CN103695414B - A kind of method of high efficiency extraction anaerobic grain sludge microorganism total serum IgE - Google Patents
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Abstract
The invention belongs to environmental molecules biology techniques field, a kind of method relating to high efficiency extraction anaerobic grain sludge microorganism total serum IgE, take activated sludge to be centrifuged, abandon supernatant, then in centrifuge tube, add sterilizing bead and stock solution I, being tightened by centrifuge tube pipe lid and be placed on vortex mixer impact, be centrifuged and abandon supernatant, phosphate buffer washs; Add stock solution II incubation; Add TRIzol, centrifuging and taking supernatant after vortex oscillation, add chloroform, vortex oscillation, room temperature is placed and centrifuging and taking supernatant, adds isopropanol, is centrifuged and abandons supernatant after-20 DEG C of placements, add 75% washing with alcohol precipitation, being centrifuged and abandon supernatant, air-dry precipitation, stock solution III dissolves nucleic acid precipitation, do you add DNase? I (RNase? free) and RNase inhibitor, RNA Purification Kit RNA is adopted. Simplify microorganism total serum IgE extraction step, it is achieved that extract the microorganism total serum IgE of anaerobic grain sludge fast and efficiently.
Description
Technical field
The invention belongs to environmental molecules biology techniques field, a kind of method being specifically related to high efficiency extraction anaerobic grain sludge microorganism total serum IgE.
Background technology
Active particle mud is by formed dark brown floccule bodys mixed in together such as the microorganism based on antibacterial and suspended material, colloidal substance, humus, wherein microorganism is the main body of biological wastewater treatment process, and the quality of they physiological statuss directly affects treatment effect. Active particle mud has bio-diversity highly and composition is extremely complex, does not contain only the organic pollution such as heavy metal, humic acid, there is also a large amount of ribonuclease (RNase), therefore extracts RNA from activated sludge and there is certain difficulty. In recent years, along with developing rapidly of Protocols in Molecular Biology, to the multiformity of Microbial Communities in Activated Sludge population, functional gene expression and treatment effect between the research of relation be increasingly subject to the attention of environmental microorganism worker.
There are some researches show, it is based primarily upon DNA level by the relevant research of molecular biology method expansion Microbial Communities in Activated Sludge to carry out, but the microbial diversity detected from DNA level, the microbial ecological analyses such as abundance are not by viability, the impact of the factor such as metabolism status and environmental condition, time of day can not be reflected, the expression analysis of the microbial population analysis carried out on rna level and gene then more can embody situation during analysis truly, thus extracting high-quality RNA is the essential condition studying biological wastewater treatment process Microbial Communities in Activated Sludge Flora dynamics and key controlling gene expression rule on gene expression dose.Extracting method about RNA, do excessive quantifier elimination report both at home and abroad, mainly include Trizol method, guanidine isothiocyanate method, phenol-SDS method etc., but these methods can only extract the RNA obtaining good quality from the materials such as general antibacterial, animal, plant, and the activated sludge of complicated component is less applicable. The environmental sample RNA extraction method reported at present is primarily directed to soil, deposit etc., and active particle mud is different from the characteristic of these environmental samples, not only Biomass is big, and there is zoogloea, and therefore these methods are not very suitable for the activated sludge of complicated component. Although abroad having bibliographical information freeze-thaw method, pearl mill method, RNA isolation kit etc. can extract RNA from the samples such as soil, settled sludge, activated sludge, but these methods have respective shortcoming, freeze-thaw method is very time-consuming, pearl mill method needs special instrument ball mill, test kit expensive, all it is difficult to use in the analysis of a large amount of sample, is rarely reported about the extracting method of RNA in activated sludge additionally, domestic. Additionally, microbiologic population's gene diversity and kind, abundance that different extracting method obtains are all different. Thus find method that is a kind of easy and simple to handle, with low cost and that be suitable for extracting active particle mud total serum IgE, reflect microbial diversity, for utilizing molecular biology method study microbial ecological, the functional gene in activated sludge and express significant comprehensively.
