阅读说明：本技术 一种用于微量复杂性物证检材dna提取纯化的试剂盒与提取方法 (Kit for extracting and purifying DNA (deoxyribonucleic acid) of trace complex material to be detected and extraction method ) 是由 朱灿灿 朱灵 胡安中 杨柯 崔俊生 邓国庆 刘勇 于 2021-07-30 设计创作，主要内容包括：本发明提供一种用于微量复杂性物证检材DNA提取纯化的试剂盒与提取方法。所述试剂盒可有效进行多年陈旧布类、铁锈、泥土、色素中DNA提取纯化,由裂解液、结合液、漂洗液1、漂洗液2、纳米磁珠和洗脱液六部分组成,该试剂盒提取DNA的方法包括以下步骤：裂解液对检材中细胞的消化、磁珠吸附DNA、磁珠分离、磁珠漂洗、DNA洗脱。本发明的创新点是在于一种特殊的试剂体系,可有效去除复杂物证检材中的抑制物如腐殖酸、多糖以及阳离子等,有利于含有铁锈、泥土、色素等复杂检材中DNA的提取,使得DNA纯度更高,完整性更好,更有利于下游的STR扩增与电泳图谱分析。(The invention provides a kit for extracting and purifying trace complex material evidence DNA and an extraction method. The kit can effectively extract and purify DNA in old cloth, rust, soil and pigment for many years, and consists of lysis solution, binding solution, rinsing solution 1, rinsing solution 2, nano magnetic beads and eluent, and the method for extracting DNA by the kit comprises the following steps: digesting cells in a test material by a lysis solution, adsorbing DNA by magnetic beads, separating the magnetic beads, rinsing the magnetic beads and eluting the DNA. The innovation point of the invention is a special reagent system, which can effectively remove inhibitors such as humic acid, polysaccharide, cation and the like in complex material to be detected, is beneficial to the extraction of DNA in complex material to be detected containing rust, soil, pigment and the like, ensures that the DNA has higher purity and better integrity, and is more beneficial to the downstream STR amplification and electrophoretic map analysis.)

1. A kit for extracting and purifying DNA of a trace complexity material, which is characterized by comprising: lysis solution, binding solution, rinsing solution 1, rinsing solution 2, nano magnetic bead suspension and eluent;

wherein the lysis solution comprises the following components: 0.5-5mM of guanidine hydrochloride, 1-5mM of sodium dodecyl sulfate, 5-50mM of EDTA, 20-100mg/ml of protease K and 2-80mM of PVP; when the lysis solution is used, the pH value is adjusted to 3.5-6 by using 0.2-1M acetic acid and 0.2-1M sodium acetate buffer solution;

the binding solution comprises the following components: 2-6M of guanidinium isothiocyanate, 0.2-1M of acetic acid and 0.2-1M, DMSO 1-100mM of sodium acetate;

the rinsing liquid 1 comprises the following components: 1-3M guanidine hydrochloride, 55 percent of absolute ethyl alcohol by volume, 0.2-1M acetic acid and 0.2-1M sodium acetate;

the rinsing liquid 2 comprises the following components: sodium acetate 0.5-2M, anhydrous alcohol 75% by volume;

in the nano magnetic bead suspension, the size of suspended nano magnetic beads is 50-100nm, the mass percentage of the nano magnetic beads is 10-30%, and 0.5-1x PBS is used as a buffer solution; the magnetic bead material comprises one or more of an iron compound, a cobalt compound and a nickel compound;

the eluent has the components of 0.1-0.5 XTE.

2. A method of making the kit of claim 1, comprising the steps of:

preparing lysis solution, binding solution, rinsing solution 1, rinsing solution 2, nano magnetic bead suspension and eluent according to the content of each component.

