CN113215149A - Mycobacterium tuberculosis RNA extraction method - Google Patents
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Abstract
The invention discloses a method for extracting mycobacterium tuberculosis RNA, belonging to the technical field of biology. The method firstly utilizes transZol UP to perform cleavage, and then utilizes TIANGEN DP419 kit to perform RNA extraction. The invention has the advantages of high purity, high concentration and high integrity, and is very suitable for large-scale extraction.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for extracting RNA of mycobacterium tuberculosis.
Background
Tuberculosis is a chronic infectious disease caused by infection with mycobacterium tuberculosis. Tubercle bacillus may invade various organs of the whole body of a human body, but mainly invades the lung, and is called pulmonary tuberculosis. Tuberculosis is a chronic and slow-growing infectious disease that is likely to occur in young people. The incubation period is 4-8 weeks. 80% of them occur in the lung, and other sites (cervical lymph, meninges, peritoneum, intestine, skin, bone) may also be infected secondarily.
The world health organization in 2018 released 1000 million new tuberculosis patients in 2017, and estimated 157 million deaths. In the new population, tuberculosis combined with HIV double infection accounts for 9% of 92 ten thousand; resistant to multiple drug tuberculosis in 56 thousands of people. Approximately 1/3 of the world's population suffers from latent tuberculosis, with a tubercle bacillus infected person having a 10% risk of falling ill for tuberculosis during life. In addition, tuberculosis is the number one killer of aids carriers.
Therefore, the detection of Mycobacterium tuberculosis is crucial, however, the extraction of Mycobacterium tuberculosis RNA is very difficult.
Disclosure of Invention
In order to solve at least one of the above technical problems, the technical solution adopted by the present invention is as follows:
the invention provides a method for extracting mycobacterium tuberculosis RNA, which comprises the following steps:
s1, obtaining mycobacterium tuberculosis;
s2, adding the mixture into a tube containing the lysate, uniformly mixing the mixture by vortex, and standing the mixture for 10 to 20min at room temperature;
s3, adding chloroform, violently shaking for 10-30sec, standing at room temperature for 1-5min, and centrifuging for 5-15 mim;
s4, sucking the water phase, slowly adding absolute ethyl alcohol with the volume 0.5 times that of the water phase, uniformly mixing, transferring the obtained solution and the precipitate into an adsorption column CR3 in a TIANGEN DP419 kit, and centrifuging for 20-40 percent;
s5, adding deproteinized liquid RW1 into an adsorption column CR3, centrifuging for 30-60sec, discarding waste liquid, and placing the adsorption column back into the collection tube;
s6, adding DNase I working solution into the center of an adsorption column CR3, and standing at room temperature for 15 min;
s7, adding deproteinized liquid RW1 into an adsorption column CR3, centrifuging for 30-60sec, discarding waste liquid, and placing the adsorption column back into the collection tube;
s8, adding a rinsing solution RW into an adsorption column CR3, standing at room temperature for 1-3min, centrifuging for 30-60sec, pouring off waste liquid, and putting the adsorption column CR3 back into the collection tube;
s9, repeating the step S8;
s10, centrifuging for 1-3min, pouring off waste liquid, placing the adsorption column CR3 at room temperature for 1-10min to thoroughly air-dry the residual rinsing liquid in the adsorption material;
s11 transferring the adsorption column CR3 into a new RNase-Free centrifuge tube, dripping 30-100RNase-Free ddH2O into the middle part of the adsorption membrane in suspension, standing at room temperature for 1-3min, and centrifuging for 1-3min to obtain RNA solution.
In some embodiments of the invention, in step S6, DNase I working solution is prepared: taking 10 mu L of DNase I stock solution, adding 70 mu L of Buffer RDD, and gently mixing uniformly, wherein the preparation of the DNase I stock solution comprises the following steps: the DNase I dry powder is dissolved in 550 mu L of RNase-Free ddH2O, and the mixture is gently mixed, subpackaged and stored at the temperature of minus 20 ℃.
