CN110951725A

CN110951725A - One-step nucleic acid extraction process based on paramagnetic particle method

Info

Publication number: CN110951725A
Application number: CN201911399484.0A
Authority: CN
Inventors: 董辉; 金维荣; 秦红友; 杨红
Original assignee: Shenyou Genomics Research Institute Nanjing Co Ltd
Current assignee: Shenyou Genomics Research Institute Nanjing Co Ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2020-04-03

Abstract

The invention relates to a one-step nucleic acid extraction process based on a paramagnetic particle method, which comprises the following steps: 1) respectively pre-packaging 6 deep-hole plates contained in 1 kit, turning over the pre-packaged deep-hole plates, uniformly mixing and centrifuging, tearing off a sealing film of the deep-hole plates, and sequentially adding a sample to be detected and protease K into the deep-hole plates of the pre-packaged split bonding solution LB; 2) extracting nucleic acid by a nucleic acid extractor; 3) after the nucleic acid extraction procedure is finished, taking out 6 deep-well plates, wherein the solution in the 6 th deep-well plate is the nucleic acid solution; the invention unifies the lysis solution and the binding solution in the true sense, and simultaneously carries out the lysis and the binding, thereby greatly improving the working efficiency, improving the concentration of nucleic acid extraction and ensuring the purity of nucleic acid purification. The whole kit does not contain any toxic substance, so that the safety of operators is ensured.

Description

One-step nucleic acid extraction process based on paramagnetic particle method

Technical Field

The invention relates to a one-step nucleic acid extraction process based on a paramagnetic particle method, and belongs to the technical field of nucleic acid separation and purification.

Background

The traditional nucleic acid extraction method comprises phenol-chloroform extraction method, alkali extraction method, high-salt precipitation, ion exchange method, magnetic bead method and the like. However, there are some disadvantages, such as using toxic reagents such as phenol and chloroform, too long extraction time, low concentration and purity of extracted DNA, semi-automation, and small flux. High-quality human genetic genes are the basis of various researches, and the implementation of downstream experiments is directly influenced by nucleic acid technology.

Aiming at the problems of complicated pretreatment, low nucleic acid concentration, low purity (polysaccharide, inorganic salt residue and the like), sample unicity, small flux and the like of the existing magnetic bead nucleic acid extraction kit, the company invents a one-step nucleic acid extraction and purification kit based on a magnetic bead method. At present, the pretreatment of the magnetic bead nucleic acid extraction kit is complicated, the sample needs to be fully cracked under the action of a cracking solution to remove impurities such as protein, lipid and the like, and then a binding solution is added into the cracked sample to bind nucleic acid, so that the operating mode greatly reduces the working efficiency and increases the experimental error. Although the CN 109504678A patent unifies the lysis solution and the binding solution, the use steps are still to lyse cells and then to bind nucleic acids, and the isopropanol in the binding solution is easy to volatilize during the high temperature incubation process, which cannot ensure the stability of the nucleic acid concentration, and causes physical harm to operators after long-term use.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a one-step nucleic acid extraction kit based on a magnetic bead method, lysis solution and binding solution of the kit are combined, and the lysis and the binding are simultaneously carried out, so that the working efficiency is greatly improved; the kit can be used for extracting and purifying the DNA of human genetic gene detection samples such as saliva, whole blood, mouth swabs and the like, and is simple to operate without switching experimental procedures. In addition, the kit is pre-distributed in a deep-hole plate, 96 samples can be processed at one time, and high-flux automatic extraction of nucleic acid is realized.