3. The method for extracting the kit for extracting and purifying the DNA of the trace complexity material as claimed in claim 1, which comprises the following steps:

1) putting the material evidence fragments to be extracted or the cotton swabs wiped into a centrifugal tube, adding lysis solution into the centrifugal tube, then adding protease K solution, mixing uniformly by vortex, and putting into a constant-temperature shaking table for warm bath;

2) after warm bath, centrifuging; taking out the supernatant fluid in a new centrifuge tube, adding binding liquid into the supernatant fluid with equal volume, adding the nano magnetic bead suspension, placing in an oscillator for continuous oscillation adsorption at room temperature;

3) after adsorption, placing the centrifuge tube on a magnetic frame, standing, adsorbing and discarding waste liquid in the centrifuge tube, and keeping magnetic beads;

4) adding a rinsing liquid 1 into a centrifuge tube, covering a centrifuge tube cover, carrying out vortex oscillation to resuspend magnetic beads, placing the magnetic beads on a magnetic frame, and sucking away liquid to retain the magnetic beads;

5) adding a rinsing liquid 2 into the centrifuge tube, covering a centrifuge tube cover, carrying out vortex oscillation to resuspend magnetic beads, placing the magnetic beads on a magnetic frame, and sucking away liquid to retain the magnetic beads;

6) drying the centrifugal tube at room temperature;

7) adding eluent, vortexing and shaking to make the magnetic beads be suspended in the eluent for storage.

4. The method as claimed in claim 3, wherein in the step 1), the cotton swab to be extracted or wiped is put into a centrifuge tube, 150 μ l of lysis solution is taken, protease K solution is added according to 5% -10% of the total volume of the lysis solution, and after vortex mixing, the lysis solution is put into a constant temperature shaking table at 56-60 ℃ for 8-30 min; in the step 1), the cotton swab to be extracted or wiped is put into a centrifuge tube, 150-.

5. The method according to claim 3, wherein in step 2), after the warm bath, centrifugation is performed at 10000rpm for 1 min; taking out supernatant, placing in a new centrifuge tube, adding binding solution into supernatant with equal volume, adding 10-20 μ l nanometer magnetic bead suspension, placing in an oscillator at room temperature, and continuously oscillating and adsorbing for 10-20 min.

6. The method as claimed in claim 3, wherein in the step 3), after the adsorption is completed, the centrifuge tube is placed on a magnetic frame and is kept still for 5-10min, waste liquid in the centrifuge tube is sucked and discarded, and magnetic beads are reserved.

7. The method as claimed in claim 3, wherein in step 4), 500-.

8. The method as claimed in claim 3, wherein in step 5), 500-.

9. The method according to claim 3, wherein in step 6), the centrifuge tube is dried at room temperature for 5-20 min.

10. The method as claimed in claim 3, wherein in step 7), 30-70 μ l of eluent is added, vortexed, and the magnetic beads are resuspended in the eluent for storage.

Technical Field

The invention relates to a kit for extracting and purifying trace complex material evidence DNA and an extraction method, belonging to the technical field of biological and medical inspection.

Background

DNA is a biological macromolecule almost contained in the organisms in the nature, each organism has specificity, each organism can be effectively and accurately identified by using a Short Tandem Repeat (STR) typing technology, and the DNA inspection technology is one of important methods used in the case detection process. Generally, the type of material evidence locked by public security department personnel at a scene is mostly of an exfoliated cell type, mainly comprises a material evidence and a wiping material evidence, and the material evidence can be old clothes and is easy to fade. Due to the complex environment of the scene, the collected material for physical evidence may contain rust, soil and other substances, and the sample usually contains a large amount of polysaccharide, humic acid and cations, so that the effective method for extracting high-purity DNA from the trace and complex material for physical evidence is difficult to perform. Currently, methods for extracting and purifying DNA are available in the market, such as a Chelex100 method, a silica bead column method, a magnetic bead method, and the like. These methods have certain disadvantages: for example, the Chelex100 method needs to clean the material evidence so that a trace amount of DNA sample is further lost; the silica bead column-crossing method is used for extracting complex DNA, and has the advantages of complex operation, low sensitivity and weak inhibition resistance; at present, although automatic DNA extraction workstations based on the principle of a paramagnetic particle method exist in the market, the high-efficiency extraction of DNA containing various inhibitors of test materials cannot be realized, and the detection rate of STR (short tandem repeat) loci is low. Although the three methods have good extraction effect on the conventional materials to be detected, the three methods cannot solve the problem of inhibition of complex materials to be detected, and the complex materials to be detected comprise: old cloth, rust, soil, pigment and the like for many years.

In view of the above, there is a need to develop a DNA extraction and purification kit with high extraction sensitivity and ultra-strong impurity removal capability, and according to the establishment of the kit system, the efficient extraction and purification of DNA in a trace amount of complex sample test materials can be realized.