In some embodiments of the invention, the rotational speed of the centrifugation is 12,000 rpm.
In some embodiments of the present invention, step S5 is preceded by: adding deproteinized solution RD into adsorption column CR3, centrifuging for 20-40sec, discarding waste liquid, and placing CR3 into collection tube.
In some embodiments of the invention, deproteinized liquid RW1 in TIANGEN DP430 was used in place of deproteinized liquid RD in TIANGEN DP 419.
In some embodiments of the invention, in step S2, the lysate is lysate RL. In some preferred embodiments of the invention, the lysis solution RL is added to β -mercaptoethanol to a final concentration of 1% prior to use.
In other embodiments of the present invention, in step S2, the lysis solution is TransZol UP.
The invention has the advantages of
Compared with the prior art, the invention has the following beneficial effects:
the invention has the advantages of high purity, high concentration and high integrity, and is very suitable for large-scale extraction.
Detailed Description
Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages herein are by weight and the testing and characterization methods used are synchronized with the filing date of the present application. Where applicable, the contents of any patent, patent application, or publication referred to in this application are incorporated herein by reference in their entirety and their equivalent family patents are also incorporated by reference, especially as they disclose definitions relating to synthetic techniques, products and process designs, polymers, comonomers, initiators or catalysts, and the like, in the art. To the extent that a definition of a particular term disclosed in the prior art is inconsistent with any definitions provided herein, the definition of the term provided herein controls.
The numerical ranges in this application are approximations, and thus may include values outside of the ranges unless otherwise specified. A numerical range includes all numbers from the lower value to the upper value, in increments of 1 unit, provided that there is a separation of at least 2 units between any lower value and any higher value. For example, if a compositional, physical, or other property (e.g., molecular weight, melt index, etc.) is recited as 100 to 1000, it is intended that all individual values, e.g., 100, 101, 102, etc., and all subranges, e.g., 100 to 166, 155 to 170, 198 to 200, etc., are explicitly recited. For ranges containing a numerical value less than 1 or containing a fraction greater than 1 (e.g., 1.1, 1.5, etc.), then 1 unit is considered appropriate to be 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digit numbers less than 10 (e.g., 1 to 5), 1 unit is typically considered 0.1. These are merely specific examples of what is intended to be expressed and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.
When used with respect to chemical compounds, the singular includes all isomeric forms and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively) unless expressly specified otherwise. In addition, unless explicitly stated otherwise, the use of the terms "a", "an" or "the" are intended to include the plural forms thereof.
The terms "comprising," "including," "having," and derivatives thereof do not exclude the presence of any other component, step or procedure, and are not intended to exclude the presence of other elements, steps or procedures not expressly disclosed herein. To the extent that any doubt is eliminated, all compositions herein containing, including, or having the term "comprise" may contain any additional additive, adjuvant, or compound, unless expressly stated otherwise. Rather, the term "consisting essentially of … …" excludes any other components, steps or processes from the scope of any of the terms hereinafter recited, insofar as such terms are necessary for performance. The term "consisting of … …" does not include any components, steps or processes not specifically described or listed. Unless explicitly stated otherwise, the term "or" refers to the listed individual members or any combination thereof.
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
In the following examples, some of the reagents used were formulated as follows:
preparing a lysis solution RL: before the operation, beta-mercaptoethanol is added into the lysis solution RL to the final concentration of 1 percent, for example, 10 mu L of beta-mercaptoethanol is added into 1mL of the lysis solution RL. The lysate is preferably prepared just before use, the prepared lysate RL can be placed for 1 month at 4 ℃, precipitates can be formed when the lysate RL is stored, and if the precipitates appear, the lysate RL is heated and dissolved for use.
Preparation of DNase I stock solution: DNase I dry powder (1500Kunitz units) was dissolved in 550. mu.L RNase-Free ddH2And O, mixing the components gently, subpackaging and storing at-20 ℃ (the storage period can be 9 months). The thawed DNase I stock was stored at 4 ℃ (for 6 weeks) and was not allowed to be frozen again.
