CN106047865B - A method of extracting total serum IgE from Chinese liquor fermentation fermented grain - Google Patents

A method of extracting total serum IgE from Chinese liquor fermentation fermented grain Download PDF

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CN106047865B
CN106047865B CN201610659247.3A CN201610659247A CN106047865B CN 106047865 B CN106047865 B CN 106047865B CN 201610659247 A CN201610659247 A CN 201610659247A CN 106047865 B CN106047865 B CN 106047865B
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杜海
徐岩
宋哲玮
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Jiangnan University
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Abstract

The method that the invention discloses a kind of to extract total serum IgE from Chinese liquor fermentation fermented grain, belongs to field of biotechnology.The present invention is directed to the particularity of white wine fermented grain sample, by combining sodium laurate (Sodium Laurate, SL) extraction process and guanidinium isothiocyanate-phenol-chloroform extraction process (Trizol), can in 2 hours rapidly extracting group microorganism total serum IgE.Using the microorganism total serum IgE yield of method extraction of the invention is high, integrality is good, purity is preferable, eukaryon and prokaryotic micro-organisms in sample can be effectively extracted.

Description

A method of extracting total serum IgE from Chinese liquor fermentation fermented grain
Technical field
The method that the present invention relates to a kind of to extract total serum IgE from Chinese liquor fermentation fermented grain, belongs to field of biotechnology.
Background technique
Under the conditions of the prior art, 95% or more microorganism can not be cultivated, this is that traditional microbiological ecology is disclosing nature Biggest obstacle in boundary's biological community structure, ecological functions and its interrelationship study.In recent years, pass through macro transcription group Method, the research environment microorganism from mRNA level in-site achieve very big success, and the result obtained is more acurrate, reliable.With To going deep into for the structure and function research of group microorganism, need to extract the total serum IgE of whole microorganisms in a certain ecosystem Basic demand as analysis group microorganism structure and function.
At present some about group microorganism method for extracting total RNA it is immediate be extract soil in group microorganism Total serum IgE, but edaphon is compared with Chinese Maotai-flavor liquor fermented wine unstrained spirits, there is very big difference.Containing abundant in soil The substances such as humic acid, polysaccharide, polyphenol, keep the microorganism total serum IgE purity extracted not high, and be widely present RNA enzyme in environment, RNA is easily degraded by it, brings many difficulties to RNA extraction.Phenolic compound is easily oxidized to quinones substance in homogenate, It is irreversibly combined with RNA, to influence isolating and purifying for total serum IgE.Many physicochemical properties of polysaccharide are similar to RNA, extracting It can be co-precipitated in the process with RNA, it is difficult to separate.It is mainly various grains in white wine fermented grain unlike the case where soil, it is main If sorghum.It is not only rich in polysaccharide polyphenol in grain, but also produces more cometabolisms during the utilization of microorganism Product.Various secondary metabolites are even more that reaction can be difficult to expect with RNA.
The extracting method of RNA is mainly guanidinium isothiocyanate-phenol-chloroform extraction process (Trizol), dodecyl sulphate at present Sodium (Sodium Dodecyl Sulfate, SDS) method, cetyl trimethylammonium bromide (Cetyltrimethylammonium Bromide, CTAB) method and hot borate method etc..Wherein SDS method is preferable for the Total RNAs extraction effect of prokaryotic micro-organisms, but needle To the eukaryotic microorganisms for having cell wall, the shell-broken effect of SDS is very bad, so not being available has protokaryon in existing eukaryon again In group's microorganism Total RNAs extraction.And the operation of CTAB method needs to preheat, the excessively high meeting of temperature is so that RNA degrading enzyme becomes active and leads Cause RNA be degraded, while CTAB method make well it is also long, it is troublesome in poeration, usually also LiCl is needed to be used cooperatively.Hot borate method is not It only needs to add protease, the reagents such as KCl, while you also being needed to preheat, temperature is excessively high also to make RNA degrading enzyme become Actively RNA is caused to be degraded.