Summary of the invention
A kind of method that it is an object of the invention to provide microorganism Total RNAs extraction that can be used for anaerobic grain sludge quick, effective, high-quality. The method has RNA extraction, and total amount is many, purity is high, palliating degradation degree is low, integrity is good, have abundant bio-diversity.
For achieving the above object, the present invention adopts following technical proposals:
A kind of method of high efficiency extraction anaerobic grain sludge microorganism total serum IgE, comprises the following steps:
1) activated sludge is taken centrifugal in sterile centrifugation tube;
2) abandon supernatant, in centrifuge tube, then add sterilizing bead and stock solution I, centrifuge tube pipe lid is tightened and is placed on vortex mixer impact 3��8min to solve flocculation and to wash mud removing humic acid, centrifugal;
3) abandon supernatant, repeat step 2) process after 1 time, wash mud 1 time with phosphate buffer PBS;
4) take the pretreated mud of step 3), add stock solution II in 30��40 DEG C of incubation 5��15min;
5) TRIzol is added, after vortex oscillation 3��8min, centrifugal;
6) taking supernatant, add chloroform, after vortex oscillation 10��20s, room temperature is placed 1��4min and is centrifuged;
7) take supernatant, add isopropanol, place 15��25min for-20 DEG C, centrifugal;
8) abandoning supernatant, after adding 75% washing with alcohol precipitation, 9000��11000r/min is centrifuged 3��8min;
9) supernatant is abandoned, air-dry precipitation, after dissolving nucleic acid precipitation with stock solution III, adds DNaseI (RNasefree) 25��35U and RNase inhibitor 35��40U, and act on 10��20min in 30��40 DEG C, adopt RNA Purification Kit RNA further.
Preferably, step 1) and 2) described being centrifuged be, 7000��9000r/min, centrifugal 3��8min under 3��8 DEG C of conditions; Preferably, step 1) and 2) described being centrifuged be, 8000r/min, centrifugal 5min under 4 DEG C of conditions.
Preferably, step 2) diameter of described sterilizing bead is 2��3mm, the addition of sterilizing bead is 0.2��0.4g/ml activated sludge; The addition of stock solution I is 1��2mL/ml activated sludge;It is furthermore preferred that the addition of sterilizing bead is 0.3g/ml activated sludge; The addition of stock solution I is 1.4mL/ml activated sludge; Preferably, in step 3), centrifuge tube pipe lid is tightened and is placed on vortex mixer impact 5min to solve flocculation and to wash mud removing humic acid,
Phosphate buffer PBS described in step 3) is phosphate buffer, and PH is 7.4, composition: NaCl137mmol/L, KCl2.7mmol/L, Na2HPO410mmol/L, KH2PO42mmol/L.
Preferably, in step 4), the addition of stock solution II is the 100 �� L/100 pretreated mud of �� L; Preferably, in 37 DEG C of incubation 10min in step 4).
Preferably, the TRIzol addition described in step 5) is 0.5��1.5mL/100 pretreated mud of �� L; It is furthermore preferred that the TRIzol addition described in step 5) is the 1mL/100 pretreated mud of �� L; TRIzol is a kind of total serum IgE extraction agent, and it is mainly composed of phenol.
Preferably, step 5), 6) and 7) described in be centrifuged be the centrifugal 8��12min of 9000��11000r/min; It is furthermore preferred that step 5), 6) and 7) described in be centrifuged as the centrifugal 10min of 10000r/min.
Preferably, in step 6), chloroform addition is 150��240 �� L/100 pretreated mud of �� L; It is furthermore preferred that in step 6), chloroform addition is the 200 �� L/100 pretreated mud of �� L.
Preferably, in step 7), quantity of isopropanol is 150��240 �� L/100 pretreated mud of �� L; It is furthermore preferred that in step 6), quantity of isopropanol is the 200 �� L/100 pretreated mud of �� L.
Preferably, the centrifugal 5min of 10000r/min in step 8).
Preferably, in step 9), the addition of stock solution III is 20��40 �� L/100 pretreated mud of �� L; It is furthermore preferred that the addition of stock solution III is the 30 �� L/100 pretreated mud of �� L in step 9);
DNaseI (RNasefree) described in step 9) is the deoxyribonuclease I without RNase; RNase inhibitor is the recombiant protein of a kind of escherichia coli expression, it is possible to combined in 1:1 ratio and RNaseA, RNaseB or RNaseC by non-competitive fashion, and suppresses the activity of these three enzyme, and RNA is not by these three enzymatic degradation in protection.
