CN113215150A - Preservation lysate and sample nucleic acid rapid extraction method based on preservation lysate - Google Patents
Preservation lysate and sample nucleic acid rapid extraction method based on preservation lysate Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
- C12N1/066—Lysis of microorganisms by physical methods
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Abstract
The invention relates to a lysate and a sample nucleic acid rapid extraction method based on the lysate, which comprises the following components in volume content: 5 to 30 percent of absolute ethyl alcohol; 70-95% of mixed aqueous solution; and taking the volume of the lysis solution as a reference, the mixed aqueous solution comprises the following solutes: 0.5-1 mol/L of ammonium chloride; 10-50 mmol/L of trihydroxymethyl aminomethane hydrochloride; 0.4-2mol/L of guanidine thiocyanate; 0.4-1 mol/L of sodium chloride; 10-50 mmol/L of ethylenediamine tetraacetic acid; 0.1-2mmol/L of tris- (2-formylethyl) phosphine hydrochloride. It comprises the following steps: (a) placing the sample in a closed container filled with the nano beads and the lysate; (b) lysing the sample; (c) incubating at room temperature; washing by centrifugation. The reagent is prepared, and chemical and physical methods are combined to realize sufficient cell lysis and nucleic acid protection.
Description
Technical Field
The invention belongs to the technical field of nucleic acid extraction, and particularly relates to a preservation lysate and a sample nucleic acid rapid extraction method based on the preservation lysate.
Background
For molecular detection based on nucleic acid amplification and sequencing technology, how to store and obtain high-quality nucleic acid in different scenes is a necessary factor for ensuring the success of downstream nucleic acid analysis. Nucleic acids in biological samples are rapidly degraded at room temperature, and particularly, the degradation of nucleic acids is severe under the condition that the biological samples are not stored at low temperature in the field. Under the condition of lacking of effective sample amount, a method for effectively obtaining high-quality nucleic acid is particularly important, and nucleic acid can be degraded by nuclease in the environment in the nucleic acid processing process; on the other hand, in the processing of infectious biological samples, these samples which are hazardous, such as SARS-CoV-2 virus in the current epidemic situation, run the risk of being released into the environment (even leading to infection). Thus, there is a need for safe and low risk nucleic acid processing methods.
The requirements of molecular detection on nucleic acid treatment are continuously improved, and the biological sample needs to be safely treated to avoid environmental pollution; i.e., it is desirable to avoid contamination with nucleases and thereby degrade the sample. The sample needs to be rapidly processed to avoid degradation caused by long-term storage. The recovery rate of nucleic acid processing samples is also an important point to be further improved; efficient nucleic acid recovery in biological samples can only be guaranteed with sufficiently high recovery rates to warrant Polymerase Chain Reaction (PCR) and sequencing based requirements.
The existing nucleic acid extraction technology is mainly based on phenol chloroform or guanidine salt organic solvent extraction method. The nucleic acid dissolved in the solution can be further purified and extracted by silica gel membrane separation column or magnetic bead adsorption. Extraction methods derived from extracting nucleic acid based on the above two-step method are numerous, such as a nucleic acid extraction kit based on a separation column, a nucleic acid extraction kit based on magnetic beads, and the like. Cells that are easier to handle (such as gram-negative bacteria without a cell wall) can be lysed directly using phenol chloroform or high concentrations of high salts to release nucleic acid material. For intractable cells (such as gram positive bacteria, eukaryotic cells, and even tissues) a pretreatment is required to lyse the cells to achieve complete release of the nucleic acid.
However, the existing nucleic acid extraction technology has the following defects: (1) cannot be used in combination with clinical requirements: clinical samples typically require the use of a transport fluid or a clinical sample stock; transport or storage of fluids may inhibit reactions required for downstream molecular detection; the concentration of the sample can be greatly diluted by the transfusion liquid or the storage liquid, so that the detection limit is seriously influenced, and false negative is caused; (2) the existing kit generally cannot completely extract the sampled sample, and only a part of the sampled sample is used for nucleic acid extraction; (3) most of used lysate reagents are limited to extraction functions, and have limited protection effect on nucleic acid; (4) most kits are only suitable for specific cells: such as those applicable only to bacteria, only to plant cells, only to animal cells, etc.
Disclosure of Invention
In order to solve the above-mentioned problems, it is an object of the present invention to provide a lysate that can be preserved.