Preparing DNase I working solution: mu.L of DNase I stock solution was taken for each reaction, 70. mu.L of Buffer RDD was added, and mixed gently.
Preparing a lysozyme working solution: final concentration of lysozyme in TE buffer (G-bacteria 400. mu.g/mL, G + bacteria 3 mg/mL).
The molecular biological experiments, which are not specifically described in the following examples, were performed according to the specific methods listed in the manual of molecular cloning, laboratory manual (fourth edition) (j. sambrook, m.r. green, 2017), or according to the kit and product instructions. Other experimental methods, unless otherwise specified, are conventional. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1 first round optimization of extraction conditions for Mycobacterium tuberculosis
1. Sample pretreatment
In this example, 9 samples were used, and the pretreatment was as follows:
RNA extraction
The lysed samples were subjected to RNA extraction using DP430(TIANGEN), referred to the manufacturer's instructions.
The lysed sample was centrifuged at 12000rpm (. about.13,400 Xg) for 2min and the supernatant was transferred to another centrifuge tube. Add 250. mu.L of absolute ethanol and mix (where precipitation may occur). The resulting solution was transferred to adsorption column CR3 (adsorption column placed in collection tube), centrifuged at 12,000rpm (. about.13,400 Xg) for 30-60sec, the waste liquid was decanted, and adsorption column CR3 was returned to the collection tube.
② 350 mul deproteinized liquid RW1 is added into the adsorption column CR3, centrifugation is carried out for 30-60sec at 12,000rpm (13,400 Xg), waste liquid is discarded, and the adsorption column is put back into the collection tube.
③ 80 mu L of DNaseI working solution is added into the center of the adsorption column CR3 and is placed for 15min at room temperature.
And fourthly, 350 mu L of deproteinized liquid RW1 is added into the adsorption column CR3, the mixture is centrifuged for 30-60sec at 12,000rpm (13,400 Xg), waste liquid is discarded, and the adsorption column is placed back into the collection tube.
Fifthly, adding 500 mu L of rinsing liquid RW (whether ethanol is added before use) into the adsorption column CR3, standing for 2min at room temperature, centrifuging at 12,000rpm (13,400 Xg) for 30-60sec, pouring off waste liquid, and returning the adsorption column CR3 to the collection tube.
Sixthly, repeating the step 5.
7.12,000rpm (-13, 400 Xg) for 2min, and the waste liquid is poured out. The adsorption column CR3 was left at room temperature for several minutes to thoroughly dry the residual rinse solution from the adsorption material.
Note that: this step is intended to remove the residual rinse liquid from the adsorption column CR 3.
Transferring the adsorption column CR3 into a new RNase-Free centrifuge tube, and dripping 50 μ L of RNase-Free H into the middle part of the adsorption membrane2O, left at room temperature for 2min, and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min to obtain an RNA solution.
The concentration of the RNA of the tubercle bacillus extracted by DP430 in the embodiment is only 3-5 ng/microliter, and cannot meet the clinical requirement.
Example 2 extraction conditions for Mycobacterium tuberculosis in a second round of optimization
1. Sample pretreatment
In this example, 6 samples were used, and the pretreatment was as follows:
RNA extraction
RL cleavage: the lysed sample was centrifuged at 12000rpm (. about.13,400 Xg) for 2min and the supernatant was transferred to another centrifuge tube. Add 250. mu.L of absolute ethanol and mix (where precipitation may occur).
TransZolUP cleavage: add 200. mu.L chloroform, cover the tube, shake vigorously for 15sec, and let stand at room temperature for 3 min. Upon centrifugation at 12,000rpm (13,400 Xg) for 10min at 4 ℃, the sample will separate into three layers: yellow organic phase, intermediate layer and colorless aqueous phase, RNA is mainly in the aqueous phase, and the volume of the aqueous phase is about 50% of the used lysis solution RZ reagent. The aqueous phase was transferred to another centrifuge tube and 0.5 volume of absolute ethanol of the aqueous phase was added slowly and mixed (where precipitation may occur).