In addition, these methods are all single for certain a kind of or a certain microorganism, it is micro- to be not particularly suited for extraction group Biological the case where especially eukaryon and prokaryotic micro-organisms mix.By fermentation fermented grain to China white wine carry out 16S and The identification of ITS amplicon finds the fermentation method in the semi-natural fermentation of China white wine, the fermentation of especially Chinese Maotai-flavor liquor Mode determines that there are the group microorganisms that type and quantity are numerous in the fermentation fermented grain of white wine, including many common and special Different eukaryon and prokaryotes, especially miscellaneous mould, yeast, bacterium, actinomyces etc..Therefore, a kind of letter is developed Just, be suitble to rapidly extracting group microorganism total serum IgE method, be especially suitable for the group microorganism of liquor fermentation process Method for extracting total RNA is necessary.
Summary of the invention
Based on the above issues, the purpose of the invention is to provide one kind to extract always from Chinese liquor fermentation fermented grain The method of RNA is extracted by joint sodium laurate (Sodium Laurate, SL) extraction process and guanidinium isothiocyanate-phenol-chloroform Method (Trizol), can in 2 hours rapidly extracting group microorganism total serum IgE.The microorganism extracted using method of the invention Total serum IgE yield is high, integrality is good, purity is preferable, can effectively extract eukaryon and prokaryotic micro-organisms in sample.
The method that total serum IgE is extracted from liquor fermentation fermented grain, steps are as follows:
(1) fermented grain sample sterile PBS buffer is suspended and the concussion of bead whirlpool is added；
(2) supernatant is taken after low-speed centrifugal, precipitating PBS buffer solution repeated washing, low-speed centrifugal merge supernatant；
(3) the supernatant high speed centrifugation for obtaining step (2) takes precipitating, freezing；
It (4) will be based on volume parts, by 15-25 parts of sodium laurate extraction buffers, 15-25 parts Trizol, 0.5-1.5 parts The extracting mixed liquor of mercaptoethanol and 0.5-1.5 parts of dithiothreitol (DTT)s (m/V) composition, mixes gently, and places on ice；
(5) sample by the freezing of step (3) is placed in the mortar of pre-cooling, pours into liquid nitrogen, is ground to powder rapidly, during which It is continuously added liquid nitrogen, RNA in process of lapping is prevented to be degraded；
(6) ground powder is moved into the Rnase-free centrifuge tube equipped with extracting mixed liquor, is slowly shaken repeatedly It is even；
(7) high speed centrifugation, Aspirate supernatant are placed in RNase-free centrifuge tube；
(8) chloroform of 0.8-1.2 times of volume (for the supernatant volume that previous step is drawn) is added, covers Pipe lid, mixes gently, and places on ice；
(9) high speed centrifugation, Aspirate supernatant are placed in Rnase-free centrifuge tube；
(10) isopropanol of 0.5-1 times of volume (for the supernatant volume that previous step is drawn) is added, mixes It is even, it places on ice；
(11) high speed centrifugation discards supernatant liquid；
(12) ethanol washing that volumetric concentration is 70-80% is added to precipitate, discards supernatant liquid after high speed centrifugation；
(13) RNase-Free ddH is added2O is to get the RNA for arriving dissolution.
In one embodiment of the present invention, PBS buffer solution concentration is 0.1mol/L (configuration method: 42.3ml 1mol/ LNaH2PO4With 57.7mL 1mol/L Na2HPO4, 121 DEG C of sterilizing 15min after being settled to 1L with deionized water).
Contain the humic acid scavenger of 0.25-0.5% (v/v) in one embodiment of the present invention, PBS buffer solution. After being added to humic acid scavenger, obtained total rna concentration concentration is constant and purity is higher.
Sample and PBS are mixed according to the ratio of 1:2 (m/v) in one embodiment of the present invention, the step (1), Bead dosage is the 1/5 of sample size；The concussion time is 2-3min.
In one embodiment of the present invention, the centrifugation time of the step (2) is 4-8min.
In one embodiment of the present invention, Rnase-free centrifuge tube is the 1.5mL used.
