as abducted women, children and the like. With the continuous perfection of legal system construction and the continuous development of DNA technology, cases and material evidence requiring DNA inspection are more and more, and forensic DNA analysis has become a conventional technology for biological material evidence inspection.
DNA is a macromolecule existing in an organism, and is easily damaged and broken in an in vitro environment to become a micromolecule, namely DNA degradation. Successful DNA analysis begins with the correct extraction, storage and submission of biological material for evidence. Forensic biological inspection materials from case sites carry important functions of providing clues for investigation and evidences for courts, and whether the extraction of the inspection materials is standard and whether the storage is proper and whether the inspection is timely directly influences the final identification result. Conventional biological samples include blood (plaque), saliva (plaque), semen (plaque), hair, bone, exfoliated cell, and the like, and in these biological samples, the content of DNA in exfoliated cell samples is low, and in addition, DNA degradation is severe in high-temperature and humid environments, which affects the quality and yield of DNA extraction, and thus, DNA analysis fails easily.
The extraction, storage and submission specifications (GA/T1162-2014) of the forensic biological material preparation set unified specification requirements for the extraction, storage and submission of the material preparation for forensic physical evidence inspection, and also provide guarantee for the success of the physical evidence inspection. For the spot type test material extracted by the wiping method, a dry-wet two-step method is usually adopted for extraction, namely, a wet cotton swab is firstly used for wiping the surface of the object of the test material, and then a dry cotton swab is used for wiping and transferring. The most commonly used reagent for wetting cotton swabs is deionized water, but studies have shown that biological samples taken by wiping with deionized water are susceptible to spoilage by microorganisms, resulting in degradation of trace amounts of DNA therein. At present, experts and scholars in the market add antibiotics into deionized water to be used as exfoliated cell collecting reagents, and the mode can effectively inhibit the damage of microorganisms to DNA, but has limitations, such as incapability of well peeling the exfoliated cells from a test material, and the like.
Therefore, there is a need to develop a biological evidence collection and preservation reagent that can effectively collect trace amounts of biological evidence DNA and provide a microenvironment that prevents DNA degradation.
Disclosure of Invention
In view of the above problems, the present invention provides a biological evidence collecting and preserving reagent. The reagent can effectively collect trace biological material evidence DNA and provide a microenvironment for avoiding DNA degradation. The reagent can replace the traditional deionized water, is used for wiping and extracting the spot biological detection material by a dry-wet two-step method, realizes the on-site DNA extraction, and has wide development prospect in the forensic field.
In order to achieve the purpose, the invention adopts the following technical scheme:
a biological evidence collection and preservation reagent, the reagent comprising Sodium Dodecyl Sulfate (SDS), ethylenediaminetetraacetic 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.
Preferably, the pH of the reagent is adjusted to 8.0.
Preferably, the reagent is used for micro-organism evidence collection and preservation and is subjected to subpackaging, sealing, sterilization and nucleic acid-free treatment, and the method comprises the following 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.
Preferably, the autoclaving process is specified as follows: the temperature is 121.3 ℃, the pressure is 103.4kPa, and the time is 20 min.
Preferably, the appropriate amount of reagent is 100. mu.l.
The reagent is (SDS-EDTA-Tris-uric acid) quadruple reagent, wherein Sodium Dodecyl Sulfate (SDS) belongs to an anionic surfactant, has excellent functions of permeation, washing, wetting, decontamination and emulsification, is easily dissolved in water and ethanol, and has a special chemical structure which can be similarly dissolved with a cell membrane phospholipid bilayer, so that a cell membrane can be damaged, and DNA in the cell membrane can be released. Ethylenediaminetetraacetic acid (EDTA), an organic compound capable of reacting with Mg2+、Ca2+、Mn2+、Fe2+Divalent metal ion binding, commonly used as metal ion chelators, due to the vast majority of nuclease active centers require Mg2+For nucleic acidsEnzyme inhibitors can inhibit the damage of nuclease to DNA. Tris (hydroxymethyl) aminomethane (Tris), an organic compound, is widely used in the preparation of buffers in biochemical and molecular biological experiments for the solubilization of nucleic acids. Uric acid (Uric acid), which is the final product of purine metabolism, is trioxypurine, is slightly soluble in water, has stable chemical properties, is easy to form crystals, and wraps DNA to prevent from being infected by microorganisms.
Compared with the prior art, the invention has the following beneficial effects:
the reagent combines four components (SDS-EDTA-Tris-uric acid) to mutually cooperate and cooperate, so that the reagent can be beneficial to stripping trace cells from biological material evidence, can effectively inhibit the damage of nuclease to DNA, and can provide a microenvironment for avoiding the degradation of the DNA.
