CN117143861A - Method for rapidly extracting total RNA of white spirit Daqu - Google Patents
Method for rapidly extracting total RNA of white spirit Daqu Download PDFInfo
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- CN117143861A CN117143861A CN202311259561.9A CN202311259561A CN117143861A CN 117143861 A CN117143861 A CN 117143861A CN 202311259561 A CN202311259561 A CN 202311259561A CN 117143861 A CN117143861 A CN 117143861A
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- 238000000034 method Methods 0.000 title claims abstract description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 50
- 239000007788 liquid Substances 0.000 claims abstract description 32
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 25
- 238000000605 extraction Methods 0.000 claims abstract description 21
- 238000005406 washing Methods 0.000 claims abstract description 12
- 239000006166 lysate Substances 0.000 claims abstract description 9
- 238000007710 freezing Methods 0.000 claims abstract description 6
- 230000008014 freezing Effects 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 24
- 239000006228 supernatant Substances 0.000 claims description 21
- 239000004570 mortar (masonry) Substances 0.000 claims description 16
- 238000002156 mixing Methods 0.000 claims description 13
- 238000005303 weighing Methods 0.000 claims description 12
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 7
- 238000000227 grinding Methods 0.000 claims description 7
- 230000001954 sterilising effect Effects 0.000 claims description 7
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- BDDLHHRCDSJVKV-UHFFFAOYSA-N 7028-40-2 Chemical compound CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O BDDLHHRCDSJVKV-UHFFFAOYSA-N 0.000 claims description 3
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 claims description 3
- 239000005977 Ethylene Substances 0.000 claims description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- 229910021538 borax Inorganic materials 0.000 claims description 3
- -1 ethylphenyl Chemical group 0.000 claims description 3
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
- 238000000197 pyrolysis Methods 0.000 claims description 3
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 3
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 abstract description 18
- 230000004151 fermentation Effects 0.000 abstract description 18
- 244000005700 microbiome Species 0.000 abstract description 18
- 238000004458 analytical method Methods 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 3
- 238000013518 transcription Methods 0.000 abstract description 2
- 230000035897 transcription Effects 0.000 abstract description 2
- 238000002123 RNA extraction Methods 0.000 description 9
- 235000019441 ethanol Nutrition 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 241000235342 Saccharomycetes Species 0.000 description 2
- XLOMVQKBTHCTTD-UHFFFAOYSA-N Zinc monoxide Chemical compound [Zn]=O XLOMVQKBTHCTTD-UHFFFAOYSA-N 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 235000014692 zinc oxide Nutrition 0.000 description 2
- 239000011787 zinc oxide Substances 0.000 description 2
- RHEXWAQFMZCXIM-UHFFFAOYSA-N NC(N)=N.N=C=S.OC1=CC=CC=C1.ClC(Cl)Cl Chemical compound NC(N)=N.N=C=S.OC1=CC=CC=C1.ClC(Cl)Cl RHEXWAQFMZCXIM-UHFFFAOYSA-N 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000013124 brewing process Methods 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003935 denaturing gradient gel electrophoresis Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- BTURAGWYSMTVOW-UHFFFAOYSA-M sodium dodecanoate Chemical compound [Na+].CCCCCCCCCCCC([O-])=O BTURAGWYSMTVOW-UHFFFAOYSA-M 0.000 description 1
- 229940082004 sodium laurate Drugs 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 235000020097 white wine Nutrition 0.000 description 1
- 235000014101 wine Nutrition 0.000 description 1
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- C12N15/09—Recombinant DNA-technology
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses a rapid extraction method of total RNA of Daqu liquor, belonging to the technical field of bioengineering. The rapid extraction method of the total RNA of the Daqu in white spirit is characterized in that the total RNA of the Daqu microorganisms can be rapidly extracted within 1 hour through liquid nitrogen freezing treatment and the developed RNA lysate treatment extraction method and finally washing by washing liquid. The method provided by the invention can be used for separating and obtaining the total RNA with high concentration and high purity from the Daqu with complex components for the first time, has high extraction speed, can be used for completing the extraction of the total RNA of the Daqu of the white spirit within 1h, can provide technical support for the analysis of transcription information of microorganisms in the Daqu of the white spirit, and has important significance for researching the generation mechanism of important substances in the fermentation process of the Daqu of the white spirit and improving the quality of the Daqu.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for rapidly extracting total RNA of Daqu liquor.