In one embodiment of the present invention, ground powder and extracting mixed liquor are according to 1:8-1:12 (m/v) Ratio addition, i.e., every 1g ground powder are added 8-12mL and extract mixed liquor.
In one embodiment of the present invention, the low-speed centrifugal refers to that 450-550rpm, high speed centrifugation refer to 11000- 13000rpm。
In one embodiment of the present invention, the sodium laurate extraction buffer: the Tris-HCl of pH 8.0 The EDTA 0.02mol/L of 0.1mol/L, NaCl0.1mol/L, pH8.0, sodium laurate 10g/L, pH 8.0.
In one embodiment of the present invention, the Trizol reagent can be Invitrogen company The TRI of Reagent, Sigma companyDeng the Trizol reagent for being also possible to above-mentioned company mixes according to a certain percentage It obtains.
In one embodiment of the present invention, step (3), (7), (9), (11) time of centrifugation be 8-15min, step (12) time of centrifugation is 2-5min.
10-25min is slowly shaken up repeatedly in one embodiment of the present invention, the step (6).
In one embodiment of the present invention, the centrifugation of step (7), (9), (11), (12) be under conditions of 0-4 DEG C into Capable.
In one embodiment of the present invention, the step (8) places 0.5-2min on ice.
In one embodiment of the present invention, the step (10) places 15-25min on ice.
In one embodiment of the present invention, the ethyl alcohol volume that step (12) is added is 1mL.
In one embodiment of the present invention, step (13) is first to be placed at room temperature for 1-2min, and 30-50uL RNase- is added Free ddH2O。
In one embodiment of the present invention, the method is specifically:
(1) 25g sample is taken to be dissolved in sterile 0.1mol/L PBS (the 42.3ml 1mol/LNaH of 50ml2PO4And 57.7ml 1mol/L Na2HPO4, 121 DEG C of sterilizing 15min after being settled to 1L with deionized water) and buffer suspension, 5 5-6mm glass are added Pearl, whirlpool shake 2-3min；
(2) 500rpm is centrifuged 5min, takes supernatant, and precipitating is washed repeatedly three times with PBS buffer solution, is collected after centrifugation in whole Clearly；
(3) supernatant 12000rpm is centrifuged 15min, discards supernatant, covers pipe lid, liquid nitrogen frozen；
(4) prepare extracting mixed liquor (by 20 parts of sodium laurate extraction buffers, 20 parts of Trizol, 1 part of mercaptoethanol and 1 Part dithiothreitol (DTT) composition), it mixes gently, places on ice；
(5) sample is placed in mortar by mortar with Liquid nitrogen precooler, pours into liquid nitrogen, is ground to powder rapidly, is during which constantly added Enter liquid nitrogen, RNA in process of lapping is prevented to be degraded；
(6) ground powder (about 0.15g) is moved into the 1.5mL Rnase-free centrifuge tube equipped with mixed liquor, instead It is multiple slowly to shake up 15-20min；
(7) it is taken out after 4 DEG C of centrifugation 10min of 12000rpm, careful Aspirate supernatant is placed in 1.5mL Rnase-free centrifugation Guan Zhong；
(8) isometric chloroform is added, covers pipe lid, mixes gently, place 1min on ice；
(9) it is taken out after 4 DEG C of centrifugation 10min of 12000rpm, careful Aspirate supernatant is placed in 1.5mL Rnase-free centrifugation Pipe；
(10) isopropanol of 0.7 times of volume is added, mixes, places 15-25min on ice；
(11) 4 DEG C of centrifugation 10min of 12000rpm, discard supernatant liquid；
(12) (v/v) ethanol washing of 1ml 75% precipitating is added, 4 DEG C of centrifugations of 12000rpm are centrifuged 3min.Liquid is poured out, It is careful not to pour out precipitating, remaining liquid is sucked out with pipette tips, is careful not to be drawn onto precipitating；
(13) it is placed at room temperature for 1-2min, 30uL RNase-Free ddH is added2O blows and beats repeatedly, mixes, sufficiently dissolves RNA。
In one embodiment of the invention, RNase-Free ddH in the step (13)2It is also protected containing RNA in O Deposit reagent and Dnase.