In order to realize the purpose, the technical scheme is as follows: a one-step nucleic acid extraction and purification kit based on a magnetic bead method is developed and comprises a pre-packaged reagent cracking binding solution LB, a washing solution W1, a washing solution W2, a washing solution W3, an eluent lowTE, a magnetic bead mixed solution and a single-tube proteinase K, wherein the components of the cracking binding solution LB are guanidine isothiocyanate 2-5 mol/L, SDS 0.5-1% (W/v), Tris-HCl 20-50 mmol/L, EDTA 5-10 mmol/L, Nacl 20-50 mmol/L, Triton X-1000.2-1.5% (v/v), Tween 200.2-1.0% (v/v) and PEG 4005-30% (v/v), and a solvent is sterile water. The washing liquid W1 comprises 2-4mol/L guanidine hydrochloride, 30-50% (v/v) ethanol and Triton X-1000.2-0.5% (v/v), and the solvent is sterile water. The washing solution W2 is PEG 60005-10% (W/v) and NaCl 20-50 mmol/L, and the solvent is sterile water. The washing solution W3 was 75-80% (v/v) ethanol. The magnetic bead mixed solution is 0.25-2ng/ml of magnetic beads and 75-80% (v/v) of ethanol; eluent lowTE: Tris-HCl 10-30mmol/L, EDTA 1-5 mmol/L. The concentration of proteinase K was 20 mg/mL.

Guanidinium isothiocyanate and SDS are denaturants, which lyse cells, proteins, polysaccharides, etc. by denaturing proteins. Tris-HCl solution provides an environment to keep the nucleic acid structure stable. EDTA as nuclease inhibitor can chelate Mg in solution²⁺、Ca²⁺And divalent metal ions. Triton X-100 and Tween 20 are nonionic surfactants which can solubilize intracellular lipids, increase the permeability of cell membranes, and facilitate the release of nucleic acids. The PEG 400 can effectively promote the magnetic beads to be combined with nucleic acid, and the PEG 400 can not volatilize under the heating condition. The invention can crack the combination liquid, which not only can crack the cells, but also can provide a proper environment for the combination of the nucleic acid and the magnetic beads, thereby reducing the operation steps and improving the quality of nucleic acid extraction.

In order that the kit can simultaneously satisfy samples such as saliva, whole blood, mouth swabs and the like, the washing liquid W1 of the kit uses high-concentration guanidine hydrochloride, and the guanidine hydrochloride can further crack proteins, polysaccharides and the like in magnetic bead-nucleic acid composites, so that the degradation of impurities such as mucopolysaccharides of saliva samples and the like is ensured, and the inhibition of saliva sample DNA to downstream experiments is reduced. Various upstream impurities including salts such as guanidine hydrochloride can be further washed out from the washing liquid W2, and the inhibition of the downstream experiment by inorganic salts is also reduced.

The nucleic acid extraction sample provided by the invention comprises saliva, whole blood and a mouth swab, and comprises the following steps:

1) respectively pre-packaging 6 deep-hole plates contained in 1 kit, inverting and uniformly mixing the pre-packaged deep-hole plates, then performing instant centrifugation for 10s, carefully tearing off a sealing film of the deep-hole plate, and sequentially adding a sample to be detected and protease K into a hole of a plate 1 (pre-packaged lysis binding solution LB);

2) setting an extraction program on a nucleic acid extractor, and then carrying out full-automatic extraction on nucleic acid;

3) and after the extraction program is operated, taking out 6 deep-well plates, wherein the solution in the 6 th deep-well plate is the nucleic acid solution.

The invention also provides the procedures of pre-dispensing the kit in the well plate mode in an automatic nucleic acid extractor such as Tiangen TGuide S96.

The magnetic rod is transferred into the magnetic bead mixed liquid of the 4 th plate to adsorb the magnetic beads;

and (3) transferring the magnetic rod with the magnetic beads to the lysis binding solution LB of the 1 st plate, lysing impurities such as cells and proteins under the action of the lysis binding solution LB, releasing nucleic acid, binding the nucleic acid to the magnetic beads under the action of a buffer solution, and descending the magnetic rod to adsorb the magnetic beads.