Background
The Chinese white spirit brewing history is long, and the unique taste is deeply favored by the masses, so that the Chinese white spirit brewing history has higher economic value. Daqu is a starter for brewing white spirit, provides saccharification force and liquefaction force for the white spirit brewing process, and is crucial to the formation of flavor in white spirit, so that the quality of Daqu directly determines the yield and quality of white spirit. The microorganisms in the Daqu mainly comprise bacteria, mould and saccharomycetes, wherein the bacteria are the main power for producing aroma, the mould mainly plays a role in saccharification, and the saccharomycetes have the capacity of alcohol and ester production. It can be seen that functional microorganisms in Daqu play an important role in brewing white spirit.
However, only 1% of microorganisms in the environment can be cultured under the experimental conditions, and the limitation results in that information of very few microorganisms in the environment can be obtained, so that the ecological distribution situation of the microorganisms in the wine cheese environment cannot be fully reflected, and along with the wide application of culture-free technology (DNA sequence homology analysis, PCR-DGGE, clone library, second generation sequencing and the like), the knowledge of the diversity and structure of the white wine Daqu microorganism community is relatively clear. At present, research on Daqu microorganisms is mainly focused on a method based on DNA level, but most detected microorganisms are in a dead or dormant state and cannot accurately reflect active microorganisms which really contribute to white spirit Daqu. In contrast, macro-transcriptome sequencing techniques (RNA level) allow for deep knowledge of the expressed genes and can accurately reveal the transcriptionally active microbial community in the environmental sample of interest.
Because RNA is of a single-stranded structure, unlike DNA, the double-stranded structure is stable; and the RNA is very much and has strong activity, whether the RNA is endogenous or exogenous, so that the RNA is very easy to degrade, and the extraction of the RNA is difficult. At present, research on analysis of active microbial communities of Daqu using methods such as macro transcriptomics based on RNA level has not been found. The patent CN106047865A adopts a sodium laurate extraction method and a guanidine isothiocyanate-phenol-chloroform extraction method to extract distilled grains of white spirit, but has high yield of extracted RNA but low purity, wherein the A260/280 value is 1.18-1.82, the 83% sample value is less than 1.8, and the A260/280 value of pure RNA is 1.8-2.2.
Therefore, a high-efficiency and rapid extraction method of total RNA of the white spirit Daqu is needed, and the method is used for analyzing white spirit Daqu microorganisms, and has important significance for researching white spirit Daqu active microorganisms, generating mechanism of important substances and improving Daqu quality.
Disclosure of Invention
Aiming at the technical problems, the invention provides a method for rapidly extracting total RNA of Daqu microorganisms in white spirit, which is characterized in that the total RNA of Daqu microorganisms can be rapidly extracted within 1 hour by liquid nitrogen freezing treatment and an RNA lysate treatment extraction method developed by combining with liquid nitrogen freezing treatment and finally washing with a washing solution.
The technical scheme adopted for solving the technical problems is as follows: the method for rapidly extracting the total RNA of the Daqu liquor comprises the following steps:
a. hammering and crushing Daqu liquor, and freezing and preserving at-80 ℃ under RNase-free condition for standby;
b. fully precooling the mortar with liquid nitrogen, then rapidly putting 3-5g of standby Daqu into the mortar, and rapidly and strongly grinding by adding liquid nitrogen;
c. and (c) scooping 0.2-0.5g of the crushed Daqu treated in the step (b) and 1ml of the lysate, putting the mixture into a centrifuge tube, uniformly mixing and centrifuging, taking supernatant, fully washing the supernatant, adding RNase-free ddH2O, and standing at room temperature to obtain the total RNA of the Daqu of the white spirit.