The present invention has the following advantages and effects:
1) easy to operate: extraction process is simple, easy to operate；Overcome existing SDS, in CTAB and hot boric acid extraction process, It needs to preheat, needs to be added batch-wise protease, the shortcomings that the reagents such as KCl, effectively prevent being heated at high temperature and be easy to accelerate RNA points The probability for solving enzyme degradation of rna, is pre-mixed
2) excellent effect: operating process, whole liquid reagents are added to RNase inhibitor in advance in operating process, all It operates at low temperature, hinders RNA as far as possible and be degraded, to enable RNA concentration in 900-1200ng/uL.
3) applied widely: be applicable in different regions, separate sources China white wine fermented grain in microorganism total serum IgE extraction, By the analysis of subsequent macro transcript profile, eukaryon and prokaryotic micro-organisms have good recovery rate (Fig. 2).
Detailed description of the invention
Fig. 1: the extraction effect under different method for extracting；Wherein swimming lane 1 is the RNA that embodiment 1 obtains, and swimming lane 2 is to implement The RNA that example 2 obtains, swimming lane 3 are the RNA that embodiment 3 obtains, and swimming lane 4 is the RNA that Trizol extraction process extracts, and swimming lane 5 is The RNA obtained using the extracting mixed liquor that mercaptoethanol is not added, swimming lane 6 are to use RNase-Free ddH2O is obtained instead of PBS RNA；
Fig. 2: macro transcriptome analysis.
Specific embodiment
Reagent configuration:
(1) sterile 0.1mol/LPBS buffer:
42.3mL 1mol/L NaH2PO4With 57.7mL 1mol/L Na2HPO4, 121 DEG C after being settled to 1L with deionized water Sterilize 15min.
(2) sodium laurate extraction buffer:
Tris-HCl(pH8.0)0.1mol/L；NaCl 0.1mol/L；EDTA(pH8.0)0.02mol/L；Sodium laurate 10g/L.NaCl 5.844g is accurately weighed, sodium laurate 10g is placed in 1000mL beaker, and 100ml Tris-HCl is added (1mol/L), 40mL EDTA (0.5mol/L) and 800mL RNase-free ddH2O is settled to salt acid for adjusting pH to 8.0 1L, room temperature preservation after sterilizing.
(3) Trizol reagent uses the TRIzol reagent of Invitrogen company.
(4) 75% (v/v) ethyl alcohol: by 7.5 parts of dehydrated alcohols and 2.5 parts of RNase-free ddH2O configuration.
Embodiment 1:
The microorganism total serum IgE of Chinese Maotai-flavor liquor fermented wine unstrained spirits is extracted in accordance with the following methods:
(1) 25g sample is taken to be dissolved in sterile 0.1mol/L PBS (the 42.3ml 1mol/LNaH of 50mL2PO4And 57.7ml1mol/ LNa2HPO4, 121 DEG C of sterilizing 15min after being settled to 1L with deionized water) and buffer suspension, 5g bead is added, whirlpool shakes 2- 3min；
(2) 500rpm is centrifuged 5min, takes supernatant, and precipitating is washed repeatedly three times with PBS buffer solution, is collected after centrifugation in whole Clearly；Remaining impurity in addition to microorganism in fermentation fermented grain is eliminated by step (1) and step (2), wrapped solid grain And it is dissolved in the polysaccharide and heteroacid of liquid；
(3) supernatant 12000rpm is centrifuged 15min, discards supernatant, covers pipe lid, liquid nitrogen frozen；
(4) prepare extracting mixed liquor (by 20 parts of sodium laurate extraction buffers, 20 parts of Trizol, 1 part of mercaptoethanol and 1 Part dithiothreitol (DTT) composition, wherein liquid is figured according to parts by volume, and solid is figured according to mass parts, and quality is than the unit of volume G/mL), mix gently, place on ice；
(5) sample is placed in mortar by mortar with Liquid nitrogen precooler, pours into liquid nitrogen, is ground to powder rapidly, is during which constantly added Enter liquid nitrogen, make microbial cell breakage, and RNA in process of lapping is prevented to be degraded；