The magnetic beads with nucleic acid are transferred into the No. 2 plate washing liquid W1 under the action of a magnetic bar, and impurities such as protein, polysaccharide and the like on the nucleic acid are washed under the action of the washing liquid W1;

the magnetic beads with nucleic acid are transferred into the 3 rd plate washing solution W2 under the action of the magnetic bar, and the redundant impurities such as saccharides, salts and the like on the nucleic acid are washed away under the action of the washing solution W2;

transferring the magnetic beads with the nucleic acid to the 4 th plate under the action of a magnetic rod, and washing away impurities on the nucleic acid under the action of 75-80% ethanol;

the magnetic beads with the nucleic acid are transferred into the 5 th washing liquid W3 under the action of the magnetic bar, and impurities on the nucleic acid are washed continuously under the action of the washing liquid W3;

transferring the magnetic beads with nucleic acid to the position above lowTE of the eluent in the column 6 under the action of a magnetic bar, drying for 5min, volatilizing residual ethanol, and then entering a plate 6 to elute the nucleic acid;

after the elution is finished, the magnetic beads are adsorbed by the magnetic bar and transferred to the 4 th plate, and the magnetic beads are discarded, so that the nucleic acid separation is realized.

The invention unifies the lysis solution and the binding solution in the true sense, and simultaneously carries out the lysis and the binding, thereby greatly improving the working efficiency, improving the concentration of nucleic acid extraction and ensuring the purity of nucleic acid purification. The whole kit does not contain any toxic substance, so that the safety of operators is ensured.

Compared with the prior art, the invention has the following beneficial effects because the technology is adopted:

(1) no toxic reagent is used, so that the life safety of an operator is ensured;

(2) the extraction efficiency is high: the sample is directly cracked and combined with the magnetic beads without any pretreatment, 96 samples can be processed at one time, the whole process is completed within 40min, the working efficiency is enhanced, and the method is economical and efficient;

(3) the automation is high: the automation degree is high, and human errors and errors are reduced;

(4) the concentration and purity of the extracted DNA are high, and the working efficiency of downstream experiments is improved.

Drawings

FIG. 1 is a DNA gel electrophoresis image after extraction of a whole blood sample;

FIG. 2 is a DNA gel electrophoresis image after extraction of a saliva sample.

Detailed Description

The invention is further elucidated with reference to the drawings and the detailed description.

Example 1: preparation method and application of whole blood nucleic acid DNA extraction kit

1. Reagent:

and (3) cracking the binding solution: 2mol/L, SDS 0.5.5% (w/v) of guanidinium isothiocyanate, 20mmol/L, EDTA10mmol/L, Nacl 50mmol/L of Tris-HCl, 200.5% (v/v) of Tween and 20% (v/v) of PEG 40026, and the solvent is sterile water.

The washing solution W1 was composed of guanidine hydrochloride 3.5mol/L, Triton X-1000.5% (v/v), ethanol 50% (v/v), and the solvent was sterile water.

Wash W2 was PEG 60005% (W/v) and NaCl 20mmol/L and the solvent was sterile water.

Wash W3 was 75% ethanol (v/v).

The magnetic bead mixture was 1ng/ml magnetic beads and 75% ethanol (v/v).

Eluent lowTE: Tris-HCl20 mmol/L, EDTA1 mmol/L.

The concentration of proteinase K was 20 mg/mL.

2. The reagent is pre-packaged to form:

subpackaging: as shown in Table 1, the lysis conjugate LB was pre-dispensed into the 96-well plate of the 1 st plate kit, the washing reagent W1 was pre-dispensed into the 96-well plate of the 2 nd plate kit, the washing reagent W2 was pre-dispensed into the 96-well plate of the 3 rd plate kit, the mixed solution of magnetic beads was pre-dispensed into the 96-well plate of the 4 th plate kit, the washing reagent W3 was pre-dispensed into the 96-well plate of the 5 th plate kit, and Low TE was pre-dispensed into the 96-well plate of the 6 th plate kit.

Sealing the film: the 96-well plate was sealed using a heat sealer.

