CN114958830A - Kit for simultaneously extracting pathogen DNA and RNA and application thereof - Google Patents
Kit for simultaneously extracting pathogen DNA and RNA and application thereof Download PDFInfo
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- CN114958830A CN114958830A CN202210737451.8A CN202210737451A CN114958830A CN 114958830 A CN114958830 A CN 114958830A CN 202210737451 A CN202210737451 A CN 202210737451A CN 114958830 A CN114958830 A CN 114958830A
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Abstract
The application relates to the technical field of nucleic acid extraction, and particularly discloses an extraction reagent and an extraction kit for simultaneously extracting pathogen DNA and RNA; the extraction reagent comprises protease K, lysis solution, magnetic bead solution, washing solution I, washing solution II and eluent; the lysis solution comprises the following components: 10-100 mmol/L Tris-HCl with the pH value of 6.0-8.0, 1-6M guanidine salt, 0.1-5 Vol% sodium dodecyl sulfate, 0.5-3 Vol% Tween 20, 1-10 mmol/L disodium ethylene diamine tetraacetate, 1-5M isopropanol and water as a solvent; the extraction kit contains the extraction reagent; the extraction reagent adopts a superparamagnetic silicon oxide nanometer magnetic bead extraction method, can be used for simultaneously extracting DNA and RNA in pathogens, and has the advantages of high efficiency and high quality of nucleic acid extraction.
Description
Technical Field
The invention belongs to the technical field of nucleic acid extraction, and particularly relates to an extraction reagent and an extraction kit for simultaneously extracting pathogen DNA and RNA.
Background
The extraction of nucleic acid from biological samples is an important process in the field of genetic engineering or clinical examination, and at present, there are many methods for extracting nucleic acid, wherein, the magnetic bead method for extracting nucleic acid can realize high-throughput and automatic operation, and has the advantages of simple operation, safety, no toxicity and the like, and is widely used at present.
The principle of extracting nucleic acid by using a magnetic bead method is that the surfaces of superparamagnetic nanoparticles are improved and surface-modified by using a nanotechnology to prepare superparamagnetic silicon oxide nanometer magnetic beads, the magnetic beads can be specifically identified and efficiently combined with nucleic acid molecules on a microscopic interface, the magnetic particles are separated from liquid under the action of an external magnetic field, and the purified nucleic acid molecules are obtained by removing the liquid and then eluting.
In the technology of extracting nucleic acid by the magnetic bead method, the related technology can singly extract DNA or RNA. However, this method enables simultaneous extraction of DNA and RNA.
Disclosure of Invention
In order to solve the problems, the invention discloses an extraction reagent and an extraction kit for extracting pathogen DNA and RNA while improving the efficiency of nucleic acid extraction.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention provides an extraction reagent for simultaneously extracting pathogen DNA and RNA, which comprises lysis solution, magnetic bead solution, washing solution I, washing solution II, eluent and proteinase K; the lysis solution comprises the following components: 10-100 mmol/L Tris-HCl with pH value of 6.0-7.0, 1-6M guanidine salt, 0.1-5 Vol% sodium dodecyl sulfate, 0.5-3 Vol% Tween 20, 1-10 mmol/L disodium ethylene diamine tetraacetate, 1-5M isopropanol and water as solvent.
Further preferably, the extraction reagent comprises lysis solution 600ul, magnetic bead solution 600ul, washing solution I800 ul, washing solution II 800ul, eluent 100ul and proteinase K20 ul.
Further, the guanidine salt is at least one selected from the group consisting of guanidine hydrochloride, guanidine nitrate, guanidine carbonate, guanidine acetate, guanidine thiocyanate and guanidine isothiocyanate. Disodium edetate is an enzyme inhibitor. Sodium lauryl sulfate is an anionic surfactant.
Further, the magnetic bead solution comprises 15-25 mg/mL of magnetic microspheres, 0.3-0.8 mol/L of sodium chloride, 5-30 mmol/L of Tris-HCl with pH of 7.0-7.5, and a solvent is water. The magnetic microspheres are superparamagnetic silicon oxide nano magnetic beads modified by specific hydroxyl groups.
Further, the washing solution I comprises 5-30 mmol/L Tris-HCl with pH of 7.0-7.5, 1-10 mmol/L disodium ethylene diamine tetraacetate, 100-200 mmol/L sodium chloride, 0.5-5M guanidinium, and 50-70 Vol% ethanol solution, and the solvent is water.
Further, the washing liquid II comprises 50-75 Vol% ethanol solution; the eluate comprises water.
Further, the proteinase K is 20 μ g/μ L proteinase K solution.