Preferably, step 9) adds DNaseI (RNasefree) 30U and RNase inhibitors 4 0U, and acts on 15min in 37 DEG C.
Wherein, all consumptive materials will process and autoclaving through DEPC. Described DEPC is pyrocarbonic acid diethyl ester, is a kind of strong but halfway RNase inhibitor.
" supernatant " as herein described refers to the supernatant.
DEPC processes water: adds 0.1% (v/v) pyrocarbonic acid diethyl ester (DEPC) in sterile distilled water, is stirred at room temperature more than 4 hours, and 121 degree of autoclavings 20 minutes cool down standby.
It is furthermore preferred that technical scheme is as follows:
Take 10mL activated sludge centrifugal 8000r/min in the sterile centrifugation tube of 15mL, 4 DEG C, 5min, after abandoning supernatant, in pipe, add 3g sterilizing bead (diameter 2��3mm) and 1.4mL stock solution I, centrifuge tube pipe lid is tightened and is placed on vortex mixer impact 5min to solve flocculation and to wash mud removing humic acid, centrifugal 8000r/min, 4 DEG C, 5min, abandons supernatant, repeats to process after 1 time, washing mud 1 time with phosphate buffer PBS, precipitation is for lysis. take a certain amount of pretreated mud sample and add the conventional stock solution II of 100 �� L after 37 DEG C of incubation 10min, add 1mLTRIzol, after vortex oscillation 5min, 10000r/min is centrifuged 10min, take supernatant and add 200 �� L chloroforms, after vortex oscillation 15s, room temperature places 2min and in the centrifugal 10min of 10000r/min, take supernatant and add equal-volume isopropanol, place 20min for-20 DEG C, 10000r/min is centrifuged 10min, abandon supernatant, after adding 75% washing with alcohol precipitation, 10000r/min is centrifuged 5min, abandon supernatant air-dry precipitation, after dissolving nucleic acid precipitation with the conventional stock solution III of 30 �� L, add DNaseI (RNasefree) 30U and RNase inhibitors 4 0U, and act on 15min in 37 DEG C, adopt RNA Purification Kit RNA further.Wherein, all consumptive materials will process and autoclaving through DEPC.
Extract RNA stock solution I: Tris-HCl50mmol/L+EDTA20mmol/L+Nacl100mmol/L+PVP1% and be configured to 100ml solution; EDTA is ethylenediaminetetraacetic acid, and Tris-HCl is three (methylol) aminomethane, and PVP is polyvinyl pyrrolidone homopolymer, is called for short stock solution I. PVP is 1% in the concentration of stock solution I. In described extraction RNA stock solution, if no special instructions, described concentration is the solute concentration extracted in RNA stock solution.
Extract RNA stock solution II: 20mg/mL lysozyme+20mg/mL E.C. 3.4.21.64+10%SDS and be configured to 100ml solution; Lysozyme is N-acetylmuramide lycanohydrlase, and E.C. 3.4.21.64 is be the highly active protein enzyme of a kind of Subtilisin enzyme, and SDS is dodecyl sodium sulfate, is called for short stock solution II.
Extract RNA stock solution III: 1.2mLDEPC process water+1mmolBSA and be configured to 100ml solution; It is addition 0.1% (v/v) pyrocarbonic acid diethyl ester (DEPC) in sterile distilled water that DEPC processes water, is stirred at room temperature more than 4 hours, and 121 degree of autoclavings 20 minutes cool down standby. BSA is bovine serum albumin. It is called for short stock solution III.
The beneficial effects of the present invention is:
1, extracting solution prepared by the present invention is lysozyme, E.C. 3.4.21.64, SDS, is beneficial to microorganism wall cracking.
2, containing BSA in extracting solution prepared by the present invention, it is possible to promote the stability of PCR reaction.
3, the extract recipe that prepared by present invention composition, is the commonly used conventional reagent of laboratory, good economy performance.