TABLE 1 Pre-dispensed reagent composition in kit

Composition of	Name (R)	Plate type/volume (ml)	Amount of reagent per well
				1 st plate	Cleavage of the binding solution LB	Deep hole plate/2.2	600ul
2 nd plate	Lotion W1	Deep hole plate/2.2	600ul
				No. 3 plate	Lotion W2	Deep hole plate/2.2	600ul
4 th plate	Magnetic bead mixed liquid	Deep hole plate 2.2	600ul
				5 th plate	Lotion W3	Deep hole plate 2.2	600ul
6 th plate	Low TE	Deep hole plate/1.0	100ul

3. Experimental procedure

1) Taking out 6 pre-packaged 96-deep-well plates, reversing and uniformly mixing for several times to resuspend magnetic beads, removing the package, lightly throwing the 96-deep-well plate to enable reagents and magnetic beads to be concentrated at the bottom of the 96-deep-well plate (a plate centrifuge can also be used, centrifuging at 500rpm for 1min), carefully tearing off an aluminum foil sealing film before use, avoiding the vibration of the 96-deep-well plate and preventing liquid from splashing.

2) Adding 200 mu L of anticoagulated whole blood sample and 20 mu L of proteinase K into the 1 st plate of the 96 deep-well plate, and placing the 96 deep-well plate (lysis binding solution LB, washing solution W1, washing solution W2, washing solution W3, magnetic bead mixed solution and Low TE) at a position corresponding to the equipment; inserting the magnetic rod sleeve into a 96-deep-hole plate (magnetic bead mixed liquid);

3) running a corresponding automatic extraction program;

TABLE 2 reaction conditions for the automated extraction procedure

4) After the automated extraction procedure was completed, the 6 th plate (Low TE) solution was the nucleic acid solution.

Example 2: preparation method and application of oral cell nucleic acid DNA extraction kit

1. Reagent: the formulation was carried out as in example 1.

2. The reagent is pre-packaged to form:

subpackaging: as shown in Table 3, the lysis conjugate LB was pre-dispensed into the 96-well plate of the 1 st plate kit, the washing reagent W1 was pre-dispensed into the 96-well plate of the 2 nd plate kit, the washing reagent W2 was pre-dispensed into the 96-well plate of the 3 rd plate kit, the mixed solution of magnetic beads was pre-dispensed into the 96-well plate of the 4 th plate kit, the washing reagent W3 was pre-dispensed into the 96-well plate of the 5 th plate kit, and Low TE was pre-dispensed into the 96-well plate of the 6 th plate kit.

Sealing the film: the 96-well plate was sealed using a heat sealer.

TABLE 3 Pre-dispensed reagent composition in kit

Composition of	Name (R)	Plate type/volume (ml)	Amount of reagent per well
				1 st plate	Cleavage of the binding solution LB	Deep hole plate/2.2	450ul
2 nd plate	Lotion W1	Deep hole plate/2.2	600ul
				No. 3 plate	Lotion W2	Deep hole plate/2.2	600ul
4 th plate	Magnetic bead mixed liquid	Deep hole plate/2.2	600ul
				5 th plate	Lotion W3	Deep hole plate/2.2	600ul
6 th plate	Low TE	Deep hole plate/1.0	80ul

3. Experimental procedure

2) Adding 450 mu L of oral cell sample and 20 mu L of proteinase K into the 1 st plate of the 96 deep-well plate, and placing the 96 deep-well plate (lysis binding solution LB, washing solution W1, washing solution W2, washing solution W3, magnetic bead mixed solution and Low TE) at the corresponding position of the device; inserting the magnetic rod sleeve into a 96-deep-hole plate (magnetic bead mixed liquid);

3) running the corresponding automated extraction program, according to table 2 in example 1;

after the automated extraction procedure was completed, the 6 th plate (Low TE) solution was the nucleic acid solution.

Example 3: comparison of two kits on whole blood DNA extraction

8 pieces of EDTA anticoagulated whole blood are taken as samples, and the kit and the control kit-GNT are respectively used^TMThe nucleic acid extraction was carried out using a blood genome DNA extraction kit (magnetic bead method). The kit of the present invention was prepared by extracting nucleic acid, GNT, according to the procedure and reaction conditions of example 1^TMThe blood genome DNA extraction kit (magnetic bead method) is used for extracting nucleic acid according to the procedure of the kit's instruction.

1) The nucleic acids extracted by the kit and the control kit of the invention were subjected to Qubit quantification to obtain the DNA concentration, and the results are shown in table 4. The concentration of the extracted product of the kit is obviously higher than that of a control kit.