The invention also provides a kit for simultaneously extracting the DNA and RNA of a pathogen, which comprises the extraction reagent.
The invention also provides an extraction method for simultaneously extracting the DNA and RNA of pathogens, which comprises the following steps:
(1) adding 200 mu L of simulated pathogen biological sample and 20 mu L of 20 mu g/mu L proteinase K solution into each well of a 96-well plate,
(2) then placing the sample into an extractor (BNP32), installing a magnetic rod protective sleeve, and operating a program, wherein the specific program is shown in the following table:
full-automatic nucleic acid extraction procedure:
| step (ii) of | Hole site | Name (R) | Mixing time | Time of magnetic attraction | Speed of mixing | Capacity (mL) | Temperature (. degree.C.) |
| 1 | 4 | Magnetic bead preparation | 00：30 | 00：30 | Fast-acting toy | 200 | 0 |
| 2 | 1 | Cracking | 04：00 | 00：30 | Fast-acting toy | 800 | 75 |
| 3 | 2 | Washing I | 00：30 | 00：30 | Fast-acting toy | 800 | 0 |
| 4 | 3 | Washing II | 01：00 | 00：30 | Fast-acting toy | 800 | 0 |
| 5 | 5 | Elution is carried out | 03：00 | 00：30 | Slow | 100 | 65 |
| 6 | 3 | Magnetic bead recovery | 00：30 | 00：00 | Fast-acting toy | 800 | 0 |
The invention has the beneficial effects that:
(1) after the kit provided by the invention is used for extracting DNA and RNA, the purity of the extracted DNA and RNA is higher.
(2) After the kit provided by the invention is used for extracting DNA and RNA, the extracted DNA and RNA have higher concentrations.
Drawings
FIG. 1 is a QPCR amplification plot for the detection of DNA viruses by nucleic acid extraction products of examples 1-5;
FIG. 2 is a QPCR amplification plot for the detection of RNA viruses from nucleic acid extraction products of examples 1-5;
FIG. 3 is a QPCR amplification plot for the detection of DNA viruses by nucleic acid extraction products in comparative examples 1-3;
FIG. 4 is a QPCR amplification plot for the detection of RNA viruses from the nucleic acid extraction products of comparative examples 1-3; .
Detailed Description
The present invention will be further illustrated with reference to the accompanying drawings and specific embodiments, which are to be understood as merely illustrative of the invention and not as limiting the scope of the invention.
In the reaction system for extracting nucleic acid by the magnetic bead method, the superparamagnetic silica nano magnetic bead (MagSilica Beads magnetic bead, mr. and biotechnology limited) modified by specific hydroxyl is adopted in the nucleic acid extraction technology, and can perform adsorption reaction with nucleic acid under the conditions of high-concentration guanidinium, sodium salt ions and low-pH solution (extraction reagent), so that nucleic acid molecules are specifically adsorbed to the surface of the magnetic bead. And transferring the magnetic beads adsorbed with the nucleic acid molecules into an eluent I and a washing solution II, and removing protein, salt ions and other small molecular compound impurities from the washing solution I and the washing solution II. After washing, the magnetic beads with the adsorbed nucleic acid molecules are transferred to an aqueous solution for elution.
The present invention will be described in detail with reference to the accompanying drawings, in which the present invention is described in detail.
In the following examples, unless otherwise specified, all the raw materials were generally commercially available.
Example 1
Nucleic acid extraction of pathogen biological samples 1
Sample preparation
Preparing a sample mother solution: respectively taking 500 mu L of high-concentration streptococcus pneumoniae (DNA) bacterial liquid and parainfluenza virus culture (RNA) to mix in a 15mL centrifuge tube, and diluting 10 times by using nuclease-free water for later use;
negative pharyngeal swab solution preparation: using a blank pharynx swab preservation solution, taking a negative pharynx swab sample of a healthy person in the preservation solution, and obtaining a negative pharynx swab solution;
preparation of a simulated pathogen biological sample: the sample stock was diluted 10-fold with the negative pharyngeal swab solution described above to prepare simulated pathogen biological samples.