In order to achieve the technical purpose, the invention provides a lysis solution, which comprises the following components in volume:
5 to 30 percent of absolute ethyl alcohol;
70-95% of a mixed aqueous solution;
and taking the volume of the lysis solution as a reference, the mixed aqueous solution comprises the following solutes:
optimally, the mixed aqueous solution comprises the following solutes in concentration based on the volume of the preserved lysate:
the invention also provides a sample nucleic acid rapid extraction method based on the lysis solution preservation, which comprises the following steps:
(a) placing the sample in a closed container filled with the nano beads and the lysate;
(b) the sample is cracked and then transferred to a nucleic acid separation column to be subjected to column chromatography, and waste liquid is poured off;
(c) putting the nucleic acid separation column in the step (b) into a micro tube, adding TE buffer solution, and incubating at room temperature; washing by centrifugation.
Preferably, in step (a), a sample is collected with a cotton swab, and then the cotton swab is inserted into a preservation tube containing the nanobead and the preservation lysate, and a cover is screwed.
Further, in the step (b), placing the preservation tube on a vortex device, and oscillating for 1.5-3 min to perform cracking; or cracking with a homogenizer at a speed of 1.5-3 m/s for 15-30 seconds.
Specifically, in the step (b), the preservation tube is placed in a centrifuge and centrifuged for 0.5-2 min at 5000-8000 rpm.
Specifically, in the step (b), the preservation tube is taken out of the centrifuge, the mixture therein is transferred to a nucleic acid separation column having a collection tube, and the nucleic acid separation column is covered with a cap at 8X 103～1.5×104g, centrifuging for 20-50 seconds at the speed of g, and pouring off the waste liquid in the collecting pipe.
Specifically, in the step (b), the washing buffer WB was added to the nucleic acid separation column at 1.0X 104～1.5×104g, centrifuging at the speed of 20-50 seconds; or optionally repeating steps (a) to (b), and pouring out the liquid in the collecting tube to obtain a product with a volume of 1.0 × 104～1.5×104g for 1.5-5 minutes.
Optimally, in the step (c), the nucleic acid separation column is placed into a micro-tube, TE buffer solution is added to the center of the white membrane of the nucleic acid separation column by a pipette, the cover is covered, and the nucleic acid separation column is incubated for 3-8 minutes at room temperature.
Further, in the step (c), the nucleic acid separation column is packed at 1.0X 104～1.5×104g for 0.5-2 minutes at a speed of g to elute the nucleic acid separation column.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages: the lysis solution is preserved by compounding raw materials with specific content, so that the lysis solution can be used for preserving a biological sample, and can also be used for lysing cells and settling nucleic acid; can be stored for 48 hours at normal temperature and can completely extract the collected sample, and is suitable for bacterial cells, plant cells, animal cells and even biological tissues. Through improvement, the reagent is also suitable for being matched with magnetic beads for extracting bacteria and viruses by a magnetic bead method.
According to the method for rapidly extracting the sample nucleic acid, the prepared reagent is combined with chemical and physical methods to realize the sufficient cell lysis and nucleic acid protection; the control of the types of nucleic acid extraction products of different microorganisms is realized by combining different types of nano beads; the sampling cotton swab, the preservation tube, the preservation lysate and the nano-beads are integrated; and the whole operation flow from sampling to nucleic acid extraction (especially the mixture of cell lysate and nano-beads) is transferred to a nucleic acid separation column together and then passes through the column, so that the operation is safe.
Drawings
FIG. 1 is a schematic flow chart of a method for rapid extraction of nucleic acid from a sample according to the present invention;
FIG. 2 is a comparison graph of the effect of extracting viral RNA between the lysate stored in example 1 and other conventional kits (comparison of electrophoresis results);
FIG. 3 is a graph showing the comparison of the E.coli extraction effect between the lysate preserved in example 1 and other conventional kits (comparison of electrophoresis results).
Detailed Description
Example 1
This example provides a preservation lysate comprising the following components by volume:
5% of absolute ethyl alcohol;
95% of mixed aqueous solution;
based on the volume of the lysate to be preserved, the mixed aqueous solution contains the following solutes in concentration:
the method for rapidly extracting the sample nucleic acid by adopting the preserved lysate comprises the following steps:
(a) placing the sample in a closed container filled with the nano beads and the lysate; specifically, a sample is collected by a cotton swab, then the cotton swab is inserted into a storage tube filled with the nano-beads and the lysis solution, and a cover is screwed (the sampling cotton swab, the storage tube, the lysis solution and the nano-beads can be combined to realize module integration);
(b) cracking the sample, transferring the sample to a nucleic acid separation column for column chromatography, and dumping waste liquid; specifically, the preservation tube is placed on a vortex device, and is vibrated for 1.5-3 min to carry out full cracking (or a homogenizer is used for carrying out full cracking, the speed is 1.5-3 m/s, and the time is 15-30 seconds); after finishing, placing the preservation tube in a centrifuge, and centrifuging for 0.5-2 min at 5000-8000 rpm; removing the preservation tube from the centrifuge, transferring the mixture therein to a nucleic acid separation column having a collection tube, covering the nucleic acid separation column with a cover at 8X 103～1.5×104g, centrifuging for 20-50 seconds at the speed of g, and pouring off the waste liquid in the collecting pipe; carefully open the lid of the nucleic acid separation column, 600. mu.l of the washing buffer WB was added to the nucleic acid separation column, followed by washing at 1.0X 104～1.5×104g, centrifuging at the speed of 20-50 seconds;
repeating the steps (a) to (b) (i.e., the above steps), pouring out the liquid in the collection tube, and returning the nucleic acid separation column to the collection tube at a rate of 1.0X 104～1.5×104g for 1.5-5 minutes.