4, the extracting solution that prepared by the present invention is nontoxic, have no irritating odor, and operates safety.
5, this invention simplifies microorganism total serum IgE extraction step, it is achieved that extract the microorganism total serum IgE of anaerobic grain sludge fast and efficiently.
6, the extracting solution extraction effect that prepared by the present invention is good, and the impurities left such as protein, phenols is less.
By comparing the evaluation indexes such as RNA yield, purity, palliating degradation degree, the amplification ability of specific gene, microbial diversity, investigate and inquire into RNA extraction method and on the impact of activated sludge Total RNAs extraction effect and finally establish one activated sludge RNA extraction method fast and effectively. Namely wash on the basis of precipitating sludge at TENP and PBS, it is respectively adopted lysozyme and TRIzol lytic activity mud antibacterial, chloroform remove the albumen in bacterial lysate and after major part DNA, isopropanol precipitating nucleic acid and DNaseI hydrolysis residual DNA, finally use centrifugal column purifying RNA further.
Result shows, this method can effectively extract high-quality flora RNA, the RNA total amount many (every g mud can extract 169.6 �� gRNA) not only extracted, purity are high, palliating degradation degree is low, integrity good, have abundant bio-diversity, and can carry out the RT-PCR amplified reaction of 16SrRNA and amcrA gene simultaneously; Compared with other method, cost performance is high, there is obvious superiority, a large amount of extractions suitable in activated sludge RNA, simultaneously, T-RFLP result proves that the RNA extraction method microbe species on analyzing sample and the impact of enrichment analysis result are relatively big, and microbiologic population's gene diversity and kind, abundance that different RNA extraction method obtains are all different. This research establishes one high quality RNA extracting method fast and effectively, will be significant in the researchs such as the monitoring change of activated sludge bacteria group motion state, bacterial metabolism function assessment, microbiologic population's chip.
Accompanying drawing explanation
Fig. 1 is the RNA electrophoretogram of embodiment 1;
Fig. 2 is the RNA electrophoretogram of embodiment 2.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1
Active particle mud sample takes from Feitian, Yucheng majoy sewage treatment plant of pharmaceutcal corporation, Ltd.
Take 10mL activated sludge centrifugal 8000r/min in the sterile centrifugation tube of 15mL, 4 DEG C, 5min, after abandoning supernatant, in pipe, add 3g sterilizing bead (diameter 2-3mm) and 1.4mL stock solution I, centrifuge tube pipe lid is tightened and is placed on vortex mixer impact 5min to solve flocculation and to wash mud removing humic acid, centrifugal 8000r/min, 4 DEG C, 5min, abandons supernatant, repeats to process after 1 time, washing mud 1 time with phosphate buffer PBS, precipitation is for lysis. take a certain amount of pretreated mud sample and add the conventional stock solution II of 100 �� L after 37 DEG C of incubation 10min, add 1mLTRIzol, after vortex oscillation 5min, 10000r/min is centrifuged 10min, take supernatant and add 200 �� L chloroforms, after vortex oscillation 15s, room temperature places 2min and in the centrifugal 10min of 10000r/min, take supernatant and add equal-volume isopropanol, place 20min for-20 DEG C, 10000r/min is centrifuged 10min, abandon supernatant, after adding 75% washing with alcohol precipitation, 10000r/min is centrifuged 5min, abandon supernatant air-dry precipitation, after dissolving nucleic acid precipitation with the conventional stock solution III of 30 �� L, add DNaseI (RNasefree) 30U and RNase inhibitors 4 0U, and act on 15min in 37 DEG C, adopt RNA Purification Kit RNA further. take 1 �� LRNA, dilute 50 times, carry out quality testing with ultraviolet spectrophotometer. take 5 �� LRNA, detect RNA band with agarose gel electrophoresis.
Total serum IgE OD260/OD280 is 1.98, and this shows that RNA pollutes without protein or phenol, and electrophoresis result shows that RNA sample band is clear, and 28S close to 2:1, illustrates that RNA mass is higher than 18SrRNA band brightness, sees Fig. 1, table 1.
Table 1RNA quality testing
Embodiment 2
Active particle mud sample takes from sewage treatment plant of bowling Po Co., Ltd of Yucheng majoy.