(6) ground powder (about 0.15g) is moved into the 1.5mL Rnase-free centrifuge tube equipped with mixed liquor, instead It is multiple slowly to shake up 15-20min；
(7) it is taken out after 4 DEG C of centrifugation 10min of 12000rpm, careful Aspirate supernatant is placed in 1.5mL Rnase-free centrifugation Guan Zhong；
(8) isometric chloroform is added, covers pipe lid, mixes gently, place 1min on ice；
(9) it is taken out after 4 DEG C of centrifugation 10min of 12000rpm, careful Aspirate supernatant is placed in 1.5mL Rnase-free centrifugation Pipe；
(10) isopropanol of 0.7 times of volume is added, mixes, places 15-25min on ice；
(11) 4 DEG C of centrifugation 10min of 12000rpm, discard supernatant liquid；
(12) (v/v) ethanol washing of 1ml 75% precipitating is added, 4 DEG C of centrifugations of 12000rpm are centrifuged 3min.Liquid is poured out, It is careful not to pour out precipitating, remaining liquid is sucked out with pipette tips, is careful not to be drawn onto precipitating；
(13) it is placed at room temperature for 1-2min, 30uL RNase-Free ddH is added2O blows and beats repeatedly, mixes, sufficiently dissolves RNA。
Embodiment 2:
The microorganism total serum IgE of Chinese Fermentation of Fen-flavor Liquors fermented grain is extracted in accordance with the following methods:
(1) it takes 25g sample to be dissolved in the sterile 0.1mol/L PBS buffer solution of 50mL to suspend, 5g bead, whirlpool concussion is added 2-3min；
(2) 550rpm is centrifuged 4min, takes supernatant, and precipitating is washed repeatedly three times with PBS buffer solution, is collected after centrifugation in whole Clearly；Remaining impurity in addition to microorganism in fermentation fermented grain is eliminated by step (1) and step (2), wrapped solid grain And it is dissolved in the polysaccharide and heteroacid of liquid；
(3) supernatant 11000rpm is centrifuged 8min, discards supernatant, covers pipe lid, liquid nitrogen frozen；
(4) prepare extracting mixed liquor (by 15 parts of sodium laurate extraction buffers, 25 parts of Trizol, 0.5 part of mercaptoethanol and 1.5 parts of dithiothreitol (DTT) compositions), it mixes gently, places on ice；
(5) sample is placed in mortar by mortar with Liquid nitrogen precooler, pours into liquid nitrogen, is ground to powder rapidly, is during which constantly added Enter liquid nitrogen, make microbial cell breakage, and RNA in process of lapping is prevented to be degraded；
(6) ground powder (about 0.15g) is moved into the 1.5mL Rnase-free centrifuge tube equipped with mixed liquor, instead It is multiple slowly to shake up 20min；
(7) it is taken out after 4 DEG C of centrifugation 12min of 12000rpm, careful Aspirate supernatant is placed in 1.5mL Rnase-free centrifugation Guan Zhong；
(8) chloroform of 0.8 times of volume is added, covers pipe lid, mixes gently, place 1min on ice；
(9) it is taken out after 4 DEG C of centrifugation 15min of 13000rpm, careful Aspirate supernatant is placed in 1.5mL Rnase-free centrifugation Pipe；
(10) isopropanol of 1 times of volume is added, mixes, places 15-25min on ice；
(11) 4 DEG C of centrifugation 8min of 11000rpm, discard supernatant liquid；
(12) 70% (v/v) ethanol washing precipitating is added, 4 DEG C of centrifugations of 12000rpm are centrifuged 3min.Liquid is poured out, is paid attention to Precipitating is not poured out, remaining liquid is sucked out with pipette tips, is careful not to be drawn onto precipitating；
(13) it is placed at room temperature for 1-2min, 30uL RNase-Free ddH is added2O blows and beats repeatedly, mixes, sufficiently dissolves RNA。
Embodiment 3:
The microorganism total serum IgE of Chinese flavor liquor fermentation fermented grain is extracted in accordance with the following methods:
(1) it takes 25g sample to be dissolved in the sterile 0.1mol/L PBS buffer solution of 50mL to suspend, 5g bead, whirlpool concussion is added 2-3min；
(2) 450rpm is centrifuged 8min, takes supernatant, and precipitating is washed repeatedly three times with PBS buffer solution, is collected after centrifugation in whole Clearly；Remaining impurity in addition to microorganism in fermentation fermented grain is eliminated by step (1) and step (2), wrapped solid grain And it is dissolved in the polysaccharide and heteroacid of liquid；
(3) supernatant 13000rpm is centrifuged 12min, discards supernatant, covers pipe lid, liquid nitrogen frozen；
(4) prepare extracting mixed liquor (25 parts of sodium laurate extraction buffers, 15 parts of Trizol, 1.5 parts of mercaptoethanols and 0.5 part of dithiothreitol (DTT) composition), it mixes gently, places on ice；
(5) sample is placed in mortar by mortar with Liquid nitrogen precooler, pours into liquid nitrogen, is ground to powder rapidly, is during which constantly added Enter liquid nitrogen, make microbial cell breakage, and RNA in process of lapping is prevented to be degraded；
(6) ground powder (about 0.15g) is moved into the 1.5mL Rnase-free centrifuge tube equipped with mixed liquor, instead It is multiple slowly to shake up 15min；
(7) it is taken out after 4 DEG C of centrifugation 8min of 11500rpm, careful Aspirate supernatant is placed in 1.5mL Rnase-free centrifugation Guan Zhong；
(8) chloroform of 1.2 times of volumes is added, covers pipe lid, mixes gently, place 1min on ice；
(9) it is taken out after 4 DEG C of centrifugation 12min of 13000rpm, careful Aspirate supernatant is placed in 1.5mL Rnase-free centrifugation Pipe；
(10) isopropanol of 0.5 times of volume is added, mixes, places 15-25min on ice；
(11) 4 DEG C of centrifugation 15min of 12000rpm, discard supernatant liquid；
(12) (v/v) ethanol washing of 1mL 80% precipitating is added, 4 DEG C of centrifugations of 12000rpm are centrifuged 3min.Liquid is poured out, It is careful not to pour out precipitating, remaining liquid is sucked out with pipette tips, is careful not to be drawn onto precipitating；
(13) it is placed at room temperature for 1-2min, 50uL RNase-Free ddH is added2O blows and beats repeatedly, mixes, sufficiently dissolves RNA。
According to electrophoresis detection result (Fig. 1) display for the RNA solution that the method for embodiment 1-3 obtains, using of the invention The total serum IgE that sodium laurate extraction process combination Trizol extraction process extracts all has two complete 28S and 18S rRNA items Band (the swimming lane 1-3 of Fig. 1), and the total serum IgE for compareing the extraction of Trizol extraction process is not generally visible (swimming lane 4 of Fig. 1), shows this hair Bright method can extract the total serum IgE of high quality.
Control:
Control 1: it using Trizol extraction process, in particular to changes the extracting mixed liquor of step (4) into Trizol and (is free of Have sodium laurate extraction buffer, dithiothreitol (DTT) and mercaptoethanol), other steps and embodiment 1 are consistent；Obtained total serum IgE The swimming lane 4 of electrophoretogram such as Fig. 1.
Control 2: mercaptoethanol is not added in extracting mixed liquor；Other steps and embodiment 1 are consistent；Obtained total serum IgE electrophoresis The swimming lane 5 of figure such as Fig. 1.In addition, dithiothreitol (DTT) is not added in extracting mixed liquor, or dithiothreitol (DTT) and sulfydryl second are not added simultaneously Alcohol, obtained total serum IgE electrophoretogram are similar with the swimming lane 5 of Fig. 1.