② RNA extraction of the lysed sample was performed using DP430(TIANGEN), and the subsequent procedures were the same as in example 1.
This example used the TransZol UP + DP430 column chromatography method, and the extracted RNA concentration was 80.9 ng/. mu.l, 260/280 ═ 2.65, 260/230 ═ 2.33.
Example 3 extraction conditions for Mycobacterium tuberculosis third round of optimization
1. Sample pretreatment
In this example, 6 samples were used, and the pretreatment was as follows:
| sample(s) | Treatment method |
| 1 | Adding tubercle bacillus into EP tube containing 1ml RZ, vortex mixing, standing at room temperature for 5min |
| 2 | Adding tubercle bacillus into EP tube containing 1ml RZ, vortex mixing, standing at room temperature for 10min |
| 3 | Adding tubercle bacillus into EP tube containing 1ml RZ, vortex mixing, standing at room temperature for 15min |
| 4 | Adding tubercle bacillus into EP tube containing 1ml TransZolUP, vortex mixing, standing at room temperature for 5min |
| 5 | Adding tubercle bacillus into EP tube containing 1ml TransZolUP, vortex mixing, standing at room temperature for 10min |
| 6 | Adding tubercle bacillus into EP tube containing 1ml TransZol UP, vortex mixing, standing at room temperature for 15min |
RNA extraction
Cracking RZ: the sample after completion of the lysis was added to 200. mu.L of chloroform, the cap was closed, shaken vigorously for 15sec, and allowed to stand at room temperature for 3 min.
TransZol UP cleavage: the sample after completion of the lysis was added to 200. mu.L of chloroform, the cap was closed, shaken vigorously for 15sec, and allowed to stand at room temperature for 3 min. Upon centrifugation at 12,000rpm (13,400 Xg) for 10min at 4 ℃, the sample will separate into three layers: yellow organic phase, intermediate layer and colorless aqueous phase, RNA is mainly in the aqueous phase, and the volume of the aqueous phase is about 50% of the used lysis solution RZ reagent. The aqueous phase was transferred to another centrifuge tube.
② slowly adding 0.5 times volume of absolute ethyl alcohol into the water phase, and uniformly mixing (at this time, precipitation may occur). The resulting solution was transferred together with the precipitate to an adsorption column CR3 and centrifuged at 12,000rpm (. about.13,400 Xg) at 4 ℃ for 30sec, and if the whole solution and mixture could not be added to the adsorption column CR3 at one time, it was transferred to an adsorption column CR3 in two portions and centrifuged at 12,000rpm (. about.13,400 Xg) at 4 ℃ for 30sec, and the waste liquid in the collection tube was discarded.
③ to the adsorption column CR3, 500. mu.L of deproteinized liquid RD (before use, whether ethanol is added or not is checked) is added, centrifuged at 4 ℃ and 12,000rpm (13,400 Xg) for 30sec, the waste liquid is discarded, and CR3 is put into a collection tube.
(iv) RNA extraction was performed on the treated sample using DP419(TIANGEN), and the subsequent procedures were the same as in example 1.