Take 10mL activated sludge centrifugal 8000r/min in the sterile centrifugation tube of 15mL, 4 DEG C, 5min, after abandoning supernatant, in pipe, add 3g sterilizing bead (diameter 2-3mm) and 1.4mL stock solution I, centrifuge tube pipe lid is tightened and is placed on vortex mixer impact 5min to solve flocculation and to wash mud removing humic acid, centrifugal 8000r/min, 4 DEG C, 5min, abandons supernatant, repeats to process after 1 time, washing mud 1 time with phosphate buffer PBS, precipitation is for lysis. take a certain amount of pretreated mud sample and add the conventional stock solution II of 100 �� L after 37 DEG C of incubation 10min, add 1mLTRIzol, after vortex oscillation 5min, 10000r/min is centrifuged 10min, take supernatant and add 200 �� L chloroforms, after vortex oscillation 15s, room temperature places 2min and in the centrifugal 10min of 10000r/min, take supernatant and add equal-volume isopropanol, place 20min for-20 DEG C, 10000r/min is centrifuged 10min, abandon supernatant, after adding 75% washing with alcohol precipitation, 10000r/min is centrifuged 5min, abandon supernatant air-dry precipitation, after dissolving nucleic acid precipitation with the conventional stock solution III of 30 �� L, add DNaseI (RNasefree) 30U and RNase inhibitors 4 0U, and act on 15min in 37 DEG C, adopt RNA Purification Kit RNA further. take 1 �� LRNA, dilute 50 times, carry out quality testing with ultraviolet spectrophotometer.Take 5 �� LRNA, detect RNA band with agarose gel electrophoresis.
Total serum IgE OD260/OD280 is 1.98, and this shows that RNA pollutes without protein or phenol, and electrophoresis result shows that RNA sample band is clear, and 28S close to 2:1, illustrates that RNA mass is higher than 18SrRNA band brightness, sees Fig. 2, table 2.
Table 2RNA quality testing
Although above-mentioned, the specific embodiment of the present invention is described; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme, those skilled in the art need not pay various amendments or deformation that creative work can make still within protection scope of the present invention.
Claims (12)
1. the method extracting anaerobic grain sludge microorganism total serum IgE, comprises the following steps:
1) activated sludge is taken centrifugal in sterile centrifugation tube,
2) abandon supernatant, in centrifuge tube, then add sterilizing bead and stock solution I, centrifuge tube pipe lid is tightened and is placed on vortex mixer impact 3��8min to solve flocculation and to wash mud removing humic acid, centrifugal; The diameter of described sterilizing bead is 2-3mm, and the addition of sterilizing bead is 0.2��0.4g/ml activated sludge; The addition of stock solution I is 1��2mL/ml activated sludge;
Step 1) and 2) described being centrifuged be, 7000��9000r/min, centrifugal 3��8min under 3��8 DEG C of conditions;
3) abandon supernatant, repeat step 2) process after 1 time, wash mud 1 time with phosphate buffer PBS;
4) step 3 is taken) pretreated mud, add stock solution II in 30��40 DEG C of incubation 5��15min;
5) TRIzol is added, after vortex oscillation 3��8min, centrifugal,
6) taking supernatant, add chloroform, after vortex oscillation 10��20s, room temperature is placed 1��4min and is centrifuged,
7) take supernatant, add isopropanol, place 15��25min for-20 DEG C, centrifugal,
8) abandoning supernatant, after adding 75% washing with alcohol precipitation, 9000��11000r/min is centrifuged 3��8min,
9) supernatant is abandoned, air-dry precipitation, after dissolving nucleic acid precipitation with stock solution III, add DNaseI25��35U and the RNase inhibitor 35��40U of RNasefree, and act on 10��20min in 30��40 DEG C, adopt RNA Purification Kit RNA further;
Wherein, stock solution I: Tris-HCl50mmol/L+EDTA20mmol/L+Nacl100mmol/L+PVP1% is configured to 100ml solution; EDTA is ethylenediaminetetraacetic acid, and Tris-HCl is three (methylol) aminomethane, and PVP is polyvinyl pyrrolidone homopolymer;
Stock solution II: 20mg/mL lysozyme+20mg/mL E.C. 3.4.21.64+10%SDS is configured to 100ml solution; Lysozyme is N-acetylmuramide lycanohydrlase, and E.C. 3.4.21.64 is be the highly active protein enzyme of a kind of Subtilisin enzyme, and SDS is dodecyl sodium sulfate;
Stock solution III: 1.2mLDEPC processes water+1mmolBSA and is configured to 100ml solution; It is addition 0.1% (v/v) pyrocarbonic acid diethyl ester (DEPC) in sterile distilled water that DEPC processes water, is stirred at room temperature more than 4 hours, and 121 degree of autoclavings 20 minutes cool down standby;
All consumptive materials will process and autoclaving through DEPC.