Control 3: RNase-Free ddH is used2O replaces the PBS buffer solution in step (1) and (2), other steps and reality It is consistent to apply example 1；The swimming lane 6 of obtained total serum IgE electrophoretogram such as Fig. 1.
1 total rna concentration of table
Concentration (ng/uL) Total content (ug/uL) A260/280
Embodiment 1 632.8 30.3 1.71
Embodiment 2 933.2 60.1 1.82
Embodiment 3 417.5 22.7 1.55
Control 1 107.6 3.8 1.43
Control 2 82.8 2.4 1.29
Control 3 54.3 1.5 1.18
The RNA that embodiment 1 is obtained carries out macro transcript profile sequencing, as a result as shown in Figure 2.The result shows that it is total to extract gained Existing combining yeast in RNA, Pichia pastoris, saccharomyces cerevisiae this kind eukaryotic microorganisms also have lactic acid bacteria, streptococcus, actinomyces This kind of prokaryotic micro-organisms illustrate that this extracting method effectively while can extract eukaryon and prokaryotic micro-organisms.And it originally mentions Taking method, there is no Preferences, can completely save the ratio of eukaryon and prokaryotic micro-organisms in group microorganism.
To sum up, the method for the invention Total RNAs extraction suitable for all kinds of white wine fermented grains, and extraction effect is good, can obtain To two complete 28S and 18S ribosomic RNA bands, the total serum IgE of eukaryotic microorganisms and prokaryotic micro-organisms can also all be extracted Out, the RNA obtained is suitable for subsequent macro transcription group analysis.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

Claims (1)

1. a kind of method for extracting total serum IgE from liquor fermentation fermented grain, which is characterized in that the step of the method is as follows:
(1) fermented grain sample sterile PBS buffer is suspended and the concussion of bead whirlpool is added；
(2) supernatant is taken after low-speed centrifugal, precipitating PBS buffer solution repeated washing, low-speed centrifugal merge supernatant；
(3) the supernatant high speed centrifugation for obtaining step (2) takes precipitating, freezing；
It (4) will be by 15-25 parts of sodium laurate extraction buffers, 15-25 parts of Trizol, 0.5-1.5 parts of mercaptoethanols and 0.5-1.5 The extracting mixed liquor of part dithiothreitol (DTT) composition, mixes gently, and places on ice, the sodium laurate extraction buffer: pH 8.0 Tris-HCl 0.1mol/L, NaCl 0.1mol/L, pH8.0 EDTA 0.02mol/L, sodium laurate 10g/L, pH 8.0；
(5) sample by the freezing of step (3) is placed in the mortar of pre-cooling, pours into liquid nitrogen, is ground to powder rapidly, during which continuous Liquid nitrogen is added, RNA in process of lapping is prevented to be degraded；
(6) ground powder is moved into the Rnase-free centrifuge tube equipped with extracting mixed liquor, is slowly shaken up repeatedly, institute Stating ground powder and extracting mixed liquor is added according to the ratio that m/V is 1:8-1:12；
(7) high speed centrifugation, Aspirate supernatant are placed in Rnase-free centrifuge tube；
(8) chloroform of 0.8-1.2 times of volume is added, covers pipe lid, mixes gently, places on ice；
(9) high speed centrifugation, Aspirate supernatant are placed in Rnase-free centrifuge tube；
(10) isopropanol of 0.5-1 times of volume is added, mixes, places on ice；
(11) high speed centrifugation discards supernatant liquid；
(12) ethanol washing that volumetric concentration is 70-80% is added to precipitate, discards supernatant liquid after high speed centrifugation 2-5min；
(13) it is first placed at room temperature for 1-2min, 30-50 μ L RNase-Free ddH is added2O to get to dissolution RNA,
The low-speed centrifugal refers to that revolving speed is 450-550rpm, and the high speed centrifugation refers to that revolving speed is 11000-13000rpm, step Suddenly centrifugation described in (7), (9), (11) or (12) is carried out under conditions of 0-4 DEG C, step (3), (7), (9) or (11) institute The time for the centrifugation stated is 8-15min.
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