The quality guarantee period of the reagent reaches more than 1 year at room temperature (25 ℃), no exogenous DNA pollution, no toxic action and no influence on subsequent DNA detection in a laboratory.
Fingerprint prints are adopted to simulate the cells shed at the crime scene, and the reagent is compared with deionized water for experiments, so that the detection rate of the reagent is high; 10 mul is dried after being collected by 100 times diluted blood cells, and is stored for 1 year at room temperature, and the detection rate of the reagent is high.
The chemical components in the reagent can be completely domesticated, and the reagent is low in price, non-toxic, harmless and environment-friendly, can greatly improve the detection rate of biological detection materials after being widely popularized, and has good social and economic benefits.
Drawings
FIG. 1 is a DNA typing map obtained by collecting a simulated test material using a qualified reagent obtained after the treatment of example 2 in example 4;
FIG. 2 is a DNA typing map obtained by collecting a dummy sample with deionized water in example 4.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1
A biological material evidence collection and preservation reagent configuration, the reagent includes Sodium Dodecyl Sulfate (SDS), Ethylene Diamine Tetraacetic Acid (EDTA), Tris and Uric acid (Uric acid). In the reagent: the mass concentration of Sodium Dodecyl Sulfate (SDS) is 10%, the molar concentration of Ethylene Diamine Tetraacetic Acid (EDTA) is 0.5mol/L, the molar concentration of Tris (hydroxymethyl) aminomethane (Tris) is 1mol/L, and the molar concentration of Uric acid (Uric acid) is 2 mol/L.
The reagent is (SDS-EDTA-Tris-uric acid) quadruple reagent, wherein Sodium Dodecyl Sulfate (SDS) belongs to an anionic surfactant, has excellent functions of permeation, washing, wetting, decontamination and emulsification, is easily dissolved in water and ethanol, and has a special chemical structure which can be similarly dissolved with a cell membrane phospholipid bilayer, so that a cell membrane can be damaged, and DNA in the cell membrane can be released. Ethylenediaminetetraacetic acid (EDTA), an organic compound capable of reacting with Mg2+、Ca2+、Mn2+、Fe2+Divalent metal ion binding, commonly used as metal ion chelators, due to the vast majority of nuclease active centers require Mg2+The compound is used as a nuclease inhibitor and can inhibit the damage of nuclease to DNA. Tris (hydroxymethyl) aminomethane (Tris), an organic compound, is widely used in the preparation of buffers in biochemical and molecular biological experiments for the solubilization of nucleic acids. Uric acid (Uric acid), which is the final product of purine metabolism, is trioxypurine, is slightly soluble in water, has stable chemical properties, is easy to form crystals, and wraps DNA to prevent from being infected by microorganisms.
Example 2
A dispensing, sealing, sterilizing and nucleic acid-free process for collecting and preserving biological evidence (as described in example 1) comprises the following steps: subpackaging the reagent with the pH value adjusted to 8.0 into nucleic acid-free reagent bottles, sealing, performing high-pressure steam sterilization treatment (temperature 121.3 ℃, pressure 103.4kPa, time 20min), extracting DNA from 100 mu l of the reagent by a magnetic bead method, amplifying by using a kit, detecting STR typing by electrophoresis, and determining that the STR typing is qualified if the STR typing is not detected.
Example 3
Fingerprint prints are adopted to simulate the cells shed at the crime scene, and the qualified reagent treated in the example 2 is compared with deionized water for experiment. After 90 fingerprint cast-off cells are respectively collected, DNA is extracted by a magnetic bead method, a kit is adopted for amplification, STR typing is detected by electrophoresis, more than 100 fluorescence units are adopted for interpretation in the experiment, statistics is not carried out when the fluorescence units are lower than the standard, and the detection rate of two groups of experiment DNA sites is calculated:
example 2 the qualified reagent after treatment collects 90 fingerprints, and the detection rate of DNA sites is as follows: 76.9 percent.
90 fingerprints are collected by deionized water, and the detection rate of DNA loci is as follows: 42.9 percent.