2. the method extracting anaerobic grain sludge microorganism total serum IgE as claimed in claim 1, it is characterised in that the addition of sterilizing bead is 0.3g/ml activated sludge; The addition of stock solution I is 1.4mL/ml activated sludge.
3. the method extracting anaerobic grain sludge microorganism total serum IgE as claimed in claim 1, it is characterized in that, step 3) in, centrifuge tube pipe lid is tightened and is placed on vortex mixer impact 5min to solve flocculation and to wash mud removing humic acid, step 4) in the addition of stock solution II be the 100 �� L/100 pretreated mud of �� L;Step 4) in 37 DEG C of incubation 10min.
4. the as claimed in claim 1 method extracting anaerobic grain sludge microorganism total serum IgE, it is characterised in that step 5) described in TRIzol addition be 0.5��1.5mL/100 pretreated mud of �� L.
5. the as claimed in claim 1 method extracting anaerobic grain sludge microorganism total serum IgE, it is characterised in that step 5) described in TRIzol addition be the 1mL/100 pretreated mud of �� L.
6. the as claimed in claim 1 method extracting anaerobic grain sludge microorganism total serum IgE, it is characterised in that step 5), 6) and 7) described in be centrifuged be that 9000��11000r/min is centrifuged 8��12min.
7. the as claimed in claim 6 method extracting anaerobic grain sludge microorganism total serum IgE, it is characterised in that step 5), 6) and 7) described in be centrifuged and be centrifuged 10min for 10000r/min.
8. the method extracting anaerobic grain sludge microorganism total serum IgE as claimed in claim 1, it is characterised in that step 6) in, chloroform addition is 150��240 �� L/100 pretreated mud of �� L;
Step 7) in, quantity of isopropanol is 150��240 �� L/100 pretreated mud of �� L;
Step 8) in the centrifugal 5min of 10000r/min.
9. the method extracting anaerobic grain sludge microorganism total serum IgE as claimed in claim 1, it is characterised in that step 6) in, chloroform addition is the 200 �� L/100 pretreated mud of �� L; Step 6) in, quantity of isopropanol is the 200 �� L/100 pretreated mud of �� L.
10. the as claimed in claim 1 method extracting anaerobic grain sludge microorganism total serum IgE, it is characterised in that step 9) in the addition of stock solution III be 20��40 �� L/100 pretreated mud of �� L.
11. the method extracting as claimed in claim 1 anaerobic grain sludge microorganism total serum IgE, it is characterised in that step 9) in the addition of stock solution III be the 30 �� L/100 pretreated mud of �� L.
12. the method extracting anaerobic grain sludge microorganism total serum IgE as claimed in claim 1, it is characterised in that step 9) middle DNaseI30U and the RNase inhibitors 4 0U adding RNasefree, and act on 15min in 37 DEG C.
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| 污泥厌氧消化工艺中病原菌VBNC状态的发生与复苏研究;姜谦 等;《准哦更难过优秀硕士学位论文全文数据库 工程科技I辑》;20131215(第S1期);全文 * |
| 活性污泥高质量RNA快速提取方法研究;金敏 等;《环境科学》;20100131;第31卷(第1期);第261页左栏倒数第1-2段，第2.1节，图1 * |
| 活性污泥高质量总RNA 提取的一种有效方法;黎秋华 等;《陕西科技大学学报》;20080831;第26卷(第4期);第69-73页 * |
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