Disclosure of Invention

The invention mainly aims to provide a method for extracting and purifying trace DNA in a complex sample, which solves the problem of extraction and purification of the DNA in the complex sample and realizes rapid, simple and high-quality extraction and purification.

In order to realize the purpose, the invention provides a kit and an extraction method for extracting and purifying DNA of a trace complex material to be detected. The kit comprises six parts of lysis solution with super strong impurity removal, binding solution for ultra-high promotion DNA and magnetic beads, rinsing solution 1, rinsing solution 2, nano magnetic bead suspension and eluent.

The invention adopts the following technical scheme:

a kit for DNA extraction and purification of a trace complexity material, the kit comprising: lysis solution, binding solution, rinsing solution 1, rinsing solution 2, nano magnetic bead suspension and eluent. The kit can effectively remove inhibitors such as humic acid, polysaccharide, cations and the like in complex material evidence materials.

Wherein the composition of the lysis solution is 0.5-5mM of guanidine hydrochloride; 1-5mM of sodium dodecyl sulfate; EDTA5-50 mM; 20-100mg/ml of protease K; PVP 2-80 mM; when the lysis solution is used, the pH value of the lysis solution is adjusted to 3.5-6 by using 0.2-1M acetic acid and 0.2-1M sodium acetate buffer solution.

The binding solution consists of 2-6M of guanidine isocyanate, 0.2-1M of acetic acid, 0.2-1M of sodium acetate and 1-100mM of DMSO.

The rinsing liquid 1 consists of 1-3M guanidine hydrochloride, 55 percent of absolute ethyl alcohol by volume, 0.2-1M acetic acid and 0.2-1M sodium acetate.

The rinsing liquid 2 consists of 0.5-2M of sodium acetate and 75 percent of absolute ethyl alcohol by volume;

in the nano magnetic bead suspension, the size of suspended nano magnetic beads is 50-100 nm. In the nano magnetic bead suspension, the mass percent of the magnetic beads is 10-30%, and 0.5-1xPBS is used as a buffer solution. The magnetic bead material includes one or more of an iron compound, a cobalt compound, and a nickel compound.

The eluent composition is 0.1-0.5 XTE.

The lysis solution has the functions of having the synergistic effect of guanidine hydrochloride, sodium dodecyl sulfate, EDTA, PVP, proteinase K, acetic acid 0.2-1M, sodium acetate 0.2-1M and buffer solution with specific working concentration, endowing the invention with good lysis function for human exfoliated cells, being suitable for extracting human DNA, ensuring the integrity of DNA structure, chelating cation, dissolving humic acid, polysaccharide and other substances, and further realizing the removal of complex sample impurities;

the binding solution has the effects of providing an effective binding environment for the DNA and the magnetic beads, wherein the addition of DMSO is favorable for promoting the magnetic beads to be free from the interference of impurities, and the magnetic beads can be effectively bound with the DNA and can be adsorbed.

The rinsing liquid is used for effectively removing impurities attached to the magnetic beads except the DNA through the operation of the rinsing liquid, so that the quality of the DNA adsorbed by the magnetic beads is further improved, and the subsequent STR amplification use is facilitated; by optimizing the concentration of ions contained in the rinsing liquid and the content of absolute ethyl alcohol, ions, proteins, polysaccharides and the like other than DNA are dissolved and washed in a gradient manner.

The nano magnetic beads are magnetic nano particles, can be effectively combined with DNA under the action of a binding solution, have the size of 50-100nm, and are dispersed in 0.5-1xPBS buffer solution.

A method of making a kit as described above, comprising the steps of:

preparing lysis solution, binding solution, rinsing solution 1, rinsing solution 2, nano magnetic bead suspension and eluent according to the content of each component.