In the step b, the mortar is sterilized at high temperature and then cooled, then placed in a box containing liquid nitrogen, and the liquid nitrogen is added into the mortar for full precooling.
Further, the amount of the added liquid nitrogen is 50-100ml, and the grinding time is not more than 1min.
In the step c, a weighing scoop is adopted to scoop the Daqu; before the weighing scoop is used, the weighing scoop is cooled after high-temperature sterilization and then is inserted into liquid nitrogen for full precooling.
Further, the high temperature sterilization temperature is 121 ℃ and the time is 30min.
In the step c, the pyrolysis liquid contains guanidine isothiocyanate with the concentration of 0.8mol/L, phenol with the concentration of 1mol/L, ethylene sugar tetraacetic acid with the concentration of 0.03mol/L, ethylphenyl polyethylene glycol with the concentration of 0.5%, beta-thioglycoll with the concentration of 0.01mol/L and sodium borate with the concentration of 0.2mol/L.
In the step c, the washing step is as follows: loading 400-500 μl of supernatant and equal volume of absolute ethanol into a centrifuge tube, mixing, centrifuging, removing supernatant, adding equal volume of isopropanol, mixing, centrifuging, removing supernatant, adding equal volume of 75% ethanol, mixing, centrifuging, removing supernatant, and washing.
Further, the above centrifuge tube was a 1.5ml RNase-free centrifuge tube.
Further, the rotational speed of centrifugation was 13000-15000rpm, the centrifugation time was 2min, and the temperature was 4 ℃.
Furthermore, the RNase-free suction head is adopted to transfer liquid, and operators need to wear medical masks in all operation steps; the operation table surface is sterilized by 75% ethanol.
The beneficial effects of the invention are as follows: the total RNA extraction method provided by the invention can be applied to aerobic and dry Daqu samples. The method is convenient, quick and efficient to operate: the extraction process is simple and easy to operate, and the defect that the existing extraction method needs heating and needs adding reagents such as chloroform, protease and the like is overcome; the operation is rapid, and the extraction of the total RNA of the Daqu can be completed within 1 h; the concentration of the extracted distilled spirit Daqu is up to 100-500 ng/mu l, the purity is high, and the A260/A280 value is 2.0-2.2.
The method provided by the invention can be used for separating and obtaining the total RNA with high concentration and high purity from the Daqu with complex components for the first time, has high extraction speed, can be used for completing the extraction of the total RNA of the Daqu of the white spirit within 1h, can provide technical support for the analysis of transcription information of microorganisms in the Daqu of the white spirit, and has important significance for researching the generation mechanism of important substances in the fermentation process of the Daqu of the white spirit and improving the quality of the Daqu.
Detailed Description
The technical scheme of the invention can be implemented in the following way.
The method for rapidly extracting the total RNA of the Daqu liquor comprises the following steps:
a. hammering and crushing Daqu liquor, and freezing and preserving at-80 ℃ under RNase-free condition for standby;
b. fully precooling the mortar with liquid nitrogen, then rapidly putting 3-5g of standby Daqu into the mortar, and rapidly and strongly grinding by adding liquid nitrogen;
c. and (c) scooping 0.2-0.5g of the crushed Daqu treated in the step (b) and 1ml of the lysate, putting the mixture into a centrifuge tube, uniformly mixing and centrifuging, taking supernatant, fully washing the supernatant, adding RNase-free ddH2O, and standing at room temperature to obtain the total RNA of the Daqu of the white spirit.
In the step b, the mortar is sterilized at high temperature and then cooled, then placed in a box containing liquid nitrogen, and the liquid nitrogen is added into the mortar for full precooling. Preferably, the liquid nitrogen is added in an amount of 50-100ml, and the grinding time is not more than 1min.