Preparation of extraction reagents
Lysate (ph 7.0): Tris-HCl (40mmol/L), guanidine hydrochloride (1M), sodium dodecyl sulfate (0.5 Vol%), Tween 20(1.5 Vol%), disodium ethylene diamine tetraacetate (70mmol/L), isopropanol (2M), and water as a solvent;
magnetic bead solution: magnetic microspheres (20mg/mL), sodium chloride (1mol/L), Tris-HCl (pH7.5, 10mmol/L), and water as a solvent; the magnetic microspheres are superparamagnetic silica nano magnetic Beads modified by specific hydroxyl groups, (MagSilica Beads magnetic Beads, Changzhou Tiandi and Biotech, Inc.);
wash I (pH 7.0): Tris-HCl (15mmol/L), disodium ethylene diamine tetraacetate (10mmol/L), sodium chloride (150 mmol/L), guanidine hydrochloride (2M), ethanol solution (60 Vol%) and water as solvent;
washing solution II: ethanol solution (65 Vol%);
eluent: nuclease-free water;
20. mu.g/. mu.L proteinase K.
Nucleic acid extraction
And (3) performing nucleic acid extraction on the prepared pathogen biological sample by using a full-automatic nucleic acid extractor of the bleeker BNP 32.
Adding 200 mu L of simulated pathogen biological sample and 20 mu L of 20 mu g/mu L of proteinase K solution into each well of a 96-well plate, then putting the sample into an extractor, installing a magnetic bar protective sleeve, and operating the program, wherein the specific program is shown in the following table 1:
table 1 full-automatic nucleic acid extraction procedure:
| step (ii) of | Hole site | Name (R) | Mixing time | Time of magnetic attraction | Speed of mixing | Capacity (mL) | Temperature (. degree.C.) |
| 1 | 4 | Magnetic bead preparation | 00：30 | 00：30 | Fast-acting toy | 200 | 0 |
| 2 | 1 | Cracking | 04：00 | 00：30 | Fast-acting toy | 800 | 75 |
| 3 | 2 | Washing I | 00：30 | 00：30 | Fast-acting toy | 800 | 0 |
| 4 | 3 | Washing II | 01：00 | 00：30 | Fast-acting toy | 800 | 0 |
| 5 | 5 | Elution is carried out | 03：00 | 00：30 | Slow | 100 | 65 |
| 6 | 3 | Magnetic bead recovery | 00：30 | 00：00 | Fast-acting toy | 800 | 0 |
Example 2
Nucleic acid extraction of pathogen biological samples 2
This example differs from example 1 in that the concentration of guanidine hydrochloride in the lysate is 5M, otherwise the same as example 1.
Example 3
Nucleic acid extraction of pathogen biological samples 3
This example differs from example 1 in that the concentration of guanidine hydrochloride in the lysate was 3M, otherwise the same as example 1.
Example 4
Nucleic acid extraction of pathogen biological samples 4
This example differs from example 1 in that the lysate has a pH of 6.0 and a guanidine hydrochloride concentration of 3M, otherwise the same as example 1.
Example 5
Nucleic acid extraction of pathogen biological samples 5
This example differs from example 1 in that the lysis solution had an isopropanol concentration of 3M and a guanidine hydrochloride concentration of 3M, and the other examples are the same as example 1.
Comparative example 1
Nucleic acid extraction of pathogen biological samples 6
This example is different from example 1 in that 5% SDS was added to the washing solution I, and the procedure of example 1 was repeated.
Comparative example 2
Nucleic acid extraction of pathogen biological samples 7
This example differs from example 1 in that 4% Triton X-100 was added to washing solution I, and the procedure was otherwise the same as in example 1.
Comparative example 3
Nucleic acid extraction of pathogen biological samples 8
This example differs from example 1 in that disodium ethylenediaminetetraacetate was not added to washing solution I, and the procedure of example 1 was otherwise the same.
The nucleic acids obtained by extraction in examples 1-5 and comparative examples 1-3 were used for qPCR detection, and the Ct values were averaged after 3 replicates were performed on each sample. The purity of the nucleic acid was determined to be 260/280nm, and the Ct value and the purity of the nucleic acid are shown in Table 2.
TABLE 2 nucleic acid purity and Ct value measured for reagents extracted in different examples
The amplification curves are shown in FIG. 1, FIG. 2, FIG. 3 and FIG. 4.
As can be seen from the data in FIGS. 1-4 and Table 2, the extraction kit of the present application has good purity, and the purity of example 4 is the best.
From the results in examples 1 and 4, it is seen that a low pH contributes to the enhanced cleavage effect and the fluorescence signal value is higher.
As seen from the data results of example 1, example 2 and example 3, the increased concentration of guanidinium salts aids in the lysis of the sample
As can be seen from the data results of comparative examples 1-3, the addition of SDS and Triton X-100 has an effect on the purity, and the addition of EDTA helps to improve the purity of the extracted nucleic acids.
It should be noted that the above-mentioned contents only illustrate the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and it is obvious to those skilled in the art that several modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations fall within the protection scope of the claims of the present invention.