The invention also provides an extraction method of the kit for extracting and purifying the DNA of the trace complexity material, which comprises the following steps:

1) putting the material evidence fragments to be extracted or the cotton swabs wiped into a centrifugal tube, adding lysis solution into the centrifugal tube, then adding protease K solution, mixing uniformly by vortex, and putting into a constant-temperature shaking table for warm bath;

2) after warm bath, centrifuging; taking out the supernatant fluid in a new centrifuge tube, adding binding liquid into the supernatant fluid with equal volume, adding the nano magnetic bead suspension, placing in an oscillator for continuous oscillation adsorption at room temperature;

3) after adsorption, placing the centrifuge tube on a magnetic frame, standing, adsorbing and discarding waste liquid in the centrifuge tube, and keeping magnetic beads;

4) adding a rinsing liquid 1 into a centrifuge tube, covering a centrifuge tube cover, carrying out vortex oscillation to resuspend magnetic beads, placing the magnetic beads on a magnetic frame, and sucking away liquid to retain the magnetic beads;

5) adding a rinsing liquid 2 into the centrifuge tube, covering a centrifuge tube cover, carrying out vortex oscillation to resuspend magnetic beads, placing the magnetic beads on a magnetic frame, and sucking away liquid to retain the magnetic beads;

6) drying the centrifugal tube at room temperature;

7) adding eluent, vortexing and shaking to make the magnetic beads be suspended in the eluent for storage.

Further, in the step 1), putting the cotton swab to be extracted or wiped into a centrifuge tube, taking 150-; preferably, in the step 1), the cotton swab to be extracted or wiped is placed into a centrifuge tube, 600 mul of lysis solution is taken, protease K solution is added according to 5% of the total volume of the lysis solution, and after vortex mixing, the lysis solution is placed into a constant temperature shaking table at 56 ℃ for 8-30 min.

Further, in the step 2), after warm bath, centrifuging at 10000rpm for 1 min; taking out supernatant, placing in a new centrifuge tube, adding binding solution into supernatant with equal volume, adding 10-20 μ l nanometer magnetic bead suspension, placing in an oscillator at room temperature, and continuously oscillating and adsorbing for 10-20 min;

further, in the step 3), after adsorption, placing the centrifugal tube on a magnetic frame, standing for 5-10min, absorbing and discarding waste liquid in the tube, and keeping magnetic beads.

Further, in step 4), 500-.

Further, in step 5), 500-.

Further, in step 6), the centrifuge tube is dried for 5-20min at room temperature.

Further, in step 7), 30-70. mu.l of eluent is added, vortexed and shaken to resuspend the magnetic beads into the eluent for storage.

Particularly, the invention also provides an extraction method of the kit for extracting and purifying the DNA of the trace physical evidence complexity test material, which comprises the following steps:

1) placing the cotton swab with the fragment with the proper size or the cotton swab wiped with the fragment to be extracted into a centrifugal tube, adding 600 mul of lysis solution into the centrifugal tube, adding protease K solution according to 5% of the total volume of the lysis solution, uniformly mixing in a vortex mode, and then placing into a constant-temperature shaking table at 56-60 ℃ for warm bath for 8-30 min;

2) centrifuging at 10000rpm for 1-5min after warm bath; taking out supernatant, placing in a new centrifuge tube, adding binding solution into supernatant with equal volume, adding 10-20 μ l nanometer magnetic bead suspension, placing in an oscillator at room temperature, and continuously oscillating and adsorbing for 10-20 min;

3) after adsorption, placing the centrifuge tube on a magnetic frame, standing for 5-10min, absorbing and discarding waste liquid in the centrifuge tube, and keeping magnetic beads;

4) adding 500-;

5) adding 500-;

6) drying the centrifuge tube at room temperature for 5-20 min;

7) adding 30-70 μ l of eluent, vortexing and shaking to resuspend the magnetic beads into the eluent for storage.

The invention can play the positive roles that: the DNA recovery efficiency is ultrahigh, and trace exfoliated cells can be efficiently recovered; the DNA extracting method has the advantages of super-strong impurity removing capability, good quality of DNA extracted from complex inspection materials such as iron rust, soil, old cloth for many years and the like, simplicity in operation, short time, capability of being matched with an automatic nucleic acid extracting instrument and effective improvement of working efficiency.

Drawings

FIG. 1 is a schematic diagram of a DNAreal-time PCR amplification curve of a complex sample extracted by different extraction methods;

FIG. 2 example 1STR profile;

FIG. 3 example 2STR profile;

FIG. 4, chelex-100, extracting the DNA STR atlas of the complex test material.

Detailed Description

According to the above-mentioned technical solutions of the invention, the invention is further described by means of the following embodiments, which are exemplary and for reference only.