The RNA results obtained in this example using DP419 Trizol cleavage were as follows:
| sample(s) | Nucleic acid concentration (ng/. mu.L) | A260/A280 | A260/A230 |
| 1 | 57.6 | 1.23 | 0.41 |
| 2 | 70.92 | 1.59 | 0.83 |
| 3 | 53.25 | 1.65 | 0.70 |
| 4 | 80.62 | 1.24 | 0.33 |
| 5 | 48.48 | 1.48 | 0.55 |
| 6 | 49.08 | 1.21 | 0.37 |
The result of the invention shows that the effect of using Trizol to perform thallus lysis is better, and using Trizol (RZ/TransZol UP) can also be used to lyse tubercle bacillus, the concentration difference obviously probably has a relation with the amount of picked bacteria, but RZ is slightly better than TransZol UP in the aspect of purity, and the purity of subsequent processes of RW1 rinsing and RW rinsing extraction is better than RD rinsing and RW rinsing. To further improve purity, the RD may be repeatedly rinsed once with DP419 kit, and then BufferRW1 in DP430 may be used instead of BufferRD in DP 419.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (8)
1. The extraction method of the mycobacterium tuberculosis RNA is characterized by comprising the following steps of:
s1, obtaining mycobacterium tuberculosis;
s2, adding the mixture into a tube containing the lysate, uniformly mixing the mixture by vortex, and standing the mixture for 10 to 20min at room temperature;
s3, adding chloroform, violently shaking for 10-30sec, standing at room temperature for 1-5min, and centrifuging for 5-15 mim;
s4, sucking the water phase, slowly adding absolute ethyl alcohol with the volume 0.5 times that of the water phase, uniformly mixing, transferring the obtained solution and the precipitate into an adsorption column CR3 in a TIANGEN DP419 kit, and centrifuging for 20-40 percent;
s5, adding deproteinized liquid RW1 into an adsorption column CR3, centrifuging for 30-60sec, discarding waste liquid, and placing the adsorption column back into the collection tube;
s6, adding DNase I working solution into the center of an adsorption column CR3, and standing at room temperature for 15 min;
s7, adding deproteinized liquid RW1 into an adsorption column CR3, centrifuging for 30-60sec, discarding waste liquid, and placing the adsorption column back into the collection tube;
s8, adding a rinsing solution RW into an adsorption column CR3, standing at room temperature for 1-3min, centrifuging for 30-60sec, pouring off waste liquid, and putting the adsorption column CR3 back into the collection tube;
s9, repeating the step S8;
s10, centrifuging for 1-3min, pouring off waste liquid, placing the adsorption column CR3 at room temperature for 1-10min to thoroughly air-dry the residual rinsing liquid in the adsorption material;
s11 transferring the adsorption column CR3 into a new RNase-Free centrifuge tube, and dripping 30-100RNase-Free ddH into the middle part of the adsorption film2And O, standing at room temperature for 1-3min, and centrifuging for 1-3min to obtain an RNA solution.
2. The method of claim 1, wherein in step S6, DNase I working solution is prepared by: taking 10 mu L of DNase I stock solution, adding 70 mu L of Buffer RDD, and gently mixing uniformly, wherein the preparation of the DNase I stock solution comprises the following steps: the DNase I dry powder was dissolved in 550. mu.L of RNase-Free ddH2And O, mixing the components gently and uniformly, subpackaging and storing at-20 ℃.
3. The method of claim 1, wherein the centrifugation is at a speed of 12,000 rpm.
4. The method according to claim 1, further comprising, before step S5: adding deproteinized solution RD into adsorption column CR3, centrifuging for 20-40sec, discarding waste liquid, and placing CR3 into collection tube.
5. The method according to claim 4, characterized in that the deproteinizing liquid RW1 from TIANGEN DP430 is used instead of the deproteinizing liquid RD from TIANGEN DP 419.
6. The method according to any one of claims 1 to 5, wherein the lysis solution is lysis solution RL in step S2.
7. The method according to claim 6, characterized in that the lysis solution RL is added with β -mercaptoethanol to a final concentration of 1% before use.
8. The method of any one of claims 1 to 5, wherein in step S2, the lysis solution is TransZol UP.
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| CN104388567A (en) * | 2014-11-27 | 2015-03-04 | 大连工业大学 | Rapid detection method for Listeria monocytogene virulence gene inlB |
| CN104694531A (en) * | 2015-03-20 | 2015-06-10 | 中国科学院昆明植物研究所 | Method for extracting total RNA from tea tissue |
| CN111607592A (en) * | 2020-06-30 | 2020-09-01 | 河南大学 | Method for extracting RNA (ribonucleic acid) in early development stage of cotton fiber cells |
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