(c) Putting the nucleic acid separation column in the step (b) into a micro tube, and adding TE buffer solutionIncubating at room temperature; washing by centrifugation; specifically, a nucleic acid separation column was put into a microtube (a new 1.5ml Eppendorf tube), 50. mu.l of TE buffer was added to the center of the white membrane of the nucleic acid separation column with a pipette, the lid was closed, and incubation was performed at room temperature for 3 to 8 minutes; separating nucleic acid column at 1.0 × 104～1.5×104g, centrifuging for 0.5-2 minutes at the speed of g to elute the DNA in the nucleic acid separation column; the purified sample nucleic acid genome DNA is obtained in an Eppendorf tube
Example 2
This example provides a lysate which is essentially the same as that of example 1, except that:
based on the volume of the lysate to be preserved, the mixed aqueous solution contains the following solutes in concentration:
example 3
This example provides a lysate which is essentially the same as that of example 1, except that:
based on the volume of the lysate to be preserved, the mixed aqueous solution contains the following solutes in concentration:
example 4
This example provides a lysate which is essentially the same as that of example 1, except that:
based on the volume of the lysate to be preserved, the mixed aqueous solution contains the following solutes in concentration:
example 5
This example provides a lysate which is essentially the same as that of example 1, except that: the composite material comprises the following components in percentage by volume:
30% of absolute ethyl alcohol;
the aqueous solution was mixed to 70%.
Example 6
This example provides a lysate which is essentially the same as that of example 1, except that: the composite material comprises the following components in percentage by volume:
10% of absolute ethyl alcohol;
the aqueous solution was mixed 90%.
Comparative example 1
This example provides a lysate which is essentially the same as that of example 1, except that: no ammonium chloride was included.
Comparative example 2
This example provides a lysate which is essentially the same as that of example 1, except that: without tris hydrochloride.
Comparative example 3
This example provides a lysate which is essentially the same as that of example 1, except that: guanidine thiocyanate is not present.
Comparative example 4
This example provides a lysate which is essentially the same as that of example 1, except that: does not contain sodium chloride.
Comparative example 5
This example provides a lysate which is essentially the same as that of example 1, except that: does not contain ethylenediamine tetraacetic acid.
Comparative example 6
This example provides a lysate which is essentially the same as that of example 1, except that: does not contain tris- (2-formylethyl) phosphine hydrochloride.
A throat swab was sampled from the preserved lysate of example 1, and simultaneously, MS2 phage carrying influenza a virus fragment was added; viral RNA was extracted according to the extraction procedure described in example 1 (magnetic bead method). Compared with virus RNA extraction kits (both medical instruments) of other two manufacturers. It can be seen that the lysate and the method of example 1 have higher extraction efficiency (see table 1) and better extraction effect (fig. 2) after nucleic acid extraction. It can be seen that the preservation of the lysate in example 1 maintained better RNA integrity, indicating that RNA degradation was effectively avoided. The extraction effects of the preserved lysates in examples 2-6 reach 60%, 72%, 45%, 10%, and 20%, respectively; however, none of the lysates of comparative examples 1-6 could extract viral RNA.
TABLE 1 comparison table of concentration of extracted viral nucleic acid from throat swab
| Extraction concentration (ng/. mu.L, Nanodrop detection result) | |
| Reagents and extraction methods in example 1 (A) | 54.2 |
| Kit extraction method of manufacturer 1 (B) | 42 |
| Kit extraction method of manufacturer 2 (C) | 30 |
The effect of the reagent on extraction of bacterial nucleic acid was also verified by taking the extraction of gram-negative bacteria Escherichia coli from the lysate in example 1 as an example. The method specifically comprises the following steps: taking 300 mu L of escherichia coli cultured overnight, centrifuging, taking supernatant, and adding 500 mu L of lysate; extracting according to the steps of an extracted column passing method and a magnetic bead method respectively, and extracting results shown in a table 2 and a figure 3; the magnetic bead method and the lysate preservation effect are better.