Example 1

In order to achieve the above object, according to one aspect of the present invention, there is provided a kit for extracting and purifying DNA from a trace-complexity material, comprising: ultra-strong impurity-removing lysis solution, ultra-high promoting DNA and magnetic bead combined solution, rinsing solution 1, rinsing solution 2, nano magnetic bead suspension and eluent,

wherein, the nano magnetic bead is a commercial nano magnetic bead (nano-30-NA, Jinzhi Zhi Biotech Co., Ltd.) of 50 nm;

the lysis solution consists of 2mM guanidine hydrochloride; 3mM of sodium dodecyl sulfate; EDTA20 mM; 20mg/ml of protease K; PVP 50 mM; adjusting the working pH value to 5 by using 0.2M acetic acid and 0.2M sodium acetate buffer solution;

the binding solution consists of guanidine isocyanate (5M), acetic acid 0.2M, sodium acetate 0.2M and DMSO (20 mM);

the rinsing liquid 1 consists of guanidine hydrochloride (2M), absolute ethyl alcohol with the volume percentage of 55 percent, acetic acid with the volume percentage of 0.2M and sodium acetate with the volume percentage of 0.2M,

the rinsing liquid 2 consists of sodium acetate (1M) and 75 percent of absolute ethyl alcohol by volume;

in the magnetic bead suspension, the particle size of nano magnetic beads is 50nm, the mass percentage of the nano magnetic beads is 10%, and 1xPBS is used as a buffer solution;

the eluent composition is 0.1-0.5 XTE;

preparing lysis solution, binding solution, rinsing solution 1, rinsing solution 2, nano magnetic bead suspension and eluent according to the contents of the components.

The extraction method of the kit for extracting and purifying the DNA of the trace complex material comprises the following steps:

1) putting fragments of old clothes to be extracted and with iron embroidery and soil mixture with proper size (1cmx2cm) into a centrifugal tube, adding 200 mu L of lysate into the centrifugal tube, adding 10 mu L of 40mg/ml proteinase K solution, uniformly mixing by vortex, and putting into a constant-temperature shaking table at 56 ℃ for 10 min;

2) centrifuging at 10000rpm for 1min after warm bath; taking out the supernatant, putting the supernatant into a new centrifuge tube, adding binding solution into the supernatant in a medium volume, adding 10 mu l of nano magnetic bead suspension, putting the suspension in an oscillator at room temperature, and continuously oscillating and adsorbing for 10 min;

3) after adsorption, placing the centrifuge tube on a magnetic frame, standing for 5min, absorbing and discarding waste liquid in the centrifuge tube, and keeping magnetic beads;

4) adding 500 mul of rinsing liquid 1 into a centrifuge tube, covering a centrifuge tube cover, carrying out vortex oscillation on the resuspended magnetic beads, placing the resuspended magnetic beads on a magnetic frame, and sucking away liquid to retain the magnetic beads;

5) adding 500 mul of rinsing liquid 2 into the centrifuge tube, covering the centrifuge tube cover, carrying out vortex oscillation on the resuspended magnetic beads, placing the magnetic beads on a magnetic frame, and sucking away liquid to retain the magnetic beads;

6) drying the centrifuge tube at room temperature for 10 min;

7) add 50. mu.l of eluent, vortex and shake to resuspend the beads into the eluent for storage.

Example 2

In order to achieve the above object, according to one aspect of the present invention, there is provided a kit for extracting and purifying DNA from a trace-complexity material, comprising: ultra-strong impurity-removing lysis solution, ultra-high promoting DNA and magnetic bead combined solution, rinsing solution 1, rinsing solution 2, nano magnetic bead suspension and eluent,

wherein, the nano magnetic bead is a commercial nano magnetic bead (prenano-30-NA, Jinqianzhi Biotechnology (Jilin) Co., Ltd.) of 50 nm;

the composition of the lysis solution is 1mM of guanidine hydrochloride; 2mM of sodium dodecyl sulfate; EDTA 10 nM; 30mg/ml of protease K; PVP 30 mM; adjusting the working pH value to 4.5 by using 0.3M acetic acid and 0.3M sodium acetate buffer solution;

the binding solution consists of guanidine isocyanate (4M), acetic acid 0.3M, sodium acetate 0.3M and DMSO (80 mM);

the rinsing liquid 1 consists of guanidine hydrochloride (3M), absolute ethyl alcohol (55 percent by volume), acetic acid (0.3M) and sodium acetate (0.3M);

the rinsing liquid 2 consists of sodium acetate (2M) and 75 percent (volume percentage) of absolute ethyl alcohol;

in the magnetic bead suspension, the particle size of nano magnetic beads is 100nm, the mass percentage of the nano magnetic beads is 20%, and 0.5xPBS is used as a buffer solution;

the eluent composition was 0.5 XTE.