Claims (8)
1. An extraction reagent for simultaneously extracting pathogen DNA and RNA is characterized by comprising lysis solution, magnetic bead solution, washing solution I, washing solution II, eluent and proteinase K; the lysis solution comprises the following components: 10-100 mmol/L Tris-HCl with pH value of 6.0-7.0, 1-6M guanidine salt, 0.1-5 Vol% sodium dodecyl sulfate, 0.5-3 Vol% Tween 20, 1-10 mmol/L disodium ethylene diamine tetraacetate, 1-5M isopropanol and water as solvent.
2. The extraction reagent for simultaneously extracting DNA and RNA of a pathogen according to claim 1, wherein the guanidine salt is one or more of guanidine hydrochloride, guanidine nitrate, guanidine carbonate, guanidine acetate, guanidine thiocyanate and guanidine isothiocyanate.
3. The extraction reagent for simultaneously extracting pathogen DNA and RNA as claimed in claim 1, wherein the magnetic bead solution comprises 15-25 mg/mL magnetic microspheres, 0.3-0.8 mol/L sodium chloride, 5-30 mmol/L Tris-HCl with pH of 7.0-7.5, and the solvent is water.
4. The extraction reagent for simultaneously extracting pathogen DNA and RNA as claimed in claim 1, wherein the washing solution I comprises 5-30 mmol/L Tris-HCl with pH 7.0-7.5, 1-10 mmol/L disodium EDTA, 100-200 mmol/L NaCl, 0.5-5M guanidinium salt, and 50-70 Vol% ethanol solution, and the solvent is water.
5. The extraction reagent for simultaneously extracting the DNA and RNA of a pathogen according to claim 1, wherein the washing solution II comprises a 50-75 Vol% ethanol solution; the eluate comprises water.
6. The reagent of claim 1, wherein the proteinase K is 20 μ g/μ L aqueous proteinase K solution.
7. A kit for simultaneously extracting DNA and RNA from a pathogen, comprising the extraction reagent of any one of claims 1 to 6.
8. An extraction method for simultaneously extracting pathogen DNA and RNA is characterized by comprising the following steps:
(1) adding 200 mu L of simulated pathogen biological sample and 20ul of 20 mu g/mu L proteinase K solution into each hole of a 96-hole plate;
(2) and then placing the sample into an extractor, installing a magnetic rod protective sleeve, and operating the program, wherein the specific program is shown in the following table.
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| Application Number | Priority Date | Filing Date | Title |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116891849A (en) * | 2023-09-11 | 2023-10-17 | 成都斯马特科技有限公司 | Kit for rapidly extracting nucleic acid from nasopharyngeal swab and extraction method |
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| US20090253903A1 (en) * | 2006-07-11 | 2009-10-08 | Aj Innuscreen Gmbh | Method for parallel isolation of viral nucleic acids |
| US20200032241A1 (en) * | 2016-09-30 | 2020-01-30 | Eiken Kagaku Kabushiki Kaisha | Nucleic acid extraction method and kit using same |
| CN111484991A (en) * | 2019-01-29 | 2020-08-04 | 中山大学达安基因股份有限公司 | Kit for high-throughput full-automatic extraction of viral nucleic acid and extraction method |
| CN114457069A (en) * | 2022-03-07 | 2022-05-10 | 江苏迅睿生物技术有限公司 | Magnetotherapeutic bead method pathogen nucleic acid extraction kit and use method thereof |
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- 2022-06-27 CN CN202210737451.8A patent/CN114958830A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090253903A1 (en) * | 2006-07-11 | 2009-10-08 | Aj Innuscreen Gmbh | Method for parallel isolation of viral nucleic acids |
| US20200032241A1 (en) * | 2016-09-30 | 2020-01-30 | Eiken Kagaku Kabushiki Kaisha | Nucleic acid extraction method and kit using same |
| CN111484991A (en) * | 2019-01-29 | 2020-08-04 | 中山大学达安基因股份有限公司 | Kit for high-throughput full-automatic extraction of viral nucleic acid and extraction method |
| CN114457069A (en) * | 2022-03-07 | 2022-05-10 | 江苏迅睿生物技术有限公司 | Magnetotherapeutic bead method pathogen nucleic acid extraction kit and use method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116891849A (en) * | 2023-09-11 | 2023-10-17 | 成都斯马特科技有限公司 | Kit for rapidly extracting nucleic acid from nasopharyngeal swab and extraction method |
| CN116891849B (en) * | 2023-09-11 | 2023-11-17 | 成都斯马特科技有限公司 | Kit for rapidly extracting nucleic acid from nasopharyngeal swab and extraction method |
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