In the step c, a weighing scoop is adopted to scoop the Daqu; before the weighing scoop is used, the weighing scoop is cooled after high-temperature sterilization and then is inserted into liquid nitrogen for full precooling.
Preferably, the high temperature sterilization temperature is 121 ℃ and the time is 30min.
In the step c, the pyrolysis liquid contains guanidine isothiocyanate with the concentration of 0.8mol/L, phenol with the concentration of 1mol/L, ethylene sugar tetraacetic acid with the concentration of 0.03mol/L, ethylphenyl polyethylene glycol with the concentration of 0.5%, beta-thioglycoll with the concentration of 0.01mol/L and sodium borate with the concentration of 0.2mol/L. The lysate of the invention can inactivate nuclease released by cells while rapidly lysing the cells, and maintain the integrity of RNA.
In the step c, the washing step is as follows: loading 400-500 μl of supernatant and equal volume of absolute ethanol into a centrifuge tube, mixing, centrifuging, removing supernatant, adding equal volume of isopropanol, mixing, centrifuging, removing supernatant, adding equal volume of 75% ethanol, mixing, centrifuging, removing supernatant, and washing.
Preferably, the centrifuge tube is a 1.5m lRNase-free centrifuge tube.
Preferably, the rotational speed of centrifugation is 13000-15000rpm, the centrifugation time is 2min, and the temperature is 4 ℃.
Preferably, the RNase-free suction head is adopted to transfer liquid, and operators need to wear medical masks in all operation steps; the operation table surface is sterilized by 75% ethanol.
The technical scheme and effect of the present invention will be further described by practical examples.
Examples
EXAMPLE 1 Total RNA extraction of wuliangye Packed in 6 days fermentation
1. Sampling
1 wuliangye bag Qu Yangpin is randomly extracted from 3 wuliangye bags Qu Qufang fermented for 6 days in each yeast room, put into a sterile bag, smashed by a hammer, put into a 50ml centrifuge tube of RNase-free, and rapidly put into a refrigerator at-80 ℃ for standby.
2. RNA extraction material preparation
Sterilizing 3 mortar and weighing spoon at 121deg.C for 30min, oven drying, cooling to room temperature, placing the mortar and weighing spoon into a foam box containing small amount of liquid nitrogen, adding 50-100ml of liquid nitrogen into the mortar, and cooling thoroughly.
3. Extraction of wuliangye bag Bao Quzong RNA after 6 days fermentation
The extraction steps are as follows:
(1) Pouring 3-5g of spare Daqu into the precooled mortar, immediately adding 50-100ml of liquid nitrogen, and rapidly and strongly grinding;
(2) After the sterilized and cooled weighing scoop is inserted into liquid nitrogen for full precooling, 0.2-0.5g of ground Daqu is scooped into a 1.5ml centrifuge tube of RNase-free containing 1ml of lysate, thus obtaining a lysate mixture containing total RNA, and the lysate mixture is centrifuged at high speed;
(3) Sucking 400-500 μl of the supernatant into a 1.5ml centrifuge tube of RNase-free;
(4) Adding absolute ethyl alcohol with the same volume, covering a tube cover, fully shaking, centrifuging at a high speed, and removing supernatant;
(5) Adding isopropyl alcohol with the same volume, covering a tube cover, fully shaking, centrifuging at a high speed, and removing supernatant;
(6) Adding 75% ethanol, mixing, centrifuging at high speed, and removing supernatant;
(7) Adding RNase-free ddH2O, and standing at room temperature for 2min to obtain total RNA.
4. Detection of wuliangye bag Bao Quzong RNA after 6 days of fermentation
The total RNA extracted was extracted by 1. Mu.l, and the purity and concentration thereof were measured by using Nanodrop one from Thermo scintilic company, and the results are shown in Table 1, and it was found that the concentration of RNA in wuliangye bag Bao Quzong after 6 days of fermentation was 344-400 ng/. Mu.l, and the A260/A280 value was 2.15-2.18, and both were 1.8-2.2, indicating that the purity and concentration of RNA were high.