Example 4
Adopting diluted blood to simulate trace biological detection materials in a crime scene, carrying out a contrast experiment on qualified reagents processed in example 2 and deionized water, respectively collecting 90 parts of simulated detection materials, standing at room temperature (25 +/-2 ℃) for 1 year, extracting DNA by a magnetic bead method, adopting kit amplification, carrying out electrophoresis detection STR typing, judging by adopting more than 100 fluorescence units in the experiment, not counting when the standard is lower than the standard, and calculating the detection rate of two groups of experimental DNA loci:
example 2 the qualified reagent after treatment collects 90 parts of simulated test materials, and the DNA site detectable rate is as follows: 100 percent, and the heterozygote balance of all sample types is good; FIG. 1 is a DNA typing map obtained by collecting a sample specimen from a test specimen using an acceptable reagent obtained after the treatment of example 2.
The deionized water is used for collecting 90 simulated test materials, the detection rate of DNA loci is 95.2%, all sample types show the degradation trend of long fragments, and FIG. 2 is a DNA typing map obtained by collecting the simulated test materials by using the deionized water.
Example 5
The reagent which passed after the treatment of example 2 was stored at room temperature (25 ℃ C.), and the influence of the storage time was examined:
Figure BDA0003515301400000041
Figure BDA0003515301400000051
from the above table, it can be seen that: the quality guarantee period of the reagent reaches more than 1 year at room temperature (25 ℃), no exogenous DNA pollution, no toxic action, no influence on laboratory DNA inspection, conformity with the standard requirements of forensic physical evidence inspection, and the technical level can reach the international advanced level.
The technical solutions provided by the embodiments of the present invention are described in detail above, and the principles and embodiments of the present invention are explained herein by using specific examples, and the descriptions of the embodiments are only used to help understanding the principles of the embodiments of the present invention; meanwhile, for a person skilled in the art, according to the embodiments of the present invention, there may be variations in the specific implementation manners and application ranges, and in summary, the content of the present description should not be construed as a limitation to 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.
CN202210162254.8A 2022-02-22 2022-02-22 Biological material evidence collecting and preserving reagent Pending CN114507710A (en)

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5756126A (en) * 1991-05-29 1998-05-26 Flinders Technologies Pty. Ltd. Dry solid medium for storage and analysis of genetic material
US5807527A (en) * 1991-05-29 1998-09-15 Flinders Technologies Pty. Ltd. Solid medium and method for DNA storage
US5972386A (en) * 1995-12-19 1999-10-26 Flinders Technologies Pty, Ltd. Dry solid medium for storage and analysis of genetic material
US20040101895A1 (en) * 1999-04-14 2004-05-27 Galina Fomovskaia Fta-coated media for use as a molecular diagnostic tool
US20060094015A1 (en) * 2002-10-04 2006-05-04 Whatman Inc. Methods and materials for using chemical compounds as a tool for nucleic acid storage on media of nucleic acid purification systems
CN101048501A (en) * 2004-09-07 2007-10-03 固德基因公司 Method for storing DNA by using chitosan, and products using the methods
CN101153263A (en) * 2007-09-07 2008-04-02 鼎生科技（北京）有限公司 Blood DNA conserving card and method for making the same
CN106029910A (en) * 2014-02-28 2016-10-12 通用电气医疗集团英国有限公司 Improvements in and relating to processed biological sample storage
CN112322697A (en) * 2020-11-09 2021-02-05 苏州乾康基因有限公司 DNA sample preservation solution and preparation method and application thereof

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5756126A (en) * 1991-05-29 1998-05-26 Flinders Technologies Pty. Ltd. Dry solid medium for storage and analysis of genetic material
US5807527A (en) * 1991-05-29 1998-09-15 Flinders Technologies Pty. Ltd. Solid medium and method for DNA storage
US5972386A (en) * 1995-12-19 1999-10-26 Flinders Technologies Pty, Ltd. Dry solid medium for storage and analysis of genetic material
US20040101895A1 (en) * 1999-04-14 2004-05-27 Galina Fomovskaia Fta-coated media for use as a molecular diagnostic tool
US20060094015A1 (en) * 2002-10-04 2006-05-04 Whatman Inc. Methods and materials for using chemical compounds as a tool for nucleic acid storage on media of nucleic acid purification systems
CN101048501A (en) * 2004-09-07 2007-10-03 固德基因公司 Method for storing DNA by using chitosan, and products using the methods
CN101153263A (en) * 2007-09-07 2008-04-02 鼎生科技（北京）有限公司 Blood DNA conserving card and method for making the same
CN106029910A (en) * 2014-02-28 2016-10-12 通用电气医疗集团英国有限公司 Improvements in and relating to processed biological sample storage
CN112322697A (en) * 2020-11-09 2021-02-05 苏州乾康基因有限公司 DNA sample preservation solution and preparation method and application thereof

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