The extraction method of the kit for extracting and purifying the DNA of the trace complex material comprises the following steps:

1) putting fragments of old clothes to be extracted and with iron mud mixture with proper size (1cmx2cm) into a centrifugal tube, adding 300 mu L of lysis solution into the centrifugal tube, adding 15 mu L of 30mg/ml protease K solution, uniformly mixing by vortex, and putting into a constant temperature table at 56 ℃ for warm bath for 15 min;

2) centrifuging at 10000rpm for 1min after warm bath; taking out the supernatant, putting the supernatant into a new centrifuge tube, adding binding solution into the supernatant in a medium volume, adding 15 mu l of nano magnetic bead suspension, putting the suspension in an oscillator at room temperature, and continuously oscillating and adsorbing for 15 min;

3) after adsorption, placing the centrifuge tube on a magnetic frame, standing for 8min, absorbing and discarding waste liquid in the centrifuge tube, and keeping magnetic beads;

4) adding 600 mul of rinsing liquid 1 into a centrifuge tube, covering a centrifuge tube cover, carrying out vortex oscillation on the resuspended magnetic beads, placing the resuspended magnetic beads on a magnetic frame, and sucking away liquid to retain the magnetic beads;

5) adding 600 mul of rinsing liquid 2 into the centrifuge tube, covering the centrifuge tube cover, carrying out vortex oscillation on the resuspended magnetic beads, placing the magnetic beads on a magnetic frame, and sucking away liquid to retain the magnetic beads;

6) drying the centrifuge tube at room temperature for 12 min;

7) add 50. mu.l of eluent, vortex and shake to resuspend the beads into the eluent for storage.

Comparative experiment: in order to illustrate the outstanding effect of extracting DNA from a complex sample test material, chelex-100 which is currently used in the market is selected as a comparison method in the experiment, and the experiment for extracting and purifying the DNA of the trace complex sample test material is carried out simultaneously with the kit.

chelex-100 extraction procedure

1) The cloth used for many years and with the same size as that in example 1 is put into deionized water for incubation for 30min at 56 ℃, the sample is released into the deionized water, and the incubation liquid is sucked out to a new 2ml centrifuge tube.

2) To the sample-containing incubation solution was added an equal volume of 10% chelex-100 and placed in a 90 ℃ constant temperature shaker at 100rpm for 1 hours.

3) After centrifugation at 12000rpm for 5min, the supernatant was taken for PCR and str amplification and electrophoresis.

The kit of the invention in the experiment of the invention is configured according to the embodiments 1 and 2, the sample is the same as the sample used by chelex-100, and DNA is digested, lysed, adsorbed, rinsed and re-suspended by magnetic beads. The same fluorescence quantitative PCR and STR electrophoresis are adopted; the results of the experiments are shown in FIGS. 1, 2, 3, 4 and Table 1. FIG. 1 is a comparison of fluorescent quantitation effects of different methods on extraction of DNA from a trace amount of complex test material; FIG. 2 is an STR map of example 1, FIG. 3 is an STR map of example 2, and FIG. 4 is an STR map of complex sample DNA extracted by chelex. Table 1 comparison of STR effect of different methods on extraction of trace complex test material DNA. The experimental result shows that the kit for extracting and purifying the DNA of the trace material evidence complex test material has outstanding advantages for extracting the DNA of the trace material evidence complex test material.

TABLE 1 comparison of STR effect of different methods on DNA extraction of trace complex test material

	Chelex-100	Example 1	Example 2
				Material for testing complex substance	Not detected out	STR locus is complete	STR locus is complete

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and the preferred embodiments are not exhaustive and do not limit the invention to the precise embodiments described. Various modifications and improvements of the technical solution of the present invention may be made by those skilled in the art without departing from the spirit of the present invention, and the technical solution of the present invention is to be covered by the protection scope defined by the claims.