TABLE 2 Table of the effect of lysis buffer preservation on the extraction of gram-negative Escherichia coli by different methods in example 1
The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (10)
1. A lysate preserving solution, comprising the following components in volume:
5 to 30 percent of absolute ethyl alcohol;
70-95% of a mixed aqueous solution;
and taking the volume of the lysis solution as a reference, the mixed aqueous solution comprises the following solutes:
2. the lysate preserving solution of claim 1, wherein the mixed aqueous solution comprises the following solutes in concentration, based on the volume of the lysate preserving solution:
3. a method for rapidly extracting nucleic acid from a sample, which is characterized in that the lysis solution for preservation as described in claim 1 or 2 is used, and comprises the following steps:
(a) placing the sample in a closed container filled with the nano beads and the lysate;
(b) the sample is cracked and then transferred to a nucleic acid separation column to be subjected to column chromatography, and waste liquid is poured off;
(c) putting the nucleic acid separation column in the step (b) into a micro tube, adding TE buffer solution, and incubating at room temperature; washing by centrifugation.
4. The method for rapidly extracting nucleic acid from a sample according to claim 3, wherein: in step (a), a sample is collected with a cotton swab, which is then inserted into a storage tube containing the nanobead and the lysate, and a lid is screwed down.
5. The method for rapidly extracting nucleic acid from a sample according to claim 4, wherein: in the step (b), placing the preservation tube on a vortex device, and oscillating for 1.5-3 min to perform cracking; or cracking with a homogenizer at a speed of 1.5-3 m/s for 15-30 seconds.
6. The method for rapidly extracting nucleic acid from a sample according to claim 5, wherein: in the step (b), the preservation tube is placed in a centrifuge and centrifuged for 0.5-2 min at 5000-8000 rpm.
7. The method for rapidly extracting nucleic acid from a sample according to claim 6, wherein: in the step (b), the storage tube is taken out from the centrifuge, the mixture therein is transferred to a nucleic acid separation column having a collection tube, and the nucleic acid separation column is covered with a cap at 8X 103～1.5×104g, centrifuging for 20-50 seconds at the speed of g, and pouring off the waste liquid in the collecting pipe.
8. The method for rapidly extracting nucleic acid from a sample according to claim 6, wherein: in the step (b), a washing buffer WB was added to the nucleic acid separation column at 1.0X 104～1.5×104g, centrifuging at the speed of 20-50 seconds; or optionally repeating steps (a) to (b), and pouring out the liquid in the collecting tube to obtain a product with a volume of 1.0 × 104～1.5×104g for 1.5-5 minutes.
9. The method for rapidly extracting nucleic acid from a sample according to claim 3, wherein: in the step (c), the nucleic acid separation column is placed in a micro tube, TE buffer solution is added to the center of a white membrane of the nucleic acid separation column through a liquid transfer machine, a cover is covered, and incubation is carried out for 3-8 minutes at room temperature.
10. The method for rapidly extracting nucleic acid from a sample according to claim 9, wherein: in the step (c), the nucleic acid separation column is packed at 1.0X 104～1.5×104g for 0.5-2 minutes at a speed of g to elute the nucleic acid separation column.
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| US20090048439A1 (en) * | 2007-08-06 | 2009-02-19 | Weisburg William G | Isolation of nucleic acids molecules using modified solid supports |
| CN108935444A (en) * | 2018-07-27 | 2018-12-07 | 广州奇辉生物科技有限公司 | A kind of RNA Sample preservation liquid and its application method |
| CN109913447A (en) * | 2019-04-04 | 2019-06-21 | 深圳市南科征途有限公司 | Dissociative DNA Extraction and enrichment kit and dissociative DNA extracting method |
| CN111808844A (en) * | 2020-07-29 | 2020-10-23 | 广州捷倍斯生物科技有限公司 | Kit for simultaneously extracting DNA and RNA and use method thereof |
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- 2021-06-24 CN CN202110706698.9A patent/CN113215150A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090048439A1 (en) * | 2007-08-06 | 2009-02-19 | Weisburg William G | Isolation of nucleic acids molecules using modified solid supports |
| CN108935444A (en) * | 2018-07-27 | 2018-12-07 | 广州奇辉生物科技有限公司 | A kind of RNA Sample preservation liquid and its application method |
| CN109913447A (en) * | 2019-04-04 | 2019-06-21 | 深圳市南科征途有限公司 | Dissociative DNA Extraction and enrichment kit and dissociative DNA extracting method |
| CN111808844A (en) * | 2020-07-29 | 2020-10-23 | 广州捷倍斯生物科技有限公司 | Kit for simultaneously extracting DNA and RNA and use method thereof |
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