EXAMPLE 2 Total RNA extraction of wuliangye Packed in 15 days fermentation
1. Sampling
1 wuliangye bag Qu Yangpin is randomly extracted from 3 wuliangye bags Qu Qufang fermented for 15 days in each yeast room, put into a sterile bag, smashed by a hammer, put into a 50ml centrifuge tube of RNase-free, and rapidly put into a refrigerator at-80 ℃ for standby.
2. RNA extraction material preparation was performed in the same manner as in example 1.
3. Extraction of wuliangye bag Bao Quzong RNA after 15 days of fermentation was performed in the same manner as in example 1.
4. Detection of wuliangye bag Bao Quzong RNA after 15 days of fermentation
The total RNA extracted was extracted by 1. Mu.l, and the purity and concentration thereof were measured by using Nanodrop one from Thermo scintilic company, and the results are shown in Table 1, and it was found that the concentration of RNA in the wuliangye bag Bao Quzong after 15 days of fermentation was 117-160 ng/. Mu.l, and the A260/A280 value was 1.95-2.00, and both were 1.8-2.2, indicating that the purity and concentration of RNA were high.
EXAMPLE 3 Total RNA extraction of wuliangye Packed in 22 day fermentation
1. Sampling
1 wuliangye bag Qu Yangpin is randomly extracted from 3 wuliangye bags Qu Qufang fermented for 22 days in each yeast room, put into a sterile bag, smashed by a hammer, put into a 50ml centrifuge tube of RNase-free, and rapidly put into a refrigerator at-80 ℃ for standby.
2. RNA extraction material preparation was performed in the same manner as in example 1.
3. Extraction of wuliangye bag Bao Quzong RNA after 22 days of fermentation was performed in the same manner as in example 1.
4. Detection of wuliangye bag Bao Quzong RNA after 22 days of fermentation
The total RNA extracted was extracted by 1. Mu.l, and the purity and concentration thereof were measured by using Nanodrop one from Thermo scintilic company, and the results are shown in Table 1, and it was found that the concentration of RNA in wuliangye bag Bao Quzong was 101.9-124.8 ng/. Mu.l, and the A260/A280 value was 1.93-2.02, both of which were 1.8-2.2, for 22 days of fermentation, indicating that the purity and concentration of RNA were high.
EXAMPLE 4 Total RNA extraction of wuliangye Packed in 30 days fermentation
1. Sampling
1 wuliangye bag Qu Yangpin is randomly extracted from 3 wuliangye bags Qu Qufang fermented for 30 days in each yeast room, put into a sterile bag, smashed by a hammer, put into a 50ml centrifuge tube of RNase-free, and rapidly put into a refrigerator at-80 ℃ for standby.
2. RNA extraction material preparation was performed in the same manner as in example 1.
3. Extraction of wuliangye bag Bao Quzong RNA after 30 days fermentation was performed in the same manner as in example 1.
4. Detection of wuliangye bag Bao Quzong RNA after 30 days of fermentation
The total RNA extracted was extracted by 1. Mu.l, and the purity and concentration thereof were measured by using Nanodrop one from Thermo scintilic company, and the results are shown in Table 1, and it was found that the concentration of the RNA in the wuliangye bag Bao Quzong after 30 days of fermentation was 103.5-122.2 ng/. Mu.l, and the A260/A280 value was 1.81-1.91, and both were 1.8-2.2, indicating that the purity and concentration of the RNA were high.
Table 1 example package Bao Quzong RNA concentration and purity measurements
The method for extracting total RNA of the white spirit Daqu is not only suitable for the strong aromatic white spirit bag Bao Qu, but also suitable for extracting total RNA in various white spirit Daqus, and the method is rapid in extraction, high in concentration and purity of the extracted total RNA of microorganisms, and capable of effectively extracting microorganisms in samples.