TABLE 4 quantitative Qubit detection table (ng/. mu.l) for nucleic acid extracted from the kit of the present invention and the control kit

Serial number	Concentration of nucleic acid extracted by the kit of the present invention	Concentration of nucleic acid extracted with control reagent
			T1	42.2	33.3
T2	62.6	60.1
			T3	53.1	51.7
T4	84.6	77.3
			T5	48.1	36.6
T6	62.3	53.7
			T7	74.9	54.2
T8	71.2	69.5

2) Detection of OD Using ultramicro Spectrophotometer₂₆₀/OD₂₈₀And D₂₆₀/OD₂₃₀Values, where 1-8 are the whole blood products extracted with the kit of the invention, 9-16 are the products extracted with the control kit, and the assay data are shown in Table 5. DNA product, OD, extracted from the kit of the invention₂₆₀/OD₂₈₀At 1.7-2.0, OD₂₆₀/OD₂₃₀In the range of 1.7-2.0; DNA product, OD, extracted from control kit₂₆₀/OD₂₈₀At 1.5-2.0, OD₂₆₀/OD₂₃₀In the range of 1.0-2.0. The purity of the nucleic acid product extracted by the kit is obviously higher than that of a control kit.

TABLE 5 detection of OD by ultramicro-spectrophotometer₂₆₀/OD₂₈₀And OD₂₆₀/OD₂₃₀Value of

Serial number	OD₂₆₀/OD₂₈₀	OD₂₆₀/OD₂₅₀	Serial number	OD₂₆₀/OD₂₈₀	OD₂₆₀/OD ₂₅₀
						1	1.82	1.92	9	1.65	1.28
2	1.84	1.72	10	1.73	1.63
						3	1.79	1.72	11	1.50	1.16
4	1.85	1.96	12	1.69	1.10
						5	1.87	1.92	13	1.52	1.27
6	1.84	1.91	14	1.60	1.15
						7	1.82	1.87	15	1.58	1.28
8	1.78	1.92	16	1.51	1.21

3) Gel electrophoresis imaging

FIG. 1 is an image of the electrophoresis gel of the above experimental results, 1-8 are the extraction products of the kit of the present invention, 9-16 are the extraction products of the control kit, and the bands are clear and bright and have no obvious degradation.

Example 4: extracting nucleic acid from saliva sample

10 saliva samples (saliva collecting tube is saliva preservation tube of Nanjing Shenyou Biotech company) are taken and are inverted and mixed evenly for several times. GNT, a kit of the invention and a control kit, respectively^TMExtracting nucleic acid by saliva DNA extraction kit (magnetic bead method). The kit of the present invention was prepared by extracting nucleic acid, GNT, according to the procedure and reaction conditions of example 2^TMThe saliva DNA extraction kit (magnetic bead method) is used for extracting nucleic acid according to the steps of the instruction manual of the kit.

1) The nucleic acid extracted by the kit and the control kit of the invention is subjected to the quantitative detection of the Qubit to obtain the nucleic acid concentration, and the results are shown in Table 6. The concentration of the extracted product of the kit is higher.

TABLE 6 quantitative test table (ng/. mu.l) for nucleic acid extracted from the kit of the present invention and the control kit

Sample number	Extraction concentration of the kit of the invention	Control kit extraction concentration
			T1	47.2	33.1
T2	50	33.5
			T3	37	22.6
T4	28.4	22.0
			T5	23.8	11.7
T6	30.28	24.1
			T7	16.21	11.1
T8	23.61	21.7
			T9	18.31	15.3
T10	45.2	30.2

2) Detection of OD Using ultramicro Spectrophotometer₂₆₀/0D₂₈₀And OD₂₆₀/OD₂₃₀The values and the detection data are shown in Table 7, wherein 1-10 are the extracted products of the kit of the invention, and 11-20 are the extracted products of the control kit. The kit of the invention extracts DNA product, OD, obtained by saliva sample₂₆₀/OD₂₈₀At 1.8-2.0, OD₂₆₀/OD₂₃₀In the range of 1.7-2.0. DNA product, OD, extracted from control kit₂₆₀/OD₂₈₀At 1.6-2.0, OD₂₆₀/OD₂₃₀In the range of 1.5-2.2. The purity of the extracted product of the kit is higher.