Claims (10)
1. The rapid extraction method of the total RNA of the Daqu liquor is characterized by comprising the following steps:
a. hammering and crushing Daqu liquor, and freezing and preserving at-80 ℃ under RNase-free condition for standby;
b. fully precooling the mortar with liquid nitrogen, then rapidly putting 3-5g of standby Daqu into the mortar, and rapidly and strongly grinding by adding liquid nitrogen;
c. scooping 0.2-0.5g of the crushed Daqu treated in the step b and 1ml of the lysate are put into a centrifuge tube for uniform mixing and centrifugation, and the supernatant is taken, fully washed and added with RNase-free ddH 2 And O, standing at room temperature to obtain the total RNA of the white spirit Daqu.
2. The method for rapidly extracting total RNA of white spirit Daqu as defined in claim 1, which is characterized in that: in the step b, the mortar is sterilized at high temperature and then cooled, then placed in a box containing liquid nitrogen, and the liquid nitrogen is added into the mortar for full precooling.
3. The method for rapidly extracting total RNA of white spirit Daqu as defined in claim 2, which is characterized in that: the amount of liquid nitrogen added is 50-100ml, and grinding time is not more than 1min.
4. The method for rapidly extracting total RNA of white spirit Daqu as defined in claim 1, which is characterized in that: in the step c, scooping the Daqu by using a weighing scoop; before the weighing scoop is used, the weighing scoop is cooled after high-temperature sterilization and then is inserted into liquid nitrogen for full precooling.
5. The method for rapidly extracting total RNA of white spirit Daqu according to claim 2 or 4, which is characterized in that: the high-temperature sterilization temperature is 121 ℃ and the time is 30min.
6. The method for rapidly extracting total RNA of white spirit Daqu as defined in claim 1, which is characterized in that: in the step c, the pyrolysis liquid contains guanidine isothiocyanate with the concentration of 0.8mol/L, phenol with the concentration of 1mol/L, ethylene sugar tetraacetic acid with the concentration of 0.03mol/L, ethylphenyl polyethylene glycol with the concentration of 0.5%, beta-thioethanol with the concentration of 0.01mol/L and sodium borate with the concentration of 0.2mol/L.
7. The method for rapidly extracting total RNA of white spirit Daqu as defined in claim 1, which is characterized in that: in the step c, the washing step is as follows: loading 400-500 μl of supernatant and equal volume of absolute ethanol into a centrifuge tube, mixing, centrifuging, removing supernatant, adding equal volume of isopropanol, mixing, centrifuging, removing supernatant, adding equal volume of 75% ethanol, mixing, centrifuging, removing supernatant, and washing.
8. The method for rapidly extracting total RNA of white spirit Daqu according to claim 1 or 7, which is characterized in that: the centrifuge tube was a 1.5ml RNase-free centrifuge tube.
9. The method for rapidly extracting total RNA of white spirit Daqu according to claim 1 or 7, which is characterized in that: the rotational speed of the centrifugation is 13000-15000rpm, the centrifugation time is 2min, and the temperature is 4 ℃.
10. The method for rapidly extracting total RNA of white spirit Daqu as defined in claim 1, which is characterized in that: transferring the liquid by using an RNase-free suction head; all the operation steps are that an operator needs to wear the medical mask; the operation table surface is sterilized by 75% ethanol.
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| CN202311259561.9A CN117143861A (en) | 2023-09-27 | 2023-09-27 | Method for rapidly extracting total RNA of white spirit Daqu |
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| CN202311259561.9A CN117143861A (en) | 2023-09-27 | 2023-09-27 | Method for rapidly extracting total RNA of white spirit Daqu |
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| CN202311259561.9A Pending CN117143861A (en) | 2023-09-27 | 2023-09-27 | Method for rapidly extracting total RNA of white spirit Daqu |
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