TABLE 7 detection of OD by ultramicro-spectrophotometer₂₆₀/OD₂₈₀And OD₂₆₀/OD₂₃₀Value of

Taking 3 mul of genome DNA solution to carry out electrophoresis detection by using 1.0% agarose gel, wherein 1-10 is the extraction product of the kit of the invention, 11-20 is the extraction product of the control kit, the result is shown in figure 2, and the band is clear and bright.

The above-mentioned embodiments are merely preferred embodiments of the present invention, and should not be construed as limiting the present invention, and the scope of the present invention should be defined by the claims, and equivalents including technical features of the claims, i.e., equivalent modifications within the scope of the present invention.

Claims

1. A one-step nucleic acid extraction process based on a magnetic bead method is characterized by comprising the following steps:

1) respectively pre-packaging 6 deep-hole plates contained in 1 kit, turning over the pre-packaged deep-hole plates, uniformly mixing and centrifuging, tearing off a sealing film of the deep-hole plates, and sequentially adding a sample to be detected and protease K into the deep-hole plates of the pre-packaged split bonding solution LB;

2) extracting nucleic acid by a nucleic acid extractor;

3) and (3) after the nucleic acid extraction procedure is finished, taking out 6 deep-well plates, wherein the solution in the 6 th deep-well plate is the nucleic acid solution.

2. The one-step nucleic acid extraction process based on the magnetic bead method as claimed in claim 1, wherein the 6 kits in the step (1) are pre-packaged as follows:

(a) filling a cracking bonding liquid LB in a first deep hole plate;

(b) filling a second deep hole plate with a washing solution W1;

(c) the third deep hole plate is filled with washing liquor W2;

(d) filling the magnetic bead mixed liquid into the fourth deep hole plate;

(e) the fifth deep hole plate is filled with washing liquid W3;

(f) the sixth deep well plate was loaded with eluent lowTE.

3. The one-step nucleic acid extraction process based on the magnetic bead method as claimed in claim 2, wherein the lysis solution LB in step (a) comprises the following components: 2-5 mol/L, SDS 0.5.5-1% (w/v) of guanidinium isothiocyanate, 5-10 mmol/L, EDTA 5-50 mmol/L, Nacl 20-50 mmol/L of Tris-HCl20, 24-1.5% (v/v) of Triton X-1000.2, 200.2-1.0% (v/v) of Tween, 5-30% (v/v) of PEG 400, and the balance of solvent.

4. The one-step nucleic acid extraction process based on the magnetic bead method as claimed in claim 2, wherein the washing solution W1 in the step (b) comprises the following components: 2-4mol/L of guanidine hydrochloride, 0.5-0.78% (v/v) of Triton X-1000.2, 30-50% (v/v) of ethanol, and the balance of solvent.

5. The one-step nucleic acid extraction process based on the magnetic bead method as claimed in claim 2, wherein the washing solution W2 in the step (c) comprises the following components: PEG 60005-10% (w/v), NaCl 20-50 mmol/L and the balance of solvent.

6. The one-step nucleic acid extraction process based on the magnetic bead method according to claim 3, 4 or 5, wherein: the solvent is sterile water.

7. The one-step nucleic acid extraction process based on the magnetic bead method according to claim 2, wherein: the washing liquid W3 in the step (e) is 75-80% (v/v) ethanol.

8. The one-step nucleic acid extraction process based on the magnetic bead method according to claim 2, wherein: the magnetic bead mixed solution in the step (d) comprises the following components: 0.25-2ng/ml of magnetic beads and 75-80% (v/v) of ethanol.

9. The one-step nucleic acid extraction process based on the magnetic bead method according to claim 2, wherein: the eluent lowTE in step (f) comprises the following components: Tris-HCl 10-30mmol/L, EDTA 1-5 mmol/L.

10. The one-step nucleic acid extraction process based on the magnetic bead method according to claim 1, wherein: each deep-well plate is provided with 96 wells.